The gain-of-function mutation in the TALK-1 K
channel (p.L114P) is associated with maturity-onset diabetes of the young (MODY). TALK-1 is a key regulator of β-cell electrical activity and ...glucose-stimulated insulin secretion. The
gene encoding TALK-1 is the most abundant and β-cell-restricted K
channel transcript. To investigate the impact of
L114P on glucose homeostasis and confirm its association with MODY, a mouse model containing the
L114P mutation was generated. Heterozygous and homozygous
L114P mice exhibit increased neonatal lethality in the C57BL/6J and the CD-1 (ICR) genetic background, respectively. Lethality is likely a result of severe hyperglycemia observed in the homozygous
L114P neonates due to lack of glucose-stimulated insulin secretion and can be reduced with insulin treatment.
L114P increased whole-cell β-cell K
currents resulting in blunted glucose-stimulated Ca
entry and loss of glucose-induced Ca
oscillations. Thus, adult
L114P mice have reduced glucose-stimulated insulin secretion and plasma insulin levels, which significantly impairs glucose homeostasis. Taken together, this study shows that the MODY-associated
L114P mutation disrupts glucose homeostasis in adult mice resembling a MODY phenotype and causes neonatal lethality by inhibiting islet insulin secretion during development. These data suggest that TALK-1 is an islet-restricted target for the treatment for diabetes.
Biomedical sciences PhDs pursue a wide range of careers inside and outside academia. However, there is little data regarding how career interests of PhD students relate to the decision to pursue ...postdoctoral training or to their eventual career outcomes. Here, we present the career goals and career outcomes of 1452 biomedical sciences PhDs who graduated from Vanderbilt University between 1997 and 2021. We categorized careers using an expanded three-tiered taxonomy and flags that delineate key career milestones. We also analyzed career goal changes between matriculation and doctoral defense, and the reasons why students became more- or less-interested in research-intensive faculty careers. We linked students' career goal at doctoral defense to whether they did a postdoc, the duration of time between doctoral defense and the first non-training position, the career area of the first non-training position, and the career area of the job at 10 years after graduation. Finally, we followed individual careers for 10 years after graduation to characterize movement between different career areas over time. We found that most students changed their career goal during graduate school, declining numbers of alumni pursued postdoctoral training, many alumni entered first non-training positions in a different career area than their goal at doctoral defense, and the career area of the first non-training position was a good indicator of the job that alumni held 10 years after graduation. Our findings emphasize that students need a wide range of career development opportunities and career mentoring during graduate school to prepare them for futures in research and research-related professions.
Brown adipose tissue (BAT) has in recent times been rediscovered in adult humans, and together with work from preclinical models, has shown to have the potential of providing a variety of positive ...metabolic benefits. These include lower plasma glucose, improved insulin sensitivity, and reduced susceptibility to obesity and its comorbidities. As such, its continued study could offer insights to therapeutically modulate this tissue to improve metabolic health. It has been reported that adipose‐specific deletion of the gene for protein kinase D1 (Prkd1) in mice enhances mitochondrial respiration and improves whole‐body glucose homeostasis. We sought to determine whether these effects were mediated specifically through brown adipocytes using a Prkd1 brown adipose tissue (BAT) Ucp1‐Cre‐specific knockout mouse model, Prkd1BKO. We unexpectedly observed that upon both cold exposure and β3‐AR agonist administration, Prkd1 loss in BAT did not alter canonical thermogenic gene expression or adipocyte morphology. We took an unbiased approach to assess whether other signaling pathways were affected. RNA from cold‐exposed mice was subjected to RNA‐Seq analysis. These studies revealed that myogenic gene expression is altered in Prkd1BKO BAT after both acute and extended cold exposure. Given that brown adipocytes and skeletal myocytes share a common precursor cell lineage expressing myogenic factor 5 (Myf5), these data suggest that loss of Prkd1 in BAT may alter the biology of mature brown adipocytes and preadipocytes in this depot. The data presented herein clarify the role of Prkd1 in BAT thermogenesis and present new avenues for the further study of Prkd1 function in BAT.
The primary goal of these studies was to determine whether loss of PRKD1 in uncoupling protein‐1 (UCP1)‐expressing adipocytes altered the β‐adrenergic receptor (β‐AR)‐stimulated brown adipose tissue (BAT) thermogenesis program since it had been previously reported that adipose‐specific deletion of the gene for Prkd1 in mice enhanced mitochondrial respiration and improved whole‐body glucose homeostasis. We find that Prkd1BKO mice show no differences in brown adipocyte thermogenic gene expression, histology, and morphology compared with their Prkd1fl/fl littermates. However, unbiased RNA‐Seq studies showed that upon cold exposure, Prkd1 loss in BAT significantly altered myogenic gene signatures in the Prkd1BKO mice.
Clonal evolution in myelodysplastic syndrome (MDS) can result in clinical progression and secondary acute myeloid leukemia (sAML). To dissect changes in clonal architecture associated with this ...progression, we performed single-cell genotyping of paired MDS and sAML samples from 18 patients. Analysis of single-cell genotypes revealed patient-specific clonal evolution and enabled the assessment of single-cell mutational cooccurrence. We discovered that changes in clonal architecture proceed via distinct patterns, classified as static or dynamic, with dynamic clonal architectures having a more proliferative phenotype by blast count fold change. Proteogenomic analysis of a subset of patients confirmed that pathogenic mutations were primarily confined to primitive and mature myeloid cells, though we also identify rare but present mutations in lymphocyte subsets. Single-cell transcriptomic analysis of paired sample sets further identified gene sets and signaling pathways involved in two cases of progression. Together, these data define serial changes in the MDS clonal landscape with clinical and therapeutic implications.
Precise clonal trajectories in MDS progression are made possible by single-cell genomic sequencing. Here we use this technology to uncover the patterns of clonal architecture and clonal evolution that drive the transformation to secondary AML. We further define the phenotypic and transcriptional changes of disease progression at the single-cell level. See related article by Menssen et al., p. 330 (31). See related commentary by Romine and van Galen, p. 270. This article is highlighted in the In This Issue feature, p. 265.
Repressive KRAB domain-containing zinc-finger proteins (KRAB-ZFPs) are abundant in mammalian genomes and contribute both to the silencing of transposable elements (TEs) and to the regulation of ...developmental stage- and cell type-specific gene expression. Here we describe studies of zinc finger protein 92 (Zfp92), an X-linked KRAB-ZFP that is highly expressed in pancreatic islets of adult mice, by analyzing global Zfp92 knockout (KO) mice. Physiological, transcriptomic and genome-wide chromatin binding studies indicate that the principal function of ZFP92 in mice is to bind to and suppress the activity of B1/Alu type of SINE elements and modulate the activity of surrounding genomic entities. Deletion of Zfp92 leads to changes in expression of select LINE and LTR retroelements and genes located in the vicinity of ZFP92-bound chromatin. The absence of Zfp92 leads to altered expression of specific genes in islets, adipose and muscle that result in modest sex-specific alterations in blood glucose homeostasis, body mass and fat accumulation. In islets, Zfp92 influences blood glucose concentration in postnatal mice via transcriptional effects on Mafb, whereas in adipose and muscle, it regulates Acacb, a rate-limiting enzyme in fatty acid metabolism. In the absence of Zfp92, a novel TE-Capn11 fusion transcript is overexpressed in islets and several other tissues due to de-repression of an IAPez TE adjacent to ZFP92-bound SINE elements in intron 3 of the Capn11 gene. Together, these studies show that ZFP92 functions both to repress specific TEs and to regulate the transcription of specific genes in discrete tissues.
Crystal structures of the Asp$^{96}$ to Asn mutant of the light-driven proton pump bacteriorhodopsin and its M photointermediate produced by illumination at ambient temperature have been determined ...to 1.8 and 2.0 angstroms resolution, respectively. The trapped photoproduct corresponds to the late M state in the transport cycle-that is, after proton transfer to Asp$^{85}$ and release of a proton to the extracellular membrane surface, but before reprotonation of the deprotonated retinal Schiff base. Its density map describes displacements of side chains near the retinal induced by its photoisomerization to 13-cis, 15-anti and an extensive rearrangement of the three-dimensional network of hydrogenbonded residues and bound water that accounts for the changed pK$_a$ values (where K$_a$ is the acid constant) of the Schiff base and Asp$^{85}$. The structural changes detected suggest the means for conserving energy at the active site and for ensuring the directionality of proton translocation.
The conversion of adenosine to inosine (A to I) by RNA editing represents a common posttranscriptional mechanism for diversification of both the transcriptome and proteome, and is a part of the ...cellular response for innate immune tolerance. Due to its preferential base-pairing with cytosine (C), inosine (I) is recognized as guanosine (G) by reverse transcriptase, as well as the cellular splicing and translation machinery. A-to-I editing events appear as A-G discrepancies between genomic DNA and cDNA sequences. Molecular analyses of RNA editing have leveraged these nucleoside differences to quantify RNA editing in ensemble populations of RNA transcripts and within individual cDNAs using high-throughput sequencing or Sanger sequencing-derived analysis of electropherogram peak heights. Here, we briefly review and compare these methods of RNA editing quantification, as well as provide experimental protocols by which such analyses may be achieved.
ASCL1, a pioneer transcription factor, is essential for neural cell differentiation and function. Previous studies have shown that Ascl1 expression is increased in pancreatic β-cells lacking ...functional KATP channels or after feeding of a high fat diet (HFD) suggesting that it may contribute to the metabolic stress response of β-cells.
We generated β-cell-specific Ascl1 knockout mice (Ascl1βKO) and assessed their glucose homeostasis, islet morphology and gene expression after feeding either a normal diet or HFD for 12 weeks, or in combination with a genetic disruption of Abcc8, an essential KATP channel component.
Ascl1 expression is increased in response to both a HFD and membrane depolarization and requires CREB-dependent Ca2+ signaling. No differences in glucose homeostasis or islet morphology were observed in Ascl1βKO mice fed a normal diet or in the absence of KATP channels. However, male Ascl1βKO mice fed a HFD exhibited decreased blood glucose levels, improved glucose tolerance, and increased β-cell proliferation. Bulk RNA-seq analysis of islets from Ascl1βKO mice from three studied conditions showed alterations in genes associated with the secretory function. HFD-fed Ascl1βKO mice showed the most extensive changes with increased expression of genes necessary for glucose sensing, insulin secretion and β-cell proliferation, and a decrease in genes associated with β-cell dysfunction, inflammation and dedifferentiation. HFD-fed Ascl1βKO mice also displayed increased expression of parasympathetic neural markers and cholinergic receptors that was accompanied by increased insulin secretion in response to acetylcholine and an increase in islet innervation.
Ascl1 expression is induced by stimuli that cause Ca2+-signaling to the nucleus and contributes in a multifactorial manner to the loss of β-cell function by promoting the expression of genes associated with cellular dedifferentiation, attenuating β-cells proliferation, suppressing acetylcholine sensitivity, and repressing parasympathetic innervation of islets. Thus, the removal of Ascl1 from β-cells improves their function in response to metabolic stress.
•Induction of Ascl1 in pancreatic β-cells requires CREB-dependent Ca2+ signaling.•Deletion of Ascl1 does not measurably alter islet function in the normal state.•β-cells lacking Ascl1 exhibit increased insulin secretion and proliferation on a HFD.•HFD-induced β-cell dedifferentiation is decreased in the absence of Ascl1.•Parasympathetic innervation is increased in mice lacking Ascl1 on a HFD.
The α-chains of the collagen superfamily are encoded with information that specifies self-assembly into fibrils, microfibrils, and networks that have diverse functions in the extracellular matrix. A ...key self-organizing step, common to all collagen types, is trimerization that selects, binds, and registers cognate α-chains for assembly of triple helical protomers that subsequently oligomerize into specific suprastructures. In this article, we review recent findings on the mechanism of chain selection and infer that terminal noncollagenous domains function as recognition modules in trimerization and are therefore key determinants of specificity in the assembly of suprastructures. This mechanism is also illustrated with computer-generated animations.
Leigh syndrome (LS) is a rare, inherited neurometabolic disorder that presents with bilateral brain lesions caused by defects in the mitochondrial respiratory chain and associated nuclear-encoded ...proteins. We generated human induced pluripotent stem cells (iPSCs) from three LS patient-derived fibroblast lines. Using whole-exome and mitochondrial sequencing, we identified unreported mutations in pyruvate dehydrogenase (GM0372, PDH; GM13411, MT-ATP6/PDH) and dihydrolipoyl dehydrogenase (GM01503, DLD). These LS patient-derived iPSC lines were viable and capable of differentiating into progenitor populations, but we identified several abnormalities in three-dimensional differentiation models of brain development. LS patient-derived cerebral organoids showed defects in neural epithelial bud generation, size and cortical architecture at 100 days. The double mutant MT-ATP6/PDH line produced organoid neural precursor cells with abnormal mitochondrial morphology, characterized by fragmentation and disorganization, and showed an increased generation of astrocytes. These studies aim to provide a comprehensive phenotypic characterization of available patient-derived cell lines that can be used to study Leigh syndrome.
PDF
