International Journal of Molecular Sciences

19 pages, 2777 KiB

Open AccessArticle

Regulation of Transcriptional Activity of Merkel Cell Polyomavirus Large T-Antigen by PKA-Mediated Phosphorylation

by Mar Falquet, Carla Prezioso, Maria Ludvigsen, Jack-Ansgar Bruun, Sara Passerini, Baldur Sveinbjørnsson, Valeria Pietropaolo and Ugo Moens

Int. J. Mol. Sci. 2023, 24(1), 895; https://doi.org/10.3390/ijms24010895 - 3 Jan 2023

Cited by 1 | Viewed by 5070

Abstract

Merkel cell polyomavirus (MCPyV) is the major cause of Merkel cell carcinoma (MCC), an aggressive skin cancer. MCPyV large T-antigen (LTag) and small T-antigen (sTag) are the main oncoproteins involved in MCPyV-induced MCC. A hallmark of MCPyV-positive MCC cells is the expression of [...] Read more.

Merkel cell polyomavirus (MCPyV) is the major cause of Merkel cell carcinoma (MCC), an aggressive skin cancer. MCPyV large T-antigen (LTag) and small T-antigen (sTag) are the main oncoproteins involved in MCPyV-induced MCC. A hallmark of MCPyV-positive MCC cells is the expression of a C-terminal truncated LTag. Protein kinase A (PKA) plays a fundamental role in a variety of biological processes, including transcription by phosphorylating and thereby regulating the activity of transcription factors. As MCPyV LTag has been shown to be phosphorylated and acts as a transcription factor for the viral early and late promoter, we investigated whether LTag can be phosphorylayted by PKA, and whether this affects the transcript activity of LTag. Using a phosphorylation prediction algorithm, serine 191, 203, and 265 were identified as putative phosphorylation sites for PKA. Mass spectrometry of in vitro PKA-phosphorylated peptides confirmed phosphorylation of S203 and S265, but not S191. Full-length LTag inhibited early and late promoter activity of MCPyV, whereas the truncated MKL2 LTag variant stimulated both promoters. Single non-phosphorylable, as well as phosphomimicking mutations did not alter the inhibitory effect of full-length LTag. However, the non-phosphorylable mutations abrogated transactivation of the MCPyV promoters by MKL2 LTag, whereas phosphomimicking substitutions restored the ability of MKL2 LTag to activate the promoters. Triple LTag and MKL2 LTag mutants had the same effect as the single mutants. Activation of the PKA signaling pathway did not enhance MCPyV promoter activity, nor did it affect LTag expression levels in MCPyV-positive Merkel cell carcinoma (MCC) cells. Our results show that phosphorylation of truncated LTag stimulates viral promoter activity, which may contribute to higher levels of the viral oncoproteins LTag and sTag. Interfering with PKA-induced LTag phosphorylation/activity may be a therapeutic strategy to treat MCPyV-positive MCC patients. Full article

(This article belongs to the Special Issue Viruses and Cancers)

► Show Figures

Figure 1

10 pages, 1233 KiB

Open AccessArticle

Myelin Basic Protein in Oligodendrocyte-Derived Extracellular Vesicles as a Diagnostic and Prognostic Biomarker in Multiple Sclerosis: A Pilot Study

by Cristina Agliardi, Franca Rosa Guerini, Milena Zanzottera, Elisabetta Bolognesi, Silvia Picciolini, Domenico Caputo, Marco Rovaris, Maria Barbara Pasanisi and Mario Clerici

Int. J. Mol. Sci. 2023, 24(1), 894; https://doi.org/10.3390/ijms24010894 - 3 Jan 2023

Cited by 15 | Viewed by 4498

Abstract

Approximately 15% of multiple sclerosis (MS) patients develop a progressive form of disease from onset; this condition (primary progressive-PP) MS is difficult to diagnose and treat, and is associated with a poor prognosis. Extracellular vesicles (EVs) of brain origin isolated from blood and [...] Read more.

Approximately 15% of multiple sclerosis (MS) patients develop a progressive form of disease from onset; this condition (primary progressive-PP) MS is difficult to diagnose and treat, and is associated with a poor prognosis. Extracellular vesicles (EVs) of brain origin isolated from blood and their protein cargoes could function as a biomarker of pathological conditions. We verified whether MBP and MOG content in oligodendrocytes-derived EVs (ODEVs) could be biomarkers of MS and could help in the differential diagnosis of clinical MS phenotypes. A total of 136 individuals (7 clinically isolated syndrome (CIS), 18 PPMS, 49 relapsing remitting (RRMS)) and 70 matched healthy controls (HC) were enrolled. ODEVs were enriched from serum by immune-capture with anti-MOG antibody; MBP and MOG protein cargoes were measured by ELISA. MBP concentration in ODEVs was significantly increased in CIS (p < 0.001), RRMS (p < 0.001) and PPMS (p < 0.001) compared to HC and was correlated with disease severity measured by EDSS and MSSS. Notably, MBP concentration in ODEVs was also significantly augmented in PPMS compared to RRMS (p = 0.004) and CIS (p = 0.03). Logistic regression and ROC analyses confirmed these results. A minimally invasive blood test measuring the concentration of MBP in ODEVs is a promising tool that could facilitate MS diagnosis. Full article

(This article belongs to the Special Issue Molecular Biomarkers in Neurological Diseases)

► Show Figures

Figure 1

Figure 1
ODEVs characterization. (A): Exo-Check™ Exosome Antibody Array on an exemplificative ODEVs lysate. In the image are visible exosomal associated markers: FLOT1 (flotillin-1), ICAM1 (intercellular adhesion molecule 1), ALIX (programmed cell death 6 interacting protein), CD81 and CD63 (tetraspanins), EpCAM (epithelial cell adhesion molecule), ANXA5 (annexin A5), TSG101 (tumor susceptibility gene 101) and controls (2 positive assay control, negative control: blank and GM130: cis-golgi matrix protein: control for cellular contamination). (B): Immuno-gold (OMGp antigen detected) TEM micrograph of an exemplificative ODEVs preparation. Scale bar: 100 nm. (C): Representative size distribution graph of nanoparticle tracking analysis (NTA) that shows size and concentration of enriched ODEVs in a sample from an RRMS patient; a frame of the video is also shown. Mean ODEVs concentration (particles/mL) ± SD and mean ODEVs diameter (nm) ± SD obtained by NTA analysis from five ODEVs samples from the three conditions (HC, PPMS, and RRMS). ANOVA tests p values are reported. Full article ">Figure 2
MBP in enriched ODEVs in HC, CIS, RR-MS, and PP-MS. (A): Multiple comparison graphs of MBP concentration in enriched ODEVs, respectively in HC, CIS, RRMS, and PPMS subjects; all data are plotted, and median and interquartile range (IQR) are reported. The reported global p values of the differences between the groups of subjects was calculated by Kruskal–Wallis test for non-parametric distributions. p values of post hoc Dwass–Steel–Critchlow–Fligner for pairwise comparisons are also reported. (B): Multiple comparison graphs of MBP concentration in enriched ODEVs, respectively in HC, (CIS + RRMS) and PP-MS subjects; all data are plotted; median and interquartile range (IQR) are reported. The reported global p values of the differences between the groups of subjects was calculated by Kruskal–Wallis test for non-parametric distributions. p values of post hoc Dwass–Steel–Critchlow–Fligner for pairwise comparisons are also reported. Full article ">Figure 3
ROC curve analysis. (A): ROC curves of MBP in enriched ODEVs: HC vs. MS (CIS + RRMS + PPMS). AUC and p value are reported. (B): ROC curves of MBP in enriched ODEVs: PPMS vs. (CIS + RRMS). AUC and p value are reported. Full article ">Figure 4
(A): Correlation between MBP in ODEVs and clinical scales. Bivariate Pearson’s correlation between MBP concentration in enriched ODEVs and EDSS. (B): Bivariate Pearson’s correlation between MBP concentration in enriched ODEVs and MSSS. Full article ">

15 pages, 3447 KiB

Open AccessArticle

Development of a Duplex LAMP Assay with Probe-Based Readout for Simultaneous Real-Time Detection of Schistosoma mansoni and Strongyloides spp. -A Laboratory Approach to Point-Of-Care

by Beatriz Crego-Vicente, Pedro Fernández-Soto, Juan García-Bernalt Diego, Begoña Febrer-Sendra and Antonio Muro

Int. J. Mol. Sci. 2023, 24(1), 893; https://doi.org/10.3390/ijms24010893 - 3 Jan 2023

Cited by 5 | Viewed by 4102

Abstract

Loop-mediated isothermal amplification (LAMP) is the most popular technology for point-of-care testing applications due its rapid, sensitive and specific detection with simple instrumentation compared to PCR-based methods. Many systems for reading the results of LAMP amplifications exist, including real-time fluorescence detection using fluorophore-labelled [...] Read more.

Loop-mediated isothermal amplification (LAMP) is the most popular technology for point-of-care testing applications due its rapid, sensitive and specific detection with simple instrumentation compared to PCR-based methods. Many systems for reading the results of LAMP amplifications exist, including real-time fluorescence detection using fluorophore-labelled probes attached to oligonucleotide sequences complementary to the target nucleic acid. This methodology allows the simultaneous detection of multiple targets (multiplexing) in one LAMP assay. A method for multiplexing LAMP is the amplification by release of quenching (DARQ) technique by using a 5′-quencher modified LAMP primer annealed to 3′-fluorophore-labelled acting as detection oligonucleotide. The main application of multiplex LAMP is the rapid and accurate diagnosis of infectious diseases, allowing differentiation of co-infecting pathogens in a single reaction. Schistosomiasis, caused among other species by Schistosoma mansoni and strongyloidiasis, caused by Strongyloides stercoralis, are the most common helminth-parasite infections worldwide with overlapping distribution areas and high possibility of coinfections in the human population. It would be of great interest to develop a duplex LAMP to detect both pathogens in the same reaction. In this study, we investigate the use of our two previously developed and well-stablished LAMP assays for S. mansoni and Strongyloides spp. DNA detection in a new duplex real-time eight-primer system based on a modified DARQ probe method that can be performed in a portable isothermal fluorimeter with minimal laboratory resources. We also applied a strategy to stabilize the duplexed DARQ-LAMP mixtures at room temperature for use as ready-to-use formats facilitating analysis in field settings as point-of-care diagnostics for schistosomiasis and strongyloidiasis. Full article

(This article belongs to the Special Issue New Strategies for the Diagnosis and Treatment of Diseases Caused by Parasites)

► Show Figures

Figure 1

Figure 1
Setting up simplex DARQ-LAMP assays. (A) Schistosoma mansoni DARQ-LAMP. (B) Strongyloides venezuelensis DARQ-LAMP. Evaluation of 15% and 10% percentages of quencher probe duplex (QPD) in relation to the total amount of unlabeled FIP and different supplementary MgSO4 concentrations (4, 6 and 8 mM) to obtain final MgSO4 concentrations of 6, 8 and 10 mM are indicated. NTC, non-template control (ultrapure water instead gDNA); RFU, relative fluorescence units. Full article ">Figure 2
Sensitivity assessment of simplex DARQ-LAMP assays using the Genie III handheld device. (A) Analytical sensitivity for Schistosoma mansoni DARQ-LAMP reaction. (B) Analytical sensitivity for Strongyloides venezuelensis DARQ-LAMP reaction. The 10-fold serial dilutions (5 ng/µL to 5 fg/µL) of gDNA from parasites are represented by different shades of green (for Sm) and blue (for Sv). NTC, non-template control (ultrapure water instead gDNA) is represented by grey lines in both cases. RFU, relative fluorescence units. Full article ">Figure 3
Duplex DARQ-LAMP for simultaneous amplification of gDNA from Schistosoma mansoni and Strongyloides venezuelensis using different combinations of gDNA concentrations as template in different volumes of master mixes. (A) Duplex DARQ-LAMP in 25 µL. (B) Duplex DARQ-LAMP in 20 µL. (C) Duplex DARQ-LAMP in 15 µL. Combinations of gDNA concentrations of the two parasites used as templates are indicated on the right panel. NTC, non-template control (ultrapure water instead gDNA). The reactions were carried out in a PCR max Eco 48 Real Time PCR System. Dotted lines indicate fluorescence reading in channel 1 for Strongyloides venezuelensis amplification (probe labelled with 6-FAM) and continuous lines indicate fluorescence reading in channel 4 for Schistosoma mansoni amplification (probe labelled with Cy5). Full article ">Figure 4
Sensitivity assessment of duplex DARQ-LAMP assay using the Genie III handheld device. (A) Analytical sensitivity for simultaneous detection of Schistosoma mansoni gDNA and Strongyloides venezuelensis gDNA using 10-fold serial dilutions. (B) Analytical sensitivity for S. venezuelensis in duplex DARQ-LAMP reaction. Only the excitation (λex = 495) and emission (λem = 520) channel 1 for fluorescence measurement of 6-FAM is represented. (C) Analytical sensitivity for S. mansoni in duplex DARQ-LAMP reaction. Only the excitation (λex = 648) and emission (λem = 668) channel 2 for fluorescence measurement of Cy5 is represented. The 10-fold serial dilutions (5 ng/µL to 5 fg/µL) of gDNA from parasites are represented by different shades of blue (for S. venezuelensis) and green (for S. mansoni). NTC, non-template control (ultrapure water instead gDNA) is represented by grey lines in both cases. RFU, relative fluorescence units. Full article ">Figure 5
Amplification time of duplex Dry-DARQ-LAMP assays as a function of storage time at ambient temperature. Amplification times of different concentrations of gDNA (10 ng/µL, 5 ng/µL and 2.5 ng/µL) from both Schistosoma mansoni (Sm) and Strongyloides venezuelensis (Sv) in duplex DARQ-LAMP assays performed with dry mixtures containing sets of primers and fluorescence labelled-probes tested at 0, 15, 30 and 60-days post-desiccation are represented. Al reactions were performed in a Genie III portable instrument. Full article ">Figure 6
Schematic illustration of customized primers and the principle of the DARQ-LAMP technique. (A) Schematic illustration of a Quencher Probe Duplex (QPD), with a 5′-quencher FIP (Q-F1c + F2 sequence) annealed to a sequence complementary to F1c labeled at the 3′ end with fluorophore (Fd). In this study, from each set of primers used, both FIP primers (comprising F1c + F2 sequences) were labelled at 5′ end by adding a dark quencher (Q-FIP): Iowa Black RQ (IAbRQ) for Schistosoma. mansoni-FIP primer and Iowa Black FQ (IAbFQ) for Strongyloides spp.-FIP primer. F1c sequences complementary probes were designed and labelled with a fluorophore at 3′ end: Cyanine 5 (Cy5) and 6-Carboxyfluorescein (6-FAM) for amplification of S. mansoni and Strongyloides spp., respectively. (B) Schematic diagram of DARQ-LAMP operation, with LAMP inner primers FIP (F1c + F2) and BIP (B1c + B2) and outer primers F3 and B3 and the QPD (Q-FIP + Fd). (1) LAMP is initiated at the F2c sequence of the DNA target, with the Fd probe turned off by hybridization to Q-FIP. This new strand is displaced by upstream synthesis from the external primer F3. (2) The BIP primer (B1c + B2) is anchored to the B2c sequence on the newly synthesized strand. (3) Synthesis from the primer annealed to the B2c sequence displaces the Fd probe. This releases the quenching generating a fluorescent signal. The newly synthesized strand is displaced by the extension from the external primer B3. (4) The resulting structure undergoes exponential amplification in the LAMP reaction. Successive initiations at primer FIP result in additional release of Fd, resulting in exponential detection of the signal. Full article ">

17 pages, 3626 KiB

Open AccessArticle

Concentration-Dependent Efficacy of Recombinant Human Bone Morphogenetic Protein-2 Using a HA/β-TCP Hydrogel Carrier in a Mini-Pig Vertebral Oblique Lateral Interbody Fusion Model

by Hye-Yeong Lee, Ji-In Kang, Hye-Lan Lee, Gwang-Yong Hwang, Keung-Nyun Kim and Yoon Ha

Int. J. Mol. Sci. 2023, 24(1), 892; https://doi.org/10.3390/ijms24010892 - 3 Jan 2023

Cited by 2 | Viewed by 2605

Abstract

Bone morphogenetic protein-2 (BMP-2) is used in the treatment of degenerative spinal disease and vertebral fractures, spine fusion, dental surgery, and facial surgery. However, high doses are associated with side effects such as inflammation and osteophytes. In this study, we performed spinal fusion [...] Read more.

Bone morphogenetic protein-2 (BMP-2) is used in the treatment of degenerative spinal disease and vertebral fractures, spine fusion, dental surgery, and facial surgery. However, high doses are associated with side effects such as inflammation and osteophytes. In this study, we performed spinal fusion surgery on mini-pigs using BMP-2 and a HA/β-TCP hydrogel carrier, and evaluated the degree of fusion and osteophyte growth according to time and dosage. Increasing the dose of BMP-2 led to a significantly higher fusion rate than was observed in the control group, and there was no significant difference between the 8-week and 16-week samples. We also found that the HA + β-TCP hydrogel combination helped maintain the rate of BMP-2 release. In conclusion, the BMP-2-loaded HA/β-TCP hydrogel carrier used in this study overcame the drawback of potentially causing side effects when used at high concentrations by enabling the sustained release of BMP-2. This method is also highly efficient, since it provides mineral matter to accelerate the fusion rate of the spine and improve bone quality. Full article

(This article belongs to the Special Issue Regeneration for Spinal Diseases 3.0)

► Show Figures

Figure 1

Figure 1
In vitro release kinetics according to the composition of the carrier and the concentration of rhBMP-2. (A) The release pattern of 2 μg and 10 μg rhBMP-2 from HA, TH, and HA + TH using ELISA. Mixing rhBMP-2 at a high concentration with HA + TH as a carrier delayed the release of rhBMP-2. (B) The amount of rhBMP-2 released at intervals of 0–1, 1–2, and 2–3 weeks. From 0 to 1 week, HA and TH released much more rhBMP-2 than HA + TH. (C) Concentration-dependent release patterns of rhBMP-2 from HA + TH. Although there was a difference in the amount of rhBMP-2 released depending on the rhBMP-2 concentration, the pattern was almost the same. Most of the rhBMP-2 was released in 0–1 weeks, and there was no significant change at 1–2 weeks. However, when 10 μg of rhBMP-2 was used, it was detected at 2–3 weeks in amounts greater than observed at 1–2 weeks, along with carrier degradation. * p < 0.05 and *** p < 0.001 indicate statistically significant differences. Full article ">Figure 2
Fusion rate and osteophyte analysis of 2-dimensional sections. (A) A schematic image of the lumbar spine and cage implanted axial section. (B) Through micro-CT, the fusion rate was analyzed and scored from 0 to 20 points. (C,D) A significant difference in fusion was clearly observed between the control (Ctrl) and BMP groups. The BMP group had a higher fusion score. (E,F) A significant difference was also found in osteophyte formation, with lower BMP concentrations corresponding to lower scores. * p < 0.05, ** p < 0.01, and *** p < 0.001 indicate statistically significant differences. Full article ">Figure 3
Trabecular bone analysis of 3-dimensional micro-CT images. (A) A 3D-reconstruction image of spine OLIF using micro-CT. (B) Trabecular bone analysis on a 3D-micro-CT image. The bone volume fraction, trabecular thickness, number, and separation were analyzed for total and new bone. In total and new bone, the bone volume fraction and trabecular number increased in proportion to the presence of rhBMP-2. However, fiber strain segregation showed an inverse pattern, decreasing with higher rhBMP-2 concentrations. However, significant differences were not found according to the concentration of rhBMP-2, and the low-rhBMP2-concentration group tended to show higher values for the bone volume fraction. * p < 0.05, ** p < 0.01, and *** p < 0.001 indicate statistically significant differences. Full article ">Figure 4
H&E staining to confirm the bone region and inflammatory reaction. (A) Overall and magnified pictures after hematoxylin and eosin (H&E) staining inside the cage of the fusion area. (B,C) Graphs analyzing the bone area (stained purple and pink). The bone area increased in proportion to the concentration of rhBMP-2 in the 8-week spine samples, but the low-concentration group (500 μg of rhBMP-2) showed higher values in the 16-week spine sample. (D) Magnified image of the fusion region. The BGM carrier was used for the synthesis of the new bone area at 8 weeks. Bone remodeling by osteoclasts was accelerated at 16 w after new bone synthesis was completed and increased in proportion to the concentration of rhBMP-2. Furthermore, inflammatory cells were recruited in the fusion region in the high-concentration group (1000 μg of rhBMP-2). * p < 0.05, ** p < 0.01, and *** p < 0.001 indicate statistically significant differences. Full article ">Figure 5
GT staining of organic and mineralized regions. (A) Overall and magnified pictures after GT staining inside the cage of the fusion area. (B,C) A graph analyzing the osteoid region (stained red and orange) and the mineralized region (stained sky blue). The low-concentration group (500 μg of rhBMP-2) had an overwhelmingly high value of the osteoid area at 8 weeks. It also showed high values of the mineralized region compared to the other group at 16 weeks. (D) Magnified images of the fusion region. In the control (Ctrl) group, the osteoid and mineralized areas formed in small proportions and were located between the BGM carrier. In the low-concentration group (500 μg of rhBMP-2), the osteoid and mineralized areas were mixed at 8 weeks, but the mineralized area increased overwhelmingly at 16 weeks. In the high-concentration group (1000 μg of rhBMP-2), only the mineralized area was observed at all periods. (E) Graphs analyzing the fusion rate and osteophytes. (D) Sagittal section of a lumbar spine from 16 weeks that was subjected to GT staining. Outside the PEEK cage, an increase in osteophytes was observed in proportion to the concentration of rhBMP-2. * p < 0.05, ** p < 0.01, and *** p < 0.001 indicate statistically significant differences. Full article ">Figure 6
VK staining of calcium-positive mineralized regions. (A) Overall and magnified pictures after VK staining inside the cage of the fusion area. (B,C) A graph analyzing calcium-positive mineralized areas (stained dark brown). The calcium-positive area increased in proportion to the concentration of rhBMP-2 in the 8-week spine samples. However, the low-concentration group (500 μg of rhBMP-2) showed high values in the 16-week spine samples. * p < 0.05 and ** p < 0.01 indicate statistically significant differences. Full article ">Figure 7
Establishment of the swine OLIF model. (A) Schematic and X-ray images of the swine oblique lateral interbody fusion (OLIF) in the lumbar spine. rhBMP-2 (0, 500 μg [1.9 mg/cc]), 1000 μg [3.8 mg/cc]) was mixed with 0.53 mL of the bone graft material (BGM). The BGM was used to fill the PEEK cage (14 × 13 × 7 mm) and implanted at the lumbar 2–3 and 4–5 levels. (B) Steps of OLIF surgery in the swine lumbar spine. Full article ">

25 pages, 3787 KiB

Open AccessReview

Bioprinting Technologies and Bioinks for Vascular Model Establishment

by Zhiyuan Kong and Xiaohong Wang

Int. J. Mol. Sci. 2023, 24(1), 891; https://doi.org/10.3390/ijms24010891 - 3 Jan 2023

Cited by 14 | Viewed by 5564

Abstract

Clinically, large diameter artery defects (diameter larger than 6 mm) can be substituted by unbiodegradable polymers, such as polytetrafluoroethylene. There are many problems in the construction of small diameter blood vessels (diameter between 1 and 3 mm) and microvessels (diameter less than 1 [...] Read more.

Clinically, large diameter artery defects (diameter larger than 6 mm) can be substituted by unbiodegradable polymers, such as polytetrafluoroethylene. There are many problems in the construction of small diameter blood vessels (diameter between 1 and 3 mm) and microvessels (diameter less than 1 mm), especially in the establishment of complex vascular models with multi-scale branched networks. Throughout history, the vascularization strategies have been divided into three major groups, including self-generated capillaries from implantation, pre-constructed vascular channels, and three-dimensional (3D) printed cell-laden hydrogels. The first group is based on the spontaneous angiogenesis behaviour of cells in the host tissues, which also lays the foundation of capillary angiogenesis in tissue engineering scaffolds. The second group is to vascularize the polymeric vessels (or scaffolds) with endothelial cells. It is hoped that the pre-constructed vessels can be connected with the vascular networks of host tissues with rapid blood perfusion. With the development of bioprinting technologies, various fabrication methods have been achieved to build hierarchical vascular networks with high-precision 3D control. In this review, the latest advances in 3D bioprinting of vascularized tissues/organs are discussed, including new printing techniques and researches on bioinks for promoting angiogenesis, especially coaxial printing, freeform reversible embedded in suspended hydrogel printing, and acoustic assisted printing technologies, and freeform reversible embedded in suspended hydrogel (flash) technology. Full article

(This article belongs to the Special Issue 3D Printing and Biomaterials for Biological and Medical Application: Recent Advances)

► Show Figures

Figure 1

15 pages, 3688 KiB

Open AccessArticle

Metabolic Signature of Energy Metabolism Alterations and Excess Nitric Oxide Production in Culture Media Correlate with Low Human Embryo Quality and Unsuccessful Pregnancy

by Romina Pallisco, Giacomo Lazzarino, Gabriele Bilotta, Francesca Marroni, Renata Mangione, Miriam Wissam Saab, Maria Violetta Brundo, Alessandra Pittalà, Giuseppe Caruso, Elena Capoccia, Giuseppe Lazzarino, Barbara Tavazzi, Pasquale Bilotta and Angela Maria Amorini

Int. J. Mol. Sci. 2023, 24(1), 890; https://doi.org/10.3390/ijms24010890 - 3 Jan 2023

Cited by 1 | Viewed by 2505

Abstract

Notwithstanding the great improvement of ART, the overall rate of successful pregnancies from implanted human embryos is definitely low. The current routine embryo quality assessment is performed only through morphological criteria, which has poor predictive capacity since only a minor percentage of those [...] Read more.

Notwithstanding the great improvement of ART, the overall rate of successful pregnancies from implanted human embryos is definitely low. The current routine embryo quality assessment is performed only through morphological criteria, which has poor predictive capacity since only a minor percentage of those in the highest class give rise to successful pregnancy. Previous studies highlighted the potentiality of the analysis of metabolites in human embryo culture media, useful for the selection of embryos for implantation. In the present study, we analyzed in blind 66 human embryo culture media at 5 days after in vitro fertilization with the aim of quantifying compounds released by cell metabolism that were not present as normal constituents of the human embryo growth media, including purines, pyrimidines, nitrite, and nitrate. Only some purines were detectable (hypoxanthine and uric acid) in the majority of samples, while nitrite and nitrate were always detectable. When matching biochemical results with morphological evaluation, it was found that low grade embryos (n = 12) had significantly higher levels of all the compounds of interest. Moreover, when matching biochemical results according to successful (n = 17) or unsuccessful (n = 25) pregnancy, it was found that human embryos from the latter group released higher concentrations of hypoxanthine, uric acid, nitrite, and nitrate in the culture media. Additionally, those embryos that developed into successful pregnancies were all associated with the birth of healthy newborns. These results, although carried out on a relatively low number of samples, indicate that the analysis of the aforementioned compounds in the culture media of human embryos is a potentially useful tool for the selection of embryos for implantation, possibly leading to an increase in the overall rate of ART. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Sperm Activation)

► Show Figures

Figure 1

Figure 1
Concentrations of hypoxanthine (A), uric acid (B), and the sum of oxypurines (C) detected in culture media of human embryos at 5 days post-fertilization. The two groups of high-grade (n = 54, empty circles) and low-grade (n = 12, red filled circles) embryos were morphologically classified according to the Gardner scale. The mean values are represented by the horizontal bars. Sum oxypurines = hypoxanthine + uric acid. * Significantly different from high grade, q < 0.0001 (A–C). Full article ">Figure 2
Concentrations of nitrite (A), nitrate (B), and nitrite + nitrate (C), as stable end-products of nitric oxide metabolism, detected in culture media of human embryos at 5 days post-fertilization. The two groups of high-grade (n = 54, empty circles) and low-grade (n = 12, red filled circles) embryos were morphologically classified according to the Gardner scale. The mean values are represented by the horizontal bars. * Significantly different from high grade, q < 0.001, (A–C). Full article ">Figure 3
Concentrations of hypoxanthine (A), uric acid (B), and the sum of oxypurines (C) detected in culture media of human embryos at 5 days post-fertilization. Embryos were divided into those with successful (n = 17, empty circles) or unsuccessful pregnancy (n = 25, red filled circles), independently on the embryo grade. The mean values are represented by the horizontal bars. Sum oxypurines = hypoxanthine + uric acid. * Significantly different from high grade, q < 0.02 (A), q < 0.01 (B), and q < 0.0005 (C). Full article ">Figure 4
Concentrations of nitrite (A), nitrate (B), and nitrite + nitrate (C), as stable end-products of nitric oxide metabolism, detected in culture media of human embryos at 5 days post-fertilization. Embryos were divided into those with successful (n = 17, empty circles) or unsuccessful pregnancy (n = 25, red filled circles), independently on the embryo grade. The mean values are represented by the horizontal bars. * Significantly different from high grade, q < 0.01 (A), q < 0.05 (B), and q < 0.003 (C). Full article ">Figure 5
Receiver Operating Characteristic (ROC) curves of hypoxanthine (A), uric acid (B), and sum oxypurines (C) determined in the culture media of the two groups of human embryos categorized according to morphological quality using the Gardner scale system. The significance of the Area Under the Curve (AUC) is indicated in each panel. The sum of oxypurines = hypoxanthine + uric acid. Full article ">Figure 6
Receiver Operating Characteristic (ROC) curves of nitrite (A), nitrate (B), and nitrite + nitrate (C) determined in the culture media of the two groups of human embryos categorized according to morphological quality using the Gardner scale system. The significance of the Area Under the Curve (AUC) is indicated in each panel. Full article ">Figure 7
Receiver Operating Characteristic (ROC) curves of the sum of oxypurines (A) and nitrite + nitrate (B) determined in culture media of two groups of human embryos categorized according to morphological quality using the Gardner scale system. The significance of the Area Under the Curve (AUC) is indicated in each panel. The sum of oxypurines = hypoxanthine + uric acid. Full article ">

15 pages, 2840 KiB

Open AccessArticle

Genome Assembly of a Relict Arabian Species of Daphnia O. F. Müller (Crustacea: Cladocera) Adapted to the Desert Life

by Waleed Hamza, Khaled M. Hazzouri, Naganeeswaran Sudalaimuthuasari, Khaled M. A. Amiri, Anna N. Neretina, Shamma E. S. Al Neyadi and Alexey A. Kotov

Int. J. Mol. Sci. 2023, 24(1), 889; https://doi.org/10.3390/ijms24010889 - 3 Jan 2023

Cited by 3 | Viewed by 3218

Abstract

The water flea Daphnia O.F. Müller 1776 (Crustacea: Cladocera) is an important model of recent evolutionary biology. Here, we report a complete genome of Daphnia (Ctenodaphnia) arabica (Crustacea: Cladocera), recently described species endemic to deserts of the United Arab Emirates. In this study, [...] Read more.

The water flea Daphnia O.F. Müller 1776 (Crustacea: Cladocera) is an important model of recent evolutionary biology. Here, we report a complete genome of Daphnia (Ctenodaphnia) arabica (Crustacea: Cladocera), recently described species endemic to deserts of the United Arab Emirates. In this study, genome analysis of D. arabica was carried out to investigate its genomic differences, complexity as well as its historical origins within the subgenus Daphnia (Ctenodaphnia). Hybrid genome assembly of D. arabica resulted in ~116 Mb of the assembled genome, with an N50 of ~1.13 Mb (BUSCO score of 99.2%). From the assembled genome, in total protein coding, 5374 tRNA and 643 rRNA genes were annotated. We found that the D. arabica complete genome differed from those of other Daphnia species deposited in the NCBI database but was close to that of D. cf. similoides. However, its divergence time estimate sets D. arabica in the Mesozoic, and our demographic analysis showed a great reduction in its genetic diversity compared to other Daphnia species. Interestingly, the population expansion in its diversity occurred during the megadrought climate around 100 Ka ago, reflecting the adaptive feature of the species to arid and drought-affected environments. Moreover, the PFAM comparative analysis highlights the presence of the important domain SOSS complex subunit C in D. arabica, which is missing in all other studied species of Daphnia. This complex consists of a few subunits (A, B, C) working together to maintain the genome stability (i.e., promoting the reparation of DNA under stress). We propose that this domain could play a role in maintaining the fitness and survival of this species in the desert environment. The present study will pave the way for future research to identify the genes that were gained or lost in this species and identify which of these were key factors to its adaptation to the harsh desert environment. Full article

(This article belongs to the Section Molecular Genetics and Genomics)

► Show Figures

Figure 1

Figure 1
(A) General views of Daphnia arabica: parthenogenetic female and ephippial female and male at copulation. (B) D. arabica whole genome assembly statistics and assembly quality in snail plot view. (C) D. arabica mitogenome map. (D) Mitogenome based phylogenetic tree of D. arabica. Full article ">Figure 2
(A) Repeat landscape annotation in D. arabica genome. (B) D. arabica whole genome characterization with black color of the pie chart reflecting the total genome not occupied with repeats. Full article ">Figure 3
(A) Whole genome shot-gun sequence-based divergence time estimation of D. arabica. (B) Pairwise genome comparison D. arabica vs. D. pulex (left) and D. arabica vs. D. sinensis (right). Full article ">Figure 4
Comparative analysis based on the Daphnia gene families. (A) Venn diagram shows the distribution of PFam gene families between five Daphnia species (D. arabica, D. pulicaria, D. pulex, D. magna and D. galeata). (B) Comparison of SOSS-C in D. arabica, D. pulicaria, D. pulex, D. magna, and D. galeata. Full article ">Figure 5
D. arabica effective population size reduction estimation. Full article ">

18 pages, 5279 KiB

Open AccessArticle

Comprehensive Profiling of ceRNA (circRNA-miRNA-mRNA) Networks in Hypothalamic-Pituitary-Mammary Gland Axis of Dairy Cows under Heat Stress

by Hanfang Zeng, Haibin Xia, Xinling Wang, Yue Wang, Jian Fang, Shujie Li, Yunfei Zhai and Zhaoyu Han

Int. J. Mol. Sci. 2023, 24(1), 888; https://doi.org/10.3390/ijms24010888 - 3 Jan 2023

Cited by 10 | Viewed by 2715

Abstract

Heat stress (HS) is directly correlated with mammary gland dysfunction and the hypothalamic-pituitary-mammary gland (HPM) axis is involved in regulating stress responses and lactation in dairy cows. Circular RNAs (circRNAs) play major roles in regulating transcription and post-transcription but their expression in the [...] Read more.

Heat stress (HS) is directly correlated with mammary gland dysfunction and the hypothalamic-pituitary-mammary gland (HPM) axis is involved in regulating stress responses and lactation in dairy cows. Circular RNAs (circRNAs) play major roles in regulating transcription and post-transcription but their expression in the HPM axis of dairy cows under HS is still unclear. In the present study, we performed RNA sequencing to identify diferentially expressed (DE) circRNAs, DE microRNAs(miRNAs) and DEmRNAs, and performed bioinformatics analysis on those in HPM axis-related tissues of heat-stressed and normal cows. A total of 1680, 1112 and 521 DEcircRNAs, 120, 493 and 108 DEmiRNAs, 274, 6475 and 3134 DEmRNAs were identified in the hypothalamic, pituitary, and mammary gland tissues, respectively. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses indicated that the MAPK signaling pathway is potentially a key pathway. Competitive endogenous RNA (ceRNA) networks related to HS response and lactation regulation were established in three tissues. In conclusion, our results indicate that HS induces differential circRNA expression profiles in HPM axis-related tissues, and the predicted ceRNA network provides a molecular basis for regulating the stress response and lactation regulation in heat-stressed dairy cows. Full article

(This article belongs to the Special Issue Advances in circRNA Biology)

► Show Figures

Figure 1

Figure 1
Differences in endocrine hormones, antioxidant enzymes and heat shock proteins between the NHS and HS groups. (A–D) serum endocrine hormones. (E) serum antioxidant enzymes. (F) serum heat shock protein. Data are presented as the mean ± SEM (n = 20 per group); * p < 0.05, *** p < 0.001. Full article ">Figure 2
Histomorphological observation of HPM-axis related tissues and neurotransmitters in hypothalamus between the NHS and HS groups. (A) Histomorphological observation of HPM-axis related tissues. HY: hypothalamus; PI: pituitary; MA: mammary gland. (B) The percentage of basophils in pituitary. (C–G) Neurotransmitters in hypothalamus. Data are presented as the mean ± SEM (n = 6 per group); *** p < 0.001. Full article ">Figure 3
Identification and characterization of circRNAs. (A) Reads align region statistics. (B) circRNAs length distribution. (C) Annotation type of circRNAs. (D) Sample violin diagram in HPMaxis-related tissues between the NHS and HS groups. Full article ">Figure 4
(A) Volcano plot of DEcircRNAs in hypothalamus. (B) Volcano plot of DEcircRNAs pituitary. (C) Volcano plot of DEcircRNAs mammary gland. Red dots represent up-regulated circRNAs and blue dots represent down-regulated circRNAs. (D) Venn diagram of DEcircRNAs in the HPM axis-related tissues between NHS and HS groups. (E) Comparison of the circRNAs expression levels determined by RNA-seq and RT-qPCR. Full article ">Figure 5
GO functional enrichment and KEGG pathway analysis on the source genes of DEcircRNAs in the hypothalamus (A,B), pituitary (C,D), and mammary gland (E,F) in dairy cows under heat stress. Full article ">Figure 6
(A) Statistics of differential expression of miRNAs. (B) Comparison of the miRNAs expression levels determined by RNA-seq and RT-qPCR. (C–F) KEGG pathway analysis on the target genes of DE miRNAs in the hypothalamus, pituitary and mammary gland in dairy cows under heat stress. Full article ">Figure 7
(A) Statistics of differential expression of mRNAs. (B) Comparison of the mRNAs expression levels determined by RNA-seq and RT-qPCR. (C–F) KEGG pathway analysis on the DE mRNAs in the hypothalamus, pituitary and mammary gland in dairy cows under heat stress. Full article ">Figure 8
ceRNA regulatory networks in HPM axis of dairy cows under heat stress. (A,B) Subnetwork of HSPH1 and GHRHR in hypothalamus. (C–F) Subnetwork of IGF1, HSP90AA1, PRL, and GH1 in pituitary. (G,H) Subnetwork of HSP90B1, HSPA1A, PRLR and IGF-1 in mammary gland. Full article ">

15 pages, 1120 KiB

Open AccessReview

Oxidative Stress and Immune Response in Melanoma: Ion Channels as Targets of Therapy

by Alessia Remigante, Sara Spinelli, Angela Marino, Michael Pusch, Rossana Morabito and Silvia Dossena

Int. J. Mol. Sci. 2023, 24(1), 887; https://doi.org/10.3390/ijms24010887 - 3 Jan 2023

Cited by 16 | Viewed by 3557

Abstract

Oxidative stress and immune response play an important role in the development of several cancers, including melanoma. Ion channels are aberrantly expressed in tumour cells and regulate neoplastic transformation, malignant progression, and resistance to therapy. Ion channels are localized in the plasma membrane [...] Read more.

Oxidative stress and immune response play an important role in the development of several cancers, including melanoma. Ion channels are aberrantly expressed in tumour cells and regulate neoplastic transformation, malignant progression, and resistance to therapy. Ion channels are localized in the plasma membrane or other cellular membranes and are targets of oxidative stress, which is particularly elevated in melanoma. At the same time, ion channels are crucial for normal and cancer cell physiology and are subject to multiple layers of regulation, and therefore represent promising targets for therapeutic intervention. In this review, we analyzed the effects of oxidative stress on ion channels on a molecular and cellular level and in the context of melanoma progression and immune evasion. The possible role of ion channels as targets of alternative therapeutic strategies in melanoma was discussed. Full article

(This article belongs to the Special Issue The Role of Reactive Oxygen Species (ROS) in the Immune System and Health)

► Show Figures

Figure 1

Figure 1
Major reactive species sources in melanocytes. The increase of reactive oxygen species (H2O2, O2−•) and/or reactive nitrogenous species (NO and ONOO−) induces severe damages to major biomolecules, resulting in DNA and protein oxidation, as well as lipoperoxidation, that compromise cellular structure and function. Consequently, these alterations can induce inflammation and can initiate tumorigenesis processes (e.g., cell proliferation and adaptive immune resistance). To maintain acceptable levels of reactive species, melanocytes cells usually increase their antioxidant systems to protect cells from oxidative stress damage and restore physiological redox balance. The redox balance in the cell is normally regulated by a complex antioxidant system. Endogenous antioxidants include catalase (CAT), superoxide dismutase (SOD), glutathione peroxidases (GPXs) and glutathione (GSH). In particular, GSH metabolism protects melanocytes from the toxic effects of H2O2 formed during melanin synthesis. GSH metabolism, therefore, appears to be critically important to the maintenance of melanocyte cell viability [<a href="#B62-ijms-24-00887" class="html-bibr">62</a>]. Instead, natural antioxidant compounds can be obtained from the diet, e.g., beta-carotene (vitamin A), alpha-ascorbic acid (vitamin C), tocopherol (vitamin E) [<a href="#B44-ijms-24-00887" class="html-bibr">44</a>]. The figure was created using BioRender.com. Full article ">Figure 2
Major hypoxia and cellular oxidative stress-dependent mechanisms involving ion channels in melanoma. Events directly linked to oxidative stress are represented in red: (1) the ER-residing STIM2 protein gates ORAI Ca2+ channels at the plasma membrane. This mechanism acts as a regulator in cancer-associated processes, such as cell migration. Oxidative stress-induced C313 sulfonylation hinders STIM2 oligomerization, thus causing inhibition of SOCE; (2) increased oxidative stress leads to up-regulation of TRP channels (e.g., TRPM2 and TRPA1) and induces an increase of intracellular Ca2+ levels, thus promoting cancer progression; (3) oxidative stress-induced increase of intracellular Ca2+content is accompanied by K+ efflux via BK channels, which preserves the ion balance and helps to maintain the Ca2+ entry, thus promoting cancer cell migration. Moreover, this oxidation condition stimulates the activation of the medium-conductance voltage-independent KCa3.1, a target implicated in the promotion of cell migration; (4) during oxidative stress, TASK-3 channel down-regulation impedes the mitochondrial activity of cancer cells. In addition, TASK-3 knockdown in melanoma cells leads to reduced viability and increased apoptosis; and (5) hypoxic melanoma cells activate a mechanism of immune evasion through the miR-192-5p efflux by connexin 43 channel (Cx43). The figure was created using BioRender.com. Full article ">

4 pages, 186 KiB

Open AccessEditorial

Research of Mitochondrial Function, Structure, Dynamics and Intracellular Organization

by Andrey V. Kuznetsov and Michael J. Ausserlechner

Int. J. Mol. Sci. 2023, 24(1), 886; https://doi.org/10.3390/ijms24010886 - 3 Jan 2023

Cited by 2 | Viewed by 2618

Abstract

Mitochondria have been recognized as the energy (in the form of ATP)-producing cell organelles, required for cell viability, survival and normal cell function [...] Full article

(This article belongs to the Special Issue Research of Mitochondrial Function, Structure, Dynamics and Intracellular Organization 2.0)

17 pages, 1022 KiB

Open AccessReview

Diagnostic and Prognostic Role of Extracellular Vesicles in Pancreatic Cancer: Current Evidence and Future Perspectives

by Alberto Nicoletti, Marcantonio Negri, Mattia Paratore, Federica Vitale, Maria Elena Ainora, Enrico Celestino Nista, Antonio Gasbarrini, Maria Assunta Zocco and Lorenzo Zileri Dal Verme

Int. J. Mol. Sci. 2023, 24(1), 885; https://doi.org/10.3390/ijms24010885 - 3 Jan 2023

Cited by 10 | Viewed by 4069

Abstract

Pancreatic cancer is one of the most aggressive tumors, with a dismal prognosis due to poor detection rates at early stages, rapid progression, post-surgical complications, and limited effectiveness of conventional oncologic therapies. There are no consistently reliable biomarkers or imaging modalities to accurately [...] Read more.

Pancreatic cancer is one of the most aggressive tumors, with a dismal prognosis due to poor detection rates at early stages, rapid progression, post-surgical complications, and limited effectiveness of conventional oncologic therapies. There are no consistently reliable biomarkers or imaging modalities to accurately diagnose, classify, and predict the biological behavior of this tumor. Therefore, it is imperative to develop new and improved strategies to detect pancreatic lesions in the early stages of cancerization with greater sensitivity and specificity. Extracellular vesicles, including exosome and microvesicles, are membrane-coated cellular products that are released in the outer environment. All cells produce extracellular vesicles; however, this process is enhanced by inflammation and tumorigenesis. Based on accumulating evidence, extracellular vesicles play a crucial role in pancreatic cancer progression and chemoresistance. Moreover, they may represent potential biomarkers and promising therapy targets. The aim of the present review is to review the current evidence on the role of extracellular vesicles in pancreatic cancer. Full article

(This article belongs to the Collection Feature Papers in Molecular Immunology)

► Show Figures

Figure 1

14 pages, 444 KiB

Open AccessReview

Microglial Activation and Priming in Alzheimer’s Disease: State of the Art and Future Perspectives

by Giulia Bivona, Matilda Iemmolo, Luisa Agnello, Bruna Lo Sasso, Caterina Maria Gambino, Rosaria Vincenza Giglio, Concetta Scazzone, Giulio Ghersi and Marcello Ciaccio

Int. J. Mol. Sci. 2023, 24(1), 884; https://doi.org/10.3390/ijms24010884 - 3 Jan 2023

Cited by 23 | Viewed by 4769

Abstract

Alzheimer’s Disease (AD) is the most common cause of dementia, having a remarkable social and healthcare burden worldwide. Amyloid β (Aβ) and protein Tau aggregates are disease hallmarks and key players in AD pathogenesis. However, it has been hypothesized that microglia can contribute [...] Read more.

Alzheimer’s Disease (AD) is the most common cause of dementia, having a remarkable social and healthcare burden worldwide. Amyloid β (Aβ) and protein Tau aggregates are disease hallmarks and key players in AD pathogenesis. However, it has been hypothesized that microglia can contribute to AD pathophysiology, as well. Microglia are CNS-resident immune cells belonging to the myeloid lineage of the innate arm of immunity. Under physiological conditions, microglia are in constant motion in order to carry on their housekeeping function, and they maintain an anti-inflammatory, quiescent state, with low expression of cytokines and no phagocytic activity. Upon various stimuli (debris, ATP, misfolded proteins, aggregates and pathogens), microglia acquire a phagocytic function and overexpress cytokine gene modules. This process is generally regarded as microglia activation and implies that the production of pro-inflammatory cytokines is counterbalanced by the synthesis and the release of anti-inflammatory molecules. This mechanism avoids excessive inflammatory response and inappropriate microglial activation, which causes tissue damage and brain homeostasis impairment. Once the pathogenic stimulus has been cleared, activated microglia return to the naïve, anti-inflammatory state. Upon repeated stimuli (as in the case of Aβ deposition in the early stage of AD), activated microglia shift toward a less protective, neurotoxic phenotype, known as “primed” microglia. The main characteristic of primed microglia is their lower capability to turn back toward the naïve, anti-inflammatory state, which makes these cells prone to chronic activation and favours chronic inflammation in the brain. Primed microglia have impaired defence capacity against injury and detrimental effects on the brain microenvironment. Additionally, priming has been associated with AD onset and progression and can represent a promising target for AD treatment strategies. Many factors (genetics, environmental factors, baseline inflammatory status of microglia, ageing) generate an aberrantly activated phenotype that undergoes priming easier and earlier than normally activated microglia do. Novel, promising targets for therapeutic strategies for AD have been sought in the field of microglia activation and, importantly, among those factors influencing the baseline status of these cells. The CX3CL1 pathway could be a valuable target treatment approach in AD, although preliminary findings from the studies in this field are controversial. The current review aims to summarize state of the art on the role of microglia dysfunction in AD pathogenesis and proposes biochemical pathways with possible targets for AD treatment. Full article

(This article belongs to the Collection Feature Papers in Molecular Neurobiology)

► Show Figures

Figure 1

28 pages, 4383 KiB

Open AccessArticle

The Prolonged Treatment of Salmonella enterica Strains with Human Serum Effects in Phenotype Related to Virulence

by Bożena Futoma-Kołoch, Michał Małaszczuk, Kamila Korzekwa, Małgorzata Steczkiewicz, Andrzej Gamian and Gabriela Bugla-Płoskońska

Int. J. Mol. Sci. 2023, 24(1), 883; https://doi.org/10.3390/ijms24010883 - 3 Jan 2023

Viewed by 5301

Abstract

Salmonella enterica as common pathogens of humans and animals are good model organisms to conduct research on bacterial biology. Because these bacteria can multiply in both the external environments and in the living hosts, they prove their wide adaptability. It has been previously [...] Read more.

Salmonella enterica as common pathogens of humans and animals are good model organisms to conduct research on bacterial biology. Because these bacteria can multiply in both the external environments and in the living hosts, they prove their wide adaptability. It has been previously demonstrated that prolonged exposition of Salmonella serotype O48 cells to normal human serum led to an increase in resistance to sera in connection with the synthesis of very long O-antigen. In this work, we have studied the phenotype connected to virulence of Salmonella enterica strains that were subjected to consecutive passages in 50% human serum from platelet-poor plasma (SPPP). We found that eight passages in SPPP may not be enough for the bacteria to become serum-resistant (S. Typhimurium ATCC 14028, S. Senftenberg). Moreover, C1q and C3c complement components bound to Salmonellae (S. Typhimurium ATCC 14028, S. Hammonia) membrane proteins, which composition has been changed after passaging in sera. Interestingly, passages in SPPP generated genetic changes within gene fljB, which translated to cells’ motility (S. Typhimurium ATCC 14028, S. Erlangen). One strain, S. Hammonia exposed to a serum developed a multi-drug resistance (MDR) phenotype and two S. Isaszeg and S. Erlangen tolerance to disinfectants containing quaternary ammonium salts (QAS). Furthermore, colonial morphotypes of the serum adaptants were similar to those produced by starter cultures. These observations suggest that overcoming stressful conditions is manifested on many levels. Despite great phenotypic diversity occurring after prolonged exposition to SPPP, morphotypes of colonies remained unchanged in basic media. This work is an example in which stable morphotypes distinguished by altered virulence can be confusing during laboratory work with life-threatening strains. Full article

(This article belongs to the Collection State-of-the-Art Molecular Microbiology in Poland)

► Show Figures

Figure 1

Figure 1
Survival of Salmonella enterica strains during eight passages in 50% human serum from platelet-poor plasma (SPPP). Black/bottom line–survival of Salmonella enterica in 50% SPPP counted as log10 of colony forming units per ml (CFU/mL−1) connected with dashed trendline formula; blue/upper line (control)–survival of bacteria in heat-inactivated (56 °C, 30 min) sera (HISPPP); SS–serum sensitive; SR–serum resistant; (A)—S. Isaszeg P8 (SR); (B)—S. Erlangen P8 (SR); (C)—S. Hammonia P8 (SR); (D)—S. Enteritidis ATCC 13076 P8 (SR); (E)—S. Typhimurium ATCC 14028 P8 (SS); (F)—S. Senftenberg P8 (SS). Full article ">Figure 2
Analysis of complement C1q (A) and C3c (B) deposition on S. enterica cells. Cells of the WT serum-sensitive (SS) strains and the serum-resistant (SR, P8) were incubated in 50% SPPP for 12 min at 37 °C. C1q and C3c components deposited on cells were identified by indirect ELISA with appropriate antibodies as described in the methods. Data are presented as the means of standard deviations of triplicate determinations from three independent experiments, after subtraction of negative control (A490 0.125); * indicates a statistically significant difference in values (p < 0.05) with respect to the data obtained for WT strains. Full article ">Figure 3
SDS-PAGE analysis of the membrane protein (MP) profiles (12 μg/10 μL per well) of the studied Salmonella enterica isolates. Definitions of abbreviations: M—protein marker (AppliChem, Darmstadt, Germany), Line 1—S. Isaszeg, Line 2—S. Erlangen, Line 3—S. Hammonia, Line 4—S. Typhimurium ATCC 14028, Line 5—S. Enteritidis ATCC 13076, Line 6—S. Senftenberg; (A) WT (serum sensitive, SS) Salmonella strains, (B) Salmonella strains passaged eight times in 50% SPPP (P8); SR—serum resistant. The bottom half of <a href="#ijms-24-00883-f003" class="html-fig">Figure 3</a> represents densitograms obtained with Image Lab. V. 4.1 (Bio-Rad, Hercules, CA, USA). Full article ">Figure 4
Dot blot C1q and C3c-binding assays performed for membrane proteins (MPs) isolated from serum-resistant (SR) and serum-sensitive (SS) strains. 1—MPs of SS S. Hammonia, 2—MPs of SR S. Hammonia P8, 3—MPs of SS S. Typhimurium ATCC 14028, 4—MPs of SS S. Typhimurium ATCC 14028 P8. MPs in concentrations of (A) (25 × diluted in H2OmiliQ, 0.24 mg/mL−1) and (B) (50 × diluted in H2OmiliQ, 0.12 mg/mL−1) were loaded onto nitrocellulose membrane. K1—positive control 1; K2—positive control 2; K3—negative control 1, instead of MPs PBS was used; K4, negative control 2—instead of SPPP PBS was used. Full article ">Figure 5
FljB profiles for Salmonella enterica. Definitions of abbreviations: WT—wild-type serum-sensitive (SS) strain, P8—strain passaged in 50% SPPP. Full article ">Figure 6
Morphology of bacterial colonies produced by serum-sensitive (SS) and serum-resistant (SR) strains. (A) Salmonella strains not passaged in SPPP, (B) Salmonella strains passaged eight times in 50% SPPP; 1—S. Erlangen, 2—S. Hammonia, 3—S. Typhimurium ATCC 14028. Full article ">Figure 7
Swarming motility assay. Definitions of abbreviations: (A)—Salmonella strains not passaged in SPPP, (B)—Salmonella strains passaged eight times in 50% SPPP; 1—S. Erlangen, 2—S. Hammonia, 3—S. Isaszeg, 4—S. Senftenberg, 5—S. Enteritidis ATCC 13076, 6—S. Typhimurium ATCC 14028. Full article ">Figure 8
Swimming motility assay. Definitions of abbreviations: (A)—the average (n = 3) diameter of bacterial cultures (WT and P8) grown on 0.3% soft agar supplemented with TTC at 28 °C and 37 °C. (B)—differences between inoculum growth of pairs S. Erlangen (SS)/S. Erlangen P8 (SR) and S. Typhimurium ATCC 14028 (SS)/S. Typhimurium ATCC 14028 P8 (SS); WT—wild-type strain (SS), P8—strain passaged eight times in 50% SPPP; * indicates a statistically significant difference in values (p < 0.05) with respect to the WT strains, SS—serum sensitive, SR—serum resistant. Full article ">Scheme 1
Chronological system of own research conducted on Salmonella strains involving steps taken in this work. Full article ">

15 pages, 3161 KiB

Open AccessArticle

PLA2G7/PAF-AH as Potential Negative Regulator of the Wnt Signaling Pathway Mediates Protective Effects in BRCA1 Mutant Breast Cancer

by Yue Liao, Susann Badmann, Fabian Kraus, Nicole Elisabeth Topalov, Doris Mayr, Thomas Kolben, Anna Hester, Susanne Beyer, Sven Mahner, Udo Jeschke, Fabian Trillsch, Bastian Czogalla and Alexander Burges

Int. J. Mol. Sci. 2023, 24(1), 882; https://doi.org/10.3390/ijms24010882 - 3 Jan 2023

Cited by 3 | Viewed by 2871

Abstract

Past studies have confirmed that aberrant activation of the Wnt/β-catenin signaling is associated with tumorigenesis and metastasis in breast cancer, while the role of platelet-activating factor acetylhydrolase (PLA2G7/PAF-AH) in this signaling pathway remains unclear. In this study, we analyze the functional impact of [...] Read more.

Past studies have confirmed that aberrant activation of the Wnt/β-catenin signaling is associated with tumorigenesis and metastasis in breast cancer, while the role of platelet-activating factor acetylhydrolase (PLA2G7/PAF-AH) in this signaling pathway remains unclear. In this study, we analyze the functional impact of PAF-AH on BRCA1 mutant breast cancer and explore its relationship to the Wnt signaling pathway. By performing immunohistochemistry, PAF-AH expression and β-catenin expression were examined in both BRCA1 WT and BRCA1 mutant breast cancer specimens. The BRCA1 mutant breast cancer cell line HCC1937 was used for in vitro experiments to assess the impact of PAF-AH on cellular functions. The intracellular distribution of β-catenin depending on PLA2G7/PAF-AH expression was investigated by immunocytochemistry. Significantly higher nuclear expression levels of PAF-AH were found in BRCA1 mutant tissue specimens than in BRCA1 WT samples. Cell viability, proliferation, and the motility rate of HCC1937 were significantly enhanced after PLA2G7 silencing, which indicated a protective role of PAF-AH in breast cancer. Nuclear PAF-AH expressed correlatedly with membranous β-catenin. PLA2G7 silencing provoked the β-catenin translocation from the membrane to the nucleus and activated Wnt signaling downstream genes. Our data showed a protective effect of high PAF-AH expression in BRCA1 mutant breast cancer. PAF-AH may achieve its protective effect by negatively regulating the Wnt pathway. In conclusion, our research sheds new light on the regulatory pathways in BRCA1 mutant breast cancer. Full article

(This article belongs to the Special Issue Breast Cancer Mechanistic Insights and Targeted Therapies)

► Show Figures

Figure 1

Figure 1
BRCA1 mutation carriers show stronger nuclear PAF-AH and membranous β-catenin staining intensity. Compared to BRCA1 WT, cases with BRCA1 mutation (n = 22; median IRScore = 3) expressed significantly more nuclear PAF-AH, the outliers in BRCA1-WT were indicated with small circle and asterisk (p < 0.001) (A). Among all cases with membranous β-catenin expression, BRCA1 mutation carriers (n = 14; median IRScore = 12) showed higher membranous expression than those without BRCA1 mutation (n = 19; median IRScore = 4) (p < 0.001) (B). Full article ">Figure 2
Only the BRCA1 negative BC cell line HCC1937 shows a relevant expression of PLA2G7/PAF-AH. Basal mRNA (qPCR; (A)) and protein (Western blot analysis; (B)) expression of PLA2G7/PAF-AH in five BC cell lines were compared to the expression in the benign breast cell line MCF10A. Significant results are indicated by asterisks (*: p ≤ 0.05, **: p ≤ 0.01), and non-significant results by diamonds (#: p > 0.05). Full article ">Figure 3
Successful downregulation of PLA2G7/PAF-AH by siRNA knockdown. The efficiency of siRNA knockdown was investigated by qPCR (A) and western blot analysis (B). The best knockdown of PLA2G7 was achieved after an incubation time of 60 h (p < 0.01; A). Concordant to the result on RNA level, western blot analysis showed a decrease in protein expression (p < 0.05), confirming a successful downregulation (B). Full article ">Figure 4
PLA2G7/PAF-AH downregulation causes cancer progression by an activation of cell viability, proliferation, and migration. In MTT assays, the viability of PLA2G7 silenced HCC1937 significantly increased (p < 0.01) (A). BrdU results (B) showed an increased proliferation capacity (p < 0.01), and wound healing assays indicated an augmented migration ability of PLA2G7 downregulated HCC1937 cells (p < 0.05) (C,D). All assays were conducted 60 h after transfection with siRNA-PLA2G7 and scaled to a control treated with scrambled siRNA. Significant results are indicated by asterisks (*: p ≤ 0.05) and double asterisks (**: p ≤ 0.001). Full article ">Figure 5
Activation of the Wnt signaling pathway by PLA2G7 silencing. After 60 h transfection with siRNA-PLA2G7, the expression of PAF-AH was significantly suppressed (A), while the expression of β-catenin shifted from the membrane to the nucleus, activating Wnt downstream genes (B). In addition, HCC1937 cells show a higher mitotic activity after gene knockdown. Full article ">Figure 6
PAF-PAFR affects the Wnt/β-catenin signaling pathway by phosphorylation. The active state of Wnt/β-catenin signaling is shown on the left. The inactive state of Wnt/β-catenin signaling and degradation of β-catenin is shown on the right. PAF–PAFR signaling and cell functions changed after activation by phosphorylation (middle). Full article ">

19 pages, 4398 KiB

Open AccessArticle

iPSC-Derived MSCs Are a Distinct Entity of MSCs with Higher Therapeutic Potential than Their Donor-Matched Parental MSCs

by Hae-Ri Lee, Soo Kim, Sungho Shin, Seon-Yeong Jeong, Dae-Won Lee, Sun-Ung Lim, Ji Yeon Kang, Mi-Young Son, Cheolju Lee, Kyung-Rok Yu, Myungshin Kim and Il-Hoan Oh

Int. J. Mol. Sci. 2023, 24(1), 881; https://doi.org/10.3390/ijms24010881 - 3 Jan 2023

Cited by 9 | Viewed by 4152

Abstract

Mesenchymal stromal cells derived from induced pluripotent stem cells (iMSCs) have been proposed as alternative sources of primary MSCs with various advantages for cell therapeutic trials. However, precise evaluation of the differences between iMSCs and primary MSCs is lacking due to individual variations [...] Read more.

Mesenchymal stromal cells derived from induced pluripotent stem cells (iMSCs) have been proposed as alternative sources of primary MSCs with various advantages for cell therapeutic trials. However, precise evaluation of the differences between iMSCs and primary MSCs is lacking due to individual variations in the donor cells, which obscure direct comparisons between the two. In this study, we generated donor-matched iMSCs from individual bone marrow-derived MSCs and directly compared their cell-autonomous and paracrine therapeutic effects. We found that the transition from primary MSCs to iMSCs is accompanied by a functional shift towards higher proliferative activity, with variations in differentiation potential in a donor cell-dependent manner. The transition from MSCs to iMSCs was associated with common changes in transcriptomic and proteomic profiles beyond the variations of their individual donors, revealing expression patterns unique for the iMSCs. These iMSC-specific patterns were characterized by a shift in cell fate towards a pericyte-like state and enhanced secretion of paracrine cytokine/growth factors. Accordingly, iMSCs exhibited higher support for the self-renewing expansion of primitive hematopoietic progenitors and more potent immune suppression of allogenic immune responses than MSCs. Our study suggests that iMSCs represent a separate entity of MSCs with unique therapeutic potential distinct from their parental MSCs, but points to the need for iMSC characterization in the individual basis. Full article

(This article belongs to the Special Issue Mesenchymal Stem Cells and Their Derived Products in Aging and Diseases)

► Show Figures

Figure 1

Figure 1
Reprogramming of bone marrow MSCs into induced pluripotent stem cells (iPSCs) and generation of iMSCs from same donor-origin iPSCs. (A) Characterization of iPSCs established from primary MSCs. Microscopic examination for morphology in bright field (BF) and expression of indicated pluripotency markers by immunofluorescence analysis. Representative images are shown. (B) Immunofluorescence analysis of tri-lineage differentiation potential of iPSCs using endodermal markers (SOX17, FOXA2), mesodermal markers (α-SMA and DESMIN) and ectodermal markers (NESTIN, TUJ1). Nuclei were stained with DAPI. (C) Karyotyping of established iPSCs by G-banding analysis confirming normal human karyotypes. (D) Schematic diagram of the process for induction of MSCs from iPSC. (E) Flow cytometry analysis of MSC markers CD34−/CD45−, CD73+, CD90+, CD105+ in MSCs and donor-matched iMSCs. The number represents each individual donor. Full article ">Figure 2
Comparison of proliferation potential between iMSCs and donor-matched primary MSCs. (A) Cumulative cell number assay of 3 lines of iMSC and donor-matched primary MSCs. Numbers represent individual donor cell origin. p = passage number. (B) Relative telomere length of individual iMSCs in comparison to donor-matched MSCs expressed as a T/S ratio (telomere/single copy) measured by real time PCR. Full article ">Figure 3
Colonogenic and differentiation potential of iMSCs in comparison with donor-matched MSCs. (A) Each individual iMSC line and their parental donor-matched MSCs were subjected to colony formation (CFU-F) assays and stained with crystal violet. Representative images for CFU-F assays (left panel) and their quantification (right) are shown (n = 3, mean ± SEM, *; p < 0.05). (B) Osteogenic differentiation of MSCs and iMSCs analyzed by Alizarin Red S staining for calcium deposition. Representative images for osteogenic differentiation (left) and quantification as determined by the absorbance at 405nm (right) are shown (n = 3, mean ± SEM, *; p < 0.05). (C) Adipogenic differentiation of MSCs and iMSCs analyzed by Oil- Red O staining for lipid droplets. Representative images for adipogenic differentiation (left) and the quantification by absorbance at 520nm (right) (n = 3, mean ± SEM, *; p < 0.05) are shown. Full article ">Figure 4
Transcriptomic changes in the transition from primary MSCs to donor-matched iMSCs. (A) PCA plot of gene expression data obtained from RNA-seq data for iMSCs (blue dots) and primary MSCs (red dots). (B) Heat map of RNA-seq transcriptome analysis for the top 300 most variable genes from iMSCs and donor-matched primary MSCs. (C) Volcano plot displaying the log2 fold changes (x axis) against the t test-derived −log10 FDR (false discovery rate) (y axis) for whole transcriptome differentially expressed between iMSCs and primary MSCs. (D) Gene ontology (GO) analysis for differentially expressed genes between iMSCs and MSCs. The top 10 most significant list is shown. Full article ">Figure 5
Molecular characterization of iMSCs in comparison with primary MSCs. (A) iMSCs and donor-matched primary MSCs were compared for expression of genes involved in the cell fate control of MSCs including genes for EMT, pluripotency and pericyte markers. Shown are normalized expression levels of transcripts in each iMSCs relative to the levels in their donor-matched primary MSCs, analyzed by quantitative RT-PCR (n = 3, mean ± SEM, *; p < 0.05). (B) Expression levels of human CD146 in iMSCs and their donor-matched primary MSCs. Shown are the representative flow cytometry profiles (left) and quantification of expression levels by mean fluorescent intensity (MFI) (right) (n = 3 for each group, mean ± SEM, *; p < 0.05). (C) Comparisons for expression levels of human NESTIN between individual iMSCs and their donor-matched parental iMSCs. The expression levels were measured by Western blot. Representative profile (upper) and quantification by image analysis (Image J) (lower) are shown. Full article ">Figure 6
Comparison of secretomes between iMSCs and donor-matched MSCs. (A) Cytokine/growth factors in secretomes analyzed by cytokine array. The serum-free supernatant from individual iMSCs and their donor-matched MSCs were harvested and subjected to analysis of cytokine/growth factors using cytokine array blots. Shown are the relative levels of each indicated cytokine/growth factor in the supernatants from iMSCs relative to the levels from their parental MSCs. (B) The secretome from each group of MSCs were analyzed by LC-MS/MS. From each secretome, proteins found in at least two independent iMSC or MSC lines were selected and the top 100 secreted proteins of these secretome were identified and compared between the iMSCs and MSCs. (C) Gene ontology of secreted proteins up-regulated in iMSCs in comparison to donor-matched MSCs. Shown are the gene clusters of up-regulated proteins in supernatants of iMSCs compared to primary MSCs (p-value < 0.05). Full article ">Figure 7
Comparison of HSC supporting activity between iMSCs and donor-matched primary MSCs. (A) Schematic illustration of experimental scheme. MSCs from each group were co-cultured with CD34+ cells for 4 days and expansion of each indicated subsets of hematopoietic progenitors were analyzed by colony assays or flow cytometry. (B) % of CD34+ cells in each co-culture condition. (C) % of CD34+90+ cells and (D) number of CD34+90+ cells after co-culture with the iMSCs and donor-matched primary MSCs (n = 6/group, mean ± SEM, *; p < 0.05). (E) Effect of co-culture on colony forming cells (CFC). Shown are the mean numbers ± SEM of CFCs and their lineages from 300 input CD34+ cells after 4 days co-culture with each MSC group (n = 6/group, mean ± SEM, *; p < 0.05). Full article ">Figure 8
Suppression of the allogenic immune reaction by iMSCs in comparison to donor-matched primary MSCs. (A) Representative flow cytometry profiles for analysis of allogenic immune responses by T-cells. Mononuclear cells (MNCs) from umbilical cord blood were stained with CFSE and stimulated with anti-CD3/CD28 microbeads and IL-2 for 6 days in the presence or absence of MSCs or iMSCs. Shown are representative flow cytometry profiles for gating T-cells and their changes in CFSE intensity. (B) Quantitative measurement of immune suppressive function of MSCs and iMSCs. The suppression of T-cell proliferation by each type of MSCs was analyzed by the decrease in mean CFSE fluorescence intensity in CD3+ or CD3+CD4+ and CD3+CD8+ cells. Shown are the mean fluorescence intensity of CFSE in T-cells of each group relative to the intensity in the unstimulated group (one-way ANOVA followed by Tukey’s test, *; p < 0.05, n = 3). Full article ">

20 pages, 5725 KiB

Open AccessArticle

Alginate Beads Containing Cerium-Doped Mesoporous Glass and Curcumin: Delivery and Stabilization of Therapeutics

by Debora Carrozza, Gianluca Malavasi, Erika Ferrari and Maria Cristina Menziani

Int. J. Mol. Sci. 2023, 24(1), 880; https://doi.org/10.3390/ijms24010880 - 3 Jan 2023

Cited by 2 | Viewed by 1940

Abstract

Cancer is a leading cause of death worldwide, its genesis and progression are caused by homeostatic errors, and reactive oxygen species play a major role in promoting aberrant cancer homeostasis. In this scenario, curcumin could be an interesting candidate due to its versatile [...] Read more.

Cancer is a leading cause of death worldwide, its genesis and progression are caused by homeostatic errors, and reactive oxygen species play a major role in promoting aberrant cancer homeostasis. In this scenario, curcumin could be an interesting candidate due to its versatile antioxidant, anti-inflammatory, anti-tumor, anti-HIV, and anti-infection properties. Nonetheless, the major problem related to its use is its poor oral bioavailability, which can be overcome by encapsulating it into small particles, such as hydrogel beads containing mesoporous silica. In this work, various systems have been synthesized: starting from mesoporous silica glasses (MGs), cerium-containing MGs have been produced; then, these systems have been loaded with 4 to 6% of curcumin. Finally, various MGs at different compositions have been included in alginate beads. In vitro studies showed that these hybrid materials enable the stabilization and effective delivery of curcumin and that a synergic effect can be achieved if Ce³⁺/Ce⁴⁺ and curcumin are both part of the beads. From swelling tests, it is possible to confirm a controlled curcumin release compartmentalized into the gastrointestinal tract. For all beads obtained, a curcumin release sufficient to achieve the antioxidant threshold has been reached, and a synergic effect of cerium and curcumin is observed. Moreover, from catalase mimetic activity tests, we confirm the well-known catalytic activity of the couple Ce³⁺/Ce⁴⁺. In addition, an extremely good radical scavenging effect of curcumin has been demonstrated. In conclusion, these systems, able to promote an enzymatic-like activity, can be used as drug delivery systems for curcumin-targeted dosing. Full article

(This article belongs to the Special Issue Biomaterials and Their Composites for Biomedical Applications and Beyond)

► Show Figures

Figure 1

18 pages, 4175 KiB

Open AccessArticle

Double Stimuli-Responsive di- and Triblock Copolymers of Poly(N-isopropylacrylamide) and Poly(1-vinylimidazole): Synthesis and Self-Assembly

by Elena Yu. Kozhunova, Anna V. Plutalova, Andrey V. Sybachin, Alexander V. Chertovich and Elena V. Chernikova

Int. J. Mol. Sci. 2023, 24(1), 879; https://doi.org/10.3390/ijms24010879 - 3 Jan 2023

Viewed by 2377

Abstract

For the first time, double stimuli-responsive properties of poly(N-isopropylacrylamide) (PNIPA) and poly(1-vinylimidazole) (PVIM) block copolymers in aqueous solutions were studied. The synthesis of PNIPA₆₀-b-PVIM₉₀ and PNIPA₂₈-b-PVIM₆₂-b-PNIPA₂₉ was performed using [...] Read more.

For the first time, double stimuli-responsive properties of poly(N-isopropylacrylamide) (PNIPA) and poly(1-vinylimidazole) (PVIM) block copolymers in aqueous solutions were studied. The synthesis of PNIPA₆₀-b-PVIM₉₀ and PNIPA₂₈-b-PVIM₆₂-b-PNIPA₂₉ was performed using reversible addition–fragmentation chain transfer (RAFT) polymerization. The polymers were characterized by size exclusion chromatography and ¹H NMR spectroscopy. The conformational behavior of the polymers was studied using dynamic light scattering (DLS) and fluorescence spectroscopy (FS). It was found that PNIPA and block copolymers conformation and ability for self-assembly in aqueous medium below and above cloud point temperature depend on the locus of hydrophobic groups derived from the RAFT agent within the chain. Additionally, the length of PVIM block, its locus in the chain and charge perform an important role in the stabilization of macromolecular micelles and aggregates below and above cloud point temperature. At 25 °C the average hydrodynamic radius (R_h) of the block copolymer particles at pH 3 is lower than at pH 9 implying the self-assembling of macromolecules in the latter case. Cloud points of PNIPA₆₀-b-PVIM₉₀ are ~43 °C and ~37 °C at a pH of 3 and 9 and of PNIPA₂₈-b-PVIM₆₂-b-PNIPA₂₉ they are ~35 °C and 31 °C at a pH of 3 and 9. Around cloud point independently of pH, the R_h value for triblock copolymer rises sharply, achieves the maximum value, then falls and reaches the constant value, while for diblock copolymer, it steadily grows after reaching cloud point. The information about polarity of microenvironment around polymer obtained by FS accords with DLS data. Full article

(This article belongs to the Special Issue Synthesis of Advanced Polymer Materials)

► Show Figures

Figure 1

17 pages, 1445 KiB

Open AccessReview

Choroid Plexus Aquaporins in CSF Homeostasis and the Glymphatic System: Their Relevance for Alzheimer’s Disease

by Cristina Municio, Laura Carrero, Desireé Antequera and Eva Carro

Int. J. Mol. Sci. 2023, 24(1), 878; https://doi.org/10.3390/ijms24010878 - 3 Jan 2023

Cited by 17 | Viewed by 6169

Abstract

The glymphatic system, a fluid-clearance pathway involved in brain waste clearance, is known to be impaired in neurological disorders, including Alzheimer’s disease (AD). For this reason, it is important to understand the specific mechanisms and factors controlling glymphatic function. This pathway enables the [...] Read more.

The glymphatic system, a fluid-clearance pathway involved in brain waste clearance, is known to be impaired in neurological disorders, including Alzheimer’s disease (AD). For this reason, it is important to understand the specific mechanisms and factors controlling glymphatic function. This pathway enables the flow of cerebrospinal fluid (CSF) into the brain and subsequently the brain interstitium, supported by aquaporins (AQPs). Continuous CSF transport through the brain parenchyma is critical for the effective transport and drainage of waste solutes, such as toxic proteins, through the glymphatic system. However, a balance between CSF production and secretion from the choroid plexus, through AQP regulation, is also needed. Thus, any condition that affects CSF homeostasis will also interfere with effective waste removal through the clearance glymphatic pathway and the subsequent processes of neurodegeneration. In this review, we highlight the role of AQPs in the choroid plexus in the modulation of CSF homeostasis and, consequently, the glymphatic clearance pathway, with a special focus on AD. Full article

(This article belongs to the Special Issue The Molecular and Cellular Mechanisms of Neurodegenerative Diseases)

► Show Figures

Figure 1

Figure 1
Illustration of choroid plexus epithelial cells (left) and tight junctions at the luminal membrane (right). CP: choroid plexus; CSF: cerebrospinal fluid; JAMs: junctional adhesion molecules; ZO: zonula occludens. Full article ">Figure 2
Brain lymphatic drainage system. (A) Glymphatic system clears solutes and waste of the brain parenchyma due to the influx of CSF from the periarterial space. Once the CSF is mixed with the ISF, they drain into the perivenous space. Part (B) shows a summary of the other brain drainage systems that transport the CSF through the meningeal lymphatic vessels and/or perineural pathways, such as olfactory nerve, to the cervical lymph nodes AQP: aquaporin; CNS: central nervous system; CSF: cerebrospinal fluid; ISF: interstitial fluid. Full article ">Figure 3
Relationship between choroid plexus and glymphatic system. In healthy conditions (above), the choroid plexus forms the BCSFB and produces CSF. CSF flows from the periarterial space to the brain parenchyma via AQP4, located in the end-feet of astrocytes. This movement is favored positive pressure of CSF production from the choroid plexus and the arterial pulse. The CSF/ISF mixture and waste products are cleared by passing into the perivenous space to be taken to the lymphatic tissues. In AD (below) the expression of AQPs decreases in choroid plexus epithelial cells causing a reduction in CSF production. The decrease in pressure exerted by the CSF together with the diminution and depolarization of AQP4 in the astrocyte end-feet, reduces the glymphatic function, preventing a correct clearance of the ISF and waste products. AQP: aquaporin; BCSFB: brain CSF barrier; CSF: cerebrospinal fluid; CP: choroid plexus; ISF: interstitial fluid. Full article ">

11 pages, 5531 KiB

Open AccessCommunication

Dihydroceramides Derived from Bacteroidetes Species Sensitize TRPV1 Channels

by Nora Ludwig, Isaac S. Demaree, Chiaki Yamada, Amilia Nusbaum, Frank C. Nichols, Fletcher A. White, Alexandru Movila and Alexander G. Obukhov

Int. J. Mol. Sci. 2023, 24(1), 877; https://doi.org/10.3390/ijms24010877 - 3 Jan 2023

Cited by 1 | Viewed by 2016

Abstract

Bacterial colonization of open wounds is common, and patients with infected wounds often report significantly elevated pain sensitivity at the wound site. Transient Receptor Potential Vanilloid Type 1 (TRPV1) channels are known to play an important role in pain signaling and may be [...] Read more.

Bacterial colonization of open wounds is common, and patients with infected wounds often report significantly elevated pain sensitivity at the wound site. Transient Receptor Potential Vanilloid Type 1 (TRPV1) channels are known to play an important role in pain signaling and may be sensitized under pro-inflammatory conditions. Bacterial membrane components, such as phosphoethanolamine dihydroceramide (PEDHC), phosphoglycerol dihydroceramide (PGDHC), and lipopolysaccharide (LPS), are released in the environment from the Gram-negative bacteria of the Bacteroidetes species colonizing the infected wounds. Here, we used intracellular calcium imaging and patch-clamp electrophysiology approaches to determine whether bacterially derived PEDHC, PGDHC, or LPS can modulate the activity of the TRPV1 channels heterologously expressed in HEK cells. We found that PEDHC and PGDHC can sensitize TRPV1 in a concentration-dependent manner, whereas LPS treatment does not significantly affect TRPV1 activity in HEK cells. We propose that sensitization of TRPV1 channels by Bacteroidetes-derived dihydroceramides may at least in part underlie the increased pain sensitivity associated with wound infections. Full article

(This article belongs to the Special Issue Targeting TRP Channels for Pain, Itch and Inflammation Relief)

► Show Figures

Figure 1

Figure 1
Bacteroidetes spp. release several membrane components into the environment, such as phosphoethanolamine dihydroceramide (PEDHC), phosphoglycerol dihydroceramide (PGDHC), and lipopolysaccharides (LPS). The upper panel of the figure shows molecular structures of the named components (LPS from Porphyromonas gingivalis, LPS-PG, is shown; [<a href="#B18-ijms-24-00877" class="html-bibr">18</a>]). The lower panel depicts a drawing of bacterially infected wound. Full article ">Figure 2
The effect of high concentration of dihydroceramides (10 μg/mL) on capsaicin-induced intracellular Ca2+ increases measured using Fura-2 fluorescence imaging. (A) F340/F380 ratio increases were greater in cells pretreated with PEDHC (10 μg/mL) compared to cells pretreated with PBS or PGDHC (PBS, n = 68; PEDHC, n = 24; PGDHC, n = 52). The gray area indicates ± SEM. The solid lines are the means of F340/F380 ratio changes over time recorded in each individual cell. The horizontal bars denote the times when the indicated compounds were present in the bath. (B) Summary of the data presented in (A). The differences between the peak values of capsaicin-induced Fura-2 F340/F380 ratio increases and the F340/F380 ratio at the baseline were determined in each tested TRPV1-HEK cell. The filled red circles represent the mean difference values for each experimental group, and the vertical red lines are the error bars (SEM). The Kruskal–Wallis one-way analysis of variance (ANOVA) on ranks test, followed by the Dunn’s post hoc multiple comparisons versus control group test, was used to determine whether there is a significant difference between the tested groups. A statistically significant difference was observed only between the PEDHC and PBS groups at this concentration (p = 0.018). Full article ">Figure 3
The effect of intermediate concentration of dihydroceramides (1 μg/mL) on capsaicin-induced intracellular Ca2+ increases measured using Fura-2 fluorescence imaging. (A) F340/F380 ratio increases were greater in cells pretreated with PGDHC (1 μg/mL) compared to cells pretreated with PBS or PEDHC (1 μg/mL) (PBS, n = 76; PEDHC, n = 83; PGDHC, n = 70). The gray area indicates ± SEM. The solid lines are the means of F340/F380 ratio changes over time recorded in each individual cell. The horizontal bars denote the times when the indicated compounds were present in the bath. (B) Summary of the data presented in (A). The differences between the peak values of capsaicin-induced F340/F380 ratio increases and the F340/F380 ratio at the baseline were determined in each tested TRPV1-HEK cell. The filled red circles represent the mean difference values for each experimental group, and the vertical red lines are the error bars (SEM). The Kruskal–Wallis one-way ANOVA on ranks test, followed by the Dunn’s post hoc multiple comparisons versus control group test, was used to determine whether there is a significant difference between the tested groups. A statistically significant difference was observed only between the PGDHC and PBS groups at this concentration (p = 0.001). Full article ">Figure 4
Low concentration of neither PGDHC (0.1 μg/mL) nor PEDHC (0.1 μg/mL) altered capsaicin-induced intracellular Ca2+ increases measured using Fura-2 fluorescence imaging. (A) F340/F380 ratio increases were not different in cells pretreated with PGDHC (0.1 μg/mL) or PEDHC (0.1 μg/mL) compared to cells pretreated with PBS (PBS, n = 60; PEDHC, n = 116; PGDHC, n = 94). The gray area indicates ± SEM. The solid lines are the means of F340/F380 ratio changes over time recorded in each individual cell. The horizontal bars denote the times when the indicated compounds were present in the bath. (B) Summary of the data presented in (A). The differences between the peak values of capsaicin-induced F340/F380 ratio increases and the F340/F380 ratio at the baseline were determined in each tested TRPV1-HEK cell. The filled red circles represent the mean difference values for each experimental group, and the vertical red lines are the error bars (SEM). The Kruskal–Wallis one-way ANOVA on ranks test was used to determine whether there is a significant difference between the tested groups. No statistically significant difference was observed between the tested groups at this concentration (p = 0.072). Full article ">Figure 5
LPS-PG (1 μg/mL and 10 μg/mL) pretreatment did not affect capsaicin-induced intracellular Ca2+ increases measured using Fura-2 fluorescence imaging in TRPV1-HEK cells. (A) F340/F380 ratio increases were not significantly different in cells pretreated with LPS-PG (1 μg/mL) or LPS-PG (10 μg/mL) compared to cells pretreated with vehicle control (PBS, n = 65; LPS-PG 10 μg/mL, n = 58; LPS-PG 1 μg/mL, n = 36). The gray area indicates ± SEM. The solid lines are the means of F340/F380 ratio changes over time recorded in each individual cell. The horizontal bars denote the times when the indicated compounds were present in the bath. (B) Summary of the data presented in (A). The differences between the peak values of capsaicin-induced F340/F380 ratio increases and the F340/F380 ratio at the baseline were determined in each tested TRPV1-HEK cell. The filled red circles represent the mean difference values for each experimental group, and the vertical red lines are the error bars (SEM). No statistically significant difference was observed between each LPS-PG group and the PBS group (Kruskal–Wallis one-way ANOVA on ranks test, p = 0.111). Full article ">Figure 6
Effect of dihydroceramides on capsaicin-activated currents in TRPV1-HEK cells. The recordings were obtained using the whole-cell voltage-clamp approach. (A–C) Sample traces of capsaicin (50 nM)-activated current time courses in TRPV1-HEK cells pretreated with either PBS, PEDHC (1 μg/mL, n = 10), or PGDHC (1 μg/mL, n = 10). The holding potential was −60 mV. The horizontal bars denote the times when the indicated compounds were present in the bath. The kinetics of capsaicin-induced current decays were variable and did not significantly differ between the tested groups. Neither PEDHC (1 μg/mL) nor PGDHC (1 μg/mL) induced any currents in TRPV1-HEK cells when each dihydroceramide was applied alone in the absence of capsaicin. (D,E) Comparison of the differences between the amplitudes of peak capsaicin (50 nM)-induced currents recorded in the presence of either PBS, PEDHC (1 μg/mL), or PGDHC (1 μg/mL) indicated with (2) in panels A–C and the currents recorded in the presence of only PBS, PEDHC (1 μg/mL), or PGDHC (1 μg/mL) at the time point indicated with (1) in panels A–C. The data were collected at a holding potential of −60 mV. Each filled black circle indicates the value for each tested cell. The filled red circles represent the mean difference values for each experimental group, and the vertical red lines are the error bars (SEM). The t-test was used to compare the data sets. Statistically significant differences were observed between the experimental groups. (F) Current–voltage relationships for capsaicin (50 nM)-induced currents in the presence of either PBS or PEDHC (1 μg/mL). The gray area indicates ± SEM. The solid lines are the means of current–voltage relationships (PBS, n = 8; PEDHC, n = 10). (G) Current–voltage relationships for capsaicin (50 nM)-induced currents in the presence of either PBS or PGDHC (1 μg/mL). The gray area indicates ± SEM. The solid lines are the means of current–voltage relationships (PBS, n = 10; PGDHC, n = 10). (H) The voltage waveform protocol used during the whole-cell voltage-clamp experiments. Full article ">Figure 7
Effect of LPS-PG on capsaicin-activated currents in TRPV1-HEK cells. (A,B) Sample traces of capsaicin (50 nM)-activated current time courses in TRPV1-HEK cells pretreated with either PBS (n = 7) or LPS-PG (1 μg/mL, n = 5). Whole-cell voltage-clamp recordings are shown. The holding potential was −60 mV. The horizontal bars denote the times when the indicated compounds were present in the bath. LPS-PG (1 μg/mL) did not induce any currents in TRPV1-HEK cells when it was added to the recording chamber alone. (C) Comparison of the differences between peak capsaicin (50 nM)-induced current amplitudes recorded in the presence of either PBS or LPS-PG (1 μg/mL) indicated with (2) in panels A–B and the currents recorded in the presence of PBS or LPS-PG (1 μg/mL) alone at a time point indicated with (1) in panels A–B. The data were collected at a holding potential of −60 mV. Each filled black circle indicates the difference value for each tested cell. The filled red circle represents the mean value for each experimental group, and the vertical red lines are the error bars (SEM). The t-test was used to compare the data sets. There was no statistically significant difference between the two experimental groups. (D) Current–voltage relationships for capsaicin (50 nM)-induced currents in the presence of either PBS or LPS-PG (1 μg/mL). The gray area indicates ± SEM. The solid lines are the means of current–voltage relationships (PBS, n = 7; LPS-PG, n = 5). (E) the voltage waveform protocol used during the whole-cell voltage-clamp experiments. Full article ">

22 pages, 6592 KiB

Open AccessArticle

Activated Leukocyte Cell Adhesion Molecule (ALCAM), a Potential ‘Seed’ and ‘Soil’ Receptor in the Peritoneal Metastasis of Gastrointestinal Cancers

by Yi Ming Yang, Lin Ye, Fiona Ruge, Ziqian Fang, Ke Ji, Andrew J. Sanders, Shuqin Jia, Chunyi Hao, Q. Ping Dou, Jiafu Ji and Wen G. Jiang

Int. J. Mol. Sci. 2023, 24(1), 876; https://doi.org/10.3390/ijms24010876 - 3 Jan 2023

Cited by 3 | Viewed by 3421

Abstract

Activated Leukocyte Cell Adhesion Molecule (ALCAM/CD166) is a cell–cell adhesion protein conferring heterotypic and homotypic interactions between cells of the same type and different types. It is aberrantly expressed in various cancer types and has been shown to be a regulator of cancer [...] Read more.

Activated Leukocyte Cell Adhesion Molecule (ALCAM/CD166) is a cell–cell adhesion protein conferring heterotypic and homotypic interactions between cells of the same type and different types. It is aberrantly expressed in various cancer types and has been shown to be a regulator of cancer metastasis. In the present study, we investigated potential roles of ALCAM in the peritoneal transcoelomic metastasis in gastrointestinal cancers, a metastatic type commonly occurred in gastro-intestinal and gynaecological malignancies and resulting in poor clinical outcomes. Specifically, we studied whether ALCAM acts as both a ‘seed’ receptor in these tumour cells and a ‘soil’ receptor in peritoneal mesothelial cells during cancer metastasis. Gastric cancer and pancreatic cancer tissues with or without peritoneal metastasis were compared for their levels of ALCAM expression. The impact of ALCAM expression in these tumours was also correlated to the patients’ clinical outcomes, namely peritoneal metastasis-free survival. In addition, cancer cells of gastric and pancreatic origins were used to create cell models with decreased or increased levels of ALCAM expression by genetic knocking down or overexpression, respectively. Human peritoneal mesothelial cells were also genetically transfected to generate cell models with different profiles of ALCAM expression. These cell models were used in the tumour-mesothelial interaction assay to assess if and how the interaction was influenced by ALCAM. Both gastric and pancreatic tumour tissues from patients who developed peritoneal metastases had higher levels of ALCAM transcript than those without. Patients who had tumours with high levels of ALCAM had a much shorter peritoneal metastasis free survival compared with those who had low ALCAM expression (p = 0.006). ALCAM knockdown of the mesothelial cell line MET5A rendered the cells with reduced interaction with both gastric cancer cells and pancreatic cancer cells. Likewise, levels of ALCAM in both human gastric and pancreatic cancer cells were also a determining factor for their adhesiveness to mesothelial cells, a process that was likely to be triggered the phosphorylation of the SRC kinase. A soluble ALCAM (sALCAM) was found to be able to inhibit the adhesiveness between cancer cells and mesothelial cells, mechanistically behaving like a SRC kinase inhibitor. ALCAM is an indicator of peritoneal metastasis in both gastric and pancreatic cancer patients. It acts as not only a potential peritoneal ‘soil’ receptor of tumour seeding but also a ‘soil’ receptor in peritoneal mesothelial cells during cancer metastasis. These findings have an important therapeutic implication for treating peritoneal transcoelomic metastases. Full article

(This article belongs to the Special Issue State-of-the-Art Molecular Oncology in UK)

► Show Figures

Figure 1

20 pages, 4778 KiB

Open AccessArticle

The Antimicrobial Peptide Cathelicidin Exerts Immunomodulatory Effects via Scavenger Receptors

by Ryo Amagai, Toshiya Takahashi, Hitoshi Terui, Taku Fujimura, Kenshi Yamasaki, Setsuya Aiba and Yoshihide Asano

Int. J. Mol. Sci. 2023, 24(1), 875; https://doi.org/10.3390/ijms24010875 - 3 Jan 2023

Cited by 7 | Viewed by 3070

Abstract

An active form of cathelicidin antimicrobial peptide, LL-37, has immunomodulatory and stimulatory effects, though the specific pathways are not clear. The purpose of this study was to identify the cellular pathways by which LL-37 amplifies the inflammation induced by damage-associated molecular patterns (DAMPs). [...] Read more.

An active form of cathelicidin antimicrobial peptide, LL-37, has immunomodulatory and stimulatory effects, though the specific pathways are not clear. The purpose of this study was to identify the cellular pathways by which LL-37 amplifies the inflammation induced by damage-associated molecular patterns (DAMPs). We performed DNA microarray, reverse transcription polymerase chain reaction, immunoblotting, and proximity ligation assays using cultured keratinocytes treated with LL-37 and/or the DAMP poly(I:C), a synthetic double-stranded RNA. In contrast to the combination of LL-37 and poly(I:C), LL-37 alone induced genes related to biological metabolic processes such as VEGFA and PTGS2 (COX-2). Inhibition of FPR2, a known receptor for cathelicidin, partially suppressed the induction of VEGFA and PTGS2. Importantly, VEGFA and PTGS2 induced by LL-37 alone were diminished by the knockdown of scavenger receptors including SCARB1 (SR-B1), OLR1 (SR-E1), and AGER (SR-J1). Moreover, LL-37 alone, as well as the combination of LL-37 and poly(I:C), showed proximity to the scavenger receptors, indicating that LL-37 acts via scavenger receptors and intermediates between them and poly(I:C). These results showed that the broad function of cathelicidin is generally dependent on scavenger receptors. Therefore, inhibitors of scavenger receptors or non-functional mock cathelicidin peptides may serve as new anti-inflammatory and immunosuppressive agents. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Skin Autoimmunity and Hypersensitivity)

► Show Figures

Graphical abstract

12 pages, 2391 KiB

Open AccessArticle

Conjugated Linoleic Acids Have Anti-Inflammatory Effects in Cultured Endothelial Cells

by Carina A. Valenzuela, Ella J. Baker, Elizabeth A. Miles and Philip C. Calder

Int. J. Mol. Sci. 2023, 24(1), 874; https://doi.org/10.3390/ijms24010874 - 3 Jan 2023

Cited by 5 | Viewed by 2203

Abstract

Conjugated linoleic acid (CLA) isomers may have a role in preventing atherosclerosis through the modulation of inflammation, particularly of the endothelium. However, whether low concentrations of CLAs are able to affect basal unstimulated endothelial cell (EC) responses is not clear. The aim of [...] Read more.

Conjugated linoleic acid (CLA) isomers may have a role in preventing atherosclerosis through the modulation of inflammation, particularly of the endothelium. However, whether low concentrations of CLAs are able to affect basal unstimulated endothelial cell (EC) responses is not clear. The aim of this study was to evaluate the effects of two CLAs (cis-9, trans-11 (CLA9,11) and trans-10, cis-12 (CLA10,12)) on the basal inflammatory responses by ECs. EA.hy926 cells (HUVEC lineage) were cultured under standard conditions and exposed to individual CLAs for 48 h. Both CLAs were incorporated into ECs in a dose-dependent manner. CLA9,11 (1 μM) significantly decreased concentrations of MCP-1 (p < 0.05), IL-6 (p < 0.05), IL-8 (p < 0.01) and RANTES (p < 0.05) in the culture medium. CLA10,12 (10 μM) decreased the concentrations of MCP-1 (p < 0.05) and RANTES (p < 0.05) but increased the concentration of IL-6 (p < 0.001). At 10 μM both CLAs increased the relative expression of the NFκβ subunit 1 gene (p < 0.01 and p < 0.05, respectively), while decreasing the relative expression of PPARα (p < 0.0001), COX-2 (p < 0.0001) and IL-6 (p < 0.0001) genes. CLA10,12 increased the relative expression of the gene encoding IκK-β at 10 μM compared with CLA9,11 (p < 0.05) and increased the relative expression of the gene encoding IκBα at 1 and 10 μM compared with linoleic acid (both p < 0.05). Neither CLA affected the adhesion of monocytes to ECs. These results suggest that low concentrations of both CLA9,11 and CLA10,12 have modest anti-inflammatory effects in ECs. Thus, CLAs may influence endothelial function and the risk of vascular disease. Nevertheless, at these low CLA concentrations some pro-inflammatory genes are upregulated while others are downregulated, suggesting complex effects of CLAs on inflammatory pathways. Full article

(This article belongs to the Special Issue Nutraceuticals in the Prevention and Treatment of Inflammatory Diseases)

► Show Figures

Figure 1

Figure 1
(A) Viability of EA.hy926 cells after preincubation for 48 h with medium a containing 0.1% of ethanol (Control) or different concentrations (1, 10 or 50 µM) of linoleic acid (LA), cis-9, trans-11 linoleic acid (CLA9,11) or trans-10, cis-12 linoleic acid (CLA10,12) followed by incubation with a medium without fatty acids for 24 h. Bars are mean ± SD of 9 samples from 3 experiments. Data were analysed using one-way ANOVA with Tukey’s post hoc test. **** p < 0.0001 vs. Control. (B,C) Microscope images of EA.hy926 cells incubated with medium containing 0.1% ethanol (B) or containing trans-10, cis-12 linoleic acid at 50 µM (C). Full article ">Figure 2
Concentrations (% of control) of MCP-1 (A), ICAM-1 (B), IL-6 (C), IL-8 (D) and RANTES (E) in the medium of EA.hy926 cells incubated for 48 h with medium containing 0.1% of ethanol (Control) or fatty acid at 1 or 10 µM, followed by incubation with a medium without fatty acids for 24 h. Bars are mean ± SD of 9 samples from 3 experiments. Data were analysed using the one-way ANOVA with Tukey’s post hoc test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; where asterisks are shown immediately above a bar they refer to the difference from the control and where asterisks are shown above a horizontal line they refer to the differences between the two groups indicated by that line. LA, linoleic acid; CLA9,11, cis-9, trans-11 linoleic acid; CLA10,12, trans-10, cis-12 linoleic acid. Full article ">Figure 3
Expression of NFκB1 (A), NFκBIA (for IκBα, (B)), IκBKB (for IκK-β, (C)), PPPAR-α (D), PTGS2 (for COX-2, (E)) and IL-6 (F) genes in EA.hy926 cells preincubated for 48 h with 1 or 10 µM of fatty acid in a medium containing 0.1% of ethanol (Control) followed by incubation with a medium without fatty acids for 6 h. Cq values were normalized by the geometric mean of reference targets (YWHAZ and RPL13A genes). Bars are mean ± SD of 9 samples from 3 experiments. Data were analysed using the one-way ANOVA with Tukey’s post hoc test. * p < 0.05, ** p < 0.01, **** p < 0.0001; where asterisks are shown immediately above a bar they refer to the difference from the control and where asterisks are shown above a horizontal line they refer to the differences between the two groups indicated by that line. LA, linoleic acid; CLA9,11, cis-9, trans-11 linoleic acid; CLA10,12, trans-10, cis-12 linoleic acid. Full article ">Figure 4
Adhesion of THP-1 cells (% of control) to EA.hy926 cells incubated for 48 h with a medium containing 0.1% of ethanol (Control) or different concentrations (1 µM (A), 10 µM (B)) of fatty acid, followed by incubation with a medium without fatty acids for 6 h and then 1 h co-incubation with THP-1 cells. Bars are mean ± SD of 9 samples from 3 experiments. Data were analysed using the one-way ANOVA with Tukey post hoc test. ** p < 0.01; *** p < 0.001; **** p < 0.0001; where asterisks are shown immediately above a bar they refer to the difference from the control and where asterisks are shown above a horizontal line they refer to the differences between the two groups indicated by that line. LA, linoleic acid; CLA9,11, cis-9, trans-11 linoleic acid; CLA10,12, trans-10, cis-12 linoleic acid. Full article ">Figure 5
Images of THP-1 cell adhesion to EA.hy926 cells. Adhesion of THP-1 cells to EA.hy926 cells without pre-incubation with fatty acid (control (A)) or with 48 h prior exposure to 10 µM linoleic acid (B), cis-9, trans-11 linoleic acid (C), trans-10, cis-12 linoleic acid (D), followed by incubation with a medium without fatty acids for 6 h and then 1 h co-incubation with calcein-labelled THP-1 cells. Attached THP-1 cells were visualised by fluorescence microscopy (Nikon Elipse Ti) at a magnification of 100× under transmitted light. Full article ">

16 pages, 3510 KiB

Open AccessArticle

Genome-Wide Association Studies of Seven Root Traits in Soybean (Glycine max L.) Landraces

by Seong-Hoon Kim, Rupesh Tayade, Byeong-Hee Kang, Bum-Soo Hahn, Bo-Keun Ha and Yoon-Ha Kim

Int. J. Mol. Sci. 2023, 24(1), 873; https://doi.org/10.3390/ijms24010873 - 3 Jan 2023

Cited by 6 | Viewed by 3746

Abstract

Soybean [Glycine max (L.) Merr.], an important oilseed crop, is a low-cost source of protein and oil. In Southeast Asia and Africa, soybeans are widely cultivated for use as traditional food and feed and industrial purposes. Given the ongoing changes in global [...] Read more.

Soybean [Glycine max (L.) Merr.], an important oilseed crop, is a low-cost source of protein and oil. In Southeast Asia and Africa, soybeans are widely cultivated for use as traditional food and feed and industrial purposes. Given the ongoing changes in global climate, developing crops that are resistant to climatic extremes and produce viable yields under predicted climatic conditions will be essential in the coming decades. To develop such crops, it will be necessary to gain a thorough understanding of the genetic basis of agronomic and plant root traits. As plant roots generally lie beneath the soil surface, detailed observations and phenotyping throughout plant development present several challenges, and thus the associated traits have tended to be ignored in genomics studies. In this study, we phenotyped 357 soybean landraces at the early vegetative (V2) growth stages and used a 180 K single-nucleotide polymorphism (SNP) soybean array in a genome-wide association study (GWAS) conducted to determine the phenotypic relationships among root traits, elucidate the genetic bases, and identify significant SNPs associated with root trait-controlling genomic regions/loci. A total of 112 significant SNP loci/regions were detected for seven root traits, and we identified 55 putative candidate genes considered to be the most promising. Our findings in this study indicate that a combined approach based on SNP array and GWAS analyses can be applied to unravel the genetic basis of complex root traits in soybean, and may provide an alternative high-resolution marker strategy to traditional bi-parental mapping. In addition, the identified SNPs, candidate genes, and diverse variations in the root traits of soybean landraces will serve as a valuable basis for further application in genetic studies and the breeding of climate-resilient soybeans characterized by improved root traits. Full article

(This article belongs to the Special Issue Comparative Genomics and Functional Genomics Analysis in Plants)

► Show Figures

Figure 1

Figure 1
Population structure of 357 soybean landraces. (A) A bar plot diagram showing the results of clustering analysis when the number of subgroups (K) = 3. The colors blue, green, and red represent separate groups with different levels of admixture. (B) A phylogenetic tree of 357 soybean landraces. (C) Principal component plot for the 357 soybean landraces. (D) Genome-wide linkage disequilibrium (LD) decay for all 357 soybean landraces. R2 indicates the squared allele frequency correlations between all pairs of SNP markers. The X-axis indicates the distance between marker pairs. Full article ">Figure 2
Manhattan and quantile-quantile (QQ) plots of the 357 soybean landraces using the MLM model. (A) Root average diameter (DIAM) and (B) link average diameter (LAD). The X-axis represents the chromosome number, and the Y-axis represents the −log10p) value. The threshold of 4.0 (Bonferroni correction) was adopted with the blue line in the Manhattan plots. The Manhattan and QQ plots are based on analyses of the association between 84,210 chromosomal SNPs and DIAM and LAD traits. Full article ">Figure 3
Manhattan and quantile-quantile (QQ) plots of the 357 soybean landraces using the MLM model. (A) Link average length (LAL) and (B) total root length (LENGTH). The X-axis represents the chromosome number and the Y-axis represents the −log10(p) value. The threshold of 4.0 (Bonferroni correction) was adopted with the blue line in the Manhattan plots. The Manhattan and QQ plots are based on analyses of the associations between 84,210 chromosomal SNPs and DIAM and LAD traits. Full article ">Figure 4
Manhattan and quantile-quantile (QQ) plots of the 357 soybean landraces using the MLM model. (A) Number of root forks (NF), (B) number of root tips (NT), and (C) root surface area (SA). The X-axis represents the chromosome number and the Y-axis represents the −log10(p) value. The threshold of 4.0 (Bonferroni correction) was adopted with the blue line in the Manhattan plots. The Manhattan plot and QQ plot is based on the association of 84,210 chromosomal SNPs with DIAM and LAD traits. Full article ">

19 pages, 4144 KiB

Open AccessArticle

Topographically Distinguished Microbiome Taxonomy and Stress-Response Genes of Royal Belum Rainforest and Raja Muda Musa Peat Swamp Revealed through Metagenomic Inquisition

by Mohd Fadzli Ahmad, Hasdianty Abdullah, Muhammad Naim Hassan, Muhammad Imran Jamaludin, Ashvini Sivam, Kazuhiro Komatsu, Irni Suhayu Sapian, Halimah Alias, Mohd Noor Mat Isa, Victor S. Kuwahara and Nor Suhaila Yaacob

Int. J. Mol. Sci. 2023, 24(1), 872; https://doi.org/10.3390/ijms24010872 - 3 Jan 2023

Cited by 2 | Viewed by 2830

Abstract

Soil ecosystems are home to a diverse range of microorganisms, but they are only partially understood because no single-cell sequencing or whole-community sequencing provides a complete picture of these complex communities. Using one of such metagenomics approaches, we succeeded in monitoring the microbial [...] Read more.

Soil ecosystems are home to a diverse range of microorganisms, but they are only partially understood because no single-cell sequencing or whole-community sequencing provides a complete picture of these complex communities. Using one of such metagenomics approaches, we succeeded in monitoring the microbial diversity and stress-response gene in the soil samples. This study aims to test whether known differences in taxonomic diversity and composition are reflected in functional gene profiles by implementing whole gene sequencing (WGS) metagenomic analysis of geographically dispersed soils from two distinct pristine forests. The study was commenced by sequencing three rainforest soil samples and three peat swamp soil samples. Soil richness effects were assessed by exploring the changes in specific functional gene abundances to elucidate physiological constraints acting on different soil systems and identify variance in functional pathways relevant to soil biogeochemical cycling. Proteobacteria shows abundances of microbial diversity for 52.15% in Royal Belum Reserved Forest and 48.28% in Raja Musa; 177 out of 1,391,841 and 449 out of 3,586,577 protein coding represent acidic stress-response genes for Royal Belum and Raja Musa, respectively. Raja Musa indicates pH 2.5, which is extremely acidic. The analysis of the taxonomic community showed that Royal Belum soils are dominated by bacteria (98% in Sungai Kooi (SK), 98% in Sungai Papan (SP), and 98% in Sungai Ruok (SR), Archaea (0.9% in SK, 0.9% in SP, and 1% in SR), and the remaining were classed under Eukaryota and viruses. Likewise, the soils of Raja Muda Musa are also dominated by bacteria (95% in Raja Musa 1 (RM1), 98% in Raja Musa 2 (RM2), and 96% in Raja Musa 3 (RM3)), followed by Archaea (4% in RM1, 1% in RM2, and 3% in RM3), and the remaining were classed under Eukaryota and viruses. This study revealed that RBFR (Royal Belum Foresr Reserve) and RMFR (Raja Musa Forest Reserve) metagenomes contained abundant stress-related genes assigned to various stress-response pathways, many of which did not show any difference among samples from both sites. Our findings indicate that the structure and functional potential of the microbial community will be altered by future environmental potential as the first glimpse of both the taxonomic and functional composition of soil microbial communities. Full article

(This article belongs to the Section Molecular Genetics and Genomics)

► Show Figures

Figure 1

13 pages, 62543 KiB

Open AccessCommunication

Microglia in Cultured Porcine Retina: Qualitative Immunohistochemical Analyses of Reactive Microglia in the Outer Retina

by Kjell Johansson and Camilla Mohlin

Int. J. Mol. Sci. 2023, 24(1), 871; https://doi.org/10.3390/ijms24010871 - 3 Jan 2023

Viewed by 2123

Abstract

A late stage of several retinal disorders is retinal detachment, a complication that results in rapid photoreceptor degeneration and synaptic damages. Experimental retinal detachment in vivo is an invasive and complicated method performed on anesthetized animals. As retinal detachment may result in visual [...] Read more.

A late stage of several retinal disorders is retinal detachment, a complication that results in rapid photoreceptor degeneration and synaptic damages. Experimental retinal detachment in vivo is an invasive and complicated method performed on anesthetized animals. As retinal detachment may result in visual impairment and blindness, research is of fundamental importance for understanding degenerative processes. Both morphological and ethical issues make the porcine retina a favorable organotypic model for studies of the degenerative processes that follow retinal detachment. In the cultured retina, photoreceptor degeneration and synaptic injuries develop rapidly and correlate with resident microglial cells’ transition into a reactive phenotype. In this immunohistochemical study, we have begun to analyze the transition of subsets of reactive microglia which are known to localize close to the outer plexiform layer (OPL) in degenerating in vivo and in vitro retina. Biomarkers for reactive microglia included P2Ry12, CD63 and CD68 and the general microglial markers were CD11b, Iba1 and isolectin B₄ (IB₄). The reactive microglia markers labeled microglia subpopulations, suggesting that protective or harmful reactive microglia may be present simultaneously in the injured retina. Our findings support the usage of porcine retina cultures for studies of photoreceptor injuries related to retinal detachment. Full article

(This article belongs to the Special Issue Advanced Research in Retina)

► Show Figures

Figure 1

Figure 1
Confocal stacks (A,B) and fluorescence micrographs (C) of normal and 5 DIV retinas. (A) In normal healthy retina, Iba1 immunoreactive microglia (green: large arrows) localize to the IPL and project long, thin processes that finally arborize along the inner aspect (small arrows) of the PSD immunoreactive photoreceptor terminals (red) in the OPL. At 5 DIV (C), retinas CD11b immunoreactive microglia (green) are evident at the IPL (large arrows), close to photoreceptor terminals in the OPL (small arrows). Thick CD11b immunoreactive processes localize close to or within the OPL (small arrows). (C) 5 DIV retina with IB4 labeled microglial cell bodies (large arrows) localized in the outer retina. Note also labeled degenerating capillaries (small arrows). DAPI counterstaining in (A,B). Abbreviations. GCL ganglion cell layer; INL inner nuclear layer; IPL inner plexiform layer; ONL outer nuclear layer; OPL outer plexiform layer; OS outer segments. Scale (A–C) 10 µm. Full article ">Figure 2
Confocal stacks (F,G) and fluorescence micrographs (A–E,H) showing P2Ry12 immunoreactivity (green) in normal and 5 DIV retina. Calbindin or PSD95 immunoreactivity (red) indicate the OPL. (A,B) P2Ry12 immunoreactivity is mainly evident in the inner parts of Müller cells. (C,E,F) Colabeling with calbindin shows that P2Ry12 immunoreactivity distributes close to horizontal cell processes at 5 DIV. (E) Structures resembling cone photoreceptor terminals occasionally expressed P2Ry12 immunoreactivity. (D,G) Juxtaposed P2Ry12 and PSD95 immunoreactivities in the OPL (yellowish hue in (D)). (H) P2Ry12 immunoreactive microglial cells (arrow) and processes in the PSD95 labeled OPL. Abbreviations as in <a href="#ijms-24-00871-f001" class="html-fig">Figure 1</a>. Scale (A–H) 10 μm. Full article ">Figure 3
Confocal stacks (C) and fluorescence micrographs (A,B,D) showing CD63 immunoreactivity (green) in normal and 5 DIV retina. Calbindin immunoreactivity and IB4 labeling (red) indicate horizontal and microglial cells, respectively. (A) Dotted CD63 immunoreactivity is evident in Müller cells in healthy retina. (B) IB4-labeled microglia close to the OPL with perinuclear CD63 immunoreactivity (large arrow). Note the presence of CD immunoreactive cell without IB4 labeling (small arrow). (C,D) High magnification of the OPL showing perinuclear CD63 immunoreactivity (arrows in C) and in IB4-labeled microglia (D). Note also labeled capillary (asterisk in (D)). (E) CD63 vesicular immunoreactivity in presumptive microglia (arrows) in ONL. Abbreviations as in <a href="#ijms-24-00871-f001" class="html-fig">Figure 1</a>. Scale (A–E) 10 μm. Full article ">Figure 4
Confocal stacks (A,C) and fluorescence micrographs (B,D) showing CD68 immunoreactivity (red) and CD11b immunoreactive microglia (green). Confocal stacks (E,F) showing CD68 immunoreactivity (red) in Iba1 immunoreactive microglia (green). (A,C) Vesicular CD 68 immunoreactivity in low and high magnification in the ONL of 5 DIV retina. (B,D) Perinuclear CD68 in a CD11b immunoreactive microglia close to the OPL. (D) CD68 immunoreactivity in microglial cell body (asterisk) and process (small arrows in B,D). (E,F) CD68 immunoreactivity in large strongly Iba1 immunoreactive microglial cell and processes (small arrows in (E,F)). Note also small microglia cell body (large arrows in E,F) displaying weak CD68 and Iba1 immunoreactivities. Abbreviations as in <a href="#ijms-24-00871-f001" class="html-fig">Figure 1</a>. Scale (A–F) 10 µm. Full article ">

12 pages, 3642 KiB

Open AccessArticle

Exploring the Potential Energy Surface of Pt₆ Sub-Nano Clusters Deposited over Graphene

by Daniel Barrena-Espés, Sergio Boneta, Victor Polo and Julen Munárriz

Int. J. Mol. Sci. 2023, 24(1), 870; https://doi.org/10.3390/ijms24010870 - 3 Jan 2023

Cited by 1 | Viewed by 1827

Abstract

Catalytic systems based on sub-nanoclusters deposited over different supports are promising for very relevant chemical transformations such as many electrocatalytic processes as the ORR. These systems have been demonstrated to be very fluxional, as they are able to change shape and interconvert between [...] Read more.

Catalytic systems based on sub-nanoclusters deposited over different supports are promising for very relevant chemical transformations such as many electrocatalytic processes as the ORR. These systems have been demonstrated to be very fluxional, as they are able to change shape and interconvert between each other either alone or in the presence of adsorbates. In addition, an accurate representation of their catalytic activity requires the consideration of ensemble effects and not a single structure alone. In this sense, a reliable theoretical methodology should assure an accurate and extensive exploration of the potential energy surface to include all the relevant structures and with correct relative energies. In this context, we applied DFT in conjunction with global optimization techniques to obtain and analyze the characteristics of the many local minima of Pt₆ sub-nanoclusters over a carbon-based support (graphene)—a system with electrocatalytic relevance. We also analyzed the magnetism and the charge transfer between the clusters and the support and paid special attention to the dependence of dispersion effects on the ensemble characteristics. We found that the ensembles computed with and without dispersion corrections are qualitatively similar, especially for the lowest-in-energy clusters, which we attribute to a (mainly) covalent binding to the surface. However, there are some significant variations in the relative stability of some clusters, which would significantly affect their population in the ensemble composition. Full article

(This article belongs to the Special Issue Multi-Metallic Systems: From Strong Cooperative Bonds to Weak M-M Interactions)

► Show Figures

Figure 1

22 pages, 5017 KiB

Open AccessArticle

Fluoroplast Doped by Ag₂O Nanoparticles as New Repairing Non-Cytotoxic Antibacterial Coating for Meat Industry

by Sergey V. Gudkov, Ruibin Li, Dmitriy A. Serov, Dmitriy E. Burmistrov, Ilya V. Baimler, Alexey S. Baryshev, Alexander V. Simakin, Oleg V. Uvarov, Maxim E. Astashev, Natalia B. Nefedova, Sergey Y. Smolentsev, Andrey V. Onegov, Mikhail A. Sevostyanov, Alexey G. Kolmakov, Mikhail A. Kaplan, Andrey Drozdov, Eteri R. Tolordava, Anastasia A. Semenova, Andrey B. Lisitsyn and Vasily N. Lednev

Int. J. Mol. Sci. 2023, 24(1), 869; https://doi.org/10.3390/ijms24010869 - 3 Jan 2023

Cited by 6 | Viewed by 3494

Abstract

Foodborne infections are an important global health problem due to their high prevalence and potential for severe complications. Bacterial contamination of meat during processing at the enterprise can be a source of foodborne infections. Polymeric coatings with antibacterial properties can be applied to [...] Read more.

Foodborne infections are an important global health problem due to their high prevalence and potential for severe complications. Bacterial contamination of meat during processing at the enterprise can be a source of foodborne infections. Polymeric coatings with antibacterial properties can be applied to prevent bacterial contamination. A composite coating based on fluoroplast and Ag₂O NPs can serve as such a coating. In present study, we, for the first time, created a composite coating based on fluoroplast and Ag₂O NPs. Using laser ablation in water, we obtained spherical Ag₂O NPs with an average size of 45 nm and a ζ-potential of −32 mV. The resulting Ag₂O NPs at concentrations of 0.001–0.1% were transferred into acetone and mixed with a fluoroplast-based varnish. The developed coating made it possible to completely eliminate damage to a Teflon cutting board. The fluoroplast/Ag₂O NP coating was free of defects and inhomogeneities at the nano level. The fluoroplast/Ag₂O NP composite increased the production of ROS (H₂O₂, OH radical), 8-oxogualnine in DNA in vitro, and long-lived active forms of proteins. The effect depended on the mass fraction of the added Ag₂O NPs. The 0.01–0.1% fluoroplast/NP Ag₂O coating exhibited excellent bacteriostatic and bactericidal properties against both Gram-positive and Gram-negative bacteria but did not affect the viability of eukaryotic cells. The developed PTFE/NP Ag₂O 0.01–0.1% coating can be used to protect cutting boards from bacterial contamination in the meat processing industry. Full article

(This article belongs to the Special Issue Biopolymers as Nanoparticles Carriers)

► Show Figures

Figure 1

15 pages, 3974 KiB

Open AccessArticle

Integrative Proteome Analysis Revels 3-Hydroxybutyrate Exerts Neuroprotective Effect by Influencing Chromatin Bivalency

by Xin-Liang Zhu, Huan Du, Lei-Lei Wang, Er-Ling Hu, Ning Li, Hai-Xia Lu, Guo-Qiang Chen and Xiao-Yun Lu

Int. J. Mol. Sci. 2023, 24(1), 868; https://doi.org/10.3390/ijms24010868 - 3 Jan 2023

Cited by 1 | Viewed by 2473

Abstract

3-hydroxybutyrate (3OHB) has been proved to act as a neuroprotective molecule in multiple neurodegenerative diseases. Here, we employed a quantitative proteomics approach to assess the changes of the global protein expression pattern of neural cells upon 3OHB administration. In combination with a disease-related, [...] Read more.

3-hydroxybutyrate (3OHB) has been proved to act as a neuroprotective molecule in multiple neurodegenerative diseases. Here, we employed a quantitative proteomics approach to assess the changes of the global protein expression pattern of neural cells upon 3OHB administration. In combination with a disease-related, protein-protein interaction network we pinpointed a hub marker, histone lysine 27 trimethylation, which is one of the key epigenetic markers in multiple neurodegenerative diseases. Integrative analysis of transcriptomic and epigenomic datasets highlighted the involvement of bivalent transcription factors in 3OHB-mediated disease protection and its alteration of neuronal development processes. Transcriptomic profiling revealed that 3OHB impaired the fate decision process of neural precursor cells by repressing differentiation and promoting proliferation. Our study provides a new mechanism of 3OHB’s neuroprotective effect, in which chromatin bivalency is sensitive to 3OHB alteration and drives its neuroprotective function both in neurodegenerative diseases and in neural development processes. Full article

(This article belongs to the Section Molecular Neurobiology)

► Show Figures

Figure 1

Figure 1
Proteomics Workflow and GSEA Analysis. (A) HT22 cells were treated with 0.2 mM of β-hydroxybutyrate (3OHB) and a mock solution, respectively. Cells were then processed for MS-based dimethyl-labeling quantitative proteomics analysis. (B) GSEA was applied to extract an enriched gene ontology map from the proteomics data. As indicated by the pink tag, pathways related to protein acetylation, neurodegenerative disease, ALS and cognition were especially highlighted, which was in good agreement with previous studies. At the same time, biological processes related to protein methylation also showed sensitive responses to 3OHB administration in normal neurons. Red dot represents up-regulated processes and pathways, blue dot represents down-regulated processes and pathways, the green line indicates shared proteins between connected processes and pathways, where thickness is proportional to the number of shared proteins. Full article ">Figure 2
Integrative analysis of the proteomics dataset pinpointed H3K27me3 and H3K4me3 as hubs. (A) Proteomics data from our study and transcriptomic data from references overlapped. Left, genes that showed a consistent increase or decrease in both RNA and protein levels are highlighted by red and blue columns, respectively; right, the network that was generated using the STRING database by loading consistently changed genes as seeds which constitute nodes of a protein interaction network. Among them, yellow nodes represent genes related to neurodegenerative disease like AD, PD and HD. (B) Up, the protein-protein interaction model used to identify hub proteins. Down, the protein-protein interaction network, was seeded with AD, PD, HD, Epilepsy, ASD, and ALS candidate proteins identified in our dataset (<a href="#app1-ijms-24-00868" class="html-app">Supplementary Table S2</a>); highlighted by red nodes. Blue nodes indicate intermediate protein-protein interactions between seed genes. Green nodes represent proteins connected to the top hub protein of high degree and/or betweenness centrality. (C) Western blot analysis (n = 3), left, revealed that 3OHB induced a significant reduction of H3K27me3 levels in the HT22 cells upon long-term treatment (at 12 h (p < 0.01) and 24 h (p < 0.001)); right, the levels of H3K4me3 in the HT22 cells were increased after 12 h of treatment (p < 0.01). (D) Western blot analysis (n = 6), left, revealed that fasting induced a similar pattern of H3K27me3 level reduction in the brains of C67 mice at 24 h (p < 0.001) comparable to that of HT22 cells; right, H3K4me3 levels showed an earlier response at 6 h of fasting (p < 0.01) than what was observed upon 3OHB treatment in HT22 cells. Data are mean ± SEM n = 3 (C); Data are mean ± SEM n = 6 mice per group (D), one-way ANOVA, Tukey’s test, ** means p < 0.01 while *** means p < 0.001 (C,D). Full article ">Figure 3
The correlation between chromatin bivalency and 3OHB-perturbed gene expression patterns highlighted that the 3OHB-sensitive transcriptional regulatory network is a promoter of the interaction between disease-related genes and 3OHB. (A) Biological processes associated with H3K27me3-H3K4me3 chromatin bivalency and processes perturbed by ketone bodies (1 mM 3OHB and 1 mM acetoacetate, 1 h) in neurons were extracted and overplayed to reveal possible correlations between chromatin bivalency and 3OHB. (B) Up, the overlapping genes were further subjected to gene ontology analysis; down, the protein-protein interaction network was seeded with the overlapping genes. Red nodes indicate seed genes and blue nodes represent intermediate protein-protein interactions between seed genes. (C) A network was generated using the STRING database by loading the overlapping genes as seeds which constitute nodes of a protein interaction network. Among them, yellow nodes represent genes related to neurodegenerative disease like AD, PD and HD. Autoregulatory transcription factors were highlighted with purple circles. Full article ">Figure 4
BHB perturbed chromatin bivalency and resulted in the alteration of neural differentiation processes. (A) Workflow of neural precursor cell isolation and 3OHB treatment for transcriptome analysis. (B) Up, immunocytochemical staining used to validate the alteration of the differentiation status of NSCs in the presence of 0.02, 0.2 and 2 mM 3OHB; down, volcano plot of the transcriptomic dataset showing changes of gene expression upon BHB administration ((5 days; n = 3 biological replicates) (p < 0.05; log2(fold change) > 0.25 or < −0.25. (C) GSEA extraction-enriched gene ontology map derived from the transcriptomic data. Red nodes indicate signaling pathways upregulated by 3OHB while blue nodes represent signaling pathways downregulated by it in NSCs. The cellular metabolic processes indicated by circles were increased by 3OHB treatment; biological processes related to neural differentiation were significantly inhibited (p < 0.001) while processes related to the cell cycle were significantly promoted by 3OHB (p < 0.001). Full article ">Figure 5
Identification and validation of abundant histone lysine hydroxybutyrylation (Kbhb) sites. (A) Western blot analysis (n = 3), left, revealed that 3OHB induced a significant increase of H2AK118bhb levels in the HT22 cells (at 6 h (p < 0.001), 12 h (p < 0.01) and 24 h (p < 0.01); right, H2AK119ub levels in the HT22 cells were significantly reduced after 3OHB treatment (p < 0.001). (B)Western blot analysis (n = 6), left, showing that fasting induced a significant increase of H2AK118bhb levels in C67 mice at 6 h of fasting (p < 0.01); right, H2AK119ub levels showed a similar pattern of significant reduction as observed in the HT22 cells (p < 0.001). Data are mean ± SEM n = 3 (A), Data are mean ± SEM n = 6 mice per group (B), one-way ANOVA, Tukey’s test, * means p < 0.05, ** means p < 0.01 while *** means p < 0.001 (A,B). Full article ">

19 pages, 2637 KiB

Open AccessArticle

Protein-Coding Region Derived Small RNA in Exosomes from Influenza A Virus–Infected Cells

by Malgorzata Kwasnik, Wojciech Socha, Bartosz Czech, Magdalena Wasiak, Jerzy Rola and Wojciech Rozek

Int. J. Mol. Sci. 2023, 24(1), 867; https://doi.org/10.3390/ijms24010867 - 3 Jan 2023

Cited by 2 | Viewed by 2480

Abstract

Exosomes may function as multifactorial mediators of cell-to-cell communication, playing crucial roles in both physiological and pathological processes. Exosomes released from virus-infected cells may contain RNA and proteins facilitating infection spread. The purpose of our study was to analyze how the small RNA [...] Read more.

Exosomes may function as multifactorial mediators of cell-to-cell communication, playing crucial roles in both physiological and pathological processes. Exosomes released from virus-infected cells may contain RNA and proteins facilitating infection spread. The purpose of our study was to analyze how the small RNA content of exosomes is affected by infection with the influenza A virus (IAV). Exosomes were isolated by ultracentrifugation after hemadsorption of virions and their small RNA content was identified using high-throughput sequencing. As compared to mock-infected controls, 856 RNA transcripts were significantly differentially expressed in exosomes from IAV-infected cells, including fragments of 458 protein-coding (pcRNA), 336 small, 28 long intergenic non-coding RNA transcripts, and 33 pseudogene transcripts. Upregulated pcRNA species corresponded mainly to proteins associated with translation and antiviral response, and the most upregulated among them were RSAD2, CCDC141 and IFIT2. Downregulated pcRNA species corresponded to proteins associated with the cell cycle and DNA packaging. Analysis of differentially expressed pseudogenes showed that in most cases, an increase in the transcription level of pseudogenes was correlated with an increase in their parental genes. Although the role of exosome RNA in IAV infection remains undefined, the biological processes identified based on the corresponding proteins may indicate the roles of some of its parts in IAV replication. Full article

(This article belongs to the Special Issue Exosomes and Extracellular Vesicles in Health and Diseases)

► Show Figures

Figure 1

Figure 1
Immunoperoxidase staining of Madin–Darby canine kidney (MDCK) cells infected with equine influenza virus (MOI of 10), (a) Mock-infected MDCK cells; (b) MDCK infected with A/equi/Kentucky 81 cells. Full article ">Figure 2
Identification and characterization of exosomes. (a) Scanning microscope photograph of purified exosomes. (b) Nanoparticle tracking analysis size distribution of exosomes isolated from mock-infected and influenza A virus (IAV)–infected MDCK cells. Red error bars indicate +/− 1 standard error of the mean. (c) Immunoblotting of exosomal marker (CD9) and IAV matrix protein (M1). Exosomes secreted by mock–infected (mock 1, 2) and IAV infected (flu 1, 2) cells; IAV–influenza virions; MDCK–mock–infected (−) and IAV infected cells (+). Full article ">Figure 3
Differentially expressed RNAs identified in exosomes from influenza-infected cells, (a) Percentage of RNA biotypes; (b) Over- and underexpressed RNA biotypes, “other”—transcripts that could not be unambiguously assigned to any biotype. Full article ">Figure 4
Protein−coding RNAs that exhibited the highest over− and underexpression in exosomes released from influenza−infected MDCK cells. Full article ">Figure 5
The interaction network of proteins encoded by transcripts overexpressed in exosomes isolated from IAV-infected MDCK cells, STRING pathway analysis. Colors indicate GO annotations: red—GO:0006412 “Biological process—Translation”; blue—GO:0009615 “Biological process—Response to virus”; green—GO:0006880 “Biological process—Intracellular sequestering of iron ion”; white—not assigned to a specific process. Full article ">

24 pages, 1312 KiB

Open AccessReview

Inflammation in Urological Malignancies: The Silent Killer

by Martina Catalano, Giandomenico Roviello, Raffaella Santi, Donata Villari, Pietro Spatafora, Ilaria Camilla Galli, Francesco Sessa, Francesco Lupo Conte, Enrico Mini, Tommaso Cai and Gabriella Nesi

Int. J. Mol. Sci. 2023, 24(1), 866; https://doi.org/10.3390/ijms24010866 - 3 Jan 2023

Cited by 7 | Viewed by 2589

Abstract

Several studies have investigated the role of inflammation in promoting tumorigenesis and cancer progression. Neoplastic as well as surrounding stromal and inflammatory cells engage in well-orchestrated reciprocal interactions to establish an inflammatory tumor microenvironment. The tumor-associated inflammatory tissue is highly plastic, capable of [...] Read more.

Several studies have investigated the role of inflammation in promoting tumorigenesis and cancer progression. Neoplastic as well as surrounding stromal and inflammatory cells engage in well-orchestrated reciprocal interactions to establish an inflammatory tumor microenvironment. The tumor-associated inflammatory tissue is highly plastic, capable of continuously modifying its phenotypic and functional characteristics. Accumulating evidence suggests that chronic inflammation plays a critical role in the development of urological cancers. Here, we review the origins of inflammation in urothelial, prostatic, renal, testicular, and penile cancers, focusing on the mechanisms that drive tumor initiation, growth, progression, and metastasis. We also discuss how tumor-associated inflammatory tissue may be a diagnostic marker of clinically significant tumor progression risk and the target for future anti-cancer therapies. Full article

(This article belongs to the Special Issue Inflammation and Cancer 2021)

► Show Figures

Figure 1

Journal Menu

Journal Browser

Int. J. Mol. Sci., Volume 24, Issue 1 (January-1 2023) – 895 articles

Further Information

Guidelines

MDPI Initiatives

Follow MDPI