High-throughput analysis of the transcriptional patterns of sexual genes in malaria

Abstract Background Plasmodium falciparum (Pf) is the leading protozoan causing malaria, the most devastating parasitic disease. To ensure transmission, a small subset of Pf parasites differentiate into the sexual forms (gametocytes). Since the abundance of these essential parasitic forms is extremely low within the human host, little is currently known about the molecular regulation of their sexual differentiation, highlighting the need to develop tools to investigate Pf gene expression during this fundamental mechanism. Methods We developed a high-throughput quantitative Reverse-Transcription PCR (RT-qPCR) platform to robustly monitor Pf transcriptional patterns, in particular, systematically profiling the transcriptional pattern of a large panel of gametocyte-related genes (GRG). Initially, we evaluated the technical performance of the systematic RT-qPCR platform to ensure it complies with the accepted quality standards for: (i) RNA extraction, (ii) cDNA synthesis and (iii) evaluation of gene expression through RT-qPCR. We then used this approach to monitor alterations in gene expression of a panel of GRG upon treatment with gametocytogenesis regulators. Results We thoroughly elucidated GRG expression profiles under treatment with the antimalarial drug dihydroartemisinin (DHA) or the metabolite choline over the course of a Pf blood cycle (48 h). We demonstrate that both significantly alter the expression pattern of PfAP2-G, the gametocytogenesis master regulator. However, they also markedly modify the developmental rate of the parasites and thus might bias the mRNA expression. Additionally, we screened the effect of the metabolites lactate and kynurenic acid, abundant in severe malaria, as potential regulators of gametocytogenesis. Conclusions Our data demonstrate that the high-throughput RT-qPCR method enables studying the immediate transcriptional response initiating gametocytogenesis of the parasites from a very low volume of malaria-infected RBC samples. The obtained data expand the current knowledge of the initial alterations in mRNA profiles of GRG upon treatment with reported regulators. In addition, using this method emphasizes that asexual parasite stage composition is a crucial element that must be considered when interpreting changes in GRG expression by RT-qPCR, specifically when screening for novel compounds that could regulate Pf sexual differentiation. Graphical Abstrac

Unexpected Transcriptional Programs Contribute to Hippocampal Memory Deficits and Neuronal Stunting after Early-Life Adversity.

Early-life adversity (ELA) is associated with lifelong memory deficits, yet the responsible mechanisms remain unclear. We impose ELA by rearing rat pups in simulated poverty, assess hippocampal memory, and probe changes in gene expression, their transcriptional regulation, and the consequent changes in hippocampal neuronal structure. ELA rats have poor hippocampal memory and stunted hippocampal pyramidal neurons associated with ~140 differentially expressed genes. Upstream regulators of the altered genes include glucocorticoid receptor and, unexpectedly, the transcription factor neuron-restrictive silencer factor (NRSF/REST). NRSF contributes critically to the memory deficits because blocking its function transiently following ELA rescues spatial memory and restores the dendritic arborization of hippocampal pyramidal neurons in ELA rats. Blocking NRSF function in vitro augments dendritic complexity of developing hippocampal neurons, suggesting that NRSF represses genes involved in neuronal maturation. These findings establish important, surprising contributions of NRSF to ELA-induced transcriptional programming that disrupts hippocampal maturation and memory function

Unexpected Transcriptional Programs Contribute to Hippocampal Memory Deficits and Neuronal Stunting after Early-Life Adversity.

Early-life adversity (ELA) is associated with lifelong memory deficits, yet the responsible mechanisms remain unclear. We impose ELA by rearing rat pups in simulated poverty, assess hippocampal memory, and probe changes in gene expression, their transcriptional regulation, and the consequent changes in hippocampal neuronal structure. ELA rats have poor hippocampal memory and stunted hippocampal pyramidal neurons associated with ~140 differentially expressed genes. Upstream regulators of the altered genes include glucocorticoid receptor and, unexpectedly, the transcription factor neuron-restrictive silencer factor (NRSF/REST). NRSF contributes critically to the memory deficits because blocking its function transiently following ELA rescues spatial memory and restores the dendritic arborization of hippocampal pyramidal neurons in ELA rats. Blocking NRSF function in vitro augments dendritic complexity of developing hippocampal neurons, suggesting that NRSF represses genes involved in neuronal maturation. These findings establish important, surprising contributions of NRSF to ELA-induced transcriptional programming that disrupts hippocampal maturation and memory function

A Regression-Based Analysis of Ribosome-Profiling Data Reveals a Conserved Complexity to Mammalian Translation

A fundamental goal of genomics is to identify the complete set of expressed proteins. Automated annotation strategies rely on assumptions about protein-coding sequences (CDSs), e.g., they are conserved, do not overlap, and exceed a minimum length. However, an increasing number of newly discovered proteins violate these rules. Here we present an experimental and analytical framework, based on ribosome profiling and linear regression, for systematic identification and quantification of translation. Application of this approach to lipopolysaccharide-stimulated mouse dendritic cells and HCMV-infected human fibroblasts identifies thousands of novel CDSs, including micropeptides and variants of known proteins, that bear the hallmarks of canonical translation and exhibit translation levels and dynamics comparable to that of annotated CDSs. Remarkably, many translation events are identified in both mouse and human cells even when the peptide sequence is not conserved. Our work thus reveals an unexpected complexity to mammalian translation suited to provide both conserved regulatory or protein-based functions.Klarman Cell Observator

Combinatorial Patterning of Chromatin Regulators Uncovered by Genome-wide Location Analysis in Human Cells

Hundreds of chromatin regulators (CRs) control chromatin structure and function by catalyzing and binding histone modifications, yet the rules governing these key processes remain obscure. Here, we present a systematic approach to infer CR function. We developed ChIP-string, a meso-scale assay that combines chromatin immunoprecipitation with a signature readout of 487 representative loci. We applied ChIP-string to screen 145 antibodies, thereby identifying effective reagents, which we used to map the genome-wide binding of 29 CRs in two cell types. We found that specific combinations of CRs colocalize in characteristic patterns at distinct chromatin environments, at genes of coherent functions, and at distal regulatory elements. When comparing between cell types, CRs redistribute to different loci but maintain their modular and combinatorial associations. Our work provides a multiplex method that substantially enhances the ability to monitor CR binding, presents a large resource of CR maps, and reveals common principles for combinatorial CR function.National Human Genome Research Institute (U.S.) Center of Excellence in Genome Science (Grant)Howard Hughes Medical InstituteNational Institutes of Health (U.S.). Pioneer AwardBurroughs Wellcome Fun

MOESM1 of Clinical impact of diabetes mellitus in patients undergoing transcatheter aortic valve replacement

Additional file 1: Table S1. Complication rates based on HbA1c levels