International Journal of Molecular Sciences

19 pages, 3350 KiB

Open AccessArticle

Tryptophan Derivatives by Saccharomyces cerevisiae EC1118: Evaluation, Optimization, and Production in a Soybean-Based Medium

by Michele Dei Cas, Ileana Vigentini, Sara Vitalini, Antonella Laganaro, Marcello Iriti, Rita Paroni and Roberto Foschino

Int. J. Mol. Sci. 2021, 22(1), 472; https://doi.org/10.3390/ijms22010472 - 5 Jan 2021

Cited by 10 | Viewed by 7920

Abstract

Given the pharmacological properti es and the potential role of kynurenic acid (KYNA) in human physiology and the pleiotropic activity of the neurohormone melatonin (MEL) involved in physiological and immunological functions and as regulator of antioxidant enzymes, this study aimed at evaluating the [...] Read more.

Given the pharmacological properti es and the potential role of kynurenic acid (KYNA) in human physiology and the pleiotropic activity of the neurohormone melatonin (MEL) involved in physiological and immunological functions and as regulator of antioxidant enzymes, this study aimed at evaluating the capability of Saccharomyces cerevisiae EC1118 to release tryptophan derivatives (dTRPs) from the kynurenine (KYN) and melatonin pathways. The setting up of the spectroscopic and chromatographic conditions for the quantification of the dTRPs in LC-MS/MS system, the optimization of dTRPs’ production in fermentative and whole-cell biotransformation approaches and the production of dTRPs in a soybean-based cultural medium naturally enriched in tryptophan, as a case of study, were included in the experimental plan. Variable amounts of dTRPs, with a prevalence of metabolites of the KYN pathway, were detected. The LC-MS/MS analysis showed that the compound synthesized at highest concentration is KYNA that reached 9.146 ± 0.585 mg/L in fermentation trials in a chemically defined medium at 400 mg/L TRP. Further experiments in a soybean-based medium confirm KYNA as the main dTRPs, whereas the other dTRPs reached very lower concentrations. While detectable quantities of melatonin were never observed, two MEL isomers were successfully measured in laboratory media. Full article

(This article belongs to the Special Issue Tryptophan in Nutrition and Health)

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S. cerevisiae EC1118 growth curve in YNBT100 medium and TRP consumption. Full article ">Figure 2
(a) Schematic view of the metabolites from the KYN pathway accumulated in YNBT100 medium and (b,c) kinetics production of the extracellular intermediates of the KYN pathway in S. cerevisiae EC1118. (d) Kinetics production of the extracellular intermediates of the MEL pathway in S. cerevisiae EC1118 in YNBT100. Full article ">Figure 3
Melatonin isomers (MI) production by S.cerevisiae EC1118. Chromatogram in (A) shows the melatonin transition (m/z 233.2 > 174.2) of a pure standard containing melatonin (MEL, rt 6.80) tryptophan ethyl ester (TEE, rt 6.11) both at a concentration of 2 ng/mL and internal standard (MEL OCD3, rt 6.84, m/z 236.2 > 177.2, black). Chromatograms in (B) show the superimposition of melatonin trace (m/z 233.2 > 174.2) from three different S. cerevisiae EC1118 fermentation supernatants at 24 h (blue), 48 h (red), and 72 h (green). In (B) is evident the absence of the melatonin peak that should be expected at the same retention time of its labeled analogue (rt 6.80, black). Among melatonin isomers in fermentation medium it was evidenced the presence of tryptophan ethyl ester (TEE, rt 6.06) and two isomers, named MI1 (rt 4.70) and MI2 (rt 5.6), with a not-elucidated structure. Full article ">Figure 4
S. cerevisiae EC1118 growth curve in YNBT400 medium and TRP consumption. Full article ">Figure 5
TRP derivatives produced by S. cerevisiae EC1118 in YNBT400 medium. Error bars indicate the standard deviation. Full article ">Figure 6
TRP derivatives produced by S. cerevisiae EC1118 in WCB experiments. Error bars indicate the standard deviation among the mean values of biological replicates. Full article ">Figure 7
TRP derivatives produced by S. cerevisiae EC1118 in fermentation experiments in YNBSOY medium. Error bars indicate the standard deviation among the means of biological replicates. Letters indicate the grouping information using the Fisher LSD Method and 95% confidence. Full article ">

29 pages, 1129 KiB

Open AccessReview

More Than Just Simple Interaction between STIM and Orai Proteins: CRAC Channel Function Enabled by a Network of Interactions with Regulatory Proteins

by Sascha Berlansky, Christina Humer, Matthias Sallinger and Irene Frischauf

Int. J. Mol. Sci. 2021, 22(1), 471; https://doi.org/10.3390/ijms22010471 - 5 Jan 2021

Cited by 21 | Viewed by 5883

Abstract

The calcium-release-activated calcium (CRAC) channel, activated by the release of Ca²⁺ from the endoplasmic reticulum (ER), is critical for Ca²⁺ homeostasis and active signal transduction in a plethora of cell types. Spurred by the long-sought decryption of the molecular nature of [...] Read more.

The calcium-release-activated calcium (CRAC) channel, activated by the release of Ca²⁺ from the endoplasmic reticulum (ER), is critical for Ca²⁺ homeostasis and active signal transduction in a plethora of cell types. Spurred by the long-sought decryption of the molecular nature of the CRAC channel, considerable scientific effort has been devoted to gaining insights into functional and structural mechanisms underlying this signalling cascade. Key players in CRAC channel function are the Stromal interaction molecule 1 (STIM1) and Orai1. STIM1 proteins span through the membrane of the ER, are competent in sensing luminal Ca²⁺ concentration, and in turn, are responsible for relaying the signal of Ca²⁺ store-depletion to pore-forming Orai1 proteins in the plasma membrane. A direct interaction of STIM1 and Orai1 allows for the re-entry of Ca²⁺ from the extracellular space. Although much is already known about the structure, function, and interaction of STIM1 and Orai1, there is growing evidence that CRAC under physiological conditions is dependent on additional proteins to function properly. Several auxiliary proteins have been shown to regulate CRAC channel activity by means of direct interactions with STIM1 and/or Orai1, promoting or hindering Ca²⁺ influx in a mechanistically diverse manner. Various proteins have also been identified to exert a modulatory role on the CRAC signalling cascade although inherently lacking an affinity for both STIM1 and Orai1. Apart from ubiquitously expressed representatives, a subset of such regulatory mechanisms seems to allow for a cell-type-specific control of CRAC channel function, considering the rather restricted expression patterns of the specific proteins. Given the high functional and clinical relevance of both generic and cell-type-specific interacting networks, the following review shall provide a comprehensive summary of regulators of the multilayered CRAC channel signalling cascade. It also includes proteins expressed in a narrow spectrum of cells and tissues that are often disregarded in other reviews of similar topics. Full article

(This article belongs to the Special Issue STIMulating Ca²⁺ Homeostasis)

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17 pages, 4463 KiB

Open AccessArticle

Apigenin, a Partial Antagonist of the Estrogen Receptor (ER), Inhibits ER-Positive Breast Cancer Cell Proliferation through Akt/FOXM1 Signaling

by Thu Ha Pham, Yann Le Page, Frédéric Percevault, François Ferrière, Gilles Flouriot and Farzad Pakdel

Int. J. Mol. Sci. 2021, 22(1), 470; https://doi.org/10.3390/ijms22010470 - 5 Jan 2021

Cited by 27 | Viewed by 6157

Abstract

Approximately 80% of breast cancer (BC) cases express the estrogen receptor (ER), and 30–40% of these cases acquire resistance to endocrine therapies over time. Hyperactivation of Akt is one of the mechanisms by which endocrine resistance is acquired. Apigenin (Api), a flavone found [...] Read more.

Approximately 80% of breast cancer (BC) cases express the estrogen receptor (ER), and 30–40% of these cases acquire resistance to endocrine therapies over time. Hyperactivation of Akt is one of the mechanisms by which endocrine resistance is acquired. Apigenin (Api), a flavone found in several plant foods, has shown beneficial effects in cancer and chronic diseases. Here, we studied the therapeutic potential of Api in the treatment of ER-positive, endocrine therapy-resistant BC. To achieve this objective, we stably overexpressed the constitutively active form of the Akt protein in MCF-7 cells (named the MCF-7/Akt clone). The proliferation of MCF-7/Akt cells is partially independent of estradiol (E2) and exhibits an incomplete response to the anti-estrogen agent 4-hydroxytamoxifen, demonstrating the resistance of these cells to hormone therapy. Api exerts an antiproliferative effect on the MCF-7/Akt clone. Api inhibits the proliferative effect of E2 by inducing G2/M phase cell cycle arrest and apoptosis. Importantly, Api inhibits the Akt/FOXM1 signaling pathway by decreasing the expression of FOXM1, a key transcription factor involved in the cell cycle. Api also alters the expression of genes regulated by FOXM1, including cell cycle-related genes, particularly in the MCF-7/Akt clone. Together, our results strengthen the therapeutic potential of Api for the treatment of endocrine-resistant BC. Full article

(This article belongs to the Special Issue Antitumor Activities of Natural Compounds From Plants)

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12 pages, 1189 KiB

Open AccessReview

Intracellular Ca²⁺ Signaling in Protozoan Parasites: An Overview with a Focus on Mitochondria

by Pedro H. Scarpelli, Mateus F. Pecenin and Celia R. S. Garcia

Int. J. Mol. Sci. 2021, 22(1), 469; https://doi.org/10.3390/ijms22010469 - 5 Jan 2021

Cited by 16 | Viewed by 4392

Abstract

Ca²⁺ signaling has been involved in controling critical cellular functions such as activation of proteases, cell death, and cell cycle control. The endoplasmatic reticulum plays a significant role in Ca²⁺ storage inside the cell, but mitochondria have long been recognized as [...] Read more.

Ca²⁺ signaling has been involved in controling critical cellular functions such as activation of proteases, cell death, and cell cycle control. The endoplasmatic reticulum plays a significant role in Ca²⁺ storage inside the cell, but mitochondria have long been recognized as a fundamental Ca²⁺ pool. Protozoan parasites such as Plasmodium falciparum, Toxoplasma gondii, and Trypanosoma cruzi display a Ca²⁺ signaling toolkit with similarities to higher eukaryotes, including the participation of mitochondria in Ca²⁺-dependent signaling events. This review summarizes the most recent knowledge in mitochondrial Ca²⁺ signaling in protozoan parasites, focusing on the mechanism involved in mitochondrial Ca²⁺ uptake by pathogenic protists. Full article

(This article belongs to the Special Issue Mitochondrial Calcium Signaling)

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14 pages, 14154 KiB

Open AccessArticle

Distinct Responses of Arabidopsis Telomeres and Transposable Elements to Zebularine Exposure

by Klára Konečná, Pavla Polanská Sováková, Karin Anteková, Jiří Fajkus and Miloslava Fojtová

Int. J. Mol. Sci. 2021, 22(1), 468; https://doi.org/10.3390/ijms22010468 - 5 Jan 2021

Cited by 7 | Viewed by 3901

Abstract

Involvement of epigenetic mechanisms in the regulation of telomeres and transposable elements (TEs), genomic regions with the protective and potentially detrimental function, respectively, has been frequently studied. Here, we analyzed telomere lengths in Arabidopsis thaliana plants of Columbia, Landsberg erecta and Wassilevskija ecotypes [...] Read more.

Involvement of epigenetic mechanisms in the regulation of telomeres and transposable elements (TEs), genomic regions with the protective and potentially detrimental function, respectively, has been frequently studied. Here, we analyzed telomere lengths in Arabidopsis thaliana plants of Columbia, Landsberg erecta and Wassilevskija ecotypes exposed repeatedly to the hypomethylation drug zebularine during germination. Shorter telomeres were detected in plants growing from seedlings germinated in the presence of zebularine with a progression in telomeric phenotype across generations, relatively high inter-individual variability, and diverse responses among ecotypes. Interestingly, the extent of telomere shortening in zebularine Columbia and Wassilevskija plants corresponded to the transcriptional activation of TEs, suggesting a correlated response of these genomic elements to the zebularine treatment. Changes in lengths of telomeres and levels of TE transcripts in leaves were not always correlated with a hypomethylation of cytosines located in these regions, indicating a cytosine methylation-independent level of their regulation. These observations, including differences among ecotypes together with distinct dynamics of the reversal of the disruption of telomere homeostasis and TEs transcriptional activation, reflect a complex involvement of epigenetic processes in the regulation of crucial genomic regions. Our results further demonstrate the ability of plant cells to cope with these changes without a critical loss of the genome stability. Full article

(This article belongs to the Special Issue Role of Epigenetic Mechanisms in Plants: From Basic to Applicative Aspects 2.0)

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Figure 1
Experimental design. Seeds of A. thaliana of Columbia-0 (Col), Landsberg erecta (Ler), and Wassilevskija (Ws) ecotypes were germinated for 7 days on a control medium (O) or a medium supplemented with 250 µM zebularine (Z), and plants were cultivated in soil without the drug (first generation, 1G). Seeds from one parental plant (P) were germinated to limit the contribution of inter-individual variability in telomere lengths. Seeds from selected 1G plants were germinated either on a medium supplemented with zebularine (Z/Z) or on a control medium (Z/O and O/O), and plants were grown in the soil (second generation, 2G). The same protocol was followed once more to get the third generation (Z/Z/Z, Z/Z/O, and O/O/O, 3G) plants. This nomenclature is used in the following text and figures. Full article ">Figure 2
Lengths of telomeres in A. thaliana plants grown from seeds exposed to the zebularine during germination. (a) Seeds of Columbia-0 (Col); (b) Landsberg erecta (Ler); (c) Wassilewskija (Ws) ecotypes were exposed to the 250 µM zebularine during germination for three generations; in parallel, progenies of zebularine plants were germinated on a control medium (for experimental design and nomenclature of samples, see <a href="#ijms-22-00468-f001" class="html-fig">Figure 1</a>). Leaves for analysis were collected from 8-week-old plants. DNA was extracted from 2 g of leaves, 5 µg of DNA was digested by MseI restriction endonuclease and analyzed by Southern hybridization using radioactively labeled telomeric probe. Each sample (line) corresponded to the leaf taken from one independently cultivated plant. P, parental plant; Z, first generation (1G) of plants grown from seedlings germinated in the presence of 250 µM zebularine; Z/Z, plants grown from seedlings germinated for two generations (2G) in the presence of 250 µM zebularine; Z/Z/Z, plants grown from seedlings germinated for three generations (3G) in the presence of 250 µM zebularine; Z/O, 2G plants grown from seedlings of 1G zebularine plants (Z) germinated on control medium; Z/Z/O, 3G plants grown from seedlings of 2G zebularine plants (Z/Z) germinated on a control medium. Black arrows indicate plants that were propagated to the subsequent generation. In 3G plants with distinct telomere phenotypes (S, short telomeres and L, long telomeres), the levels of transposable elements (TEs) transcripts, TE methylation and methylation of telomeric cytosines were analyzed (Figures 3–5). M, DNA fragment size marker; GeneRuler 1 kb DNA Ladder (Thermo Fisher Scientific). In the right panels, lengths of telomeres were presented using a box and whisker plot with the bottom part (black) and the top part (white) representing the lower and upper quartiles, respectively, separated by the median. The ends of whiskers represent the minimal and maximal telomere lengths reflecting the range of the hybridization signal. Full article ">Figure 3
Relative levels of transcripts of selected TEs in seedlings of A. thaliana plants exposed repeatedly to 250 µM zebularine during germinations and in leaves of plants grown from these seedlings. RNA was extracted using the NucleoSpin RNA kit (Macherey-Nagel) from 100 mg of 7-day-old seedlings and leaves of 8-week-old plants. Relative levels of transcripts in Z/Z/Z 7-day-old seedlings (7ds) and leaves were compared to levels detected in control organs (7ds O/O/O, leaf O/O/O/, for experimental design and nomenclature of samples, see <a href="#ijms-22-00468-f001" class="html-fig">Figure 1</a>). S, plants with short telomeres and L, plants with long telomeres (see <a href="#ijms-22-00468-f002" class="html-fig">Figure 2</a>a). Signals of TE transcripts were related to that of the ubiquitin10 reference gene, levels of TE transcripts in 7ds O/O/O samples were arbitrarily chosen as 1. Data were evaluated by two sample F-test and t-test. * p < 0.05, ** p < 0.01, and *** p < 0.001. Note the logarithmic scale at the y axis. Full article ">Figure 4
SPM11 transposon methylation analyzed by bisulfite sequencing. Methylation of 44 cytosines located in an amplified 336 bp region (6 cytosines in the CG sequence context, 14 cytosines in the CHG, 24 cytosines in the CHH, H = A, T, C) was evaluated by the CyMATE software [<a href="#B27-ijms-22-00468" class="html-bibr">27</a>]. Five to ten clones were analyzed per sample; mean values of methylated cytosines and standard deviations are presented. O/O/O, leaf of the control Col plant; Z/Z/Z L, leaf of 3G plant with long telomeres grown from seedlings exposed to zebularine during germinations; Z/Z/Z S, leaf of 3G plant with short telomeres grown from seedlings exposed to zebularine during germinations (see <a href="#ijms-22-00468-f002" class="html-fig">Figure 2</a>a). For experimental design and nomenclature of samples, see <a href="#ijms-22-00468-f001" class="html-fig">Figure 1</a>. Full article ">Figure 5
Relative levels of methylated cytosines in telomeric repeats in the first and third generations of 7-day-old seedlings (7ds) of A. thaliana plants germinated on control medium or exposed to 250 µM zebularine, and in leaves of plants grown from these seedlings in soil. Mixed samples were analyzed; DNAs were isolated from 1 g of seedlings cultivated on three Petri dishes and 2 g of leaves of three plants grown from these seedlings, except of individual 3G zebularine plants with short and long telomeres (leaf Z/Z/Z S, leaf Z/Z/Z L). For bisulfite conversion, DNAs were mixed at an equimolar ratio. Bisulfite-modified DNAs were hybridized with the oligonucleotide probe reflecting fraction of telomeres with methylated cytosines (a), and the probe complementary to the G-strand of telomeres to determine loading (b). The same membrane was sequentially hybridized with both probes. PC, positive control, genomic DNA from Col leaves non-converted by sodium bisulfite; NC, negative control, pUC19 plasmid DNA. (c) Relative methylation of cytosines in telomeric repeats. Intensities of hybridization signals in (a,b) were evaluated by the MultiGauge software (FujiFilm) and expressed as the methylation/loading ratio. Signal ratios in respective control samples of the first generation (7ds O, leaf O) were arbitrarily taken as 1. For experimental design and nomenclature of samples, see <a href="#ijms-22-00468-f001" class="html-fig">Figure 1</a>. Full article ">

13 pages, 5397 KiB

Open AccessArticle

AtPiezo Plays an Important Role in Root Cap Mechanotransduction

by Xianming Fang, Beibei Liu, Qianshuo Shao, Xuemei Huang, Jia Li, Sheng Luan and Kai He

Int. J. Mol. Sci. 2021, 22(1), 467; https://doi.org/10.3390/ijms22010467 - 5 Jan 2021

Cited by 23 | Viewed by 5375

Abstract

Plants encounter a variety of mechanical stimuli during their growth and development. It is currently believed that mechanosensitive ion channels play an essential role in the initial perception of mechanical force in plants. Over the past decade, the study of Piezo, a mechanosensitive [...] Read more.

Plants encounter a variety of mechanical stimuli during their growth and development. It is currently believed that mechanosensitive ion channels play an essential role in the initial perception of mechanical force in plants. Over the past decade, the study of Piezo, a mechanosensitive ion channel in animals, has made significant progress. It has been proved that the perception of mechanical force in various physiological processes of animals is indispensable. However, little is still known about the function of its homologs in plants. In this study, by investigating the function of the AtPiezo gene in the model plant Arabidopsis thaliana, we found that AtPiezo plays a role in the perception of mechanical force in plant root cap and the flow of Ca²⁺ is involved in this process. These findings allow us to understand the function of AtPiezo from the perspective of plants and provide new insights into the mechanism of plant root cap in response to mechanical stimuli. Full article

(This article belongs to the Section Molecular Plant Sciences)

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Figure 1
Evolutionary Analysis of Piezo in Plants. (A) Maximum likelihood unrooted phylogenetic tree of Piezo homologs. Green branches belong to the plant kingdom. (B) Predicted topology of the AtPiezo monomer. Beam and CTD domains are indicated in black. (C) The alignment of beam, inner helix and CTD domains of AtPiezo and its orthologs. Protein multiple sequence alignment of AtPiezo (Arabidopsis thaliana), GmPiezo1a (Glycine max), OsPiezo (Oryza sativa Japonica Group), ZmPiezo (Zea mays), PpPiezo1a (Physcomitrella patens), OlPiezo (Ostreococcus lucimarinus), MmPiezo1 (Mus musculus), and HmPiezo1 (Homo sapiens). The highly conserved amino acids among Piezo orthologues are highlighted in yellow. The consensus is with a threshold of >50%. Full article ">Figure 2
Expression patterns of AtPiezo in Arabidopsis. (A) Histochemical GUS staining is shown in 0.5-day-old, 2-day-old, and 10-day-old seedlings. Bars = 0.5 mm. (B) Longitudinal and cross sections of the root cap in different positions of pAtPiezo::GUS plant. Bars = 50 μm and 20 μm. (C) The expression of pAtPiezo::NLS-YFP in the root cap. Bars = 50 μm. (D) RT-PCR analysis of AtPiezo transcripts in different tissues. Full article ">Figure 3
atpiezo mutants exhibit reduced rooting ability. (A) Schematic diagram of AtPiezo gene structure. Exons are indicated by black boxes. The black lines represent introns. Untranslated regions are indicated by gray boxes. The insertion sites of two T-DNA insertion alleles are indicated by the black solid triangles. The allele of delete fragment is indicated by the black hollow triangle. Bar = 1000 bp. (B) Representative images of WT and atpiezo mutants grown on 1/2MS medium five days after germination, Bar = 1 cm. (C) Representative images of WT and atpiezo mutants grown in the soil 55 days after germination. Bar = 5 cm. (D) The rooting phenotype of Col-0, piezo-1, piezo-2 and piezo-c1 horizontally grown on the agar medium. Five-day-old seedlings of different plant lines were grown on the medium containing 0.7%, 0.8%, or 0.9% agar. White solid arrows indicate the seedlings rooting in the medium. Bar = 1 cm. (E) The rate of WT and atpiezo mutants foraging into the medium five days after germination. Data are presented as mean ± SD (n = 6 plates, more than 30 seedlings in each plate) and analyzed with two-way ANOVA and Tukey’s multiple comparison test (Different lowercase letters indicate significant differences at p < 0.05). Full article ">Figure 4
The atpiezo mutant plants show altered growth status inside the medium. (A) Representative images of growth status of primary roots of 7-day-old plants in the agar medium. White solid arrows indicate the helixes at primary roots. Black solid arrows indicate the positions where the root tips reach. Bar = 1 cm. (B) The rate of helical roots of WT and atpiezo mutants in the media with different agar concentrations. Data are presented as mean ± SD (n = 6 plates, more than 10 seedlings in each plate) and analyzed with two-way ANOVA and Tukey’s multiple comparison test (Different lowercase letters indicate significant differences at p < 0.05). (C) The distance from the root tip to the medium surface of WT and atpiezo mutants in the media with different agar concentrations. Boxplots span the first to the third quartiles of the data. A line in the box represents the median (n > 20). The results were analyzed with two-way ANOVA and Tukey’s multiple comparison test (Different lowercase letters indicate significant differences at p < 0.05). Full article ">Figure 5
AtPiezo affects the shape of root caps in response to mechanical forces. (A) Schematic diagram of root cap index. The index is the ratio of the width across the QC and the length from QC to the root tip. (B) Root cap shape of Col-0, piezo1, and piezo-2 grown on the medium with 0.8% agar. The root cap of 4-day-old plants that vertically grew on the agar medium (Vertical). The root cap of the plants that horizontally grew for 24 h followed by vertical growth for three days on the medium (Horizontal). Bar = 50 μm. (C) Statistic analysis of root cap index for different plants under vertical and horizontal growth conditions. Boxplots span the first to the third quartiles of the data. A line in the box represents the median (n = 30). The results were analyzed with two-way ANOVA and Tukey’s multiple comparison test (Different lowercase letters indicate significant differences at p < 0.05). Full article ">Figure 6
AtPiezo affects the Ca2+ flux of plant root cap. (A) Schematic diagram of the position for Ca2+ flux measurement using NMT. (B) The net Ca2+ fluxes in Col-0, piezo-1, and piezo-c1 seedlings that were grown on medium for five days. Boxplots span the first to the third quartiles of the data. A line in the box represents the median (n = 9). The results were analyzed with one-way ANOVA and Tukey’s multiple comparison test (Different lowercase letters indicate significant differences at p < 0.05) (C) Real-time kinetics of Ca2+ flow in the tip of root cap. Data are presented as mean ± SE (n = 5). Full article ">

20 pages, 343 KiB

Open AccessReview

Determination of Intraprostatic and Intratesticular Androgens

by Markéta Šimková, Jiří Heráček, Pavel Drašar and Richard Hampl

Int. J. Mol. Sci. 2021, 22(1), 466; https://doi.org/10.3390/ijms22010466 - 5 Jan 2021

Cited by 5 | Viewed by 3799

Abstract

Androgens represent the main hormones responsible for maintaining hormonal balance and function in the prostate and testis. As they are involved in prostate and testicular carcinogenesis, more detailed information of their active concentration at the site of action is required. Since the introduction [...] Read more.

Androgens represent the main hormones responsible for maintaining hormonal balance and function in the prostate and testis. As they are involved in prostate and testicular carcinogenesis, more detailed information of their active concentration at the site of action is required. Since the introduction of the term intracrinology as the local formation of active steroid hormones from inactive precursors of the adrenal gland, mainly dehydroepiandrosterone (DHEA) and DHEA-S, it is evident that blood circulating levels of sex steroid hormones need not reflect their actual concentrations in the tissue. Here, we review and critically evaluate available methods for the analysis of human intraprostatic and intratesticular steroid concentrations. Since analytical approaches have much in common in both tissues, we discuss them together. Preanalytical steps, including various techniques for separation of the analytes, are compared, followed by the end-point measurement. Advantages and disadvantages of chromatography-mass spectrometry (LC-MS, GC-MS), immunoanalytical methods (IA), and hybrid (LC-IA) are discussed. Finally, the clinical information value of the determined steroid hormones is evaluated concerning differentiating between patients with cancer or benign hyperplasia and between patients with different degrees of infertility. Adrenal-derived 11-oxygenated androgens are mentioned as perspective prognostic markers for these purposes. Full article

(This article belongs to the Special Issue Candidate Biomarkers in Pathophysiology and Diagnostics of Endocrinopathies)

21 pages, 4503 KiB

Open AccessArticle

Prion-Associated Neurodegeneration Causes Both Endoplasmic Reticulum Stress and Proteasome Impairment in a Murine Model of Spontaneous Disease

by Alicia Otero, Marina Betancor, Hasier Eraña, Natalia Fernández Borges, José J. Lucas, Juan José Badiola, Joaquín Castilla and Rosa Bolea

Int. J. Mol. Sci. 2021, 22(1), 465; https://doi.org/10.3390/ijms22010465 - 5 Jan 2021

Cited by 13 | Viewed by 4021

Abstract

Prion diseases are a group of neurodegenerative disorders that can be spontaneous, familial or acquired by infection. The conversion of the prion protein PrP^C to its abnormal and misfolded isoform PrP^Sc is the main event in the pathogenesis of prion diseases [...] Read more.

Prion diseases are a group of neurodegenerative disorders that can be spontaneous, familial or acquired by infection. The conversion of the prion protein PrP^C to its abnormal and misfolded isoform PrP^Sc is the main event in the pathogenesis of prion diseases of all origins. In spontaneous prion diseases, the mechanisms that trigger the formation of PrP^Sc in the central nervous system remain unknown. Several reports have demonstrated that the accumulation of PrP^Sc can induce endoplasmic reticulum (ER) stress and proteasome impairment from the early stages of the prion disease. Both mechanisms lead to an increment of PrP aggregates in the secretory pathway, which could explain the pathogenesis of spontaneous prion diseases. Here, we investigate the role of ER stress and proteasome impairment during prion disorders in a murine model of spontaneous prion disease (TgVole) co-expressing the Ub^G76V-GFP reporter, which allows measuring the proteasome activity in vivo. Spontaneously prion-affected mice showed a significantly higher accumulation of the PKR-like ER kinase (PERK), the ER chaperone binding immunoglobulin protein (BiP/Grp78), the ER protein disulfide isomerase (PDI) and the Ub^G76V-GFP reporter than age-matched controls in certain brain areas. The upregulation of PERK, BiP, PDI and ubiquitin was detected from the preclinical stage of the disease, indicating that ER stress and proteasome impairment begin at early stages of the spontaneous disease. Strong correlations were found between the deposition of these markers and neuropathological markers of prion disease in both preclinical and clinical mice. Our results suggest that both ER stress and proteasome impairment occur during the pathogenesis of spontaneous prion diseases. Full article

(This article belongs to the Section Molecular Neurobiology)

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14 pages, 979 KiB

Open AccessReview

Metabolome-Driven Regulation of Adenovirus-Induced Cell Death

by Anastasia Laevskaya, Anton Borovjagin, Peter S. Timashev, Maciej S. Lesniak and Ilya Ulasov

Int. J. Mol. Sci. 2021, 22(1), 464; https://doi.org/10.3390/ijms22010464 - 5 Jan 2021

Cited by 4 | Viewed by 4383

Abstract

A viral infection that involves virus invasion, protein synthesis, and virion assembly is typically accompanied by sharp fluctuations in the intracellular levels of metabolites. Under certain conditions, dramatic metabolic shifts can result in various types of cell death. Here, we review different types [...] Read more.

A viral infection that involves virus invasion, protein synthesis, and virion assembly is typically accompanied by sharp fluctuations in the intracellular levels of metabolites. Under certain conditions, dramatic metabolic shifts can result in various types of cell death. Here, we review different types of adenovirus-induced cell death associated with changes in metabolic profiles of the infected cells. As evidenced by experimental data, in most cases changes in the metabolome precede cell death rather than represent its consequence. In our previous study, the induction of autophagic cell death was observed following adenovirus-mediated lactate production, acetyl-CoA accumulation, and ATP release, while apoptosis was demonstrated to be modulated by alterations in acetate and asparagine metabolism. On the other hand, adenovirus-induced ROS production and ATP depletion were demonstrated to play a significant role in the process of necrotic cell death. Interestingly, the accumulation of ceramide compounds was found to contribute to the induction of all the three types of cell death mentioned above. Eventually, the characterization of metabolite analysis could help in uncovering the molecular mechanism of adenovirus-mediated cell death induction and contribute to the development of efficacious oncolytic adenoviral vectors. Full article

(This article belongs to the Special Issue Autophagy in Cancer Progression and Therapeutics)

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Figure 1
Schematic representation of the tentative cell death mechanism operating through a ceramide-mediated mitophagy. Adenoviral infection induces ceramide accumulation through de novo synthesis from palmitoyl CoA and serine [<a href="#B19-ijms-22-00464" class="html-bibr">19</a>]. Subsequently, ceramide mediates the LC3 binding to the mitochondrial membrane and promotes lethal mitophagy leading to autophagic cell death [<a href="#B25-ijms-22-00464" class="html-bibr">25</a>]. Full article ">Figure 2
Calcium influx contributes to the induction of necrotic cell death. Calcium influx is promoted by many factors, including endoplasmic reticulum stress, acidosis (due to lactate accumulation) [<a href="#B70-ijms-22-00464" class="html-bibr">70</a>,<a href="#B71-ijms-22-00464" class="html-bibr">71</a>], and ATP depletion (particularly due to assembly of virions and RIPK1-dependent inhibition of ANT in the mitochondrial membrane mediated by ceramide accumulation). Subsequently, an elevated concentration of calcium induces ROS production through increasing mitochondrial permeability [<a href="#B56-ijms-22-00464" class="html-bibr">56</a>,<a href="#B72-ijms-22-00464" class="html-bibr">72</a>,<a href="#B73-ijms-22-00464" class="html-bibr">73</a>]. A simultaneous decrease in ATP levels and the induced ROS production result in necrotic cell death. Abbreviations: ER—endoplasmic reticulum, ANT—adenosine nucleotide translocase, MCI—mitochondrial complex I, LDH—lactate dehydrogenase, “+”—increase effect. Full article ">Figure 3
Metabolome fluctuations that trigger different types of cell death. Elevated acetate levels stimulating uptake of glucose and lactate along with asparagine deficiency can be linked to the induction of apoptosis. Calcium influx, production of reactive oxygen species (ROS), increased glucose uptake and lactate secretion as well as an elevation in acetyl-CoA concentration are all capable of triggering autophagic cell death. In addition, ROS production, calcium elevation and ATP depletion are known to induce necroptosis. Furthermore, accumulation of ceramide species is capable of triggering all three types of cell death described in this review. Up arrow means it increases and down arrow means it decreases. Full article ">

16 pages, 1694 KiB

Open AccessReview

The Association between Hepatic Encephalopathy and Diabetic Encephalopathy: The Brain-Liver Axis

by So Yeong Cheon and Juhyun Song

Int. J. Mol. Sci. 2021, 22(1), 463; https://doi.org/10.3390/ijms22010463 - 5 Jan 2021

Cited by 22 | Viewed by 7545

Abstract

Hepatic encephalopathy (HE) is one of the main consequences of liver disease and is observed in severe liver failure and cirrhosis. Recent studies have provided significant evidence that HE shows several neurological symptoms including depressive mood, cognitive dysfunction, impaired circadian rhythm, and attention [...] Read more.

Hepatic encephalopathy (HE) is one of the main consequences of liver disease and is observed in severe liver failure and cirrhosis. Recent studies have provided significant evidence that HE shows several neurological symptoms including depressive mood, cognitive dysfunction, impaired circadian rhythm, and attention deficits as well as motor disturbance. Liver disease is also a risk factor for the development of diabetes mellitus. Diabetic encephalopathy (DE) is characterized by cognitive dysfunction and motor impairment. Recent research investigated the relationship between metabolic changes and the pathogenesis of neurological disease, indicating the importance between metabolic organs and the brain. Given that a diverse number of metabolites and changes in the brain contribute to neurologic dysfunction, HE and DE are emerging types of neurologic disease. Here, we review significant evidence of the association between HE and DE, and summarise the common risk factors. This review may provide promising therapeutic information and help to design a future metabolic organ-related study in relation to HE and DE. Full article

(This article belongs to the Special Issue Oxidative Stress and Inflammation in Chronic Diseases)

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34 pages, 8154 KiB

Open AccessArticle

The Thyroid Hormone Transporter Mct8 Restricts Cathepsin-Mediated Thyroglobulin Processing in Male Mice through Thyroid Auto-Regulatory Mechanisms That Encompass Autophagy

by Vaishnavi Venugopalan, Alaa Al-Hashimi, Maren Rehders, Janine Golchert, Vivien Reinecke, Georg Homuth, Uwe Völker, Mythili Manirajah, Adam Touzani, Jonas Weber, Matthew S. Bogyo, Francois Verrey, Eva K. Wirth, Ulrich Schweizer, Heike Heuer, Janine Kirstein and Klaudia Brix

Int. J. Mol. Sci. 2021, 22(1), 462; https://doi.org/10.3390/ijms22010462 - 5 Jan 2021

Cited by 8 | Viewed by 4594

Abstract

The thyroid gland is both a thyroid hormone (TH) generating as well as a TH responsive organ. It is hence crucial that cathepsin-mediated proteolytic cleavage of the precursor thyroglobulin is regulated and integrated with the subsequent export of TH into the blood circulation, [...] Read more.

The thyroid gland is both a thyroid hormone (TH) generating as well as a TH responsive organ. It is hence crucial that cathepsin-mediated proteolytic cleavage of the precursor thyroglobulin is regulated and integrated with the subsequent export of TH into the blood circulation, which is enabled by TH transporters such as monocarboxylate transporters Mct8 and Mct10. Previously, we showed that cathepsin K-deficient mice exhibit the phenomenon of functional compensation through cathepsin L upregulation, which is independent of the canonical hypothalamus-pituitary-thyroid axis, thus, due to auto-regulation. Since these animals also feature enhanced Mct8 expression, we aimed to understand if TH transporters are part of the thyroid auto-regulatory mechanisms. Therefore, we analyzed phenotypic differences in thyroid function arising from combined cathepsin K and TH transporter deficiencies, i.e., in Ctsk^-/-/Mct10^-/-, Ctsk^-/-/Mct8^-/y, and Ctsk^-/-/Mct8^-/y/Mct10^-/-. Despite the impaired TH export, thyroglobulin degradation was enhanced in the mice lacking Mct8, particularly in the triple-deficient genotype, due to increased cathepsin amounts and enhanced cysteine peptidase activities, leading to ongoing thyroglobulin proteolysis for TH liberation, eventually causing self-thyrotoxic thyroid states. The increased cathepsin amounts were a consequence of autophagy-mediated lysosomal biogenesis that is possibly triggered due to the stress accompanying intrathyroidal TH accumulation, in particular in the Ctsk^-/-/Mct8^-/y/Mct10^-/- animals. Collectively, our data points to the notion that the absence of cathepsin K and Mct8 leads to excessive thyroglobulin degradation and TH liberation in a non-classical pathway of thyroid auto-regulation. Full article

(This article belongs to the Special Issue Thyroid Hormone and Thyroid Hormone-Related Compounds: Molecular Pathways and Effects)

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24 pages, 2925 KiB

Open AccessArticle

Intermittent Hypoxic Conditioning Rescues Cognition and Mitochondrial Bioenergetic Profile in the Triple Transgenic Mouse Model of Alzheimer’s Disease

by Sónia C. Correia, Nuno J. Machado, Marco G. Alves, Pedro F. Oliveira and Paula I. Moreira

Int. J. Mol. Sci. 2021, 22(1), 461; https://doi.org/10.3390/ijms22010461 - 5 Jan 2021

Cited by 14 | Viewed by 4999

Abstract

The lack of effective disease-modifying therapeutics to tackle Alzheimer’s disease (AD) is unsettling considering the actual prevalence of this devastating neurodegenerative disorder worldwide. Intermittent hypoxic conditioning (IHC) is a powerful non-pharmacological procedure known to enhance brain resilience. In this context, the aim of [...] Read more.

The lack of effective disease-modifying therapeutics to tackle Alzheimer’s disease (AD) is unsettling considering the actual prevalence of this devastating neurodegenerative disorder worldwide. Intermittent hypoxic conditioning (IHC) is a powerful non-pharmacological procedure known to enhance brain resilience. In this context, the aim of the present study was to investigate the potential long-term protective impact of IHC against AD-related phenotype, putting a special focus on cognition and mitochondrial bioenergetics and dynamics. For this purpose, six-month-old male triple transgenic AD mice (3×Tg-AD) were submitted to an IHC protocol for two weeks and the behavioral assessment was performed at 8.5 months of age, while the sacrifice of mice occurred at nine months of age and their brains were removed for the remaining analyses. Interestingly, IHC was able to prevent anxiety-like behavior and memory and learning deficits and significantly reduced brain cortical levels of amyloid-β (Aβ) in 3×Tg-AD mice. Concerning brain energy metabolism, IHC caused a significant increase in brain cortical levels of glucose and a robust improvement of the mitochondrial bioenergetic profile in 3×Tg-AD mice, as mirrored by the significant increase in mitochondrial membrane potential (ΔΨm) and respiratory control ratio (RCR). Notably, the improvement of mitochondrial bioenergetics seems to result from an adaptative coordination of the distinct but intertwined aspects of the mitochondrial quality control axis. Particularly, our results indicate that IHC favors mitochondrial fusion and promotes mitochondrial biogenesis and transport and mitophagy in the brain cortex of 3×Tg-AD mice. Lastly, IHC also induced a marked reduction in synaptosomal-associated protein 25 kDa (SNAP-25) levels and a significant increase in both glutamate and GABA levels in the brain cortex of 3×Tg-AD mice, suggesting a remodeling of the synaptic microenvironment. Overall, these results demonstrate the effectiveness of the IHC paradigm in forestalling the AD-related phenotype in the 3×Tg-AD mouse model, offering new insights to AD therapy and forcing a rethink concerning the potential value of non-pharmacological interventions in clinical practice. Full article

(This article belongs to the Section Biochemistry)

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19 pages, 4475 KiB

Open AccessArticle

STAT3 Is an Upstream Regulator of Granzyme G in the Maternal-To-Zygotic Transition of Mouse Embryos

by Huan Ou-Yang, Shinn-Chih Wu, Li-Ying Sung, Shiao-Hsuan Yang, Shang-Hsun Yang, Kowit-Yu Chong and Chuan-Mu Chen

Int. J. Mol. Sci. 2021, 22(1), 460; https://doi.org/10.3390/ijms22010460 - 5 Jan 2021

Cited by 7 | Viewed by 3722

Abstract

The maternal-to-zygotic transition (MZT), which controls maternal signaling to synthesize zygotic gene products, promotes the preimplantation development of mouse zygotes to the two-cell stage. Our previous study reported that mouse granzyme g (Gzmg), a serine-type protease, is required for the MZT. In this [...] Read more.

The maternal-to-zygotic transition (MZT), which controls maternal signaling to synthesize zygotic gene products, promotes the preimplantation development of mouse zygotes to the two-cell stage. Our previous study reported that mouse granzyme g (Gzmg), a serine-type protease, is required for the MZT. In this study, we further identified the maternal factors that regulate the Gzmg promoter activity in the zygote to the two-cell stage of mouse embryos. A full-length Gzmg promoter from mouse genomic DNA, FL-pGzmg (−1696~+28 nt), was cloned, and four deletion constructs of this Gzmg promoter, Δ1-pGzmg (−1369~+28 nt), Δ2-pGzmg (−939~+28 nt), Δ3-pGzmg (−711~+28 nt) and Δ4-pGzmg (−417~+28 nt), were subsequently generated. Different-sized Gzmg promoters were used to perform promoter assays of mouse zygotes and two-cell stage embryos. The results showed that Δ4-pGzmg promoted the highest expression level of the enhanced green fluorescent protein (EGFP) reporter in the zygotes and two-cell embryos. The data suggested that time-specific transcription factors upregulated Gzmg by binding cis-elements in the −417~+28-nt Gzmg promoter region. According to the results of the promoter assay, the transcription factor binding sites were predicted and analyzed with the JASPAR database, and two transcription factors, signal transducer and activator of transcription 3 (STAT3) and GA-binding protein alpha (GABPα), were identified. Furthermore, STAT3 and GABPα are expressed and located in zygote pronuclei and two-cell nuclei were confirmed by immunofluorescence staining; however, only STAT3 was recruited to the mouse zygote pronuclei and two-cell nuclei injected with the Δ4-pGzmg reporter construct. These data indicated that STAT3 is a maternal transcription factor and may upregulate Gzmg to promote the MZT. Furthermore, treatment with a STAT3 inhibitor, S3I-201, caused mouse embryonic arrest at the zygote and two-cell stages. These results suggest that STAT3, a maternal protein, is a critical transcription factor and regulates Gzmg transcription activity in preimplantation mouse embryos. It plays an important role in the maternal-to-zygotic transition during early embryonic development. Full article

(This article belongs to the Special Issue Regulation of Gene Expression During Embryonic Development)

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21 pages, 3704 KiB

Open AccessArticle

Characterization of Urine Stem Cell-Derived Extracellular Vesicles Reveals B Cell Stimulating Cargo

by Asmaa A. Zidan, Mohammed Al-Hawwas, Griffith B. Perkins, Ghada M. Mourad, Catherine J. M. Stapledon, Larisa Bobrovskaya, Xin-Fu Zhou and Plinio R. Hurtado

Int. J. Mol. Sci. 2021, 22(1), 459; https://doi.org/10.3390/ijms22010459 - 5 Jan 2021

Cited by 18 | Viewed by 4906

Abstract

Elucidation of the biological functions of extracellular vesicles (EVs) and their potential roles in physiological and pathological processes is an expanding field of research. In this study, we characterized USC–derived EVs and studied their capacity to modulate the human immune response in vitro. [...] Read more.

Elucidation of the biological functions of extracellular vesicles (EVs) and their potential roles in physiological and pathological processes is an expanding field of research. In this study, we characterized USC–derived EVs and studied their capacity to modulate the human immune response in vitro. We found that the USC–derived EVs are a heterogeneous population, ranging in size from that of micro–vesicles (150 nm–1 μm) down to that of exosomes (60–150 nm). Regarding their immunomodulatory functions, we found that upon isolation, the EVs (60–150 nm) induced B cell proliferation and IgM antibody secretion. Analysis of the EV contents unexpectedly revealed the presence of BAFF, APRIL, IL–6, and CD40L, all known to play a central role in B cell stimulation, differentiation, and humoral immunity. In regard to their effect on T cell functions, they resembled the function of mesenchymal stem cell (MSC)–derived EVs previously described, suppressing T cell response to activation. The finding that USC–derived EVs transport a potent bioactive cargo opens the door to a novel therapeutic avenue for boosting B cell responses in immunodeficiency or cancer. Full article

(This article belongs to the Special Issue Interaction of Nanomaterials with the Immune System)

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Urine stem cells (USCs) characterization; (A) Phase–contrast light micrograph (LM) at passage zero showing stellate shaped cells with membrane projection reaching nearby cells (original magnification ×100, scale bar = 100 µm). (B) hematoxylin and eosin staining of USCs at passage three, showing basophilic stellate cells with perinuclear eosinophilia, large central nuclei, and cytoplasmic projections Actively dividing cells were occasionally seen represented by kissing cells (arrow) (original magnification ×400, scale bar = 20 µm). (C) Periodic acid–Schiff (PAS) staining of USCs at passage three, showing perinuclear magenta color (original magnification ×400, scale bar = 20 µm). (D) Toluidine Blue staining of USC pellet following chondrogenic differentiation showing blue staining proteoglycan–rich extracellular matrix (original magnification ×40, scale bar = 200 µm). (E) Oil Red O staining of the induced adipogenic cells showing red staining of intracellular lipid (original magnification ×400, scale bar = 20 µm). (F) Alizarin Red staining of induced osteogenic cells showing a dark orange discoloration of the calcification nodules (original magnification ×100, scale bar = 100 µm). (G) A representative sample of immune phenotypical characterization of 105 USCs (shaded curve) showing positive expression of CD105, CD73, and negative for HLA–DR, CD34, CD45, and CD14. Full article ">Figure 2
Transmission electron microscopy (TEM) characterization of USCs, and extracellular vesicles EVs morphology and Biogenesis; (A) USCs TEM where different cell structures can be visualized such as the nucleus (N), abundant mitochondria (M), rough endoplasmic reticulum (rER), lysosomes (ly), pools of dense granules (DG) and multivesicular body (MVB); magnified in (B), where biogenesis of exosomes can be observed in the three phases described, endosomes (Endo), MVB containing intraluminal vesicles of different sizes (Asterix) and fusing of MVB to the membrane with exosome release (Mic. Mag. ×13,000). The cell membrane exhibits multiple projections, membrane budding forming vesicles (solid square) and micro–vesicles (large dotted square, illustrated in (C) containing dense granules varying in size, shape, and content, (Mic. Mag. ×2900). (C–E) The pictures show the heterogenicity in sizes and density, varying dense granules containing larger vesicles between 150 nm–1µm, typical for micro–vesicles (C–E), and homogenous less granular vesicles < 150 nm, typical for exosomes (E,F), (Mic. Mag. ×2900). (F) Vesicle enclosed by USC membrane, mimic endocytosis (Mic. Mag. ×18,500) (Lead citrate/Uranyl acetate stain). Full article ">Figure 3
Characterization of USCs–derived EVs; (A,B) TEM negative staining of the isolated EVs showing cup–shaped vesicles with an average size of 110 nm visualized at lower magnification (A; Mic. Mag. ×23,000) and higher magnification (B; Mic. Mag. ×30,000) (2%Uranyl Acetate). (C) Histogram presentation of 19 EVs pellets collected from 1.6 × 107 USCs analyzed for size and concentration, showing an average size of 122 nm and an average concentration of 1.90925 × 1011. (D) Size distribution curve of USCs isolated EVs particle concentration (×107) vs. particle size mode, measured by nanoparticle tracking analysis (NTA) showing the average of the six technical replicate measurements for each exosome isolation by NanosightS300. (E) Western blot of USCs cell lysate and isolated the EVs for CD63, CD81, TSG101 antibodies as positive markers for EVs and Cytochrome C as mitochondrial membrane marker (cellular Marker) and negative marker for EVs. (F) Immune gold staining of the isolated vesicles for CD63 using 6 nm gold nanoparticles (2% Uranyl Acetate, scale bar = 100nm, Mic. Mag. ×30,000 (upper left) and ×23,000 rest). Full article ">Figure 4
The effect of USCs EVs on B cells; (A) CD69 (early activation marker) expression on B cells population showing increased expression with EV co-culture. (B,C) Proliferation assay of the % proliferating B cells as the result of EVs co-culture in (B) resting and (C) CpGB–stimulated conditions showing significant enhancement of proliferation in both conditions in response to EV co-culture (n = 5). (D–F) Confocal microscopy images of DAPI (4′,6-diamidino-2-phenylindole) stained purified B cells (D) co-cultured with labeled EVs (E) for 24 h, (F) showing the presence of labeled EVs inside the cytoplasm and in aggregates attached to the cell surface (Mic. Magnification ×600, scale bar = 10 µm). (G) A representative sample of flow cytometry analysis of CD69+ B cell population for FITC labeled EVs uptake versus control. (H) Antibody analysis of the supernatant showing a significant increase of IgM with EV co-culture (n = 6). Values are presented as Mean ± SEM, p-values were determined by Paired Student’s t–tests. * p < 0.05, ** p < 0.01. Full article ">Figure 5
Cytokines Analysis of the EVs; (A) Cytokines analysis of EV co-culture supernatant run by Biolegend plex assay shows a significant increase in IL6, CD40L, IL10, and TNFα in response to EV co-culture (n = 3). (B) Heat map of the EV lysates, PBMCs, and the co-culture for cytokines concentration generated by Bio–legendplex software. (C) Cytokines analysis of 10 µg/mL EV lysates shows the expression of considerable amounts of IL–6, BAFF, APRIL, CD40L, and traces of IL–10, TNFα, and IFN–γ (n = 3). (D) Immune gold staining of the isolated vesicles for CD40L using 6 nm gold nanoparticles (Mic. Mag. ×23,000, 2% Uranyl Acetate). (E) Confocal microscope image of DAPI (blue) stained USC after permeabilization for CD40L mAb (red) (Mic. Mag. ×600, scale bar = 20 µm). (F) Confocal microscope image of DAPI (blue) stained USCs after staining with BAFFR mAb (green) (Mic. Mag. ×600, scale bar = 50 µm). (G) Flow cytometry analysis of 104 USCs for BAFFR expression showing 99% positive cells (green shaded histogram) in relation to the isotype control (non–shaded histogram), representative sample. Values are presented as mean ± SEM and p–values were determined by paired Student’s t–tests. Cytokines data are prior to log treatment. * p < 0.05, ** p < 0.01. Full article ">Figure 6
USCs EVs effect on T cells in response to anti–CD3/CD28 stimulation; (A) Effect of the EVs on the proliferation of T cells in the presence of anti–CD3/CD28–bead stimulation showing significant suppression of the T cell proliferation (represented by % proliferating cells, n = 5). Values are presented as mean ± SEM, and p-values were determined by paired t–test analysis. ** p < 0.01. (B–D) A representative sample of the viability assay of CD3+ (T cell) population where (B) control T cells viability, (C) viability in response to EVs co-culture. (D) Viability in response to EV co-culture in the presence of anti–CD3/CD28 beads. Note the decrease in the viable population in the activated T cells co-cultured with EVs. Full article ">

13 pages, 3227 KiB

Open AccessArticle

Increasing Oxygen Partial Pressures Induce a Distinct Transcriptional Response in Human PBMC: A Pilot Study on the “Normobaric Oxygen Paradox”

by Deborah Fratantonio, Fabio Virgili, Alessandro Zucchi, Kate Lambrechts, Tiziana Latronico, Pierre Lafère, Peter Germonpré and Costantino Balestra

Int. J. Mol. Sci. 2021, 22(1), 458; https://doi.org/10.3390/ijms22010458 - 5 Jan 2021

Cited by 44 | Viewed by 4457

Abstract

The term “normobaric oxygen paradox” (NOP), describes the response to the return to normoxia after a hyperoxic event, sensed by tissues as oxygen shortage, and resulting in up-regulation of the Hypoxia-inducible factor 1α (HIF-1α) transcription factor activity. The molecular characteristics of this response [...] Read more.

The term “normobaric oxygen paradox” (NOP), describes the response to the return to normoxia after a hyperoxic event, sensed by tissues as oxygen shortage, and resulting in up-regulation of the Hypoxia-inducible factor 1α (HIF-1α) transcription factor activity. The molecular characteristics of this response have not been yet fully characterized. Herein, we report the activation time trend of oxygen-sensitive transcription factors in human peripheral blood mononuclear cells (PBMCs) obtained from healthy subjects after one hour of exposure to mild (MH), high (HH) and very high (VHH) hyperoxia, corresponding to 30%, 100%, 140% O₂, respectively. Our observations confirm that MH is perceived as a hypoxic stress, characterized by the activation of HIF-1α and Nuclear factor (erythroid-derived 2)-like 2 (NRF2), but not Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB). Conversely, HH is associated to a progressive loss of NOP response and to an increase in oxidative stress leading to NRF2 and NF-kB activation, accompanied by the synthesis of glutathione (GSH). After VHH, HIF-1α activation is totally absent and oxidative stress response, accompanied by NF-κB activation, is prevalent. Intracellular GSH and Matrix metallopeptidase 9 (MMP-9) plasma levels parallel the transcription factors activation pattern and remain elevated throughout the observation time. In conclusion, our study confirms that, in vivo, the return to normoxia after MH is sensed as a hypoxic trigger characterized by HIF-1α activation. On the contrary, HH and VHH induce a shift toward an oxidative stress response, characterized by NRF2 and NF-κB activation in the first 24 h post exposure. Full article

(This article belongs to the Special Issue Cellular Oxygen Homeostasis)

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HIF-1α nuclear translocation following 1 h hyperoxia. (a) Mild hyperoxia (30% O2); (b) high hyperoxia (100% O2); (c) very high hyperoxia (140% O2) before and after the recovery to normoxic conditions. Above histograms, the picture shows a representative western blot analysis. Results are expressed as fold change (mean ± SEM) in comparison to baseline (0), which was set at 1. * p < 0.05, ** p < 0,01; for one-way ANOVA followed by Dunnett’s post hoc test. Full article ">Figure 2
NRF2 nuclear translocation following 1 h hyperoxia. (a) Mild hyperoxia (30% O2); (b) high hyperoxia (100% O2); (c) very high hyperoxia (140% O2) before and after the recovery to normoxic conditions. Above histograms, the picture shows a representative western blot analysis. Results are expressed as fold change (mean ± SEM) in comparison to baseline (0), which was set at 1. * p < 0.05, ** p < 0,01; for one-way ANOVA followed by Dunnett’s post hoc test. Full article ">Figure 3
NF-κB (p65 subunit) nuclear translocation following 1 h hyperoxia. (a) Mild hyperoxia (30% O2); (b) high hyperoxia (100% O2); (c) very high hyperoxia (140% O2) before and after the recovery to normoxic conditions. Above histograms, the picture shows a representative western blot analysis. Results are expressed as fold change (mean ± SEM) in comparison to baseline (0), which was set at 1. * p < 0.05, ** p < 0,01; for one-way ANOVA followed by Dunnett’s post hoc test. Full article ">Figure 4
Changes in intracellular glutathione levels in human PBMC from different groups of healthy subjects after one hour of oxygen exposure in (a) mild (30% O2), (b) high (100% O2) and (c) very high hyperoxia (140% O2). The histograms display the nmole of GSH for mg of protein (mean ± SEM) in comparison to baseline (Time 0), which was set at 10. * p < 0.05 , ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. base line before oxygen exposure (time 0), for one-way ANOVA followed by Dunnett’s post hoc test. Full article ">Figure 5
Zymographic analysis of matrix metallo-proteinase (MMPs) in human plasma from different groups of healthy subjects after one hour of oxygen exposure in (a) mild (30% O2), (b) high (100% O2) and (c) very high hyperoxia (140% O2). The pictures show a representative zymograph gel for all the subjects submitted to the analysis. The histograms display the percentage (mean ± SEM) in comparison to baseline (Time 0), which was set at 10. * p < 0.05 vs. base line before oxygen exposure (Time 0), for one-way ANOVA followed by Dunnett’s post hoc test. Full article ">

14 pages, 3814 KiB

Open AccessArticle

Delivery of the Radionuclide ¹³¹I Using Cationic Fusogenic Liposomes as Nanocarriers

by Rejhana Kolašinac, Dirk Bier, Laura Schmitt, Andriy Yabluchanskiy, Bernd Neumaier, Rudolf Merkel and Agnes Csiszár

Int. J. Mol. Sci. 2021, 22(1), 457; https://doi.org/10.3390/ijms22010457 - 5 Jan 2021

Cited by 10 | Viewed by 3801

Abstract

Liposomes are highly biocompatible and versatile drug carriers with an increasing number of applications in the field of nuclear medicine and diagnostics. So far, only negatively charged liposomes with intercalated radiometals, e.g., ⁶⁴Cu, ^99mTc, have been reported. However, the process of [...] Read more.

Liposomes are highly biocompatible and versatile drug carriers with an increasing number of applications in the field of nuclear medicine and diagnostics. So far, only negatively charged liposomes with intercalated radiometals, e.g., ⁶⁴Cu, ^99mTc, have been reported. However, the process of cellular uptake of liposomes by endocytosis is rather slow. Cellular uptake can be accelerated by recently developed cationic liposomes, which exhibit extraordinarily high membrane fusion ability. The aim of the present study was the development of the formulation and the characterization of such cationic fusogenic liposomes with intercalated radioactive [¹³¹I]I⁻ for potential use in therapeutic applications. The epithelial human breast cancer cell line MDA-MB-231 was used as a model for invasive cancer cells and cellular uptake of [¹³¹I]I⁻ was monitored in vitro. Delivery efficiencies of cationic and neutral liposomes were compared with uptake of free iodide. The best cargo delivery efficiency (~10%) was achieved using cationic fusogenic liposomes due to their special delivery pathway of membrane fusion. Additionally, human blood cells were also incubated with cationic control liposomes and free [¹³¹I]I⁻. In these cases, iodide delivery efficiencies remained below 3%. Full article

(This article belongs to the Special Issue Functionalized Liposomes)

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Chemical structures of the lipid molecules forming cationic fusogenic (A) and control endocytic liposomes (B). Preparation of iodide-loaded liposomes (C). After evaporation of the organic solvent, the dry lipid mixture was rehydrated in an iodide-containing buffer whereby liposomes containing iodide isotopes formed spontaneously between the lamellas. Iodide loading efficiency was determined on liposomes separated from the free solution by centrifugation. Full article ">Figure 2
Representative curves of the hydrodynamic diameter (A) and zeta potential distributions (B) of cationic fusogenic (FL) and control endocytic liposomes (EL) with and without [127I]I− incubation. Hypothetical structures of the same liposomes based on the physicochemical characterization (C). Note that the same liposomal structures are hypothesized in the presence of both [131I]I− as well as [127I]I−. Full article ">Figure 3
Absolute activity of free [131I]I− radionuclide solution, as well as the activity of [131I]I− intercalated into cationic fusogenic (FL) and control endocytic liposomal (EL) pellet (index p) and the activity of free [131I]I− in the supernatant (A). The uptake efficiency of free [131I]I− was determined using two different iodide concentrations, the initial one (131I−), and one quarter of this concentration (131I−/4), which was approximately identical to the amount of intercalated iodide in FLs. Activity changes of [131I]I− in phosphate buffered saline (PBS) and liposomal supernatants (B) as well as in liposomal pellets (C) were monitored over 16 days. Statistical significances were considered as follows: p < 0.001 (***), p < 0.01 (**). Full article ">Figure 4
Delivery efficiencies of [131I]I− using liposomes as carrier particles and free [131I]I- from supernatants and neat solutions as controls to MDA MB-231 cancer cell line (A), human blood cells (B), primary neuronal cells (C), and Chinese hamster ovary (CHO) cells (D). Data are presented as mean (SD). N = 3. Statistical significances were considered as follows: p < 0.001 (***), p < 0.01 (**), p < 0.05 (*). Full article ">Figure 5
Monitoring the incorporation of cationic fusogenic (FL) and control endocytic liposomal (EL) pellet (index p) and supernatant (index s) containing [127I]I− into MDA MB-231 cancer cells and human blood cells by microscopy. The fluorescence signal of the lipid tracer DiR (red) and the cell nuclei staining of DAPI (blue) are shown in overlays. Phase contrast images reveal healthy cell morphologies. Scale bars, 20 µm. Full article ">

29 pages, 1356 KiB

Open AccessReview

Emerging Options for the Diagnosis of Bacterial Infections and the Characterization of Antimicrobial Resistance

by Simone Rentschler, Lars Kaiser and Hans-Peter Deigner

Int. J. Mol. Sci. 2021, 22(1), 456; https://doi.org/10.3390/ijms22010456 - 5 Jan 2021

Cited by 35 | Viewed by 10219

Abstract

Precise and rapid identification and characterization of pathogens and antimicrobial resistance patterns are critical for the adequate treatment of infections, which represent an increasing problem in intensive care medicine. The current situation remains far from satisfactory in terms of turnaround times and overall [...] Read more.

Precise and rapid identification and characterization of pathogens and antimicrobial resistance patterns are critical for the adequate treatment of infections, which represent an increasing problem in intensive care medicine. The current situation remains far from satisfactory in terms of turnaround times and overall efficacy. Application of an ineffective antimicrobial agent or the unnecessary use of broad-spectrum antibiotics worsens the patient prognosis and further accelerates the generation of resistant mutants. Here, we provide an overview that includes an evaluation and comparison of existing tools used to diagnose bacterial infections, together with a consideration of the underlying molecular principles and technologies. Special emphasis is placed on emerging developments that may lead to significant improvements in point of care detection and diagnosis of multi-resistant pathogens, and new directions that may be used to guide antibiotic therapy. Full article

(This article belongs to the Special Issue Analysing Bacterial Infection, Microbiome Characterics and Small Molecules in Diagnostics: Emerging Options)

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Typical timeframes required for techniques in current use for the diagnosis of bacterial infections. The classical cultivation of biological specimens, in combination with biochemical characterization, requires ~42 h as a minimum estimate. Replacement of biochemical methods with MALDI-TOF mass spectrometry (MS) reduces this timeframe significantly. The use of nucleic acid testing (NAT) bypasses the initial cultivation of clinical specimens, and thus reduces the timeframes to fewer than 4 h. However, NAT methods are sequence-dependent and involve only a limited number of primer-combinations; as such, these methods require a priori knowledge of the suspected pathogen(s). The use of NGS-based methods eliminates the need for any a priori knowledge of a suspected pathogen, although typical timeframes are increased. Full article ">Figure 2
Possible shortcuts for bacterial pathogen identification from patient specimens, using MALDI-TOF MS. Typically, specimens are cultivated and identified via biochemical approaches. By MALDI-TOF-based analysis of single colonies obtained during the first round of cultivation, long turnaround times obligatory for biochemical characterization, can be bypassed. Furthermore, patient specimens can be directly analyzed by purification and concentration of bacterial cells, further shortening turnaround time. However, in case of polymicrobial infections, single bacteria need to be further separated, increasing the time to result. As polymicrobial infections cannot be ruled out in most clinical cases, a further separation of single species should be integrated into routine workflows. Full article ">Figure 3
Illustration of different technologies for pathogen identification, suitable for point-of-care (POC) application. General approaches can be divided into antigen detection (top) and nucleic acid testing (bottom). Lateral Flow Immunoassays, for example, are readily applicable for detection of pathogen specific antigens, multiplexing-approaches for identification of several different species can be realized by incorporating different fluorescence labels (e.g., quantum dots). Furthermore, Plasmonic Biosensors show a great potential for POC applications, as sensitivity can be drastically increased and sample treatment can be avoided [<a href="#B105-ijms-22-00456" class="html-bibr">105</a>]. Another emerging technology for POC application is the Whispering Gallery Mode sensor technology, attracting much attention over the past decade. Here, the binding of molecules to the resonators surface can be detected as a change of the effective refractive index. Although the WGM technology displays a promising candidate, there are currently still several challenges hindering transformation into the clinical environment [<a href="#B78-ijms-22-00456" class="html-bibr">78</a>]. In case of approaches for POC applicable nucleic acid testing, several variable approaches are present. In general, shorter time periods during the amplification step can be achieved via implementation of paper-based (e.g., isothermal amplification [<a href="#B19-ijms-22-00456" class="html-bibr">19</a>]) or micro-fluidic- based (e.g., micro-fluidic PCR [<a href="#B106-ijms-22-00456" class="html-bibr">106</a>]) approaches. For detection of the amplicon, several different technologies can readily be used, including Nucleic Acid Lateral Flow Assays or intercalating dyes. Full article ">Figure 4
Overview of current and promising technologies suitable for antimicrobial resistance profiling. Beside classical culture-based approaches (not included), currently available commercial solutions include PCR multiplex panels (e.g., Biofire®-FilmArray® panels (biomérieux)), MALDI-TOF MS (MTB-STAR Assays (Bruker Daltonics, Inc.)), biochemical tests (e.g., RAPIDEC® CARBA NP test (biomérieux)) and protein marker tests (e.g., RESIST-3 O.K.N. K-SeT (Coris BioConcept, Gembloux, Belgium)). Further promising technological advances have been made in the field of Next-generation Sequencing (e.g., [<a href="#B174-ijms-22-00456" class="html-bibr">174</a>]) adaption of biochemical tests to faster electrochemical formats (e.g., [<a href="#B167-ijms-22-00456" class="html-bibr">167</a>]) or development of electrochemical sensors for bacterial growth (e.g., [<a href="#B154-ijms-22-00456" class="html-bibr">154</a>]). Several of these approaches are suspected to result in commercially available solutions for antimicrobial resistance profiling in the future. Full article ">

22 pages, 5055 KiB

Open AccessReview

FUT8 Alpha-(1,6)-Fucosyltransferase in Cancer

by Kayla Bastian, Emma Scott, David J. Elliott and Jennifer Munkley

Int. J. Mol. Sci. 2021, 22(1), 455; https://doi.org/10.3390/ijms22010455 - 5 Jan 2021

Cited by 84 | Viewed by 10466

Abstract

Aberrant glycosylation is a universal feature of cancer cells that can impact all steps in tumour progression from malignant transformation to metastasis and immune evasion. One key change in tumour glycosylation is altered core fucosylation. Core fucosylation is driven by fucosyltransferase 8 (FUT8), [...] Read more.

Aberrant glycosylation is a universal feature of cancer cells that can impact all steps in tumour progression from malignant transformation to metastasis and immune evasion. One key change in tumour glycosylation is altered core fucosylation. Core fucosylation is driven by fucosyltransferase 8 (FUT8), which catalyses the addition of α1,6-fucose to the innermost GlcNAc residue of N-glycans. FUT8 is frequently upregulated in cancer, and plays a critical role in immune evasion, antibody-dependent cellular cytotoxicity (ADCC), and the regulation of TGF-β, EGF, α3β1 integrin and E-Cadherin. Here, we summarise the role of FUT8 in various cancers (including lung, liver, colorectal, ovarian, prostate, breast, melanoma, thyroid, and pancreatic), discuss the potential mechanisms involved, and outline opportunities to exploit FUT8 as a critical factor in cancer therapeutics in the future. Full article

(This article belongs to the Special Issue Glycobiology of Cancer)

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20 pages, 7599 KiB

Open AccessArticle

RNA-Seq Provides New Insights into the Molecular Events Involved in “Ball-Skin versus Bladder Effect” on Fruit Cracking in Litchi

by Jun Wang, Xiao Fang Wu, Yong Tang, Jian Guo Li and Ming Lei Zhao

Int. J. Mol. Sci. 2021, 22(1), 454; https://doi.org/10.3390/ijms22010454 - 5 Jan 2021

Cited by 17 | Viewed by 3537

Abstract

Fruit cracking is a disorder of fruit development in response to internal or external cues, which causes a loss in the economic value of fruit. Therefore, exploring the mechanism underlying fruit cracking is of great significance to increase the economic yield of fruit [...] Read more.

Fruit cracking is a disorder of fruit development in response to internal or external cues, which causes a loss in the economic value of fruit. Therefore, exploring the mechanism underlying fruit cracking is of great significance to increase the economic yield of fruit trees. However, the molecular mechanism underlying fruit cracking is still poorly understood. Litchi, as an important tropical and subtropical fruit crop, contributes significantly to the gross agricultural product in Southeast Asia. One important agricultural concern in the litchi industry is that some famous varieties with high economic value such as ‘Nuomici’ are susceptible to fruit cracking. Here, the cracking-susceptible cultivar ‘Nuomici’ and cracking-resistant cultivar ‘Huaizhi’ were selected, and the samples including pericarp and aril during fruit development and cracking were collected for RNA-Seq analysis. Based on weighted gene co-expression network analysis (WGCNA) and the “ball-skin versus bladder effect” theory (fruit cracking occurs upon the aril expanding pressure exceeds the pericarp strength), it was found that seven co-expression modules genes (1733 candidate genes) were closely associated with fruit cracking in ‘Nuomici’. Importantly, we propose that the low expression level of genes related to plant hormones (Auxin, Gibberellins, Ethylene), transcription factors, calcium transport and signaling, and lipid synthesis might decrease the mechanical strength of pericarp in ‘Nuomici’, while high expression level of genes associated with plant hormones (Auxin and abscisic acid), transcription factors, starch/sucrose metabolism, and sugar/water transport might increase the aril expanding pressure, thereby resulting in fruit cracking in ‘Nuomici’. In conclusion, our results provide comprehensive molecular events involved in the “ball-skin versus bladder effect” on fruit cracking in litchi. Full article

(This article belongs to the Section Molecular Plant Sciences)

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13 pages, 928 KiB

Open AccessReview

Macrophage Autophagy and Silicosis: Current Perspective and Latest Insights

by Shiyi Tan and Shi Chen

Int. J. Mol. Sci. 2021, 22(1), 453; https://doi.org/10.3390/ijms22010453 - 5 Jan 2021

Cited by 59 | Viewed by 6416

Abstract

Silicosis is an urgent public health problem in many countries. Alveolar macrophage (AM) plays an important role in silicosis progression. Autophagy is a balanced mechanism for regulating the cycle of synthesis and degradation of cellular components. Our previous study has shown that silica [...] Read more.

Silicosis is an urgent public health problem in many countries. Alveolar macrophage (AM) plays an important role in silicosis progression. Autophagy is a balanced mechanism for regulating the cycle of synthesis and degradation of cellular components. Our previous study has shown that silica engulfment results in lysosomal rupture, which may lead to the accumulation of autophagosomes in AMs of human silicosis. The excessive accumulation of autophagosomes may lead to apoptosis in AMs. Herein, we addressed some assumptions concerning the complex function of autophagy-related proteins on the silicosis pathogenesis. We also recapped the molecular mechanism of several critical proteins targeting macrophage autophagy in the process of silicosis fibrosis. Furthermore, we summarized several exogenous chemicals that may cause an aggravation or alleviation for silica-induced pulmonary fibrosis by regulating AM autophagy. For example, lipopolysaccharides or nicotine may have a detrimental effect combined together with silica dust via exacerbating the blockade of AM autophagic degradation. Simultaneously, some natural product ingredients such as atractylenolide III, dioscin, or trehalose may be the potential AM autophagy regulators, protecting against silicosis fibrosis. In conclusion, the deeper molecular mechanism of these autophagy targets should be explored in order to provide feasible clues for silicosis therapy in the clinical setting. Full article

(This article belongs to the Section Molecular Pathology, Diagnostics, and Therapeutics)

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The general silicosis pathological mechanism. The attack of silica dust triggers the phagocytosis of alveolar macrophages (AMs). However, the lysosomal membrane of AM will be disrupted by the H-bonding reaction, causing AM apoptosis. Notably, AM apoptosis may be regulated by the mitochondria apoptotic pathway, Fas apoptotic pathway, nuclear factor kappa-B (NF-κB) apoptotic pathway, and p53 apoptotic pathway. Apoptotic AM secrets a series of inflammatory factors. Eventually, the proliferation, activation, and migration of fibroblasts synthesize and release collagen, resulting in silicosis fibrosis. Full article ">Figure 2
Determined mechanism of silicosis pathogenesis mediated by macrophage autophagy. (a) Normally, the pre-autophagosomal structure (PAS) begins to engulf cytosolic components, following which the signal of autophagy is activated. Subsequently, the autophagosomal membrane expands, matures, and closes gradually. Once closed, the autophagosome will fuse with the lysosome to form the autophagolysosome, degrading its wrapped contents. Several autophagy-related proteins act critically, indicating effect during the process of autophagy. Sequestosome 1 (p62/SQSTM1) interacts with ubiquitinated substrates, participating in the subsequent process of autophagic degradation. Microtubule-associated protein 1A/1B-light chain 3-I (LC3-I) conjugates with phosphatidylethanolamine to form LC3-II, involved in the expansion, maturation, and closure of autophagosome. Additionally, lysosome-associated membrane protein (LAMP) is attached to the surface of the lysosome. (b) When invading macrophages (especially alveolar macrophages) excessively, silica dust will cause autophagosomes to accumulate and lysosomes to disrupt (i.e., the dysfunction of the autophagy-lysosomal system). (c) Silica dust invades macrophages (especially alveolar macrophages) via a class A scavenger receptor (SR-A) and causes the blockade of autophagic degradation. The impairment of autophagy function caused by silica further leads to excessive macrophage apoptosis by decreasing the level of BCL2 and increasing the level of BCL2-Associated X (Bax). The inflammation and subsequent silicosis fibrosis occur eventually. Notably, nuclear transfer of nuclear factor kappa-B (NF-κB) may be an important link between autophagy and apoptosis in silicosis. In this pathological progression, disruption of the autophagy-lysosomal system induces macrophage apoptosis through the activation of NACHT-, LRR-, and PYD domain-containing protein 3 (NALP3). NALP3 also increases macrophage apoptosis via the nuclear transfer of NF-κB. Meanwhile, activation of BCL2-binding component 3 (BBC3) or Monocyte chemoattractant protein-1-induced protein 1 (MCPIP1) promotes macrophage apoptosis through exacerbating the autophagic degradation. However, the functional role of Toll-like receptor 4 (TLR4) is still controversial. Full article ">

12 pages, 3317 KiB

Open AccessArticle

Intracerebroventricular Treatment with 2-Hydroxypropyl-β-Cyclodextrin Decreased Cerebellar and Hepatic Glycoprotein Nonmetastatic Melanoma Protein B (GPNMB) Expression in Niemann–Pick Disease Type C Model Mice

by Madoka Fukaura, Yoichi Ishitsuka, Seiichi Shirakawa, Naoki Ushihama, Yusei Yamada, Yuki Kondo, Toru Takeo, Naomi Nakagata, Keiichi Motoyama, Taishi Higashi, Hidetoshi Arima, Yuki Kurauchi, Takahiro Seki, Hiroshi Katsuki, Katsumi Higaki, Muneaki Matsuo and Tetsumi Irie

Int. J. Mol. Sci. 2021, 22(1), 452; https://doi.org/10.3390/ijms22010452 - 5 Jan 2021

Cited by 20 | Viewed by 4584

Abstract

Niemann–Pick disease type C (NPC) is a recessive hereditary disease caused by mutation of the NPC1 or NPC2 gene. It is characterized by abnormality of cellular cholesterol trafficking with severe neuronal and hepatic injury. In this study, we investigated the potential of glycoprotein [...] Read more.

Niemann–Pick disease type C (NPC) is a recessive hereditary disease caused by mutation of the NPC1 or NPC2 gene. It is characterized by abnormality of cellular cholesterol trafficking with severe neuronal and hepatic injury. In this study, we investigated the potential of glycoprotein nonmetastatic melanoma protein B (GPNMB) to act as a biomarker reflecting the therapeutic effect of 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) in an NPC mouse model. We measured serum, brain, and liver expression levels of GPNMB, and evaluated their therapeutic effects on NPC manifestations in the brain and liver after the intracerebroventricular administration of HP-β-CD in Npc1 gene-deficient (Npc1^−/−) mice. Intracerebroventricular HP-β-CD inhibited cerebellar Purkinje cell damage in Npc1^−/− mice and significantly reduced serum and cerebellar GPNMB levels. Interestingly, we also observed that the intracerebral administration significantly reduced hepatic GPNMB expression and elevated serum ALT in Npc1^−/− mice. Repeated doses of intracerebroventricular HP-β-CD (30 mg/kg, started at 4 weeks of age and repeated every 2 weeks) drastically extended the lifespan of Npc1^−/− mice compared with saline treatment. In summary, our results suggest that GPNMB level in serum is a potential biomarker for evaluating the attenuation of NPC pathophysiology by intracerebroventricular HP-β-CD treatment. Full article

(This article belongs to the Special Issue Advances in Knowledge in Niemann-Pick Disease Type C: Facts and Perspectives- 2nd Edition)

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Potential of the intracerebroventricular administration of 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) to protect against reductions in body weight (A) and cerebellar Purkinje cells (B and C) in Npc1−/− mice. HP-β-CD was administered at 21.4 μmol/kg in mice at 4 and 6 weeks of age. We recorded the changes in body weight once a week from 4 to 8 weeks old. We collected samples from the whole brain, liver, and blood at 8 weeks and 3 days of age and analyzed them. Histological images of the mouse cerebellum (B). Scale bar: 100 μm. Quantitative evaluation of calbindin-positive cells (C). Data are presented as the mean ± S.E. (n = 8–11). *** p < 0.001 compared with the wild-type group. ### p < 0.001 compared with the saline group. Full article ">Figure 2
Suppression of serum soluble glycoprotein nonmetastatic melanoma protein B (sGPNMB) levels in Npc1−/− mice treated with 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) intracerebroventricularly. Levels of sGPNMB in mouse serum were assayed by ELISA. Data are presented as the mean ± S.E. (n = 8–11). *** p < 0.001 compared with the wild-type group. ### p < 0.001 compared with the saline group. Full article ">Figure 3
Intracerebroventricular treatment with 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) attenuated the abnormal increase of glycoprotein nonmetastatic melanoma protein B (GPNMB) expression in Npc1−/− mice. Assays of GPNMB levels in brain using ELISA (A) and immunohistochemical staining of GPNMB (B) were performed. Scale bar: 100 μm. Data are presented as the mean ± S.E. (n = 4–7). *** p < 0.01 compared with the wild-type group. ### p < 0.001 compared with the saline group. Full article ">Figure 4
Attenuating effects of intracerebroventricular 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) treatment on liver manifestations in Npc1−/− mice. Glycoprotein nonmetastatic melanoma protein B (GPNMB) levels in liver homogenate (A), GPNMB immunohistochemistry in liver (B), serum alanine aminotransferase (ALT) levels (C), and hematoxylin-and-eosin (H&E)-stained hepatic histology (D) are shown. Scale bar: 100 μm for GPNMB and H&E staining. Each bar represents the mean ± S.E.M. (n = 8–11). *** p < 0.001 compared with the wild-type group. # p < 0.05, ### p < 0.001 compared with the saline group. Full article ">Figure 5
Life-prolonging effect of intracerebroventricular administration of 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) in Npc1−/− mice. HP-β-CD treatment (21.4 μmol/kg) was started at 4 weeks of age, with repeated injection into the mice every 2 weeks. Kaplan–Meier survival curves for the Npc1−/− mice are shown (n = 4 for the saline-treated group (circle) and n = 7 for the HP-β-CD (biweekly) group (square)). A significant difference between the two groups was identified by the log-rank test (p < 0.01). Full article ">

15 pages, 1802 KiB

Open AccessArticle

Impairment of IGF-1 Signaling and Antioxidant Response Are Associated with Radiation Sensitivity and Mortality

by Saeed Y. Aghdam, Doreswamy Kenchegowda, Gregory P. Holmes-Hampton, Maria Moroni and Sanchita P. Ghosh

Int. J. Mol. Sci. 2021, 22(1), 451; https://doi.org/10.3390/ijms22010451 - 5 Jan 2021

Cited by 5 | Viewed by 2578

Abstract

Following exposure to high doses of ionizing radiation, diverse strains of vertebrate species will manifest varying levels of radiation sensitivity. To understand the inter-strain cellular and molecular mechanisms of radiation sensitivity, two mouse strains with varying radiosensitivity (C3H/HeN, and CD2F1), were exposed to [...] Read more.

Following exposure to high doses of ionizing radiation, diverse strains of vertebrate species will manifest varying levels of radiation sensitivity. To understand the inter-strain cellular and molecular mechanisms of radiation sensitivity, two mouse strains with varying radiosensitivity (C3H/HeN, and CD2F1), were exposed to total body irradiation (TBI). Since Insulin-like Growth Factor-1 (IGF-1) signaling pathway is associated with radiosensitivity, we investigated the link between systemic or tissue-specific IGF-1 signaling and radiosensitivity. Adult male C3H/HeN and CD2F1 mice were irradiated using gamma photons at Lethal Dose-70/30 (LD_70/30), 7.8 and 9.35 Gy doses, respectively. Those mice that survived up to 30 days post-irradiation, were termed the survivors. Mice that were euthanized prior to 30 days post-irradiation due to deteriorated health were termed decedents. The analysis of non-irradiated and irradiated survivor and decedent mice showed that inter-strain radiosensitivity and post-irradiation survival outcomes are associated with activation status of tissue and systemic IGF-1 signaling, nuclear factor erythroid 2–related factor 2 (Nrf2) activation, and the gene expression profile of cardiac mitochondrial energy metabolism pathways. Our findings link radiosensitivity with dysregulation of IGF-1 signaling, and highlight the role of antioxidant gene response and mitochondrial function in radiation sensitivity. Full article

(This article belongs to the Section Molecular Toxicology)

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Irradiation scheme for the CD2F1 and C3H/HeN adult male mice. Animals were irradiated at LD70/30 dose. The day of irradiation was considered as day 0; animals that underwent unscheduled euthanasia due to severe health deterioration before day 30 post-irradiation were considered as decedents. Animals that survived from radiation exposure were euthanized on day 30 post-irradiation (scheduled euthanasia). Full article ">Figure 2
Serum IGF-1 and Nitric Oxide (NO) levels in sham and LD70/30 irradiated CD2F1 and C3H/HeN adult male mice. (A) ELISA analysis of serum IGF-1 levels in sham, irradiated decedent (Decd.) and irradiated survivor (Surv.) mice. n = 7 for sham animals of both strains, n = 8 for decedent animals of both strains, n = 3 for survivors of both strains. (B) Serum nitric oxide levels in sham, irradiated decedent (Decd.) and irradiated survivor (Surv.) mice. n = 8 for sham animals of both strains, n = 8 for CD2F1 decedents, n = 10 for C3H/HeN decedents, n = 3 for survivors of both strains. Data analyzed by student’s t-test. Results presented as mean ± SEM, *** p < 0.001 and **** p < 0.0001, p > 0.05 considered as ‘not significant’ (ns). Full article ">Figure 3
Western blot analysis of IGF-1R, Akt and Nrf2 activation in sham and LD70/30 irradiated CD2F1 and C3H/HeN adult male mice. (A,B) Analysis of phosphorylated IGF-1 receptor (p.IGF1R, Tyr1135/1136), total IGF-1R, phosphorylated Akt (p.Akt, Ser473), total Akt, phosphorylated Nrf2 (p.Nrf2, Ser40 residue), β–Tubulin and β–Actin in heart protein extracts from sham, irradiated decedent (Decd.) and irradiated survivor (Surv.) mice. (C,D) Quantification of the IGF-1R and Nrf2 activation. The Y axis in graphs represents the ratios of the respective measured pixel intensities of Western blot bands. Total number of analyzed samples from both CD2F1 and C3H strains: sham—n = 6; decedent—n = 6; survivor n = 3. Data analyzed by Student’s t-test and are presented as mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001, p > 0.05 considered as ‘not significant’ (ns). Full article ">Figure 4
Peroxide and ATP levels in sham and LD70/30 irradiated survivor and decedent heart samples from CD2F1 and C3H/HeN mice. Graphs representing the heart peroxide (A) and ATP (B) levels measured in sham and irradiated CD2F1 and C3H/HeN mice. Total number of analyzed samples: n = 8 for sham and irradiated decedent mice and n = 3 for irradiated survivors. Data analyzed by Student’s t-test, presented as mean ± SEM, * p < 0.05, *** p < 0.001 and p > 0.05, ns—not significant. Full article ">Figure 5
PCR array analysis of genes involved in nitric oxide (NO), oxidative stress and mitochondrial energy metabolism in heart samples from sham and LD70/30 irradiated CD2F1 and C3H/HeN mice. Quantitative PCR array analysis showing the fold changes in the expression of NO (A,B) oxidative stress (C,D) and mitochondrial energy metabolism (E,F) mRNA levels in heart tissues from sham, irradiated decedent and irradiated survivor mice. The Y axis shows the relative fold changes in expression of the genes in irradiated survivors and decedents compared with the sham. Only those genes showing changes of three-fold or higher, either in the survivors or decedents are shown. The dotted line indicates 3-fold upregulation or downregulation in the graphs. Number of samples for both CD2F1 and C3H strains: Sham (control)—n = 7; decedent—n = 8; survivors—n = 3. Full article ">Figure 6
Western blot analysis of IGF-1R, Akt and Nrf2 in lung and kidney samples from sham and LD70/30 irradiated CD2F1 and C3H/HeN adult male mice. (A) Analysis of phosphorylated IGF-1 receptor (p.IGF1R, Tyr1135/1136), total IGF-1R, phosphorylated Akt (p.Akt, Ser473), total Akt and (C) phosphorylated Nrf2 (p.Nrf2, Ser40) in lung protein extracts from sham, irradiated decedent (Decd.) and irradiated survivor (Surv.) mice. β–Actin used as loading control. (B,D) Quantification of IGF-1R and Nrf2 activation in the lung samples. IGF-1R activation is determined by calculating the ratio of the pixel intensity of p.IGF-1R to total IGF-1R levels. Nrf2 activation is determined by calculating the ratio of pixel intensity of the p.Nrf2 to respective β–Actin band. (E) Western blot analysis of phosphorylated IGF-1 receptor (p.IGF1R, Tyr1135/1136), total IGF-1R and total Akt in kidney protein extracts from sham, irradiated decedent (Decd.) and irradiated survivor (Surv.) mice. GAPDH is used as loading control. Total number of analyzed samples for both strains: n = 6 for sham, n = 6 for decedents, n = 3 for survivors. Results analyzed by Student’s t-test; presented as mean ± SEM, * p < 0.05, *** p < 0.001 and p > 0.05 is considered as ‘not significant’ (ns). Full article ">

16 pages, 4667 KiB

Open AccessArticle

CHID1 Is a Novel Prognostic Marker of Non-Small Cell Lung Cancer

by Olga V. Kovaleva, Madina A. Rashidova, Daria V. Samoilova, Polina A. Podlesnaya, Rasul M. Tabiev, Valeria V. Mochalnikova and Alexei Gratchev

Int. J. Mol. Sci. 2021, 22(1), 450; https://doi.org/10.3390/ijms22010450 - 5 Jan 2021

Cited by 8 | Viewed by 4029

Abstract

There is an urgent need for identification of new prognostic markers and therapeutic targets for non-small cell lung cancer (NSCLC). In this study, we evaluated immune cells markers in 100 NSCLC specimens. Immunohistochemical analysis revealed no prognostic value for the markers studied, except [...] Read more.

There is an urgent need for identification of new prognostic markers and therapeutic targets for non-small cell lung cancer (NSCLC). In this study, we evaluated immune cells markers in 100 NSCLC specimens. Immunohistochemical analysis revealed no prognostic value for the markers studied, except CD163 and CD206. At the same time, macrophage markers iNOS and CHID1 were found to be expressed in tumor cells and associated with prognosis. We showed that high iNOS expression is a marker of favorable prognosis for squamous cell lung carcinoma (SCC), and NSCLC in general. Similarly, high CHID1 expression is a marker of good prognosis in adenocarcinoma and in NSCLC in general. Analysis of prognostic significance of a high CHID1/iNOS expression combination showed favorable prognosis with 20 months overall survival of patients from the low CHID1/iNOS expression group. For the first time, we demonstrated that CHID1 can be expressed by NSCLC cells and its high expression is a marker of good prognosis for adenocarcinoma and NSCLC in general. At the same time, high expression of iNOS in tumor cells is a marker of good prognosis in SCC. When used in combination, CHID1 and iNOS show a very good prognostic capacity for NSCLC. We suggest that in the case of lung cancer, tumor-associated macrophages are likely ineffective as a therapeutic target. At the same time, macrophage markers expressed by tumor cells may be considered as targets for anti-tumor therapy or, as in the case of CHID1, as potential anti-tumor agents. Full article

(This article belongs to the Special Issue The New Molecular Strategies under Development in Thoracic Oncology)

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13 pages, 2481 KiB

Open AccessArticle

Tranexamic Acid Promotes Murine Bone Marrow-Derived Osteoblast Proliferation and Inhibits Osteoclast Formation In Vitro

by Anke Baranowsky, Jessika Appelt, Kristina Tseneva, Shan Jiang, Denise Jahn, Serafeim Tsitsilonis, Karl-Heinz Frosch and Johannes Keller

Int. J. Mol. Sci. 2021, 22(1), 449; https://doi.org/10.3390/ijms22010449 - 5 Jan 2021

Cited by 8 | Viewed by 2722

Abstract

Despite modern surgical trauma care, bleeding contributes to one-third of trauma-related death. A significant improvement was obtained through the introduction of tranexamic acid (TXA), which today is widely used in emergency and elective orthopedic surgery to control bleeding. However, concerns remain regarding potential [...] Read more.

Despite modern surgical trauma care, bleeding contributes to one-third of trauma-related death. A significant improvement was obtained through the introduction of tranexamic acid (TXA), which today is widely used in emergency and elective orthopedic surgery to control bleeding. However, concerns remain regarding potential adverse effects on bone turnover and regeneration. Therefore, we employed standardized cell culture systems including primary osteoblasts, osteoclasts, and macrophages to evaluate potential effects of TXA on murine bone cells. While osteoblasts derived from calvarial digestion were not affected, TXA increased cell proliferation and matrix mineralization in bone marrow-derived osteoblasts. Short-term TXA treatment (6 h) failed to alter the expression of osteoblast markers; however, long-term TXA stimulation (10 days) was associated with the increased expression of genes involved in osteoblast differentiation and extracellular matrix synthesis. Similarly, whereas short-term TXA treatment did not affect gene expression in terminally differentiated osteoclasts, long-term TXA stimulation resulted in the potent inhibition of osteoclastogenesis. Finally, in bone marrow-derived macrophages activated with LPS, simultaneous TXA treatment led to a reduced expression of inflammatory cytokines and chemokines. Collectively, our study demonstrates a differential action of TXA on bone cells including osteoanabolic, anti-resorptive, and anti-inflammatory effects in vitro which suggests novel treatment applications. Full article

(This article belongs to the Special Issue Different Strategies for Osteogenic Differentiation and Bone Regeneration)

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TXA does not affect the differentiation or function of calvaria-derived osteoblasts. (a) Representative alizarin red stainings of calvaria-derived osteoblasts from WT mice differentiated in the presence of indicated concentrations of TXA for 10 days in osteogenic medium. Scale bar 10 mm. The quantification of extracellular matrix mineralization is indicated below. (b) qRT-PCR expression analysis for the indicated genes in calvaria-derived osteoblasts at day 10 of osteogenic differentiation, stimulated with TXA (1 mg/mL) during the entire course of cell differentiation. (c) qRT-PCR expression analysis for the indicated genes in the same samples. (d) MTT proliferation assay of calvaria-derived osteoblasts stimulated with 1 mg/mL TXA for 6 h at the indicated days of differentiation. For (a–d), n = 4–6 independent cultures per group were used, as indicated by individual data points. Data presented are means ± SD. Gene abbreviations: runt-related transcription factor 2 (Runx2), osterix (Sp7), alkaline phosphatase (Alpl), alpha-1 type I collagen (Col1a1), osteocalcin (Bglap), sclerostin (Sost), Rankl (Tnfsf11), osteoprotegerin (Tnfrsf11b). Full article ">Figure 2
TXA promotes extracellular matrix mineralization. (a) Representative alizarin red stainings of bone marrow-derived osteoblasts from WT mice differentiated in the presence of indicated concentrations of TXA for 10 days in osteogenic medium. Scale bar 10 mm. The quantification of extracellular matrix mineralization is indicated below. (b) qRT-PCR expression analysis for the indicated genes in bone marrow-derived osteoblasts at day 10 of osteogenic differentiation, stimulated with TXA (1 mg/mL) during the entire course of cell differentiation. (c,d) qRT-PCR expression analysis for the indicated genes in bone marrow-derived osteoblasts at day 2 (c) and day 10 (d) of osteogenic differentiation, stimulated with TXA for 6 h at the indicated concentrations after serum starvation overnight. (e) qRT-PCR expression analysis for the indicated genes in the same cells at day 2 or (f) day 10 of differentiation. For (a–f), n = 3–4 independent cultures per group were used. Data presented are means ± SD. Gene abbreviations: runt-related transcription factor 2 (Runx2), osterix (Sp7), alkaline phosphatase (Alpl), alpha-1 type I collagen (Col1a1), osteocalcin (Bglap), sclerostin (Sost), Rankl (Tnfsf11), osteoprotegerin (Tnfrsf11b). Full article ">Figure 3
TXA enhances cell proliferation of bone marrow-derived osteoblasts. MTT proliferation assay of bone marrow-derived osteoblasts stimulated with 1 mg/mL TXA for 6 h at the indicated time points; n = 3–5 independent cultures per group were used. Data presented are means ± SD. Full article ">Figure 4
Inhibition of early osteoclastogenesis through TXA. (a) TRAP activity staining of WT bone marrow cells at day 7 of differentiation, cultured in the presence of M-CSF and RANKL and continuous exposure to vehicle or TXA at the indicated concentrations. Scale bars = 50 μm. The quantification of osteoclast numbers per viewing field is depicted on the left (Ocl.N./VF). (b) TRAP activity staining of WT bone marrow cells at day 7 of differentiation, cultured in the presence of M-CSF and RANKL, and exposure to vehicle or TXA at the indicated concentrations only at day 1 of osteoclast differentiation. The quantification of osteoclast numbers per viewing field is depicted on the left (Ocl.N./VF). (c) TRAP activity staining of WT osteoclasts at day 7 of differentiation, cultured in the presence of M-CSF and RANKL and exposed to vehicle or TXA at the indicated concentrations for 24 h. (d) MTT proliferation assay of WT osteoclasts at day 7 of differentiation stimulated with the indicated concentrations of TXA for 6 h. (e) qRT-PCR expression analysis for the indicated genes in bone marrow-derived osteoclasts at day 7, stimulated with TXA (1 mg/mL) during the entire course of cell differentiation. (f) qRT-PCR expression analysis for the indicated genes in bone marrow-derived osteoclasts at the indicated stages of cell differentiation, stimulated with TXA (1 mg/mL) for 6 h. (g) qRT-PCR expression analysis for the indicated genes in bone marrow-derived osteoclasts at day 5 of differentiation, stimulated with TXA (1 mg/mL) during the entire course of cell differentiation. For (a–g), n = 3–4 independent cultures per group were used. Data presented are means ± SD. Gene abbreviations: chloride channel 7 (Clcn7), nuclear factor kappa B subunit 1 (Nfkb1), receptor activator of nf-κb (Tnfrsf11a), tartrate-resistant acid phosphatase (Acp5), cathepsin K (Ctsk). Full article ">Figure 5
Modulation of cytokine responses through TXA in resting and activated macrophages. (a) qRT-PCR expression analysis for the indicated genes in bone marrow-derived macrophages at day 7 of differentiation, stimulated with TXA (1 mg/mL) for 6 h. (b) qRT-PCR expression analysis for the indicated genes in bone marrow-derived macrophages at day 7 of differentiation, activated with 1 μg/mL LPS and simultaneously co-stimulated with TXA (1 mg/mL) for 6 h. (c) MTT proliferation assay of bone marrow-derived macrophages stimulated with the indicated concentrations of TXA. (d) Representative images of macrophage migration assays using Ibidi chambers of the indicated groups and time points. Scale bars = 50 μm. Blue lines indicate spreading cell fronts. The quantification of cell migration is shown on the right. For (a–d), n = 3–5 independent macrophage cultures per group were used as indicated with individual data points. Data presented are means ± SD. Gene abbreviations: interleukin-1 alpha (Il1a), interleukin-1 beta (Il1b), interleukin-4 (Il4), interleukin-6 (Il6), interleukin-10 (Il10), tumor necrosis factor alpha (Tnfa), CC-chemokine ligand 5 (Ccl5), cluster of differentiation 14 (Cd14), interleukin-1 receptor antagonist (Il1ra), inducible NO synthase (iNos), toll-like receptor 4 (Tlr4), transforming growth factor beta (Tgfb). Full article ">

13 pages, 1860 KiB

Open AccessArticle

KRAS Promoter G-Quadruplexes from Sequences of Different Length: A Physicochemical Study

by Federica D’Aria, Bruno Pagano, Luigi Petraccone and Concetta Giancola

Int. J. Mol. Sci. 2021, 22(1), 448; https://doi.org/10.3390/ijms22010448 - 5 Jan 2021

Cited by 8 | Viewed by 3563

Abstract

DNA G-quadruplexes (G4s) form in relevant genomic regions and intervene in several biological processes, including the modulation of oncogenes expression, and are potential anticancer drug targets. The human KRAS proto-oncogene promoter region contains guanine-rich sequences able to fold into G4 structures. Here, by [...] Read more.

DNA G-quadruplexes (G4s) form in relevant genomic regions and intervene in several biological processes, including the modulation of oncogenes expression, and are potential anticancer drug targets. The human KRAS proto-oncogene promoter region contains guanine-rich sequences able to fold into G4 structures. Here, by using circular dichroism and differential scanning calorimetry as complementary physicochemical methodologies, we compared the thermodynamic stability of the G4s formed by a shorter and a longer version of the KRAS promoter sequence, namely 5′-AGGGCGGTGTGGGAATAGGGAA-3′ (KRAS 22RT) and 5′-AGGGCGGTGTGGGAAGAGGGAAGAGGGGGAGG-3′ (KRAS 32R). Our results show that the unfolding mechanism of KRAS 32R is more complex than that of KRAS 22RT. The different thermodynamic stability is discussed based on the recently determined NMR structures. The binding properties of TMPyP4 and BRACO-19, two well-known G4-targeting anticancer compounds, to the KRAS G4s were also investigated. The present physicochemical study aims to help in choosing the best G4 target for potential anticancer drugs. Full article

(This article belongs to the Special Issue The Polymorphic World of G-Quadruplexes: From Structural Insights to Functional Activity)

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Figure 1
Schematic representation of the Nuclease-Hypersensitive Element (NHE) region of the KRAS gene promoter. Full article ">Figure 2
(a) Circular dichroism (CD) spectra of KRAS 22RT (black line) and KRAS 32R (red line). CD melting (black line) and annealing (red line) profiles of (b) KRAS 22RT and (c) KRAS 32R at 0.5 °C min−1. The buffer solution was 20 mM KH2PO4/K2HPO4 with 60 mM KCl and 0.1 mM EDTA at pH 7.0. Full article ">Figure 3
Experimental differential scanning calorimetry (DSC) profiles (black line) and van ’t Hoff calculated curves based on the two-states model (red line) for (a) KRAS 22RT and (b) KRAS 32R. The buffer solution was 20 mM KH2PO4/K2HPO4 with 60 mM KCl and 0.1 mM EDTA at pH 7.0. Full article ">Figure 4
DSC profile of KRAS 32R (black line) and the best fitting curve (red line) obtained by the thermodynamic model described in <a href="#sec4dot4dot1-ijms-22-00448" class="html-sec">Section 4.4.1</a>. Full article ">Figure 5
CD melting curves of (a) KRAS 22RT and (c) KRAS 32R in the absence (black line) and in the presence of 1 (red line) and 2 equivalents of TMPyP4 (blue line). CD melting curves of (b) KRAS 22RT and (d) KRAS 32R in the absence (black line) and in the presence of 1 (red line) and 2 equivalents (blue line) of BRACO-19. The buffer solution was 20 mM KH2PO4/K2HPO4 with 60 mM KCl and 0.1 mM EDTA at pH 7.0. Full article ">Figure 6
DSC profiles of KRAS 22RT (a) and KRAS 32R (c) in the absence (black line) and presence (red line) of 1 equivalent of TMPyP4. DSC profiles of KRAS 22RT (b) and KRAS 32R (d) in the absence (black line) and presence (red line) of 1 equivalent of BRACO-19. The buffer solution was 20 mM KH2PO4/K2HPO4 with 60 mM KCl and 0.1 mM EDTA at pH 7.0. Full article ">

18 pages, 5717 KiB

Open AccessArticle

Evolution of A bHLH Interaction Motif

by Peter S. Millard, Birthe B. Kragelund and Meike Burow

Int. J. Mol. Sci. 2021, 22(1), 447; https://doi.org/10.3390/ijms22010447 - 5 Jan 2021

Cited by 5 | Viewed by 4095

Abstract

Intrinsically disordered proteins and regions with their associated short linear motifs play key roles in transcriptional regulation. The disordered MYC-interaction motif (MIM) mediates interactions between MYC and MYB transcription factors in Arabidopsis thaliana that are critical for constitutive and induced glucosinolate (GLS) biosynthesis. [...] Read more.

Intrinsically disordered proteins and regions with their associated short linear motifs play key roles in transcriptional regulation. The disordered MYC-interaction motif (MIM) mediates interactions between MYC and MYB transcription factors in Arabidopsis thaliana that are critical for constitutive and induced glucosinolate (GLS) biosynthesis. GLSs comprise a class of plant defense compounds that evolved in the ancestor of the Brassicales order. We used a diverse set of search strategies to discover additional occurrences of the MIM in other proteins and in other organisms and evaluate the findings by means of structural predictions, interaction assays, and biophysical experiments. Our search revealed numerous MIM instances spread throughout the angiosperm lineage. Experiments verify that several of the newly discovered MIM-containing proteins interact with MYC TFs. Only hits found within the same transcription factor family and having similar characteristics could be validated, indicating that structural predictions and sequence similarity are good indicators of whether the presence of a MIM mediates interaction. The experimentally validated MIMs are found in organisms outside the Brassicales order, showing that MIM function is broader than regulating GLS biosynthesis. Full article

(This article belongs to the Special Issue Protein Intrinsic Disorder in Plant Biology)

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12 pages, 1361 KiB

Open AccessArticle

A Theoretical and Experimental Study on the Potential Luminescent and Biological Activities of Diaminodicyanoquinodimethane Derivatives

by Edison Rafael Jiménez, Manuel Caetano, Nelson Santiago, F. Javier Torres, Thibault Terencio and Hortensia Rodríguez

Int. J. Mol. Sci. 2021, 22(1), 446; https://doi.org/10.3390/ijms22010446 - 5 Jan 2021

Cited by 2 | Viewed by 3412

Abstract

Recently, several studies have demonstrated that diaminodicyanoquinone derivatives (DADQs) could present interesting fluorescence properties. Furthermore, some DADQs under the solid state are capable of showing quantum yields that can reach values of 90%. Besides, the diaminodiacyanoquinone core represents a versatile building block propense [...] Read more.

Recently, several studies have demonstrated that diaminodicyanoquinone derivatives (DADQs) could present interesting fluorescence properties. Furthermore, some DADQs under the solid state are capable of showing quantum yields that can reach values of 90%. Besides, the diaminodiacyanoquinone core represents a versatile building block propense either to modification or integration into different systems to obtain and provide them unique photophysical features. Herein, we carried out a theoretical study on the fluorescence properties of three different diaminodicyanoquinodimethane systems. Therefore, time-dependent density functional theory (TD-DFT) was used to obtain the values associated with the dipole moments, oscillator strengths, and the conformational energies between the ground and the first excited states of each molecule. The results suggest that only two of the three studied systems possess significant luminescent properties. In a further stage, the theoretical insights were confirmed by means of experimental measurements, which not only retrieved the photoluminescence of the DADQs, but also suggest a preliminary and promising antibacterial activity of these systems. Full article

(This article belongs to the Section Molecular Biophysics)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Molecular structure of the target molecules. Full article ">Figure 2
Comparison between the optimized ground state (blue) and excited state (red) structures. (a) frontal view of molecule 1; (b) lateral view of molecule 1; (c) frontal view of molecule 2; (d) lateral view of molecule 2; (e) frontal view of molecule 3; (f) lateral view of molecule 3. Full article ">Figure 3
Synthetic route starting with the activation of TCNQ, to obtain molecules 1 and 2. Full article ">Figure 4
Recorded emission spectra in all the visible range using a diode laser class 3R of 405 nm. (a) Emission spectra and its proper deconvolution of molecule 1; (b) Emission spectra and its proper deconvolution of molecule 2. Full article ">Figure 5
Growth curves for the OD600 tests performed to five different test tubes, blank (growth curve under standard conditions); Solvent (growth curve under the presence of the used solvent 5:5 water/DMSO); Comp.b (growth curve under the presence of PTCNQ) ); Comp.1 (growth curve under the presence of compound 1); Comp.2 (growth curve under the presence of compound 2); Comp c (growth curve under the presence of compound N-(4-nitrophenyl)ethylenediamine)). Full article ">

14 pages, 1821 KiB

Open AccessArticle

Switching between Ultrafast Pathways Enables a Green-Red Emission Ratiometric Fluorescent-Protein-Based Ca²⁺ Biosensor

by Longteng Tang, Shuce Zhang, Yufeng Zhao, Nikita D. Rozanov, Liangdong Zhu, Jiahui Wu, Robert E. Campbell and Chong Fang

Int. J. Mol. Sci. 2021, 22(1), 445; https://doi.org/10.3390/ijms22010445 - 5 Jan 2021

Cited by 11 | Viewed by 4654

Abstract

Ratiometric indicators with long emission wavelengths are highly preferred in modern bioimaging and life sciences. Herein, we elucidated the working mechanism of a standalone red fluorescent protein (FP)-based Ca²⁺ biosensor, REX-GECO1, using a series of spectroscopic and computational methods. Upon 480 nm [...] Read more.

Ratiometric indicators with long emission wavelengths are highly preferred in modern bioimaging and life sciences. Herein, we elucidated the working mechanism of a standalone red fluorescent protein (FP)-based Ca²⁺ biosensor, REX-GECO1, using a series of spectroscopic and computational methods. Upon 480 nm photoexcitation, the Ca²⁺-free biosensor chromophore becomes trapped in an excited dark state. Binding with Ca²⁺ switches the route to ultrafast excited-state proton transfer through a short hydrogen bond to an adjacent Glu80 residue, which is key for the biosensor’s functionality. Inspired by the 2D-fluorescence map, REX-GECO1 for Ca²⁺ imaging in the ionomycin-treated human HeLa cells was achieved for the first time with a red/green emission ratio change (ΔR/R₀) of ~300%, outperforming many FRET- and single FP-based indicators. These spectroscopy-driven discoveries enable targeted design for the next-generation biosensors with larger dynamic range and longer emission wavelengths. Full article

(This article belongs to the Special Issue Bioluminescent and Fluorescent Proteins: Molecular Mechanisms and Modern Applications)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Steady-state electronic spectra of REX-GECO1. The pH-dependent (pH = 3.5–11) UV/Visible absorption spectra of the Ca2+ (a) free and (b) bound biosensors exhibit different patterns. The horizontal dashed line in (b) highlights spectral changes across pH = 5. (c) Modeled chromophore (CRO) and key residues inside the Ca2+-bound REX-GECO1 biosensor. 2D-fluorescence spectra for the Ca2+ (d) free and (e) bound REX-GECO1 in pH = 7 buffer show considerable differences. The ground-state electronic absorption spectra are displayed vertically in white curves. Full article ">Figure 2
Femtosecond transient absorption (fs-TA) spectroscopy elucidates the excited-state pathways of REX-GECO1. 2D-contour plots for the Ca2+ (a) free and (b) bound biosensors in pH = 7 buffer upon 480 nm excitation show prominent electronic bands that rise and decay within hundreds of picoseconds. Intensity dynamics of representative 10-nm band regions are correspondingly plotted in (c,d), respectively. Full article ">Figure 3
Quantum calculations (a) and molecular dynamics simulations (b) indicate a more twisted structure of the Ca2+ free than bound REX-GECO1 in the excited and ground states. Two key dihedral angles of the chromophore are shown in the (a) inset. On the density contour plots in (b), angles from the Ca2+-bound R-GECO1 (progenitor of REX-GECO1) crystal structure are marked (black crosses). Full article ">Figure 4
Emission-ratiometric imaging of the REX-GECO1 biosensor. (a) Confocal images of HeLa cells expressing REX-GECO1 before (low Ca2+) and after (high Ca2+) ionomycin treatment. Scale bar = 100 μm. (b) Time-lapse Ca2+ imaging shown by the red/green fluorescence intensity ratio change (ΔR/R0) after 480 nm excitation. Gray lines: single-cell traces. Red line with pink shade: mean ± s.e.m. Full article ">

11 pages, 2405 KiB

Open AccessArticle

PACAP for Retinal Health: Model for Cellular Aging and Rescue

by Etelka Pöstyéni, Andrea Kovács-Valasek, Viktória Dénes, Adrienn Mester, György Sétáló, Jr. and Róbert Gábriel

Int. J. Mol. Sci. 2021, 22(1), 444; https://doi.org/10.3390/ijms22010444 - 5 Jan 2021

Cited by 12 | Viewed by 2598

Abstract

Retinal aging is the result of accumulating molecular and cellular damage with a manifest decline in visual functions. Somatostatin (SST) and pituitary adenylate cyclase-activating polypeptide (PACAP) have been implicated in neuroprotection through regulating disparate aspects of neuronal activity (survival, proliferation and renewal). The [...] Read more.

Retinal aging is the result of accumulating molecular and cellular damage with a manifest decline in visual functions. Somatostatin (SST) and pituitary adenylate cyclase-activating polypeptide (PACAP) have been implicated in neuroprotection through regulating disparate aspects of neuronal activity (survival, proliferation and renewal). The aim of the present study was to validate a transgenic model for SST-expressing amacrine cells and to investigate the chronic effect of PACAP on the aging of SSTergic and dopaminergic cells of the retina. SST-tdTomato transgenic mice that were 6, 12 and 18 months old were treated intravitreally with 100 pmol of PACAP every 3 months. The density of SST and dopaminergic amacrine cells was assessed in whole-mounted retinas. Cells displaying the transgenic red fluorescence were identified as SST-immunopositive amacrine cells. By comparing the three age groups. PACAP treatment was shown to induce a moderate elevation of cell densities in both the SST and dopaminergic cell populations in the 12- and 18-month-old animals. By contrast, the control untreated and saline-treated retinas showed a minor cell loss. In conclusion, we report a reliable transgenic model for examining SSTergic amacrine cells. The fundamental novelty of this study is that PACAP could increase the cell density in matured retinal tissue, anticipating new therapeutic potential in age-related pathological processes. Full article

(This article belongs to the Special Issue Peptides for Health Benefits 2020)

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Figure 1
SST and TH immunohistochemistry in retinal whole mounts and sections in 6-month-old mouse retina. tdTomato-expressing cells (a) and TH-immunopositive neurons (b) in retinal whole mount. Co-localization of anti-SST antibody (labeled with AF 488-green) and red autofluorescent cells appeared as yellow in the merged image (c). Co-localization was not observed between SST cells (red) and TH immunopositive cells (green) (d). INL—Inner nuclear layer; IPL—Inner plexiform layer. Scale bar in (a) is valid for (b). Full article ">Figure 2
Distributions of SST (red) and TH (green) cells are shown in central (a,b) and peripheral (c,d) retina of 6-month-old mice as well as in saline-treated (a,c) and PACAP-treated (b,d) whole mounts. Scale bar is identical for all pictures. Full article ">Figure 3
Distributions of SST (red) and TH (green) cells are shown in central (a,b) and peripheral (c,d) retina of 12-month-old mice. An increase in the density of both cell populations is clearly seen in the PACAP-treated (b,d) whole mount compared to saline-treated (a,c) retina. Scale bar is identical for all pictures. Full article ">Figure 4
Distributions of SST (red) and TH (green) cells are shown in central (a,b) and peripheral (c,d) retina of 18-month-old mice. The increase in the SST and TH cell density is even more enhanced in the PACAP-treated (b,d) whole mount compared to saline-treated (a,c) retina. The arrows point to tdTomato-expressing glia-like cells. Scale bar is identical for all pictures. Full article ">Figure 5
The SST (a,c)- and TH (b,d)-positive cell densities in central (a,b) and peripheral retinas (c,d) in different groups. Data presented as mean ± SEM, where * means p < 0.05 and ** means p < 0.01, compared to the cell densities of the 6-month-old PACAP-treated group. Full article ">

16 pages, 2248 KiB

Open AccessArticle

Temporomandibular Joint Osteoarthritis: Regenerative Treatment by a Stem Cell Containing Advanced Therapy Medicinal Product (ATMP)—An In Vivo Animal Trial

by Robert Köhnke, Marcus Oliver Ahlers, Moritz Alexander Birkelbach, Florian Ewald, Michael Krueger, Imke Fiedler, Björn Busse, Max Heiland, Tobias Vollkommer, Martin Gosau, Ralf Smeets and Rico Rutkowski

Int. J. Mol. Sci. 2021, 22(1), 443; https://doi.org/10.3390/ijms22010443 - 5 Jan 2021

Cited by 22 | Viewed by 5796

Abstract

Temporomandibular joint osteoarthritis (TMJ-OA) is a chronic degenerative disease that is often characterized by progressive impairment of the temporomandibular functional unit. The aim of this randomized controlled animal trial was a comparative analysis regarding the chondroregenerative potency of intra-articular stem/stromal cell therapy. Four [...] Read more.

Temporomandibular joint osteoarthritis (TMJ-OA) is a chronic degenerative disease that is often characterized by progressive impairment of the temporomandibular functional unit. The aim of this randomized controlled animal trial was a comparative analysis regarding the chondroregenerative potency of intra-articular stem/stromal cell therapy. Four weeks after combined mechanical and biochemical osteoarthritis induction in 28 rabbits, therapy was initiated by a single intra-articular injection, randomized into the following groups: Group 1: AB Serum (ABS); Group 2: Hyaluronic acid (HA); Group 3: Mesenchymal stromal cells (STx.); Group 4: Mesenchymal stromal cells in hyaluronic acid (HA + STx.). After another 4 weeks, the animals were euthanized, followed by histological examination of the removed joints. The histological analysis showed a significant increase in cartilage thickness in the stromal cell treated groups (HA + STx. vs. ABS, p = 0.028; HA + ST.x vs. HA, p = 0.042; STx. vs. ABS, p = 0.036). Scanning electron microscopy detected a similar heterogeneity of mineralization and tissue porosity in the subchondral zone in all groups. The single intra-articular injection of a stem cell containing, GMP-compliant advanced therapy medicinal product for the treatment of iatrogen induced osteoarthritis of the temporomandibular joint shows a chondroregenerative effect. Full article

(This article belongs to the Special Issue Optimization of Biomaterials for Reconstructive and Regenerative Medicine)

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Figure 1
To objectify the cartilage thickness measurements, a line grid (black) was used to define random measuring points on the cartilage surface. At these points the thickness measurement (yellow) was finally taken, each right angled to an imaginary tangent (dotted). Full article ">Figure 2
Boxplot analysis showing the underlying distribution of final cartilage thickness vs. the four treatment groups. Circles represent outliers with more than 1.5 times interquartile range. Significant group differences are marked (***). Full article ">Figure 3
(A–D). Histological analysis 4 weeks after intra-articular injection therapy (1 = Safranin-O, 2 = Picrosirius red, 3 = Picrosirius red (polarized)). (A1–A3) representing group 1 (ABS), (B1–B3) group 2 (HA), (C1–C3) group 3 (STx.), and (D1–D3) group 4 (STx. + HA). Collagen I on cartilage surface could be visualized much better in group 3 (STx.) than in the other groups (Picrosirius red: red; Picrosirius red (polarized): yellow-orange), indicating a comparatively higher cartilage regeneration. A statistical significance analysis was not performed. Overall, no significant increased or decreased GAG accumulation could be detected in any of the groups using Safranin-O staining. Full article ">Figure 4
Representative back-scattered images of temporomandibular joint (TMJ) specimens of all groups. (A–D) The images show a cross-sectional view of the TMJ, whereby darker pixels correspond to a low degree of mineralization and brighter pixels correspond to higher degree of mineralization. The articular cartilage (AC) is located on top of the subchondral mineralized zone (SMZ) which is composed of calcified cartilage containing chondrocyte lacunae and subchondral bone containing osteocyte lacunae. (E) In the calcified cartilage zone, the ratio of mineralized area per tissue area, an inverse measure of tissue porosity, was similar between the groups. (F) The heterogeneity of mineralization, assessed as the full-width-half-maximum of the gray value histogram, was similar between groups. Full article ">

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Int. J. Mol. Sci., Volume 22, Issue 1 (January-1 2021) – 472 articles

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