The rockefeller university

Background: One-third of people living with dementia (PLWD) have highly fragmented care (i.e., care spread across many ambulatory providers without a dominant provider). It is unclear whether PLWD with fragmented care and their caregivers perceive gaps in communication among the providers involved and whether any such gaps are perceived as benign inconveniences or as clinically meaningful, leading to adverse events. We sought to determine the frequency of perceived gaps in communication (coordination) among providers and the frequency of self-reported adverse events attributed to poor coordination. Methods: We conducted a cross-sectional study in the context of a Medicare accountable care organization (ACO) in New York in 2022-2023. We included PLWD who were attributed to the ACO, had fragmented care in the past year by claims (reversed Bice-Boxerman Index >= 0.86), and were in a pragmatic clinical trial on care management. We used an existing survey instrument to determine perceptions of care coordination and perceptions of four adverse events (repeat tests, drug-drug interactions, emergency department visits, and hospital admissions). ACO care managers collected data by telephone, using clinical judgment to determine whether each survey respondent was the patient or a caregiver. We used descriptive statistics to summarize results. Results: Of 167 eligible PLWD, surveys were completed for 97 (58.1%). Of those, 88 (90.7%) reported having >1 ambulatory visit and >1 ambulatory provider and were thus at risk for gaps in care coordination and included in the analysis. Of those, 23 respondents were patients (26.1%) and 64 were caregivers (72.7%), with one respondent's role missing. Overall, 57% of respondents reported a problem (or "gap") in the coordination of care and, separately, 18% reported an adverse event that they attributed to poor care coordination. Conclusion: Gaps in coordination of care for PLWD are reported to be very common and often perceived as hazardous.

The complex carbohydrate structures decorating human proteins and lipids, also called glycans, are abundantly present at cell surfaces and in the secretome. Glycosylation is vital for biological processes including cell-cell recognition, immune responses, and signaling pathways. Therefore, the structural and functional characterization of the human glycome is gaining more and more interest in basic biochemistry research and in the context of developing new therapies, diagnostic tools, and biotechnology applications. For glycomics to reach its full potential in these fields, it is critical to appreciate the specific factors defining the function of the human glycome. Here, we review the glycosyltransferases (the writers) that form the glycome and the glycan-binding proteins (the readers) with an essential role in decoding glycan functions. While abundantly present throughout different cells and tissues, the function of specific glycosylation features is highly dependent on their context. In this review, we highlight the relevance of studying the glycome in the context of specific carrier proteins, cell types, and subcellular locations. With this, we hope to contribute to a richer understanding of the glycome and a more systematic approach to identifying the roles of glycosylation in human physiology.

Most cases of herpes simplex virus 1 (HSV-1) encephalitis (HSE) remain unexplained1,2. Here, we report on two unrelated people who had HSE as children and are homozygous for rare deleterious variants of TMEFF1, which encodes a cell membrane protein that is preferentially expressed by brain cortical neurons. TMEFF1 interacts with the cell-surface HSV-1 receptor NECTIN-1, impairing HSV-1 glycoprotein D- and NECTIN-1-mediated fusion of the virus and the cell membrane, blocking viral entry. Genetic TMEFF1 deficiency allows HSV-1 to rapidly enter cortical neurons that are either patient specific or derived from CRISPR-Cas9-engineered human pluripotent stem cells, thereby enhancing HSV-1 translocation to the nucleus and subsequent replication. This cellular phenotype can be rescued by pretreatment with type I interferon (IFN) or the expression of exogenous wild-type TMEFF1. Moreover, ectopic expression of full-length TMEFF1 or its amino-terminal extracellular domain, but not its carboxy-terminal intracellular domain, impairs HSV-1 entry into NECTIN-1-expressing cells other than neurons, increasing their resistance to HSV-1 infection. Human TMEFF1 is therefore a host restriction factor for HSV-1 entry into cortical neurons. Its constitutively high abundance in cortical neurons protects these cells from HSV-1 infection, whereas inherited TMEFF1 deficiency renders them susceptible to this virus and can therefore underlie HSE. A study of two childhood cases of herpes simplex encephalitis shows that TMEFF1 interacts with the HSV-1 cell-surface receptor NECTIN-1, preventing HSV-1 from fusing with the cell membrane and entering cortical neurons.

Severe defects in human IFN gamma immunity predispose individuals to both Bacillus Calmette-Gu & eacute;rin disease and tuberculosis, whereas milder defects predispose only to tuberculosis1. Here we report two adults with recurrent pulmonary tuberculosis who are homozygous for a private loss-of-function TNF variant. Neither has any other clinical phenotype and both mount normal clinical and biological inflammatory responses. Their leukocytes, including monocytes and monocyte-derived macrophages (MDMs) do not produce TNF, even after stimulation with IFN gamma. Blood leukocyte subset development is normal in these patients. However, an impairment in the respiratory burst was observed in granulocyte-macrophage colony-stimulating factor (GM-CSF)-matured MDMs and alveolar macrophage-like (AML) cells2 from both patients with TNF deficiency, TNF- or TNFR1-deficient induced pluripotent stem (iPS)-cell-derived GM-CSF-matured macrophages, and healthy control MDMs and AML cells differentiated with TNF blockers in vitro, and in lung macrophages treated with TNF blockers ex vivo. The stimulation of TNF-deficient iPS-cell-derived macrophages with TNF rescued the respiratory burst. These findings contrast with those for patients with inherited complete deficiency of the respiratory burst across all phagocytes, who are prone to multiple infections, including both Bacillus Calmette-Gu & eacute;rin disease and tuberculosis3. Human TNF is required for respiratory-burst-dependent immunity to Mycobacterium tuberculosis in macrophages but is surprisingly redundant otherwise, including for inflammation and immunity to weakly virulent mycobacteria and many other infectious agents. Human TNF is required for respiratory-burst-dependent immunity to Mycobacterium tuberculosis in macrophages but seems to be largely redundant physiologically.

Background. Development of a screening assay for the clinical use of broadly neutralizing antibodies (bnAbs) is a priority for HIV therapy and cure initiatives. Methods. We assessed the PhenoSense Monoclonal Antibody Assay (Labcorp-Monogram Biosciences), which is Clinical Laboratory Improvement Amendments (CLIA) validated and has been used prospectively and retrospectively in multiple recent bnAb clinical trials. Results. When performed on plasma and longitudinal peripheral blood mononuclear cell samples (before and during antiretroviral therapy, respectively), as sourced from a recent clinical trial, the PhenoSense assay produced robust reproducibility, concordance across sample types, and expected ranges in the susceptibility measures of bnAbs in clinical development. When applied retrospectively to baseline samples from 3 recent studies, the PhenoSense assay correlated with published laboratory-based study evaluations, but baseline bnAb susceptibility was not consistently predictive of durable virus suppression. Assessment of assay feasibility in 4 recent clinical studies provides estimates of assay success rate and processing time. Conclusions. The PhenoSense Monoclonal Antibody Assay provides reproducible bnAb susceptibility measurements across relevant sample types yet is not consistently predictive of virus suppression. Logistical and operational assay requirements can affect timely clinical trial conduct. These results inform bnAb studies in development.

Patients with severe West Nile virus and SARS-CoV-2 infections deserve accurate diagnosis of underlying diseases, determining possible anti-interferon autoantibody production, since they must receive antiviral and immunological therapies to enhance antiviral response.The current study aimed to investigate determinants of severity in a previously healthy patient who experienced 2 life-threatening infections, from West Nile Virus (WNV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV2). During coronavirus disease 2019 (COVID-19) hospitalization he was diagnosed with a thymoma, retrospectively identified as already present at the time of WNV infection. Heterozygosity for p.Pro554Ser in the TLR3 gene, which increases susceptibility to severe COVID-19, and homozygosity for CCR5 c.554_585del, associated with severe WNV infection, were found. Neutralizing anti-interferon (IFN)-alpha and anti-IFN-omega autoantibodies were detected, likely induced by the underlying thymoma and increasing susceptibility to both severe COVID-19 pneumonia and West Nile encephalitis.

Immune responses need to be regulated to prevent autoimmunity. CRISPR-Cas systems provide adaptive immunity in prokaryotes through the acquisition of short DNA sequences from invading viruses (bacteriophages), known as spacers. Spacers are inserted into the CRISPR locus and serve as templates for the transcription of guides used by RNA-guided nucleases to recognize complementary nucleic acids of the invaders and start the CRISPR immune response. In type II-A CRISPR systems, Cas9 uses the guide RNA to cleave target DNA sequences in the genome of infecting phages, and the tracrRNA to bind the promoter of cas genes and repress their transcription. We previously isolated a Cas9 mutant carrying the I473F substitution that increased the frequency of spacer acquisition by 2-3 orders of magnitude, leading to a fitness cost due to higher levels of autoimmunity. Here, we investigated the molecular basis underlying these findings. We found that the I473F mutation decreases the association of Cas9 to tracrRNA, limiting its repressor function, leading to high levels of expression of cas genes, which in turn increase the strength of the type II-A CRISPR-Cas immune response. We obtained similar results for a related type II-A system, and therefore our findings highlight the importance of the interaction between Cas9 and its tracrRNA cofactor in tuning the immune response to balanced levels that enable phage defense but avoid autoimmunity. Graphical Abstract

During formation of the transcription-competent open complex (RPo) by bacterial RNA polymerases (RNAPs), transient intermediates pile up before overcoming a rate-limiting step. Structural descriptions of these interconversions in real time are unavailable. To address this gap, here we use time-resolved cryogenic electron microscopy (cryo-EM) to capture four intermediates populated 120 ms or 500 ms after mixing Escherichia coli sigma 70-RNAP and the lambda PR promoter. Cryo-EM snapshots revealed that the upstream edge of the transcription bubble unpairs rapidly, followed by stepwise insertion of two conserved nontemplate strand (nt-strand) bases into RNAP pockets. As the nt-strand 'read-out' extends, the RNAP clamp closes, expelling an inhibitory sigma 70 domain from the active-site cleft. The template strand is fully unpaired by 120 ms but remains dynamic, indicating that yet unknown conformational changes complete RPo formation in subsequent steps. Given that these events likely describe DNA opening at many bacterial promoters, this study provides insights into how DNA sequence regulates steps of RPo formation. Time-resolved cryo-EM captured transient intermediates during E. coli RNAP promoter melting, revealing conformational changes affecting stepwise transcription bubble opening. Results inform how DNA sequence controls bacterial transcription initiation.

Conventional optical microscopy imaging of obligate intracellular bacteria is hampered by the small size of bacterial cells, tight clustering exhibited by some bacterial species and challenges relating to labelling such as background from host cells, a lack of validated reagents, and a lack of tools for genetic manipulation. In this study, we imaged intracellular bacteria from the species Orientia tsutsugamushi (Ot) using five different fluorescence microscopy techniques: standard confocal, Airyscan confocal, instant Structured Illumination Microscopy (iSIM), three-dimensional Structured Illumination Microscopy (3D-SIM) and Stimulated Emission Depletion Microscopy (STED). We compared the ability of each to resolve bacterial cells in intracellular clumps in the lateral (xy) axis, using full width half-maximum (FWHM) measurements of a labelled outer membrane protein (ScaA) and the ability to detect small, outer membrane vesicles external to the cells. Comparing the techniques readily available to us (above), 3D-SIM microscopy, in combination with the shortest-wavelength dyes, was found overall to give the best lateral resolution. We next compared the ability of each technique to sufficiently resolve bacteria in the axial (z) direction and found 3D-STED to be the most successful method for this. We then combined this 3D-STED approach with a custom 3D cell segmentation and analysis pipeline using the open-source, deep learning software, Cellpose to segment the cells and subsequently the commercial software Imaris to analyse their 3D shape and size. Using this combination, we demonstrated differences in bacterial shape, but not their size, when grown in different mammalian cell lines. Overall, we compare the advantages and disadvantages of different super-resolution microscopy techniques for imaging this cytoplasmic obligate intracellular bacterium based on the specific research question being addressed.

Background Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease with a considerable disease burden. Existing treatment options are limited and often suboptimal; a high unmet need exists for effective targeted therapies.Objectives To explore the effects of spesolimab treatment in patients with HS.Methods This randomized double-blind placebo-controlled proof-of-clinical-concept (PoCC) study was conducted at 25 centres across 12 countries from 3 May 2021 to 21 April 2022. Patients had moderate-to-severe HS for >= 1 year before enrolment. Patients were randomized (2 : 1) to receive a loading dose of 3600-mg intravenous spesolimab (1200 mg at weeks 0, 1 and 2) or matching placebo, followed by maintenance with either 1200-mg subcutaneous spesolimab every 2 weeks from weeks 4 to 10 or matching placebo. The primary endpoint was the percentage change from baseline in total abscess and inflammatory nodule (AN) count at week 12. Secondary endpoints were the absolute change from baseline in the International Hidradenitis Suppurativa Severity Score System (IHS4), percentage change from baseline in draining tunnel (dT) count, the proportion of patients achieving a dT count of 0, absolute change from baseline in the revised Hidradenitis Suppurativa Area and Severity Index (HASI-R), the proportion of patients achieving Hidradenitis Suppurativa Clinical Response (HiSCR50), the proportion of patients with >= 1 flare (all at week 12) and patient-reported outcomes.Results In this completed trial, randomized patients (n = 52) received spesolimab (n = 35) or placebo (n = 17). The difference vs. placebo in least squares mean is reported. At week 12, the percentage change in total AN count was similar between treatment arms: -4.1% [95% confidence interval (CI) -31.7 to 23.4]. There was greater numerical improvement in the spesolimab arm, as measured by IHS4 (13.9, 95% CI -25.6 to -2.3); percentage change from baseline in dT count (-96.6%, 95% CI -154.5 to -38.8); and the proportion of patients achieving a dT count of 0 (18.3%, 95% CI -7.9 to 37.5). Spesolimab treatment also improved HASI-R and HiSCR50 vs. placebo. Spesolimab demonstrated a favourable safety profile, similar to that observed in trials in other diseases.Conclusions This exploratory PoCC study supports the development of spesolimab as a new therapeutic option in HS. Graphical Abstract Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease that affects approximately 0.4% to 1% of people worldwide. HS mainly affects areas where skin touches skin and can result in painful lumps and abscesses. Tunnel-shaped structures often form below the skin and discharge pus and can greatly affect a person's quality of life.In this study, we tested a drug called 'spesolimab' as a treatment for people with moderate-to-severe HS. Spesolimab is a medicine in development that affects the immune system. This 12-week study included 52 adults who had moderate-to-severe HS for at least 1 year, from North America, Europe and Australia. People who took part were selected at random to receive either spesolimab or placebo. Thirty-five people received spesolimab and 17 received placebo into a vein once a week for 3 weeks, starting at week 0. They then received four injections of spesolimab or placebo under the skin once every 2 weeks until week 10. The number of lumps and abscesses, tunnels and a score based on their combination, called the International Hidradenitis Suppurativa Severity Score System (IHS4), were evaluated before spesolimab or placebo were given, and at week 12.We found that spesolimab and the placebo had a similar effect on the number of lumps and abscesses. However, more people treated with spesolimab showed improvements in tunnels and IHS4 score than those who received the placebo. The safety of spesolimab was favourable, similar to when spesolimab has been used in studies of other diseases. Our findings support further research into the use of spesolimab as a medicine for HS. This 12-week study explored the effects of spesolimab, which inhibits interleukin (IL)-36 signalling, in patients with moderate-to-severe hidradenitis suppurativa for at least 1 year. Patients received spesolimab (n = 35) or placebo (n = 17) intravenously once weekly for 3 weeks starting at week 0. Patients then received four subcutaneous injections of spesolimab or placebo once every 2 weeks until week 10. At week 12, the percentage change from baseline in total abscess and inflammatory nodule count was similar between the treatment arms. However, spesolimab did improve IHS4, percentage change from baseline in draining tunnel count, HASI-R and HiSCR50 vs. placebo. Furthermore, spesolimab demonstrated a favourable safety profile, similar to that observed in trials in other diseases.