The rockefeller university

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.

T follicular helper (Tfh) cells specialize in helping B cells and are therefore critical contributors to the generation of humoral immunity. Tfh cells aid immunoglobulin class-switch recombination and support the germinal center response, thereby promoting immunoglobulin affinity maturation and the generation of humoral immune memory. Although their primary function is to promote B cell responses, Tfh cells also display phenotypic and functional diversity determined by the immunological and spatial contexts from which they emerge. We review recent advances in understanding the heterogeneity within Tfh cell subsets along with their differentiation and migratory trajectory, the phenotypes they adopt, their ontological relationships with one another, and their function in their respective environments.

Although the majority of annotated new genes in a given genome appear to have arisen from duplication-related mechanisms, recent studies have shown that genes can also originate de novo from ancestrally nongenic sequences. Investigating de novo-originated genes offers rich opportunities to understand the origin and functions of new genes, their regulatory mechanisms, and the associated evolutionary processes. Such studies have uncovered unexpected and intriguing facets of gene origination, offering novel perspectives on the complexity of the genome and gene evolution. In this review, we provide an overview of the research progress in this field, highlight recent advancements, identify key technical and conceptual challenges, and underscore critical questions that remain to be addressed.

Methyl-CpG-binding protein 2 (MeCP2) is an essential chromatin-binding protein whose mutations cause Rett syndrome (RTT), a severe neurological disorder that primarily affects young females. The canonical view of MeCP2 as a DNA methylation-dependent transcriptional repressor has proven insufficient to describe its dynamic interaction with chromatin and multifaceted roles in genome organization and gene expression. Here we used single-molecule correlative force and fluorescence microscopy to directly visualize the dynamics of wild-type and RTT-causing mutant MeCP2 on DNA. We discovered that MeCP2 exhibits distinct one-dimensional diffusion kinetics when bound to unmethylated versus CpG methylated DNA, enabling methylation-specific activities such as co-repressor recruitment. We further found that, on chromatinized DNA, MeCP2 preferentially localizes to nucleosomes and stabilizes them from mechanical perturbation. Our results reveal the multimodal behavior of MeCP2 on chromatin that underlies its DNA methylation- and nucleosome-dependent functions and provide a biophysical framework for dissecting the molecular pathology of RTT mutations. Using single-molecule techniques, the authors find that the methyl-CpG-binding protein MeCP2, whose mutations cause Rett syndrome, exhibits distinctive behaviors when bound to nucleosomes versus free DNA, thus directing its multifaceted functions on chromatin.

Bacteria have adapted to phage predation by evolving a vast assortment of defence systems 1 . Although anti-phage immunity genes can be identified using bioinformatic tools, the discovery of novel systems is restricted to the available prokaryotic sequence data 2 . Here, to overcome this limitation, we infected Escherichia coli carrying a soil metagenomic DNA library 3 with the lytic coliphage T4 to isolate clones carrying protective genes. Following this approach, we identified Brig1, a DNA glycosylase that excises alpha-glucosyl-hydroxymethylcytosine nucleobases from the bacteriophage T4 genome to generate abasic sites and inhibit viral replication. Brig1 homologues that provide immunity against T-even phages are present in multiple phage defence loci across distinct clades of bacteria. Our study highlights the benefits of screening unsequenced DNA and reveals prokaryotic DNA glycosylases as important players in the bacteria-phage arms race. A screen utilizing an environmental DNA library in Escherichia coli is used to identify Brig1, a previously unknown anti-phage defence system with homologues across distinct clades of bacteria.

Working memory, the process through which information is transiently maintained and manipulated over a brief period, is essential for most cognitive functions 1-4 . However, the mechanisms underlying the generation and evolution of working-memory neuronal representations at the population level over long timescales remain unclear. Here, to identify these mechanisms, we trained head-fixed mice to perform an olfactory delayed-association task in which the mice made decisions depending on the sequential identity of two odours separated by a 5 s delay. Optogenetic inhibition of secondary motor neurons during the late-delay and choice epochs strongly impaired the task performance of the mice. Mesoscopic calcium imaging of large neuronal populations of the secondary motor cortex (M2), retrosplenial cortex (RSA) and primary motor cortex (M1) showed that many late-delay-epoch-selective neurons emerged in M2 as the mice learned the task. Working-memory late-delay decoding accuracy substantially improved in the M2, but not in the M1 or RSA, as the mice became experts. During the early expert phase, working-memory representations during the late-delay epoch drifted across days, while the stimulus and choice representations stabilized. In contrast to single-plane layer 2/3 (L2/3) imaging, simultaneous volumetric calcium imaging of up to 73,307 M2 neurons, which included superficial L5 neurons, also revealed stabilization of late-delay working-memory representations with continued practice. Thus, delay- and choice-related activities that are essential for working-memory performance drift during learning and stabilize only after several days of expert performance. Delay- and choice-related activities that are essential for working-memory performance drift during learning and stabilize only after several days of expert performance.

Single-cell sequencing has characterized cell state heterogeneity across diverse healthy and malignant tissues. However, the plasticity or heritability of these cell states remains largely unknown. To address this, we introduce PATH (phylogenetic analysis of trait heritability), a framework to quantify cell state heritability versus plasticity and infer cell state transition and proliferation dynamics from single-cell lineage tracing data. Applying PATH to a mouse model of pancreatic cancer, we observed heritability at the ends of the epithelial-to-mesenchymal transition spectrum, with higher plasticity at more intermediate states. In primary glioblastoma, we identified bidirectional transitions between stem- and mesenchymal-like cells, which use the astrocyte-like state as an intermediary. Finally, we reconstructed a phylogeny from single-cell whole-genome sequencing in B cell acute lymphoblastic leukemia and delineated the heritability of B cell differentiation states linked with genetic drivers. Altogether, PATH replaces qualitative conceptions of plasticity with quantitative measures, offering a framework to study somatic evolution. Phylogenetic analysis of trait heritability (PATH) applies phylogenetic correlations to single-cell lineage tracing data, quantifying cell state plasticity and transition probabilities. PATH offers insights into cell state heritability and transition dynamics in cancers.