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Macrophages infected with Gram-negative bacteria expressing Type III secretion system (T3SS) activate the NLRC4 inflammasome, resulting in Gasdermin D (GSDMD)-dependent, but GSDME independent IL-1β secretion and pyroptosis. Here we examine inflammasome signaling in neutrophils infected with Pseudomonas aeruginosa strain PAO1 that expresses the T3SS effectors ExoS and ExoT. IL-1β secretion by neutrophils requires the T3SS needle and translocon proteins and GSDMD. In macrophages, PAO1 and mutants lacking ExoS and ExoT ( ΔexoST ) require NLRC4 for IL-1β secretion. While IL-1β release from ΔexoST infected neutrophils is also NLRC4-dependent, infection with PAO1 is instead NLRP3-dependent and driven by the ADP ribosyl transferase activity of ExoS. Genetic and pharmacologic approaches using MCC950 reveal that NLRP3 is also essential for bacterial killing and disease severity in a murine model of P. aeruginosa corneal infection (keratitis). Overall, these findings reveal a function for ExoS ADPRT in regulating inflammasome subtype usage in neutrophils versus macrophages and an unexpected role for NLRP3 in P. aeruginosa keratitis. Bacterial infection of immune cells can result in engagement of different immunological pathways. Here the authors show that a Pseudomonas aeruginosa type three secretion system exoenzyme is linked to the differential selection of inflammasome usage between macrophages and neutrophils.

The current study explores the videos of eight popular transgender YouTubers. A content analysis was conducted to examine the themes present in videos, the specific issues discussed, and the educational value for viewers. In particular, the present research was interested in the amount of videos that contain transgender-specific content, and in the types of issues—both general and in terms of gender transition—about which transgender YouTubers converse. Videos were also analyzed in relation to self-identified type of transgender individual (e.g. male-to-female [MTF] or female-to-male [FTM]), and significant differences were found in both the amount of transgender-specific content as well as the educational value of videos. This article positions transgender YouTube content as an educational tool that can both help serve as guidance for transgender viewers, as well as increase mainstream audiences’ understanding of transgender persons, subjects, and struggles.

Subarachnoid hemorrhage (SAH) can lead to devastating neurological outcomes, and there are few pharmacologic treatments available for treating this condition. Both animal and human studies provide evidence of inflammation being a driving force behind the pathology of SAH, leading to both direct brain injury and vasospasm, which in turn leads to ischemic brain injury. Several inflammatory mediators that are elevated after SAH have been studied in detail. While there is promising data indicating that blocking these factors might benefit patients after SAH, there has been little success in clinical trials. One of the key factors that complicates clinical trials of SAH is the variability of the initial injury and subsequent inflammatory response. It is likely that both genetic and environmental factors contribute to the variability of patients’ post-SAH inflammatory response and that this confounds trials of anti-inflammatory therapies. Additionally, systemic inflammation from other conditions that affect patients with SAH could contribute to brain injury and vasospasm after SAH. Continuing work on biomarkers of inflammation after SAH may lead to development of patient-specific anti-inflammatory therapies to improve outcome after SAH.

The streptothricin natural product mixture (also known as nourseothricin) was discovered in the early 1940s, generating intense initial interest because of excellent gram-negative activity. Here, we establish the activity spectrum of nourseothricin and its main components, streptothricin F (S-F, 1 lysine) and streptothricin D (S-D, 3 lysines), purified to homogeneity, against highly drug-resistant, carbapenem-resistant Enterobacterales (CRE) and Acinetobacter baumannii . For CRE, the MIC 50 and MIC 90 for S-F and S-D were 2 and 4 μM, and 0.25 and 0.5 μM, respectively. S-F and nourseothricin showed rapid, bactericidal activity. S-F and S-D both showed approximately 40-fold greater selectivity for prokaryotic than eukaryotic ribosomes in in vitro translation assays. In vivo, delayed renal toxicity occurred at >10-fold higher doses of S-F compared with S-D. Substantial treatment effect of S-F in the murine thigh model was observed against the otherwise pandrug-resistant, NDM-1-expressing Klebsiella pneumoniae Nevada strain with minimal or no toxicity. Cryo-EM characterization of S-F bound to the A . baumannii 70S ribosome defines extensive hydrogen bonding of the S-F steptolidine moiety, as a guanine mimetic, to the 16S rRNA C1054 nucleobase ( Escherichia coli numbering) in helix 34, and the carbamoylated gulosamine moiety of S-F with A1196, explaining the high-level resistance conferred by corresponding mutations at the residues identified in single rrn operon E . coli . Structural analysis suggests that S-F probes the A-decoding site, which potentially may account for its miscoding activity. Based on unique and promising activity, we suggest that the streptothricin scaffold deserves further preclinical exploration as a potential therapeutic for drug-resistant, gram-negative pathogens.

Neonatal intraventricular hemorrhage (IVH) is a major complication of preterm birth, yet how developmental stage influences the brain’s response to injury remains unclear. We performed single-nucleus RNA sequencing on rat brains 24 h after IVH at postnatal day 2 (PND2) or day 5 (PND5) to define transcriptional responses across cell types. We identified 42 distinct cell populations and found that PND5 brains exhibited a markedly stronger immune and inflammatory response to IVH, with a threefold increase in differentially expressed genes compared to PND2. Microglia were the most perturbed cell type at both stages, showing increased oxidative stress and polarization toward both pro- and anti-inflammatory phenotypes at PND5. Ligand-receptor and regulon analysis revealed a shift from reparative IGF2 and TGF-β signaling at PND2 to proinflammatory Wnt signaling and activation of Runx1 and Stat5 at PND5. These findings highlight the importance of developmental timing in shaping the neuroimmune response to IVH and identify potential stage-specific therapeutic targets.

Covalent drug discovery efforts are growing rapidly but have major unaddressed limitations. These include high false positive rates during hit-to-lead identification; the inherent uncoupling of covalent drug concentration and effect [i.e., uncoupling of pharmacokinetics (PK) and pharmacodynamics (PD)]; and a lack of bioanalytical and modeling methods for determining PK and PD parameters. We present a covalent drug discovery workflow that addresses these limitations. Our bioanalytical methods are based upon a mass spectrometry (MS) assay that can measure the percentage of drug-target protein conjugation (% target engagement) in biological matrices. Further we develop an i ntact protein PK/PD model ( i PK/PD) that outputs PK parameters (absorption and distribution) as well as PD parameters (mechanism of action, protein metabolic half-lives, dose, regimen, effect) based on time-dependent target engagement data. Notably, the i PK/PD model is applicable to any measurement (e.g., bottom-up MS and other drug binding studies) that yields % of target engaged. A Decision Tree is presented to guide researchers through the covalent drug development process. Our bioanalytical methods and the Decision Tree are applied to two approved drugs (ibrutinib and sotorasib); the most common plasma off-target, human serum albumin; three protein targets (KRAS, BTK, SOD1), and to a promising SOD1-targeting ALS drug candidates. Robust bioanalytical and modeling methods are needed for covalent drug discovery. Here, the authors demonstrate a mass spectrometry (MS) assay to measure target engagement of any drug-target protein complex, a universal PK/PD model for covalent drugs, and a decision tree to guide research.

The budding yeast spindle pole body (SPB) is anchored in the nuclear envelope so that it can simultaneously nucleate both nuclear and cytoplasmic microtubules. During SPB duplication, the newly formed SPB is inserted into the nuclear membrane. The mechanism of SPB insertion is poorly understood but likely involves the action of integral membrane proteins to mediate changes in the nuclear envelope itself, such as fusion of the inner and outer nuclear membranes. Analysis of the functional domains of the budding yeast SUN protein and SPB component Mps3 revealed that most regions are not essential for growth or SPB duplication under wild-type conditions. However, a novel dominant allele in the P-loop region, MPS3-G186K, displays defects in multiple steps in SPB duplication, including SPB insertion, indicating a previously unknown role for Mps3 in this step of SPB assembly. Characterization of the MPS3-G186K mutant by electron microscopy revealed severe over-proliferation of the inner nuclear membrane, which could be rescued by altering the characteristics of the nuclear envelope using both chemical and genetic methods. Lipid profiling revealed that cells lacking MPS3 contain abnormal amounts of certain types of polar and neutral lipids, and deletion or mutation of MPS3 can suppress growth defects associated with inhibition of sterol biosynthesis, suggesting that Mps3 directly affects lipid homeostasis. Therefore, we propose that Mps3 facilitates insertion of SPBs in the nuclear membrane by modulating nuclear envelope composition.

Background Intraventricular hemorrhage (IVH) remains a major complication in preterm infants with lifelong sequelae. There is no effective treatment for IVH other than supportive care and surgery for post-hemorrhagic hydrocephalus. We previously reported that the innate neuroimmune response in an animal model of IVH was dependent on developmental stage, only occurring in older animals. Methods This study utilized a lysed-blood injection model of IVH in rats. This model specifically captures the effects of blood products released by IVH on brain parenchyma. We performed RNAseq and differential gene expression analysis on CD11b/c-positive cells in the brain (microglia/macrophages) to define gene expression in innate immune cells after IVH. We examined CD68 expression, a marker of activated microglia/infiltrating macrophages, in the periventricular white matter after IVH over 90 days. Using IBA1 staining with skeletonized branch analysis and secondary individual cell Sholl analysis, we characterized morphological changes in innate immune cells after IVH. Glial fibrillary protein (GFAP) staining was used to assess astrogliosis and chronic glial scar formation after IVH. We also examined CD68 expression in brain samples from human infants with or without IVH. Results RNAseq of isolated innate immune cells showed significant differences in cytokine-mediated gene expression at 24 h in IVH versus control animals. CD68 expression in white matter decreased overall with time and was elevated at 7 days in the IVH group compared to controls. IBA1 labeling, when analyzed across all time points, showed significant changes to microglial/macrophage branch number, branch area, and soma area after IVH. Sholl analysis of individual IBA1 labeled cells showed an effect of time but not IVH on microglial/macrophage morphology. At the chronic timepoint of 90 days, IVH induced astrogliosis at the margin of the lateral ventricle. A brain sample from a human infant with IVH showed increased CD68 expression throughout the occipital cortex compared with a non-IVH control, indicating immune activation in brain parenchyma after IVH. Conclusions Intraventricular blood products induce a robust innate immune response shortly after injection. RNAseq and CD68 counts are more sensitive to differences between groups than morphological immune cell analysis. Gliosis at the edge of ependyma occurs over time. These results help establish the timeline of inflammation after IVH to better define the window for treating IVH-associated inflammation and subsequent brain injury.

While many scholars have explored the sharing of nude photographs one-to-one (i.e., sexting), few have examined the sharing of nudity in a one-to-many context. The current study examined the sharing of nude photographs on Reddit, framing the practice as an act of disinhibited online behavior. A survey ( n  = 628) was conducted to assess whether Redditors levels of sensation seeking, self-esteem, perceived attractiveness, and narcissism would be related to whether or not they posted nude photographs on the site. Results indicated that posting nudity on Reddit was significantly associated with higher perceived attractiveness and narcissism, but not sensation seeking or self-esteem. The role of gender and sexual orientation in the posting of nudity online was also assessed, and an overrepresentation of nude content produced by females and bisexual persons, as well as an underrepresentation of nude content produced by males and heterosexuals, was found. Findings are discussed in relation to self-concept, sexual health, and the online disinhibition effect.