The role of the mitochondrial trans-sulfuration in cerebro-cardio renal dysfunction during trisomy down syndrome.

One in 700 children is born with the down syndrome (DS). In DS, there is an extra copy of X chromosome 21 (trisomy). Interestingly, the chromosome 21 also contains an extra copy of the cystathionine beta synthase (CBS) gene. The CBS activity is known to contribute in mitochondrial sulfur metabolism via trans-sulfuration pathway. We hypothesize that due to an extra copy of the CBS gene there is hyper trans-sulfuration in DS. We believe that understanding the mechanism of hyper trans-sulfuration during DS will be important in improving the quality of DS patients and towards developing new treatment strategies. We know that folic acid "1-carbon" metabolism (FOCM) cycle transfers the "1-carbon" methyl group to DNA (H3K4) via conversion of s-adenosyl methionine (SAM) to s-adenosyl homocysteine (SAH) by DNMTs (the gene writers). The demethylation reaction is carried out by ten-eleven translocation methylcytosine dioxygenases (TETs; the gene erasers) through epigenetics thus turning the genes off/on and opening the chromatin by altering the acetylation/HDAC ratio. The S-adenosyl homocysteine hydrolase (SAHH) hydrolyzes SAH to homocysteine (Hcy) and adenosine. The Hcy is converted to cystathionine, cysteine and hydrogen sulfide (H2S) via CBS/cystathioneγ lyase (CSE)/3-mercaptopyruvate sulfurtransferase (3MST) pathways. Adenosine by deaminase is converted to inosine and then to uric acid. All these molecules remain high in DS patients. H2S is a potent inhibitor of mitochondrial complexes I-IV, and regulated by UCP1. Therefore, decreased UCP1 levels and ATP production can ensue in DS subjects. Interestingly, children born with DS show elevated levels of CBS/CSE/3MST/Superoxide dismutase (SOD)/cystathionine/cysteine/H2S. We opine that increased levels of epigenetic gene writers (DNMTs) and decreased in gene erasers (TETs) activity cause folic acid exhaustion, leading to an increase in trans-sulphuration by CBS/CSE/3MST/SOD pathways. Thus, it is important to determine whether SIRT3 (inhibitor of HDAC3) can decrease the trans-sulfuration activity in DS patients. Since there is an increase in H3K4 and HDAC3 via epigenetics in DS, we propose that sirtuin-3 (Sirt3) may decrease H3K4 and HDAC3 and hence may be able to decrease the trans-sulfuration in DS. It would be worth to determine whether the lactobacillus, a folic acid producing probiotic, mitigates hyper-trans-sulphuration pathway in DS subjects. Further, as we know that in DS patients the folic acid is exhausted due to increase in CBS, Hcy and re-methylation. In this context, we suggest that folic acid producing probiotics such as lactobacillus might be able to improve re-methylation process and hence may help decrease the trans-sulfuration pathway in the DS patients.

Role of circadian clock system in the mitochondrial trans-sulfuration pathway and tissue remodeling.

Previous studies from our laboratory revealed that the gaseous molecule hydrogen sulfide (H2S), a metabolic product of epigenetics, involves trans-sulfuration pathway for ensuring metabolism and clearance of homocysteine (Hcy) from body, thereby mitigating the skeletal muscle's pathological remodeling. Although the master circadian clock regulator that is known as brain and muscle aryl hydrocarbon receptor nuclear translocator like protein 1 (i.e., BMAL 1) is associated with S-adenosylhomocysteine hydrolase (SAHH) and Hcy metabolism but how trans-sulfuration pathway is influenced by the circadian clock remains unexplored. We hypothesize that alterations in the functioning of circadian clock during sleep and wake cycle affect skeletal muscle's biology. To test this hypothesis, we measured serum matrix metalloproteinase (MMP) activities using gelatin gels for analyzing the MMP-2 and MMP-9. Further, employing casein gels, we also studied MMP-13 that is known to be influenced by the growth arrest and DNA damage-45 (GADD45) protein during sleep and wake cycle. The wild type and cystathionine ß synthase-deficient (CBS-/+) mice strains were treated with H2S and subjected to measurement of trans-sulfuration factors from skeletal muscle tissues. The results suggested highly robust activation of MMPs in the wake mice versus sleep mice, which appears somewhat akin to the "1-carbon metabolic dysregulation", which takes place during remodeling of extracellular matrix during muscular dystrophy. Interestingly, the levels of trans-sulfuration factors such as CBS, cystathionine γ lyase (CSE), methyl tetrahydrofolate reductase (MTHFR), phosphatidylethanolamine N-methyltransferase (PEMT), and Hcy-protein bound paraoxonase 1 (PON1) were attenuated in CBS-/+ mice. However, treatment with H2S mitigated the attenuation of the trans-sulfuration pathway. In addition, levels of mitochondrial peroxisome proliferator-activated receptor-gamma coactivator 1-α (PGC 1-α) and mitofusin-2 (MFN-2) were significantly improved by H2S intervention. Our findings suggest participation of the circadian clock in trans-sulfuration pathway that affects skeletal muscle remodeling and mitochondrial regeneration.

Simulation of COVID-19 symptoms in a genetically engineered mouse model: implications for the long haulers.

The ongoing pandemic (also known as coronavirus disease-19; COVID-19) by a constantly emerging viral agent commonly referred as the severe acute respiratory syndrome corona virus 2 or SARS-CoV-2 has revealed unique pathological findings from infected human beings, and the postmortem observations. The list of disease symptoms, and postmortem observations is too long to mention; however, SARS-CoV-2 has brought with it a whole new clinical syndrome in "long haulers" including dyspnea, chest pain, tachycardia, brain fog, exercise intolerance, and extreme fatigue. We opine that further improvement in delivering effective treatment, and preventive strategies would be benefited from validated animal disease models. In this context, we designed a study, and show that a genetically engineered mouse expressing the human angiotensin converting enzyme 2; ACE-2 (the receptor used by SARS-CoV-2 agent to enter host cells) represents an excellent investigative resource in simulating important clinical features of the COVID-19. The ACE-2 mouse model (which is susceptible to SARS-CoV-2) when administered with a recombinant SARS-CoV-2 spike protein (SP) intranasally exhibited a profound cytokine storm capable of altering the physiological parameters including significant changes in cardiac function along with multi-organ damage that was further confirmed via histological findings. More importantly, visceral organs from SP treated mice revealed thrombotic blood clots as seen during postmortem examination. Thus, the ACE-2 engineered mouse appears to be a suitable model for studying intimate viral pathogenesis thus paving the way for identification, and characterization of appropriate prophylactics as well as therapeutics for COVID-19 management.

Porphyromonas gingivalis induces cardiovascular dysfunction.

Porphyromonas gingivalis (P. gingivalis) is one of the most responsible periodontopathogenic bacteria in the development of periodontal disease (PD); however, its role in the development of other diseases still needs to be understood, specially its implications in the causation of cardiovascular pathogenesis. The aim of this study is to determine whether there is a direct association between P. gingivalis-induced PD with that of the development of cardiovascular disease, and whether a long-term administration of probiotic(s) could help improve the cardiovascular disease outcome. To test this hypothesis, we employed four different experimental groups of mice, designated as: Group I: Wild-type (WT) mice (C57BL/6J); Group II: Lactobacillus rhamnosus GG (LGG) (WT mice treated with a probiotic; LGG), Group III: PD (WT mice treated with P. gingivalis), and Group IV: PD + LGG (WT mice treated with P. gingivalis and LGG). PD was created by injecting 2 µL (i.e., 20 µg) of P. gingivalis lipopolysaccharide (LPS) intragingivally between the 1st and 2nd mandibular molars, two times a week for a total period of 6 weeks. The PD (LGG) intervention was done orally employing 2.5 × 105 CFU/day for a continuous period of 12 weeks. Immediately before the mice were sacrificed, echocardiography of the heart was performed, and after sacrifice, we collected serum samples, hearts, and the periodontal tissue. Histological assessment, cytokine analysis, and zymography of the cardiac tissue were performed. Results revealed inflammation of the heart muscle in the PD group that was marked by infiltration of neutrophils and monocytes, followed by fibrosis. Cytokine analysis of the mice sera revealed significantly elevated levels of tumor necrosis factor-α, IL-1ß, IL-6, and IL-17A in the PD group along with LPS-binding protein, and CD14. Most importantly, we observed elevated levels of P. gingivalis mRNAs in the heart tissues of PD mice. Zymographic analysis demonstrated matrix remodeling as revealed by increasing content of MMP-9 in the heart tissues of PD mice. Interestingly, LGG treatment was able to mitigate most of the pathological effects. The findings suggest that P. gingivalis could lead to cardiovascular system disorder and that probiotic intervention could alleviate, and most likely prevent bacteremia and its harmful effect(s) on the cardiovascular function.

Renal Denervation Helps Preserve the Ejection Fraction by Preserving Endocardial-Endothelial Function during Heart Failure.

Renal denervation (RDN) protects against hypertension, hypertrophy, and heart failure (HF); however, it is not clear whether RDN preserves ejection fraction (EF) during heart failure (HFpEF). To test this hypothesis, we simulated a chronic congestive cardiopulmonary heart failure (CHF) phenotype by creating an aorta-vena cava fistula (AVF) in the C57BL/6J wild type (WT) mice. Briefly, there are four ways to create an experimental CHF: (1) myocardial infarction (MI), which is basically ligating the coronary artery by instrumenting and injuring the heart; (2) trans-aortic constriction (TAC) method, which mimics the systematic hypertension, but again constricts the aorta on top of the heart and, in fact, exposes the heart; (3) acquired CHF condition, promoted by dietary factors, diabetes, salt, diet, etc., but is multifactorial in nature; and finally, (4) the AVF, which remains the only one wherein AVF is created ~1 cm below the kidneys in which the aorta and vena cava share the common middle-wall. By creating the AVF fistula, the red blood contents enter the vena cava without an injury to the cardiac tissue. This model mimics or simulates the CHF phenotype, for example, during aging wherein with advancing age, the preload volume keeps increasing beyond the level that the aging heart can pump out due to the weakened cardiac myocytes. Furthermore, this procedure also involves the right ventricle to lung to left ventricle flow, thus creating an ideal condition for congestion. The heart in AVF transitions from preserved to reduced EF (i.e., HFpEF to HFrEF). In fact, there are more models of volume overload, such as the pacing-induced and mitral valve regurgitation, but these are also injurious models in nature. Our laboratory is one of the first laboratories to create and study the AVF phenotype in the animals. The RDN was created by treating the cleaned bilateral renal artery. After 6 weeks, blood, heart, and renal samples were analyzed for exosome, cardiac regeneration markers, and the renal cortex proteinases. Cardiac function was analyzed by echocardiogram (ECHO) procedure. The fibrosis was analyzed with a trichrome staining method. The results suggested that there was a robust increase in the exosomes' level in AVF blood, suggesting a compensatory systemic response during AVF-CHF. During AVF, there was no change in the cardiac eNOS, Wnt1, or ß-catenin; however, during RDN, there were robust increases in the levels of eNOS, Wnt1, and ß-catenin compared to the sham group. As expected in HFpEF, there was perivascular fibrosis, hypertrophy, and pEF. Interestingly, increased levels of eNOS suggested that despite fibrosis, the NO generation was higher and that it most likely contributed to pEF during HF. The RDN intervention revealed an increase in renal cortical caspase 8 and a decrease in caspase 9. Since caspase 8 is protective and caspase 9 is apoptotic, we suggest that RDN protects against the renal stress and apoptosis. It should be noted that others have demonstrated a role of vascular endothelium in preserving the ejection by cell therapy intervention. In the light of foregoing evidence, our findings also suggest that RDN is cardioprotective during HFpEF via preservation of the eNOS and accompanied endocardial-endothelial function.

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.

Sustained Inhibition of NF-κB Activity Mitigates Retinal Vasculopathy in Diabetes.

This study investigated the effects of long-term NF-κB inhibition in mitigating retinal vasculopathy in a type 1 diabetic mouse model (Akita, Ins2Akita). Akita and wild-type (C57BL/6J) male mice, 24 to 26 weeks old, were treated with or without a selective inhibitor of NF-κB, 4-methyl-N1-(3-phenyl-propyl) benzene-1,2-diamine (JSH-23), for 4 weeks. Treatment was given when the mice were at least 24 weeks old. Metabolic parameters, key inflammatory mediators, blood-retinal barrier junction molecules, retinal structure, and function were measured. JSH-23 significantly lowered basal glucose levels and intraocular pressure in Akita. It also mitigated vascular remodeling and microaneurysms significantly. Optical coherence tomography of untreated Akita showed thinning of retinal layers; however, treatment with JSH-23 could prevent it. Electroretinogram demonstrated that A- and B-waves in Akita were significantly smaller than in wild type mice, indicating that JSH-23 intervention prevented loss of retinal function. Protein levels and gene expression of key inflammatory mediators, such as NOD-like receptor family pyrin domain-containing 3, intercellular adhesion molecule-1, inducible nitric oxide synthase, and cyclooxygenase-2, were decreased after JSH-23 treatment. At the same time, connexin-43 and occludin were maintained. Vision-guided behavior also improved significantly. The results show that reducing inflammation could protect the diabetic retina and its vasculature. Findings appear to have broader implications in treating not only ocular conditions but also other vasculopathies.

COVID-19 Mimics Pulmonary Dysfunction in Muscular Dystrophy as a Post-Acute Syndrome in Patients.

Although progressive wasting and weakness of respiratory muscles are the prominent hallmarks of Duchenne muscular dystrophy (DMD) and long-COVID (also referred as the post-acute sequelae of COVID-19 syndrome); however, the underlying mechanism(s) leading to respiratory failure in both conditions remain unclear. We put together the latest relevant literature to further understand the plausible mechanism(s) behind diaphragm malfunctioning in COVID-19 and DMD conditions. Previously, we have shown the role of matrix metalloproteinase-9 (MMP9) in skeletal muscle fibrosis via a substantial increase in the levels of tumor necrosis factor-α (TNF-α) employing a DMD mouse model that was crossed-bred with MMP9-knockout (MMP9-KO or MMP9-/-) strain. Interestingly, recent observations from clinical studies show a robust increase in neopterin (NPT) levels during COVID-19 which is often observed in patients having DMD. What seems to be common in both (DMD and COVID-19) is the involvement of neopterin (NPT). We know that NPT is generated by activated white blood cells (WBCs) especially the M1 macrophages in response to inducible nitric oxide synthase (iNOS), tetrahydrobiopterin (BH4), and tetrahydrofolate (FH4) pathways, i.e., folate one-carbon metabolism (FOCM) in conjunction with epigenetics underpinning as an immune surveillance protection. Studies from our laboratory, and others researching DMD and the genetically engineered humanized (hACE2) mice that were administered with the spike protein (SP) of SARS-CoV-2 revealed an increase in the levels of NPT, TNF-α, HDAC, IL-1ß, CD147, and MMP9 in the lung tissue of the animals that were subsequently accompanied by fibrosis of the diaphragm depicting a decreased oscillation phenotype. Therefore, it is of interest to understand how regulatory processes such as epigenetics involvement affect DNMT, HDAC, MTHFS, and iNOS that help generate NPT in the long-COVID patients.

Multi-organ damage by covid-19: congestive (cardio-pulmonary) heart failure, and blood-heart barrier leakage.

Corona virus disease-19 (covid-19) is caused by a coronavirus that is also known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and is generally characterized by fever, respiratory inflammation, and multi-organ failure in susceptible hosts. One of the first things during inflammation is the response by acute phase proteins coupled with coagulation. The angiotensinogen (a substrate for hypertension) is one such acute phase protein and goes on to explain an association of covid-19 with that of angiotensin-converting enzyme-2 (ACE2, a metallopeptidase). Therefore, it is advisable to administer, and test the efficacy of specific blocker(s) of angiotensinogen such as siRNAs or antibodies to covid-19 subjects. Covid-19 activates neutrophils, macrophages, but decreases T-helper cells activity. The metalloproteinases promote the activation of these inflammatory immune cells, therefore; we surmise that doxycycline (a metalloproteinase inhibitor, and a safer antibiotic) would benefit the covid-19 subjects. Along these lines, an anti-acid has also been suggested for mitigation of the covid-19 complications. Interestingly, there are three primary vegetables (celery, carrot, and long-squash) which are alkaline in their pH-range as compared to many others. Hence, treatment with fresh juice (without any preservative) from these vegies or the antioxidants derived from purple carrot and cabbage together with appropriate anti-coagulants may also help prevent or lessen the detrimental effects of the covid-19 pathological outcomes. These suggested remedies might be included in the list of putative interventions that are currently being investigated towards mitigating the multi-organ damage by Covid-19 during the ongoing pandemic.

Epigenetic memory: gene writer, eraser and homocysteine.

Naturally chromatin remodeling is highly organized, consisting of histone acetylation (opening/relaxation of the compact chromatin structure), DNA methylation (inhibition of the gene expression activity) and sequence rearrangement by shifting. All this is essentially required for proper "in-printing and off-printing" of genes thus ensuring the epigenetic memory process. Any imbalance in ratios of DNA methyltransferase (DNMT, gene writer), fat-mass obesity-associated protein (FTO, gene eraser) and product (function) homocysteine (Hcy) could lead to numerous diseases. Interestingly, a similar process also happens in stem cells during embryogenesis and development. Despite gigantic unsuccessful efforts undertaken thus far toward the conversion of a stem cell into a functional cardiomyocyte, there has been hardly any study that shows successful conversion of a stem cell into a multinucleated cardiomyocyte. We have shown nuclear hypertrophy during heart failure, however; the mechanism(s) of epigenetic memory, regulation of genes during fertilization, embryogenesis, development and during adulthood remain far from understanding. In addition, there may be a connection of aging, loosing of the memory leading to death, and presumably to reincarnation. This review highlights some of these pertinent issues facing the discipline of biology as a whole today.

Regulation of the parental gene GRM4 by circGrm4 RNA transcript and glutamate-mediated neurovascular toxicity in eyes.

Epigenetic memory plays crucial roles in gene regulation. It not only modulates the expression of specific genes but also has ripple effects on transcription as well as translation of other genes. Very often an alteration in expression occurs either via methylation or demethylation. In this context, "1-carbon metabolism" assumes a special significance since its dysregulation by higher levels of homocysteine; Hcy (known as hyperhomocysteinemia; HHcy), a byproduct of "1-Carbon Metabolism" during methionine biosynthesis leads to serious implications in cardiovascular, renal, cerebrovascular systems, and a host of other conditions. Currently, the circular RNAs (circRNAs) generated via non-canonical back-splicing events from the pre-mRNA molecules are at the center stage for their essential roles in diseases via their epigenetic manifestations. We recently identified a circular RNA transcript (circGRM4) that is significantly upregulated in the eye of cystathionine ß-synthase-deficient mice. We also discovered a concurrent over-expression of the mGLUR4 receptor in the eyes of these mice. In brief, circGRM4 is selectively transcribed from its parental mGLUR4 receptor gene (GRM4) functions as a "molecular-sponge" for the miRNAs and results into excessive turnover of the mGLUR4 receptor in the eye in response to extremely high circulating glutamate concentration. We opine that this epigenetic manifestation potentially predisposes HHcy people to retinovascular malfunctioning.

Hyperhomocysteinemia: an instigating factor for periodontal disease.

Hyperhomocysteinemia (HHcy) affects bone remodeling, since a destructive process in cortical alveolar bone has been linked to it; however, the mechanism remains at large. HHcy increases proinflammatory cytokines viz. TNF-α, IL-1b, IL-6, and IL-8 that leads to a cascade that negatively impacts methionine metabolism and homocysteine cycling. Further, chronic inflammation decreases vitamins B12, B6, and folic acid that are required for methionine homocysteine homeostasis. This study aims to investigate a HHcy mouse model (cystathionine ß-synthase deficient, CBS+/-) for studying the potential pathophysiological changes, if any, in the periodontium (gingiva, periodontal ligament, cement, and alveolar bone). We compared the periodontium side-by-side in the CBS+/- model with that of the wild-type (C57BL/6J) mice. Histology and histomorphometry of the mandibular bone along with gene expression analyses were carried out. Also, proangiogenic proteins and metalloproteinases were studied. To our knowledge, this research shows, for the first time, a direct connection between periodontal disease during CBS deficiency, thereby suggesting the existence of disease drivers during the hyperhomocysteinemic condition. Our findings offer opportunities to develop diagnostics/therapeutics for people who suffer from chronic metabolic disorders like HHcy.

Protecting the aging eye with hydrogen sulfide.

Research demonstrates that senescence is associated with tissue and organ dysfunction, and the eye is no exception. Sequelae arising from aging have been well defined as distinct clinical entities and vision impairment has significant psychosocial consequences. Retina and adjacent tissues like retinal pigmented epithelium and choroid are the key structures that are required for visual perception. Any structural and functional changes in retinal layers and blood retinal barrier could lead to age-related macular degeneration, diabetic retinopathy, and glaucoma. Further, there are significant oxygen gradients in the eye that can lead to excessive reactive oxygen species, resulting in endoplasmic reticulum and mitochondrial stress response. These radicals are source of functional and morphological impairment in retinal pigmented epithelium and retinal ganglion cells. Therefore, ocular diseases could be summarized as disturbance in the redox homeostasis. Hyperhomocysteinemia is a risk factor and causes vascular occlusive disease of the retina. Interestingly, hydrogen sulfide (H2S) has been proven to be an effective antioxidant agent, and it can help treat diseases by alleviating stress and inflammation. Concurrent glutamate excitotoxicity, endoplasmic reticulum stress, and microglia activation are also linked to stress; thus, H2S may offer additional interventional strategy. A refined understanding of the aging eye along with H2S biology and pharmacology may help guide newer therapies for the eye.

High-methionine diet in skeletal muscle remodeling: epigenetic mechanism of homocysteine-mediated growth retardation.

Epigenetic DNA methylation (1-carbon metabolism) is crucial for gene imprinting/off-printing that ensures epigenetic memory but also generates a copious amount of homocysteine (Hcy), unequivocally. That is why during pregnancy, expectant mothers are recommended "folic acid" preemptively to avoid birth defects in the young ones because of elevated Hcy levels (i.e., hyperhomocysteinemia (HHcy)). As we know, children born with HHcy have several musculoskeletal abnormalities, including growth retardation. Here, we focus on the gut-dysbiotic microbiome implication(s) that we believe instigates the "1-carbon metabolism" and HHcy causing growth retardation along with skeletal muscle abnormalities. We test our hypothesis whether high-methionine diet (HMD) (an amino acid that is high in red meat), a substrate for Hcy, can cause skeletal muscle and growth retardation, and treatment with probiotics (PB) to mitigate skeletal muscle dysfunction. To test this, we employed cystathionine ß-synthase, CBS deficient mouse (CBS+/-) fed with/without HMD and with/without a probiotic (Lactobacillus rhamnosus) in drinking water for 16 weeks. Matrix metalloproteinase (MMP) activity, a hallmark of remodeling, was measured by zymography. Muscle functions were scored via electric stimulation. Our results suggest that compared to the wild-type, CBS+/- mice exhibited reduced growth phenotype. MMP-2 activity was robust in CBS+/- and HMD effects were successfully attenuated by PB intervention. Electrical stimulation magnitude was decreased in CBS+/- and CBS+/- treated with HMD. Interestingly; PB mitigated skeletal muscle growth retardation and atrophy. Collectively, results imply that individuals with mild/moderate HHcy seem more prone to skeletal muscle injury and its dysfunction.

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.

Mechanism of Blood-Heart-Barrier Leakage: Implications for COVID-19 Induced Cardiovascular Injury.

Although blood-heart-barrier (BHB) leakage is the hallmark of congestive (cardio-pulmonary) heart failure (CHF), the primary cause of death in elderly, and during viral myocarditis resulting from the novel coronavirus variants such as the severe acute respiratory syndrome novel corona virus 2 (SARS-CoV-2) known as COVID-19, the mechanism is unclear. The goal of this project is to determine the mechanism of the BHB in CHF. Endocardial endothelium (EE) is the BHB against leakage of blood from endocardium to the interstitium; however, this BHB is broken during CHF. Previous studies from our laboratory, and others have shown a robust activation of matrix metalloproteinase-9 (MMP-9) during CHF. MMP-9 degrades the connexins leading to EE dysfunction. We demonstrated juxtacrine coupling of EE with myocyte and mitochondria (Mito) but how it works still remains at large. To test whether activation of MMP-9 causes EE barrier dysfunction, we hypothesized that if that were the case then treatment with hydroxychloroquine (HCQ) could, in fact, inhibit MMP-9, and thus preserve the EE barrier/juxtacrine signaling, and synchronous endothelial-myocyte coupling. To determine this, CHF was created by aorta-vena cava fistula (AVF) employing the mouse as a model system. The sham, and AVF mice were treated with HCQ. Cardiac hypertrophy, tissue remodeling-induced mitochondrial-myocyte, and endothelial-myocyte contractions were measured. Microvascular leakage was measured using FITC-albumin conjugate. The cardiac function was measured by echocardiography (Echo). Results suggest that MMP-9 activation, endocardial endothelial leakage, endothelial-myocyte (E-M) uncoupling, dyssynchronous mitochondrial fusion-fission (Mfn2/Drp1 ratio), and mito-myocyte uncoupling in the AVF heart failure were found to be rampant; however, treatment with HCQ successfully mitigated some of the deleterious cardiac alterations during CHF. The findings have direct relevance to the gamut of cardiac manifestations, and the resultant phenotypes arising from the ongoing complications of COVID-19 in human subjects.

Impaired Folate-Mediated One-Carbon Metabolism in Type 2 Diabetes, Late-Onset Alzheimer's Disease and Long COVID.

Impaired folate-mediated one-carbon metabolism (FOCM) is associated with many pathologies and developmental abnormalities. FOCM is a metabolic network of interdependent biosynthetic pathways that is known to be compartmentalized in the cytoplasm, mitochondria and nucleus. Currently, the biochemical mechanisms and causal metabolic pathways responsible for the initiation and/or progression of folate-associated pathologies have yet to be fully established. This review specifically examines the role of impaired FOCM in type 2 diabetes mellitus, Alzheimer's disease and the emerging Long COVID/post-acute sequelae of SARS-CoV-2 (PASC). Importantly, elevated homocysteine may be considered a biomarker for impaired FOCM, which is known to result in increased oxidative-redox stress. Therefore, the incorporation of hyperhomocysteinemia will be discussed in relation to impaired FOCM in each of the previously listed clinical diseases. This review is intended to fill gaps in knowledge associated with these clinical diseases and impaired FOCM. Additionally, some of the therapeutics will be discussed at this early time point in studying impaired FOCM in each of the above clinical disease states. It is hoped that this review will allow the reader to better understand the role of FOCM in the development and treatment of clinical disease states that may be associated with impaired FOCM and how to restore a more normal functional role for FOCM through improved nutrition and/or restoring the essential water-soluble B vitamins through oral supplementation.

Cardioprotective effects of high-intensity interval training are mediated through microRNA regulation of mitochondrial and oxidative stress pathways.

Human studies have shown high-intensity interval training (HIIT) has beneficial cardiovascular effects and is typically more time-efficient compared with traditional endurance exercise. The main goal of this study is to show the potential molecular and functional cardiovascular benefits of HIIT compared with endurance training (ET). Three groups of mice were used including sedentary-control, ET mice, and HIIT mice groups. Results indicated ejection fraction was increased in HIIT compared with ET while fractional shortening was increased in the HIIT group compared with both groups. Blood flow of the abdominal aorta was increased in both exercise groups compared with control. Increases in cross-sectional area and mitochondrial and antioxidative markers in HIIT compared with control were observed, along with several microRNAs. These findings indicate HIIT has specific cardiac-protective effects and may be a viable alternative to traditional ET as a cardiovascular preventative medicine intervention.

Dysbiotic 1-carbon metabolism in cardiac muscle remodeling.

Unless there is a genetic defect/mutation/deletion in a gene, the causation of a given disease is chronic dysregulation of gut metabolism. Most of the time, if not always, starts within the gut; that is what we eat. Recent research shows that the imbalance between good versus bad microbial population, especially in the gut, causes systemic diseases. Thus, an appropriate balance of the gut microbiota (eubiosis over dysbiosis) needs to be maintained for normal health (Veeranki and Tyagi, 2017, Journal of Cellular Physiology, 232, 2929-2930). However, during various diseases such as metabolic syndrome, inflammatory bowel disease, diabetes, obesity, and hypertension the dysbiotic gut environment tends to prevail. Our research focuses on homocysteine (Hcy) metabolism that occupies a center-stage in many biochemically relevant epigenetic mechanisms. For example, dysbiotic bacteria methylate promoters to inhibit gene activities. Interestingly, the product of the 1-carbon metabolism is Hcy, unequivocally. Emerging studies show that host resistance to various antibiotics occurs due to inverton promoter inhibition, presumably because of promoter methylation. This results from modification of host promoters by bacterial products leading to loss of host's ability to drug compatibility and system sensitivity. In this study, we focus on the role of high methionine diet (HMD), an ingredient rich in red meat and measure the effects of a probiotic on cardiac muscle remodeling and its functions. We employed wild type (WT) and cystathionine beta-synthase heterozygote knockout (CBS+/- ) mice with and without HMD and with and without a probiotic; PB (Lactobacillus) in drinking water for 16 weeks. Results indicate that matrix metalloproteinase-2 (MMP-2) activity was robust in CBS+/- fed with HMD and that it was successfully attenuated by the PB treatment. Cardiomyocyte contractility and ECHO data revealed mitigation of the cardiac dysfunction in CBS+/- + HMD mice treated with PB. In conclusion, our data suggest that probiotics can potentially reverse the Hcy-meditated cardiac dysfunction.

Genes and genetics in hyperhomocysteinemia and the "1-carbon metabolism": implications for retinal structure and eye functions.

Homocysteine (Hcy), a sulfur-containing nonproteinogenic amino acid, is generated as a metabolic intermediate. Hcy constitutes an important part of the "1-carbon metabolism" during methionine turnover. Elevated levels of Hcy known as hyperhomocysteinemia (HHcy) results from vitamin B deficiency, lack of exercise, smoking, excessive alcohol intake, high-fat and methionine-rich diet, and the underlying genetic defects. These factors directly affect the "1-carbon metabolism (methionine-Hcy-folate)" of a given cell. In fact, the Hcy levels are determined primarily by dietary intake, vitamin status, and the genetic blueprint of the susceptible individual. Although Hcy performs an important role in cellular functions, genetic alterations in any of the key enzymes responsible for the "1-carbon metabolism" could potentially upset the metabolic cycle, thus causing HHcy environment in susceptible people. As such, HHcy relates to several clinical conditions like atherosclerosis, myocardial infarction, stroke, cognitive impairment, dementia, Parkinson's disease, multiple sclerosis, epilepsy, and ocular disorders, among others. This article summarizes the findings from our laboratory and public database regarding genetics of HHcy and its effects on ocular disorders, their respective management during dysregulation of the 1-carbon metabolism.