Charting the cellular biogeography in colitis reveals fibroblast trajectories and coordinated spatial remodeling.

Gut inflammation involves contributions from immune and non-immune cells, whose interactions are shaped by the spatial organization of the healthy gut and its remodeling during inflammation. The crosstalk between fibroblasts and immune cells is an important axis in this process, but our understanding has been challenged by incomplete cell-type definition and biogeography. To address this challenge, we used multiplexed error-robust fluorescence in situ hybridization (MERFISH) to profile the expression of 940 genes in 1.35 million cells imaged across the onset and recovery from a mouse colitis model. We identified diverse cell populations, charted their spatial organization, and revealed their polarization or recruitment in inflammation. We found a staged progression of inflammation-associated tissue neighborhoods defined, in part, by multiple inflammation-associated fibroblasts, with unique expression profiles, spatial localization, cell-cell interactions, and healthy fibroblast origins. Similar signatures in ulcerative colitis suggest conserved human processes. Broadly, we provide a framework for understanding inflammation-induced remodeling in the gut and other tissues.

Advances in Neuro-Oncological Imaging: An Update on Diagnostic Approach to Brain Tumors.

This study delineates the pivotal role of imaging within the field of neurology, emphasizing its significance in the diagnosis, prognostication, and evaluation of treatment responses for central nervous system (CNS) tumors. A comprehensive understanding of both the capabilities and limitations inherent in emerging imaging technologies is imperative for delivering a heightened level of personalized care to individuals with neuro-oncological conditions. Ongoing research in neuro-oncological imaging endeavors to rectify some limitations of radiological modalities, aiming to augment accuracy and efficacy in the management of brain tumors. This review is dedicated to the comparison and critical examination of the latest advancements in diverse imaging modalities employed in neuro-oncology. The objective is to investigate their respective impacts on diagnosis, cancer staging, prognosis, and post-treatment monitoring. By providing a comprehensive analysis of these modalities, this review aims to contribute to the collective knowledge in the field, fostering an informed approach to neuro-oncological care. In conclusion, the outlook for neuro-oncological imaging appears promising, and sustained exploration in this domain is anticipated to yield further breakthroughs, ultimately enhancing outcomes for individuals grappling with CNS tumors.

Charting the cellular biogeography in colitis reveals fibroblast trajectories and coordinated spatial remodeling.

Gut inflammation involves contributions from immune and non-immune cells, whose interactions are shaped by the spatial organization of the healthy gut and its remodeling during inflammation. The crosstalk between fibroblasts and immune cells is an important axis in this process, but our understanding has been challenged by incomplete cell-type definition and biogeography. To address this challenge, we used MERFISH to profile the expression of 940 genes in 1.35 million cells imaged across the onset and recovery from a mouse colitis model. We identified diverse cell populations; charted their spatial organization; and revealed their polarization or recruitment in inflammation. We found a staged progression of inflammation-associated tissue neighborhoods defined, in part, by multiple inflammation-associated fibroblasts, with unique expression profiles, spatial localization, cell-cell interactions, and healthy fibroblast origins. Similar signatures in ulcerative colitis suggest conserved human processes. Broadly, we provide a framework for understanding inflammation-induced remodeling in the gut and other tissues.

The Impact of Hospitalist Switch Day on Length of Stay.

BACKGROUND AND OBJECTIVE: Hospitalist practices around the country switch service on different days of the week. It is unclear whether switching clinical service later in the week is associated with an increase in length of stay (LOS). The aim of this study was to examine the association between service switch day for hospitalists at an academic medical center and LOS. METHODS: A single-center, cross-sectional study examined 4284 discharges from hospitalist staffed general internal medicine ward teams over a 1-year period between July 2018 and June 2019. Hospitalist service switch day changed from Tuesday to Thursday on January 1, 2019. The period between July 1, 2018, and December 31, 2018, was defined as the pre-switch time, while January 1, 2019, to June 30, 2019, was defined as the post-switch period. We calculated the LOS in days for patients discharged from hospitalist general internal medicine teams in the 2 periods. Generalized linear models were used to examine the association between attending switch day and LOS while adjusting for demographic factors, payer status, markers of severity of illness, and hospital or discharge-level confounders. RESULTS: There was no difference in mean LOS for patients discharged in the pre-switch time (6 days) period versus patients discharged in the post-switch time (6.03 days) (difference of means 0.03 days, 95% confidence interval -0.04 to 0.09, P value .37). CONCLUSIONS: Change in attending switch day from earlier in the week to later in the week is not associated with an increase in LOS. Other factors such as group preference and institutional needs should drive service switch day selection for hospitalist groups.

The deubiquitinase USP44 promotes Treg function during inflammation by preventing FOXP3 degradation.

The transcription factor forkhead box P3 (FOXP3) is essential for the development of regulatory T cells (Tregs) and their function in immune homeostasis. Previous studies have shown that in natural Tregs (nTregs), FOXP3 can be regulated by polyubiquitination and deubiquitination. However, the molecular players active in this pathway, especially those modulating FOXP3 by deubiquitination in the distinct induced Treg (iTreg) lineage, remain unclear. Here, we identify the ubiquitin-specific peptidase 44 (USP44) as a novel deubiquitinase for FOXP3. USP44 interacts with and stabilizes FOXP3 by removing K48-linked ubiquitin modifications. Notably, TGF-ß induces USP44 expression during iTreg differentiation. USP44 co-operates with USP7 to stabilize and deubiquitinate FOXP3. Tregs genetically lacking USP44 are less effective than their wild-type counterparts, both in vitro and in multiple in vivo models of inflammatory disease and cancer. These findings suggest that USP44 plays an important role in the post-translational regulation of Treg function and is thus a potential therapeutic target for tolerance-breaking anti-cancer immunotherapy.

Human and mouse activin genes: Divergent expression of activin A protein variants and identification of a novel heparan sulfate-binding domain in activin B.

Activins are members of the transforming growth factor-ß (TGF-ß) superfamily of signaling proteins and were originally identified as components of follicular fluid. The proteins are now known to play critical roles in numerous normal and pathological processes and conditions, but less is clear about the relationships between their gene organization and protein variant expression and structure. The four human and mouse activin (Act) genes, termed INHßA, INHßB, INHßC and INHßE, differ in exon numbers. Human INHßA is the most complex with 7 exons and elicits production of three Act A variants (Act A X1, X2 and X3) differing in their pro-region, as we showed previously. Here we further analyzed the mouse INHßA gene and found that its 4 exons encode for a single open reading frame (mouse Act A), corresponding to the shortest human Act A X3 variant. Activins are synthesized and secreted as large complexes made of a long pro-region and a short mature C- terminal ligand and are known to interact with the heparan sulfate (HS) chains of cell surface and matrix proteoglycans. Human Act A X1 and X2 variants do have a HS-binding domain (HBD) with Cardin/Weintraub traits in their pro-region, while the X3 variant does not as shown previously. We found that the mouse Act A lacks a HBD as well. However, we identified a typical HBD in the pro-region of both mouse and human Act B, and synthetic peptides containing that domain interacted with immobilized HS and cell surface with nanomolar affinity. In sum, human and mouse Act A genes elicit expression of different variant sets, while there is concordance in Act B protein expression, reflecting possible evolutionary diversity in function of, and responses to, these signaling proteins in the two species.

Identification and characterization of a novel heparan sulfate-binding domain in Activin A longest variants and implications for function.

Activins regulate numerous processes including inflammation and are synthesized as precursors consisting of a long N-terminal pro-region and a mature protein. Genomic human databases currently list three activin A (Act A) variants termed X1, X2 and X3. The X3 variant is the shortest, lacks N-terminal segments present in X1 and X2, and has been the focus of most past literature. Here, we asked whether these variants are expressed by human cells and tissues and what structural features are contained within their pro-regions. Human monocytic-like cells THP1 and U937 expressed X1 and X2 variants after exposure to phorbol ester or granulocyte-macrophage colony-stimulating factor, while X2 transcripts were present in placenta. Expression vectors encoding full length X2 or X3 variants resulted in production and secretion of biologically active Act A from cultured cells. Previous studies reported a putative HS-binding domain (HBD) in the X3 pro-region. Here, we identified a novel HBD with consensus HS-binding motifs near the N-terminal end of X1 and X2 pro-regions. Peptides encompassing this new domain interacted with substrate-bound HS with nanomolar affinity, while peptides from putative X3 HBD did not. In good agreement, full length X2 pro-region interacted with heparin-agarose, while the X3 pro-region did not. In sum, our study reveals that Act A variants are expressed by inflammatory cells and placenta and yield biological activity. The high affinity HBD in X1 and X2 pro-region and its absence in X3 could greatly influence overall Act A distribution, availability and activity in physiological and pathological circumstances.

Domains with highest heparan sulfate-binding affinity reside at opposite ends in BMP2/4 versus BMP5/6/7: Implications for function.

Signaling proteins, including bone morphogenetic proteins (BMPs), specifically interact with heparan sulfate (HS). These interactions regulate protein distribution and function and are largely mediated by domains rich in basic amino acids. The N-terminal region of BMP2 and BMP4 contains one such domain with a typical Cardin-Weintraub (CW) motif, but it is unclear whether the same occurs in BMP5, BMP6, and BMP7 that constitute a separate evolutionary subgroup. Peptides spanning the N-terminal domain of BMP2/4 interacted with substrate-bound HS with nanomolar affinity, but peptides spanning BMP5/6/7 N-terminal domain did not. We re-examined the entire BMP5/6/7 sequences and identified a novel CW-like motif at their C terminus. Peptides spanning this domain displayed high-affinity HS binding, but corresponding BMP2/4 C-terminal peptides did not, likely because of acidic or noncharged residue substitutions. Peptides pre-assembled into NeutrAvidin tetramers displayed the same exact binding selectivity of respective monomers but bound HS with greater affinity. Tests of possible peptide biological activities showed that the HS-binding N-terminal BMP2/4 and C-terminal BMP5/6/7 peptides stimulated chondrogenesis in vitro, potentially by freeing endogenous BMPs. Thus, HS interactions appear largely ascribable to domains at opposite ends of BMP2/4 versus BMP5/6/7, reiterating the evolutionary distance of these BMP subgroups and possible functional diversification.

Heparan sulfate antagonism alters bone morphogenetic protein signaling and receptor dynamics, suggesting a mechanism in hereditary multiple exostoses.

Hereditary multiple exostoses (HME) is a pediatric disorder caused by heparan sulfate (HS) deficiency and is characterized by growth plate-associated osteochondromas. Previously, we found that osteochondroma formation in mouse models is preceded by ectopic bone morphogenetic protein (BMP) signaling in the perichondrium, but the mechanistic relationships between BMP signaling and HS deficiency remain unclear. Therefore, we used an HS antagonist (surfen) to investigate the effects of this HS interference on BMP signaling, ligand availability, cell-surface BMP receptor (BMPR) dynamics, and BMPR interactions in Ad-293 and C3H/10T1/2 cells. As observed previously, the HS interference rapidly increased phosphorylated SMAD family member 1/5/8 levels. FACS analysis and immunoblots revealed that the cells possessed appreciable levels of endogenous cell-surface BMP2/4 that were unaffected by the HS antagonist, suggesting that BMP2/4 proteins remained surface-bound but became engaged in BMPR interactions and SMAD signaling. Indeed, surface mobility of SNAP-tagged BMPRII, measured by fluorescence recovery after photobleaching (FRAP), was modulated during the drug treatment. This suggested that the receptors had transitioned to lipid rafts acting as signaling centers, confirmed for BMPRII via ultracentrifugation to separate membrane subdomains. In situ proximity ligation assays disclosed that the HS interference rapidly stimulates BMPRI-BMPRII interactions, measured by oligonucleotide-driven amplification signals. Our in vitro studies reveal that cell-associated HS controls BMP ligand availability and BMPR dynamics, interactions, and signaling, and largely restrains these processes. We propose that HS deficiency in HME may lead to extensive local BMP signaling and altered BMPR dynamics, triggering excessive cellular responses and osteochondroma formation.

Screening of a Haloferax volcanii Transposon Library Reveals Novel Motility and Adhesion Mutants.

Archaea, like bacteria, use type IV pili to facilitate surface adhesion. Moreover, archaeal flagella-structures required for motility-share a common ancestry with type IV pili. While the characterization of archaeal homologs of bacterial type IV pilus biosynthesis components has revealed important aspects of flagellum and pilus biosynthesis and the mechanisms regulating motility and adhesion in archaea, many questions remain. Therefore, we screened a Haloferax volcanii transposon insertion library for motility mutants using motility plates and adhesion mutants, using an adapted air-liquid interface assay. Here, we identify 20 genes, previously unknown to affect motility or adhesion. These genes include potential novel regulatory genes that will help to unravel the mechanisms underpinning these processes. Both screens also identified distinct insertions within the genomic region lying between two chemotaxis genes, suggesting that chemotaxis not only plays a role in archaeal motility, but also in adhesion. Studying these genes, as well as hypothetical genes hvo_2512 and hvo_2876-also critical for both motility and adhesion-will likely elucidate how these two systems interact. Furthermore, this study underscores the usefulness of the transposon library to screen other archaeal cellular processes for specific phenotypic defects.