Expression of \u3cem\u3espoT\u3c/em\u3e in \u3cem\u3eBorrelia burgdorferi\u3c/em\u3e during Serum Starvation

Borrelia burgdorferi, the causative agent of Lyme disease, is transmitted by the tick Ixodes scapularis. A 2.9-kb fragment containing a putative spoT gene was isolated from B. burgdorferi genomic DNA by PCR amplification and cloned into a pBAD24 vector. The cloned gene complemented Escherichia coli mutant strain CF1693, which contains deletions of both the relA and spoT genes. The spoT gene in E. coli encodes a bifunctional enzyme capable of synthesizing and degrading (p)ppGpp, which mediates the stringent response during carbon source starvation. B. burgdorferi has been reported to have a stress response to serum starvation. Thin-layer chromatography was used to detect (p)ppGpp extracted from H3 32PO4-labeled B. burgdorferi cells starved for serum in RPMI. B. burgdorferi spoT gene expression was characterized during fatty acid starvation. Northern analysis of spoT revealed detectable message at 2.5 min of starvation in RPMI. Expression of spoT during serum starvation increased _6-fold during the 30 min that starvation conditions were maintained. Further, expression of spoT decreased when serum was added to serum-starved cells. Reverse transcriptase PCR (RT-PCR) was used to detect spoT mRNA from ~106 cells starved for serum in RPMI for 2.5 to 30 min or incubated in tick saliva for 15 min. Northern blot analysis suggests that spoT transcript was ~900 nucleotides in length. RT-PCR amplification of the transcript using several sets of primers confirmed this finding. Additionally, a truncated clone containing only the first 950 bp of the 2,001-bp spoT open reading frame was able to complement E. coli CF1693. The data suggest that B. burgdorferi exhibits a stringent response to serum starvation and during incubation in tick saliva

The genomic landscape of balanced cytogenetic abnormalities associated with human congenital anomalies

Despite the clinical significance of balanced chromosomal abnormalities (BCAs), their characterization has largely been restricted to cytogenetic resolution. We explored the landscape of BCAs at nucleotide resolution in 273 subjects with a spectrum of congenital anomalies. Whole-genome sequencing revised 93% of karyotypes and demonstrated complexity that was cryptic to karyotyping in 21% of BCAs, highlighting the limitations of conventional cytogenetic approaches. At least 33.9% of BCAs resulted in gene disruption that likely contributed to the developmental phenotype, 5.2% were associated with pathogenic genomic imbalances, and 7.3% disrupted topologically associated domains (TADs) encompassing known syndromic loci. Remarkably, BCA breakpoints in eight subjects altered a single TAD encompassing MEF2C, a known driver of 5q14.3 microdeletion syndrome, resulting in decreased MEF2C expression. We propose that sequence-level resolution dramatically improves prediction of clinical outcomes for balanced rearrangements and provides insight into new pathogenic mechanisms, such as altered regulation due to changes in chromosome topology

Expression of spoT in Borrelia burgdorferi during Serum Starvation

Borrelia burgdorferi, the causative agent of Lyme disease, is transmitted by the tick Ixodes scapularis. A 2.9-kb fragment containing a putative spoT gene was isolated from B. burgdorferi genomic DNA by PCR amplification and cloned into a pBAD24 vector. The cloned gene complemented Escherichia coli mutant strain CF1693, which contains deletions of both the relA and spoT genes. The spoT gene in E. coli encodes a bifunctional enzyme capable of synthesizing and degrading (p)ppGpp, which mediates the stringent response during carbon source starvation. B. burgdorferi has been reported to have a stress response to serum starvation. Thin-layer chromatography was used to detect (p)ppGpp extracted from H(3)(32)PO(4)-labeled B. burgdorferi cells starved for serum in RPMI. B. burgdorferi spoT gene expression was characterized during fatty acid starvation. Northern analysis of spoT revealed detectable message at 2.5 min of starvation in RPMI. Expression of spoT during serum starvation increased ∼6-fold during the 30 min that starvation conditions were maintained. Further, expression of spoT decreased when serum was added to serum-starved cells. Reverse transcriptase PCR (RT-PCR) was used to detect spoT mRNA from ∼10(6) cells starved for serum in RPMI for 2.5 to 30 min or incubated in tick saliva for 15 min. Northern blot analysis suggests that spoT transcript was ∼900 nucleotides in length. RT-PCR amplification of the transcript using several sets of primers confirmed this finding. Additionally, a truncated clone containing only the first 950 bp of the 2,001-bp spoT open reading frame was able to complement E. coli CF1693. The data suggest that B. burgdorferi exhibits a stringent response to serum starvation and during incubation in tick saliva

Conversion of human fibroblasts to angioblast-like progenitor cells

International audienceLineage conversion of one somatic cell type to another is an attractive approach for generating specific human cell types. Lineage conversion can be direct, in the absence of proliferation and multipotent progenitor generation, or indirect, by the generation of expandable multipotent progenitor states. We report the development of a reprogramming methodology in which cells transition through a plastic intermediate state, induced by brief exposure to reprogramming factors, followed by differentiation. We use this approach to convert human fibroblasts to mesodermal progenitor cells, including by non-integrative approaches. These progenitor cells demonstrated bipotent differentiation potential and could generate endothelial and smooth muscle lineages. Differentiated endothelial cells exhibited neo-angiogenesis and anastomosis in vivo. This methodology for indirect lineage conversion to angioblast-like cells adds to the armamentarium of reprogramming approaches aimed at the study and treatment of ischemic pathologies

Implementation of Germline Testing for Prostate Cancer: Philadelphia Prostate Cancer Consensus Conference 2019

PURPOSE: Germline testing (GT) is a central feature of prostate cancer (PCA) treatment, management, and hereditary cancer assessment. Critical needs include optimized multigene testing strategies that incorporate evolving genetic data, consistency in GT indications and management, and alternate genetic evaluation models that address the rising demand for genetic services. METHODS: A multidisciplinary consensus conference that included experts, stakeholders, and national organization leaders was convened in response to current practice challenges and to develop a genetic implementation framework. Evidence review informed questions using the modified Delphi model. The final framework included criteria with strong (\u3e 75%) agreement (Recommend) or moderate (50% to 74%) agreement (Consider). RESULTS: Large germline panels and somatic testing were recommended for metastatic PCA. Reflex testing-initial testing of priority genes followed by expanded testing-was suggested for multiple scenarios. Metastatic disease or family history suggestive of hereditary PCA was recommended for GT. Additional family history and pathologic criteria garnered moderate consensus. Priority genes to test for metastatic disease treatment included BRCA2, BRCA1, and mismatch repair genes, with broader testing, such as ATM, for clinical trial eligibility. BRCA2 was recommended for active surveillance discussions. Screening starting at age 40 years or 10 years before the youngest PCA diagnosis in a family was recommended for BRCA2 carriers, with consideration in HOXB13, BRCA1, ATM, and mismatch repair carriers. Collaborative (point-of-care) evaluation models between health care and genetic providers was endorsed to address the genetic counseling shortage. The genetic evaluation framework included optimal pretest informed consent, post-test discussion, cascade testing, and technology-based approaches. CONCLUSION: This multidisciplinary, consensus-driven PCA genetic implementation framework provides novel guidance to clinicians and patients tailored to the precision era. Multiple research, education, and policy needs remain of importance