Improved outcomes and staging in non-small-cell lung cancer guided by a molecular assay.

There remains a critical need for improved staging of non-small-cell lung cancer, as recurrence and mortality due to undetectable metastases at the time of surgery remain high even after complete resection of tumors currently categorized as 'early stage.' A 14-gene quantitative PCR-based expression profile has been extensively validated to better identify patients at high-risk of 5-year mortality after surgical resection than conventional staging - mortality that almost always results from previously undetectable metastases. Furthermore, prospective studies now suggest a predictive benefit in disease-free survival when the assay is used to guide adjuvant chemotherapy decisions in early-stage non-small-cell lung cancer patients.

Lay abstract There is a need for improvement in the way early-stage non-small-cell lung cancers are staged and treated because many patients with 'early-stage' disease suffer high rates of cancer recurrence after surgery. In recent years, a specialized test has been developed to allow better characterization of a tumor's risk of recurrence based on the genes being expressed by tumor cells. Use of this test, in conjunction with standard staging methods, is better able to identify patients at high risk of cancer recurrence after surgery. Evidence suggests that giving chemotherapy to patients at high risk of recurrence after surgery reduces recurrence rates and improves long-term patient survival.

Variants in tubule epithelial regulatory elements mediate most heritable differences in human kidney function.

Kidney disease is highly heritable; however, the causal genetic variants, the cell types in which these variants function, and the molecular mechanisms underlying kidney disease remain largely unknown. To identify genetic loci affecting kidney function, we performed a GWAS using multiple kidney function biomarkers and identified 462 loci. To begin to investigate how these loci affect kidney function, we generated single-cell chromatin accessibility (scATAC-seq) maps of the human kidney and identified candidate cis -regulatory elements (cCREs) for kidney podocytes, tubule epithelial cells, and kidney endothelial, stromal, and immune cells. Kidney tubule epithelial cCREs explained 58% of kidney function SNP-heritability and kidney podocyte cCREs explained an additional 6.5% of SNP-heritability. In contrast, little kidney function heritability was explained by kidney endothelial, stromal, or immune cell-specific cCREs. Through functionally informed fine-mapping, we identified putative causal kidney function variants and their corresponding cCREs. Using kidney scATAC-seq data, we created a deep learning model (which we named ChromKid) to predict kidney cell type-specific chromatin accessibility from sequence. ChromKid and allele specific kidney scATAC-seq revealed that many fine-mapped kidney function variants locally change chromatin accessibility in tubule epithelial cells. Enhancer assays confirmed that fine-mapped kidney function variants alter tubule epithelial regulatory element function. To map the genes which these regulatory elements control, we used CRISPR interference (CRISPRi) to target these regulatory elements in tubule epithelial cells and assessed changes in gene expression. CRISPRi of enhancers harboring kidney function variants regulated NDRG1 and RBPMS expression. Thus, inherited differences in tubule epithelial NDRG1 and RBPMS expression may predispose to kidney disease in humans. We conclude that genetic variants affecting tubule epithelial regulatory element function account for most SNP-heritability of human kidney function. This work provides an experimental approach to identify the variants, regulatory elements, and genes involved in polygenic disease.

Epithelial zonation along the mouse and human small intestine defines five discrete metabolic domains.

A key aspect of nutrient absorption is the exquisite division of labour across the length of the small intestine, with individual nutrients taken up at different proximal:distal positions. For millennia, the small intestine was thought to comprise three segments with indefinite borders: the duodenum, jejunum and ileum. By examining the fine-scale longitudinal transcriptional patterns that span the mouse and human small intestine, we instead identified five domains of nutrient absorption that mount distinct responses to dietary changes, and three regional stem cell populations. Molecular domain identity can be detected with machine learning, which provides a systematic method to computationally identify intestinal domains in mice. We generated a predictive model of transcriptional control of domain identity and validated the roles of Ppar-δ and Cdx1 in patterning lipid metabolism-associated genes. These findings represent a foundational framework for the zonation of absorption across the mammalian small intestine.

Epithelial TNF controls cell differentiation and CFTR activity to maintain intestinal mucin homeostasis.

The gastrointestinal tract relies on the production, maturation, and transit of mucin to protect against pathogens and to lubricate the epithelial lining. Although the molecular and cellular mechanisms that regulate mucin production and movement are beginning to be understood, the upstream epithelial signals that contribute to mucin regulation remain unclear. Here, we report that the inflammatory cytokine tumor necrosis factor (TNF), generated by the epithelium, contributes to mucin homeostasis by regulating both cell differentiation and cystic fibrosis transmembrane conductance regulator (CFTR) activity. We used genetic mouse models and noninflamed samples from patients with inflammatory bowel disease (IBD) undergoing anti-TNF therapy to assess the effect of in vivo perturbation of TNF. We found that inhibition of epithelial TNF promotes the differentiation of secretory progenitor cells into mucus-producing goblet cells. Furthermore, TNF treatment and CFTR inhibition in intestinal organoids demonstrated that TNF promotes ion transport and luminal flow via CFTR. The absence of TNF led to slower gut transit times, which we propose results from increased mucus accumulation coupled with decreased luminal fluid pumping. These findings point to a TNF/CFTR signaling axis in the adult intestine and identify epithelial cell-derived TNF as an upstream regulator of mucin homeostasis.

Epithelial zonation along the mouse and human small intestine defines five discrete metabolic domains.

A key aspect of nutrient absorption is the exquisite division of labor across the length of the small intestine, with individual classes of micronutrients taken up at different positions. For millennia, the small intestine was thought to comprise three segments with indefinite borders: the duodenum, jejunum, and ileum. By examining fine-scale longitudinal segmentation of the mouse and human small intestines, we identified transcriptional signatures and upstream regulatory factors that define five domains of nutrient absorption, distinct from the three traditional sections. Spatially restricted expression programs were most prominent in nutrient-absorbing enterocytes but initially arose in intestinal stem cells residing in three regional populations. While a core signature was maintained across mice and humans with different diets and environments, domain properties were influenced by dietary changes. We established the functions of Ppar-ẟ and Cdx1 in patterning lipid metabolism in distal domains and generated a predictive model of additional transcription factors that direct domain identity. Molecular domain identity can be detected with machine learning, representing the first systematic method to computationally identify specific intestinal regions in mice. These findings provide a foundational framework for the identity and control of longitudinal zonation of absorption along the proximal:distal small intestinal axis.

Tissue-specific mitochondrial HIGD1C promotes oxygen sensitivity in carotid body chemoreceptors.

Mammalian carotid body arterial chemoreceptors function as an early warning system for hypoxia, triggering acute life-saving arousal and cardiorespiratory reflexes. To serve this role, carotid body glomus cells are highly sensitive to decreases in oxygen availability. While the mitochondria and plasma membrane signaling proteins have been implicated in oxygen sensing by glomus cells, the mechanism underlying their mitochondrial sensitivity to hypoxia compared to other cells is unknown. Here, we identify HIGD1C, a novel hypoxia-inducible gene domain factor isoform, as an electron transport chain complex IV-interacting protein that is almost exclusively expressed in the carotid body and is therefore not generally necessary for mitochondrial function. Importantly, HIGD1C is required for carotid body oxygen sensing and enhances complex IV sensitivity to hypoxia. Thus, we propose that HIGD1C promotes exquisite oxygen sensing by the carotid body, illustrating how specialized mitochondria can be used as sentinels of metabolic stress to elicit essential adaptive behaviors.

A human mutation in STAT3 promotes type 1 diabetes through a defect in CD8+ T cell tolerance.

Naturally occurring cases of monogenic type 1 diabetes (T1D) help establish direct mechanisms driving this complex autoimmune disease. A recently identified de novo germline gain-of-function (GOF) mutation in the transcriptional regulator STAT3 was found to cause neonatal T1D. We engineered a novel knock-in mouse incorporating this highly diabetogenic human STAT3 mutation (K392R) and found that these mice recapitulated the human autoimmune diabetes phenotype. Paired single-cell TCR and RNA sequencing revealed that STAT3-GOF drives proliferation and clonal expansion of effector CD8+ cells that resist terminal exhaustion. Single-cell ATAC-seq showed that these effector T cells are epigenetically distinct and have differential chromatin architecture induced by STAT3-GOF. Analysis of islet TCR clonotypes revealed a CD8+ cell reacting against known antigen IGRP, and STAT3-GOF in an IGRP-reactive TCR transgenic model demonstrated that STAT3-GOF intrinsic to CD8+ cells is sufficient to accelerate diabetes onset. Altogether, these findings reveal a diabetogenic CD8+ T cell response that is restrained in the presence of normal STAT3 activity and drives diabetes pathogenesis.

Single-cell multiomics defines tolerogenic extrathymic Aire-expressing populations with unique homology to thymic epithelium.

The autoimmune regulator (Aire), a well-defined transcriptional regulator in the thymus, is also found in extrathymic Aire-expressing cells (eTACs) in the secondary lymphoid organs. eTACs are hematopoietic antigen-presenting cells and inducers of immune tolerance, but their precise identity has remained unclear. Here, we use single-cell multiomics, transgenic murine models, and functional approaches to define eTACs at the transcriptional, genomic, and proteomic level. We find that eTACs consist of two similar cell types: CCR7+ Aire-expressing migratory dendritic cells (AmDCs) and an Airehi population coexpressing Aire and retinoic acid receptor­related orphan receptor γt (RORγt) that we term Janus cells (JCs). Both JCs and AmDCs have the highest transcriptional and genomic homology to CCR7+ migratory dendritic cells. eTACs, particularly JCs, have highly accessible chromatin and broad gene expression, including a range of tissue-specific antigens, as well as remarkable homology to medullary thymic epithelium and RANK-dependent Aire expression. Transgenic self-antigen expression by eTACs is sufficient to induce negative selection and prevent autoimmune diabetes. This transcriptional, genomic, and functional symmetry between eTACs (both JCs and AmDCs) and medullary thymic epithelium­the other principal Aire-expressing population and a key regulator of central tolerance­identifies a core program that may influence self-representation and tolerance across the spectrum of immune development.

An Evidence-Based Model for the Successful Treatment of Flank and Lateral Abdominal Wall Hernias.

BACKGROUND: Lateral abdominal wall defects are a significant contributor to patient morbidity and mortality in the United States. Reconstruction involving flank hernias and bulges is relatively scarce in the literature despite its serious consequences. The authors aim to identify an objective approach for the evaluation and successful repair of defects of the lateral abdominal wall. METHODS: A retrospective analysis was carried out on patients presenting for open repair of a lateral wall defect performed by a single surgeon. Over a 5-year period, there were 29 consecutive patients with a mean follow-up period of 21.2 months. Patient demographics including body mass index, number of hernia defects, number of previous repairs/abdominal operations, defect size, operative time, blood loss, and complications (e.g., recurrence/bulge, seroma, hematoma, wound infection, persistent pain, skin breakdown, and fascial dehiscence) were collected. RESULTS: Patients who underwent flank hernia repairs using an inlay/underlay nonbridged technique with the use of acellular dermal matrix had low recurrence and overall complication rates. Only one patient (3.4 percent) had a recurrence at follow-up, and another patient (3.4 percent) had developed a bulge. CONCLUSIONS: The authors' data indicate successful results when their technique is applied. Proper patient selection is essential, along with a thorough understanding of anatomy and techniques for successful reconstruction. The authors recommend using an inlay (preferred) or underlay repair with acellular dermal matrix to reinforce the surrounding musculofascial closure. This technique, in conjunction with the authors' holistic abdominal wall reconstruction protocol, has optimized outcomes and identified a successful multidisciplinary strategy for the reconstruction of lateral wall defects. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.