Human TSC2 Mutant Cells Exhibit Aberrations in Early Neurodevelopment Accompanied by Changes in the DNA Methylome.

Tuberous Sclerosis Complex (TSC) is a debilitating developmental disorder characterized by a variety of clinical manifestations. While benign tumors in the heart, lungs, kidney, and brain are all hallmarks of the disease, the most severe symptoms of TSC are often neurological, including seizures, autism, psychiatric disorders, and intellectual disabilities. TSC is caused by loss of function mutations in the or genes and consequent dysregulation of signaling via mechanistic Target of Rapamycin Complex 1 (mTORC1).

Genomic dissection and mutation-specific target discovery for breast cancer PIK3CA hotspot mutations.

Background: Recent advancements in high-throughput genomics and targeted therapies have provided tremendous potential to identify and therapeutically target distinct mutations associated with cancers. However, to date the majority of targeted therapies are used to treat all functional mutations within the same gene, regardless of affected codon or phenotype.

Results: In this study, we developed a functional genomic analysis workflow with a unique isogenic cell line panel bearing two distinct hotspot PIK3CA mutations, E545K and H1047R, to accurately identify targetable differences between mutations within the same gene.

Human gene regulatory evolution is driven by the divergence of regulatory element function in both cis and trans.

Gene regulatory divergence between species can result from cis-acting local changes to regulatory element DNA sequences or global trans-acting changes to the regulatory environment. Understanding how these mechanisms drive regulatory evolution has been limited by challenges in identifying trans-acting changes. We present a comprehensive approach to directly identify cis- and trans-divergent regulatory elements between human and rhesus macaque lymphoblastoid cells using assay for transposase-accessible chromatin coupled to self-transcribing active regulatory region (ATAC-STARR) sequencing.

Endocrine islet β-cell subtypes with differential function are derived from biochemically distinct embryonic endocrine islet progenitors that are regulated by maternal nutrients.

Endocrine islet b cells comprise heterogenous cell subsets. Yet when/how these subsets are produced and how stable they are remain unknown. Addressing these questions is important for preventing/curing diabetes, because lower numbers of b cells with better secretory function is a high risk of this disease.

Enhancer-promoter activation by the Kaposi sarcoma-associated herpesvirus episome maintenance protein LANA.

Higher-order genome structure influences the transcriptional regulation of cellular genes through the juxtaposition of regulatory elements, such as enhancers, close to promoters of target genes. While enhancer activation has emerged as an important facet of Kaposi sarcoma-associated herpesvirus (KSHV) biology, the mechanisms controlling enhancer-target gene expression remain obscure. Here, we discover that the KSHV genome tethering protein latency-associated nuclear antigen (LANA) potentiates enhancer-target gene expression in primary effusion lymphoma (PEL), a highly aggressive B cell lymphoma causally associated with KSHV.

Polycomb repressor complex 2 suppresses interferon-responsive MHC-II expression in melanoma cells and is associated with anti-PD-1 resistance.

Background: Despite the remarkable success of immunotherapy in treating melanoma, understanding of the underlying mechanisms of resistance remains limited. Emerging evidence suggests that upregulation of tumor-specific major histocompatibility complex-II (tsMHC-II) serves as a predictive marker for the response to anti-programmed death-1 (PD-1)/programmed death ligand 1 (PD-L1) therapy in various cancer types. The genetic and epigenetic pathways modulating tsMHC-II expression remain incompletely characterized.

Genomic dissection and mutation-specific target discovery for breast cancer hotspot mutations.

Background: Recent advancements in high-throughput genomics and targeted therapies have provided tremendous potential to identify and therapeutically target distinct mutations associated with cancers. However, to date the majority of targeted therapies are used to treat all functional mutations within the same gene, regardless of affected codon or phenotype.

Results: In this study, we developed a functional genomic analysis workflow with a unique isogenic cell line panel bearing two distinct hotspot mutations, E545K and H1047R, to accurately identify targetable differences between mutations within the same gene.

Cross-tissue patterns of DNA hypomethylation reveal genetically distinct histories of cell development.

Background: Establishment of DNA methylation (DNAme) patterns is essential for balanced multi-lineage cellular differentiation, but exactly how these patterns drive cellular phenotypes is unclear. While > 80% of CpG sites are stably methylated, tens of thousands of discrete CpG loci form hypomethylated regions (HMRs). Because they lack DNAme, HMRs are considered transcriptionally permissive, but not all HMRs actively regulate genes.

Opposite modulation of functional recovery following contusive spinal cord injury in mice with oligodendrocyte-selective deletions of Atf4 and Chop/Ddit3.

The integrated stress response (ISR)-activated transcription factors ATF4 and CHOP/DDIT3 may regulate oligodendrocyte (OL) survival, tissue damage and functional impairment/recovery in white matter pathologies, including traumatic spinal cord injury (SCI). Accordingly, in OLs of OL-specific RiboTag mice, Atf4, Chop/Ddit3 and their downstream target gene transcripts were acutely upregulated at 2, but not 10, days post-contusive T9 SCI coinciding with maximal loss of spinal cord tissue. Unexpectedly, another, OL-specific upregulation of Atf4/Chop followed at 42 days post-injury.

Human gene regulatory evolution is driven by the divergence of regulatory element function in both and .

Gene regulatory divergence between species can result from -acting local changes to regulatory element DNA sequences or global -acting changes to the regulatory environment. Understanding how these mechanisms drive regulatory evolution has been limited by challenges in identifying acting changes. We present a comprehensive approach to directly identify and divergent regulatory elements between human and rhesus macaque lymphoblastoid cells using ATAC-STARR-seq.

Human SMARCA5 is continuously required to maintain nucleosome spacing.

Genetic models suggested that SMARCA5 was required for DNA-templated events including transcription, DNA replication, and DNA repair. We engineered a degron tag into the endogenous alleles of SMARCA5, a catalytic component of the imitation switch complexes in three different human cell lines to define the effects of rapid degradation of this key regulator. Degradation of SMARCA5 was associated with a rapid increase in global nucleosome repeat length, which may allow greater chromatin compaction.

ATAC-STARR-seq reveals transcription factor-bound activators and silencers within chromatin-accessible regions of the human genome.

Massively parallel reporter assays (MPRAs) test the capacity of putative gene regulatory elements to drive transcription on a genome-wide scale. Most gene regulatory activity occurs within accessible chromatin, and recently described methods have combined assays that capture these regions-such as assay for transposase-accessible chromatin using sequencing (ATAC-seq)-with self-transcribing active regulatory region sequencing (STARR-seq) to selectively assay the regulatory potential of accessible DNA (ATAC-STARR-seq). Here, we report an integrated approach that quantifies activating and silencing regulatory activity, chromatin accessibility, and transcription factor (TF) occupancy with one assay using ATAC-STARR-seq.

Role of circadian rhythms in pathogenesis of acute CNS injuries: Insights from experimental studies.

A wide range of physiological processes show circadian oscillations that are critical for organismal homeostasis. Consequently, disruption of such rhythmicity contributes to the pathogenesis of various chronic diseases. The occurrence, severity, and resolution of acute injuries to the central nervous system may also be modulated by circadian rhythms and/or anti-rhythmic disruptions.

The glucose error in arterial sampling: assessing staff awareness and the effect of sampling technique.

Background: Using a dextrose-containing solution, instead of normal saline, to maintain the patency of an arterial cannula results in the admixture of glucose in line samples. This can misguide the clinician down an inappropriate treatment pathway for hyperglycaemia.

Methods: Following a near-miss and subsequent educational and training efforts at our institution, we conducted two simulations: (1) to observe whether 20 staff would identify a 5% dextrose/0.

Limited changes in locomotor recovery and unaffected white matter sparing after spinal cord contusion at different times of day.

The circadian gene expression rhythmicity drives diurnal oscillations of physiological processes that may determine the injury response. While outcomes of various acute injuries are affected by the time of day at which the original insult occurred, such influences on recovery after spinal cord injury (SCI) are unknown. We report that mice receiving moderate, T9 contusive SCI at ZT0 (zeitgeber time 0, time of lights on) and ZT12 (time of lights off) showed similar hindlimb function recovery in the Basso mouse scale (BMS) over a 6 week post-injury period.

SLC5A3-Dependent Myo-inositol Auxotrophy in Acute Myeloid Leukemia.

An enhanced requirement for nutrients is a hallmark property of cancer cells. Here, we optimized an genetic screening strategy in acute myeloid leukemia (AML), which led to the identification of the myo-inositol transporter SLC5A3 as a dependency in this disease. We demonstrate that SLC5A3 is essential to support a myo-inositol auxotrophy in AML.

Transcriptional Silencing of Confers a Dependency on Fanconi Anemia Proteins in Acute Myeloid Leukemia.

Hundreds of genes become aberrantly silenced in acute myeloid leukemia (AML), with most of these epigenetic changes being of unknown functional consequence. Here, we demonstrate how gene silencing can lead to an acquired dependency on the DNA repair machinery in AML. We make this observation by profiling the essentiality of the ubiquitination machinery in cancer cell lines using domain-focused CRISPR screening, which revealed Fanconi anemia (FA) proteins UBE2T and FANCL as unique dependencies in AML.

British Association of Critical Care Nurses: Evidence-based consensus paper for oral care within adult critical care units.

Background: Patients who are critically ill are at increased risk of hospital acquired pneumonia and ventilator associated pneumonia. Effective evidence based oral care may reduce the incidence of such iatrogenic infection.

Aim: To provide an evidence-based British Association of Critical Care Nurses endorsed consensus paper for best practice relating to implementing oral care, with the intention of promoting patient comfort and reducing hospital acquired pneumonia and ventilator associated pneumonia in critically ill patients.

ATAC-Me Captures Prolonged DNA Methylation of Dynamic Chromatin Accessibility Loci during Cell Fate Transitions.

DNA methylation of enhancers is dynamic, cell-type specific, and vital for cell fate progression. However, current models inadequately define its role within the hierarchy of gene regulation. Analysis of independent datasets shows an unanticipated overlap between DNA methylation and chromatin accessibility at enhancers of steady-state stem cells, suggesting that these two opposing features might exist concurrently.

Neurog3-Independent Methylation Is the Earliest Detectable Mark Distinguishing Pancreatic Progenitor Identity.

In the developing pancreas, transient Neurog3-expressing progenitors give rise to four major islet cell types: α, β, δ, and γ; when and how the Neurog3 cells choose cell fate is unknown. Using single-cell RNA-seq, trajectory analysis, and combinatorial lineage tracing, we showed here that the Neurog3 cells co-expressing Myt1 (i.e.

Molecular regulation of peripheral B cells and their progeny in immunity.

Mature B lymphocytes are crucial components of adaptive immunity, a system essential for the evolutionary fitness of mammals. Adaptive lymphocyte function requires an initially naïve cell to proliferate extensively and its progeny to have the capacity to assume a variety of fates. These include either terminal differentiation (the long-lived plasma cell) or metastable transcriptional reprogramming (germinal center and memory B cells).

Unintentional endotracheal tube cuff deflation during routine checks: a simulation study.

Background: Endotracheal tube cuff pressures should be maintained between 20 and 30 cm H O to prevent the aspiration of subglottic secretions past the cuff. Guidance recommends regular monitoring of the cuff pressure, performed using a handheld manometer/inflator. Poor technique can lead to transient deflation of the cuff, leading to the bolus aspiration of upper respiratory tract secretions, tracheal colonization and, ultimately, ventilator-associated pneumonia.