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Infection of host cells by Toxoplasma gondii is an active process, which is regulated by secretion of microneme (MICs) and rhoptry proteins (ROPs and RONs) from specialized organelles in the apical pole of the parasite. MIC1, MIC4 and MIC6 assemble into an adhesin complex secreted on the parasite surface that functions to promote infection competency. MIC1 and MIC4 are known to bind terminal sialic acid residues and galactose residues, respectively and to induce IL-12 production from splenocytes. Here we show that rMIC1- and rMIC4-stimulated dendritic cells and macrophages produce proinflammatory cytokines, and they do so by engaging TLR2 and TLR4. This process depends on sugar recognition, since point mutations in the carbohydrate-recognition domains (CRD) of rMIC1 and rMIC4 inhibit innate immune cells activation. HEK cells transfected with TLR2 glycomutants were selectively unresponsive to MICs. Following in vitro infection, parasites lacking MIC1 or MIC4, as well as expressing MIC proteins with point mutations in their CRD, failed to induce wild-type (WT) levels of IL-12 secretion by innate immune cells. However, only MIC1 was shown to impact systemic levels of IL-12 and IFN-γ in vivo. Together, our data show that MIC1 and MIC4 interact physically with TLR2 and TLR4 N-glycans to trigger IL-12 responses, and MIC1 is playing a significant role in vivo by altering T. gondii infection competency and murine pathogenesis.

Leptospirosis is a neglected, widespread zoonosis caused by pathogenic species of the genus Leptospira, and is responsible for 60,000 deaths per year. Pathogenic mechanisms of leptospirosis remain poorly understood mainly because targeted mutations or gene silencing in pathogenic Leptospira continues to be inherently inefficient, laborious, costly and difficult to implement. In addition, pathogenic leptospires are highly fastidious and the selection of mutants on solid agar media can take up to 6 weeks. The catalytically inactive Cas9 (dCas9) is an RNA-guided DNA-binding protein from the Streptococcus pyogenes CRISPR/Cas system and can be used for gene silencing, in a strategy termed CRISPR interference (CRISPRi). Here, this technique was employed to silence genes encoding major outer membrane proteins of pathogenic L. interrogans . Conjugation protocols were optimized using the newly described HAN media modified for rapid mutant recovery at 37 °C in 3% CO 2 within 8 days. Complete silencing of LipL32 and concomitant and complete silencing of both LigA and LigB outer membrane proteins were achieved, revealing for the first time that Lig proteins are involved in pathogenic Leptospira serum resistance. Gene silencing in pathogenic leptospires and rapid mutant recovery will facilitate novel studies to further evaluate and understand pathogenic mechanisms of leptospirosis.

Agonist interaction with Toll-like receptors (TLRs) induces T cell-mediated immunity, which is effective against intracellular pathogens. Consequently, TLR agonists are being tried as immunomodulatory agents. The lectin ArtinM targets TLR2 N-glycans on macrophages, induces cytokines production, and promotes T helper-1 immunity, a process that culminates in resistance to several parasitic and fungal infections in vivo . Because co-receptors influence agonist binding to TLRs, we investigated whether CD14 is required for macrophage activation induced by ArtinM. Macrophages from wild-type mice stimulated by ArtinM not only produced cytokines but also had the following activation profile: (i) expression of M1 polarization markers; (ii) nitrite oxide production; (iii) cellular migration; (iv) enhanced phagocytic and fungicide activity; (v) modulation of TLR2 expression; and (vi) activation of NF-κB pathway. This activation profile induced by ArtinM was evaluated in macrophages lacking CD14 that showed none of the ArtinM effects. We demonstrated by immunoprecipitation and sugar inhibition assays the physical interaction of ArtinM, TLR2, and CD14, which depends on recognition of the trimannoside that constitutes the core of N-glycans. Thus, our study showed that CD14 is critical for ArtinM-induced macrophage activation, providing fundamental insight into the design of anti-infective therapies based on carbohydrate recognition.

Environmental enrichment (EE) exerts powerful effects on brain physiology, and is widely used as an experimental and therapeutic tool. Typical EE paradigms are multifactorial, incorporating elements of physical exercise, environmental complexity, social interactions and stress, however the specific contributions of these variables have not been separable using conventional housing paradigms. Here, we evaluated the impacts of these individual variables on adult hippocampal neurogenesis by using a novel \"Alternating EE\" paradigm. For 4 weeks, adult male CD1 mice were alternated daily between two enriched environments; by comparing groups that differed in one of their two environments, the individual and combinatorial effects of EE variables could be resolved. The Alternating EE paradigm revealed that (1) voluntary running for 3 days/week was sufficient to increase both mitotic and post-mitotic stages of hippocampal neurogenesis, confirming the central importance of exercise; (2) a complex environment (comprised of both social interactions and rotated inanimate objects) had no effect on neurogenesis itself, but enhanced depolarization-induced c-Fos expression (attributable to social interactions) and buffered stress-induced plasma corticosterone levels (attributable to inanimate objects); and (3) neither social isolation, group housing, nor chronically increased levels of plasma corticosterone had a prolonged impact on neurogenesis. Mouse strain, handling and type of running apparatus were tested and excluded as potential confounding factors. These findings provide valuable insights into the relative effects of key EE variables on adult neurogenesis, and this \"Alternating EE\" paradigm represents a useful tool for exploring the contributions of individual EE variables to mechanisms of neural plasticity.

Recent technological advances have enabled massively parallel chromatin profiling with scATAC-seq (single-cell assay for transposase accessible chromatin by sequencing). Here we present ATAC with select antigen profiling by sequencing (ASAP-seq), a tool to simultaneously profile accessible chromatin and protein levels. Our approach pairs sparse scATAC-seq data with robust detection of hundreds of cell surface and intracellular protein markers and optional capture of mitochondrial DNA for clonal tracking, capturing three distinct modalities in single cells. ASAP-seq uses a bridging approach that repurposes antibody:oligonucleotide conjugates designed for existing technologies that pair protein measurements with single-cell RNA sequencing. Together with DOGMA-seq, an adaptation of CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing) for measuring gene activity across the central dogma of gene regulation, we demonstrate the utility of systematic multi-omic profiling by revealing coordinated and distinct changes in chromatin, RNA and surface proteins during native hematopoietic differentiation and peripheral blood mononuclear cell stimulation and as a combinatorial decoder and reporter of multiplexed perturbations in primary T cells. Chromatin accessibility, gene expression and protein levels are measured in the same single cell.

The Dzyaloshinskii-Moriya interaction (DMI) has drawn much attention, as it stabilizes magnetic chirality, with important implications in fundamental and applied research. This antisymmetric exchange interaction is induced by the broken inversion symmetry at interfaces or in noncentrosymmetric lattices. Significant interfacial DMIs are often found at magnetic/heavy-metal interfaces with large spin-orbit coupling. Recent studies have shown promise for induced DMI at interfaces involving light elements such as carbon (graphene) or oxygen. Here, we report direct observation of induced DMI by chemisorption of the lightest element, hydrogen, on a ferromagnetic layer at room temperature, which is supported by density functional theory calculations. We further demonstrate a reversible chirality transition of the magnetic domain walls due to the induced DMI via hydrogen chemisorption and desorption. These results shed new light on the understanding of DMI in low atomic number materials and the design of novel chiral spintronics and magneto-ionic devices.

IntroductionCariprazine is one of the most recent innovations in neuropsychopharmacology, with evidence for its efficacy in affective and psychotic spectrum disorders.ObjectivesTo present a case that highlights cariprazine’s potential use outside the regulatory approved indications.MethodsCase report using CARE guidelines and a narrative review.ResultsWe present the case of a 41-year-old male readmitted to a psychiatric inpatient unit due to three months of mutism and withdrawal. At admission, the patient did not communicate verbally or in writing, but he complied with simple orders, and his consciousness remained unimpaired. He scored 11 points on the Bush-Francis Catatonia Rating Scale (BFCRS), indicating immobility, mutism, staring, withdrawal, ambitendency, and automatic obedience. We observed psychomotor retardation and indirect signs of a depressive mood, including the omega sign. His medical history included ongoing psychiatric treatment since the age of 30, with two prior admissions to an acute inpatient unit. At the time of admission, he was treated with olanzapine 20 mg/day, lorazepam 2 mg/day (recently downtitrated), venlafaxine 150 mg/day, and bupropion 150 mg/day. At the start of the current episode, the patient’s diagnosis was uncertain, with previous descriptions of psychotic, affective, and catatonic features. Due to suspicion of catatonia, we administered a high dose of lorazepam (8 mg/day), resulting in a partial response with a 4-point reduction in the BFCRS. We discontinued bupropion, increased venlafaxine to 225 mg, and switched from olanzapine to cariprazine using a taper, washout, and switch strategy. Psychotic symptoms briefly appeared when the patient was not taking a dopamine D2-receptor modulatory drug. We identified mild possible adverse drug reactions, including akathisia, transient insomnia, and daytime sleepiness. At a dose of 6 mg/day of cariprazine, we observed complete remission of catatonia (BFCRS=0) and significant improvement in affective and psychotic symptoms. The patient was discharged home with diagnoses of catatonia and schizoaffective disorder, prescribed 6mg/day of cariprazine, 225mg/day of venlafaxine, and 2,5mg/day of lorazepam. At the 6-month follow-up, the patient continues to exhibit clinical stability.ConclusionsThis case emphasizes the safety and potential effectiveness of cariprazine in treating catatonia within the context of schizoaffective disorder. We consider that the partial agonist properties of cariprazine could theoretically reduce the risk of exacerbating catatonia, a risk typically associated with full D2-receptor antagonists. Other mechanisms of action, such as D3 partial agonism, may also contribute to the improvement or at least the non-aggravation of catatonic symptoms. Cariprazine’s mood-stabilizing properties make it a promising off-label choice for treating schizoaffective disorder, especially when catatonic features are present.Disclosure of InterestNone Declared

Leptospirosis is a worldwide zoonosis caused by pathogenic bacteria of the genus Leptospira , which also includes free-living saprophyte strains. Many aspects of leptospiral basic biology and virulence mechanisms remain unexplored mainly due to the lack of effective genetic tools available for these bacteria. Recently, the type II CRISPR/Cas system from Streptococcus pyogenes has been widely used as an efficient genome engineering tool in bacteria by inducing double-strand breaks (DSBs) in the desired genomic targets caused by an RNA-guided DNA endonuclease called Cas9, and the DSB repair associated machinery. In the present work, plasmids expressing heterologous S. pyogenes Cas9 in L. biflexa cells were generated, and the enzyme could be expressed with no apparent toxicity to leptospiral cells. However, L. biflexa cells were unable to repair RNA-guided Cas9-induced DSBs. Thus, we used a catalytically dead Cas9 (dCas9) to obtain gene silencing rather than disruption, in a strategy called CRISPR interference (CRISPRi). We demonstrated complete gene silencing in L. biflexa cells when both dCas9 and single-guide RNA (sgRNA) targeting the coding strand of the β-galactosidase gene were expressed simultaneously. Furthermore, when the system was applied for silencing the dnaK gene, no colonies were recovered, indicating that DnaK protein is essential in Leptospira . In addition, flagellar motor switch FliG gene silencing resulted in reduced bacterial motility. To the best of our knowledge, this is the first work applying the CRISPRi system in Leptospira and spirochetes in general, expanding the tools available for understanding leptospiral biology.

The defining features of Alzheimer’s disease (AD) include alterations in protein aggregation, immunity, lipid metabolism, synapses, and learning and memory. Of these, lipid abnormalities are the least understood. Here, we investigate the role of Stearoyl-CoA desaturase (SCD), a crucial regulator of fatty acid desaturation, in AD pathogenesis. We show that inhibiting brain SCD activity for 1-month in the 3xTg mouse model of AD alters core AD-related transcriptomic pathways in the hippocampus, and that it concomitantly restores essential components of hippocampal function, including dendritic spines and structure, immediate-early gene expression, and learning and memory itself. Moreover, SCD inhibition dampens activation of microglia, key mediators of spine loss during AD and the main immune cells of the brain. These data reveal that brain fatty acid metabolism links AD genes to downstream immune, synaptic, and functional impairments, identifying SCD as a potential target for AD treatment. Alzheimer’s disease (AD) is characterized by lipid abnormalities which are not well understood. Here, the authors investigate the role of Stearoyl-CoA desaturase (SCD) in a mouse model of AD. They show that inhibiting SCD activity induces major brain and immune cell transcriptional changes and restores dendritic structure and learning and memory.

Expanding the genetic toolkit for  Leptospira  spp. is a crucial step toward advancing our understanding of the biology and virulence of these atypical bacteria. Pathogenic  Leptospira are responsible for over 1 million human leptospirosis cases annually and significantly impact domestic animals. Bovine leptospirosis causes substantial financial losses due to abortion, stillbirths, and suboptimal reproductive performance. The advent of the CRISPR/Cas9 system has marked a turning point in genetic manipulation, with applications across multiple  Leptospira  species. However, incorporating controlled protein expression into existing genetic tools could further expand their utility. We developed and demonstrated the functionality of IPTG-inducible heterologous protein expression in  Leptospira  spp. This system was applied for regulated expression of dead Cas9 (dCas9) to generate knockdown mutants, and Cas9 to produce knockout mutants by inducing double-strand breaks (DSB) into desired targets. IPTG-induced dCas9 expression enabled validation of essential genes and non-coding RNAs. Additionally, IPTG-controlled Cas9 expression combined with a constitutive non-homologous end-joining (NHEJ) system allowed for successful recovery of knockout mutants, even in the absence of IPTG. These newly controlled protein expression systems will advance studies on the basic biology and virulence of Leptospira , as well as facilitate knockout mutant generation for improved veterinary vaccines.