Evidence for feasibility of fetal trophoblastic cell-based noninvasive prenatal testing.

OBJECTIVE: The goal was to develop methods for detection of chromosomal and subchromosomal abnormalities in fetal cells in the mother's circulation at 10-16 weeks' gestation using analysis by array comparative genomic hybridization (CGH) and/or next-generation sequencing (NGS). METHOD: Nucleated cells from 30 mL of blood collected at 10-16 weeks' gestation were separated from red cells by density fractionation and then immunostained to identify cytokeratin positive and CD45 negative trophoblasts. Individual cells were picked and subjected to whole genome amplification, genotyping, and analysis by array CGH and NGS. RESULTS: Fetal cells were recovered from most samples as documented by Y chromosome PCR, short tandem repeat analysis, array CGH, and NGS including over 30 normal male cells, one 47,XXY cell from an affected fetus, one trisomy 18 cell from an affected fetus, nine cells from a trisomy 21 case, three normal cells and one trisomy 13 cell from a case with confined placental mosaicism, and two chromosome 15 deletion cells from a case known by CVS to have a 2.7 Mb de novo deletion. CONCLUSION: We believe that this is the first report of using array CGH and NGS whole genome sequencing to detect chromosomal abnormalities in fetal trophoblastic cells from maternal blood. © 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

A multiplexed siRNA screening strategy to identify genes in the PARP pathway.

Gene silencing by RNA interference has become a powerful tool to help identify genes that regulate biological processes. However, the complexity of the biology probed and the incomplete validation of the reagents used make it difficult to interpret the results of genome-wide siRNA screens. To address this challenge and maximize the return on the efforts required for validating genomic screen hits, the screening strategy must be designed to increase the robustness of the primary screening hits and include assays that inform on the mechanism of action of the knocked-down transcripts. Here, we describe the implementation of a small interfering RNA (siRNA) screen to identify genes that sensitize the effect of poly-(ADP ribose)-polymerase (PARP) inhibitor on cell survival. In the strategy we designed for the primary screen, two biological activities, apoptosis and cell viability, were measured simultaneously at different time points in the presence and absence of a PARP inhibitor (PARPi). The multiplexed assay allowed us to identify PARPi sensitizers induced by both caspase-dependent and independent mechanisms. The multiplexed screening strategy yielded robust primary hits with significant enrichment for DNA repair genes, which were further validated using relevant high-content imaging assays and confirmation of transcript knockdown by real-time PCR (rtPCR).

MicroRNA-like off-target transcript regulation by siRNAs is species specific.

siRNAs mediate sequence-specific gene silencing in cultured mammalian cells but also silence unintended transcripts. Many siRNA off-target transcripts match the guide-strand "seed region," similar to the way microRNAs match their target sites. The extent to which this seed-matched, microRNA-like, off-target silencing affects the specificity of therapeutic siRNAs in vivo is currently unknown. Here, we compare microRNA-like off-target regulations in mouse liver in vivo with those seen in cell culture for a series of therapeutic candidate siRNAs targeting Apolipoprotein B (APOB). Each siRNA triggered regulation of consistent microRNA-like off-target transcripts in mouse livers and in cultured mouse liver tumor cells. In contrast, there was only random overlap between microRNA-like off-target transcripts from cultured human and mouse liver tumor cells. Therefore, siRNA therapeutics may trigger microRNA-like silencing of many unintended targets in vivo, and the potential toxicities caused by these off-target gene regulations cannot be accurately assessed in rodent models.

Abundance of the delta subunit of RNA polymerase is linked to the virulence of Streptococcus agalactiae.

Group B streptococcus (GBS) remains a major cause of morbidity and mortality among newborn children. The bacterium is a commensal organism colonizing the rectum and the gastrointestinal and urogenital tracts of adults, but it can be transmitted to neonates by an ascending infection of the maternal genital tract or during parturition. We previously reported that a transposon insertion disrupting rpoE resulted in the decreased survival of the mutant in the neonatal rat sepsis model of GBS infection. rpoE encodes the delta protein, a subunit of RNA polymerase (RNAP) that has been characterized in Bacillus species. In this study, we confirm the association of the delta protein with purified GBS RNAP and show that it is expressed in strains representing all nine serotypes. Flow cytometric analysis of a reporter strain containing a transcriptional fusion of the rpoE promoter to gfp revealed that, in vitro, this gene is continuously expressed. Analysis of delta expression in the transposon mutant by quantitative Western blotting revealed a 10-fold reduction in relative abundance (which was linked to the attenuation in virulence that was observed for this mutant) compared to that for the wild-type strain. These data suggest that a minimum intracellular concentration of delta is necessary for this organism to cause disease.

The Delta subunit of RNA polymerase is required for virulence of Streptococcus agalactiae.

Group B streptococci (GBS) remain the most significant bacterial pathogen causing neonatal sepsis, pneumonia, and meningitis in the United States despite the chemoprophylaxis strategies for preventing infection recommended by the Centers for Disease Control and Prevention. Using signature-tagged transposon mutagenesis to screen for novel virulence factors, we identified the rpoE gene as essential for development of sepsis in a neonatal rat model of GBS infection. An rpoE allelic replacement mutant displayed attenuated virulence in the sepsis model of infection identical to that of the transposon mutant, confirming linkage of the phenotype to the mutation in rpoE. The rpoE mutants also displayed increased sensitivity to killing in whole-blood bactericidal assays, which may explain the attenuated virulence. The mutants were otherwise phenotypically identical to the wild-type strain, including growth rate in plasma, indicating that a growth defect is not responsible for the attenuated virulence. rpoE is found only in gram-positive bacterial species and encodes the delta peptide, a subunit of RNA polymerase. Previous in vitro studies in other bacteria suggest that the delta peptide plays a role in maintaining transcriptional fidelity by blocking RNA polymerase binding at all but the strongest promoters, thereby inhibiting initiation of transcription. Despite the availability of rpoE mutants for several gram-positive bacterial species, a role for the peptide in vivo has not been defined, though it has been postulated that the delta peptide may be important for long-term survival in vitro or during growth phase transitions. Our data represent the first report of a phenotype relevant to virulence for rpoE mutants.

Penicillin-binding proteins in Streptococcus agalactiae: a novel mechanism for evasion of immune clearance.

Group B streptococci (GBS) remain the most significant bacterial pathogen causing neonatal sepsis, pneumonia and meningitis in the USA despite CDC-recommended chemoprophylaxis strategies for preventing infection. To cause infection pathogens such as GBS must evade recognition and clearance by the host's immune system. Strategies for avoidance of opsonization and phagocytic killing include elaboration of antiopsonophagocytic capsules and surface proteins. During screening for mutants of GBS that were attenuated for virulence in a neonatal rat sepsis model, we identified a mutant with a transposon insertion in the ponA gene. ponA encodes an extra-cytoplasmic penicillin-binding protein PBP1a, a newly identified virulence trait for GBS that promotes resistance to phagocytic killing independent of capsular polysaccharide. Complementation analysis in vivo and in vitro confirmed that the altered phenotypes observed in the mutant were due to the transposon insertion in ponA. Deletion of PBP1a does not affect C3 deposition on GBS suggesting that mechanism by which PBP1a protects GBS from phagocytic killing is distinct from the antiopsonic activity of capsular polysaccharide. This is the first report describing expression of an antiphagocytic surface protein by GBS and represents a novel mechanism for evasion of immune recognition and clearance that may explain the decreased virulence observed in Gram-positive bacterial species for penicillin-binding protein mutants.