Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem-cell self-renewal

The canonical Wnt/β-catenin signaling pathway governs diverse developmental, homeostatic and pathologic processes. Palmitoylated Wnt ligands engage cell surface Frizzled (Fzd) receptors and Lrp5/6 co-receptors enabling β-catenin nuclear translocation and Tcf/Lef-dependent gene transactivation1–3. Mutations in Wnt downstream signaling components have revealed diverse functions presumptively attributed to Wnt ligands themselves, although direct attribution remains elusive, as complicated by redundancy between 19 mammalian Wnts and 10 Fzds1 and Wnt hydrophobicity2,3. For example, individual Wnt ligand mutations have not revealed homeostatic phenotypes in the intestinal epithelium4, an archetypal canonical Wnt pathway-dependent rapidly self-renewing tissue whose regeneration is fueled by proliferative crypt Lgr5+ intestinal stem cells (ISCs)5–9. R-spondin ligands (Rspo1–4) engage distinct Lgr4-6 and Rnf43/Znrf3 receptor classes10–13, markedly potentiate canonical Wnt/β-catenin signaling and induce intestinal organoid growth in vitro and Lgr5+ ISCs in vivo8,14–17. However, the interchangeability, functional cooperation and relative contributions of Wnt versus Rspo ligands to in vivo canonical Wnt signaling and ISC biology remain unknown. Here, we deconstructed functional roles of Wnt versus Rspo ligands in the intestinal crypt stem cell niche. We demonstrate that the default fate of Lgr5+ ISCs is lineage commitment, escape from which requires both Rspo and Wnt ligands. However, gain-of-function studies using Rspo versus a novel non-lipidated Wnt analog reveal qualitatively distinct, non-interchangeable roles for these ligands in ISCs. Wnts are insufficient to induce Lgr5+ ISC self-renewal, but rather confer a basal competency by maintaining Rspo receptor expression that enables Rspo to actively drive and specify the extent of stem cell expansion. This functionally non-equivalent yet cooperative interplay between Wnt and Rspo ligands establishes a molecular precedent for regulation of mammalian stem cells by distinct priming and self-renewal factors, with broad implications for precision control of tissue regeneration

Cartilage Endplate Thickness Variation Measured by Ultrashort Echo-Time MRI Is Associated With Adjacent Disc Degeneration.

Study designA magnetic resonance imaging study of human cadaver spines.ObjectiveTo investigate associations between cartilage endplate (CEP) thickness and disc degeneration.Summary of background dataDamage to the CEP is associated with spinal injury and back pain. However, CEP morphology and its association with disc degeneration have not been well characterized.MethodsTen lumbar motion segments with varying degrees of disc degeneration were harvested from six cadaveric spines and scanned with magnetic resonance imaging in the sagittal plane using a T2-weighted two-dimensional (2D) sequence, a three-dimensional (3D) ultrashort echo-time (UTE) imaging sequence, and a 3D T1ρ mapping sequence. CEP thicknesses were calculated from 3D UTE image data using a custom, automated algorithm, and these values were validated against histology measurements. Pfirrmann grades and T1ρ values in the disc were assessed and correlated with CEP thickness.ResultsThe mean CEP thickness calculated from UTE images was 0.74 ± 0.04 mm. Statistical comparisons between histology and UTE-derived measurements of CEP thickness showed significant agreement, with the mean difference not significantly different from zero (P = 0.32). Within-disc variation of T1ρ (standard deviation) was significantly lower for Pfirrmann grade 4 than Pfirrmann grade 3 (P < 0.05). Within-disc variation of T1ρ and adjacent CEP thickness heterogeneity (coefficient of variation) had a significant negative correlation (r = -0.65, P = 0.04). The standard deviation of T1ρand the mean CEP thickness showed a moderate positive correlation (r = 0.40, P = 0.26).ConclusionThis study demonstrates that quantitative measurements of CEP thickness measured from UTE magnetic resonance imaging are associated with disc degeneration. Our results suggest that variability in CEP thickness and T1ρ, rather than their mean values, may serve as valuable diagnostic markers for disc degeneration.Level of evidenceN/A

Hyperpolarized [1-13C] Glutamate: A Metabolic Imaging Biomarker of IDH1 Mutational Status in Glioma

Mutations of the isocitrate dehydrogenase 1 (IDH1) gene are among the most prevalent in low-grade glioma and secondary glioblastoma, represent an early pathogenic event, and are associated with epigenetically driven modulations of metabolism. Of particular interest is the recently uncovered relationship between the IDH1 mutation and decreased activity of the branched-chain amino acid transaminase 1 (BCAT1) enzyme. Noninvasive imaging methods that can assess BCAT1 activity could therefore improve detection of mutant IDH1 tumors and aid in developing and monitoring new targeted therapies. BCAT1 catalyzes the transamination of branched-chain amino acids while converting α-ketoglutarate (α-KG) to glutamate. Our goal was to use (13)C magnetic resonance spectroscopy to probe the conversion of hyperpolarized [1-(13)C] α-KG to hyperpolarized [1-(13)C] glutamate as a readout of BCAT1 activity. We investigated two isogenic glioblastoma lines that differed only in their IDH1 status and performed experiments in live cells and in vivo in rat orthotopic tumors. Following injection of hyperpolarized [1-(13)C] α-KG, hyperpolarized [1-(13)C] glutamate production was detected both in cells and in vivo, and the level of hyperpolarized [1-(13)C] glutamate was significantly lower in mutant IDH1 cells and tumors compared with their IDH1-wild-type counterparts. Importantly however, in our cells the observed drop in hyperpolarized [1-(13)C] glutamate was likely mediated not only by a drop in BCAT1 activity, but also by reductions in aspartate transaminase and glutamate dehydrogenase activities, suggesting additional metabolic reprogramming at least in our model. Hyperpolarized [1-(13)C] glutamate could thus inform on multiple mutant IDH1-associated metabolic events that mediate reduced glutamate production

Do HIV-Infected Women Want to Discuss Reproductive Plans with Providers, and Are Those Conversations Occurring?

The purpose of the study is to assess frequency and determinants of discussions between HIV-infected women and their HIV providers about childbearing plans, and to identify unmet need for reproductive counseling. We conducted a cross-sectional, audio computer-assisted self-interview (ACASI) among 181 predominately African American HIV-infected women of reproductive age receiving HIV clinical care in two urban health clinics. We used descriptive statistics to identify unmet need for reproductive counseling by determining the proportion of women who want to, but have not, discussed future reproductive plans with their primary HIV care provider. Multivariate analysis determined which factors were associated with general and personalized discussions about pregnancy. Of the 181 women interviewed, 67% reported a general discussion about pregnancy and HIV while 31% reported a personalized discussion about future childbearing plans with their provider. Of the personalized discussions, 64% were patient initiated. Unmet reproductive counseling needs were higher for personalized discussions about future pregnancies (56%) than general discussions about HIV and pregnancy (23%). Younger age was the most powerful determinant of provider communication about pregnancy. A significant proportion of HIV-infected women want to talk about reproductive plans with their HIV provider; however, many have not. HIV care providers and gynecologists can address this unmet communication need by discussing reproductive plans with all women of childbearing age so that preconception counseling can be provided when appropriate. Providers will miss opportunities to help women safely plan pregnancy if they only discuss reproductive plans with younger patients

Batch effects in single-cell RNA-sequencing data are corrected by matching mutual nearest neighbors.

Large-scale single-cell RNA sequencing (scRNA-seq) data sets that are produced in different laboratories and at different times contain batch effects that may compromise the integration and interpretation of the data. Existing scRNA-seq analysis methods incorrectly assume that the composition of cell populations is either known or identical across batches. We present a strategy for batch correction based on the detection of mutual nearest neighbors (MNNs) in the high-dimensional expression space. Our approach does not rely on predefined or equal population compositions across batches; instead, it requires only that a subset of the population be shared between batches. We demonstrate the superiority of our approach compared with existing methods by using both simulated and real scRNA-seq data sets. Using multiple droplet-based scRNA-seq data sets, we demonstrate that our MNN batch-effect-correction method can be scaled to large numbers of cells