Deep sequencing unveils altered cardiac miRNome in congenital heart disease.

Congenital heart disease (CHD) surges from fetal cardiac dysmorphogenesis and chiefly contributes to perinatal morbidity and cardiovascular disease mortality. A continual rise in prevalence and prerequisite postoperative disease management creates need for better understanding and new strategies to control the disease. The interaction between genetic and non-genetic factors roots the multifactorial status of this disease, which remains incompletely explored. The small non-coding microRNAs (miRs, miRNAs) regulate several biological processes via post-transcriptional regulation of gene expression. Abnormal expression of miRs in developing and adult heart is associated with anomalous cardiac cell differentiation, cardiac dysfunction, and cardiovascular diseases. Here, we attempt to discover the changes in cardiac miRNA transcriptome in CHD patients over those without CHD (non-CHD) and find its role in CHD through functional annotation. This study explores the miRNome in three most commonly occurring CHD subtypes, namely atrial septal defect (ASD), ventricular septal defect (VSD), and tetralogy of fallot (TOF). We found 295 dysregulated miRNAs through high-throughput sequencing of the cardiac tissues. The bioinformatically predicted targets of these differentially expressed miRs were functionally annotated to know they were entailed in cell signal regulatory pathways, profoundly responsible for cell proliferation, survival, angiogenesis, migration and cell cycle regulation. Selective miRs (hsa-miR-221-3p, hsa-miR-218-5p, hsa-miR-873-5p) whose expression was validated by qRT-PCR, have been reported for cardiogenesis, cardiomyocyte proliferation, cardioprotection and cardiac dysfunction. These results indicate that the altered miRNome to be responsible for the disease status in CHD patients. Our data expand the existing knowledge on the epigenetic changes in CHD. In future, characterization of these cardiac-specific miRs will add huge potential to understand cardiac development, function, and molecular pathogenesis of heart diseases with a prospect of epigenetic manipulation for cardiac repair.

Low frequency magnetic force augments hepatic differentiation of mesenchymal stem cells on a biomagnetic nanofibrous scaffold.

Liver tissue engineering using polymeric nanofibrous scaffold and stem cells holds great promises for treating end-stage liver failures. The aim of this study was to evaluate hepatic trans-differentiation potential of human mesenchymal stem cells (hMSCs) on a biomagnetic electrospun nanofibrous scaffold fabricated from a blend of poly-L-lactide (PLLA), collagen and fibrin-rich blood clot, under the influence of a low frequency magnetic field. The scaffold was characterized for surface properties, biochemical and biomechanical parameters and bio-magnetic behaviour. Cell proliferation assay revealed that the scaffold was suitable for hMSCs adhesion and proliferation. Hepatic trans-differentiation potential of hMSCs was augmented on nanofibrous scaffold in magnetic field exposure group compared to control groups, as evident by strong expression of hepatocyte specific markers, albumin release, urea synthesis and presence of an inducible cytochrome P450 system. Our results conclude that biomagnetic scaffold of PLLA/collagen/blood clot augments hepatic trans-differentiation of hMSCs under magnetic field influence.

Biodegradable nanofiber coated human umbilical cord as nerve scaffold for sciatic nerve regeneration in albino Wistar rats.

BACKGROUND: Human umbilical cord (hUC) is encompassed by a mucoid connective tissue called Wharton's jelly (WJ), made of hyaluronic acid, collagen, and stromal cells to support the blood vessels of hUC. This study was aimed to determine the in vitro neuronal differentiation of WJ-derived mesenchymal stem cells (WJMSCs), and in vivo axonal regeneration potential of nanofiber coated human Wharton's jelly as a neuronal graft after sciatic nerve injury in immunosuppressed albino Wistar rats. MATERIALS AND METHODS: Wharton's jelly-derived mesenchymal stem cells could be differentiated to neuron-like cells by inducing with neuronic supplementing media. The test animal's axotomized nerves were implanted with trimmed human umbilical cord devoid of vascularity and nanocoated with electro-spun poly-l-lactic acid nanofibers. The control animals were bridged with native sciatic nerve reversed and sutured. Post-surgical functional recovery was studied by walking track, pinprick, muscle weight, and sweating quantification. At the end of the 4th week, the animals were euthanized, and magnetoneurography was performed. The explanted grafts were quantified by immunohistochemistry for immuno-rejection, neural scarring, neural adhesion axon regeneration, fibre diameter, myelin thickness, and G-ratio. The sciatic function index values were similar by walking track analysis for both the test and control groups. RESULTS: The animals had functional and sensation recovery by the end of 2 weeks. No mortality, signs of inflammation, and acute immune rejection were observed post-surgery. CONCLUSIONS: The hUCWJ devoid of vascular elements can be a perfect peripheral nerve graft, and we hypothesis that the cryopreserved hUC could be an ideal resource for axonal regeneration in the future.

16S rRNA sequencing as a diagnostic tool in the identification of culture-negative endocarditis in surgically treated patients.

BACKGROUND AND AIM OF THE STUDY: Infective endocarditis (IE) is a worldwide problem, and at least one-third of cases are culture-negative despite the use of appropriate laboratory techniques. METHODS: A broad-range polymerase chain reaction (PCR) amplification was performed of the 16S rRNA gene, followed by single-strand sequencing for 26 surgically removed heart valves from patients with culture-negative endocarditis who had undergone valve repair or replacement. RESULTS: Two of the 26 patients were PCR-positive, and sequencing of the amplicon identified the etiological agent. Gram-stained smears of the heart valves were positive in both cases. Three of the remaining 24 cases which were negative by PCR also showed the presence of micro-organisms in Gram-stained smears. CONCLUSION: The study results emphasize that, in suspected IE cases when there is no growth in culture, a combination of microscopy and 16S rRNA sequencing can be used to identify the pathogen in excised valvular tissue.

Advanced imaging and digitization of preserved heart specimens using virtual reality - A primer.

Introduction: Preserved congenital heart specimens are an important component of training professionals working with children and adults with congenital heart disease. They are curated in few institutions worldwide and not freely accessible. This was a proof-of-concept project to explore the use of advanced cardiac imaging modalities (computed tomography [CT] and magnetic resonance imaging [MRI]) and virtual reality (VR) simulation to assess the feasibility and identify the best method of imaging curated cardiac pathology specimens. Methods: Seven specimens in glass jars with formalin, with varied anatomic lesions, from a curated collection were imaged using MRI and high-dose CT to compare the fidelity of models created via each modality. Three-dimensional (3D) models were created and loaded into a VR headset and viewed in virtual space. Two independent physicians performed a "virtual dissection" and scored the resultant models. Results: The highest fidelity and tissue characterization of more delicate structures was achieved with T2 spoiled gradient-echo sequences on MRI (median score of 4 out of 5). CT (median score of 3), while excellent for external anatomy, lost some fidelity with delicate internal anatomy, even at high-radiation doses. No specimens were damaged. Conclusions: We believe that in vitro heart specimens can be easily scanned with high fidelity at a relatively low cost, without causing damage, using high-dose CT and MRI. The ability to "walk through" different chambers of the heart makes the understanding of anatomy easy and intuitive. VR and 3D printing are technologies that could be easily adapted to digitize preserved heart specimens, making it globally accessible for teaching and training purposes.

Genetic variants on apolipoprotein gene cluster influence triglycerides with a risk of coronary artery disease among Indians.

Apolipoprotein C3 and apolipoprotien A5 are proteins coded from the APOA1/C3/A4/A5 gene cluster. Sst I polymorphism on apolipoprotein C3 and -1131C polymorphism of apolipoprotien A5 are key variants involved in triglyceride metabolism and cause a significant cardio-metabolic risk. Here, we have evaluated these two variants for their roles in coronary artery disease in patients of the Indian population. The apolipoprotein gene cluster variants were analysed in 416 angiographically determined coronary artery disease patients and matched 416 controls using polymerase chain reaction-restriction fragment length polymorphism. The characteristics of the study subjects were analyzed statistically for their association with the polymorphisms. The alleles were combined as haplotypes and their combined risks were evaluated. The minor allele genotypes of both apolipoprotein C3 (S2) and apolipoprotien A5 (C) had a significant risk for coronary artery disease. The S2 allele genotyped patients had a significantly increased triglyceride level (P < 0.001) and increased triglycerides were observed among both patient and control CC genotype carriers. We identified the haplotype S2/C with a significant increased risk (P < 0.001) to coronary artery disease with increased levels of circulating triglycerides compared to other haplotypes in patients. We conclude that the variants on apolipoprotein C3 and apolipoprotien A5 modulate serum triglyceride levels and increase the risk of coronary artery disease.

Isolated congenital pulmonary regurgitation with right ventricular outflow tract aneurysm--a rare variant of Uhl's anomaly.

Isolated absent pulmonary valve syndrome is a very rare entity. We report the case of a four-year-old boy who had congenital absent pulmonary valve with a thin and aneurysmal right ventricular infundibulum. The histological picture was suggestive of Uhl's anomaly. This association of partial right ventricular Uhl's anomaly with absent pulmonary valve syndrome has not been described before. We discuss the embryological and clinical significance of this association.

Fabrication and Electrospinning of 3D Biodegradable Poly-l-Lactic Acid (PLLA) Nanofibers for Clinical Application.

Poly-L-lactic acid (PLLA) is a biodegradable synthetic polyester synthesized by polymerization or polycondensation. PLLA hydrolytically degrades into lactic acid, a biocompatible metabolic by-product, making it suitable for clinical applications. PLLA scaffolds or nanofibers have been used in various regenerative medicine and drug delivery applications. These scaffolds impart biocompatible properties of high surface area, hydrophobicity, native extracellular properties, and mechanical strength for an organ system. Moreover, PLLA nanofibers hold great promise as drug delivery systems, where fabrication parameters and drug-PLA compatibility greatly affect the drug release kinetics. In this chapter, we present the protocols to fabricate, electrospinning, and validation of 3D PLLA nanofibrous scaffolds for tissue engineering application and offer perspectives on their future use.

A multiplex PCR technique to characterize human bone marrow derived mesenchymal stem cells.

Human mesenchymal stem cells (MSCs), with capacity to differentiate into adipocytes, osteoblasts and chondrocytes, offer potential for the development of novel treatments. A critical question in MSCs biology is whether this cell population possesses a relatively uniform differentiation capability or is comprised of distinct subsets of progenitors committed to differentiate in particular pathways. To quantify the changes during growth of MSCs, we analyzed the mesenchymal phenotype and differentiation ability using a multi-marker PCR with six primer sets specific for CD73, CD90, CD105, CD166, CD45 and beta-actin allowing a gel-based differential detection of the PCR products. To determine degree of variability of MSCs populations in terms of proliferation, cell proliferation assays were performed on expanded MSCs up to the sixth passage. At each passage, the osteogenic and adipogenic differentiation potentials of MSCs were verified by culture in inductive media. RT-PCR and cytochemical analysis revealed that, despite the loss of multipotentiality during expansion, certain markers remain expressed, indicating that these markers are unlikely to be reflective of the MSC's true 'stem cell' nature. Our results suggest that decrease in the expression of MSCs specific markers correlates with down-regulation of proliferation ability and differentiation efficiency of MSCs.

Left ventricular epicardial hydatid cyst compressing the left circumflex artery.

Cardiac hydatid is a rare disease with varied presentation. We report a unique case of left ventricular epicardial hydatid cyst causing left circumflex artery compression. Cardiac hydatids have to be surgically treated on diagnosis because of the high risk of catastrophic rupture. We discuss the surgical principles and the other adjuncts to avoid recurrence.

3D Printing to Model Surgical Repair of Complex Congenitally Corrected Transposition of the Great Arteries.

We report the use of three-dimensional (3D) modeling to plan surgery for physiologic repair of congenitally corrected transposition of the great arteries with pulmonary atresia, dextrocardia, and complex intra cardiac anatomy. Based on measurements made from the 3D printed model of the actual patient's anatomy, we anticipated using a composite valved conduit (Dacron tube graft, decellularized bovine jugular vein, and aortic homograft) to establish left ventricle-to-pulmonary artery continuity with relief of stenosis involving the pulmonary artery confluence and bilateral branch pulmonary arteries.

GSK-3 Inhibitors: A Double-Edged Sword? - An Update on Tideglusib.

GSK-3 inhibitors are an emerging tool for clinical interventions in human diseases and represent a niche area in combinational therapy. They possess diverse facets in applications of nervous system disorders, Type 2 diabetes, regenerative medicine and cancer. However, conflicting reports suggest the controversial role of GSK-3 inhibitors in cancers. This review aims to highlight the rise of GSK-3 inhibitors as tools for molecular-targeted research and its shift to a promising drug candidate. The review also focuses on key GSK-3 inhibitors and their roles in cancer and regenerative medicine with special emphasis to tideglusib. In addition, the decisive roles of GSK-3 in various molecular pathways will be concisely reviewed. Finally, this review concludes the emergence of GSK-3 inhibitors as a 'double-edged sword' in the treatment against human diseases cautioning researchers about the potential ramifications of off-target pharmacological effects.

In-stent restenosis: surgical and histopathological perspective.

Background In-stent restenosis has been recognized as a distinct clinical entity that warrants a repeat procedure either in the form of percutaneous reintervention or coronary artery bypass grafting. Multivessel grafting with endarterectomy and explantation of the stent is rarely performed, with few cases reported in the literature. We aim to study the pathomorphology of the stent-vascular interface in coronary vessels undergoing in-stent restenosis. Methods Over a period of 3 years, 3 patients who had undergone angioplasty for diffuse coronary artery disease developed in-stent restenosis and were advised coronary artery bypass. The mean age was 53 years, the average time from the previous intervention was 77 months. Coronary endarterectomy with stent removal and concomitant multivessel coronary artery bypass was performed. Results Histology showed significant proliferation of the well-endothelialized intima as the reason for in-stent restenosis. There were no signs of local thrombus formation or increased inflammatory activity in any of the specimens. After coronary artery bypass, all patients were asymptomatic at a mean follow-up of 32 months. Conclusion Coronary endarterectomy with stent explantation and multivessel coronary artery bypass is a procedure that requires attention because the need is increasing due to the rise in the number of angioplasties. The complexity of this procedure increases to the extent that the adventitia is involved during stent explantation.

MitraClip and mitral annuloplasty device as a bridge to transplantation.

A 42-year-old man with dilated cardiomyopathy and endstage heart failure was evaluated for heart transplantation. He received a MitraClip and Carillon annuloplasty device for functional mitral regurgitation as palliation for his heart failure. Subsequently, he underwent successful heart transplantation.

Right ventricular outflow tract obstruction: a quest for ideal management.

Management of right ventricular outflow tract obstruction has undergone much change over the last century. Techniques described in the literature include anatomical repairs and the use of various patches, conduits, and innovative grafts. However, many of these approaches require reoperations or catheter-based interventions, leading to increased morbidity, mortality, and cost. The search for the ideal long-lasting conduit continues and there are new techniques on the horizon, using genetic engineering and nanotechnology. This review discusses the evolution of various techniques for repair of right ventricular outflow tract obstruction, past and current conduits, as well as ongoing research.

Coronary artery to pulmonary artery fistula with dual origin--embryological, clinical and surgical significance.

Coronary artery fistulae are rare anomalies encountered in 0.1-0.2% of angiographic series. We recently encountered a patient evaluated for mitral valve disease who incidentally had bilateral coronary artery fistulae detected on preoperative angiogram. These fistulae drained into the pulmonary artery. She underwent successful transpulmonary closure of the fistula along with mitral valve repair. We discuss the embryological basis of this anomaly and the clinical as well as surgical significance.

Role of Antioxidants in Horse Serum-mediated Vasculitis in Swine: Potential Relevance to Early Treatment in Mitigation of Coronary Arteritis in Kawasaki Disease.

BACKGROUND: Horse serum-induced immune complex coronary vasculitis in swine is the first experimental model to mimic most of the pictures of Kawasaki disease. Immune complex mechanism has been implicated as one of the possible mechanisms in the pathogenesis of vasculitis in Kawasaki disease. Antioxidants have a significant role in the reduction of cardiovascular diseases in both human and animal studies. We tried giving vitamins A, E, and C to treat immune complex vasculitis, in the hope of mitigating coronary vasculitis in Kawasaki disease. METHODS: Our study group consisted of 30 pure bred male piglets of 2-3 months of age, and they were divided into test and control groups. The test (AEC) group (n = 20) received two doses of horse serum, 10 mL (0.65 g protein)/kg body weight at 5-day intervals, and oral vitamins A, E, and C once daily for 14 days. The control group (n = 10) was further divided into the saline group (n = 3) receiving two doses of normal saline and the horse serum group (n = 7) receiving two doses of horse serum at 5-day intervals. Piglets were observed for the rashes and coronary artery dimensions. RESULTS: Both the AEC and the control horse serum group developed rashes after horse serum infusions, but the AEC group developed significantly fewer rashes, and no rashes were seen in the saline group. The control horse serum group (mean ± standard deviation = 2.13 ± 0.72) showed significant coronary artery dilatation, whereas there was no significant dilatation in the AEC group (mean ± standard deviation = 0.81 ± 0.58) or the control saline group (p = 0.002). CONCLUSION: Serum sickness is a prototype of immune complex vasculitis, and the severity can be ameliorated with antioxidants. A trial of therapeutic dosages of vitamins A, E, and C in acute phase of Kawasaki disease, may be effective in mitigation of coronary artery lesion in addition to intravenous immunoglobulin and aspirin.

Correlative Studies Unravelling the Possible Mechanism of Cell Death in Tideglusib-Treated Human Ovarian Teratocarcinoma-Derived PA-1 Cells.

This study aims to unravel the use of GSK-3 inhibitors as viable apoptotic inducers for teratocarcinoma-derived ovarian PA-1 cells. MTT assay was carried out to assess inhibitory concentrations of LiCl and TDG. AO/EB staining and Hoechst 33258 staining were employed to assess the damage. Mitochondrial membrane potential (ΔΨm) and ROS generation were assessed with IC50 concentrations of LiCl and TDG. Tumor-related genes (p53, p21, IL-8, TNF-α, MMP-2, Fas-L, Cox-2, and caspase-3) were assessed with 1/4 IC50, 1/2 IC50, IC50 concentrations by semi-quantitative RT- PCR. Cell cycle analysis was performed with IC50 concentration of LiCl and TDG. Western blot analysis was performed for caspase-3, caspase-7, caspase-9, PARP to estimate the possible damage induced by GSK-3 inhibitors and regulation of GSK-3ß, pGSK-3ß, Cox-2. GSK-3 inhibitors demonstrated a concentration and time-dependent reduction in cell viability, exhibiting significant ROS generation and reduced ΔΨm at their IC50 values. Substantial concentration-dependent gene expression changes with significant upregulation of P21, Cox-2, TNF-α, caspase-3, Fas-L were observed. Protein expression of caspase-3 caspase-7, caspase-9, PARP exhibited significant cleavage in LiCl and TDG-treated cells. Protein expression of Cox-2 was significantly increased in IC50 concentration of TDG. Cell cycle analysis showed significant accumulation of cells at sub-G0-G1.

Novel mitochondrial DNA mutations in a rare variety of hypertrophic cardiomyopathy.

We report a rare case of a 65 year old male with mid left ventricular cavity obstruction which is an uncommon form of hypertrophic cardiomyopathy with cytogenetic analysis revealing novel mutations in mitochondrial nucleic acid.

Tideglusib induces apoptosis in human neuroblastoma IMR32 cells, provoking sub-G0/G1 accumulation and ROS generation.

Neuroblastoma is the most common tumor amongst children amounting to nearly 15% of cancer deaths. This cancer is peculiar in its characteristics, exhibiting differentiation, maturation and metastatic transformation leading to poor prognosis and low survival rates among children. Chemotherapy, though toxic to normal cells, has shown to improve the survival of the patient with emphasis given more towards targeting angiogenesis. Recently, Tideglusib was designed as an 'Orphan Drug' to target the neurodegenerative Alzheimer's disease and gained significant momentum in its function during clinical trials. Duffy et al. recently reported a reduction in cell viability of human IMR32 neuroblastoma cells when treated with Tideglusib at varying concentrations. We investigated the effects of Tideglusib, at various concentrations, compared to Lithium chloride at various concentrations, on IMR32 cells. Lithium, a known GSK-3 inhibitor, was used as a standard to compare the efficiency of Tideglusib in a dose-dependent manner. Cell viability was assessed by MTT assay. The stages of apoptosis were evaluated by AO/EB staining and nuclear damage was determined by Hoechst 33258 staining. Reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm) were assessed by DCFDA dye and Rhodamine-123 dye, respectively. Tideglusib reported a significant dose-dependent increase in pro-apoptotic proteins (PARP, Caspase-9, Caspase-7, Caspase-3) and tumor-related genes (FasL, TNF-α, Cox-2, IL-8, Caspase-3). Anti-GSK3 ß, pGSK3 ß, Bcl-2, Akt-1, p-Akt1 protein levels were observed with cells exposed to Tideglusib and Lithium chloride. No significant dose-dependent changes were observed for the mRNA expression of collagenase MMP-2, the tumor suppressor p53, or the cell cycle protein p21. Our study also reports Tideglusib reducing colony formation and increasing the level of sub-G0/G1 population in IMR32 cells. Our investigations report the significance of Tideglusib as a promising apoptotic inducer in human neuroblastoma IMR32 cells. Our study also reports that LiCl reduced cell viability in IMR32 cells inducing apoptosis mediated by ROS generation.