Kidney Organoids Generated Using an Allelic Series of NPHS2 Point Variants Reveal Distinct Intracellular Podocin Mistrafficking.

BACKGROUND: NPHS2 variants are the most common cause of steroid-resistant nephrotic syndrome in children >1 month old. Missense NPHS2 variants were reported to cause mistrafficking of the encoded protein, PODOCIN, but this conclusion was on the basis of overexpression in some nonpodocyte cell lines. METHODS: We generated a series of human induced pluripotent stem cell (iPSC) lines bearing pathogenic missense variants of NPHS2 , encoding the protein changes p.G92C, p.P118L, p.R138Q, p.R168H, and p.R291W, and control lines. iPSC lines were also generated from a patient with steroid-resistant nephrotic syndrome (p.R168H homozygote) and a healthy heterozygous parent. All lines were differentiated into kidney organoids. Immunofluorescence assessed PODOCIN expression and subcellular localization. Podocytes were transcriptionally profiled and PODOCIN-NEPHRIN interaction interrogated. RESULTS: All variant lines revealed reduced levels of PODOCIN protein in the absence of reduced transcription. Although wild-type PODOCIN localized to the membrane, distinct variant proteins displayed unique patterns of subcellular protein trafficking, some unreported. P118L and R138Q were preferentially retained in the endoplasmic reticulum (ER); R168H and R291W accumulated in the Golgi. Podocyte profiling demonstrated minimal disease-associated transcriptional change. All variants displayed podocyte-specific apoptosis, which was not linked to ER stress. NEPHRIN-PODOCIN colocalization elucidated the variant-specific effect on NEPHRIN association and hence NEPHRIN trafficking. CONCLUSIONS: Specific variants of endogenous NPHS2 result in distinct subcellular PODOCIN localization within organoid podocytes. Understanding the effect of each variant on protein levels and localization and the effect on NEPHRIN provides additional insight into the pathobiology of NPHS2 variants. PODCAST: This article contains a podcast at https://dts.podtrac.com/redirect.mp3/www.asn-online.org/media/podcast/JASN/2023_01_05_JASN2022060707.mp3.

3'-UTRs and the Control of Protein Expression in Space and Time.

The noncoding elements of an mRNA influence multiple aspects of its fate. For example, 3'-UTRs serve as physical and sequence-based information hubs that direct the time, place, and level of translation of the protein encoded in cis, but often also have additional roles in trans. Understanding the information content of 3'-UTRs has been a challenge. Bioinformatic searches for motifs, such as those that encode the polyadenylation signal or microRNA seed regions, are simple enough, but rarely do these inferred positions in genomes correlate well with the actual sites chosen by the relevant nanomachines in living cells. This is almost certainly due to three-dimensional complexity of RNA, the physical states of which are recognized by RNA-binding proteins that serve to read and interpret the information content. Here, we follow the 3'-UTR-mediated posttranscriptional metabolism of mRNA in the germline of the nematode worm Caenorhabditis elegans. While many areas still require the clarification only detailed fundamental research can provide, this model system can serve as a basis of 3'-mediated regulatory control for elaboration in more complex metazoan systems.

Active maintenance of CD8+ T cell naïvety through regulation of global genome architecture.

The differentiation of naïve CD8+ cytotoxic T lymphocytes (CTLs) into effector and memory states results in large scale changes in transcriptional and phenotypic profiles. Little is known about how large-scale changes in genome organisation reflect or underpin these transcriptional programs. We utilised Hi-C to map changes in the spatial organisation of long-range genome contacts within naïve, effector and memory virus-specific CD8+ T cells. We observed that the architecture of the naive CD8+ T cell genome was distinct from effector and memory genome configurations with extensive changes within discrete functional chromatin domains. However, deletion of the BACH2 or SATB1 transcription factors was sufficient to remodel the naïve chromatin architecture and engage transcriptional programs characteristic of differentiated cells. This suggests that the chromatin architecture within naïve CD8+ T cells is preconfigured to undergo autonomous remodelling upon activation, with key transcription factors restraining differentiation by actively enforcing the unique naïve chromatin state.

Active maintenance of CD8+ T cell naivety through regulation of global genome architecture.

The differentiation of naive CD8+ T lymphocytes into cytotoxic effector and memory CTL results in large-scale changes in transcriptional and phenotypic profiles. Little is known about how large-scale changes in genome organization underpin these transcriptional programs. We use Hi-C to map changes in the spatial organization of long-range genome contacts within naive, effector, and memory virus-specific CD8+ T cells. We observe that the architecture of the naive CD8+ T cell genome is distinct from effector and memory genome configurations, with extensive changes within discrete functional chromatin domains associated with effector/memory differentiation. Deletion of BACH2, or to a lesser extent, reducing SATB1 DNA binding, within naive CD8+ T cells results in a chromatin architecture more reminiscent of effector/memory states. This suggests that key transcription factors within naive CD8+ T cells act to restrain T cell differentiation by actively enforcing a unique naive chromatin state.

Parallel use of human stem cell lung and heart models provide insights for SARS-CoV-2 treatment.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) primarily infects the respiratory tract, but pulmonary and cardiac complications occur in severe coronavirus disease 2019 (COVID-19). To elucidate molecular mechanisms in the lung and heart, we conducted paired experiments in human stem cell-derived lung alveolar type II (AT2) epithelial cell and cardiac cultures infected with SARS-CoV-2. With CRISPR-Cas9-mediated knockout of ACE2, we demonstrated that angiotensin-converting enzyme 2 (ACE2) was essential for SARS-CoV-2 infection of both cell types but that further processing in lung cells required TMPRSS2, while cardiac cells required the endosomal pathway. Host responses were significantly different; transcriptome profiling and phosphoproteomics responses depended strongly on the cell type. We identified several antiviral compounds with distinct antiviral and toxicity profiles in lung AT2 and cardiac cells, highlighting the importance of using several relevant cell types for evaluation of antiviral drugs. Our data provide new insights into rational drug combinations for effective treatment of a virus that affects multiple organ systems.

A Morphometric Analysis of Commonly Used Craniometric Approaches for Freehand Ventriculoperitoneal Shunting.

BACKGROUND: Ventricular catheter tip position is a predictor for ventriculoperitoneal shunt survival. Cannulation is often performed freehand, but there is limited consensus on the best craniometric approach. OBJECTIVE: To determine the accuracy of localizing craniometric entry sites and to identify which is associated with optimal catheter placement. METHODS: This is a retrospective analysis of adult patients who underwent ventriculoperitoneal shunting. The approaches were categorized as Kocher's, Keen's, Frazier's and Dandy's points as well as the parieto-occipital point. An accurately sited burr hole was within 10 mm from standard descriptions. Optimal catheter tip position was defined as within the ipsilateral frontal horn. RESULTS: A total of 110 patients were reviewed, and 58% (65/110) of burr holes were accurately sited. Keen's point was the most correctly identified (65%, 11/17), followed by Kocher's point (65%, 37/57) and Frazier's point (60%, 3/5). Predictors for accurate localization were Keen's point (odds ratio 0.3; 95% CI: 01-0.9) and right-sided access (odds ratio 0.4; 95% CI: 0.1-0.9). Sixty-three percent (69/110) of catheters were optimally placed with Keen's point (adjusted odds ratio 0.04; 95% CI: 0.01-0.67), being the only independent factor. Thirteen patients (12%) required shunt revision at a mean duration of 10 ± 25 mo. Suboptimal catheter tip position was the only independent determinant for revision (adjusted odds ratio 0.11; 95% CI: 0.01-0.98). CONCLUSION: This is the first study to compare the accuracy of freehand ventricular cannulation of standard craniometric entry sites for adult patients. Keen's point was the most accurately sited and was a predictor for optimal catheter position. Catheter tip location, not the entry site, predicted shunt survival.

Human pluripotent stem cell-derived macrophages host Mycobacterium abscessus infection.

Human macrophages are a natural host of many mycobacterium species, including Mycobacterium abscessus (M. abscessus), an emerging pathogen affecting immunocompromised and cystic fibrosis patients with few available treatments. The search for an effective treatment is hindered by the lack of a tractable in vitro intracellular infection model. Here, we established a reliable model for M. abscessus infection using human pluripotent stem cell-derived macrophages (hPSC-macrophages). hPSC differentiation permitted reproducible generation of functional macrophages that were highly susceptible to M. abscessus infection. Electron microscopy demonstrated that M. abscessus was present in the hPSC-macrophage vacuoles. RNA sequencing analysis revealed a time-dependent host cell response, with differing gene and protein expression patterns post-infection. Engineered tdTOMATO-expressing hPSC-macrophages with GFP-expressing mycobacteria enabled rapid image-based high-throughput analysis of intracellular infection and quantitative assessment of antibiotic efficacy. Our study describes the first to our knowledge hPSC-based model for M. abscessus infection, representing a novel and accessible system for studying pathogen-host interaction and drug discovery.

Current and Emerging Trends in Diabetes Care and Education: 2021 National Practice and Workforce Survey.

PURPOSE: The purpose of the National Practice Survey is to understand current trends related to the diabetes care and education specialist's integration into the full care team beyond formal diabetes self-management education and support services. METHODS: The 2021 National Practice Survey (NPS2021) contained 61 questions for all respondents with an additional 56 questions pertaining to specific diabetes care and education segments. An anonymous survey was administered online to respondents who are diabetes care and education specialists or a part of the diabetes care team. Email lists were obtained from the Association of Diabetes Care & Education Specialists (ADCES) and the Certification Board for Diabetes Care and Education (CBDCE). Approximately 39,258 emails were sent, and 3357 were undeliverable, with 3797 surveys completed between February 9 to April 6, 2021, resulting in an 11% response rate. The response rate may have been affected by the COVID-19 public health emergency. RESULTS: Diabetes care and education specialists represent an interprofessional specialty of nurses, dietitians, physicians, pharmacists, health educators, and others. Many respondents reported holding either certification as a Certified Diabetes Care and Education Specialist (CDCES) or being Board Certified in Advanced Diabetes Management (BC-ADM). In addition, there appears to be a slight increase in those trained as a Lifestyle Coach to provide the National Diabetes Prevention Program (CDC Recognized National DPP) compared to NPS2017. Most respondents reported being Caucasian/White (84%), followed by Hispanic or Latinx (7%) and African American/Black and Asian/Asian American (at 4% each), like in previous surveys. Respondents reported diverse care delivery models, including traditional and nontraditional services, and expanded models of care such as population health/risk stratification models, the Chronic Care Model, Accountable Care Organizations, managed care, and others. CONCLUSION: The NPS2021 describes DCES workforce opportunities and challenges. Identifying and addressing those that impact the specialty's sustainability, diversity, and growth will guide strategies for the future workforce and their practice settings. Opportunities identified include embracing diabetes community care coordinators for person-centered delivery of care and education services and supporting frontline health care team members to increase competence and expertise in the prevention of type 2 diabetes, diabetes care, and education/support for related chronic diseases. In addition, as health care evolves, it creates opportunities for the DCESs to demonstrate a broader, key role as part of the diabetes care team.

Parallel use of pluripotent human stem cell lung and heart models provide new insights for treatment of SARS-CoV-2.

SARS-CoV-2 primarily infects the respiratory tract, but pulmonary and cardiac complications occur in severe COVID-19. To elucidate molecular mechanisms in the lung and heart, we conducted paired experiments in human stem cell-derived lung alveolar type II (AT2) epithelial cell and cardiac cultures infected with SARS-CoV-2. With CRISPR- Cas9 mediated knock-out of ACE2, we demonstrated that angiotensin converting enzyme 2 (ACE2) was essential for SARS-CoV-2 infection of both cell types but further processing in lung cells required TMPRSS2 while cardiac cells required the endosomal pathway. Host responses were significantly different; transcriptome profiling and phosphoproteomics responses depended strongly on the cell type. We identified several antiviral compounds with distinct antiviral and toxicity profiles in lung AT2 and cardiac cells, highlighting the importance of using several relevant cell types for evaluation of antiviral drugs. Our data provide new insights into rational drug combinations for effective treatment of a virus that affects multiple organ systems. One-sentence summary: Rational treatment strategies for SARS-CoV-2 derived from human PSC models.

Genetic and pharmacological evidence for kinetic competition between alternative poly(A) sites in yeast.

Most eukaryotic mRNAs accommodate alternative sites of poly(A) addition in the 3' untranslated region in order to regulate mRNA function. Here, we present a systematic analysis of 3' end formation factors, which revealed 3'UTR lengthening in response to a loss of the core machinery, whereas a loss of the Sen1 helicase resulted in shorter 3'UTRs. We show that the anti-cancer drug cordycepin, 3' deoxyadenosine, caused nucleotide accumulation and the usage of distal poly(A) sites. Mycophenolic acid, a drug which reduces GTP levels and impairs RNA polymerase II (RNAP II) transcription elongation, promoted the usage of proximal sites and reversed the effects of cordycepin on alternative polyadenylation. Moreover, cordycepin-mediated usage of distal sites was associated with a permissive chromatin template and was suppressed in the presence of an rpb1 mutation, which slows RNAP II elongation rate. We propose that alternative polyadenylation is governed by temporal coordination of RNAP II transcription and 3' end processing and controlled by the availability of 3' end factors, nucleotide levels and chromatin landscape.

A cis-regulatory-directed pipeline for the identification of genes involved in cardiac development and disease.

BACKGROUND: Congenital heart diseases are the major cause of death in newborns, but the genetic etiology of this developmental disorder is not fully known. The conventional approach to identify the disease-causing genes focuses on screening genes that display heart-specific expression during development. However, this approach would have discounted genes that are expressed widely in other tissues but may play critical roles in heart development. RESULTS: We report an efficient pipeline of genome-wide gene discovery based on the identification of a cardiac-specific cis-regulatory element signature that points to candidate genes involved in heart development and congenital heart disease. With this pipeline, we retrieve 76% of the known cardiac developmental genes and predict 35 novel genes that previously had no known connectivity to heart development. Functional validation of these novel cardiac genes by RNAi-mediated knockdown of the conserved orthologs in Drosophila cardiac tissue reveals that disrupting the activity of 71% of these genes leads to adult mortality. Among these genes, RpL14, RpS24, and Rpn8 are associated with heart phenotypes. CONCLUSIONS: Our pipeline has enabled the discovery of novel genes with roles in heart development. This workflow, which relies on screening for non-coding cis-regulatory signatures, is amenable for identifying developmental and disease genes for an organ without constraining to genes that are expressed exclusively in the organ of interest.

The YEATS Domain Histone Crotonylation Readers Control Virulence-Related Biology of a Major Human Pathogen.

Identification of multiple histone acylations diversifies transcriptional control by metabolism, but their functions are incompletely defined. Here we report evidence of histone crotonylation in the human fungal pathogen Candida albicans. We define the enzymes that regulate crotonylation and show its dynamic control by environmental signals: carbon sources, the short-chain fatty acids butyrate and crotonate, and cell wall stress. Crotonate regulates stress-responsive transcription and rescues C. albicans from cell wall stress, indicating broad impact on cell biology. The YEATS domain crotonylation readers Taf14 and Yaf9 are required for C. albicans virulence, and Taf14 controls gene expression, stress resistance, and invasive growth via its chromatin reader function. Blocking the Taf14 C terminus with a tag reduced virulence, suggesting that inhibiting Taf14 interactions with chromatin regulators impairs function. Our findings shed light on the regulation of histone crotonylation and the functions of the YEATS proteins in eukaryotic pathogen biology and fungal infections.

Coordination of Cell Cycle Progression and Mitotic Spindle Assembly Involves Histone H3 Lysine 4 Methylation by Set1/COMPASS.

Methylation of histone H3 lysine 4 (H3K4) by Set1 complex/COMPASS is a hallmark of eukaryotic chromatin, but it remains poorly understood how this post-translational modification contributes to the regulation of biological processes like the cell cycle. Here, we report a H3K4 methylation-dependent pathway in Saccharomyces cerevisiae that governs toxicity toward benomyl, a microtubule destabilizing drug. Benomyl-sensitive growth of wild-type cells required mono- and dimethylation of H3K4 and Pho23, a PHD-containing subunit of the Rpd3L complex. Δset1 and Δpho23 deletions suppressed defects associated with ipl1-2 aurora kinase mutant, an integral component of the spindle assembly checkpoint during mitosis. Benomyl resistance of Δset1 strains was accompanied by deregulation of all four tubulin genes and the phenotype was suppressed by tub2-423 and Δtub3 mutations, establishing a genetic link between H3K4 methylation and microtubule function. Most interestingly, sine wave fitting and clustering of transcript abundance time series in synchronized cells revealed a requirement for Set1 for proper cell-cycle-dependent gene expression and Δset1 cells displayed delayed entry into S phase. Disruption of G1/S regulation in Δmbp1 and Δswi4 transcription factor mutants duplicated both benomyl resistance and suppression of ipl1-2 as was observed with Δset1 Taken together our results support a role for H3K4 methylation in the coordination of cell-cycle progression and proper assembly of the mitotic spindle during mitosis.