TRIAL REGISTRATION:ClinicalTrials.gov NCT03548818.

BACKGROUND AND AIMS:Maternal obesity increases the risk of the paediatric form of metabolic dysfunction-associated steatotic liver disease (MASLD), affecting up to 30% of youth, but the developmental origins remain poorly understood. METHODS:Using a Japanese macaque model, we investigated the impact of maternal Western-style diet (mWSD) or chow diet followed by postweaning WSD (pwWSD) or chow diet focusing on bile acid (BA) homeostasis and hepatic fibrosis in livers from third-trimester fetuses and 3-year-old juvenile offspring. RESULTS:Juveniles exposed to mWSD had increased hepatic collagen I/III content and stellate cell activation in portal regions. mWSD increased transcriptional signatures of FXR activation, while pwWSD impaired FXR pathway genes and increased liver BA content. Both mWSD and pwWSD increased serum BA concentrations. Notably, mWSD-exposed juvenile offspring had increased periportal CK19 expression and cholangiocyte gene expression supporting proliferation compared with maternal chow-exposed offspring. Fetuses exposed to mWSD had increased CK19 expression and hepatic BAs which correlated positively with periportal collagen deposition and negatively with markers of fetal oxygenation. In juvenile offspring, increased serum BAs correlated positively with hepatic oxidative stress and portal fibrosis without elevated liver enzymes. CONCLUSIONS:mWSD is associated with hallmarks of paediatric MASLD including portal bile ductular reaction, portal fibrosis and dysregulated BA homeostasis. These conditions begin in utero and persist in juvenile offspring regardless of their postweaning diet. These findings implicate changes in BA metabolism that may drive developmental programming of MASLD in juvenile offspring beginning in utero.

Developing biomarkers to quantitatively monitor disease-specific T cell activity is crucial for assessing type 1 diabetes (T1D) progression and evaluating immunotherapies. This study presents an approach using V-gene targeted sequencing to quantify T cell receptor (TCR) clonotypes as biomarkers for pathogenic T cells in T1D. We identified "public" TCR clonotypes shared among multiple non-obese diabetic (NOD) mice and human organ donors, with a subset expressed exclusively by islet antigen-reactive T cells in those with T1D. Employing V-gene targeted sequencing of only TCRs containing TRAV16/16D allowed quantitative detection of the public islet antigen-reactive TCR clonotypes in peripheral blood of NOD mice. Frequencies of these public TCR clonotypes distinguished prediabetic NOD mice from those protected from diabetes. In human islets, public TCR clonotypes identical to preproinsulin-specific clones were exclusively found in T1D donors. This quantifiable TCR sequencing approach uncovered public, disease-specific clonotypes in T1D, providing biomarker candidates to monitor pathogenic T cell frequencies in blood for assessing disease activity and therapeutic response.

Background The Yellowfin tuna ( Thunnus albacares ) is a large tuna exploited by major fisheries in tropical and subtropical waters of all oceans except the Mediterranean Sea. Genomic studies of population structure, adaptive variation or of the genetic basis of phenotypic traits are needed to inform fisheries management but are currently limited by the lack of a reference genome for this species. Here we report a draft genome assembly and a linkage map for use in genomic studies of T. albacares . Methods and results Illumina and PacBio SMRT sequencing were used in combination to generate a hybrid assembly that comprises 743,073,847 base pairs contained in 2,661 scaffolds. The assembly has a N50 of 351,587 and complete and partial BUSCO scores of 86.47% and 3.63%, respectively. Double-digest restriction associated DNA (ddRAD) was used to genotype the 2 parents and 164 of their F1 offspring resulting from a controlled breeding cross, retaining 19,469 biallelic single nucleotide polymorphism (SNP) loci. The SNP loci were used to construct a linkage map that features 24 linkage groups that represent the 24 chromosomes of yellowfin tuna. The male and female maps span 1,243.8 cM and 1,222.9 cM, respectively. The map was used to anchor the assembly in 24 super-scaffolds that contain 79% of the yellowfin tuna genome. Gene prediction identified 46,992 putative genes 20,203 of which could be annotated via gene ontology. Conclusions The draft reference will be valuable to interpret studies of genome wide variation in T. albacares and other Scombroid species.

Aims In the Treatment Options for Type 2 Diabetes in Adolescents and Youth (TODAY) study, an intervention trial followed by an observational phase, half the participants reached the primary outcome [hemoglobin A1c (HbA1c) ≥ 8% for at least 6 months] within 4 years, which was associated with a decrease in C-peptide oral disposition index (oDI). We aimed to identify circulating microRNA (miRNA) species associated with a decline in beta cell function. Methods Following a preliminary survey of select participants using nCounter Human v3 miRNA Panel (NanoString Technologies), polymerase chain reaction analyses were carried out for 17 miRNAs from 365 participants from samples at baseline, 24, 60, 96, and 120 months. Results Using a backward selection approach, 4 baseline miRNA log2 fold-changes independently predicted treatment failure; however, baseline HbA1c was higher in those with treatment failure. Three baseline miRNA log2 fold-changes remained significant predictors of this C-peptide oDI decline ≥20% (P < .05). Increased levels of miRNA-155 [odds ratio (OR): 1.2, 95% confidence interval (CI): 1.1-1.4] and miRNA-130b (OR:1.3, 95% CI: 1.0-1.7) were associated with oDI decline, while decreased levels of miRNA-126 (OR: 0.6, 95% CI: .4-.8) were associated with oDI decline. miRNA-122 was negatively correlated with C-peptide oDI at baseline and 24 months (R = 0.22, P < .01 and R = 0.19, P < .01, respectively) and positively correlated with proinsulin at baseline, 24, and 60 months (R = 0.26, P < 0.01, R = 0.26, P < .01, R = 0.18, P < .01, respectively). Conclusion The miRNA species associated with beta cell function are associated with alterations in cellular metabolism and apoptosis, suggesting that differences in baseline abundance may serve as circulating markers of beta cell dysfunction and provide potential mechanistic insights into the aggressive nature of youth-onset type 2 diabetes.

To implant in the uterus, mammalian embryos form blastocysts comprising trophectoderm (TE) surrounding an inner cell mass (ICM), confined to the polar region by the expanding blastocoel. The mode of implantation varies between species. Murine embryos maintain a single layered TE until they implant in the characteristic thick deciduum, whereas human blastocysts attach via polar TE directly to the uterine wall. Using immunofluorescence (IF) of rapidly isolated ICMs, blockade of RNA and protein synthesis in whole embryos, or 3D visualization of immunostained embryos, we provide evidence of multi-layering in human polar TE before implantation. This may be required for rapid uterine invasion to secure the developing human embryo and initiate formation of the placenta. Using sequential fluorescent labeling, we demonstrate that the majority of inner TE in human blastocysts arises from existing outer cells, with no evidence of conversion from the ICM in the context of the intact embryo.

Encoded by the NBPF gene family, Olduvai (formerly DUF1220) protein domains have undergone the largest human lineage-specific copy number expansion of any coding region in the genome. Olduvai copy number shows a linear relationship with several brain size-related measures and cortical neuron number among primates and with normal and disease-associated (micro- and macrocephaly) variation in brain size in human populations. While Olduvai domains have been shown to promote proliferation of neural stem cells, the mechanism underlying such effects has remained unclear. Here, we investigate the function of Olduvai by transcriptome and proteome analyses of cells overexpressing NBPF1, a gene encoding 7 Olduvai domains. Our results from both RNAseq and mass spectrometry approaches suggest a potential downregulation of mitochondria. In our proteomics study, a Gene Ontology (GO) enrichment analysis for the downregulated proteins revealed a striking overrepresentation of the biological process related to the mitochondrial electron transport chain (p value: 1.81e-11) and identified deregulation of the NADH dehydrogenase activity (p value: 2.43e-11) as the primary molecular function. We verify the reduction of apparent mitochondria via live-cell imaging experiments. Given these and previous Olduvai findings, we suggest that the Olduvai-mediated, dosage-dependent reduction in available energy via mitochondrial downregulation may have resulted in a developmental slowdown such that the neurogenic window among primates, and most extremely in humans, was expanded over a greater time interval, allowing for production of greater numbers of neurons and a larger brain. We further suggest that such a slowdown may extend to other developmental processes that also exhibit neotenic features.

Diffuse midline gliomas (DMGs) are devastating brain tumors that occur primarily in children. The salient feature of these tumors is the presence of a H3K27M mutation (K27M), associated with the worst prognosis. We identified the cell surface antigen CD99 as notably expressed in DMGs, particularly in K27M+DMGs. We found that the increased expression of CD99 in K27M+DMGs was a result of the onco-histone K27M mutation. In K27M+DMG cells, CD99 inactivation impaired tumor growth by inducing cell differentiation, indicating an oncogenic role of CD99 enabled by blocking differentiation. We then developed a novel therapeutic anti-CD99 chimeric antibody, 10D1, with a membrane-proximal binding epitope, and evaluated its antitumor efficacy in preclinical models of K27M+DMG. 10D1 suppressed DMG growth in vitro and in vivo by inducing apoptosis. When combined with radiation treatment, 10D1 exhibited improved antitumor efficiency and xenograft survival, providing a strong justification for its clinical development as a therapy for DMGs.Statement of Significance This study emphasizes that CD99 overexpression occurs due to the H3K27M mutation in Diffuse Midline Gliomas (DMGs). This heightened expression suppresses apoptosis, inhibits differentiation, and induces radio-resistance in DMGs. This research justifies using a novel CD99 antibody alone or combined with radiation therapy in human pediatric clinical trials.### Competing Interest StatementIB, KM, DW, JM, ND, SLC, JD, BB, ZN, KJ, ND, AG, AP, and AD report no affiliations with or involvement in any organization or entity with any financial interest in the subject matter or materials discussed in this manuscript. SV and RV are patent holders for 10D1 and co-founders of Vinasa Oncology.

Upstream regulators predicted in Ingenuity Pathway Analysis. All significant p<0.01) patient sample and cell line upstream regulators predicted to have changed after exposure to BRAFi, organized by tab. Analysis was conducted per-patient, comparing pre- and post-BRAFi RNA sequencing.

Purpose:Nitric oxide (NO) is recognized as an important biological mediator that controls several physiological functions, and evidence is now emerging that this molecule may play a significant role in the postnatal control of ocular growth and myopia development. We therefore sought to understand the role that nitric oxide plays in visually-guided ocular growth in order to gain insight into the underlying mechanisms of this process. Methods:Choroids were incubated in organ culture in the presence of the NO donor, PAPA-NONOate (1.5 mM). Following RNA extraction, bulk RNA-seq was used to quantify and compare choroidal gene expression in the presence and absence of PAPA-NONOate. We used bioinformatics to identify enriched canonical pathways, predicted diseases and functions, and regulatory effects of NO in the choroid. Results:Upon treatment of normal chick choroids with the NO donor, PAPA-NONOate, we identified a total of 837 differentially expressed genes (259 upregulated genes, 578 down-regulated genes) compared with untreated controls. Among these, the top five upregulated genes were LSMEM1, STEAP4, HSPB9, and CCL19, and the top five down-regulated genes were CDCA3, SMC2, a novel gene (ENSALGALG00000050836), an uncharacterized gene (LOC107054158), and SPAG5. Bioinformatics predicted that NO treatment will activate pathways involved in cell and organismal death, necrosis, and cardiovascular system development, and inhibit pathways involved in cell proliferation, cell movement, and gene expression. Conclusions:The findings reported herein may provide insight into possible effects of NO in the choroid during visually regulated eye growth, and help to identify targeted therapies for the treatment of myopia and other ocular diseases.

Introduction Fetal alcohol spectrum disorder (FASD), a life-long condition resulting from prenatal alcohol exposure (PAE), is associated with structural brain anomalies and neurobehavioral differences. Evidence from longitudinal neuroimaging suggest trajectories of white matter microstructure maturation are atypical in PAE. We aimed to further characterize longitudinal trajectories of developmental white matter microstructure change in children and adolescents with PAE compared to typically-developing Controls using diffusion-weighted Neurite Orientation Dispersion and Density Imaging (NODDI). Materials and methods Participants: Youth with PAE ( n = 34) and typically-developing Controls ( n = 31) ages 8–17 years at enrollment. Participants underwent formal evaluation of growth and facial dysmorphology. Participants also completed two study visits (17 months apart on average), both of which involved cognitive testing and an MRI scan (data collected on a Siemens Prisma 3 T scanner). Age-related changes in the orientation dispersion index (ODI) and the neurite density index (NDI) were examined across five corpus callosum (CC) regions defined by tractography. Results While linear trajectories suggested similar overall microstructural integrity in PAE and Controls, analyses of symmetrized percent change (SPC) indicated group differences in the timing and magnitude of age-related increases in ODI (indexing the bending and fanning of axons) in the central region of the CC, with PAE participants demonstrating atypically steep increases in dispersion with age compared to Controls. Participants with PAE also demonstrated greater increases in ODI in the mid posterior CC (trend-level group difference). In addition, SPC in ODI and NDI was differentially correlated with executive function performance for PAE participants and Controls, suggesting an atypical relationship between white matter microstructure maturation and cognitive function in PAE. Discussion Preliminary findings suggest subtle atypicality in the timing and magnitude of age-related white matter microstructure maturation in PAE compared to typically-developing Controls. These findings add to the existing literature on neurodevelopmental trajectories in PAE and suggest that advanced biophysical diffusion modeling (NODDI) may be sensitive to biologically-meaningful microstructural changes in the CC that are disrupted by PAE. Findings of atypical brain maturation-behavior relationships in PAE highlight the need for further study. Further longitudinal research aimed at characterizing white matter neurodevelopmental trajectories in PAE will be important.

We previously demonstrated gradual loss of epiblast during diapause in embryos lacking components of the LIF/IL6 receptor. Here we explore requirement for the downstream signalling transducer and activator of transcription, STAT3 and its target, TFCP2L1, in maintenance of naïve pluripotency. Unlike conventional markers, such as NANOG, which remains high in epiblast until implantation, both STAT3 and TFCP2L1 proteins decline during blastocyst expansion, but intensify in the embryonic region after induction of diapause, as observed visually and confirmed using our novel image analysis tool, consistent with our previous transcriptional expression data. Embryos lacking STAT3 or TFCP2L1, underwent catastrophic loss of most of the inner cell mass during the first few days of diapause, implicating involvement of signals in addition to LIF/IL6 for sustaining naïve pluripotency in vivo. By blocking MEK/ERK signalling from the morula stage we could derive embryonic stem cells with high efficiency from STAT3 null embryos, but not those lacking TFCP2L1, suggesting a hitherto unknown additional role for this essential STAT3 target in transition from embryo to embryonic stem cells in vitro. Summary Statement Inducing diapause in mouse embryos demonstrates that STAT3 and TFCP2L1 are essential for self-renewal of the epiblast, but only TFCP2L1 is required for derivation of embryonic stem cells.

Maternal overnutrition increases inflammatory and metabolic disease risk in postnatal offspring. This consti-tutes a major public health concern due to increasing prevalence of these diseases, yet mechanisms remain unclear. Here, using nonhuman primate models, we show that maternal Western-style diet (mWSD) exposure is associated with persistent pro-inflammatory phenotypes at the transcriptional, metabolic, and functional levels in bone marrow-derived macrophages (BMDMs) from 3-year-old juvenile offspring and in hematopoi-etic stem and progenitor cells (HSPCs) from fetal and juvenile bone marrow and fetal liver. mWSD exposure is also associated with increased oleic acid in fetal and juvenile bone marrow and fetal liver. Assay for transpo-sase-accessible chromatin with sequencing (ATAC-seq) profiling of HSPCs and BMDMs from mWSD-exposed juveniles supports a model in which HSPCs transmit pro-inflammatory memory to myeloid cells beginning in utero. These findings show that maternal diet alters long-term immune cell developmental programming in HSPCs with proposed consequences for chronic diseases featuring altered immune/inflam-matory activation across the lifespan.

Blackfin tuna (Thunnus atlanticus) is a small tuna distributed in the western Atlantic Ocean where it is exploited by growing recreational and commercial regional fisheries. In this work, genome-wide genetic variation was analysed to investigate the occurrence of stock subdivision. A de novo assembly of the blackfin tuna genome was generated using Illumina paired-end sequencing data and applied as a reference for population genomic analysis of specimens from nine localities (average sample size per locality n = 72) spanning most of the blackfin tuna distribution range. A total of 2139 single-nucleotide polymorphisms were discovered and genotyped using the double-digest restriction associated DNA sequencing. Pairwise exact homogeneity tests were significant in 24 out of 36 population pairs and significant spatial autocorrelation of genotypes was observed for specimens collected within 2250 km of each other. However, divergence among locality samples was very low (pairwise F-ST range 0.0002-0.0025) and significant temporal variations were detected in localities sampled multiple times. Approaches to detect cryptic groups de novo were unsuccessful. Additional sampling is warranted to determine if multiple stocks need to be defined for management and assess temporal and spatial patterns of gene flow connecting them.

BACKGROUND:Neurocognitive deficits are common in pediatric brain tumor survivors. The use of single nucleotide polymorphism (SNP) analysis in DNA repair genes may identify children treated with radiation therapy for brain tumors at increased risk for treatment toxicity and adverse neurocognitive outcomes. MATERIALS:The Human 660W-Quad v1.0 DNA BeadChip analysis (Illumina) was used to evaluate 1048 SNPs from 59 DNA repair genes in 46 subjects. IQ testing was measured by the Wechsler Intelligence Scale for Children. Linear regression was used to identify the 10 SNPs with the strongest association with IQ scores while adjusting for radiation type. RESULTS:The low vs high IQ patient cohorts were well matched for time from first treatment to most recent IQ, first treatment age, sex, and treatments received. 5 SNPs on 3 different genes (CYP29, XRCC1, and BRCA1) and on 3 different chromosomes (10, 19, and 17) had the strongest association with most recent IQ score that was not modified by radiation type. Furthermore, 5 SNPs on 4 different genes (WRN, NR3C1, ERCC4, RAD51L1) on 4 different chromosomes (8, 5, 16, 14) had the strongest association with change in IQ independent of radiation type, first IQ, and years between IQ measures. CONCLUSIONS:SNPs offer the potential to predict adverse neurocognitive outcomes in pediatric brain tumor survivors. Our results require validation in a larger patient cohort. Improving the ability to identify children at risk of treatment related neurocognitive deficits could allow for better treatment stratification and early cognitive interventions.

ALK and ROS1 patient specific data

Supplementary Table 2. Significant genes with Q value < 0.01 in the MDA-MB-468 cell line.

Read depths for ALK and ROS1 used in NGS

ABSTRACT:Background: Severe injury can provoke systemic processes that lead to organ dysfunction, and hemolysis of both native and transfused red blood cells (RBCs) may contribute. Hemolysis can release erythrocyte proteins, such as hemoglobin and arginase-1, the latter with the potential to disrupt arginine metabolism and limit physiologic NO production. We aimed to quantify hemolysis and arginine metabolism in trauma patients and measure association with injury severity, transfusions, and outcomes. Methods: Blood was collected from injured patients at a level I trauma center enrolled in the COMBAT (Control of Major Bleeding After Trauma) trial. Proteomics and metabolomics were performed on plasma fractions through liquid chromatography coupled with mass spectrometry. Abundances of erythrocyte proteins comprising a hemolytic profile as well as haptoglobin, l -arginine, ornithine, and l -citrulline (NO surrogate marker) were analyzed at different timepoints and correlated with transfusions and adverse outcomes. Results: More critically injured patients, nonsurvivors, and those with longer ventilator requirement had higher levels of hemolysis markers with reduced l -arginine and l -citrulline. In logistic regression, elevated hemolysis markers, reduced l -arginine, and reduced l -citrulline were significantly associated with these adverse outcomes. An increased number of blood transfusions were significantly associated with elevated hemolysis markers and reduced l -arginine and l -citrulline independently of New Injury Severity Score and arterial base excess. Conclusions: Severe injury induces intravascular hemolysis, which may mediate postinjury organ dysfunction. In addition to native RBCs, transfused RBCs can lyse and may exacerbate trauma-induced hemolysis. Arginase-1 released from RBCs may contribute to the depletion of l -arginine and the subsequent reduction in the NO necessary to maintain organ perfusion.

Livestock workers experience an increased burden of bioaerosol-induced respiratory disease including a high prevalence of rhinosinusitis. Dairy operations generate bioaerosols spanning the inhalable size fraction (0-100 mu m) containing bacterial constituents such as endotoxin. Particles with an aerodynamic diameter between 10 and 100 mu m are known to deposit in the nasopharyngeal region and likely affect the upper respiratory tract. We evaluated the effectiveness of a hypertonic saline nasal lavage in reducing inflammatory responses in dairy workers from a high-volume dairy operation. Inhalable personal breathing zone samples and pre-/post-shift nasal lavage samples from each participant over five consecutive days were collected. The treatment group (n = 5) received hypertonic saline while the control group (n = 5) received normotonic saline. Personal breathing zone samples were analyzed for particulate concentrations and endotoxin using gravimetric and enzymatic methods, respectively. Pro- and anti-inflammatory cytokines (i.e., IL-8, IL-10, and TNF-alpha) were measured from nasal lavage samples using a multiplex assay. Inhalable dust concentrations ranged from 0.15 to 1.9 mg/m(3). Concentrations of both pro- and anti-inflammatory cytokines, specifically IL-6, IL-8, and IL-10, were significantly higher in the treatment group compared to the control group (p < 0.02, p < 0.04, and p < 0.01, respectively). Further analysis of IL-10 anti-inflammatory indicates a positive association between hypertonic saline administration and IL-10 production. This pilot study demonstrates that hypertonic saline nasal lavages were successful in upregulating anti-inflammatory cytokines to support larger interventional studies.