Temporal Modulation of DNA Methylation and Gene Expression in Monolayer and 3D Spheroids of Dental Pulp Stem Cells during Osteogenic Differentiation: A Comparative Study.

BACKGROUND: Human mesenchymal stem cells are being used for various regenerative applications in past decades. This study chronicled a temporal profile of the transcriptional pattern and promoter methylation status of the osteogenic related gene in dental pulp stem cells (DPSCs) derived from 3-dimensional spheroid culture (3D) vis a vis 2-dimensional (2D) monolayer culture upon osteogenic induction. METHODS: Biomimetic properties of osteogenesis were determined by alkaline phosphatase assay and alizarin red staining. Gene expression and promoter methylation status of osteogenic genes such as runt-related transcription factor-2, collagen1α1, osteocalcin (OCN), and DLX5 (distal-homeobox) were performed by qPCR assay and bisulfite sequencing, respectively. Furthermore, µ-Computed tomography (micro-CT) was performed to examine the new bone formation in critical-sized rat calvarial bone defect model. RESULTS: Our results indicated a greater inclination of spheroid culture-derived DPSCs toward osteogenic lineage than the monolayer culture. The bisulfite sequencing of the promoter region of osteogenic genes revealed sustenance of low methylation levels in DPSCs during the progression of osteogenic differentiation. However, the significant difference in the methylation pattern between 2D and 3D derived DPSCs were identified only for OCN gene promoter. We observed differences in the mRNA expression pattern of epigenetic writers such as DNA methyltransferases (DNMTs) and methyl-cytosine dioxygenases (TET) between the two culture conditions. Further, the DPSC spheroids showed enhanced new bone formation ability in an animal model of bone defect compared to the cells cultivated in a 2D platform which further substantiated our in-vitro observations. CONCLUSION: The distinct cellular microenvironment induced changes in DNA methylation pattern and expression of epigenetic regulators such as DNMTs and TETs genes may lead to increase expression of osteogenic markers in 3D spheroid culture of DPSCs which make DPSCs spheroids suitable for osteogenic regeneration compared to monolayers.

TBK1 and GABARAP family members suppress Coxsackievirus B infection by limiting viral production and promoting autophagic degradation of viral extracellular vesicles.

Host-pathogen dynamics are constantly at play during enteroviral infection. Coxsackievirus B (CVB) is a common juvenile enterovirus that infects multiple organs and drives inflammatory diseases including acute pancreatitis and myocarditis. Much like other enteroviruses, CVB is capable of manipulating host machinery to hijack and subvert autophagy for its benefit. We have previously reported that CVB triggers the release of infectious extracellular vesicles (EVs) which originate from autophagosomes. These EVs facilitate efficient dissemination of infectious virus. Here, we report that TBK1 (Tank-binding kinase 1) suppresses release of CVB-induced EVs. TBK1 is a multimeric kinase that directly activates autophagy adaptors for efficient cargo recruitment and induces type-1 interferons during viral-mediated STING recruitment. Positioning itself at the nexus of pathogen elimination, we hypothesized that loss of TBK1 could exacerbate CVB infection due to its specific role in autophagosome trafficking. Here we report that infection with CVB during genetic TBK1 knockdown significantly increases viral load and potentiates the bulk release of viral EVs. Similarly, suppressing TBK1 with small interfering RNA (siRNA) caused a marked increase in intracellular virus and EV release, while treatment in vivo with the TBK1-inhibitor Amlexanox exacerbated viral pancreatitis and EV spread. We further demonstrated that viral EV release is mediated by the autophagy modifier proteins GABARAPL1 and GABARAPL2 which facilitate autophagic flux. We observe that CVB infection stimulates autophagy and increases the release of GABARAPL1/2-positive EVs. We conclude that TBK1 plays additional antiviral roles by inducing autophagic flux during CVB infection independent of interferon signaling, and the loss of TBK1 better allows CVB-laden autophagosomes to circumvent lysosomal degradation, increasing the release of virus-laden EVs. This discovery sheds new light on the mechanisms involved in viral spread and EV propagation during acute enteroviral infection and highlights novel intracellular trafficking protein targets for antiviral therapy.

Asporin, an extracellular matrix protein, is a beneficial regulator of cardiac remodeling.

Heart failure is accompanied by adverse cardiac remodeling involving extracellular matrix (ECM). Cardiac ECM acts as a major reservoir for many proteins including growth factors, cytokines, collagens, and proteoglycans. Activated fibroblasts during cardiac injury can alter the composition and activity of these ECM proteins. Through unbiased analysis of a microarray dataset of human heart tissue comparing normal hearts (n = 135) to hearts with ischemic cardiomyopathy (n = 94), we identified Asporin (ASPN) as the top differentially regulated gene (DEG) in ischemic cardiomyopathy; its gene-ontology terms relate closely to fibrosis and cell death. ASPN is a Class I small leucine repeat protein member implicated in cancer, osteoarthritis, and periodontal ligament mineralization. However, its role in cardiac remodeling is still unknown. Here, we initially confirmed our big dataset analysis through cells, mice, and clinical atrial biopsy samples to demonstrate increased Aspn expression after pressure overload or cardiac ischemia/reperfusion injury. We tested the hypothesis that Aspn, being a TGFß1 inhibitor, can attenuate fibrosis in mouse models of cardiac injury. We found that Aspn is released by cardiac fibroblasts and attenuates TGFß signaling. Moreover, Aspn-/- mice displayed increased fibrosis and decreased cardiac function after pressure overload by transverse aortic constriction (TAC) in mice. In addition, Aspn protected cardiomyocytes from hypoxia/reoxygenation-induced cell death and regulated mitochondrial bioenergetics in cardiomyocytes. Increased infarct size after ischemia/reperfusion injury in Aspn-/- mice confirmed Aspn's contribution to cardiomyocyte viability. Echocardiography revealed greater reduction in left ventricular systolic function post-I/R in the Aspn-/- animals compared to wild type. Furthermore, we developed an ASPN-mimic peptide using molecular modeling and docking which when administered to mice prevented TAC-induced fibrosis and preserved heart function. The peptide also reduced infarct size after I/R in mice, demonstrating the translational potential of ASPN-based therapy. Thus, we establish the role of ASPN as a critical ECM molecule that regulates cardiac remodeling to preserve heart function.

Role of Oncofetal Protein CR-1 as a Potential Tumor Marker for Oral Squamous Cell Carcinoma.

Cripto-1 (CR-1) is an oncofetal protein with its role as a key factor in early process of carcinoma has been evaluated in cases of various cancers. However, very few studies have reported its role in oral cancer, which is the sixth most common cancer around the world, particularly with high prevalence in developing countries. Oral squamous cell carcinoma (OSCC) is the most predominant (90%) of all the histological types of oral cancer. Late detection, associated with increased morbidity and mortality, is mainly attributed to non-availability of a suitable biomarker for the disease. In the present pilot study, we have evaluated the role of soluble CR-1, in serum as a potential tumor marker for OSCC. CR-1 was estimated using sandwich ELISA in serum samples of 50 biopsy proven OSCC patients (pre and post treatment) along with age and gender matched healthy controls. Immunohistochemistry was also done in corresponding tumor tissue sections to check the expression of CR-1. Pre-treatment CR-1 was found to be 2.25-fold higher in serum of OSCC patients as compared to control (p < 0.0001***), which was reduced to 1.6 folds post treatment (p = 0.0006***). CR-1 levels were comparatively higher in early stage of disease. Upon IHC 80% of the cases were found to be positive for CR-1. This study provides evidence that serum levels of CR-1 are elevated in patients of Oral Squamous Cell Carcinoma, which decrease post treatment. Also, the association of expression of protein with tumor progression predicts CR-1 as a molecule that can be further evaluated as a potential tumor maker in OSCC.

Attenuation of Adverse Postinfarction Left Ventricular Remodeling with Empagliflozin Enhances Mitochondria-Linked Cellular Energetics and Mitochondrial Biogenesis.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors such as empagliflozin are known to reduce the risk of hospitalizations related to heart failure irrespective of diabetic state. Meanwhile, adverse cardiac remodeling remains the leading cause of heart failure and death in the USA. Thus, understanding the mechanisms that are responsible for the beneficial effects of SGLT2 inhibitors is of the utmost relevance and importance. Our previous work illustrated a connection between adverse cardiac remodeling and the regulation of mitochondrial turnover and cellular energetics using a short-acting glucagon-like peptide-1 receptor agonist (GLP1Ra). Here, we sought to determine if the mechanism of the SGLT2 inhibitor empagliflozin (EMPA) in ameliorating adverse remodeling was similar and/or to identify what differences exist, if any. To this end, we administered permanent coronary artery ligation to induce adverse remodeling in wild-type and Parkin knockout mice and examined the progression of adverse cardiac remodeling with or without EMPA treatment over time. Like GLP1Ra, we found that EMPA affords a robust attenuation of PCAL-induced adverse remodeling. Interestingly, unlike the GLP1Ra, EMPA does not require Parkin to improve/maintain mitochondria-related cellular energetics and afford its benefits against developing adverse remodeling. These findings suggests that further investigation of EMPA is warranted as a potential path for developing therapy against adverse cardiac remodeling for patients that may have Parkin and/or mitophagy-related deficiencies.

Pancreas-specific deletion of protein kinase D attenuates inflammation, necrosis, and severity of acute pancreatitis.

BACKGROUND: Protein kinase D (PKD) family, which includes PKD/PKD1, PKD2, and PKD3, has been increasingly implicated in the regulation of multiple cellular functions and human diseases. We recently reported that pharmacologic inhibition of PKD ameliorated the pathologic responses and severity of pancreatitis. However, to further investigate the importance of PKD family members in pancreatitis, it is necessary to explore the effects of pancreas-specific genetic inhibition of PKD isoform on pathology of pancreatitis. METHODS: We generated a mouse model (referred as PKD3Δpanc mice) with pancreas-specific deletion of PKD3, the predominant PKD isoform in mouse pancreatic acinar cells, by crossing Pkd3flox/flox mice with Pdx1-Cre transgenic mice which express Cre recombinase under the control of the mouse Pdx1 promoter. Pancreas-specific deletion of the PKD3 gene and PKD3 protein was confirmed by PCR and Western blot analysis. Experimental pancreatitis was induced in PKD3Δpanc and Pkd3flox/flox (control mice) littermates by intraperitoneal injections of cerulein or L-arginine. RESULTS: Compared to the control mice, PKD3Δpanc mice displayed significant attenuation in inflammation, necrosis, and severity of pancreatitis in both experimental models. PKD3Δpanc mice had markedly decreased NF-κB and trypsinogen activation, pancreatic mRNA expression of multiple inflammatory molecules, and the receptor-interacting protein kinase 1 (RIP1) activation in pancreatitis. PKD3Δpanc mice also had less pancreatic ATP depletion, increased pro-survival Bcl-2 family protein expression, and autophagy promotion. CONCLUSION: With PKD3Δpanc mouse model, we further demonstrated that PKD plays a critical role in pathobiological process of pancreatitis and PKD constitutes a novel therapeutic target to treat this disorder.

A randomised trial of vitamin D in acute-stage allergic bronchopulmonary aspergillosis complicating asthma.

Vitamin D has been demonstrated to have an immunomodulatory role in cystic fibrosis with allergic bronchopulmonary aspergillosis (ABPA). Herein, we evaluate supplemental vitamin D in acute-stage ABPA complicating asthma. Thirty subjects were randomised to receive either prednisolone (n = 15, control) or prednisolone and oral vitamin D (n = 15, intervention arm). Serum vitamin D levels were significantly higher in the intervention versus the control arm, following therapy. The mean decline in total IgE at 2 months (primary outcome) was 10% higher in the intervention arm than the control arm; however, this was not statistically significant (48.6% vs 38.1%, P = 0.27). The percentage decline in total IgE after 4 and 6 months of randomisation was also similar in the two arms. There was no difference in asthma exacerbations (0 vs 2, intervention vs control; P = 0.16). No ABPA exacerbation occurred in either arm. The other outcomes including the Th2 (IL-4, IL-6 and IL-10) and Th17 (IL-17) cytokine levels were similar in the two groups. None of the participants experienced hypervitaminosis D. There was no significant improvement in the clinical or immunological outcomes following vitamin D supplementation. A larger trial is required to ascertain the role of vitamin D in ABPA complicating asthma [Clinicaltrials.gov: NCT03133299].