Biology subjects

Balaji, S.

Publications and source records attributed to Balaji, S..

22 records · Page 2Linked to original sources

bioRxiv2020

Phenotypic Differences in Adult and Fetal Dermal Fibroblast Responses to Mechanical Tension

ObjectiveFetal regenerative wound healing is characterized by hyaluronan(HA)-rich microenvironment and fibroblasts that produce pericellular matrices(PCM) abundant in high molecular weight HA. Recent studies showed that while small wounds in fetal skin heal regeneratively, large wounds heal with fibrosis. We posit large wounds generate higher mechanical tension which alters HA metabolism in the fetal fibroblasts and lead to a pro-fibrotic phenotype. ApproachC57BL/6J murine fetal (FFB; E14.5) and adult (AFB; 8wk) dermal fibroblasts were subjected to +/-10% tonic strain. Changes in PCM, HA enzymes and molecular weight, and fibrotic gene expression were measured. ResultsFFB pericellular matrix reduced upon exposure to increased tension, and the HA profile shifted from high to lower molecular weight. Under static conditions, AFB had higher expression of HA synthases (HAS) 1 and 2 and degradation enzymes KIAA1199, HYAL1, and TMEM2 than FFB, suggesting more HA turnover in AFB. Tension resulted in an increase in HAS1, HAS3, KIAA1199, and HYAL2 expression and a decrease in HAS2 and TMEM2 expression in FFB. CD26, a marker associated with scar production, increased in FFB under tension, along with altered fibrotic gene expression profile and reorganized cytoskeletal f-actin and increased -SMA that resembled AFB. InnovationThis study elucidates the differences in how biomechanical tension alters HA metabolism and fibrotic phenotype of FFB vs AFB, providing further understanding of the fetal regenerative wound healing phenotype. ConclusionUnderstanding the intrinsic differences in HA metabolism and fibrotic phenotype among FFB and AFB in response to wound mechanical stimuli may yield new insights to promote regenerative wound healing.

molecular biology↗

bioRxiv2020

Verification of genetic engineering in yeasts with nanopore whole genome sequencing

Yeast genomes can be assembled from sequencing data, but genome integrations and episomal plasmids often fail to be resolved with accuracy, completeness, and contiguity. Resolution of these features is critical for many synthetic biology applications, including strain quality control and identifying engineering in unknown samples. Here, we report an integrated workflow, named Prymetime, that uses sequencing reads from inexpensive NGS platforms, assembly and error correction software, and a list of synthetic biology parts to achieve accurate whole genome sequences of yeasts with engineering annotated. To build the workflow, we first determined which sequencing methods and software packages returned an accurate, complete, and contiguous genome of an engineered S. cerevisiae strain with two similar plasmids and an integrated pathway. We then developed a sequence feature annotation step that labels synthetic biology parts from a standard list of yeast engineering sequences or from a custom sequence list. We validated the workflow by sequencing a collection of 15 engineered yeasts built from different parent S. cerevisiae and nonconventional yeast strains. We show that each integrated pathway and episomal plasmid can be correctly assembled and annotated, even in strains that have part repeats and multiple similar plasmids. Interestingly, Prymetime was able to identify deletions and unintended integrations that were subsequently confirmed by other methods. Furthermore, the whole genomes are accurate, complete, and contiguous. To illustrate this clearly, we used a publicly available S. cerevisiae CEN.PK113 reference genome and the accompanying reads to show that a Prymetime genome assembly is equivalent to the reference using several standard metrics. Finally, we used Prymetime to resequence the nonconventional yeasts Y. lipolytica Po1f and K. phaffii CBS 7435, producing an improved genome assembly for each strain. Thus, our workflow can achieve accurate, complete, and contiguous whole genome sequences of yeast strains before and after engineering. Therefore, Prymetime enables NGS-based strain quality control through assembly and identification of engineering features.

synthetic biology↗

bioRxiv2020

Filamentous Bacteriophage Delay Healing of Pseudomonas-Infected Wounds

We have identified a novel role for filamentous bacteriophage in the delayed healing associated with chronic Pseudomonas aeruginosa (Pa) wound infections. Pf phage delays wound re-epithelialization in the absence of live Pa, indicative that Pf effects on wound healing are independent of Pa pathogenesis. Pf phage directly inhibits autocrine signaling of CXCL1 (KC) to impede keratinocyte migration and wound re-epithelization. In agreement with these studies, a prospective cohort study of 36 human patients with chronic Pa wound infections revealed that wounds infected with Pf positive strains of Pa took longer to heal and were more likely to increase in size compared to wounds infected with Pf negative strains. Together, these data implicate Pf phage in the delayed wound healing associated with Pa infection through direct manipulation of mammalian target cells. We propose that Pf phage may have potential as a biomarker and therapeutic target in delayed wound healing.

microbiology↗

bioRxiv2019

IL-10 Promotes Endothelial Progenitor Cell Driven Wound Neovascularization and Enhances Healing via STAT3.

Evidence from prior studies of cutaneous trauma, burns, and chronic diabetic wound repair demonstrates that endothelial progenitor cells (EPCs) contribute to de novo angiogenesis, anti-inflammatory reactions, tissue regeneration, and remodeling. We have shown that IL-10, a potent anti-inflammatory cytokine, promotes regenerative tissue repair in an adult model of dermal scar formation via the regulation of fibroblast-specific hyaluronan synthesis in a STAT3 dependent manner. While it is known that IL-10 drives EPC recruitment and neovascularization after myocardial infarction, its specific mode of action, particularly in dermal wound healing and neovascularization in both control and diabetic wounds remains to be defined. Here we show that IL-10 promotes EPC recruitment into the dermal wound microenvironment to facilitate neovascularization and wound healing of control and diabetic (db/db) wounds via vascular endothelial growth factor (VEGF) and stromal-cell derived factor 1 (SDF-1) signaling. Inducible skin-specific STAT3 knockout (KO) mice were studied to determine whether the impact of IL-10 on the neovascularization and wound healing is STAT3 dependent. We found that IL-10 treatment significantly promotes dermal wound healing with enhanced wound closure, robust granulation tissue formation and neovascularization. This was associated with elevated wound EPC counts as well as increased VEGF and high SDF-1 levels in control mice, an effect that was abrogated in STAT3 KO transgenic mice. These findings were supported in vitro, wherein IL-10-enhanced VEGF and SDF-1 synthesis in primary murine dermal fibroblasts. IL-10-conditioned fibroblast media was shown to promote sprouting and network formation in aortic ring assays. We conclude that overexpression of IL-10 in the wound-specific milieu recruits EPCs and promote neovascularization, which occurs in a STAT3-dependent manner via regulation of VEGF and SDF-1 levels. Collectively, our studies demonstrate that IL-10 increases EPC recruitment leading to enhanced neovascularization and healing of dermal wounds.

physiology↗