Neuroanatomical distribution of fluorophores within adult RXFP3 Cre-tdTomato/YFP mouse brain.

Relaxin-family peptide 3 receptor (RXFP3) is activated by relaxin-3 in the brain to influence arousal and related functions, such as feeding and stress responses. Two transgenic mouse lines have recently been developed that co-express different fluorophores within RXFP3-expressing neurons: either yellow fluorescent protein (YFP; RXFP3-Cre/YFP mice) or tdTomato (RXFP3-Cre/tdTomato mice). To date, the characteristics of neurons that express RXFP3-associated fluorophores in these mice have only been investigated in the bed nucleus of the stria terminalis and the hypothalamic arcuate nucleus. To better determine the utility of these fluorophore-expressing mice for further research, we characterised the neuroanatomical distribution of fluorophores throughout the brain of these mice and compared this to the published distribution of Rxfp3 mRNA (detected by in situ hybridisation) in wildtype mice. Coronal sections of RXFP3-Cre/YFP (n = 8) and RXFP3-Cre/tdTomato (n = 8) mouse brains were imaged, and the density of fluorophore-expressing cells within various brain regions/nuclei was qualitatively assessed. Comparisons with our previously reported RXFP3 mRNA distribution revealed that of 212 brain regions that contained either fluorophore or RXFP3 mRNA, approximately half recorded densities that were within two qualitative measurements of each other (on a 9-point scale), including hippocampal dentate gyrus and amygdala subregions. However, many brain areas with likely non-authentic, false-positive, or false-negative fluorophore expression were also detected, including the cerebellum. Therefore, this study provides a guide to which brain regions should be prioritized for future study of RXFP3 in these mice, to better understand the neuroanatomy and function of this intriguing, neuronal peptide receptor.

Synthesizing 3D Multi-Contrast Brain Tumor MRIs Using Tumor Mask Conditioning.

Data scarcity and data imbalance are two major challenges in training deep learning models on medical images, such as brain tumor MRI data. The recent advancements in generative artificial intelligence have opened new possibilities for synthetically generating MRI data, including brain tumor MRI scans. This approach can be a potential solution to mitigate the data scarcity problem and enhance training data availability. This work focused on adapting the 2D latent diffusion models to generate 3D multi-contrast brain tumor MRI data with a tumor mask as the condition. The framework comprises two components: a 3D autoencoder model for perceptual compression and a conditional 3D Diffusion Probabilistic Model (DPM) for generating high-quality and diverse multi-contrast brain tumor MRI samples, guided by a conditional tumor mask. Unlike existing works that focused on generating either 2D multi-contrast or 3D single-contrast MRI samples, our models generate multi-contrast 3D MRI samples. We also integrated a conditional module within the UNet backbone of the DPM to capture the semantic class-dependent data distribution driven by the provided tumor mask to generate MRI brain tumor samples based on a specific brain tumor mask. We trained our models using two brain tumor datasets: The Cancer Genome Atlas (TCGA) public dataset and an internal dataset from the University of Texas Southwestern Medical Center (UTSW). The models were able to generate high-quality 3D multi-contrast brain tumor MRI samples with the tumor location aligned by the input condition mask. The quality of the generated images was evaluated using the Fréchet Inception Distance (FID) score. This work has the potential to mitigate the scarcity of brain tumor data and improve the performance of deep learning models involving brain tumor MRI data.

Validation of a Tamil Version of the Five Facet Mindfulness Questionnaire Using Rasch Analysis.

Background: Various assessment tools that explore and assess mindfulness are available. Keeping in view both the origin of and the literature surrounding mindfulness assessment tools, this study aimed to evaluate the workability of one widely researched tool, the Five Facet Mindfulness Questionnaire (FFMQ), for establishing cross-cultural generalizability and utility in the Indian context. Methods: We recruited 303 adults over 18 with proficiency in the Tamil language and no history of significant neurological trauma and/or psychiatric history. They completed a version of the 39-item FFMQ, which we had translated into Tamil (FFMQ-T). The psychometric properties of this scale were tested using the Partial-Credit model of Rasch analysis. Results: Iterative Rasch analysis could not resolve consistent misfit of the Observe facet items. Using a subtest approach, a higher-order fit of the FFMQ-T could be achieved after the deletion of additional items from each of the remaining four facets. The resulting final model for the FFMQ-T questionnaire was a four-factor solution with 22 items. Conclusions: This study concluded the usability of the new 22-item FFMQ-T. These results are not dissimilar to the other versions in similar populations, such as the Hindi version of the FFMQ. The ordinal-to-interval conversion tables provided here ensure that the FFMQ-T can be used with enhanced precision and parametric statistics.

Structural basis of chemokine recognition by the class A3 tick evasin EVA-ACA1001.

Ticks produce chemokine-binding proteins, known as evasins, in their saliva to subvert the host's immune response. Evasins bind to chemokines and thereby inhibit the activation of their cognate chemokine receptors, thus suppressing leukocyte recruitment and inflammation. We recently described subclass A3 evasins, which, like other class A evasins, exclusively target CC chemokines but appear to use a different binding site architecture to control target selectivity among CC chemokines. We now describe the structural basis of chemokine recognition by the class A3 evasin EVA-ACA1001. EVA-ACA1001 binds to almost all human CC chemokines and inhibits receptor activation. Truncation mutants of EVA-ACA1001 showed that, unlike class A1 evasins, both the N- and C-termini of EVA-ACA1001 play minimal roles in chemokine binding. To understand the structural basis of its broad chemokine recognition, we determined the crystal structure of EVA-ACA1001 in complex with the human chemokine CCL16. EVA-ACA1001 forms backbone-backbone interactions with the CC motif of CCL16, a conserved feature of all class A evasin-chemokine complexes. A hydrophobic pocket in EVA-ACA1001, formed by several aromatic side chains and the unique disulfide bond of class A3 evasins, accommodates the residue immediately following the CC motif (the "CC + 1 residue") of CCL16. This interaction is shared with EVA-AAM1001, the only other class A3 evasins characterized to date, suggesting it may represent a common mechanism that accounts for the broad recognition of CC chemokines by class A3 evasins.

Investigating the spatiotemporal patterns and clustering of attendances for mental health services to inform policy and resource allocation in Thailand.

BACKGROUND: Mental illness poses a substantial global public health challenge, including in Thailand, where exploration of access to mental health services is limited. The spatial and temporal dimensions of mental illness in the country are not extensively studied, despite the recognized association between poor mental health and socioeconomic inequalities. Gaining insights into these dimensions is crucial for effective public health interventions and resource allocation. METHODS: This retrospective study analyzed mental health service utilization data in Thailand from 2015 to 2023. Temporal trends in annual numbers of individuals visiting mental health services by diagnosis were examined, while spatial pattern analysis employed Moran's I statistics to assess autocorrelation, identify small-area clustering, and hotspots. The implications of our findings for mental health resource allocation and policy were discussed. RESULTS: Between 2015 and 2023, mental health facilities documented a total of 13,793,884 visits. The study found anxiety, schizophrenia, and depression emerged as the top three illnesses for mental health visits, with an increase in patient attendance following the onset of the COVID-19 outbreak. Spatial analysis identified areas of significance for various disorders across different regions of Thailand. Positive correlations between certain disorder pairs were found in specific regions, suggesting shared risk factors or comorbidities. CONCLUSIONS: This study highlights spatial and temporal variations in individuals visiting services for different mental disorders in Thailand, shedding light on service gaps and socioeconomic issues. Addressing these disparities requires increased attention to mental health, the development of appropriate interventions, and overcoming barriers to accessibility. The findings provide a baseline for policymakers and stakeholders to allocate resources and implement culturally responsive interventions to improve mental health outcomes.

Healthy Minds Index: A brief measure of the core dimensions of well-being.

We developed a self-report measure of psychological well-being for teens and adults, the Healthy Minds Index, based on a novel theory that four trainable pillars underlie well-being: awareness, connection, insight, and purpose. Ninety-seven items were developed and revised by experts and guided by qualitative testing with teens (n = 32; average age = 16.0 years). After assessing the internal validity and factor structure in teens (n = 1607; average age = 16.7 years) and adults (n = 420; average age = 45.6 years), we reduced the survey to 17 items. We then validated the factor structure, internal and convergent and divergent validity, and retest reliability of the 17-item Healthy Minds Index in two new teen samples (study 1: n = 1492, average age = 15.7 years; study 2: n = 295, average age = 16.1 years), and one adult sample (n = 285; average age = 45.3 years). The Healthy Minds Index demonstrated adequate validity and provided a comprehensive measure of a novel theory of psychological well-being that includes two domains not found in other conceptualizations of this construct-awareness and insight. This measure will be invaluable for primary research on well-being and as a translational tool to assess the impact and efficacy of widely used behavioral training programs on these core dimensions of wellbeing.

Novel Scorpion Toxin ω-Buthitoxin-Hf1a Selectively Inhibits Calcium Influx via CaV3.3 and CaV3.2 and Alleviates Allodynia in a Mouse Model of Acute Postsurgical Pain.

Venom peptides have evolved to target a wide range of membrane proteins through diverse mechanisms of action and structures, providing promising therapeutic leads for diseases, including pain, epilepsy, and cancer, as well as unique probes of ion channel structure-function. In this work, a high-throughput FLIPR window current screening assay on T-type CaV3.2 guided the isolation of a novel peptide named ω-Buthitoxin-Hf1a from scorpion Hottentotta franzwerneri crude venom. At only 10 amino acid residues with one disulfide bond, it is not only the smallest venom peptide known to target T-type CaVs but also the smallest structured scorpion venom peptide yet discovered. Synthetic Hf1a peptides were prepared with C-terminal amidation (Hf1a-NH2) or a free C-terminus (Hf1a-OH). Electrophysiological characterization revealed Hf1a-NH2 to be a concentration-dependent partial inhibitor of CaV3.2 (IC50 = 1.18 µM) and CaV3.3 (IC50 = 0.49 µM) depolarized currents but was ineffective at CaV3.1. Hf1a-OH did not show activity against any of the three T-type subtypes. Additionally, neither form showed activity against N-type CaV2.2 or L-type calcium channels. The three-dimensional structure of Hf1a-NH2 was determined using NMR spectroscopy and used in docking studies to predict its binding site at CaV3.2 and CaV3.3. As both CaV3.2 and CaV3.3 have been implicated in peripheral pain signaling, the analgesic potential of Hf1a-NH2 was explored in vivo in a mouse model of incision-induced acute post-surgical pain. Consistent with this role, Hf1a-NH2 produced antiallodynia in both mechanical and thermal pain.

Proteogenomic characterization of difficult-to-treat breast cancer with tumor cells enriched through laser microdissection.

BACKGROUND: Breast cancer (BC) is the most commonly diagnosed cancer and the leading cause of cancer death among women globally. Despite advances, there is considerable variation in clinical outcomes for patients with non-luminal A tumors, classified as difficult-to-treat breast cancers (DTBC). This study aims to delineate the proteogenomic landscape of DTBC tumors compared to luminal A (LumA) tumors. METHODS: We retrospectively collected a total of 117 untreated primary breast tumor specimens, focusing on DTBC subtypes. Breast tumors were processed by laser microdissection (LMD) to enrich tumor cells. DNA, RNA, and protein were simultaneously extracted from each tumor preparation, followed by whole genome sequencing, paired-end RNA sequencing, global proteomics and phosphoproteomics. Differential feature analysis, pathway analysis and survival analysis were performed to better understand DTBC and investigate biomarkers. RESULTS: We observed distinct variations in gene mutations, structural variations, and chromosomal alterations between DTBC and LumA breast tumors. DTBC tumors predominantly had more mutations in TP53, PLXNB3, Zinc finger genes, and fewer mutations in SDC2, CDH1, PIK3CA, SVIL, and PTEN. Notably, Cytoband 1q21, which contains numerous cell proliferation-related genes, was significantly amplified in the DTBC tumors. LMD successfully minimized stromal components and increased RNA-protein concordance, as evidenced by stromal score comparisons and proteomic analysis. Distinct DTBC and LumA-enriched clusters were observed by proteomic and phosphoproteomic clustering analysis, some with survival differences. Phosphoproteomics identified two distinct phosphoproteomic profiles for high relapse-risk and low relapse-risk basal-like tumors, involving several genes known to be associated with breast cancer oncogenesis and progression, including KIAA1522, DCK, FOXO3, MYO9B, ARID1A, EPRS, ZC3HAV1, and RBM14. Lastly, an integrated pathway analysis of multi-omics data highlighted a robust enrichment of proliferation pathways in DTBC tumors. CONCLUSIONS: This study provides an integrated proteogenomic characterization of DTBC vs LumA with tumor cells enriched through laser microdissection. We identified many common features of DTBC tumors and the phosphopeptides that could serve as potential biomarkers for high/low relapse-risk basal-like BC and possibly guide treatment selections.

A distinct, high-affinity, alkaline phosphatase facilitates occupation of P-depleted environments by marine picocyanobacteria.

Marine picocyanobacteria of the genera Prochlorococcus and Synechococcus, the two most abundant phototrophs on Earth, thrive in oligotrophic oceanic regions. While it is well known that specific lineages are exquisitely adapted to prevailing in situ light and temperature regimes, much less is known of the molecular machinery required to facilitate occupancy of these low-nutrient environments. Here, we describe a hitherto unknown alkaline phosphatase, Psip1, that has a substantially higher affinity for phosphomonoesters than other well-known phosphatases like PhoA, PhoX, or PhoD and is restricted to clade III Synechococcus and a subset of high light I-adapted Prochlorococcus strains, suggesting niche specificity. We demonstrate that Psip1 has undergone convergent evolution with PhoX, requiring both iron and calcium for activity and likely possessing identical key residues around the active site, despite generally very low sequence homology. Interrogation of metagenomes and transcriptomes from TARA oceans and an Atlantic Meridional transect shows that psip1 is abundant and highly expressed in picocyanobacterial populations from the Mediterranean Sea and north Atlantic gyre, regions well recognized to be phosphorus (P)-deplete. Together, this identifies psip1 as an important oligotrophy-specific gene for P recycling in these organisms. Furthermore, psip1 is not restricted to picocyanobacteria and is abundant and highly transcribed in some α-proteobacteria and eukaryotic algae, suggesting that such a high-affinity phosphatase is important across the microbial taxonomic world to occupy low-P environments.

Bladder Management and Continence in Girls With Cloacal Malformation After 3 Years of Age.

BACKGROUND: Multiple factors impact ability to achieve urinary continence in cloacal malformation including common channel (CC) and urethral length and presence of spinal cord abnormalities. Few publications describe continence rates and bladder management in this population. We evaluated our cohort of patients with cloacal malformation to describe the bladder management and continence outcomes. METHODS: We reviewed a prospectively collected database of patients with cloacal malformation managed at our institution. We included girls ≥3 years (y) of age and evaluated their bladder management methods and continence. Dryness was defined as <1 daytime accident per week. Incontinent diversions with both vesicostomy and enterovesicostomy were considered wet. RESULTS: A total of 152 patients were included. Overall, 93 (61.2%) are dry. Nearly half (47%) voided via urethra, 65% of whom were dry. Twenty patients (13.1%) had incontinent diversions. Over 40% of the cohort performed clean intermittent catheterization (CIC), approximately half via urethra and half via abdominal channel. Over 80% of those performing CIC were dry. In total, 12.5% (n = 19) required bladder augmentation (BA). CC length was not associated with dryness (p = 0.076), need for CIC (p = 0.253), or need for abdominal channel (p = 0.497). The presence of a spinal cord abnormality was associated with need for CIC (p = 0.0117) and normal spine associated with ability to void and be dry (p = 0.004) CONCLUSIONS: In girls ≥ 3 y of age with cloacal malformation, 61.2% are dry, 65% by voiding via urethra and 82% with CIC. 12.5% require BA. Further investigation is needed to determine anatomic findings associated with urinary outcomes. LEVEL OF EVIDENCE: IV.

Contrasting consequences of podocyte insulin-like growth factor 1 receptor inhibition.

Insulin signaling to the glomerular podocyte via the insulin receptor (IR) is critical for kidney function. In this study we show that near-complete knockout of the closely related insulin-like growth factor 1 receptor (IGF1R) in podocytes is detrimental, resulting in albuminuria in vivo and podocyte cell death in vitro. In contrast, partial podocyte IGF1R knockdown confers protection against doxorubicin-induced podocyte injury. Proteomic analysis of cultured podocytes revealed that while near-complete loss of podocyte IGF1R results in the downregulation of mitochondrial respiratory complex I and DNA damage repair proteins, partial IGF1R inhibition promotes respiratory complex expression. This suggests that altered mitochondrial function and resistance to podocyte stress depends on the level of IGF1R suppression, the latter determining whether receptor inhibition is protective or detrimental. Our work suggests that the partial suppression of podocyte IGF1R could have therapeutic benefits in treating albuminuric kidney disease.

Targeting cellular cathepsins inhibits hepatitis E virus entry.

BACKGROUND AND AIMS: The hepatitis E virus (HEV) is estimated to be responsible for 70,000 deaths annually, yet therapy options remain limited. In the pursuit of effective antiviral therapies, targeting viral entry holds promise and has proven effective for other viruses. However, the precise mechanisms and host factors required during HEV entry remain unclear. Cellular proteases have emerged as host factors required for viral surface protein activation and productive cell entry by many viruses. Hence, we investigated the functional requirement and therapeutic potentials of cellular proteases during HEV infection. APPROACH AND RESULTS: Using our established HEV cell culture model and subgenomic HEV replicons, we found that blocking lysosomal cathepsins (CTS) with small molecule inhibitors, impedes HEV infection without affecting replication. Most importantly, the pan-cathepsin inhibitor K11777 suppressed HEV infections with an EC50 of ~ 0.01 nM. Inhibition by K11777, devoid of notable toxicity in hepatoma cells, was also observed in HepaRG and primary human hepatocytes. Furthermore, through time-of-addition and RNAscope experiments, we confirmed that HEV entry is blocked by inhibition of cathepsins. Cathepsin L (CTSL) knockout cells were less permissive to HEV, suggesting that CTSL is critical for HEV infection. Finally, we observed cleavage of the glycosylated ORF2 protein and virus particles by recombinant CTSL. CONCLUSIONS: In summary, our study highlights the pivotal role of lysosomal cathepsins, especially CTSL, in the HEV entry process. The profound anti-HEV efficacy of the pan-cathepsin inhibitor, K11777, especially with its notable safety profile in primary cells, further underscores its potential as a therapeutic candidate.

The natural history and genotype-phenotype correlations of TMPRSS3 hearing loss: an international, multi-center, cohort analysis.

TMPRSS3-related hearing loss presents challenges in correlating genotypic variants with clinical phenotypes due to the small sample sizes of previous studies. We conducted a cross-sectional genomics study coupled with retrospective clinical phenotype analysis on 127 individuals. These individuals were from 16 academic medical centers across 6 countries. Key findings revealed 47 unique TMPRSS3 variants with significant differences in hearing thresholds between those with missense variants versus those with loss-of-function genotypes. The hearing loss progression rate for the DFNB8 subtype was 0.3 dB/year. Post-cochlear implantation, an average word recognition score of 76% was observed. Of the 51 individuals with two missense variants, 10 had DFNB10 with profound hearing loss. These 10 all had at least one of 4 TMPRSS3 variants predicted by computational modeling to be damaging to TMPRSS3 structure and function. To our knowledge, this is the largest study of TMPRSS3 genotype-phenotype correlations. We find significant differences in hearing thresholds, hearing loss progression, and age of presentation, by TMPRSS3 genotype and protein domain affected. Most individuals with TMPRSS3 variants perform well on speech recognition tests after cochlear implant, however increased age at implant is associated with worse outcomes. These findings provide insight for genetic counseling and the on-going design of novel therapeutic approaches.

Microbial communities change along the 300 km length of the Grand River for extreme high- and low-flow regimes.

The Grand River watershed is the largest catchment in southern Ontario. The river's northern and southern sections are influenced by agriculture, whereas central regions receive wastewater effluent and urban runoff. To characterize in-river microbial communities, as they relate to spatial and environmental factors, we conducted two same-day sampling events along the entire 300 km length of the river, representing contrasting flow seasons (high flow spring melt and low flow end of summer). Through high-throughput sequencing of 16S rRNA genes, we assessed the relationship between river microbiota and spatial and physicochemical variables. Flow season had a greater impact on communities than spatial or diel effects and profiles diverged with distance between sites under both flow conditions, but low-flow profiles exhibited higher beta diversity. High-flow profiles showed greater species richness and increased presence of soil and sediment taxa, which may relate to increased input from terrestrial sources. Total suspended solids, dissolved inorganic carbon, and distance from headwaters significantly explained microbial community variation during the low-flow event, whereas conductivity, sulfate, and nitrite were significant explanatory factors for spring melt. This study establishes a baseline for the Grand River's microbial community, serving as a foundation for modeling the microbiology of anthropogenically impacted freshwater systems affected by lotic processes.

The first two blastomeres contribute unequally to the human embryo.

Retrospective lineage reconstruction of humans predicts that dramatic clonal imbalances in the body can be traced to the 2-cell stage embryo. However, whether and how such clonal asymmetries arise in the embryo is unclear. Here, we performed prospective lineage tracing of human embryos using live imaging, non-invasive cell labeling, and computational predictions to determine the contribution of each 2-cell stage blastomere to the epiblast (body), hypoblast (yolk sac), and trophectoderm (placenta). We show that the majority of epiblast cells originate from only one blastomere of the 2-cell stage embryo. We observe that only one to three cells become internalized at the 8-to-16-cell stage transition. Moreover, these internalized cells are more frequently derived from the first cell to divide at the 2-cell stage. We propose that cell division dynamics and a cell internalization bottleneck in the early embryo establish asymmetry in the clonal composition of the future human body.

Double-negative B cells and DNASE1L3 colocalise with microbiota in gut-associated lymphoid tissue.

Intestinal homeostasis is maintained by the response of gut-associated lymphoid tissue to bacteria transported across the follicle associated epithelium into the subepithelial dome. The initial response to antigens and how bacteria are handled is incompletely understood. By iterative application of spatial transcriptomics and multiplexed single-cell technologies, we identify that the double negative 2 subset of B cells, previously associated with autoimmune diseases, is present in the subepithelial dome in health. We show that in this location double negative 2 B cells interact with dendritic cells co-expressing the lupus autoantigens DNASE1L3 and C1q and microbicides. We observe that in humans, but not in mice, dendritic cells expressing DNASE1L3 are associated with sampled bacteria but not DNA derived from apoptotic cells. We propose that fundamental features of autoimmune diseases are microbiota-associated, interacting components of normal intestinal immunity.

Evidence of reassortment of avian influenza A (H2) viruses in Brazilian shorebirds.

Influenza A viruses of the H2 subtype represent a zoonotic and pandemic threat to humans due to a lack of widespread specific immunity. Although A(H2) viruses that circulate in wild bird reservoirs are distinct from the 1957 pandemic A(H2N2) viruses, there is concern that they could impact animal and public health. There is limited information on AIVs in Latin America, and next to nothing about H2 subtypes in Brazil. In the present study, we report the occurrence and genomic sequences of two influenza A viruses isolated from wild-caught white-rumped sandpipers (Calidris fuscicollis). One virus, identified as A(H2N1), was isolated from a bird captured in Restinga de Jurubatiba National Park (PNRJ, Rio de Janeiro), while the other, identified as A(H2N2), was isolated from a bird captured in Lagoa do Peixe National Park (PNLP, Rio Grande do Sul). DNA sequencing and phylogenetic analysis of the obtained sequences revealed that each virus belonged to distinct subtypes. Furthermore, the phylogenetic analysis indicated that the genomic sequence of the A(H2N1) virus isolated from PNRJ was most closely related to other A(H2N1) viruses isolated from North American birds. On the other hand, the A(H2N2) virus genome recovered from the PNLP-captured bird exhibited a more diverse origin, with some sequences closely related to viruses from Iceland and North America, and others showing similarity to virus sequences recovered from birds in South America. Viral genes of diverse origins were identified in one of the viruses, indicating local reassortment. This suggests that the extreme South of Brazil may serve as an environment conducive to reassortment between avian influenza virus lineages from North and South America, potentially contributing to an increase in overall viral diversity.