Studying Human Pathogenic Cryptococcus Gattii Lineages by Utilizing Simple Sequence Repeats to Create Diagnostic Markers and Analyzing Diversity.

In this study, we compared the occurrence, relative abundance (RA), and density (RD) of simple sequence repeats (SSRs) among the lineages of human pathogenic Cryptococcus gattii using an in-silico approach to gain a deeper understanding of the structure and evolution of their genomes. C. gattii isolate MF34 showed the highest RA and RD of SSRs in both the genomic and transcriptomic sequences, followed by isolate WM276. In both the genomic (50%) and transcriptomic (65%) sequences, trinucleotide SSRs were the most common SSR class. A motif conservation study found that the isolates had stronger conservation (56.1%) of motifs, with isolate IND107 having the most (5.7%) unique motifs. We discovered the presence of SSRs in genes that are directly or indirectly associated with disease using gene enrichment analysis. Isolate-specific unique motifs identified in this study could be utilized as molecular probes for isolate identification. To improve genetic resources among C. gattii isolates, 6499 primers were developed. These genomic resources developed in this study could help with diversity analysis and the development of isolate-specific markers.

Diabetic Covid-19 severity: Impaired glucose tolerance and pathologic bone loss.

Diabetes mellitus (DM), hypertension, and cardiovascular diseases (CVDs) are the leading chronic comorbidities that enhance the severity and mortality of COVID-19 cases. However, SARS-CoV-2 mediated deregulation of diabetes pathophysiology and comorbidity that links the skeletal bone loss remain unclear. We used both streptozocin-induced type 2 diabetes (T2DM) mouse and hACE2 transgenic mouse to enable SARS-CoV-2-receptor binding domain (RBD) mediated abnormal glucose metabolism and bone loss phenotype in mice. The data demonstrate that SARS-CoV-2-RBD treatment in pre-existing diabetes conditions in hACE2 (T2DM + RBD) mice results in the aggravated osteoblast inflammation and downregulation of Glucose transporter 4 (Glut4) expression via upregulation of miR-294-3p expression. The data also found increased fasting blood glucose and reduced insulin sensitivity in the T2DM + RBD condition compared to the T2DM condition. Femoral trabecular bone mass loss and bone mechanical quality were further reduced in T2DM + RBD mice. Mechanistically, silencing of miR-294 function improved Glut4 expression, glucose metabolism, and bone formation in T2DM + RBD + anti-miR-294 mice. These data uncover the previously undefined role of SARS-CoV-2-RBD treatment mediated complex pathological symptoms of diabetic COVID-19 mice with abnormal bone metabolism via a miRNA-294/Glut4 axis. Therefore, this work would provide a better understanding of the interplay between diabetes and SARS-CoV-2 infection.

Hydrogen sulfide prevents ethanol-induced ZO-1 CpG promoter hypermethylation-dependent vascular permeability via miR-218/DNMT3a axis.

Ethanol (ET) causes cerebrovascular dysfunction by altering homocysteine (Hcy) metabolism and by causing oxidative stress. However, there are no strategies to prevent ET-induced epigenetic deregulation of tight junction protein (hyper-methylation) and endothelial cell permeability to date. Hydrogen sulfide (H2 S) has an antioxidative, antiapoptotic, and anti-inflammatory effect. Here, we investigated the protective role of H2 S in ET-induced endothelial permeability through epigenetic changes in mouse brain endothelial cells (bEnd3). The bEnd3 cells were exposed to 50 mM ET treatment in the presence or absence of 50 µM NaHS (H2 S donor). The result demonstrates that ET-induced cellular toxicity increased intracellular Hcy levels, which further intensified mitochondrial dysfunction and energy defects. Using miScript microRNA (miRNA) polymerase chain reaction array-based screening, we identified a particular miRNA, miR-218, as a novel target of ET-induced DNA methyltransferase-3a (DNMT3a) activation. miR-218 influences CpG island methylation of the zonula occludens 1 (ZO-1) promoter in the endothelial cells. We discovered that ET suppressed miR-218 levels and induced endothelial permeability via DNMT3a-mediated ZO-1 hyper-methylation. Treatment with mito-TEMPO (mitochondria-targeted antioxidant), 5'-azacitidine (DNMT inhibitor), or miR-218 overexpression was shown to protect endothelial cells against ET-induced permeability. Also, bEnd3 cells pretreated with NaHS attenuated ET-induced vascular permeability and prevented CpG island methylation at the promoter. In conclusion, our data provide evidence that H2 S treatment protects vascular integrity from ET-induced stress by mitigating CpG (ZO-1 promoter) DNA hyper-methylation. This finding uncovers a new mechanistic understanding of NaHS/H2 S, that may have therapeutic potential in preventing or diminishing ET-induced brain vascular permeability and dysfunction induced by alcoholism.

Allyl sulfide promotes osteoblast differentiation and bone density via reducing mitochondrial DNA release mediated Kdm6b/H3K27me3 epigenetic mechanism.

Age-associated bone loss or osteoporosis is a common clinical manifestation during aging (AG). The mechanism underlying age-associated osteoblast dysfunction induced by oxidative damage in the mitochondria and loss of bone density remains elusive. Here, we demonstrated the effect of allyl sulfide (AS), a natural organosulfur compound, on mitochondrial (mt) function in bone marrow-derived mesenchymal stem cells (BMMSCs) and bone density in AG mice. The data demonstrate that AS treatment in AG mice promotes BMMSCs differentiation and mineralization via inhibition of mitochondrial oxidative damage. The data also indicate that AG related mito-damage was associated with reduced mitochondrial biogenesis and oxidative phosphorylation, and release of a greater concentration of mtDNA. Furthermore, the data showed that mtDNA caused histone H3K27 demethylase inhibition, KDM6B, and subsequent inflammation by unbalancing mitochondrial redox homeostasis. KDM6B overexpression in AG BMMSCs or AS administration in AG mice restores osteogenesis and bone density in vitro and in vivo. Mechanistically, AS or the mitochondrial-specific antioxidant Mito-TEMPO increased KDM6B expression and upregulated the expression of Runx2 in BMMSCs, probably via epigenetic inhibition of H3K27me3 methylation at the promoter. These data uncover the previously undefined role of AS mediated prevention of mtDNA release, promoting osteogenesis and bone density via an epigenetic mechanism. Therefore, AS could be a potential drug target for the treatment of aging-associated osteoporosis.

Gut microbiota and the periodontal disease: role of hyperhomocysteinemia.

Periodontal disease is one of the most common conditions resulting from poor oral hygiene and is characterized by a destructive process in the periodontium that essentially includes gingiva, alveolar mucosa, cementum, periodontal ligament, and alveolar bone. Notably, the destructive event in the alveolar bone has been linked to homocysteine (Hcy) metabolism; however, it has not been fully investigated. Therefore; the implication of Hcy towards initiation, progression, and maintenance of the periodontal disease remains incompletely understood. Higher levels of Hcy (also known as hyperhomocysteinemia (HHcy)) exerts deleterious effects on gum health and teeth in distinct ways. Firstly, increased production of proinflammatory cytokines such as TNF-α, IL-1ß, IL-6, and IL-8 leads to an inflammatory cascade of events that affect methionine (Met) and Hcy metabolism (i.e., 1-carbon metabolism) leading to HHcy. Secondly, metabolic dysregulation during chronic medical conditions increases systemic inflammation leading to a decrease in vitamins, more specifically B6, B12, and folic acid, that play important roles as cofactors in Hcy metabolism. Also, given the folate level in the HHcy state that is important during dysbiosis, these two conditions appear to be intimately related, and in this context, HHcy-induced dysbiosis may be one of the potential causes of periodontal disease. This paper sums up the link between periodontitis and HHcy, with a special emphasis on the "oral-gut microbiome axis" and the potential probiotic intervention towards warding off some of the serious periodontal disease conditions.

Hydrogen sulfide attenuates homocysteine-induced osteoblast dysfunction by inhibiting mitochondrial toxicity.

Homocysteine (Hcy) is detrimental to bone health in a mouse model of diet-induced hyperhomocysteinemia (HHcy). However, little is known about Hcy-mediated osteoblast dysfunction via mitochondrial oxidative damage. Hydrogen sulfide (H2 S) has potent antioxidant, anti-inflammatory, and antiapoptotic effects. In this study, we hypothesized that the H2 S mediated recovery of osteoblast dysfunction by maintaining mitochondrial biogenesis in Hcy-treated osteoblast cultures in vitro. MC3T3-E1 osteoblastic cells were exposed to Hcy treatment in the presence or absence of an H2 S donor (NaHS). Cell viability, osteogenic differentiation, reactive oxygen species (ROS) production were determined. Mitochondrial DNA copy number, adenosine triphosphate (ATP) production, and oxygen consumption were also measured. Our results demonstrated that administration of Hcy increases the intracellular Hcy level and decreases intracellular H2 S level and expression of the cystathionine ß-synthase/Cystathionine γ-lyase system, thereby inhibiting osteogenic differentiation. Pretreatment with NaHS attenuated Hcy-induced mitochondrial toxicity (production of total ROS and mito-ROS, ratio of mitochondrial fission (DRP-1)/fusion (Mfn-2)) and restored ATP production and mitochondrial DNA copy numbers as well as oxygen consumption in the osteoblast as compared with the control, indicating its protective effects against Hcy-induced mitochondrial toxicity. In addition, NaHS also decreased the release of cytochrome c from the mitochondria to the cytosol, which induces cell apoptosis. Finally, flow cytometry confirmed that NaHS can rescue cells from apoptosis induced by Hcy. Our studies strongly suggest that NaHS has beneficial effects on mitochondrial toxicity, and could be developed as a potential therapeutic agent against HHcy-induced mitochondrial dysfunction in cultured osteoblasts in vitro.

Hyperhomocysteinemia induced endothelial progenitor cells dysfunction through hyper-methylation of CBS promoter.

Bone marrow (BM)-derived endothelial progenitor cells (EPCs) are the key players in angiogenesis and vascular function. Cystathionine-ß-synthase (CBS), an H2S-generating enzyme in methionine metabolism, regulates the function of these EPCs. This study aims to examine whether CBS hyper-methylation contributes to the bone marrow endothelial progenitor cell (BM-EPCs) function and subsequent bone blood flow in mice fed with a high methionine diet (HMD). Bone marrow (BM) cells were collected from HMD and control mice, differentiated into BM-EPCs, and were characterized by acLDL-DiI labeling. CBS mRNA expression was analyzed by real-time PCR, and the global methylation status and methylation of the CBS promoter were detected by nuclear 5-mC assay and methylation-specific PCR (qMSP) respectively. The result reveals that CBS promoter in BM-EPCs from HMD mice was hyper-methylated and the methylation level was, indeed, negatively correlated with CBS mRNA and angiogenic function of BM-EPCs. In addition, global methylation (5-mC) and DNA methyltransferase-1 (DNMT1) expression were increased in HMD condition. In vitro study also shows that HMD induced hyperhomocysteinemia (HHcy) impaired both adhesion and angiogenesis properties of BM-EPCs, accompanied by higher methylation level of CBS promoter that compared to control. Furthermore, bone blood flow was found to be decreased in HMD mice as compared to wild-type mice. To dissect the epigenetic mechanism, we also administrated DNMT inhibitor, 5-azacytidine (5-Aza) to HMD mice. The administration of 5-Aza in HMD mice restored the CBS expression, EPC mediated angiogenesis and blood flow by reducing abnormal DNA hyper-methylation. In conclusion, HHcy dismantles BM-EPCs function and bone blood flow through the hyper-methylation of the CBS promoter in HMD fed mice.

Hydrogen sulfide alleviates hyperhomocysteinemia-mediated skeletal muscle atrophy via mitigation of oxidative and endoplasmic reticulum stress injury.

Although hyperhomocysteinemia (HHcy) occurs because of the deficiency in cystathionine-ß-synthase (CBS) causing skeletal muscle dysfunction, it is still unclear whether this effect is mediated through oxidative stress, endoplasmic reticulum (ER) stress, or both. Nevertheless, there is no treatment option available to improve HHcy-mediated muscle injury. Hydrogen sulfide (H2S) is an antioxidant compound, and patients with CBS mutation do not produce H2S. In this study, we hypothesized that H2S mitigates HHcy-induced redox imbalance/ER stress during skeletal muscle atrophy via JNK phosphorylation. We used CBS+/- mice to study HHcy-mediated muscle atrophy, and treated them with sodium hydrogen sulfide (NaHS; an H2S donor). Proteins and mRNAs were examined by Western blots and quantitative PCR. Proinflammatory cytokines were also measured. Muscle mass and strength were studied via fatigue susceptibility test. Our data revealed that HHcy was detrimental to skeletal mass, particularly gastrocnemius and quadriceps muscle weight. We noticed that oxidative stress was reversed by NaHS in homocysteine (Hcy)-treated C2C12 cells. Interestingly, ER stress markers (GRP78, ATF6, pIRE1α, and pJNK) were elevated in vivo and in vitro, and NaHS mitigated these effects. Additionally, we observed that JNK phosphorylation was upregulated in C2C12 after Hcy treatment, but NaHS could not reduce this effect. Furthermore, inflammatory cytokines IL-6 and TNF-α were higher in plasma from CBS as compared with wild-type mice. FOXO1-mediated Atrogin-1 and MuRF-1 upregulation were attenuated by NaHS. Functional studies revealed that NaHS administration improved muscle fatigability in CBS+/- mice. In conclusion, our work provides evidence that NaHS is beneficial in mitigating HHcy-mediated skeletal injury incited by oxidative/ER stress responses.

Tetrahydrocurcumin ameliorates homocysteine-mediated mitochondrial remodeling in brain endothelial cells.

Homocysteine (Hcy) causes endothelial dysfunction by inducing oxidative stress in most neurodegenerative disorders. This dysfunction is highly correlated with mitochondrial dynamics such as fusion and fission. However, there are no strategies to prevent Hcy-induced mitochondrial remodeling. Tetrahydrocurcumin (THC) is an anti-inflammatory and anti-oxidant compound. We hypothesized that THC may ameliorates Hcy-induced mitochondria remodeling in mouse brain endothelial cells (bEnd3) cells. bEnd3 cells were exposed to Hcy treatment in the presence or absence of THC. Cell viability and autophagic cell death were measured with MTT and MDC staining assay. Reactive oxygen species (ROS) production was determined using DCFH-DA staining by confocal microscopy. Autophagy flux was assessed using a conventional GFP-microtubule-associated protein 1 light chain 3 (LC3) dot assay. Interaction of phagophore marker LC-3 with mitochondrial receptor NIX was observed by confocal imaging. Mitochondrial fusion and fission were evaluated by western blot and RT-PCR. Our results demonstrated that Hcy resulted in cell toxicity in a dose-dependent manner and supplementation of THC prevented the detrimental effects of Hcy on cell survival. Furthermore, Hcy also upregulated fission marker (DRP-1), fusion marker (Mfn2), and autophagy marker (LC-3). Finally, we observed that Hcy activated mitochondrial specific phagophore marker (LC-3) and co-localized with the mitochondrial receptor NIX, as viewed by confocal microscopy. Pretreatment of bEnd3 with THC (15 µM) ameliorated Hcy-induced oxidative damage, mitochondrial fission/fusion, and mitophagy. Our studies strongly suggest that THC has beneficial effects on mitochondrial remodeling and could be developed as a potential therapeutic agent against hyperhomocysteinemia (HHcy) induced mitochondrial dysfunction.

Inflammation, oxidative stress, and higher expression levels of Nrf2 and NQO1 proteins in the airways of women chronically exposed to biomass fuel smoke.

The study was carried out to examine whether chronic exposure to smoke during daily household cooking with biomass fuel (BMF) elicits changes in airway cytology and expressions of Nrf2 (nuclear factor erythroid 2 [NF-E2]-related factor 2 [Nrf2]), Keap1 (Kelch-like erythroid-cell-derived protein with CNC homology [ECH]-associated protein 1), and NQO1 (NAD(P)H:quinone oxidoreductase 1) proteins in the airways. For this, 282 BMF-using women (median age 34 year) and 236 age-matched women who cooked with liquefied petroleum gas (LPG) were enrolled. Particulate matter with diameters of < 10 µm (PM10) and < 2.5 µm (PM2.5) were measured in indoor air with real-time laser photometer. Routine hematology, sputum cytology, Nrf2, Keap1, NQO1, and generation of reactive oxygen species (ROS) along with the levels of superoxide dismutase (SOD) and catalase were measured in both groups. PM10 and PM2.5 levels were significantly higher in BMF-using households compared to LPG. Compared with LPG users, BMF users had 32% more leukocytes in circulation and their sputa were 1.4-times more cellular with significant increase in absolute number of neutrophils, lymphocytes, eosinophils, and alveolar macrophages, suggesting airway inflammation. ROS generation was 1.5-times higher in blood neutrophils and 34% higher in sputum cells of BMF users while erythrocyte SOD was 31% lower and plasma catalase was relatively unchanged, suggesting oxidative stress. In BMF users, Keap1 expression was reduced, the percentage of AEC with nuclear expression of Nrf2 was two- to three-times more, and NQO1 level in sputum cell lysate was two-times higher than that of LPG users. In conclusion, cooking with BMF was associated with Nrf2 activation and elevated NQO1 protein level in the airways. The changes may be adaptive cellular response to counteract biomass smoke-elicited oxidative stress and inflammation-related tissue injury in the airways.

High methionine, low folate and low vitamin B6/B12 (HM-LF-LV) diet causes neurodegeneration and subsequent short-term memory loss.

Methionine is an essential amino acid found in rich quantities in average American diet such as meats, fish and eggs. Excessive consumption of such food often exceeds the normal requirement of the methionine in our body; which found to be related to the development of neurodegenerative disorders. However, the mechanistic pathways of methionine's influence on the brain are unclear. The present study is focus on the effects of high methionine, low folate and low vitamin B6/B12 (HM-LF-LV) diet on the dysfunction of neuronal and vascular specific markers in the brain. C57BL6/J male mice (8-10 week old) were fed with HM-LF-LV diet for a 6 week period. Cognitive function of mice was determine by measuring short-term memory using a Novel Object Recognition test (NORT). Neuronal dysfunction were evaluate by measuring the levels of Neuronal nuclear antigen (NeuN), Neuron-specific-enolase (NSE) and Fluoro-jade C(FJC) fluorescence; while cerebrovascular disruption were evaluate by assessing levels of endothelial junction proteins Vascular Endothelial-Cadherin (VE-Cadherin) and Claudin-5 in harvested brain tissue. Cerebrovascular permeability was assess by evaluating microvascular leakage of fluorescently labeled albumin in vivo. Endothelial and Neuronal Nitric Oxide Synthase (eNOS, nNOS) regulation and vascular inflammation (ICAM: intercellular adhesion molecules) were also evaluate in brain tissue. All assessments were conduct at weekly intervals throughout the study duration. NORT showed a significant temporal decrease in short-term memory of mice fed on HM-LF-LV diet for 6 weeks compared to the wild-type control group. Our experimental data showed that neuronal dysfunction (decreased NeuN levels and increased FJC positive neurons in brain) was more prominent in HM-LF-LV diet fed mice compared to normal diet fed control mice. In experimental mice, cerebrovascular disruption was found to be elevated as evident from increased pial venular permeability (microvascular leakage) and decreased in VE-Cadherin expression compared to control. Slight decrease in nNOS and increase in eNOS in experimental mice suggest a trend towards the decrease in potential for neuronal development due to the long-term HM-LF-LV diet fed. Collectively, our results suggest that a diet containing high methionine, low folate and low vitamin B6/B12 results in increased neuronal degeneration and vascular dysfunction, leading to short-term memory loss. Interestingly, significant neuronal damage precedes vascular dysfunction.

Dementia-like pathology in type-2 diabetes: A novel microRNA mechanism.

Although type-2 diabetes (T2D) has been reported to increase the risk of cognitive dysfunction and dementia, the underlying mechanisms remain unclear. Dementia-like pathology is attributed to the accumulation of cellular prion protein (PrPc) which plays a role in cognitive dysfunction. However, its involvement and regulation in diabetic dementia-like pathology is not well understood. Using T2D db/db (leptin receptor knockout) mice subjected to object recognition and Y-maze behavioral tests, we determined that short-term memory was compromised and that the mice displayed abrupt spontaneous behaviour compared to db/m control mice. MicroRNA analysis using qRT2-PCR array demonstrated a significant reduction in the transcript expression of microRNA-146a (miR-146a) in the brain of T2D db/db mice as compared to db/m controls. The sequence matching tools validated the binding of miR-146a to a conserved domain of the PrPc gene. Administration of mouse brain endothelial cell-derived exosomes (BECDEs) loaded with miR-146a into the brain's ventricle of T2D db/db mice attenuated brain PrPc levels and restored short-term memory function though not significant. Also, we observed hyperphosphorylation of tau through decreased expression of glycogen synthase kinase-3 in T2D db/db brains that regulates microtubule organization and memory function. We conclude that underexpression of miR-146a upregulates PrPc production in T2D db/db mice and the delivery of BECDEs loaded with a miR-146a can down regulate PrPc levels and restore short term memory function up to a certain extent.

Metabolic engineering of Escherichia coli W3110 strain by incorporating genome-level modifications and synthetic plasmid modules to enhance L-Dopa production from glycerol.

L-Tyrosine which is one of the terminal metabolites of highly regulated aromatic amino-acid biosynthesis pathway in Escherichia coli is a precursor for synthesis of L-Dopa. In this study, we report over production of L-Dopa by enhancing expression of rate limiting isoenzyme of shikimate kinase (aroL), chorismate synthase (aroC), aromatic-amino-acid aminotransferase (tyrB) and 3-phosphoshikimate 1-carboxyvinyltransferase (aroA) form a plasmid module harboring five enzymes under two inducible promoters converting shikimate to tyrosine. 4-hydroxyphenylacetate-3-hydrolase (hpaBC) which converts L-Tyrosine to L-Dopa was expressed constitutively from a separate plasmid module. Feedback deregulated expression of 3-Deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase (aroG*) replacing wild type aroG under its natural promoter led to enhancement of L-Dopa production. Deletion of transcriptional repressor tyrR and links to other competing pathways improved titers of L-Dopa. We focused on having a balanced flux by constitutive expression of pathway enzymes from plasmid constructs rather than achieving higher amounts of catalytic protein by induction. We observed glycerol when used as a carbon source for the final strain led to low acid production. The best performing strain led to decoupling of acid production and product formation in bioreactor. Fed batch analysis of the final strain led to 12.5 g/L of L-Dopa produced in bioreactor.

Identification of genes associated with stress tolerance in moth bean [Vigna aconitifolia (Jacq.) Marechal], a stress hardy crop.

Moth bean is the most drought and heat tolerant cultigens among Asian Vigna. We performed comparative transcriptome analysis of moth bean cultivar "Marumoth" under control and stress condition. De novo transcriptome assembly was carried out by using Velvet followed by Oases softwares. Differential expression analyses, SSR identification and validation and mapping of pathways and transcription factors were conducted. A total of 179,979 and 201,888 reads were generated on Roche 454 platform and 48,617,205 and 45,449,053 reads were generated on ABI Solid platform for the control and stressed samples. Combined assembly from Roche and ABI Solid platforms generated 16,090 and 15,096 transcripts for control and stressed samples. We found 1287 SSRs and 5606 transcripts involved in 179 pathways. The 55 transcription factor families represented 19.42% of total mothbean transcripts. In expression profiling, ten transcripts were found to be up-regulated and 41 down-regulated while 490 showed no major change under moisture stress condition. Stress inducible genes like Catalase, Cyt P450 monooxygenase, heat shock proteins (HSP 90 and HSP 70), oxidoreductase, protein kinases, dehydration responsive protein (DRP), universal stress protein and ferridoxin NADH oxidoreductase genes were up-regulated in stressed sample. Genes which might be involved in moisture stress tolerance in moth bean were identified and these might be useful for stress tolerance breeding in moth bean and other related crops.

Homocysteine as a Pathological Biomarker for Bone Disease.

In the last few decades, perturbation in methyl-group and homocysteine (Hcy) balance have emerged as independent risk factors in a number of pathological conditions including neurodegenerative disease, cardiovascular dysfunction, cancer development, autoimmune disease, and kidney disease. Recent studies report Hcy to be a newly recognized risk factor for osteoporosis. Elevated Hcy levels are known to modulate osteoclastgenesis by causing detrimental effects on bone via oxidative stress induced metalloproteinase-mediated extracellular matrix degradation and decrease in bone blood flow. Evidence from previous studies also suggests that the decreased chondrocytes mediated bone mineralization in chick limb-bud mesenchymal cells and during the gestational period of ossification in rat model. However, Hcy imbalance and its role in bone loss, regression in vascular invasion, and osteoporosis, are not clearly understood. More investigations are required to explore the complex interplay between Hcy imbalance and onset of bone disease progression. This article reviews the current body of knowledge on regulation of Hcy mediated oxidative stress and its role in bone remodeling, vascular blood flow and progression of bone disease. J. Cell. Physiol. 232: 2704-2709, 2017. © 2016 Wiley Periodicals, Inc.

Cerebrovascular disorders caused by hyperfibrinogenaemia.

KEY POINTS: Hyperfibrinogenaemia (HFg) results in vascular remodelling, and fibrinogen (Fg) and amyloid ß (Aß) complex formation is a hallmark of Alzheimer's disease. However, the interconnection of these effects, their mechanisms and implications in cerebrovascular diseases are not known. Using a mouse model of HFg, we showed that at an elevated blood level, Fg increases cerebrovascular permeability via mainly caveolar protein transcytosis. This enhances deposition of Fg in subendothelial matrix and interstitium making the immobilized Fg a readily accessible substrate for binding Aß and cellular prion protein (PrPC ), the protein that is thought to have a greater effect on memory than Aß. We showed that enhanced formation of Fg-Aß and Fg-PrPC complexes are associated with reduction in short-term memory. The present study delineates a new mechanistic pathway for vasculo-neuronal dysfunctions found in inflammatory cardiovascular and cerebrovascular diseases associated with an elevated blood level of Fg. ABSTRACT: Many cardiovascular diseases are associated with inflammation and as such are accompanied by an increased blood level of fibrinogen (Fg). Besides its well-known prothrombotic effects Fg seems to have other destructive roles in developing microvascular dysfunction that include changes in vascular reactivity and permeability. Increased permeability of brain microvessels has the most profound effects as it may lead to cerebrovascular remodelling and result in memory reduction. The goal of the present study was to define mechanisms of cerebrovascular permeability and associated reduction in memory induced by elevated blood content of Fg. Genetically modified, transgenic hyperfibrinogenic (HFg) mice were used to study cerebrovascular transcellular and paracellular permeability in vivo. The extent of caveolar formation and the role of caveolin-1 signalling were evaluated by immunohistochemistry (IHC) and Western blot (WB) analysis in brain samples from experimental animals. Formation of Fg complexes with amyloid ß (Aß) and with cellular prion protein (PrPC ) were also assessed with IHC and WB analysis. Short-term memory of mice was assessed by novel object recognition and Y-maze tests. Caveolar protein transcytosis was found to have a prevailing role in overall increased cerebrovascular permeability in HFg mice. These results were associated with enhanced formation of caveolae. Increased formation of Fg-PrPC and Fg-Aß complexes were correlated with reduction in short-term memory in HFg mice. Using the model of hyperfibrinogenaemia, the present study shows a novel mechanistic pathway of inflammation-induced and Fg-mediated reduction in short-term memory.

Inhibition of MMP-9 attenuates hypertensive cerebrovascular dysfunction in Dahl salt-sensitive rats.

Hypertensive cerebropathy is a pathological condition associated with cerebral edema and disruption of the blood-brain barrier. However, the molecular pathways leading to this condition remains obscure. We hypothesize that MMP-9 inhibition can help reducing blood pressure and endothelial disruption associated with hypertensive cerebropathy. Dahl salt-sensitive (Dahl/SS) and Lewis rats were fed with high-salt diet for 6 weeks and then treated without and with GM6001 (MMP inhibitor). Treatment of GM6001 (1.2 mg/kg body weight) was administered through intraperitoneal injections on alternate days for 4 weeks. GM6001 non-administered groups were given vehicle (0.9% NaCl in water) treatment as control. Blood pressure was measured by tail-cuff method. The brain tissues were analyzed for oxidative/nitrosative stress, vascular MMP-9 expression, and tight junction proteins (TJPs). GM6001 treatment significantly reduced mean blood pressure in Dahl/SS rats which was significantly higher in vehicle-treated Dahl/SS rats. MMP-9 expression and activity was also considerably reduced in GM6001-treated Dahl/SS rats, which was otherwise notably increased in vehicle-treated Dahl/SS rats. Similarly MMP-9 expression in cerebral vessels of GM6001-treated Dahl/SS rats was also alleviated, as devised by immunohistochemistry analysis. Oxidative/nitrosative stress was significantly higher in vehicle-treated Dahl/SS rats as determined by biochemical estimations of malondialdehyde, nitrite, reactive oxygen species, and glutathione levels. RT-PCR and immunohistochemistry analysis further confirmed considerable alterations of TJPs in hypertensive rats. Interestingly, GM6001 treatment significantly ameliorated oxidative/nitrosative stress and TJPs, which suggest restoration of vascular integrity in Dahl/SS rats. These findings determined that pharmacological inhibition of MMP-9 in hypertensive Dahl-SS rats attenuate high blood pressure and hypertension-associated cerebrovascular pathology.

Homocysteine, Alcoholism, and Its Potential Epigenetic Mechanism.

Alcohol is the most socially accepted addictive drug. Alcohol consumption is associated with some health problems such as neurological, cognitive, behavioral deficits, cancer, heart, and liver disease. Mechanisms of alcohol-induced toxicity are presently not yet clear. One of the mechanisms underlying alcohol toxicity has to do with its interaction with amino acid homocysteine (Hcy), which has been linked with brain neurotoxicity. Elevated Hcy impairs with various physiological mechanisms in the body, especially metabolic pathways. Hcy metabolism is predominantly controlled by epigenetic regulation such as DNA methylation, histone modifications, and acetylation. An alteration in these processes leads to epigenetic modification. Therefore, in this review, we summarize the role of Hcy metabolism abnormalities in alcohol-induced toxicity with epigenetic adaptation and their influences on cerebrovascular pathology.

Hydrogen Sulfide Epigenetically Attenuates Homocysteine-Induced Mitochondrial Toxicity Mediated Through NMDA Receptor in Mouse Brain Endothelial (bEnd3) Cells.

Previously we have shown that homocysteine (Hcy) caused oxidative stress and altered mitochondrial function. Hydrogen sulfide (H2S) has potent anti-inflammatory, anti-oxidative, and anti-apoptotic effects. Therefore, in the present study we examined whether H2S ameliorates Hcy-induced mitochondrial toxicity which led to endothelial dysfunction in part, by epigenetic alterations in mouse brain endothelial cells (bEnd3). The bEnd3 cells were exposed to 100 µM Hcy treatment in the presence or absence of 30 µM NaHS (donor of H2S) for 24 h. Hcy-activate NMDA receptor and induced mitochondrial toxicity by increased levels of Ca(2+), NADPH-oxidase-4 (NOX-4) expression, mitochondrial dehydrogenase activity and decreased the level of nitrate, superoxide dismutase (SOD-2) expression, mitochondria membrane potentials, ATP production. To confirm the role of epigenetic, 5'-azacitidine (an epigenetic modulator) treatment was given to the cells. Pretreatment with NaHS (30 µM) attenuated the Hcy-induced increased expression of DNMT1, DNMT3a, Ca(2+), and decreased expression of DNMT3b in bEND3 cells. Furthermore, NaHS treatment also mitigated mitochondrial oxidative stress (NOX4, ROS, and NO) and restored ATP that indicates its protective effects against mitochondrial toxicity. Additional, NaHS significantly alleviated Hcy-induced LC3-I/II, CSE, Atg3/7, and low p62 expression which confirm its effect on mitophagy. Likewise, NaHS also restored level of eNOS, CD31, VE-cadherin and ET-1 and maintains endothelial function in Hcy treated cells. Molecular inhibition of NMDA receptor by using small interfering RNA showed protective effect whereas inhibition of H2S production by propargylglycine (PG) (inhibitor of enzyme CSE) showed mitotoxic effect. Taken together, results demonstrate that, administration of H2S protected the cells from HHcy-induced mitochondrial toxicity and endothelial dysfunction.

Enhanced hepatitis B virus (HBV) pre-genomic RNA levels and higher transcription efficiency of defective HBV genomes.

Defective hepatitis B virus (dHBV) particles contain genomes corresponding to singly spliced HBV RNA. A limited number of studies show that dHBV is present in all chronically HBV-infected patients. Clinical studies have linked dHBV and dHBV gene products to high virus loads and liver damage. The replication characteristics of dHBV genomes remain poorly understood. We found that the splice donor/acceptor sites critical for the formation of dHBV genomes are conserved across HBV genotypes. We report a novel method to create dHBV constructs from corresponding wild-type (WT) HBV constructs. We assessed the transcriptional characteristics of the dHBV constructs with those of the corresponding WT construct using a cell culture model. Interestingly, dHBV constructs had higher pre-genomic RNA levels, transcription efficiency, HBV e antigen levels and intracellular HBV core antigen levels compared with the corresponding WT HBV constructs. Our findings highlight previously unrecognized fundamental molecular characteristics of dHBV genomes and their potential role in the pathogenesis of HBV infection.