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RationaleAtrial fibrillation (AF) genome-wide association studies (GWAS) identified significant associations for rs1152591 and linked variants in the SYNE2 gene encoding the nesprin-2 protein that connects the nuclear membrane with the cytoskeletonObjectiveDetermine the effects of the AF-associated rs1152591 and rs1152595, two linked intronic single nucleotide polymorphisms (SNPs), on SYNE2 expression and investigate the mechanisms for their association with AF.Methods and ResultsRNA sequencing of human left atrial appendage (LAA) tissues indicated that rs1152591 and rs1152595 were significantly associated with the expressions of SYNE2α1, a short mRNA isoform, without an effect on the expression of the full-length SYNE2 mRNA. SYNE2α1 mRNA uses an alternative transcription start site and encodes an N-terminal deleted 62 kDa nesprin-2α1 isoform, which can act as a dominant-negative on nuclear-cytoskeleton connectivity. Western blot and qPCR assays confirmed that AF risk alleles of both SNPs were associated with lower expression of nesprin-2α1 in human LAA tissues. Reporter gene transfections demonstrated that the risk vs. reference alleles of rs1152591 and rs1152595 had decreased enhancer activity. SYNE2 siRNA knockdown (KD) or nesprin-2α1 overexpression studies in human stem cell-derived induced cardiomyocytes (iCMs) resulted in ~12.5 % increases in the nuclear area compared to controls (pConclusionsAF-associated SNPs rs1152591 and rs1152595 downregulate the expression of SYNE2α1, increasing nuclear-cytoskeletal connectivity and nuclear stiffness. The resulting increase in mechanical stress may play a role in the development of AF.

Transseptal puncture is a routinely performed interventional cardiology procedure for an array of cardiac diseases. We aimed to review the current status of available devices and techniques of transseptal puncture with consideration to specific interventions. Except for a few modifications, devices for transseptal puncture technique has not changed much compared to when it was first described almost 60 years ago. For difficult transseptal puncture, a few newer techniques such as radio frequency needle puncture system have been used but there is lack of robust clinical study. Advanced imaging, such as intracardiac echocardiography and transesophageal echocardiography, has been found to make transseptal puncture safer. A new transseptal approach that incorporates 3D non-fluoroscopic catheter tracking systems has shown promising results in two human studies. While various modifications in the transseptal technique tailored to the specific interventions have improved procedural safety, further improvement in existing devices focusing on distinct procedure might be needed in the future.

Myocardial infarction results in loss of cardiac cell types, inflammation, extracellular matrix (ECM) degradation, and fibrotic scar. Transplantation of bone marrow-derived mesenchymal stem cells (BM-MSCs) is being explored as they could differentiate into cardiomyocyte-like cells, integrate into host tissue, and enhance resident cell activity. The ability of these cells to restore lost ECM, remodel the inflammatory scar tissue, and repair the injured myocardium remains unexplored. We here elucidated the synthesis and deposition of ECM (e.g., elastin, sulfated glycosaminoglycans, hyaluronan, collagen type III, laminin, fibrillin, lysyl oxidase, and nitric oxide synthases), matrix metalloproteinases (MMPs) and their inhibitors (TIMPs), and other secretome (cytokines, chemokines, and growth factors) in adult human BM-MSC spheroid cultures within three-dimensional collagen gels. The roles of species-specific type I collagen and 5-azacytadine were assessed over a 28-day period. Results revealed that human collagen (but not rat-derived) suppressed MSC proliferation and survival, and MSCs synthesized and released a variety of ECM proteins and secretome over the 28 days. Matrix deposition is at least an order of magnitude lower than their release levels at every time point, most possibly due to elevated MMP levels and interleukins with a concomitant decrease in TIMPs. Matrix synthesis over the 28-day period was fitted to a competitive inhibition form of Michaelis-Menten kinetics, and the production and decay rates of ECM, MMPs, and TIMPs, along with the kinetic model parameters quantified. Such an integrated experimental and modelling approach would help elucidate the critical roles of various parameters (e.g., cell encapsulation and delivery vehicles) in stem cell-based transplantation therapies.

We establish the total amino acids (AA) concentration in wash water as an alternative indicator of free chlorine (FC) levels, and develop a model to predict FC concentration based on modeling the reaction kinetics of chlorine and amino acids. Using single wash of iceberg lettuce, green cabbage, and carrots, we report the first in situ apparent reaction rate β between FC and amino acids in the range of 15.3 – 16.6 M-1. s-1 and an amplification factor γ in the range of 11.52 - 11.94 for these produce. We also report strong linear correlations between AA levels and produce-to-water ratio (R2 = 0.87), and between chemical oxygen demand (COD) and AA concentrations (R2 = 0.87). The values of the parameters γ and β of the model were validated in continuous wash experiments of chopped iceberg lettuce, and predicted the FC (R2 = 0.96) and AA (R2 = 0.92) levels very well.

Mammalian central nervous system has a limited ability for self-repair under diseased or injury conditions. Repair strategies focused on exogenously delivering autologous neural stem cells (NSCs) to replace lost neuronal populations and axonal pathways in situ, and promote endogenous repair mechanisms are gaining traction. Successful outcomes are contingent on selecting an appropriate delivery vehicle for injecting cells that promotes cell retention and survival, elicits differentiation to desired lineages, and enhances axonal outgrowth upon integration into the host tissue. Hydrogels made of varying compositions of collagen, laminin, hyaluronic acid (HA) and chondroitin sulfate proteoglycan (CSPG) were developed, with no external crosslinking agents, to mimic the native extracellular matrix composition. The physical (porosity, pore-size, gel integrity, swelling ratio, enzymatic degradation), mechanical (viscosity, storage and loss moduli, Young's modulus, creep, stress-relaxation) and biological (cell survival, differentiation, neurite outgrowth, integrin expression) characteristics of these hydrogels were assessed. These hydrogels exhibited excellent injectability, retained gel integrity, and matched the mechanical moduli of native brain tissue, possibly due to natural collagen fibril polymerization and physical-crosslinking between HA molecules and collagen fibrils. Depending on the composition, these hydrogels promoted cell survival, neural differentiation and neurite outgrowth, as evident from immunostaining and western blots. These cellular outcomes were facilitated by cellular binding via α6, β1, and CD44 surface integrins to these hydrogels. Results attest to the utility of uncrosslinked, ECM-mimicking hydrogels to deliver NSCs for tissue engineering and regenerative medicine applications. This article is protected by copyright. All rights reserved.

Regeneration following spinal cord injury (SCI) is challenging in part due to the modified tissue composition and organization of the resulting glial and fibrotic scar regions. Inhibitory cell types and biochemical cues present in the scar have received attention as therapeutic targets to promote regeneration. However, altered Young’s modulus of the scar as a readout for potential impeding factors for regeneration are not as well-defined, especially in vivo. Although the decreased Young’s modulus of surrounding tissue at acute stages post-injury is known, the causation and outcomes at chronic time points remain largely understudied and controversial, which motivates this work. This study assessed the glial and fibrotic scar tissue’s Young’s modulus and composition (scar morphometry, cell identity, extracellular matrix (ECM) makeup) that contribute to the tissue’s stiffness. The spatial Young’s modulus of a chronic (~18-wks, post-injury) hemi-section, including the glial and fibrotic regions, were significantly less than naïve tissue (~ 200 Pa; p < 0.0001). The chronic scar contained cystic cavities dispersed in areas of dense nuclei packing. Abundant CNS cell types such as astrocytes, oligodendrocytes, and neurons were dysregulated in the scar, while epithelial markers such as vimentin were upregulated. The key ECM components in the CNS, namely sulfated proteoglycans (sPGs), were significantly downregulated following injury with concomitant upregulation of unsulfated glycosaminoglycans (GAGs) and hyaluronic acid (HA), likely altering the foundational ECM network that contributes to tissue stiffness. Our results reveal the Young’s modulus of the chronic SCI scar as well as quantification of contributing elastic components that can provide a foundation for future study into their role in tissue repair and regeneration.

The assessment of neurotoxicity has been performed traditionally with animals. However, in vivo studies are highly expensive and time-consuming, and often do not correlate to human outcomes. Thus, there is a need for cost-effective, high-throughput, highly predictive alternative in vitro test methods based on early markers of mechanisms of toxicity. High-content imaging (HCI) assays performed on three-dimensionally (3D) cultured cells could provide better understanding of the mechanism of toxicity needed to predict neurotoxicity in humans. However, current 3D cell culture systems lack the throughput required for screening neurotoxicity against a large number of chemicals. Therefore, we have developed miniature 3D neural stem cell (NSC) culture on a unique 384-pillar plate, which is complementary to conventional 384-well plates. Mitochondrial membrane impairment, intracellular glutathione level, cell membrane integrity, DNA damage, and apoptosis have been tested against 3D-cultured ReNcell VM on the 384-pillar plate with four model compounds rotenone, 4-aminopyridine, digoxin, and topotecan. The HCI assays performed in 3D-cultured ReNcell VM on the 384-pillar plates were highly robust and reproducible as indicated by the average Z′ factor of 0.6 and CV values around 12%. From concentration-response curves and IC(50) values, mitochondrial membrane impairment appears to be the early stage marker of cell death by the compounds.

In this study, we developed a novel produce-specific mechanistic model to predict free chlorine (FC) dynamics during washing of disk-cut carrots, cut cabbage, and cut iceberg lettuce, in 3 L and 50–100 L tanks, and of shredded iceberg lettuce in 3200 L pilot-plant trials. Ranges for two key parameters: β (L mg−1 min−1) the apparent reaction rate constant of FC with produce constituents, and γ , the fraction of the increase of chemical oxygen demand (COD) contributing to the reaction, were determined at the 3 L scale. For disk carrots β ∈ [ 0.05 , 0.09 ] and γ ∈ [ 0.054 , 0.078 ] , for cut cabbage β ∈ [ 0.05 , 0.10 ] and γ ∈ [ 0.09 , 0.12 ] , and for cut iceberg lettuce β ∈ [ 0.03 , 0.06 ] and γ ∈ [ 0.07 , 0.14 ] . Taking values from these ranges the model was able to consistently predict experimental FC dynamics (decay and replenishment), indicating robustness of the apparent reaction rate constants across scales. Comparing sequential changes in COD with turbidity and total dissolved solids (TDS) relative to produce washing rates, our results also illustrate that turbidity and TDS may not be reliable predictors of FC decay rates across produce types and experimental scales. In concert with future experiments, these models could serve as important tools aimed at validating FC compliance within operational limits as well as guiding large-scale commercial experiments focused on improving chlorine management strategies relevant for industry.

Microfluidic platforms offer a variety of advantages including improved heat transfer, low working volumes, ease of scale-up, and stronger user control on operating parameters. However, flow within microfluidic channels occurs at low Reynolds number (Re), which makes mixing difficult to accomplish. Adding V-shaped ridges to channel walls, a pattern called the staggered herringbone design (SHB), alleviates this problem by introducing transverse flow patterns that enable enhanced mixing. Building on our prior work, we here developed a microfluidic mixer utilizing the SHB geometry and characterized using CFD simulations and complimentary experiments. Specifically, we investigated the performance of this type of mixer for unequal species diffusivities and inlet flows. A channel design with SHB ridges was simulated in COMSOL Multiphysics® software under a variety of operating conditions to evaluate its mixing capabilities. The device was fabricated using soft-lithography techniques to experimentally visualize the mixing process. Mixing within the device was enabled by injecting fluorescent dyes through the device and imaging using a confocal microscope. The device was found to efficiently mix fluids rapidly, based on both simulations and experiments. Varying Re or species diffusion coefficients had a weak effect on the mixing profile, due to the laminar flow regime and insufficient residence time, respectively. Mixing effectiveness increased as the species flow rate ratio increased. Fluid flow patterns visualized in confocal microscope images for selective cases were strikingly similar to CFD results, suggesting that the simulations serve as good predictors of device performance. This SHB mixer design would be a good candidate for further implementation as a microfluidic reactor.

Influence of plant growth regulator sprays on corm production and post-harvest life of two gladiolus cultivars, Darshan and Dhiraj, was investigated for two consecutive years, 2008-09 and 2009-10. Growth regulators, viz., gibberellic acid (100 and 150ppm), tri-iodo benzoic acid (TIBA) (50 and 100ppm), 2-chloro, 4-pyridyl phenyl urea (CPPU) (2.5 and 5ppm) and brassinosteroid (BR) (5 and 10ppm) were sprayed at the 3rd and 6thleaf stage. Cv. Darshan recorded maximum number of large cormels per plant and cormel weight, while, cv. Dhiraj recorded maximum number of small cormels per plant in treatments of pre-harvest foliar sprays with plant growth regulators. Foliar sprays of BR (10ppm) and GA3 (150ppm) significantly increased number of corms produced per plant, corm size, corm weight, and propagation coefficient. Number of large cormels and total number of cormels per plant were significantly higher in BR (10ppm), followed by TIBA (100ppm). BR (10ppm) and TIBA (100ppm) produced maximum number of small cormels per plant. Weight of cormels per plant was maximum in BR (10ppm) and GA3 (150ppm). Post-harvest studies revealed that cv. Darshan recorded maximum diameter of second fully-opened floret and higher vase-life than cv. Dhiraj with pre-harvest foliar spray of plant growth regulators. Pre-harvest foliar spray of GA3 (150ppm), BR (10ppm) and CPPU (5ppm) induced earliest first-floret opening and recorded maximum value for number-of-floretsopen- at-a-time per spike, diameter of second fully-opened floret, and vase-life. Foliar spray of BR (10ppm) and GA3 (150ppm) at 3rd and 6th leaf stage can be recommended for large-scale multiplication of quality planting material and longer vase-life of flower spikes, respectively, in gladiolus.

Human heart has endogenous regenerative capability; however, the intrinsic repair mechanism is not sufficient to overcome the impact placed by adverse pathological conditions, such as myocardial infarction (MI). In such circumstances, the damaged tissue initiates a series of remodeling process which results in the deterioration of structural, functional, and mechanical properties of the myocardium. To address such adverse conditions, clinical approaches ranging from surgical interventions, pharmaceutical drugs, and device implantation are administered which have played significant role in reducing the mortality rate. However, these approaches do not replace the lost cardiomyocytes, or restore the degraded structure-function relationship of the myocardium. In this aspect, cell-based therapy has gained substantial interest as a potential clinical approach for myocardial regeneration; however this method is impeded by lower graft retention and poor cell viability. To overcome these limitations, biomaterials are being developed as "trojan horses", i.e., vehicles for homing and deploying cells, and as matrices for delivering specific biological, mechanical, and chemical cues intended for tissue regeneration. Similarly, several candidate drugs, potent synthetic and biological molecules, and advanced drug delivery systems are being examined to provide exogenous cues in a controlled fashion to the diseased myocardium. In this article, we review biomaterials-based drug delivery systems for myocardial regeneration, specifically on the applications of hydrogels, microgels, nanoparticles, and nanofibers in the field. The prime focus of the article is on nanofibers-based drug delivery systems that is gaining considerable attention as a biomimetic pharmacological approach. We highlight literature on fabrication methods of self-assembling and electrospun nanofibers, drug incorporation methods and release kinetics, and in vitro and in vivo outcomes from nanofiber-based drug delivery systems in cardiac regeneration.

Endogenous adult cardiac regenerative machinery is not capable of replacing the lost cells following myocardial infarction, often leading to permanent alterations in structure-function-mechanical properties. Regenerative therapies based on delivering autologous stem cells within an appropriate 3D milieu could meet such demand, by enabling homing and directed differentiation of the transplanted cells into lost specialized cell populations. Since type I collagen is the predominant cardiac tissue matrix protein, we here optimized the 3D niche which could promote time-dependent evolution of cardiomyogenesis from human bone marrow-derived mesenchymal stem cells (BM-MSC). 3D collagen gel physical and mechanical characteristics were assessed using SEM and AFM, respectively, while the standalone and combined effects of collagen concentration, culture duration, and 5-azacytidine (aza) dose on the phenotype and genotype of MSC spheroids were quantified using immunofluorescence labeling and RT-PCR analysis. Increasing collagen concentration led to a significant increase in Young's modulus (p < 0.01) but simultaneous decrease in the mean pore size, resulting in stiffer gels. Spheroid formation significantly modulated MSC differentiation and genotype, mostly due to better cell-cell interactions. Among the aza dosages tested, 10 μM appears to be optimal, while 3 mg/ml gels resulted in significantly lower cell viability compared to 1 or 2 mg/ml gels. Stiffer gels (2 and 3 mg/ml) and exposure to 10 μM aza upregulated early and late cardiac marker expressions in a time-dependent fashion. On the other hand, cell-cell signaling within the MSC spheroids seem to have a strong role in influencing mature cardiac markers expression, since neither aza nor gel stiffness seem to significantly improve their expression. Western blot analysis suggested that canonical Wnt/β-catenin signaling pathway might be primarily mediating the observed benefits of aza on cardiac differentiation of MSC spheroids. In conclusion, 2 mg/ml collagen and 10 μM aza appears to offer optimal 3D microenvironment in terms of cell viability and time-dependent evolution of cardiomyogenesis from human BM-MSCs, with significant applications in cardiac tissue engineering and stem cell transplantation for regenerating lost cardiac tissue.

Neural progenitor cell (NPC) fate is influenced by a variety of biological cues elicited from the surrounding microenvironment and recent studies suggest their possible role in pediatric glioblastoma multiforme (GBM) development. Since a few GBM cells also display NPC characteristics, it is not clear whether NPCs transform to tumor cell phenotype leading to the onset of GBM formation, or NPCs migrate to developing tumor sites in response to paracrine signaling from GBM cells. Elucidating the paracrine interactions between GBM cells and NPCs in vivo is challenging due to the inherent complexity of the CNS. Here, we investigated the interactions between human NPCs (ReNcell) and human pediatric GBM-derived cells (SJ-GBM2) using a Transwell® coculture setup to assess the effects of GBM cells on ReNcells (cytokine and chemokine release, viability, phenotype, differentiation, migration). Standalone ReNcell or GBM cultures served as controls. Qualitative and quantitative results from ELISA®, Live/Dead® and BrdU assays, immunofluorescence labeling, western blot analysis, and scratch test suggests that although ReNcell viability remained unaffected in the presence of pediatric GBM cells, their morphology, phenotype, differentiation patterns, neurite outgrowth, migration patterns (average speed, distance, number of cells) and GSK-3β expression were significantly influenced. The cumulative distance migrated by the cells in each condition was fit to Furth's formula, derived formally from Ornstein-Uhlenbeck process. ReNcell differentiation into neural lineage was compromised and astrogenesis promoted within cocultures. Such coculture platform could be extended to identify the specific molecules contributing to the observed phenomena, to investigate whether NPCs could be transplanted to replace lesions of excised tumor sites, and to elucidate the underlying molecular pathways involved in GBM-NPC interactions within the tumor microenvironment.

A full-term female neonate with ectrodactyly, polycystic kidney disease, mandibular hypoplasia, uterus unicornis, bronchogenic cyst and spina bifida is reported. The features are compared with other cases of limb and renal abnormalities reported in the literature. This case along with similar cases reported by Halal et al. (1980), Evans et al. (2000); Tobias et al. (2001); Phadke and Manisha (2006); John (2007); and Girisha et al. (2012) delineates acrorenal mandibular syndrome as a distinct entity.

A major onset of heart failure is myocardial infarction, which causes the myocardium to lose cardiomyocytes and transform into a scar tissue. Since mammalian infarcted cardiac tissue has a limited ability to regenerate, alternative strategies including implantation of tissue-engineered scaffolds at the site of damaged myocardium have been explored. The goal is to enable in situ cardiac reconstruction at the injured myocardium site, replace the lost cardiomyocytes, deliver the required biomolecules, and remodel the extracellular matrix (ECM). ECM synthesis and deposition by cardiomyocytes within such scaffolds remains categorically unexplored. Here, we investigated the survival, ECM synthesis and deposition, and matrix metalloproteinases (MMPs) release by cardiomyocytes within three-dimensional (3D) substrates. Rat cardiomyocytes were cultured for three weeks within two structurally different substrates: 3D collagen hydrogels or polycaprolactone (PCL) nanofibrous scaffolds. The concentration and composition of the hydrogels was varied, while PCL nanofibers were surface-modified with various ECM proteins. Results showed that myocyte attachment and survival was higher within collagen hydrogels, while myocyte alignment and beating was noted only within PCL scaffolds. Total protein synthesis by myocytes within PCL scaffolds was significantly higher compared to that within collagen hydrogels, although more protein was deposited as matrix within hydrogels. Significant ECM synthesis and matrix deposition, TIMP-1, and MMP release were noted within modified collagen hydrogels and PCL nanofiber scaffolds. These results were qualitatively confirmed by imaging techniques. Results attest to the prominent role of scaffold composition and architecture in influencing cardiomyocyte phenotype, matrix synthesis and cytokines release, with significant applications in cardiac tissue remodeling strategies.