Tracking cell-type-specific temporal dynamics in human and mouse brains.

Progenitor cells are critical in preserving organismal homeostasis, yet their diversity and dynamics in the aged brain remain underexplored. We introduced TrackerSci, a single-cell genomic method that combines newborn cell labeling and combinatorial indexing to characterize the transcriptome and chromatin landscape of proliferating progenitor cells in vivo. Using TrackerSci, we investigated the dynamics of newborn cells in mouse brains across various ages and in a mouse model of Alzheimer's disease. Our dataset revealed diverse progenitor cell types in the brain and their epigenetic signatures. We further quantified aging-associated shifts in cell-type-specific proliferation and differentiation and deciphered the associated molecular programs. Extending our study to the progenitor cells in the aged human brain, we identified conserved genetic signatures across species and pinpointed region-specific cellular dynamics, such as the reduced oligodendrogenesis in the cerebellum. We anticipate that TrackerSci will be broadly applicable to unveil cell-type-specific temporal dynamics in diverse systems.

A Genome-wide Framework for Mapping Gene Regulation via Cellular Genetic Screens.

Over one million candidate regulatory elements have been identified across the human genome, but nearly all are unvalidated and their target genes uncertain. Approaches based on human genetics are limited in scope to common variants and in resolution by linkage disequilibrium. We present a multiplex, expression quantitative trait locus (eQTL)-inspired framework for mapping enhancer-gene pairs by introducing random combinations of CRISPR/Cas9-mediated perturbations to each of many cells, followed by single-cell RNA sequencing (RNA-seq). Across two experiments, we used dCas9-KRAB to perturb 5,920 candidate enhancers with no strong a priori hypothesis as to their target gene(s), measuring effects by profiling 254,974 single-cell transcriptomes. We identified 664 (470 high-confidence) cis enhancer-gene pairs, which were enriched for specific transcription factors, non-housekeeping status, and genomic and 3D conformational proximity to their target genes. This framework will facilitate the large-scale mapping of enhancer-gene regulatory interactions, a critical yet largely uncharted component of the cis-regulatory landscape of the human genome.

Accurate classification of BRCA1 variants with saturation genome editing.

Variants of uncertain significance fundamentally limit the clinical utility of genetic information. The challenge they pose is epitomized by BRCA1, a tumour suppressor gene in which germline loss-of-function variants predispose women to breast and ovarian cancer. Although BRCA1 has been sequenced in millions of women, the risk associated with most newly observed variants cannot be definitively assigned. Here we use saturation genome editing to assay 96.5% of all possible single-nucleotide variants (SNVs) in 13 exons that encode functionally critical domains of BRCA1. Functional effects for nearly 4,000 SNVs are bimodally distributed and almost perfectly concordant with established assessments of pathogenicity. Over 400 non-functional missense SNVs are identified, as well as around 300 SNVs that disrupt expression. We predict that these results will be immediately useful for the clinical interpretation of BRCA1 variants, and that this approach can be extended to overcome the challenge of variants of uncertain significance in additional clinically actionable genes.

CRISPR/Cas9-Mediated Scanning for Regulatory Elements Required for HPRT1 Expression via Thousands of Large, Programmed Genomic Deletions.

The extent to which non-coding mutations contribute to Mendelian disease is a major unknown in human genetics. Relatedly, the vast majority of candidate regulatory elements have yet to be functionally validated. Here, we describe a CRISPR-based system that uses pairs of guide RNAs (gRNAs) to program thousands of kilobase-scale deletions that deeply scan across a targeted region in a tiling fashion ("ScanDel"). We applied ScanDel to HPRT1, the housekeeping gene underlying Lesch-Nyhan syndrome, an X-linked recessive disorder. Altogether, we programmed 4,342 overlapping 1 and 2 kb deletions that tiled 206 kb centered on HPRT1 (including 87 kb upstream and 79 kb downstream) with median 27-fold redundancy per base. We functionally assayed programmed deletions in parallel by selecting for loss of HPRT function with 6-thioguanine. As expected, sequencing gRNA pairs before and after selection confirmed that all HPRT1 exons are needed. However, HPRT1 function was robust to deletion of any intergenic or deeply intronic non-coding region, indicating that proximal regulatory sequences are sufficient for HPRT1 expression. Although our screen did identify the disruption of exon-proximal non-coding sequences (e.g., the promoter) as functionally consequential, long-read sequencing revealed that this signal was driven by rare, imprecise deletions that extended into exons. Our results suggest that no singular distal regulatory element is required for HPRT1 expression and that distal mutations are unlikely to contribute substantially to Lesch-Nyhan syndrome burden. Further application of ScanDel could shed light on the role of regulatory mutations in disease at other loci while also facilitating a deeper understanding of endogenous gene regulation.

Practical problems in the posthumous retrieval of sperm.

This communication discusses the practical problems that arise during the collection and processing of sperm that have been retrieved posthumously. It is based on a small group, namely the last six men from whom we carried out posthumous retrieval. The reason for each retrieval, the method of that retrieval, the assessment of the sperm retrieved, the subsequent viability of the sperm and their storage method are discussed. The many ethical and legal problems that arise both before and after posthumous sperm retrieval are huge in their complexity. Therefore, they will not be discussed here and this communication will be limited to the practical aspects of posthumous sperm retrieval. The purpose of this communication is to make some suggestions that will facilitate such collections. The whole subject of posthumous sperm collection is gaining increasing clinical importance and has begun to interest the media as demonstrated by the recent national coverage in an Australian newspaper.

Hit-to-lead optimization and kinase selectivity of imidazo[1,2-a]quinoxalin-4-amine derived JNK1 inhibitors.

As the result of a rhJNK1 HTS, the imidazo[1,2-a]quinoxaline 1 was identified as a 1.6 µM rhJNK1 inhibitor. Optimization of this compound lead to AX13587 (rhJNK1 IC50=160 nM) which was co-crystallized with JNK1 to identify key molecular interactions. Kinase profiling against 125+ kinases revealed AX13587 was an inhibitor of JNK, MAST3, and MAST4 whereas its methylene homolog AX14373 (native JNK1 IC50=47 nM) was a highly specific JNK inhibitor.

6-Position optimization of tricyclic 4-quinolone-based inhibitors of glycogen synthase kinase-3ß: discovery of nitrile derivatives with picomolar potency.

We previously disclosed tricylic, 6-carboxylic acid-bearing 4-quinolones as GSK-3ß inhibitors. Herein we discuss the optimization of this series to yield a series of more potent 6-nitrile analogs with insignificant anti-microbial activity. Finally, kinase profiling indicated that members of this class were highly specific GSK-3 inhibitors.

Oxytocin-like receptor expression in evolutionarily conserved nodes of a vocal network associated with male courtship in a teleost fish.

Neuropeptides, including oxytocin-like peptides, are a conserved group of hormones that regulate a wide range of social behaviors, including vocal communication. In the current study, we evaluate whether putative brain sites for the actions of isotocin (IT), the oxytocin (OT) homolog of teleost fishes are associated with vocal courtship and circuitry in the plainfin midshipman fish (Porichthys notatus). During the breeding season, nesting males produce advertisement calls known as "hums" to acoustically court females at night and attract them to nests. We first identify IT receptor (ITR) mRNA in evolutionarily conserved regions of the forebrain preoptic area (POA), anterior hypothalamus (AH), and midbrain periaqueductal gray (PAG), and in two topographically separate populations within the hindbrain vocal pattern generator- duration-coding vocal prepacemaker (VPP) and amplitude-coding vocal motor nuclei (VMN) that also innervate vocal muscles. We also verify that ITR expression overlaps known distribution sites of OT-like immunoreactive fibers. Next, using phosphorylated ribosomal subunit 6 (pS6) as a marker for activated neurons, we demonstrate that ITR-containing neurons in the anterior parvocellular POA, AH, PAG, VPP, and VMN are activated in humming males. Posterior parvocellular and magno/gigantocellular divisions of the POA remain constitutively active in nonhumming males that are also in a reproductive state. Together with prior studies of midshipman fish and other vertebrates, our findings suggest that IT-signaling influences male courtship behavior, in part, by acting on brain regions that broadly influence behavioral state (POA) as well as the initiation (POA and PAG) and temporal structure (VPP and VMN) of advertisement hums.

Synthesis and structure-activity relationship of 4-quinolone-3-carboxylic acid based inhibitors of glycogen synthase kinase-3ß.

The synthesis, GSK-3ß inhibitory activity, and anti-microbial activity of bicyclic and tricyclic derivatives of the 5,7-diamino-6-fluoro-4-quinolone-3-carboxylic acid scaffold were studied. Kinase selectivity profiling indicated that members of this class were potent and highly selective GSK-3 inhibitors.

Allopurinol reverses mercaptopurine-induced hypoglycemia in patients with acute lymphoblastic leukemia.

Fasting hypoglycemia is a known complication of mercaptopurine (6MP) maintenance therapy for acute lymphoblastic leukemia (ALL). It is associated with high levels of the methylated metabolite 6-methyl-mercaptopurine (6MMP). Symptoms of hypoglycemia include morning tremulousness, nausea and vomiting. We have previously shown that switching 6MP dosing from evening to morning resolved hypoglycemia by reducing 6MMP; however, the reduction of 6MMP was only transient, potentially resulting in return of hypoglycemia. In children and adults with Crohn's disease, co-prescribing allopurinol with 6MP blocks the activity of thiopurine methytransferase (TPMT), reducing 6MMP and improving its tolerance. As a consequence of inhibiting TPMT, 6MP is shunted toward the production of 6-thioguanine nucleotide (6TGN), which will result in pancytopenia if the dose of 6MP is not reduced. We demonstrate that allopurinol with a reduced dose of 6MP in two patients with ALL and 6MMP-associated hypoglycemia resulted in a complete and sustained suppression of 6MMP and rapid reversal of hypoglycemia and its symptoms.

Caregivers' experiences of feeding children with cerebral palsy: a systematic review protocol of qualitative evidence.

REVIEW QUESTION/OBJECTIVE: The overall objective of this systematic review is to identify, critically appraise and synthesize the literature regarding the feeding experiences of caregivers who care for children with cerebral palsy. The specific review question is: What are the experiences of caregivers feeding children with cerebral palsy?