International Journal of Molecular Sciences

15 pages, 7162 KiB

Open AccessArticle

Jatrorrhizine Hydrochloride Suppresses RANKL-Induced Osteoclastogenesis and Protects against Wear Particle-Induced Osteolysis

by Hui Li, Jing Wang, Qiwen Sun, Gang Chen, Shengnan Sun, Xuemei Ma, Haiwen Qiu, Xuerong Liu, Liangyi Xu and Mei Liu

Int. J. Mol. Sci. 2018, 19(11), 3698; https://doi.org/10.3390/ijms19113698 - 21 Nov 2018

Cited by 21 | Viewed by 4112

Abstract

Wear particle-induced aseptic prosthetic loosening is a major complication associated with total joint arthroplasty (TJA). A growing body of evidence suggests that receptor activator of nuclear factor κ-B ligand (RANKL)-stimulated osteoclastogenesis and bone resorption are responsible for peri-implant loosening. Thus, agents which attenuate [...] Read more.

Wear particle-induced aseptic prosthetic loosening is a major complication associated with total joint arthroplasty (TJA). A growing body of evidence suggests that receptor activator of nuclear factor κ-B ligand (RANKL)-stimulated osteoclastogenesis and bone resorption are responsible for peri-implant loosening. Thus, agents which attenuate excessive osteoclast differentiation and function have been considered to offer therapeutic potential for prolonging the life of TJA implants. Jatrorrhizine hydrochloride (JH), a major protoberberine alkaloid isolated from the traditional Chinese herb Coptis chinensis, has been reported to have antimicrobial, antitumor, and antihypercholesterolemic and neuroprotective activities. However, its effects on osteoclast biology remain unknown. Here, we found that JH inhibited RANKL-induced osteoclast formation and bone resorption in vitro and exerted protection against titanium (Ti) particle-induced osteolysis in vivo. Biochemical analysis demonstrated that JH suppressed RANKL-induced activation of MAPKs (p38 and ERK) which down-regulated the production of NFATc1 and NFATc1-regulated osteoclastic marker genes, such as TRAP, CTR and CTSK. Collectively, our findings suggest that JH may be a promising anti-osteoclastogenesis agent for treating periprosthetic osteolysis or other osteoclast-related osteolytic diseases. Full article

(This article belongs to the Section Molecular Pharmacology)

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Figure 1

Figure 1
JH inhibits Ti particle-induced bone destruction in a mouse calvarial model. (A) Representative micro-CT 3D reconstructed images were obtained from different groups. (B) Bone mineral density (BMD) and bone volume/tissue volume (BV/TV) were determined using the CTan program (SkyScan). All the data were shown as mean ± SD; ## p < 0.01 and ### p < 0.001 versus sham group, * p < 0.05, ** p < 0.01 and *** p < 0.001 versus vehicle-treated Ti particle-induced group; n = 7 per group. Full article ">Figure 2
Histological staining and histomorphometric assessment of the inhibitory actions of JH on Ti particle-induced osteolysis. (A) Representative micrographs staining for H&E and tartrate-resistant acid phosphatase (TRAP). The regions marked by the black frames were enlarged and shown in the corresponding bottom panels. (B) The percentage of total porosity obtained from the H&E-stained sections. (C) The bone area (mm2) and TRAP+ osteoclast number in TRAP-stained sections were calculated and analyzed as described in the <a href="#sec4-ijms-19-03698" class="html-sec">Section 4</a>. All data were shown as mean ± SD; ### p < 0.001 versus sham group, ** p < 0.01 and *** p < 0.001 versus vehicle-treated Ti particle-induced group. Full article ">Figure 3
JH inhibits osteoclast formation in vitro. (A) A representative image showing the inhibitory effect of JH on osteoclast formation. BMM cells were treated with different concentrations of JH in the presence of RANKL (100 ng·mL−1) for 5 days and then TRAP staining was performed. The number and area of TRAP+ multinucleated cells with more than three nuclei were shown in (B), n = 3, * p < 0.05, ** p <0.01 and *** p < 0.001 relative to RANKL-treated, JH-untreated controls. OC No: osteoclast number. (C) To define the different stages of osteoclastogenesis, TRAP staining was performed on d0–d5 of RANKL induction (d0, the day RNAKL was added; d1, RANKL induction for 1 day; d2, RANKL induction for 2 days; and so on). (D) BMMs were cultured in the presence of RANKL, and JH (5 µM) was added in different times. At the end of 5 days, TRAP staining was performed. (Ctrl group: RANKL induction for 5 days without JH; Pre Treat group: JH pre-treatment for 12 h and RANKL induction for 5 days; Early Treat group: Co-treatment with JH and RANKL for 2 days and RANKL continued induction for 3 days; Late Treat group: RANKL induction for 2 days and subsequent co-treatment with JH and RANKL for 3 days; Early + Late Treat group: Co-treatment with JH and RANKL for 5 days; Pre + Early + Late Treat group: JH treatment for 12 h and subsequent co-treatment with JH and RANKL for 5 days) (E) The area and number of TRAP+ osteoclasts (≥3 nuclei) from (D) were quantified (n = 3). All data were shown as mean ± SD; * p < 0.05, ** p <0.01 and *** p < 0.001 relative to RANKL-treated, JH-untreated controls. (F) Effect of JH on cell viability was measured using a MTS assay. (G) BMMs were exposed to the indicated concentrations of JH for 24 h, and then cell apoptosis was determined using flow cytometry. Full article ">Figure 4
JH inhibits osteoclast-mediated bone resorption. (A) Representative TRAP staining images showing no cytotoxic effect of JH on mature osteoclasts. (B) The number of TRAP+ osteoclasts (≥ 3 nuclei) from (A) were quantified (n = 3). (C) Representative scanning electron microscopy (SEM) images showing the inhibitory effect of JH on osteoclast-mediated bone resorption. (D) Resorption area relative to the total area of bone was measured using ImageJ (n = 3). (E) Representative immunofluorescence staining images showing the inhibitory effect of JH on F-actin ring formation. (F) The number and size of intact actin rings from (E) were quantified (n = 3). * p < 0.05, ** p < 0.01 and *** p < 0.001 relative to RANKL-treated, JH-untreated controls. Full article ">Figure 5
JH suppresses RANKL-induced osteoclast-specific gene expression during osteoclastogenesis. BMMs were treated with RANKL and varying concentrations of JH for 5 days. The levels of osteoclast-specific marker genes were analyzed by real-time PCR, normalized to β-actin expression (n = 3). All data were shown as mean ± SD; ** p < 0.01 and *** p < 0.001 relative to RANKL-treated, JH-untreated controls. CTSK: cathepsin K; CTR: calcitonin receptor; TRAP: tartrate-resistant acid phosphatase. Full article ">Figure 6
JH attenuates RANKL-stimulated activations of MAPKs (p38 and ERK). (A,C) After pretreatment with or without JH for 1 h, BMM cells were stimulated with RANKL (100 ng∙mL−1) for 15 min in (A), and 0, 1, 3, 5 days in (C). Western blotting was performed to examine the levels of p-p38, total p38, p-ERK1/2, total ERK1/2, p-JNK, total JNK, IκBα, NFATc1, c-Fos and GAPDH. (B,D) The ratios of p-p38/p38, p-ERK/ERK, NFATc1/GAPDH and c-Fos/GAPDH were quantified and calculated using ImageJ software (n = 3). All data were shown as mean ± SD; * p < 0.05, ** p < 0.01 and *** p < 0.001 relative to RANKL-treated, JH-untreated controls. ### p < 0.001 relative to RANKL-untreated, JH-untreated controls. Full article ">

14 pages, 3443 KiB

Open AccessArticle

Medium-Chain Triglycerides Attenuate Liver Injury in Lipopolysaccharide-Challenged Pigs by Inhibiting Necroptotic and Inflammatory Signaling Pathways

by Lin Zhang, Xiuying Wang, Shaokui Chen, Shuhui Wang, Zhixiao Tu, Guolong Zhang, Huiling Zhu, Xiangen Li, Jianglin Xiong and Yulan Liu

Int. J. Mol. Sci. 2018, 19(11), 3697; https://doi.org/10.3390/ijms19113697 - 21 Nov 2018

Cited by 22 | Viewed by 6039

Abstract

This study was conducted to investigate whether medium-chain triglycerides (MCTs) attenuated lipopolysaccharide (LPS)-induced liver injury by down-regulating necroptotic and inflammatory signaling pathways. A total of 24 pigs were randomly allotted to four treatments in a 2 × 2 factorial design including diet (0 [...] Read more.

This study was conducted to investigate whether medium-chain triglycerides (MCTs) attenuated lipopolysaccharide (LPS)-induced liver injury by down-regulating necroptotic and inflammatory signaling pathways. A total of 24 pigs were randomly allotted to four treatments in a 2 × 2 factorial design including diet (0 and 4% MCTs) and immunological challenge (saline and LPS). After three weeks of feeding with or without 4% MCTs, pigs were challenged with saline or LPS. MCTs led to a significant increase in eicosapentaenoic acid, docosahexaenoic acid and total (n-3) polyunsaturated fatty acid concentrations. MCTs attenuated LPS-induced liver injury as indicated by an improvement in liver histomorphology and ultrastructural morphology of hepatocytes, a reduction in serum alanine aminotransferase and alkaline phosphatase activities as well as an increase in claudin-1 protein expression. In addition, MCTs also reduced serum tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 concentrations, liver TNF-α and IL-1β mRNA expression and protein concentrations and enhanced liver heat shock protein 70 protein expression in LPS-challenged pigs. Moreover, MCTs decreased mRNA expression of receptor-interacting serine/threonine-protein kinase (RIP) 3, mixed-lineage kinase domain-like protein (MLKL) and phosphoglycerate mutase 5 and inhibited MLKL phosphorylation in the liver. Finally, MCTs decreased liver mRNA expression of toll-like receptor (TLR) 4, nucleotide-binding oligomerization domain protein (NOD) 1 and multiple downstream signaling molecules. MCTs also suppressed LPS-induced p38 mitogen-activated protein kinase (MAPK) phosphorylation and increased extracellular signal-related kinase 1/2 phosphorylation in the liver. These results indicated that MCTs are capable of attenuating LPS-induced liver damage by suppressing hepatic necroptotic (RIP1/RIP3/MLKL) and inflammatory (TLR4/NOD1/p38 MAPK) signaling pathways. Full article

(This article belongs to the Special Issue Liver Damage and Repair)

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Effect of medium-chain triglyceride (MCT) supplementation on liver morphology after 4-h LPS challenge in weanling pigs. Pigs were subjected to a 2 × 2 factorial study fed with or without 4% MCTs for 21 days, followed by an intraperitoneal injection of saline or LPS on day 22. The representative liver histological sections from the four treatment groups were collected at 4 h after LPS challenge and stained with hematoxylin and eosin: (A) control diet and saline group, (B) 4% MCT diet and saline group, (C) control diet and LPS-challenged group and (D) 4% MCT diet and LPS-challenged group. While significant morphologic changes associated with liver injury, such as hepatocyte karyolysis (a), karyopyknosis (b), inflammatory cell infiltration (c) and disordered hepatic cell cords arrangement were observed in Panels C and D, significant attenuation of liver injury was observed in Panel D. Original magnification: 400×. Scale bars = 22.4 μm. Full article ">Figure 2
Effect of medium-chain triglyceride (MCT) supplementation on hepatocyte ultrastructure after 4-h LPS challenge in weanling pigs. Pigs were subjected to a 2 × 2 factorial study fed with or without 4% MCTs for 21 days, followed by an intraperitoneal injection of saline or LPS on day 22. The representative hepatocyte ultrastructural images from the four treatment groups were collected at 4 h after LPS challenge and processed for electron microscopy examination: (A) control diet and saline group, mitochondria (a), original magnification: 1700 ×, scale bars = 2 μm; (B) 4% MCT diet and saline group, mitochondria (a), original magnification: 1700 ×, scale bars = 2 μm; (C) control diet and LPS-challenged group, mitochondria (a), autophagosome (b), original magnification: 1700 ×, scale bars = 2 μm; (D) 4% MCT diet and LPS-challenged group, mitochondria (a), original magnification: 1700 ×, scale bars = 2 μm; (E) control diet and saline group, mitochondria (a), endoplasmic reticulum (c), original magnification: 5000 ×, scale bars = 1 μm; (F) 4% MCT diet and saline group, mitochondria (a), endoplasmic reticulum (c), original magnification: 5000 ×, scale bars = 1 μm; (G) control diet and LPS-challenged group, mitochondrial dissolution (d), endoplasmic reticulum expansion (e), nuclear deformation, nuclear membrane rupture and chromatin overflow (f), original magnification: 5000 ×, scale bars = 1 μm. (H) 4% MCT diet and LPS-challenged group, mild mitochondrial swelling (g), endoplasmic reticulum expansion (e) and integral nuclear membrane, original magnification: 5000 ×, scale bars = 1 μm. Full article ">Figure 3
Effect of medium-chain triglyceride (MCT) supplementation on claudin-1 (a) and heat shock protein 70 (HSP70) (b) protein expression in the liver after 4-h LPS challenge in pigs. The bands are the representative Western blot images (c). Values are mean and SE, n = 6 (1 pig/pen). Means with different letters differ significantly (P < 0.05). All data for protein expression were acquired using Western blot. Values for relative claudin-1 and HSP70 expression were normalized for β-actin. CO-S, pigs fed the control diet and injected with saline; MCTs-S, pigs fed MCTs and injected with saline; CO-LPS, pigs fed the control diet and challenged with LPS; MCTs-LPS, pigs fed MCTs and challenged with LPS. Full article ">Figure 4
Effect of medium-chain triglyceride (MCT) supplementation on concentrations of serum (a–c) and liver (d–f) proinflammatory cytokines after 4-h LPS challenge in pigs. Values are mean and SE, n = 6 (1 pig/pen). Means with different letters differ significantly (P < 0.05). CO-S, pigs fed the control diet and injected with saline; MCTs-S, pigs fed MCTs and injected with saline; CO-LPS, pigs fed the control diet and challenged with LPS; MCTs-LPS, pigs fed MCTs and challenged with LPS. ND, not detectable, low than the minimum detectable doses of TNF-α and IL-6. Full article ">Figure 5
Effect of medium-chain triglyceride (MCT) supplementation on liver p38 (a,b) and extracellular signal-related kinase 1/2 (ERK1/2) (c,d) phosphorylation levels after 4-h LPS challenge in pigs. The bands are the representative Western blot images (e). Values are mean and SE, n = 6 (1 pig/pen). All data for protein expression were acquired using Western blot. Phosphorylated forms of p38 and ERK1/2 were normalized to the total amount of each protein. CO-S, pigs fed the control diet and injected with saline; MCTs-S, pigs fed MCTs and injected with saline; CO-LPS, pigs fed the control diet and challenged with LPS; MCTs-LPS, pigs fed MCTs and challenged with LPS. AU, arbitrary units. Full article ">Figure 6
Effect of medium-chain triglyceride (MCT) supplementation on protein expression of key signaling molecules in necroptosis after 4-h LPS challenge in pigs (a–d). The bands are the representative Western blot images (e). Values are mean and SE, n = 6 (1 pig/pen). All data for protein expression were acquired using Western blot. Values for relative RIP1 and RIP3 expression were normalized against β-actin and the phosphorylated form of MLKL was normalized with total protein content of MLKL. CO-S, pigs fed the control diet and injected with saline; MCTs-S, pigs fed MCTs and injected with saline; CO-LPS, pigs fed the control diet and challenged with LPS; MCTs-LPS, pigs fed MCTs and challenged with LPS. RIP, receptor-interacting serine/threonine-protein kinase; MLKL, mixed-lineage kinase domain-like protein; AU, arbitrary units. Full article ">

13 pages, 462 KiB

Open AccessArticle

Uric Acid and Xanthine Levels in Pregnancy Complicated by Gestational Diabetes Mellitus—The Effect on Adverse Pregnancy Outcomes

by Anna Pleskacova, Vendula Bartakova, Katarina Chalasova, Lukas Pacal, Katerina Kankova and Josef Tomandl

Int. J. Mol. Sci. 2018, 19(11), 3696; https://doi.org/10.3390/ijms19113696 - 21 Nov 2018

Cited by 16 | Viewed by 4190

Abstract

Uric acid (UA) levels are associated with many diseases including those related to lifestyle. The aim of this study was to evaluate the influence of clinical and anthropometric parameters on UA and xanthine (X) levels during pregnancy and postpartum in women with physiological [...] Read more.

Uric acid (UA) levels are associated with many diseases including those related to lifestyle. The aim of this study was to evaluate the influence of clinical and anthropometric parameters on UA and xanthine (X) levels during pregnancy and postpartum in women with physiological pregnancy and pregnancy complicated by gestational diabetes mellitus (GDM), and to evaluate their impact on adverse perinatal outcomes. A total of 143 participants were included. Analyte levels were determined by HPLC with ultraviolet detection (HPLC-UV). Several single-nucleotide polymorphisms (SNPs) in UA transporters were genotyped using commercial assays. UA levels were higher within GDM women with pre-gestational obesity, those in high-risk groups, and those who required insulin during pregnancy. X levels were higher in the GDM group during pregnancy and also postpartum. Positive correlations between UA and X levels with body mass index (BMI) and glycemia levels were found. Gestational age at delivery was negatively correlated with UA and X levels postpartum. Postpartum X levels were significantly higher in women who underwent caesarean sections. Our data support a possible link between increased UA levels and a high-risk GDM subtype. UA levels were higher among women whose glucose tolerance was severely disturbed. Mid-gestational UA and X levels were not linked to adverse perinatal outcomes. Full article

(This article belongs to the Special Issue Risk Factors and Molecular Mechanisms of Gestational Diabetes)

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11 pages, 1423 KiB

Open AccessReview

Prenatal Programming of Neuroendocrine System Development by Lipopolysaccharide: Long-Term Effects

by Marina Izvolskaia, Viktoria Sharova and Liudmila Zakharova

Int. J. Mol. Sci. 2018, 19(11), 3695; https://doi.org/10.3390/ijms19113695 - 21 Nov 2018

Cited by 24 | Viewed by 4647

Abstract

Various stress factors during critical periods of fetal development modulate the epigenetic mechanisms controlling specific genes, which can affect the structure and function of physiological systems. Maternal immune stress by bacterial infection simulated by lipopolysaccharide (LPS) in an experiment is considered to be [...] Read more.

Various stress factors during critical periods of fetal development modulate the epigenetic mechanisms controlling specific genes, which can affect the structure and function of physiological systems. Maternal immune stress by bacterial infection simulated by lipopolysaccharide (LPS) in an experiment is considered to be a powerful programming factor of fetal development. Studies of the molecular mechanisms controlling the formation and functioning of physiological systems are in the pilot stage. LPSs are the most potent natural inflammation factors. LPS-induced increases in fetal levels of pro- and anti-inflammatory cytokines can affect brain development and have long-term effects on behavior and neuroendocrine functions. The degradation of serotonergic neurons induced by LPS in the fetus is attributed to the increased levels of interleukin (IL)-6 and tumor necrosis factor (TNFα) as well as to anxiety and depression in children. Dopamine deficiency causes dysthymia, learning disability, and Parkinson’s disease. According to our data, an LPS-induced increase in the levels of IL-6, leukemia inhibitory factor (LIF), and monocyte chemotactic protein (MCP-1) in maternal and fetal rats during early pregnancy disturbs the development and functioning of gonadotropin-releasing hormone production and reproductive systems. It is important to note the high responsiveness of epigenetic developmental mechanisms to many regulatory factors, which offers opportunities to correct the defects. Full article

(This article belongs to the Section Biochemistry)

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6 pages, 1491 KiB

Open AccessEditorial

International Journal of Molecular Science 2018 Best Paper Award

by International Journal of Molecular Science Editorial Office

Int. J. Mol. Sci. 2018, 19(11), 3694; https://doi.org/10.3390/ijms19113694 - 21 Nov 2018

Cited by 3 | Viewed by 7811

Abstract

The Editors of the International Journal of Molecular Sciences have established the Best Paper Award to recognize the most outstanding articles published in the areas of molecular biology, molecular physics, and chemistry that have been published in the International Journal of Molecular Sciences [...] Read more.

The Editors of the International Journal of Molecular Sciences have established the Best Paper Award to recognize the most outstanding articles published in the areas of molecular biology, molecular physics, and chemistry that have been published in the International Journal of Molecular Sciences. [...] Full article

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21 pages, 2466 KiB

Open AccessArticle

Experimental Combined Immunotherapy of Tumours with Major Histocompatibility Complex Class I Downregulation

by Adrianna Grzelak, Ingrid Polakova, Jana Smahelova, Julie Vackova, Lucie Pekarcikova, Ruth Tachezy and Michal Smahel

Int. J. Mol. Sci. 2018, 19(11), 3693; https://doi.org/10.3390/ijms19113693 - 21 Nov 2018

Cited by 4 | Viewed by 4383

Abstract

Combined immunotherapy constitutes a novel, advanced strategy in cancer treatment. In this study, we investigated immunotherapy in the mouse TC-1/A9 model of human papillomavirus type 16 (HPV16)-associated tumors characterized by major histocompatibility complex class I (MHC-I) downregulation. We found that the induction of [...] Read more.

Combined immunotherapy constitutes a novel, advanced strategy in cancer treatment. In this study, we investigated immunotherapy in the mouse TC-1/A9 model of human papillomavirus type 16 (HPV16)-associated tumors characterized by major histocompatibility complex class I (MHC-I) downregulation. We found that the induction of a significant anti-tumor response required a combination of DNA vaccination with the administration of an adjuvant, either the synthetic oligodeoxynucleotide ODN1826, carrying immunostimulatory CpG motifs, or α-galactosylceramide (α-GalCer). The most profound anti-tumor effect was achieved when these adjuvants were applied in a mix with a one-week delay relative to DNA immunization. Combined immunotherapy induced tumor infiltration with various subsets of immune cells contributing to tumor regression, of which cluster of differentiation (CD) 8⁺ T cells were the predominant subpopulation. In contrast, the numbers of tumor-associated macrophages (TAMs) were not markedly increased after immunotherapy but in vivo and in vitro results showed that they could be repolarized to an anti-tumor M1 phenotype. A blockade of T cell immunoglobulin and mucin-domain containing-3 (Tim-3) immune checkpoint had a negligible effect on anti-tumor immunity and TAMs repolarization. Our results demonstrate a benefit of combined immunotherapy comprising the activation of both adaptive and innate immunity in the treatment of tumors with reduced MHC-I expression. Full article

(This article belongs to the Special Issue Cancer Immunology and Immunotherapy)

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Comparison of the anti-tumor effects induced after the administration of CpG ODN1826 and α-GalCer either alone or in a mix in the non-immunized and immunized mice. Animals (n = 5) were injected s.c. with TC-1/A9 cells and immunized 3 times by a gene gun with either the empty pBSC plasmid (referred to as non-immunized mice, A–C) or pBSC/PADRE.E7GGG (immunized mice, D–F). Vaccine adjuvants ODN1826 (A,D), α-GalCer (B,E), or a mix of ODN1826 and α-GalCer (C,F) were administered on the same days as DNA vaccines. Some groups received a monoclonal antibody against Tim-3. No. of mice with a tumor/no. of mice in the group is indicated. Bars: ±SEM; *** p < 0.001, **** p < 0.0001. Statistical significance refers to the comparison with the group immunized with the PADRE.E7GGG gene. The experiment was repeated with similar results. Full article ">Figure 2
The effects of different dosage and timing protocols. Mice (n = 5) were injected with TC-1/A9 cells and immunized by a gene gun. Mice received combinations of ODN1826, α-GalCer and α-Tim-3 3 times on the days of immunization (A), 5 times with two additional doses on days 13 and 17 (B) and 3 times with a one-week delay following DNA immunization (i.e., on days 10, 13 and 17) (C). Bars: ±SEM; ** p < 0.01, *** p < 0.001, **** p < 0.0001. Statistical significance refers to the comparison with the group immunized with the PADRE.E7GGG gene. The experiment was repeated with similar results. Full article ">Figure 3
Tumor-infiltrating immune cells and their role in tumor growth. Analysis of tumor-infiltrating cells was performed by flow cytometry (A,B). Mice (n = 4) were injected with tumor cells and immunized by a gene gun. Vaccine adjuvants and anti-Tim-3 were administered on the same days as the DNA vaccines. Tumor cells were isolated on day 12 from non-treated tumors and on days 14–18 from treated tumors and stained with fluorochrome-labeled antibodies. (A) Frequencies of CD45+ and CD3+ cells, Treg (CD4+CD25+Foxp3+) and Nrp1+ Treg cells. Statistical significance refers to the comparison with the non-treated (pBSC) group. (B) Overview of the mean percentages of the major subpopulations of tumor-infiltrating cells in total cells. (C) The effect of in vivo depletion of immune cells and neutralization of IFN-γ on the anti-tumor response induced by immunotherapies with ODN1826 or α-GalCer in the immunized mice (n = 5). Vaccine adjuvants were injected on the days of immunization. Statistical significance refers to the comparison with the group that was immunized with the PADRE.E7GGG gene and received an adjuvant. Bars: ±SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Full article ">Figure 4
Activation of CD8+ T cells by combined immunotherapy and characterization of tumor-infiltrating CD8+ T cells. (A) Analysis of activated CD8+ cells by an ELISPOT assay. Mice (n = 3) were immunized by a gene gun on days 3, 6 and 10 and inoculated with ODN1826, α-GalCer and anti-Tim-3 on the days of immunization (D3) or with a one-week delay following DNA immunization (D10). Eight days after the last immunization, mononuclear cells were prepared from pooled splenocytes, restimulated with peptides and IFN-γ-producing-cells were detected. Columns, mean of triplicate samples; bars, ± SEM. The experiment was repeated with similar results. (B) Analysis of intratumoral CD8+ T cells by flow cytometry. The experiment was performed as in <a href="#ijms-19-03693-f003" class="html-fig">Figure 3</a>. Columns, mean of four samples; bars, ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001. Statistical significance refers to the comparison with the non-treated (pBSC) group. Full article ">Figure 5
Characterization of TAMs. After immunotherapy, TAMs were isolated from tumors and analyzed by flow cytometry. The experiment was performed as in <a href="#ijms-19-03693-f003" class="html-fig">Figure 3</a>. (A) Gating of an MHC-II marker. (B) Overview of mean percentages of TAM subpopulations distinguished by MHC-II expression. (C) Frequencies of iNOS+, TNF-α+ and Tim-3+ MΦs; columns, mean of four samples. TAMs were also isolated from non-treated tumors and stimulated in vitro. The nitrite (D) and TNF-α (E) concentrations were measured in the supernatants by Griess reagent and ELISA test, respectively. pMΦs were used for comparison. Columns, mean of 3 independent experiments. Bars, ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Statistical significance refers to the comparison with the non-treated (pBSC)/unstimulated group. Full article ">Figure 6
In vitro modulation of iNOS and arginase activity in the co-culture of TAMs with TC-1/A9 cells. Co-cultures as well as control cells, i.e., TAMs and TC-1/A9 cells alone, were stimulated for 44 h. The nitrite concentration was determined by Griess reagent (A) and urea was quantified by the microplate method (B). Columns, mean of three independent experiments; bars ± SEM; **** p < 0.0001. Statistical significance refers to the comparison of co-cultures with TAMs alone. Full article ">Figure 7
Analysis of gene expression in tumors by RT-qPCR. The experiment was performed as in <a href="#ijms-19-03693-f003" class="html-fig">Figure 3</a>. Columns, mean of 4–6 samples; bars, ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Relative expression and statistical significance refer to the comparisons with the non-treated (pBSC) group. The inserted graph shows the correlation between Ifng and Ido1 expression. Full article ">

13 pages, 1790 KiB

Open AccessArticle

Metformin Inhibits Migration and Invasion by Suppressing ROS Production and COX2 Expression in MDA-MB-231 Breast Cancer Cells

by Chandler Schexnayder, Kiera Broussard, Demitrius Onuaguluchi, Anthony Poché, Moamen Ismail, LeFontae McAtee, Shawn Llopis, Amber Keizerweerd, Harris McFerrin and Christopher Williams

Int. J. Mol. Sci. 2018, 19(11), 3692; https://doi.org/10.3390/ijms19113692 - 21 Nov 2018

Cited by 38 | Viewed by 6278

Abstract

Background: Several mechanisms of action have been proposed to explain the apparent antineoplastic functions of metformin, many of which are observed at high concentrations that may not be reflective of achievable tissue concentrations. We propose that metformin at low concentrations functions to inhibit [...] Read more.

Background: Several mechanisms of action have been proposed to explain the apparent antineoplastic functions of metformin, many of which are observed at high concentrations that may not be reflective of achievable tissue concentrations. We propose that metformin at low concentrations functions to inhibit ROS production and inflammatory signaling in breast cancer, thereby reducing metastasis. Methods: Using the highly invasive MDA-MB-231 breast carcinoma model, we ascertained the impact of metformin on cell viability by DNA content analysis and fluorescent dye exclusion. Migration and invasion assays were performed using a modified Boyden chamber assay and metastasis was ascertained using the chorioallantoic membrane (CAM) assay. PGE2 production was measured by Enzyme-Linked Immunosorbent Assay (ELISA). COX2 and ICAM1 levels were determined by flow cytometry immunoassay. Results: Metformin acutely decreased cell viability and caused G2 cell cycle arrest only at high concentrations (10 mM). At 100 µM, however, metformin reduced ICAM1 and COX2 expression, as well as reduced PGE2 production and endogenous mitochondrial ROS production while failing to significantly impact cell viability. Consequently, metformin inhibited migration, invasion in vitro and PGE2-dependent metastasis in CAM assays. Conclusion: At pharmacologically achievable concentrations, metformin does not drastically impact cell viability, but inhibits inflammatory signaling and metastatic progression in breast cancer cells. Full article

(This article belongs to the Section Biochemistry)

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Metformin fails to directly impact breast cancer cell viability at pharmacologically relevant concentrations. (A) MDA-MB-231 breast cancer cells were cultured in the presence or absence of metformin for 72 h. Nuclear DNA content was assessed by propidium iodide staining, and analyzed by flow cytometry. The proportion of cells in each phase is depicted in (B). In (C), cells were cultured in presence or absence of metformin for 48 h, followed by incubation in Sytox Red cell toxicity stain. The dye is excluded from viable cells whose membranes are not compromised (indicated in red). Representative figures are shown. Each assay was performed in quadruplicate. N.S. denotes no significant difference as compared to control. Full article ">Figure 2
Metformin alleviates endogenous ROS production and subsequent pJNK activation. MDA-MB-231 cells cultured in the presence (red) or absence (black) of metformin (100 µM) for 48 h and subsequently stained with MitoSOX mitochondrial superoxide detection indicator. Fluorescence intensity was ascertained by flow cytometry and was normalized to untreated control. Studies were performed in triplicate, with 10,000 events each, and compared using Student’s t-test, * p ≤ 0.05. Full article ">Figure 3
Metformin represses expression of pro-inflammatory markers in breast cancer. (A) MDA-MB-231 cells were incubated with or without metformin for 3 days and levels of PGE2 in the culture supernatant measured by competitive ELISA. MDA-MB-231 breast cancer cells were cultured in the presence or absence of metformin for 48 h after which cells were fixed and immunofluorescently stained for (B) COX2 or (C) ICAM1 protein expression. Staining intensity was measured by flow cytometry and normalized to control for comparison (right of histogram). Flow cytometry assays were performed in quadruplicate with 10,000 events registered per replicate. ELISA was performed with 4 technical repeats on 2 experiments. Significance was determined using Student’s t-test, where * p ≤ 0.05. Full article ">Figure 4
Metformin attenuates breast cancer cell migration, invasion, and metastasis. (A) MDA-MB-231 cells were pre-exposed to metformin for 48 h, collected, and stained with CellTracker Green fluorescent stain. Stained cells were ceded in the upper chamber of a Boyden chamber plate in the absence (B), or the presence (C) of Matrigel coating. The number of transmigratory/invading cells in response to chemoattractant (DMEM with 10% FBS) were enumerated by flow cytometry and normalized to control. (D) Anti-metastatic action of metformin is reversed by PGE2. MDA-MB-231 cells were labeled with CellTracker Green (10 μM) and seeded onto mesh squares placed on the chorioallantoic membrane (CAM) of 10-day old chicken embryos grown ex ovo. After 3 days, portions of the CAM-containing human cells were removed, fixed, stained with DAPI and visualized by fluorescence microscopy using an inverted laser confocal microscope with Z-axis control. Images were taken every 1 μm for approximately 100 μm, and the invasion maximum was defined by the leading edge of fluorescent cell invasion. Significance was determined by ANOVA and Tukey’s post hoc analysis, **** p ≤ 0.01, * p ≤ 0.05. Full article ">Figure 5
COX2 expression has prognostic significance in basal, but not Luminal A intrinsic breast carcinoma subtypes. (A) Interrogation of public microarray repositories using KMPLOT online analysis tool (KMPLOT.COM), DMFS was compared between those with high COX2 expression to those with low COX2 expression, in patients with either basal or luminal breast cancer subtypes. The median time of DMFS in the same cohort is summarized in (B). Full article ">Figure 6
Model for metformin anti-neoplastic activity. In this model, superoxide is generated in cancer cells during mitochondrial respiration as a by-product of NADH reduction in cancer cells. ROS activity leads to COX2 and ICAM1 expression, as well as induction of other inflammatory mediators, resulting in increased metastatic potential in tumor cells. Metformin disrupts the function of complex I, thereby preventing ROS generation and attenuating metastasis. Full article ">

33 pages, 1933 KiB

Open AccessReview

Insect Transcription Factors: A Landscape of Their Structures and Biological Functions in Drosophila and beyond

by Zhaojiang Guo, Jianying Qin, Xiaomao Zhou and Youjun Zhang

Int. J. Mol. Sci. 2018, 19(11), 3691; https://doi.org/10.3390/ijms19113691 - 21 Nov 2018

Cited by 39 | Viewed by 9144

Abstract

Transcription factors (TFs) play essential roles in the transcriptional regulation of functional genes, and are involved in diverse physiological processes in living organisms. The fruit fly Drosophila melanogaster, a simple and easily manipulated organismal model, has been extensively applied to study the [...] Read more.

Transcription factors (TFs) play essential roles in the transcriptional regulation of functional genes, and are involved in diverse physiological processes in living organisms. The fruit fly Drosophila melanogaster, a simple and easily manipulated organismal model, has been extensively applied to study the biological functions of TFs and their related transcriptional regulation mechanisms. It is noteworthy that with the development of genetic tools such as CRISPR/Cas9 and the next-generation genome sequencing techniques in recent years, identification and dissection the complex genetic regulatory networks of TFs have also made great progress in other insects beyond Drosophila. However, unfortunately, there is no comprehensive review that systematically summarizes the structures and biological functions of TFs in both model and non-model insects. Here, we spend extensive effort in collecting vast related studies, and attempt to provide an impartial overview of the progress of the structure and biological functions of current documented TFs in insects, as well as the classical and emerging research methods for studying their regulatory functions. Consequently, considering the importance of versatile TFs in orchestrating diverse insect physiological processes, this review will assist a growing number of entomologists to interrogate this understudied field, and to propel the progress of their contributions to pest control and even human health. Full article

(This article belongs to the Special Issue Transcriptional Regulation: Molecules, Involved Mechanisms and Misregulation)

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Full article ">Figure 1
The insect transcription factor (TF) atlas, including the well-studied TFs and their related references collected in the review (See <a href="#app1-ijms-19-03691" class="html-app">Table S1</a> for more detailed information about these TFs, and we apologize to researchers whose work could not be discussed and cited in the main text due to space limitations). As yet, diverse TFs have been documented in at least nine different insect orders including Diptera, Hemiptera, Lepidoptera, Coleoptera, Orthoptera, Thysanoptera, Blattaria, Neuroptera, and Hymenoptera. Different insect species are also denoted by colored circles on the vertical line. Full article ">Figure 2
The main structures of insect TFs. The pie chart represents the statistical ratio of the Drosophila TFs in different superclasses based on Hammonds’ study [<a href="#B22-ijms-19-03691" class="html-bibr">22</a>]. (A) Structures of homeodomain TFs. Homeodomain: a typical homeodomain contains a short N-terminal arm facilitating DNA binding and four α-helices with a helix II-turn-helix III structure that is responsible for DNA binding and recognition; Hox protein: in addition to the homeodomain, a Hox protein typically contains a YPWM motif in the N-terminus mediating protein dimerization; Paired-like protein: in addition to the homeodomain, some paired-like proteins include an N-terminal octapeptide (OP) motif, and some contain a C-terminal OAR motif that can be involved in transcriptional activation; Paired-domain protein: paired-domain proteins carry an N-terminal paired box with a helix-turn-helix (HTH) structure that mediates DNA binding, and some proteins have a full-length or truncated homeodomain in the C-terminus, as well as an OP motif between the paired box and the homeodomain to mediate protein dimerization; POU (Pit-Oct-Unc) protein: POU proteins have a POU-specific domain with an HTH structure in the N-terminus that contributes to the generation of high-affinity DNA binding, and the homeodomain in the C-terminus is responsible for DNA binding; LIM protein: LIM proteins possess two LIM domains with zinc finger (ZF) structures upstream of the homeodomain that mainly mediate protein-protein interactions. (B) Structures of basic DBD TFs. The basic leucine zipper (bZIP) protein: bZIP proteins harbor a basic DNA-binding domain (DBD) for specific DNA recognition and binding, and a leucine zipper domain in the C-terminus for protein dimerization and DNA binding. The leucine zipper domain forms dextrorotatory α-helixes, and a leucine appears in the seventh position of every seven amino acids; thus, an adjacent leucine appears every two turns on the same side of the helix. The basic helix-loop-helix (bHLH) protein: bHLH proteins are composed of a basic DBD in the N-terminus, followed by an HLH domain. The basic DBD accounts for DNA motif recognition and binding and facilitates protein dimerization. In the HLH domain, two hydrophilic and lipophilic α-helices are separated by a loop to form an HLH structure mediating protein dimerization and contributing to DNA binding. (C) Structures of the ZF TFs. C2H2 ZF protein: the C2H2 ZF protein has multiple connected ZF DBDs. In the root of every ZF, two cysteines and two histidines link Zn2+ to form a tetrahedron. Nuclear receptor (NR) with C4 ZF: NR contains a C4-type ZF region as a DBD that consists of eight conserved cysteine residues coordinated with two Zn2+ to form two ZFs with a tetrahedral coordination structure. The first ZF provides DNA-binding specificity, and the second ZF has a weak dimerization interface, allowing for dimerization of the receptor molecule. In addition, a ligand-binding domain is typically found in the C-terminus and functions as the main dimerization region. Full article ">Figure 3
Establishment of the embryonic anterior–posterior (A-P) axis in Drosophila. The diagram is adapted from Gilbert’s works [<a href="#B40-ijms-19-03691" class="html-bibr">40</a>,<a href="#B41-ijms-19-03691" class="html-bibr">41</a>]. (A) Regulatory hierarchy of the formation of Drosophila A-P axis patterning. The maternal effect proteins Bicoid (Bcd) and Caudal (Cad) form a concentration gradient along the A-P axis and generate specific positional information to activate the expression of the gap gene hunchback (Hb). Hh further initiates the proper expression of other gap genes along the A-P axis. Gap proteins subsequently activate the expression of pair-rule genes, which form seven stripes perpendicular to the A-P axis and divide those discontinuous regions defined by the gap gene into body segments. The pair-rule proteins then regulate the expression of segment polarity genes in specific cells of each somite, and their 14 expression stripes establish the boundaries of parasegments. Finally, each segment is characterized by specifically expressed Hox genes. (B) The concentration gradient of the maternal effect proteins Bcd and Cad along the A-P axis in the early cleavage embryo. (C) The concentration changes in gap genes along the A-P axis. (D) Regulation of expression of the pair-rule gene even-skipped (eve) in seven stripes. The above region represents a partial promoter of the eve gene that contains five different enhancers responsible for the distinct stripes. The lower part illustrates how TFs regulate eve expression in different stripes. The black box represents the TF, the green characters indicate the enhancer of eve, and the orange circles display the cells expressing eve. The vertical bars with the letters “A” and “P” denote the anterior and posterior boundaries of the eve stripe, respectively, and the numbers show the number of eve bands. (E) Regulation of a repetitive unit of sloppy paired 1 (slp1) stripe [<a href="#B42-ijms-19-03691" class="html-bibr">42</a>]. The 14-stripe patterning constitutes seven repetitive units, each containing odd-numbered and even-numbered parasegments. The odd-numbered parasegment consists of two types of cells: two type I cells in the posterior half do not express slp1 and two type II cells in the posterior half that express slp1. The even-numbered parasegment also contains two types of cells: type III cells that do not express slp1 in the first half and the latter type IV cells expressing slp1. The expression of slp1 in different cell types is regulated by different pair-rule proteins in a specific combination to regulate the proximal early stripe element (PESE) or the distal early stripe element (DESE) of slp1. The colored hexagons indicate the pair-rule proteins, the orange quadrants represent the enhancer, the gray ovals exhibit the cells that do not express slp1, the blue ovals denote the cells expressing slp1, the gray rectangles with “No” show no slp1 strips, and the blue rectangles represent slp1 strips. TF abbreviations: Hb: Hunchback; Gt: Giant; Kr: Krüppel; Kni: Knirps; Tll: Tailess; Zld: Zelda; Opa: Odd-paired; Ftz: Fushi tarazu; X: represents an as yet unidentified Factor X. Full article ">Figure 4
The 20-hydroxyecdysone (20E) and juvenile hormone (JH) signaling pathways regulating insect molting, metamorphosis, and reproduction. (A) Regulation of ecdysone titer by TFs. Changes in the ecdysone titers of insects are synergistically controlled by the synthesis and degradation of ecdysone. A low level of ecdysone promotes TFs to specifically regulate the Halloween genes in PG, thereby increasing steroidogenesis. A high level of ecdysone drives TFs to repress the expression of Halloween genes and activate the expression of degradative enzyme genes to directly degrade ecdysone, thus decreasing ecdysteroid titers. (B) Regulation of insect reproduction and metamorphosis by JH signaling pathway-related TFs. The white hexagon on the left shows TFs regulating the synthesis and degradation of JHs; the central hexagon represents JH binding to the JH receptor Met to regulate the transcription of downstream target genes; the orange hexagons display the JH/Methoprene-tolerant (Met) complex regulates downstream targets to control insect reproduction; the green hexagons indicate metamorphosis; and the purple gradient ellipses denote the cofactors of Met. Full article ">

19 pages, 2892 KiB

Open AccessArticle

Gramicidin Lateral Distribution in Phospholipid Membranes: Fluorescence Phasor Plots and Statistical Mechanical Model

by István P. Sugár, Alexander P. Bonanno and Parkson Lee-Gau Chong

Int. J. Mol. Sci. 2018, 19(11), 3690; https://doi.org/10.3390/ijms19113690 - 21 Nov 2018

Cited by 4 | Viewed by 2946

Abstract

When using small mole fraction increments to study gramicidins in phospholipid membranes, we found that the phasor dots of intrinsic fluorescence of gramicidin D and gramicidin A in dimyristoyl-sn-glycero-3-phosphocholine (DMPC) unilamellar and multilamellar vesicles exhibit a biphasic change with peptide content [...] Read more.

When using small mole fraction increments to study gramicidins in phospholipid membranes, we found that the phasor dots of intrinsic fluorescence of gramicidin D and gramicidin A in dimyristoyl-sn-glycero-3-phosphocholine (DMPC) unilamellar and multilamellar vesicles exhibit a biphasic change with peptide content at 0.143 gramicidin mole fraction. To understand this phenomenon, we developed a statistical mechanical model of gramicidin/DMPC mixtures. Our model assumes a sludge-like mixture of fluid phase and aggregates of rigid clusters. In the fluid phase, gramicidin monomers are randomly distributed. A rigid cluster is formed by a gramicidin dimer and DMPC molecules that are condensed to the dimer, following particular stoichiometries (critical gramicidin mole fractions, X_cr including 0.143). Rigid clusters form aggregates in which gramicidin dimers are regularly distributed, in some cases, even to superlattices. At X_cr, the size of cluster aggregates and regular distributions reach a local maximum. Before a similar model was developed for cholesterol/DMPC mixtures (Sugar and Chong (2012) J. Am. Chem. Soc. 134, 1164–1171) and here the similarities and differences are discussed between these two models. Full article

(This article belongs to the Section Molecular Biophysics)

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23 pages, 2466 KiB

Open AccessArticle

Synergistic Effects of Weightlessness, Isoproterenol, and Radiation on DNA Damage Response and Cytokine Production in Immune Cells

by Maria Moreno-Villanueva, Alan H. Feiveson, Stephanie Krieger, AnneMarie Kay Brinda, Gudrun Von Scheven, Alexander Bürkle, Brian Crucian and Honglu Wu

Int. J. Mol. Sci. 2018, 19(11), 3689; https://doi.org/10.3390/ijms19113689 - 21 Nov 2018

Cited by 17 | Viewed by 4504

Abstract

The implementation of rotating-wall vessels (RWVs) for studying the effect of lack of gravity has attracted attention, especially in the fields of stem cells, tissue regeneration, and cancer research. Immune cells incubated in RWVs exhibit several features of immunosuppression including impaired leukocyte proliferation, [...] Read more.

The implementation of rotating-wall vessels (RWVs) for studying the effect of lack of gravity has attracted attention, especially in the fields of stem cells, tissue regeneration, and cancer research. Immune cells incubated in RWVs exhibit several features of immunosuppression including impaired leukocyte proliferation, cytokine responses, and antibody production. Interestingly, stress hormones influence cellular immune pathways affected by microgravity, such as cell proliferation, apoptosis, DNA repair, and T cell activation. These pathways are crucial defense mechanisms that protect the cell from toxins, pathogens, and radiation. Despite the importance of the adrenergic receptor in regulating the immune system, the effect of microgravity on the adrenergic system has been poorly studied. Thus, we elected to investigate the synergistic effects of isoproterenol (a sympathomimetic drug), radiation, and microgravity in nonstimulated immune cells. Peripheral blood mononuclear cells were treated with the sympathomimetic drug isoproterenol, exposed to 0.8 or 2 Gy γ-radiation, and incubated in RWVs. Mixed model regression analyses showed significant synergistic effects on the expression of the β2-adrenergic receptor gene (ADRB2). Radiation alone increased ADRB2 expression, and cells incubated in microgravity had more DNA strand breaks than cells incubated in normal gravity. We observed radiation-induced cytokine production only in microgravity. Prior treatment with isoproterenol clearly prevents most of the microgravity-mediated effects. RWVs may be a useful tool to provide insight into novel regulatory pathways, providing benefit not only to astronauts but also to patients suffering from immune disorders or undergoing radiotherapy. Full article

(This article belongs to the Special Issue Adaptation of Living Organisms in Space: From Mammals to Plants)

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Cell recovery and cell viability after 24 h incubation in rotating-wall vessels (RWVs): (A) Change in the initial cell concentration with incubation time at a rotation speed of 10 rpm (6 independent experiments); (B) cell recovery of nontreated and treated cells over experimental conditions before and after 24 h incubation in RWVs rotating at 8.5 rpm (18 independent experiments); and (C) the percentage of live nontreated cells that remained alive after 24 h incubation rotating at a speed of 8.5 rpm (20 independent experiments). Full article ">Figure 2
Apoptosis rate (calculated as indicated in <a href="#ijms-19-03689-t001" class="html-table">Table 1</a>) and residual DNA strand breaks after 24 h incubation in RWVs in µg or 1g. Apoptosis rate is represented by the mean transformed values (A) and (B) and DNA strand breaks are means expressed in Gy dose equivalent (C). Nontreated cells (C), isoproterenol (Iso) and radiation (R) treated cells. Error bars represent +1 SEM. Statistical analysis are summarized in <a href="#ijms-19-03689-t001" class="html-table">Table 1</a>. Full article ">Figure 3
Radiation effects on gene expression relative to non-irradiated cells incubated in (A) 1g and (B) µg. Cells were irradiated either with 0.8 Gy (light bars) or 2 Gy (dark bars) and immediately incubated in 1g (grey bars) or µg (blue bars) for 24 h. Error bars represent SEM. Asterisks represent significant differences in gene expression in cells irradiated with 0.8 Gy compared to cells irradiated with 2 Gy. Statistical method: Krieger. p-value threshold: 0.018 after controlling the FDR (false-discovery rate—see statistical methods) to 5%. Full article ">Figure 4
Cytokine concentration in cell culture medium. Cells were irradiated 2 Gy and subsequently incubated in 1g or µg. After 24 h cytokine concentration was measured. Radiation induced cytokine production in µg but not in 1g. Isoproterenol treatment prior to radiation prevented the production of all cytokines. Bars represent mean + 1 SEM from 10 independent experiments. The synergistic effect of isoproterenol and radiation in µg (Iso × R(µg)) was significant for all four cytokines. Statistical analyses are summarized in <a href="#ijms-19-03689-t004" class="html-table">Table 4</a> and <a href="#ijms-19-03689-t005" class="html-table">Table 5</a>. Full article ">Figure 5
Association between changes in cytokines and corresponding changes in gene expression over the eight experimental conditions for the group of samples irradiated with 2 Gy. BAX, CASP3, PCNA, LIG4, and MDM2 gene expressions were positively associated, while AKT1, TP53, PARP1, OGG1, and APXE1 were negatively associated with cytokines. Full article ">Figure 6
Schematic representation of the treatment conditions. Cells were distributed in eight RCCSVs. Isoproterenol treatment (Iso) and radiation exposure (R) were performed separately and in combination (Iso + R). For the combined treatment, cells were irradiated for 2 min immediately after 2 min isoproterenol (Iso) treatment. Vessels were placed in an incubator and rotated at 8.5 rpm on a vertical (Earth gravity = 1g) or on a horizontal axis (simulated microgravity = µg). Vessels with nontreated cells (C) were incubated in 1g and in µg. After 24 h cells were recovered from the vessels and analyses were performed (DSB = DNA strand breaks). Full article ">

15 pages, 2565 KiB

Open AccessReview

The Question of a Role for Statins in Age-Related Macular Degeneration

by Marina Roizenblatt, Nara Naranjit, Mauricio Maia and Peter L. Gehlbach

Int. J. Mol. Sci. 2018, 19(11), 3688; https://doi.org/10.3390/ijms19113688 - 21 Nov 2018

Cited by 22 | Viewed by 5257

Abstract

Age-related macular degeneration (AMD) is the leading cause of irreversible central vision loss in patients over the age of 65 years in industrialized countries. Epidemiologic studies suggest that high dietary fat intake is a risk factor for the development and progression of both [...] Read more.

Age-related macular degeneration (AMD) is the leading cause of irreversible central vision loss in patients over the age of 65 years in industrialized countries. Epidemiologic studies suggest that high dietary fat intake is a risk factor for the development and progression of both vascular and retinal disease. These, and other associations, suggest a hypothesis linking elevated cholesterol and AMD progression. It follows, therefore, that cholesterol-lowering medications, such as statins, may influence the onset and progression of AMD. However, the findings have been inconclusive as to whether statins play a role in AMD. Due to the significant public health implications of a potential inhibitory effect of statins on the onset and progression of AMD, it is important to continually evaluate emerging findings germane to this question. Full article

(This article belongs to the Special Issue Molecular Biology of Age-Related Macular Degeneration (AMD))

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14 pages, 477 KiB

Open AccessArticle

Hierarchical Clustering of DNA k-mer Counts in RNAseq Fastq Files Identifies Sample Heterogeneities

by Wolfgang Kaisers , Holger Schwender and Heiner Schaal

Int. J. Mol. Sci. 2018, 19(11), 3687; https://doi.org/10.3390/ijms19113687 - 21 Nov 2018

Cited by 6 | Viewed by 4485

Abstract

We apply hierarchical clustering (HC) of DNA k-mer counts on multiple Fastq files. The tree structures produced by HC may reflect experimental groups and thereby indicate experimental effects, but clustering of preparation groups indicates the presence of batch effects. Hence, HC of DNA [...] Read more.

We apply hierarchical clustering (HC) of DNA k-mer counts on multiple Fastq files. The tree structures produced by HC may reflect experimental groups and thereby indicate experimental effects, but clustering of preparation groups indicates the presence of batch effects. Hence, HC of DNA k-mer counts may serve as a diagnostic device. In order to provide a simple applicable tool we implemented sequential analysis of Fastq reads with low memory usage in an R package (seqTools) available on Bioconductor. The approach is validated by analysis of Fastq file batches containing RNAseq data. Analysis of three Fastq batches downloaded from ArrayExpress indicated experimental effects. Analysis of RNAseq data from two cell types (dermal fibroblasts and Jurkat cells) sequenced in our facility indicate presence of batch effects. The observed batch effects were also present in reads mapped to the human genome and also in reads filtered for high quality (Phred > 30). We propose, that hierarchical clustering of DNA k-mer counts provides an unspecific diagnostic tool for RNAseq experiments. Further exploration is required once samples are identified as outliers in HC derived trees. Full article

(This article belongs to the Section Biochemistry)

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18 pages, 3500 KiB

Open AccessArticle

Interactions Between Spermine-Derivatized Tentacle Porphyrins and The Human Telomeric DNA G-Quadruplex

by Navin C. Sabharwal, Jessica Chen, Joo Hyun (June) Lee, Chiara M. A. Gangemi, Alessandro D'Urso and Liliya A. Yatsunyk

Int. J. Mol. Sci. 2018, 19(11), 3686; https://doi.org/10.3390/ijms19113686 - 21 Nov 2018

Cited by 18 | Viewed by 4828

Abstract

G-rich DNA sequences have the potential to fold into non-canonical G-Quadruplex (GQ) structures implicated in aging and human diseases, notably cancers. Because stabilization of GQs at telomeres and oncogene promoters may prevent cancer, there is an interest in developing small molecules that selectively [...] Read more.

G-rich DNA sequences have the potential to fold into non-canonical G-Quadruplex (GQ) structures implicated in aging and human diseases, notably cancers. Because stabilization of GQs at telomeres and oncogene promoters may prevent cancer, there is an interest in developing small molecules that selectively target GQs. Herein, we investigate the interactions of meso-tetrakis-(4-carboxysperminephenyl)porphyrin (TCPPSpm4) and its Zn(II) derivative (ZnTCPPSpm4) with human telomeric DNA (Tel22) via UV-Vis, circular dichroism (CD), and fluorescence spectroscopies, resonance light scattering (RLS), and fluorescence resonance energy transfer (FRET) assays. UV-Vis titrations reveal binding constants of 4.7 × 10⁶ and 1.4 × 10⁷ M⁻¹ and binding stoichiometry of 2–4:1 and 10–12:1 for TCPPSpm4 and ZnTCPPSpm4, respectively. High stoichiometry is supported by the Job plot data, CD titrations, and RLS data. FRET melting indicates that TCPPSpm4 stabilizes Tel22 by 36 ± 2 °C at 7.5 eq., and that ZnTCPPSpm4 stabilizes Tel22 by 33 ± 2 °C at ~20 eq.; at least 8 eq. of ZnTCPPSpm4 are required to achieve significant stabilization of Tel22, in agreement with its high binding stoichiometry. FRET competition studies show that both porphyrins are mildly selective for human telomeric GQ vs duplex DNA. Spectroscopic studies, combined, point to end-stacking and porphyrin self-association as major binding modes. This work advances our understanding of ligand interactions with GQ DNA. Full article

(This article belongs to the Special Issue A Commemorative Issue in Honor of Professor Nick Hadjiliadis: Metal Complex Interactions with Nucleic Acids and/or DNA)

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14 pages, 415 KiB

Open AccessReview

Biotherapeutics: Challenges and Opportunities for Predictive Toxicology of Monoclonal Antibodies

by Dale E. Johnson

Int. J. Mol. Sci. 2018, 19(11), 3685; https://doi.org/10.3390/ijms19113685 - 21 Nov 2018

Cited by 33 | Viewed by 7109

Abstract

Biotherapeutics are a rapidly growing portion of the total pharmaceutical market accounting for almost one-half of recent new drug approvals. A major portion of these approvals each year are monoclonal antibodies (mAbs). During development, non-clinical pharmacology and toxicology testing of mAbs differs from [...] Read more.

Biotherapeutics are a rapidly growing portion of the total pharmaceutical market accounting for almost one-half of recent new drug approvals. A major portion of these approvals each year are monoclonal antibodies (mAbs). During development, non-clinical pharmacology and toxicology testing of mAbs differs from that done with chemical entities since these biotherapeutics are derived from a biological source and therefore the animal models must share the same epitopes (targets) as humans to elicit a pharmacological response. Mechanisms of toxicity of mAbs are both pharmacological and non-pharmacological in nature; however, standard in silico predictive toxicological methods used in research and development of chemical entities currently do not apply to these biotherapeutics. Challenges and potential opportunities exist for new methodologies to provide a more predictive program to assess and monitor potential adverse drug reactions of mAbs for specific patients before and during clinical trials and after market approval. Full article

(This article belongs to the Special Issue Frontiers in Drug Toxicity Prediction)

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18 pages, 5260 KiB

Open AccessArticle

Functional Biological Activity of Sorafenib as a Tumor-Treating Field Sensitizer for Glioblastoma Therapy

by Yunhui Jo, Eun Ho Kim, Sei Sai, Jin Su Kim, Jae-Min Cho, Hyeongi Kim, Jeong-Hwa Baek, Jeong-Yub Kim, Sang-Gu Hwang and Myonggeun Yoon

Int. J. Mol. Sci. 2018, 19(11), 3684; https://doi.org/10.3390/ijms19113684 - 21 Nov 2018

Cited by 45 | Viewed by 5232

Abstract

Glioblastoma, the most common primary brain tumor in adults, is an incurable malignancy with poor short-term survival and is typically treated with radiotherapy along with temozolomide. While the development of tumor-treating fields (TTFields), electric fields with alternating low and intermediate intensity has facilitated [...] Read more.

Glioblastoma, the most common primary brain tumor in adults, is an incurable malignancy with poor short-term survival and is typically treated with radiotherapy along with temozolomide. While the development of tumor-treating fields (TTFields), electric fields with alternating low and intermediate intensity has facilitated glioblastoma treatment, clinical outcomes of TTFields are reportedly inconsistent. However, combinatorial administration of chemotherapy with TTFields has proven effective for glioblastoma patients. Sorafenib, an anti-proliferative and apoptogenic agent, is used as first-line treatment for glioblastoma. This study aimed to investigate the effect of sorafenib on TTFields-induced anti-tumor and anti-angiogenesis responses in glioblastoma cells in vitro and in vivo. Sorafenib sensitized glioblastoma cells to TTFields, as evident from significantly decreased post-TTFields cell viability (p < 0.05), and combinatorial treatment with sorafenib and TTFields accelerated apoptosis via reactive oxygen species (ROS) generation, as evident from Poly (ADP-ribose) polymerase (PARP) cleavage. Furthermore, use of sorafenib plus TTFields increased autophagy, as evident from LC3 upregulation and autophagic vacuole formation. Cell cycle markers accumulated, and cells underwent a G2/M arrest, with an increased G0/G1 cell ratio. In addition, the combinatorial treatment significantly inhibited tumor cell motility and invasiveness, and angiogenesis. Our results suggest that combination therapy with sorafenib and TTFields is slightly better than each individual therapy and could potentially be used to treat glioblastoma in clinic, which requires further studies. Full article

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

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21 pages, 6366 KiB

Open AccessArticle

Metabolic Changes of Amino Acids and Flavonoids in Tea Plants in Response to Inorganic Phosphate Limitation

by Santosh KC, Meiya Liu, Qunfeng Zhang, Kai Fan, Yuanzhi Shi and Jianyun Ruan

Int. J. Mol. Sci. 2018, 19(11), 3683; https://doi.org/10.3390/ijms19113683 - 21 Nov 2018

Cited by 32 | Viewed by 13313

Abstract

The qualities of tea (Camellia sinensis) are not clearly understood in terms of integrated leading molecular regulatory network mechanisms behind inorganic phosphate (Pi) limitation. Thus, the present work aims to elucidate transcription factor-dependent responses of quality-related metabolites and the expression of [...] Read more.

The qualities of tea (Camellia sinensis) are not clearly understood in terms of integrated leading molecular regulatory network mechanisms behind inorganic phosphate (Pi) limitation. Thus, the present work aims to elucidate transcription factor-dependent responses of quality-related metabolites and the expression of genes to phosphate (P) starvation. The tea plant organs were subjected to metabolomics analysis by GC×GC-TOF/MS and UPLC-Q-TOF/MS along with transcription factors and 13 metabolic genes by qRT-PCR. We found P starvation upregulated SPX2 and the change response of Pi is highly dependent on young shoots. This led to increased change in abundance of carbohydrates (fructose and glucose), amino acids in leaves (threonine and methionine), and root (phenylalanine, alanine, tryptophan, and tyrosine). Flavonoids and their glycosides accumulated in leaves and root exposed to P limitation was consistent with the upregulated expression of anthocyanidin reductase (EC 1.3.1.77), leucoanthocyanidin dioxygenase (EC 1.4.11.19) and glycosyltransferases (UGT78D1, UGT78D2 and UGT57L12). Despite the similar kinetics and high correlation response of Pi and SPX2 in young shoots, predominating theanine and other amino acids (serine, threonine, glutamate, valine, methionine, phenylalanine) and catechin (EGC, EGCG and CG) content displayed opposite changes in response to Pi limitation between Fengqing and Longjing-43 tea cultivars. Full article

(This article belongs to the Special Issue Plant Metabolism in Crops: A Systems Biology Perspective)

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Full article ">Figure 1
Fold change (log2[−P/+P]) of primary metabolites and P4Ha in response to P starvation. Fengqing (left column) and Longjing-43 (right column) and from the top to the bottom rows are young shoots (first row), leaves (middle row) and root (button row). YS, L and R represents young shoots, leaves, and root, respectively. Metabolites and metabolic genes inside background color box represents different biosynthesis pathway. The solid arrow shows direct and dotted arrow represents speculated steps in the pathway. The data depicted from <a href="#app1-ijms-19-03683" class="html-app">Table S1</a> and Table 5, positive as increase and negative as decrease in fold change. On the false color scale red indicates increase; blue and green indicates decrease in metabolites and gene. aP4H means prolyl 4-hydroxylase. Full article ">Figure 2
Fold change (log2[−P/+P]) of secondary metabolites and metabolic genes in response to P starvation. Fengqing (left column) and Longjing-43 (right column) and from the top to the bottom rows are young shoots (first row), leaves (middle row) and root (button row). YS, L and R represents young shoots, leaves, and root, respectively. Metabolites and metabolic genes inside background color box represents different biosynthesis pathway. The solid arrow shows direct and dotted arrow represents speculated steps in the pathway. The data depicted from <a href="#app1-ijms-19-03683" class="html-app">Table S2</a> and Table 5, positive as increase and negative as decrease in fold change. On the false color scale red indicates increase; blue and green indicates decrease in metabolites and gene. ANR, anthocyanin reductase; LDOX, leucoanthocyanidin dioxygenase; UGT57L12, flavanol 7-O glycosyltransferase; UGT78D2, flavanol 3-O-glucosyltransferase 2; UGT78D1, flavanol 3-O-glycoside L-rhamnosyl transferase 1. Full article ">Figure 3
Correlations between fold change (log2[−P/+P]) of selected significantly changed (<math display="inline"><semantics> <mrow> <mi>p</mi> <mo><</mo> </mrow> </semantics></math> 0.05, t-test) metabolites in young shoots of Fengqing and Longjing-43. Red and blue colors indicate positive and negative coefficients and line thickness indicate correlation strength. Full article ">Figure 4
Linear correlations between the fold changes (log2[−P/+P]) of PHO1, PHR1 and SPX2. The expression of Pi concentration in young shoots (A,D), leaves (B,E) and root (C,F) of cultivars Fengqing (A–C) and Longjing-43 (D–F). Full article ">Figure 5
Heat map of correlations between fold change (log2[−P/+P]) of selected significantly changed (<math display="inline"><semantics> <mrow> <mi>p</mi> <mo><</mo> </mrow> </semantics></math> 0.05, t-test) metabolites and metabolic genes in young shoots. Red and blue colors indicate positive and negative coefficients and their scales indicate values of Fengqing (A) and Longjing-43 (B). Full article ">

9 pages, 1531 KiB

Open AccessCommunication

A Cysteine-Reactive Small Photo-Crosslinker Possessing Caged-Fluorescence Properties: Binding-Site Determination of a Combinatorially-Selected Peptide by Fluorescence Imaging/Tandem Mass Spectrometry

by Kazuki Yatabe, Masaru Hisada, Yudai Tabuchi and Masumi Taki

Int. J. Mol. Sci. 2018, 19(11), 3682; https://doi.org/10.3390/ijms19113682 - 21 Nov 2018

Cited by 1 | Viewed by 4450

Abstract

To determine the binding-site of a combinatorially-selected peptide possessing a fluoroprobe, a novel cysteine reactive small photo-crosslinker that can be excited by a conventional long-wavelength ultraviolet handlamp (365 nm) was synthesized via Suzuki coupling with three steps. The crosslinker is rationally designed, not [...] Read more.

To determine the binding-site of a combinatorially-selected peptide possessing a fluoroprobe, a novel cysteine reactive small photo-crosslinker that can be excited by a conventional long-wavelength ultraviolet handlamp (365 nm) was synthesized via Suzuki coupling with three steps. The crosslinker is rationally designed, not only as a bioisostere of the fluoroprobe, but as a caged-fluorophore, and the photo-crosslinked target protein became fluorescent with a large Stokes-shift. By introducing the crosslinker to a designated sulfhydryl (SH) group of a combinatorially-selected peptide, the protein-binding site of the targeted peptide was deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/fluorescence imaging followed by matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry (MALDI-TOF-MS/MS) analysis. Full article

(This article belongs to the Section Physical Chemistry and Chemical Physics)

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Determining the binding-site of a combinatorially-selected peptide using a rationally designed photo-crosslinker, which is a bioisostere of the solvatochromic fluoroprobe present in the parent peptide. Irradiation with UV light simultaneously crosslinks the fluorophore to the protein binding site and uncages the fluorescence property by forming an intramolecular charge transfer (ICT) structure. This facilitates the rapid deduction of the binding site of the peptide using SDS-PAGE with fluorescence imaging followed by tandem MS analysis. The dotted line stands for the fluorescent color of each probe. Full article ">Figure 2
Overall scheme for the synthesis of the cysteine-reactive small photo-crosslinker 3. Reagents and conditions: (i) K3PO4, XPhos Pd G2, 1,4-dioxane/H2O, reflux, 2 hours; 1.7 M HCl (dichloromethane/ethyl acetate), room temperature, 24 hours, 77%; (ii) NaNO2, NaN3, 3.4 M aqueous HCl, 0 °C, 3 hours, 84%; (iii) N-Bromosuccinimide (NBS), TsOH monohydrate, MeCN, 35 °C, 24 hours, 33%. Full article ">Figure 3
Specific conjugation between glutathione-S-transferase (GST) and the caged binder, confirmed by 15% SDS-PAGE/fluorescence imaging. GST (blue arrow) was visualized by CBB staining (left panel), and the binder-conjugated GST was visualized by fluorescence in the same gel (right panel). For the fluorescence imaging, the excitation wavelength was 405 nm, and a band-pass filter (605 nm) was used for the detection. Plus (+) and minus (-) stand for presence and absence of the suggested molecules (i.e., GST or the binder), respectively. Full article ">Figure 4
(A) Identification of trypsinized peptide fragments derived from the covalent-binder-conjugated GST by MALDI-TOF-MS analysis (lower panel). As a negative control, pristine GST was also trypsinized, and the resulting fragments were also analyzed under the same conditions (upper panel). A newly appeared fragment in the presence of the covalent binder is highlighted by a blue arrow. (B) MS/MS spectra of the newly appeared fragment. All the detected fragments were consistent with theoretical m/z values of the represented structure; b- and y-ions are highlighted by using blue and red colors, respectively. The peptide fragment of LTQSMAIIR was derived from a constituent of the glutathione binding pocket of GST protein; judging from the peak intensity of the remaining non-crosslinked fragment (lower panel in A), the crosslinking reaction yield was estimated to be a few percent. M* and C* mean conjugated methionine and cysteine, respectively; the conjugation is highlighted by a thick red line. Full article ">Figure 5
Molecular docking simulation of the caged binder (shown as a stick; C, N, O and S atoms are highlighted by cyan, blue, red and yellow colors, respectively) to GST (PDB ID: 1UA5) using the sievgene of myPresto; the best docking model with a lowest binding energy of −12.0 kcal/mol was presented. The azido group (i.e., N3) in the caged fluorophore and conjugated methionine in GST were colored in blue (double-lined) and red, respectively. GST was shown as a cartoon with side chains as a line description. Including this lowest model, 23 independent models out of the 30 separate poses resulted that the azido group was also closely located to the methionine. Full article ">

20 pages, 2275 KiB

Open AccessReview

Biological Roles of Ornithine Aminotransferase (OAT) in Plant Stress Tolerance: Present Progress and Future Perspectives

by Alia Anwar, Maoyun She, Ke Wang, Bisma Riaz and Xingguo Ye

Int. J. Mol. Sci. 2018, 19(11), 3681; https://doi.org/10.3390/ijms19113681 - 21 Nov 2018

Cited by 51 | Viewed by 7283

Abstract

Plant tolerance to biotic and abiotic stresses is complicated by interactions between different stresses. Maintaining crop yield under abiotic stresses is the most daunting challenge for breeding resilient crop varieties. In response to environmental stresses, plants produce several metabolites, such as proline (Pro), [...] Read more.

Plant tolerance to biotic and abiotic stresses is complicated by interactions between different stresses. Maintaining crop yield under abiotic stresses is the most daunting challenge for breeding resilient crop varieties. In response to environmental stresses, plants produce several metabolites, such as proline (Pro), polyamines (PAs), asparagine, serine, carbohydrates including glucose and fructose, and pools of antioxidant reactive oxygen species. Among these metabolites, Pro has long been known to accumulate in cells and to be closely related to drought, salt, and pathogen resistance. Pyrroline-5-carboxylate (P5C) is a common intermediate of Pro synthesis and metabolism that is produced by ornithine aminotransferase (OAT), an enzyme that functions in an alternative Pro metabolic pathway in the mitochondria under stress conditions. OAT is highly conserved and, to date, has been found in all prokaryotic and eukaryotic organisms. In addition, ornithine (Orn) and arginine (Arg) are both precursors of PAs, which confer plant resistance to drought and salt stresses. OAT is localized in the cytosol in prokaryotes and fungi, while OAT is localized in the mitochondria in higher plants. We have comprehensively reviewed the research on Orn, Arg, and Pro metabolism in plants, as all these compounds allow plants to tolerate different kinds of stresses. Full article

(This article belongs to the Special Issue Mechanisms of Drought Stress Tolerance in Plants)

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Conservation of the ornithine aminotransferase (OAT) enzyme among prokaryotes and eukaryotes: (A) Maximum likelihood phylogenetic tree showing the conservation of OAT enzymes from prokaryotes to higher plants. The tree was constructed using MEGA 6 software with the bootstrap method. Accession numbers of the species used in the study are as follows: Bacillus subtilis (NP-391914.1), Streptomyces avermitilis (Q82HT8), Bacillus velezensis (ABS76054.1), Mycobacterium avium (AAS04411.1), Aspergillus nidulans (Q92413), Saccharomyces cerevisiae (P07991), Neurospora crassa (Q7RX93), Aspergillus lacticoffeatus (XP_025460070), Arabidopsis thaliana (OAO92185), Brassica napus (NP_001303219.1), Glycine max (XP_003531161.1), and Brachypodium distachyon (KQK13994.1). (B) Differences in the Glu pathway of Pro synthesis among prokaryotic and eukaryotic organisms. γ-GK: γ-glutamyl-kinase; γ-Glu: γ-glutamyl-phosphate; GSADH: glutamic-γ-semi-aldehyde dehydrogenase. Full article ">Figure 2
Proline, ornithine, and arginine metabolism and transport in plants. An illustration of the components of the proline (Pro) and arginine (Arg) metabolic pathways that have been identified to date. Data were taken from previously published papers [<a href="#B53-ijms-19-03681" class="html-bibr">53</a>,<a href="#B54-ijms-19-03681" class="html-bibr">54</a>,<a href="#B55-ijms-19-03681" class="html-bibr">55</a>,<a href="#B56-ijms-19-03681" class="html-bibr">56</a>,<a href="#B57-ijms-19-03681" class="html-bibr">57</a>,<a href="#B58-ijms-19-03681" class="html-bibr">58</a>]. Most data were obtained from the model plant A. thaliana, but it is hypothesized that this pathway is the same in related plant species. The Pro metabolic pathway is depicted by black lines, the blue lines show the Arg pathway, and the green line shows the ornithine (Orn) pathway, which is further described in <a href="#ijms-19-03681-f003" class="html-fig">Figure 3</a>. Solid lines show cellular pathways while the dotted lines show the intracellular transport of metabolic products. Enzyme transporter proteins are depicted in blue. Glu: glutamate; Arg: arginine; Orn: ornithine; Gln: glutamine; ARG: arginase; Cit: citrulline; OTC: ornithine transcarbamylase; AS: arginosuccinate synthetase; AL: arginosuccinate lyase; ProDH: Pro dehydrogenase; Spe: spermidine; BAC: basic amino acid transporter involved in Arg and Orn exchange; ?: predicted transporters. Full article ">Figure 3
Linear and cyclic ornithine synthesis pathways linked to the arginine metabolic pathway in plants. Arg biosynthesis is divided into two parts and nine discrete steps. In the first part of the pathway, Orn is synthesized from glutamate (Glu), and in the second part, Arg is synthesized from Orn. The first four steps are distinct from the Orn pathway, while the fifth and sixth steps, known as the cyclic and linear pathways, respectively, are also included in the Orn pathway but take different routes. The last three steps (second part) are known as the Arg pathway, which is illustrated in <a href="#ijms-19-03681-f002" class="html-fig">Figure 2</a>. NGS2: N-acetylglutamine synthase; NAGK: N-acetyl glutamate kinase; NAGPR: N-acetylglutamate-5-phosphate reductase; NAOAT: N-acetylornithine aminotransferase; NAOGAcT: N-acetylornithine-glutamate acetyltransferase; NAGK/PII (a plastid localized protein) double-headed arrow: regulatory interaction between the NAGK and PII proteins; NAOD: N-acetylornithine deacetylase. Full article ">Figure 4
Model for the activation of stress-related genes under different stress environments in plants. There are two groups of stresses: (A) abiotic and (B) biotic. This diagram shows the up-regulation and down-regulation of the OAT, pyrroline-5-carboxylate dehydrogenase (P5CDH), pyrroline-5-carboxylate synthase (P5CS), pyrroline-5-carboxylate reductase (P5CR), and Pro dehydrogenase (ProDH) enzymes under different stress conditions. Red indicates up-regulation, blue indicates down-regulation, and the thickness of each arrow shows the extent of up- or down-regulation. Yellow dots indicate Pro, which accumulates either in the cytosol or chloroplast depending on the nature of stress. Dotted red lines show activation of P5CR mediated Pro biosynthesis while dotted black lines show normal interacellular transportation of P5C/GSA. In A1 and A2, down-regulation of ProDH1 results in Pro accumulation during drought and salinity stress. During drought stress, all Pro biosynthesis enzymes are up-regulated and the catabolic pathway is down-regulated to favor Pro biosynthesis. During salinity stress, P5CDH is down-regulated and OAT is up-regulated to increase Pro biosynthesis. In A3 when exogenous Pro is supplied all stress-related genes are up-regulated. In B1 and B2, increased Pro catabolism due to up-regulation of ProDH causes the production of reactive oxygen species (ROS), thus activating defense mechanisms during avirulent and non-host pathogen resistance. In B3, induction of P5CDH expression by virulent pathogens prevents pyrroline-5-carboxylate (P5C) accumulation in mitochondria, and activation of the ProDH gene results in moderate levels of Pro accumulation, reducing cell death during infection. Additionally, OAT expression is increased during non-host pathogen resistance, causing increased production of ROS, which in turn activates the hypersensitive response and other defense responses. PCD: programmed cell death. Full article ">Figure 4 Cont.
Model for the activation of stress-related genes under different stress environments in plants. There are two groups of stresses: (A) abiotic and (B) biotic. This diagram shows the up-regulation and down-regulation of the OAT, pyrroline-5-carboxylate dehydrogenase (P5CDH), pyrroline-5-carboxylate synthase (P5CS), pyrroline-5-carboxylate reductase (P5CR), and Pro dehydrogenase (ProDH) enzymes under different stress conditions. Red indicates up-regulation, blue indicates down-regulation, and the thickness of each arrow shows the extent of up- or down-regulation. Yellow dots indicate Pro, which accumulates either in the cytosol or chloroplast depending on the nature of stress. Dotted red lines show activation of P5CR mediated Pro biosynthesis while dotted black lines show normal interacellular transportation of P5C/GSA. In A1 and A2, down-regulation of ProDH1 results in Pro accumulation during drought and salinity stress. During drought stress, all Pro biosynthesis enzymes are up-regulated and the catabolic pathway is down-regulated to favor Pro biosynthesis. During salinity stress, P5CDH is down-regulated and OAT is up-regulated to increase Pro biosynthesis. In A3 when exogenous Pro is supplied all stress-related genes are up-regulated. In B1 and B2, increased Pro catabolism due to up-regulation of ProDH causes the production of reactive oxygen species (ROS), thus activating defense mechanisms during avirulent and non-host pathogen resistance. In B3, induction of P5CDH expression by virulent pathogens prevents pyrroline-5-carboxylate (P5C) accumulation in mitochondria, and activation of the ProDH gene results in moderate levels of Pro accumulation, reducing cell death during infection. Additionally, OAT expression is increased during non-host pathogen resistance, causing increased production of ROS, which in turn activates the hypersensitive response and other defense responses. PCD: programmed cell death. Full article ">

10 pages, 3608 KiB

Open AccessArticle

Evolution and Expression Characteristics of Receptor-Like Cytoplasmic Protein Kinases in Maize, Rice and Arabidopsis

by Mingxia Fan, Wenjuan Ma, Chen Liu, Chunyu Zhang, Suwen Wu, Meiming Chen, Kuichen Liu, Fengchun Cai and Feng Lin

Int. J. Mol. Sci. 2018, 19(11), 3680; https://doi.org/10.3390/ijms19113680 - 21 Nov 2018

Cited by 11 | Viewed by 3768

Abstract

Receptor-like cytoplasmic protein kinases (RLCKs) are involved in various activities in plant growth and development. We have totally identified 162, 160, and 402 RLCK genes in maize, rice, and Arabidopsis genomes, respectively. Phylogenetic analyses divided 724 RLCK genes into 15 subfamilies and similar [...] Read more.

Receptor-like cytoplasmic protein kinases (RLCKs) are involved in various activities in plant growth and development. We have totally identified 162, 160, and 402 RLCK genes in maize, rice, and Arabidopsis genomes, respectively. Phylogenetic analyses divided 724 RLCK genes into 15 subfamilies and similar structural patterns of kinase activity sites and functional sites were observed within the subfamilies. Furthermore, the structural patterns of intron/exon in the same subfamilies were similar, implicating their close evolutionary relationship. Chromosome distribution indicated that segmental duplication of RLCK genes might be a major mechanism contributing to the expansion of the RLCK superfamilies in maize, rice, and Arabidopsis, respectively. The analysis of the synteny relationship and gene structure indicated that the evolution of most RLCKs in maize were prior to rice and Arabidopsis. Most of the ratio of Ka/Ks is inferior to one, suggesting that RLCK genes have experienced the negative selection in maize, rice and Arabidopsis. Duplication time revealed that the maize was the earliest emergence among these three species. The expression profiles showed that there are some specifically expressed RLCK genes in maize root, leaf, ear, and tassel. These specific expression genes may participate in the developmental regulation of these maize tissues. Our results will be useful in providing new insights into evolution of RLCKs and revealing the regulatory network of maize, rice, and Arabidopsis development. Full article

(This article belongs to the Section Molecular Plant Sciences)

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Figure 1

Figure 1
The phylogram and conserved kinase domain analysis of receptor-like cytoplasmic protein kinases (RLCKs) in maize, rice, and Arabidopsis. (a) The RLCK phylogram of maize, rice, and Arabidopsis. The pinkish purple, yellow, and blue were used to show the RLCK genes from maize, rice, and Arabidopsis, respectively. (b) The conserved kinase domain of RLCKs in each group. The pink letters were the predicted kinase activity sites of RLCKs. (c) The RLCK conservative motifs in each group. Full article ">Figure 2
Circos map of RLCK genes in maize, rice and Arabidopsis. The outer two circles were proportional chromosomes for maize, rice, and Arabidopsis in megabase (Mb) units. The third circle represented each RLCK gene distribution on chromosomes. Lines below each chromosome represent the intron numbers of RLCK genes (orange > 5 introns, black = 5 introns, blue < 5 introns). Innermost colored lines represent synteny gene pairs among the genomes of maize, rice, and Arabidopsis. Full article ">Figure 3
The distribution and frequency of Ka/Ks values. The purple, blue, and orange colors represent the maize, rice, and Arabidopsis, respectively. (a), (b) and (c) show the Ka/Ks distribution. The line in black is the boundary of Ka/Ks = 1. (d), (e), and (f) represent the frequency distribution of Ka/Ks < 1. Full article ">Figure 4
RLCK gene distribution on chromosomes in maize (a), rice (b), and Arabidopsis (c). The lines in purple represent gene clusters. Full article ">Figure 5
Maize RLCK gene hierarchial clustering. On the left, the color scale indicates the log2 values and the hierarchial clustering displays four expression patterns marked by different colors. The different development periods of leaf, root, ear, and tassel in maize are showed on the bottom. Full article ">

12 pages, 2576 KiB

Open AccessArticle

Effect of Pretreatment with the NADPH Oxidase Inhibitor Apocynin on the Therapeutic Efficacy of Human Placenta-Derived Mesenchymal Stem Cells in Intracerebral Hemorrhage

by Saehong Min, Ok Joon Kim, Jinkun Bae and Tae Nyoung Chung

Int. J. Mol. Sci. 2018, 19(11), 3679; https://doi.org/10.3390/ijms19113679 - 21 Nov 2018

Cited by 18 | Viewed by 4707

Abstract

Several studies have demonstrated the beneficial effect of mesenchymal stem cells (MSCs) on intracerebral hemorrhage (ICH). Enhancement of the therapeutic efficacy of MSCs in ICH is necessary, considering the diseases high association with mortality and morbidity. Various preconditioning methods to enhance the beneficial [...] Read more.

Several studies have demonstrated the beneficial effect of mesenchymal stem cells (MSCs) on intracerebral hemorrhage (ICH). Enhancement of the therapeutic efficacy of MSCs in ICH is necessary, considering the diseases high association with mortality and morbidity. Various preconditioning methods to enhance the beneficial properties of MSCs have been introduced. We suggested apocynin, a well-known nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, as a novel preconditioning regimen to enhance the therapeutic efficacy of MSCs in ICH. Rat ICH models were made using bacterial collagenase. 24 h after ICH induction, the rats were randomly divided into apocynin-preconditioned MSC-treated (Apo-MSC), naïve MSC-treated and control groups. Hematoma volume, brain edema, and degenerating neuron count were compared at 48 h after the ICH induction. The expression of tight junction proteins (occludin, zona occludens [ZO]-1) were also compared. Hematoma size, hemispheric enlargement and degenerating neuron count were significantly lower in the Apo-MSC group than in the naïve MSC group (p = 0.004, 0.013 and 0.043, respectively), while the expression of occludin was higher (p = 0.024). Apocynin treatment enhances the therapeutic efficacy of MSCs in ICH in the acute stage, through the improvement of the beneficial properties of MSCs, such as neuroprotection and the reinforcement of endovascular integrity of cerebral vasculature. Full article

(This article belongs to the Special Issue Role of NADPH Oxidase on Neuron Death or on Neurogenesis)

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Effect of apocynin-preconditioned human placenta-derived mesenchymal stem cells (Apo-MSCs) and naïve mesenchymal stem cells (MSCs) on hematoma volume and brain edema in the rats at 48 h after the induction of an intracranial hemorrhage (ICH). (a) Representative images of cresyl violet staining, depicting the coronal whole-brain section at rostral-caudal levels from +2.04 to −5.52 from the bregma. Unstained area inside brain parenchyma represents hematoma lesion. Scale bar = 1 mm. (b) The bar graphs represent the hematoma volume of the Apo-MSCs, naïve MSCs and vehicle treated groups at 48 h after ICH induction. The volume of hematoma is expressed as the proportion of total brain area (%). (c) The bar graphs represent hemispheric enlargement of the Apo-MSCs, naïve MSCs and vehicle treated groups at 48 h after ICH induction. The hemispheric enlargement is expressed as the percentage of increase in hemispheric size comparing with that of the contralateral hemisphere. Data are mean + standard deviation (SD). * p < 0.05, *** p < 0.001. Full article ">Figure 2
Effect of apocynin-preconditioned human placenta-derived mesenchymal stem cells (Apo-MSCs) and naïve MSCs on the peri-hematoma neuronal death in the rats at 48 h after the induction of an intracranial hemorrhage (ICH). (a) The location of core hemorrhagic regions at 0.2 mm from the bregma. Each number represents a region of interest to be analyzed. (b) Fluorescence images reveal the degenerating neurons in the peri-hematoma region at 24 h after the induction of an ICH. Degenerating neurons are detected by Fluoro-Jade C (FJC) staining (green). Each number represents a region of interest defined at <a href="#ijms-19-03679-f002" class="html-fig">Figure 2</a>a. Scale bar = 20 μm. (c) The bar graphs represent the count of FJC-positive neurons in the peri-hematoma region from the Apo-MSCs, naïve MSCs and vehicle treated groups at 48 h after ICH induction. Data are mean +SD. * p < 0.05, ** p < 0.01. Full article ">Figure 3
Effect of apocynin-preconditioned human placenta-derived mesenchymal stem cells (Apo-MSCs) and naïve MSCs on the expression of tight junction proteins in the rats at 48 h after the induction of an intracranial hemorrhage (ICH). (a) Results of western blotting of occludin and ZO-1 at 48 h after ICH induction. (b) Amount of exosome production. Bar graphs indicate the level of occludin (c) and ZO-1 (d) expression measured by the densitometric analysis of the bands. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. Data are mean +SD. * p < 0.05, ** p < 0.01, *** p < 0.001. Full article ">Figure 4
Conceptual illustrations of the experimental protocol. (a) Schematic diagrams of a rat intracranial hemorrhage (ICH) model. ICH was induced by the infusion of bacterial collagenase type IV (0.1 U, 1 μL) into the striatum. (b) Brief timeline of the experimental procedures. Full article ">

18 pages, 14201 KiB

Open AccessArticle

Anti-inflammatory and Neuroprotective Effects of Fungal Immunomodulatory Protein Involving Microglial Inhibition

by Wen-Ying Chen, Cheng-Yi Chang, Jian-Ri Li, Jiaan-Der Wang, Chih-Cheng Wu, Yu-Hsiang Kuan, Su-Lan Liao, Wen-Yi Wang and Chun-Jung Chen

Int. J. Mol. Sci. 2018, 19(11), 3678; https://doi.org/10.3390/ijms19113678 - 21 Nov 2018

Cited by 21 | Viewed by 4601

Abstract

Microglia polarization of classical activation state is crucial to the induction of neuroinflammation, and has been implicated in the pathogenesis of numerous neurodegenerative diseases. Fungal immunomodulatory proteins are emerging health-promoting natural substances with multiple pharmacological activities, including immunomodulation. Herein, we investigated the anti-inflammatory [...] Read more.

Microglia polarization of classical activation state is crucial to the induction of neuroinflammation, and has been implicated in the pathogenesis of numerous neurodegenerative diseases. Fungal immunomodulatory proteins are emerging health-promoting natural substances with multiple pharmacological activities, including immunomodulation. Herein, we investigated the anti-inflammatory and neuroprotective potential of fungal immunomodulatory protein extracted from Ganoderma microsporum (GMI) in an in vitro rodent model of primary cultures. Using primary neuron/glia cultures consisting of neurons, astrocytes, and microglia, a GMI showed an alleviating effect on lipopolysaccharide (LPS)/interferon-γ (IFN-γ)-induced inflammatory mediator production and neuronal cell death. The events of neuroprotection caused by GMI were accompanied by the suppression of Nitric Oxide (NO), Tumor Necrosis Factor-α (TNF-α), Interleukin-1β (IL-1β), and Prostaglandin E2 (PGE2) production, along with the inhibition of microglia activation. Mechanistic studies showed that the suppression of microglia pro-inflammatory polarization by GMI was accompanied by the resolution of oxidative stress, the preservation of protein tyrosine phosphatase and serine/threonine phosphatase activity, and the reduction of NF-κB, AP-1, cyclic AMP response element-binding protein (CREB), along with signal transducers and activators of transcription (Stat1) transcriptional activities and associated upstream activators. These findings suggest that GMI may have considerable potential towards the treatment of neuroinflammation-mediated neurodegenerative diseases. Full article

(This article belongs to the Special Issue Natural Anti-Inflammatory Agents 2018)

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16 pages, 599 KiB

Open AccessReview

Targeting Smoothened as a New Frontier in the Functional Recovery of Central Nervous System Demyelinating Pathologies

by Alice Del Giovane and Antonella Ragnini-Wilson

Int. J. Mol. Sci. 2018, 19(11), 3677; https://doi.org/10.3390/ijms19113677 - 20 Nov 2018

Cited by 13 | Viewed by 5179

Abstract

Myelin sheaths on vertebrate axons provide protection, vital support and increase the speed of neuronal signals. Myelin degeneration can be caused by viral, autoimmune or genetic diseases. Remyelination is a natural process that restores the myelin sheath and, consequently, neuronal function after a [...] Read more.

Myelin sheaths on vertebrate axons provide protection, vital support and increase the speed of neuronal signals. Myelin degeneration can be caused by viral, autoimmune or genetic diseases. Remyelination is a natural process that restores the myelin sheath and, consequently, neuronal function after a demyelination event, preventing neurodegeneration and thereby neuron functional loss. Pharmacological approaches to remyelination represent a promising new frontier in the therapy of human demyelination pathologies and might provide novel tools to improve adaptive myelination in aged individuals. Recent phenotypical screens have identified agonists of the atypical G protein-coupled receptor Smoothened and inhibitors of the glioma-associated oncogene 1 as being amongst the most potent stimulators of oligodendrocyte precursor cell (OPC) differentiation in vitro and remyelination in the central nervous system (CNS) of mice. Here, we discuss the current state-of-the-art of studies on the role of Sonic Hedgehog reactivation during remyelination, referring readers to other reviews for the role of Hedgehog signaling in cancer and stem cell maintenance. Full article

(This article belongs to the Special Issue Hedgehog Signaling)

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1 pages, 158 KiB

Open AccessCorrection

Correction: Serwotka-Suszczak, A. M. et al. A Conjugate Based on Anti-HER2 Diaffibody and Auristatin E Targets HER2-Positive Cancer Cells. Int. J. Mol. Sci. 2017, 18, 401

by Anna M. Serwotka-Suszczak, Alicja M. Sochaj-Gregorczyk, Jerzy Pieczykolan, Daniel Krowarsch, Filip Jelen and Jacek Otlewski

Int. J. Mol. Sci. 2018, 19(11), 3676; https://doi.org/10.3390/ijms19113676 - 20 Nov 2018

Viewed by 2542

Abstract

It has been brought to our attention that the affiliation of Dr. Jerzy Pieczykolan at the time when he was responsible for the work described in the paper [...] Full article

16 pages, 1436 KiB

Open AccessCase Report

Microduplication of 15q13.3 and Microdeletion of 18q21.32 in a Patient with Moyamoya Syndrome

by Francesca Luisa Sciacca, Ambra Rizzo, Gloria Bedini, Fioravante Capone, Vincenzo Di Lazzaro, Sara Nava, Francesco Acerbi, Davide Rossi Sebastiano, Simona Binelli, Giuseppe Faragò, Andrea Gioppo, Marina Grisoli, Maria Grazia Bruzzone, Paolo Ferroli, Chiara Pantaleoni, Luigi Caputi, Jesus Vela Gomez, Eugenio Agostino Parati and Anna Bersano

Int. J. Mol. Sci. 2018, 19(11), 3675; https://doi.org/10.3390/ijms19113675 - 20 Nov 2018

Cited by 5 | Viewed by 4618

Abstract

Moyamoya angiopathy (MA) is a cerebrovascular disease determining a progressive stenosis of the terminal part of the internal carotid arteries (ICAs) and their proximal branches and the compensatory development of abnormal “moyamoya” vessels. MA occurs as an isolated cerebral angiopathy (so-called moyamoya disease) [...] Read more.

Moyamoya angiopathy (MA) is a cerebrovascular disease determining a progressive stenosis of the terminal part of the internal carotid arteries (ICAs) and their proximal branches and the compensatory development of abnormal “moyamoya” vessels. MA occurs as an isolated cerebral angiopathy (so-called moyamoya disease) or in association with various conditions (moyamoya syndromes) including several heritable conditions such as Down syndrome, neurofibromatosis type 1 and other genomic defects. Although the mechanism that links MA to these genetic syndromes is still unclear, it is believed that the involved genes may contribute to the disease susceptibility. Herein, we describe the case of a 43 years old woman with bilateral MA and peculiar facial characteristics, having a 484-kb microduplication of the chromosomal region 15q13.3 and a previously unreported 786 kb microdeletion in 18q21.32. This patient may have a newly-recognized genetic syndrome associated with MA. Although the relationship between these genetic variants and MA is unclear, our report would contribute to widening the genetic scenario of MA, in which not only genic mutation, but also genome unbalances are possible candidate susceptibility factors. Full article

(This article belongs to the Section Molecular Neurobiology)

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Figure 1

Figure 1
MRI and angiographic images of MA of our case. Axial T2-weighted MRI (A,B) exhibits only a few vascular voids in the Sylvian fissures (white circles), due to a reduced flow in both the middle cerebral arteries. A well-demarcated ischemic stroke in the left nucleo-capsular region (white arrowhead) and an infarct involving the left frontal lobe (white asterisk) are also noticeable. Axial angio-CT (C) shows proliferation and enlargement of the lenticulostriate arteries (white arrows) in the basal ganglia. Right and left common carotid arteries’ angiograms (D–E) demonstrate a distal stenosis of the right internal carotid artery (black asterisk), with development of prominent collateral vessels, giving the characteristic puff of smoke appearance of moyamoya disease, and an occlusion of the left internal carotid artery, distally to the ophthalmic artery (black arrowhead). Left vertebral artery angiogram (F) indicates leptomeningeal cortical anastomosis from both the posterior cerebral arteries to the parietal and temporal lobes (black arrows). Full article ">Figure 2
Patient’s array comparative genomic hybridization (aCGH) data result. The patient carried: (panel A) a 484 Kb microduplication in the chromosomal region 15q13.3 (the blue bar in the chromosome track), from 32065077 to 32539670 chromosome coordinate, zoomed in the probes track as indicated by purple lines, and (panel B) a 786 Kb microdeletion in the 18q21.32 region (the blue bar in the chromosome), from 58014483 to 58816361 chromosome coordinate, zoomed in the probes track as indicated by purple lines. Duplication and deletion are revealed by a disclosure of at least 3 probes from the +0.4 (duplication)/−0.6 (deletion range) of Log2ratio value in X axis (normal values are included in the range from +0.30, green line, to −0.30, red line; probed duplicated and deleted in the patient are yellow in the figure, while green dots are balanced probes. In the track “unbalance”, the patient’s duplication in panel A and the patient’s deletion in panel B are indicated with a green and red bars respectively. Genes are represented as blue strips in the genes truck, and genes associated with pathology are colored in green. Full article ">Figure 3
Proband and proband’s mother’s photos. Front and profile photos of the proband (upper panels) and her mother (lower panels). The proband shows peculiar characteristics such as a long narrowing face with bifrontal prominence, low frontal hairline, arched eyebrows, downslanting palpebral fissures, hypertelorism, broad nasal tip, upturned nares, and large and low-set ears. The mother is similar, but not having low frontal hairline and hypertelorism. Full article ">

23 pages, 835 KiB

Open AccessReview

YAP/TAZ Signaling as a Molecular Link between Fibrosis and Cancer

by Satoshi Noguchi, Akira Saito and Takahide Nagase

Int. J. Mol. Sci. 2018, 19(11), 3674; https://doi.org/10.3390/ijms19113674 - 20 Nov 2018

Cited by 181 | Viewed by 14474

Abstract

Tissue fibrosis is a pathological condition that is associated with impaired epithelial repair and excessive deposition of extracellular matrix (ECM). Fibrotic lesions increase the risk of cancer in various tissues, but the mechanism linking fibrosis and cancer is unclear. Yes-associated protein (YAP) and [...] Read more.

Tissue fibrosis is a pathological condition that is associated with impaired epithelial repair and excessive deposition of extracellular matrix (ECM). Fibrotic lesions increase the risk of cancer in various tissues, but the mechanism linking fibrosis and cancer is unclear. Yes-associated protein (YAP) and the transcriptional coactivator with PDZ-binding motif (TAZ) are core components of the Hippo pathway, which have multiple biological functions in the development, homeostasis, and regeneration of tissues and organs. YAP/TAZ act as sensors of the structural and mechanical features of the cell microenvironment. Recent studies have shown aberrant YAP/TAZ activation in both fibrosis and cancer in animal models and human tissues. In fibroblasts, ECM stiffness mechanoactivates YAP/TAZ, which promote the production of profibrotic mediators and ECM proteins. This results in tissue stiffness, thus establishing a feed-forward loop of fibroblast activation and tissue fibrosis. In contrast, in epithelial cells, YAP/TAZ are activated by the disruption of cell polarity and increased ECM stiffness in fibrotic tissues, which promotes the proliferation and survival of epithelial cells. YAP/TAZ are also involved in the epithelial–mesenchymal transition (EMT), which contributes to tumor progression and cancer stemness. Importantly, the crosstalk with transforming growth factor (TGF)-β signaling and Wnt signaling is essential for the profibrotic and tumorigenic roles of YAP/TAZ. In this article, we review the latest advances in the pathobiological roles of YAP/TAZ signaling and their function as a molecular link between fibrosis and cancer. Full article

(This article belongs to the Special Issue Links between Fibrogenesis and Cancer: Mechanistic and Therapeutic Challenges)

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13 pages, 2628 KiB

Open AccessArticle

Sedoheptulose-1,7-Bisphosphatase is Involved in Methyl Jasmonate- and Dark-Induced Leaf Senescence in Tomato Plants

by Fei Ding, Meiling Wang and Shuoxin Zhang

Int. J. Mol. Sci. 2018, 19(11), 3673; https://doi.org/10.3390/ijms19113673 - 20 Nov 2018

Cited by 33 | Viewed by 4536

Abstract

Leaf senescence represents the final stage of leaf development and is regulated by diverse internal and environmental factors. Jasmonates (JAs) have been demonstrated to induce leaf senescence in several species; however, the mechanisms of JA-induced leaf senescence remain largely unknown in tomato plants [...] Read more.

Leaf senescence represents the final stage of leaf development and is regulated by diverse internal and environmental factors. Jasmonates (JAs) have been demonstrated to induce leaf senescence in several species; however, the mechanisms of JA-induced leaf senescence remain largely unknown in tomato plants (Solanum lycopersicum). In the present study, we tested the hypothesis that sedoheptulose-1,7-bisphosphatase (SBPase), an enzyme functioning in the photosynthetic carbon fixation in the Calvin–Benson cycle, was involved in methyl jasmonate (MeJA)- and dark-induced leaf senescence in tomato plants. We found that MeJA and dark induced senescence in detached tomato leaves and concomitantly downregulated the expression of SlSBPASE and reduced SBPase activity. Furthermore, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9)-mediated mutagenesis of SlSBPASE led to senescence-associated characteristics in slsbpase mutant plants, including loss of chlorophyll, repressed photosynthesis, increased membrane ion leakage, and enhanced transcript abundance of senescence-associated genes. Collectively, our data suggest that repression of SBPase by MeJA and dark treatment plays a role in JA- and dark-induced leaf senescence. Full article

(This article belongs to the Section Molecular Plant Sciences)

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Figure 1

Figure 1
Methyl jasmonate (MeJA) induced tomato leaf senescence. (A) Phenotypes of detached tomato leaves treated with MeJA, bar = 1 cm. (B) Relative chlorophyll contents of tomato leaves treated with or without MeJA. The chlorophyll content in the leaves without MeJA treatment was set to 100%, and the relative chlorophyll content in the leaves treated with MeJA was calculated accordingly. The values presented are means ± SDs (n = 3). Asterisks indicate significant difference at ** p < 0.01 between leaves treated with MeJA and control leaves. Full article ">Figure 2
MeJA downregulated SlSBPASE expression, reduced sedoheptulose-1,7-bisphosphatase (SBPase) activity, and decreased carbon assimilation rates in detached tomato leaves treated with MeJA for 4 d. (A) Analysis of SlSBPASE expression level. The expression level in the leaves without MeJA treatment for day 1 was set to 1, and the relative expression levels in the rest of samples were calculated accordingly. (B) Quantification of SBPase activity. (C) CO2 assimilation rates. The values presented are means ± SDs (n = 3). Asterisks indicate significant difference at ** p < 0.01 between leaves treated with MeJA and control leaves. Full article ">Figure 3
MeJA upregulated the expression of SAG (Solyc02g076910.2) and SEN (Solyc12g008460.1). (A) Transcript abundance of SAG in detached tomato leaves treated with or without MeJA for 4 d. (B) Transcript abundance of SEN in detached tomato leaves treated with or without MeJA for 4 d. The expression level in the leaves without MeJA treatment was set to 1, and the relative expression level in the leaves with MeJA treatment was calculated accordingly. The values presented are means ± SDs (n = 3). Asterisks indicate significant difference at ** p < 0.01 between leaves treated with MeJA and control leaves. Full article ">Figure 4
Downregulation of SlSBPASE was involved in dark-induced senescence. (A) Relative chlorophyll contents of tomato leaves treated with or without dark. The chlorophyll content in the leaves without dark treatment was set to 100%, and the relative chlorophyll content in the leaves treated with dark was calculated accordingly. (B) Transcript abundance of SlSBPASE in tomato leaves with or without dark treatment. The expression level in the leaves without dark treatment was set to 1, and the relative expression level in the leaves with dark treatment was calculated accordingly. (C) SBPase activity in tomato leaves with or without dark treatment. The values presented are means ± SDs (n = 3). Asterisks indicate significant difference at ** p < 0.01 between leaves treated with dark and control leaves. Full article ">Figure 5
Mutation of SlSBPASE led to senescence-associated characteristics in slsbpase mutant plants. (A) Phenotypes of slsbpase mutant plants and their wild type counterparts. (B) Relative chlorophyll contents of mutant and wild type leaves. The chlorophyll content in the wild type was set to 100%, and the relative chlorophyll content in the mutant was calculated accordingly. (C) CO2 assimilation rates in the mutant and wild type plants. (D) Membrane ion leakage in the leaves of mutant and wild type plants. The ion leakage in the mutant was set to 100%, and the ion leakage in the wild type was calculated accordingly. The values presented are means ± SDs (n = 3). Asterisks indicate significant difference at ** p < 0.01 between slsbpase mutant plants and their wild type counterparts. Full article ">Figure 6
Mutation of SlSBPASE upregulated the expression of SAG (SENESCENCE-ASSOCIATED GENE, Solyc02g076910.2) and SEN (SENESCENCE, Solyc12g008460.1), and downregulated the expression of CAB1 (chlorophyll a/b-binding protein 1, Solyc02g070970.1) and RBCS (Rubisco small subunit, Solyc07g017950.2) in slsbpase mutant plants. Transcript abundance of SAG (A), SEN (B), CAB1 (C), and RBCS (D) in the leaves of slsbpase mutant and wild type plants. The expression levels in the wild type leaves were set to 1, and the relative expression levels in the mutant leaves were calculated accordingly. The values presented are means ± SDs (n = 3). Asterisks indicate significant difference at ** p < 0.01 between slsbpase mutant plants and their wild type counterparts. Full article ">

30 pages, 1810 KiB

Open AccessReview

Epithelial-Mesenchymal Transition and Metastasis under the Control of Transforming Growth Factor β

by Yutaro Tsubakihara and Aristidis Moustakas

Int. J. Mol. Sci. 2018, 19(11), 3672; https://doi.org/10.3390/ijms19113672 - 20 Nov 2018

Cited by 124 | Viewed by 10216

Abstract

Metastasis of tumor cells from primary sites of malignancy to neighboring stromal tissue or distant localities entails in several instances, but not in every case, the epithelial-mesenchymal transition (EMT). EMT weakens the strong adhesion forces between differentiated epithelial cells so that carcinoma cells [...] Read more.

Metastasis of tumor cells from primary sites of malignancy to neighboring stromal tissue or distant localities entails in several instances, but not in every case, the epithelial-mesenchymal transition (EMT). EMT weakens the strong adhesion forces between differentiated epithelial cells so that carcinoma cells can achieve solitary or collective motility, which makes the EMT an intuitive mechanism for the initiation of tumor metastasis. EMT initiates after primary oncogenic events lead to secondary secretion of cytokines. The interaction between tumor-secreted cytokines and oncogenic stimuli facilitates EMT progression. A classic case of this mechanism is the cooperation between oncogenic Ras and the transforming growth factor β (TGFβ). The power of TGFβ to mediate EMT during metastasis depends on versatile signaling crosstalk and on the regulation of successive waves of expression of many other cytokines and the progressive remodeling of the extracellular matrix that facilitates motility through basement membranes. Since metastasis involves many organs in the body, whereas EMT affects carcinoma cell differentiation locally, it has frequently been debated whether EMT truly contributes to metastasis. Despite controversies, studies of circulating tumor cells, studies of acquired chemoresistance by metastatic cells, and several (but not all) metastatic animal models, support a link between EMT and metastasis, with TGFβ, often being a common denominator in this link. This article aims at discussing mechanistic cases where TGFβ signaling and EMT facilitate tumor cell dissemination. Full article

(This article belongs to the Special Issue Epithelial-Mesenchymal Transition (EMT))

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Full article ">Figure 1
The trajectory of differentiation change is pictorially graphed with epithelial cells on the left, hybrid E/M cells in the middle, and mesenchymal cells on the right hand of the arrow. The EMT and MET are also depicted as gradients of molecular and phenotypic change at the top of the figure and inside the main trajectory. Specific molecular and cellular attributes of epithelial and mesenchymal cells are listed on top of the relevant cell types. Important cell surface antigens are also drawn on the plasma membrane of each cell in a different color, in order to mark the molecular progression from an epithelial to a mesenchymal phenotype and the intermediate stages. At the bottom, the photomicrograph shows a mixed population of epithelial (red) and mesenchymal (dark grey) Py2T mouse breast cancer cells that have undergone EMT followed by MET. An additional cell model depicts a possible hybrid E/M cell that expresses EpCAM, CD61, and N-cadherin (N-Cad) as revealed in certain studies of circulating tumor cells. The features of EMT that are relevant to cancer are listed on the right. Full article ">Figure 2
Basic TGFβ signaling diagram along with a program of TGFβ-regulated genes that contribute to the EMT. Left side, the extracellular dimeric TGFβ ligand is shown to bind to its plasma membrane receptors, the type II and type I receptors (each drawn as a dimer), causing trans-phosphorylation (circled P) of the type I receptor by the type II receptor. The type II receptor phosphorylates the polarity protein Par6, which recruits the ubiquitin ligase Smurf1 and regulates RhoA-dependent actin assembly and tight junction disassembly. The type I receptor also recruits the ubiquitin ligases TRAF4 and TRAF6, which activate the MAP-kinase pathway by ubiquitination, leading to the transcription factor phosphorylation. The type I receptor kinase also phosphorylates R-Smads, which form complexes with the Co-Smad, Smad4. In the nucleus, Smad complexes and cooperating transcription factors bound to various genes, along with co-repressors (co-activators) either repress the expression of epithelial genes or induce the expression of mesenchymal genes. Right side, a summary of the TGFβ-regulated EMT program divided into seven subprograms, each enlisting only a small representative example of genes that are involved in the EMT and cell motility. Signaling flow is indicated by black arrows (positive flow) and red T-bars (negative regulation); on the gene transcriptional start site, a red T-bar indicates negative regulation and a blue arrow indicates positive regulation of transcription. Full article ">Figure 3
The non-coding RNAs regulate the EMT. Epithelial (left) and mesenchymal (right) genes are listed, the latter being transcriptionally induced by TGFβ signaling (thick arrow). Thin T-bars indicate the negative regulation of EMT-TFs by miRNAs and inversely, the negative regulation of miRNA expression by the EMT-TFs or lncRNAs. Negative transcriptional regulation is shown with blue T-bars, whereas negative regulation of mRNA translation and stability by miRNAs is shown with red T-bars. Full article ">

24 pages, 322 KiB

Open AccessReview

It’s Hard to Avoid Avoidance: Uncoupling the Evolutionary Connection between Plant Growth, Productivity and Stress “Tolerance”

by Albino Maggio, Ray A. Bressan, Yang Zhao, Junghoon Park and Dae-Jin Yun

Int. J. Mol. Sci. 2018, 19(11), 3671; https://doi.org/10.3390/ijms19113671 - 20 Nov 2018

Cited by 22 | Viewed by 4670

Abstract

In the last 100 years, agricultural developments have favoured selection for highly productive crops, a fact that has been commonly associated with loss of key traits for environmental stress tolerance. We argue here that this is not exactly the case. We reason that [...] Read more.

In the last 100 years, agricultural developments have favoured selection for highly productive crops, a fact that has been commonly associated with loss of key traits for environmental stress tolerance. We argue here that this is not exactly the case. We reason that high yield under near optimal environments came along with hypersensitization of plant stress perception and consequently early activation of stress avoidance mechanisms, such as slow growth, which were originally needed for survival over long evolutionary time periods. Therefore, mechanisms employed by plants to cope with a stressful environment during evolution were overwhelmingly geared to avoid detrimental effects so as to ensure survival and that plant stress “tolerance” is fundamentally and evolutionarily based on “avoidance” of injury and death which may be referred to as evolutionary avoidance (EVOL-Avoidance). As a consequence, slow growth results from being exposed to stress because genes and genetic programs to adjust growth rates to external circumstances have evolved as a survival but not productivity strategy that has allowed extant plants to avoid extinction. To improve productivity under moderate stressful conditions, the evolution-oriented plant stress response circuits must be changed from a survival mode to a continued productivity mode or to avoid the evolutionary avoidance response, as it were. This may be referred to as Agricultural (AGRI-Avoidance). Clearly, highly productive crops have kept the slow, reduced growth response to stress that they evolved to ensure survival. Breeding programs and genetic engineering have not succeeded to genetically remove these responses because they are polygenic and redundantly programmed. From the beginning of modern plant breeding, we have not fully appreciated that our crop plants react overly-cautiously to stress conditions. They over-reduce growth to be able to survive stresses for a period of time much longer than a cropping season. If we are able to remove this polygenic redundant survival safety net we may improve yield in moderately stressful environments, yet we will face the requirement to replace it with either an emergency slow or no growth (dormancy) response to extreme stress or use resource management to rescue crops under extreme stress (or both). Full article

(This article belongs to the Section Molecular Plant Sciences)

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16 pages, 3094 KiB

Open AccessArticle

Functionalized Keratin as Nanotechnology-Based Drug Delivery System for the Pharmacological Treatment of Osteosarcoma

by Elisa Martella, Claudia Ferroni, Andrea Guerrini, Marco Ballestri, Marta Columbaro, Spartaco Santi, Giovanna Sotgiu, Massimo Serra, Davide Maria Donati, Enrico Lucarelli, Greta Varchi and Serena Duchi

Int. J. Mol. Sci. 2018, 19(11), 3670; https://doi.org/10.3390/ijms19113670 - 20 Nov 2018

Cited by 33 | Viewed by 6910

Abstract

Osteosarcoma therapy might be moving toward nanotechnology-based drug delivery systems to reduce the cytotoxicity of antineoplastic drugs and improve their pharmacokinetics. In this paper, we present, for the first time, an extensive chemical and in vitro characterization of dual-loaded photo- and chemo-active keratin [...] Read more.

Osteosarcoma therapy might be moving toward nanotechnology-based drug delivery systems to reduce the cytotoxicity of antineoplastic drugs and improve their pharmacokinetics. In this paper, we present, for the first time, an extensive chemical and in vitro characterization of dual-loaded photo- and chemo-active keratin nanoparticles as a novel drug delivery system to treat osteosarcoma. The nanoparticles are prepared from high molecular weight and hydrosoluble keratin, suitably functionalized with the photosensitizer Chlorin-e6 (Ce6) and then loaded with the chemotherapeutic drug Paclitaxel (PTX). This multi-modal PTX-Ce6@Ker nanoformulation is prepared by both drug-induced aggregation and desolvation methods, and a comprehensive physicochemical characterization is performed. PTX-Ce6@Ker efficacy is tested on osteosarcoma tumor cell lines, including chemo-resistant cells, using 2D and 3D model systems. The single and combined contributions of PTX and Ce6 is evaluated, and results show that PTX retains its activity while being vehiculated through keratin. Moreover, PTX and Ce6 act in an additive manner, demonstrating that the combination of the cytostatic blockage of PTX and the oxidative damage of ROS upon light irradiation have a far superior effect compared to singularly administered PTX or Ce6. Our findings provide the proof of principle for the development of a novel, nanotechnology-based drug delivery system for the treatment of osteosarcoma. Full article

(This article belongs to the Special Issue Current Advances in Soft Tissue and Bone Sarcoma)

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Full article ">Figure 1
Synthesis and characterization on PTX-Ce6@ker nanoparticles. (A) Schematic representation of PTX-Ce6@ker synthesis through the (i) drug-induced aggregation (ag), and (ii) desolvation (ds) methods; (B,C) Trasmission Electron Microscopy (TEM) micrograph of PTX-Ce6@kerag; (D) PTX-Ce6@kerag colloidal stability in Phosphate buffered solution (PBS) at 37 °C. Results are expressed as means of three independent experiments; (E) release profiles of PTX-Ce6@kerag and PTX-Ce6@kerds performed at 37 °C under stirring for 24 h. All results are expressed as the mean ± SD (from at least three independent experiments performed in triplicate). Full article ">Figure 2
Sensitivity of osteosarcoma (OS) cell lines to free Paclitaxel (PTX), and PTX loaded onto keratin nanoparticles. The graphs show the WST-1 assay performed on OS cell lines exposed to PTX (black circles), PTX@kerag (blue squares), PTX@kerds (red triangles), PTX-Ce6@kerag (pink squares), or PTX-Ce6@kerds (green triangles). All results are expressed as the mean ± SD (from at least three independent experiments performed in triplicate). Full article ">Figure 3
PTX-Ce6@kerag localization and the effect of PTX on MG63 cells. (A–C) Representative confocal images of MG63 treated for 24 h with PTX-Ce6@kerag and immunostained with EEA1, Lamp-1, and lysosomes, respectively (green channel in all pictures). The nuclei staining is shown in blue, the Ce6 signal in red, and colocalization in white; (D) Phalloidin (green channel) and β-tubulin (red channel) stainings were evaluated on MG63 at the end of the 24 h of treatment with Ce6@ker or PTX-Ce6@kerag. Images are representative of at least three independent experiments. Scale bar: 20 µm. Full article ">Figure 4
Impact of medium dosage of PTX-Ce6@Kerag on OS cells’ viability in a 2D system, and the additive effect of PTX and photodynamic therapy (PDT) on OS cell viability. OS cell lines were treated for 24 h with Ce6@ker, PTX@kerag, or PTX-Ce6@kerag at medium concentration. The graphs show the Alamar blue assay performed immediately after keratin nanoparticle treatments (−PDT), and 24 h after irradiation of the same samples (+PDT). Data, normalized to untreated cells (Ctrl) at the first time-point, are expressed as the mean ± SD (N = 2 biological replicates; N = 3 technical replicates) and analyzed using the one-way ANOVA test, and Tukey’s multiple comparison test as a post-test. Results were considered to be statistically significant at p values < 0.05 (*** p values < 0.001). Full article ">Figure 5
Impact of keratin nanoformulation on chemoresistant SaOS-2/DX580 cells. (A,B) SaOS-2 and SaOS-2/DX580 were treated for 24 h with Ce6 or PTX-Ce6@ker at a [Ce6] concentration of 3.35 µM. (A) Representative confocal microscopy images of cells treated with Ce6 or PTX-Ce6@kerag. Scale bar: 25 µm. (B) the graphs show the Ce6 fluorescence after internalization of the photosensitizer by itself (blue line) or loaded into keratin nanoparticles (red line) quantified by flow cytometry analysis (Control, black line). (C) the graphs show the Alamar blue assay on SaoOS-2/DX580 after 24 h treatment with PTX, PTX@kerag, or PTX-Ce6@kerag at an equivalent concentration of [PTX] of 13.4 µM (High) and 24 h after irradiation (+PDT). All data are normalized to untreated cells (Ctrl) and expressed as the mean ± SD (from at least two independent experiments performed in triplicate) and analyzed using a one-way ANOVA test, and Tukey’s multiple comparison test as a post-test. Results were considered to be statistically significant at p values < 0.05 (*** p values < 0.001). Full article ">Figure 6
Impact of keratin nanoformulation on 3D OS tumor model. (A) Schematic representation of cell spheroid formation and treatment with PTX-Ce6@kerag at a [Ce6] concentration of 6.7 µM; (B, C) the graphs show the Cell Titer Glo assay performed 1 day (B) or 6 days (C) after keratin nanoparticle treatment and −/+ irradiation (−/+PDT). The results are normalized to Ctrl at day 1 (no treated cells) and expressed as mean ± SD (N = 6 technical replicates and N = 2 individual replicates experiments) using the one-way ANOVA test, and Tukey’s multiple comparison test as a post-test. Results were considered to be statistically significant at p values < 0.05. * p-values < 0.05, ** p-values < 0.01 and *** p-values < 0.001; (D) transmission electron microscopy of treated MG63 spheroids with PTX@kerag and PTX-Ce6@kerag one day after irradiation (+PDT). The black squares highlight the mitocondrial alterations after nanoparticle treatment, compared to Ctrl spheroids. The white square in PTX@kerag highlights the necrosis area, and the red star in PTX-Ce6@kerag -PDT highlights the vesicle containing semidigested keratin nanoparticles. Scale bar: 2 µm. Full article ">

14 pages, 539 KiB

Open AccessReview

Central Nervous System Responses to Simulated Galactic Cosmic Rays

by Egle Cekanaviciute, Susanna Rosi and Sylvain V. Costes

Int. J. Mol. Sci. 2018, 19(11), 3669; https://doi.org/10.3390/ijms19113669 - 20 Nov 2018

Cited by 76 | Viewed by 6650

Abstract

In preparation for lunar and Mars missions it is essential to consider the challenges to human health that are posed by long-duration deep space habitation via multiple stressors, including ionizing radiation, gravitational changes during flight and in orbit, other aspects of the space [...] Read more.

In preparation for lunar and Mars missions it is essential to consider the challenges to human health that are posed by long-duration deep space habitation via multiple stressors, including ionizing radiation, gravitational changes during flight and in orbit, other aspects of the space environment such as high level of carbon dioxide, and psychological stress from confined environment and social isolation. It remains unclear how these stressors individually or in combination impact the central nervous system (CNS), presenting potential obstacles for astronauts engaged in deep space travel. Although human spaceflight research only within the last decade has started to include the effects of radiation transmitted by galactic cosmic rays to the CNS, radiation is currently considered to be one of the main stressors for prolonged spaceflight and deep space exploration. Here we will review the current knowledge of CNS damage caused by simulated space radiation with an emphasis on neuronal and glial responses along with cognitive functions. Furthermore, we will present novel experimental approaches to integrate the knowledge into more comprehensive studies, including multiple stressors at once and potential translation to human functions. Finally, we will discuss the need for developing biomarkers as predictors for cognitive decline and therapeutic countermeasures to prevent CNS damage and the loss of cognitive abilities. Full article

(This article belongs to the Special Issue Oxidative Stress and Damage in the Space Environment: Physiological Ground and Flight Parallels)

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