Transcript assembly from RNA-seq reads is a critical step in gene expression and subsequent functional analyses. Here we present PsiCLASS, an accurate and efficient transcript assembler based on an approach that simultaneously analyzes multiple RNA-seq samples. PsiCLASS combines mixture statistical models for exonic feature selection across multiple samples with splice graph based dynamic programming algorithms and a weighted voting scheme for transcript selection. PsiCLASS achieves significantly better sensitivity-precision tradeoff, and renders precision up to 2-3 fold higher than the StringTie system and Scallop plus TACO, the two best current approaches. PsiCLASS is efficient and scalable, assembling 667 GEUVADIS samples in 9 h, and has robust accuracy with large numbers of samples.

Nasal gene expression profiling is a new approach to investigate the airway epithelium as a biomarker to study the activity and treatment responses of obstructive pulmonary diseases. We investigated to what extent gene expression profiling of nasal brushings is similar to that of bronchial brushings. We performed genome wide gene expression profiling on matched nasal and bronchial epithelial brushes from 77 respiratory healthy individuals. To investigate differences and similarities among regulatory modules, network analysis was performed on correlated, differentially expressed and smoking-related genes using Gaussian Graphical Models. Between nasal and bronchial brushes, 619 genes were correlated and 1692 genes were differentially expressed (false discovery rate <0.05, |Fold-change|>2). Network analysis of correlated genes showed pro-inflammatory pathways to be similar between the two locations. Focusing on smoking-related genes, cytochrome-P450 pathway related genes were found to be similar, supporting the concept of a detoxifying response to tobacco exposure throughout the airways. In contrast, cilia-related pathways were decreased in nasal compared to bronchial brushes when focusing on differentially expressed genes. Collectively, while there are substantial differences in gene expression between nasal and bronchial brushes, we also found similarities, especially in the response to the external factors such as smoking.

Dissemination of tumour cells to the bone marrow is an early event in breast cancer, however cells may lie dormant for many years before bone metastases develop. Treatment for bone metastases is not curative, therefore new adjuvant therapies which prevent the colonisation of disseminated cells into metastatic lesions are required. There is evidence that cancer stem cells (CSCs) within breast tumours are capable of metastasis, but the mechanism by which these colonise bone is unknown. Here, we establish that bone marrow-derived IL1β stimulates breast cancer cell colonisation in the bone by inducing intracellular NFkB and CREB signalling in breast cancer cells, leading to autocrine Wnt signalling and CSC colony formation. Importantly, we show that inhibition of this pathway prevents both CSC colony formation in the bone environment, and bone metastasis. These findings establish that targeting IL1β-NFKB/CREB-Wnt signalling should be considered for adjuvant therapy to prevent breast cancer bone metastasis.

Chondroitin sulfate (CS) is the most abundant glycosaminoglycan (GAG) in articular cartilage and the loss of CS-GAG occurs early in OA. As a major component of perichondral matrix interacting directly with chondrocytes, the active turnover of CS can affect to break the homeostasis of chondrocytes. Here we employ CS-based 3-dimensional (3D) hydrogel scaffold system to investigate how the degradation products of CS affect the catabolic phenotype of chondrocytes. The breakdown of CS-based ECM by the chondroitinase ABC (ChABC) resulted in a hypertrophy-like morphologic change in chondrocytes, which was accompanied by catabolic phenotypes, including increased MMP-13 and ADAMTS5 expression, nitric oxide (NO) production and oxidative stress. The inhibition of Toll-like receptor 2 (TLR2) or TLR4 with OxPAPC (TLR2 and TLR4 dual inhibitor) and LPS-RS (TLR4-MD2 inhibitor) ameliorated these catabolic phenotypes of chondrocytes by CS-ECM degradation, suggesting a role of CS breakdown products as damage-associated molecular patterns (DAMPs). As downstream signals of TLRs, MAP kinases, NF-kB, NO and STAT3-related signals were responsible for the catabolic phenotypes of chondrocytes associated with ECM degradation. NO in turn reinforced the activation of MAP kinases as well as NFkB signaling pathway. Thus, these results propose that the breakdown product of CS-GAG can recapitulate the catabolic phenotypes of OA.

Effective drug delivery is restricted by pathophysiological barriers in solid tumors. In human pancreatic adenocarcinoma, poorly-permeable blood vessels limit the intratumoral permeation and penetration of chemo or nanotherapeutic drugs. New and clinically viable strategies are urgently sought to breach the neoplastic barriers that prevent effective drug delivery. Here, we present an original idea to boost drug delivery by selectively knocking down the tumor vascular barrier in a human pancreatic cancer model. Clinical radiation activates the tumor endothelial-targeted gold nanoparticles to induce a physical vascular damage due to the high photoelectric interactions. Active modulation of these tumor neovessels lead to distinct changes in tumor vascular permeability. Noninvasive MRI and fluorescence studies, using a short-circulating nanocarrier with MR-sensitive gadolinium and a long-circulating nanocarrier with fluorescence-sensitive nearinfrared dye, demonstrate more than two-fold increase in nanodrug delivery, post tumor vascular modulation. Functional changes in altered tumor blood vessels and its downstream parameters, particularly, changes in Ktrans (permeability), Kep (flux rate), and Ve (extracellular interstitial volume), reflect changes that relate to augmented drug delivery. The proposed dual-targeted therapy effectively invades the tumor vascular barrier and improve nanodrug delivery in a human pancreatic tumor model and it may also be applied to other nonresectable, intransigent tumors that barely respond to standard drug therapies.

Medial ganglionic eminence (MGE)-derived somatostatin (SST)+ and parvalbumin (PV)+ cortical interneurons (CINs), have characteristic molecular, anatomical and physiological properties. However, mechanisms regulating their diversity remain poorly understood. Here, we show that conditional loss of the Tuberous Sclerosis Complex (TSC) gene, Tsc1, which inhibits the mammalian target of rapamycin (MTOR), causes a subset of SST+ CINs, to express PV and adopt fast-spiking (FS) properties, characteristic of PV+ CINs. Milder intermediate phenotypes also occur when only one allele of Tsc1 is deleted. Notably, treatment of adult mice with rapamycin, which inhibits MTOR, reverses the phenotypes. These data reveal novel functions of MTOR signaling in regulating PV expression and FS properties, which may contribute to TSC neuropsychiatric symptoms. Moreover, they suggest that CINs can exhibit properties intermediate between those classically associated with PV+ or SST+ CINs, which may be dynamically regulated by the MTOR signaling.

Cerebral cavernous malformations (CCMs) is a microvascular disorder in the central nervous system. Despite tremendous efforts, the causal genetic mutation in some CCM patients has not be identified, raising the possibility of an unknown CCM locus. The CCM2/MGC4607 gene has been identified as one of three known genes causing CCMs. In this report, we defined a total of 29 novel exons and 4 novel promoters in CCM2 genomic structure and subsequently identified a total of 50 new alternative spliced isoforms of CCM2 which eventually generated 22 novel protein isoforms. Genetic analysis of CCM2 isoforms revealed that the CCM2 isoforms can be classified into two groups based on their alternative promoters and alternative start codon exons. Our data demonstrated that CCM2 isoforms not only are specific in their subcellular compartmentation but also have distinct cellular expression patterns among various tissues and cells, indicating the pleiotropic cellular roles of CCM2 through their multiple isoforms. In fact, the complexity of the CCM2 genomic structure was reflected by the multiple layers of regulation of CCM2 expression patterns. At the transcriptional level, it is accomplished by alternative promoters, alternative splicing, and multiple transcriptional start sites and termination sites; while at the translational level, it is carried out with various cellular functions with a distinguishable CCM2 protein group pattern, specified abundance and composition of selective isoforms in a cell and tissue specific fashion. Through experimentation, we discovered a unique phosphotyrosine binding (PTB) domain, namely atypical phosphotyrosine binding (aPTB) domain. Some long CCM2 isoform proteins contain both classes of PTB domains, making them a dual PTB domain-containing protein. Both CCM1 and CCM3 can bind competitively to this aPTB domain, indicating CCM2 as the cornerstone for CCM signaling complex (CSC).

The association between several Single Nucleotide Polymorphisms (SNPs) within the transcription factor 7-like 2 (TCF7L2) gene and Type 2 Diabetes (T2D) as well as additional T2D-related traits is well established. Since alteration in total and regional brain volumes are consistent findings among T2D individuals, we studied the association of four T2D susceptibility SNPS within TCF7L2 (rs7901695, rs7903146, rs11196205, and rs12255372) with volumes of white matter hyperintensities (WMH), gray matter, and regional volumes of amygdala and hippocampus obtained from structural MRI among 191 T2D elderly Jewish individuals. Under recessive genetic model (controlling for age, sex and intracranial volume), we found that for all four SNPs, carriers of two copies of the T2D risk allele (homozygous genotype) had significantly smaller amygdalar volume: rs7901695- CC genotype vs. CT + TT genotypes, p = 0.002; rs7903146-TT vs. TC + CC, p = 0.003; rs11196205- CC vs. CG + GG, p = 0.0003; and rs12255372- TT vs. TG + GG, p = 0.003. Adjusting also for T2D-related covariates, body mass index (BMI), and ancestry did not change the results substantively (rs7901695, p = 0.003; rs7903146, p = 0.005; rs11196205, p = 0.001; and rs12255372, p = 0.005). Conditional analysis demonstrated that only rs11196205 was independently associated with amygdalar volume at a significant level. Separate analysis of left and right amygdala revealed stronger results for left amygdalar volume. Taken together, we report association of TCF7L2 SNPs with amygdalar volume among T2D elderly Jewish patients. Further studies in other populations are required to support these findings and reach more definitive conclusions.

Tree swallows (Tachycineta bicolor) are one of the most commonly studied wild birds in North America. They have advanced numerous research areas, including life history, physiology, and organismal responses to global change; however, transcriptomic resources are scarce. To further advance the utility of this system for biologists across disciplines, we generated a transcriptome for the tree swallow using six tissues (brain, blood, ovary, spleen, liver, and muscle) collected from breeding females. We de novo assembled 207,739 transcripts, which we aligned to 14,717 high confidence protein-coding genes. We then characterized each tissue with regard to its unique genes and processes and applied this transcriptome to two fundamental questions in evolutionary biology and endocrinology. First, we analyzed 3,015 single-copy orthologs and identified 46 genes under positive selection in the tree swallow lineage, including those with putative links to adaptations in this species. Second, we analyzed tissue-specific expression patterns of genes involved in sex steroidogenesis and processing. Enzymes capable of synthesizing these behaviorally relevant hormones were largely limited to the ovary, whereas steroid binding genes were found in nearly all other tissues, highlighting the potential for local regulation of sex steroid-mediated traits. These analyses provide new insights into potential sources of phenotypic variation in a free-living female bird and advance our understanding of fundamental questions in evolutionary and organismal biology.

To evaluate changes in peripapillary microvascular parameters in diabetes mellitus (DM) patients using optical coherence tomography angiography (OCTA). Seventy-one diabetic patients (40 in the no diabetic retinopathy [DR] group and 31 in the non-proliferative DR [NPDR] group) and 50 control subjects. OCTA (Zeiss HD-OCT 5000 with AngioPlex) 6 × 6 mm scans centered on the optic disc were analyzed. Peripapillary vessel density (VD), perfusion density (PD) in superficial capillary plexus (SCP) were automatically calculated. The average macular ganglion cell-inner plexiform layer (mGC-IPL) and peripapillary retinal nerve fiber layer (pRNFL) thicknesses of the no DR and NPDR groups were significantly thinner than those of the control group. The no DR and NPDR groups showed lower peripapillary VD and PD in SCP compared with the control group. Using univariate regression analyses, the average mGC-IPL thickness, the pRNFL thickness, the no DR group and NPDR group were significant factors that affected the peripapillary VD and PD in SCP. Multivariate regression analyses showed that the grade of DR was a significant factor affecting the peripapillary VD and PD in SCP. OCTA revealed that peripapillary microvascular parameters in the no DR and NPDR groups were lower than those of normal controls. The peripapillary VD and PD in SCP were correlated with the mGC-IPL thickness, the pRNFL thickness, and the no DR and NPDR groups. Changes in peripapillary OCTA parameters may help with understanding the pathophysiology of DM and evaluating a potentially valuable biomarker for patients with subclinical DR.

Pseudomonas aeruginosa, an opportunistic pathogen of humans, uses quorum sensing (QS) to regulate the production of extracellular products that can benefit all members of the population. P. aeruginosa can police QS-deficient cheaters by producing hydrogen cyanide, which is also QS regulated; however, the mechanism by which cooperators selectively protect themselves from the toxicity of cyanide remained unresolved. Here, we show that a cyanide-insensitive terminal oxidase encoded by cioAB provides resistance to cyanide, but only in QS-proficient strains. QS-deficient cheaters do not activate cioAB transcription. QS-mediated regulation of cioAB expression depends on production of both cyanide by cooperators (which is QS regulated) and reactive oxygen species (ROS) from cheaters (which is not QS regulated). This type of regulatory system allows cooperating populations to respond, via ROS, to the presence of cheaters, and might allow them to defer the substantial metabolic cost of policing until cheaters are present in the population.

Short tandem repeats (STRs) have been implicated in a variety of complex traits in humans. However, genome-wide studies of the effects of STRs on gene expression thus far have had limited power to detect associations and provide insights into putative mechanisms. Here, we leverage whole-genome sequencing and expression data for 17 tissues from the Genotype-Tissue Expression Project to identify more than 28,000 STRs for which repeat number is associated with expression of nearby genes (eSTRs). We use fine-mapping to quantify the probability that each eSTR is causal and characterize the top 1,400 fine-mapped eSTRs. We identify hundreds of eSTRs linked with published genome-wide association study signals and implicate specific eSTRs in complex traits, including height, schizophrenia, inflammatory bowel disease and intelligence. Overall, our results support the hypothesis that eSTRs contribute to a range of human phenotypes, and our data should serve as a valuable resource for future studies of complex traits.

The early stages of type 1 diabetes (T1D) are characterized by local autoimmune inflammation and progressive loss of insulin-producing pancreatic β cells. Here we show that exposure to proinflammatory cytokines reveals a marked plasticity of the β-cell regulatory landscape. We expand the repertoire of human islet regulatory elements by mapping stimulus-responsive enhancers linked to changes in the β-cell transcriptome, proteome and three-dimensional chromatin structure. Our data indicate that the β-cell response to cytokines is mediated by the induction of new regulatory regions as well as the activation of primed regulatory elements prebound by islet-specific transcription factors. We find that T1D-associated loci are enriched with newly mapped cis-regulatory regions and identify T1D-associated variants disrupting cytokine-responsive enhancer activity in human β cells. Our study illustrates how β cells respond to a proinflammatory environment and implicate a role for stimulus response islet enhancers in T1D.

Here, we explored the structure of the 'Parenting Strategies Questionnaire', a new scale designed to measure parenting strategies for problem behaviour in ASD. We then examined links between child behaviour and parenting in a sample of 222 predominantly-UK parents of ASD children exhibiting behaviour found difficult or challenging. Analysis revealed three parenting subscales: Accommodation, Reinforcement Approaches and Reducing Uncertainty. Both Accommodation and Reducing Uncertainty were linked to child problem behaviour. Child factors explained up to 29% of the variance in Accommodation, with Socially Inflexible Non-compliance the strongest predictor, and up to 24% of the variance in Reducing Uncertainty, with Intolerance of Uncertainty the strongest predictor. Child factors were not related to Reinforcement Approaches. Longitudinal studies investigating these relationships are needed.

Pilocytic astrocytoma (PA) is the most common pediatric glioma, arising from a single driver MAPK pathway alteration. Classified as a grade I tumor according to the 2016 WHO classification, prognosis is excellent with a 10-year survival rate > 95% after surgery. However, rare cases present with anaplastic features, including an unexpected high mitotic/proliferative index, thus posing a diagnostic and therapeutic challenge. Based on small histomolecular series and case reports, such tumors arising at the time of diagnosis or recurrence have been designated by many names including pilocytic astrocytoma with anaplastic features (PAAF). Recent DNA methylation-profiling studies performed mainly on adult cases have revealed that PAAF exhibit a specific methylation signature, thus constituting a distinct methylation class from typical PA [methylation class anaplastic astrocytoma with piloid features-(MC-AAP)]. However, the diagnostic and prognostic significance of MC-AAP remains to be determined in children. We performed an integrative work on the largest pediatric cohort of PAAF, defined according to strict criteria: morphology compatible with the diagnosis of PA, with or without necrosis, ≥ 4 mitoses for 2.3 mm2, and MAPK pathway alteration. We subjected 31 tumors to clinical, imaging, morphological and molecular analyses, including DNA methylation profiling. We identified only one tumor belonging to the MC-AAP (3%), the others exhibiting a methylation profile typical for PA (77%), IDH-wild-type glioblastoma (7%), and diffuse leptomeningeal glioneuronal tumor (3%), while three cases (10%) did not match to a known DNA methylation class. No significant outcome differences were observed between PAAF with necrosis versus no necrosis (p = 0.07), or with 4-6 mitoses versus 7 or more mitoses (p = 0.857). Our findings argue that the diagnostic histomolecular criteria established for anaplasia in adult PA are not of diagnostic or prognostic value in a pediatric setting. Further extensive and comprehensive integrative studies are necessary to accurately define this exceptional entity in children.

Adeno-associated viruses (AAV) have become one of the most promising tools for gene transfer in clinics. Among all the serotypes, AAV9 has been described as the most efficient for cardiac transduction. In order to achieve optimal therapeutic delivery in heart disease, we have explored AAV9 transduction efficiency in an infarcted heart using different routes of administration and promoters, including a cardiac-specific one. AAV9 vectors carrying luciferase or green fluorescence protein under the control of the ubiquitous elongation-factor-1-alpha or the cardiac-specific troponin-T (TnT) promoters were administered by intramyocardial or intravenous injection, either in healthy or myocardial-infarcted mice. The transduction efficacy and specificity, the time-course expression, and the safety of each vector were tested. High transgene expression levels were found in the heart, but not in the liver, of mice receiving AAV-TnT, which was significantly higher after intramyocardial injection regardless of ischemia-induction. On the contrary, high hepatic transgene expression levels were detected with the elongation-factor-1-alpha-promoter, independently of the administration route and heart damage. Moreover, tissue-specific green fluorescence protein expression was found in cardiomyocytes with the TnT vector, whereas minimal cardiac expression was detected with the ubiquitous one. Interestingly, we found that myocardial infarction greatly increased the transcriptional activity of AAV genomes. Our findings show that the use of cardiac promoters allows for specific and stable cardiac gene expression, which is optimal and robust when intramyocardially injected. Furthermore, our data indicate that the pathological status of the tissue can alter the transcriptional activity of AAV genomes, an aspect that should be carefully evaluated for clinical applications.

DNA methylation patterns in the blood, genetic risk scores (GRSs), and environmental risk factors can potentially improve breast cancer (BC) risk prediction. We assessed the individual and joint predictive performance of methylation, GRS, and environmental risk factors for BC incidence in a prospective cohort study. In a cohort of 5462 women aged 50-75 from Germany, 101 BC cases were identified during 14 years of follow-up and were compared to 263 BC-free controls in a nested case-control design. Three previously suggested methylation risk scores (MRSs) based on methylation of 423, 248, and 131 cytosine-phosphate-guanine (CpG) loci, and a GRS based on the risk alleles from 269 recently identified single nucleotide polymorphisms were constructed. Additionally, multiple previously proposed environmental risk scores (ERSs) were built based on environmental variables. Areas under the receiver operating characteristic curves (AUCs) were estimated for evaluating BC risk prediction performance. MRS and ERS showed limited accuracy in predicting BC incidence, with AUCs ranging from 0.52 to 0.56 and from 0.52 to 0.59, respectively. The GRS predicted BC incidence with a higher accuracy (AUC = 0.61). Adjusted odds ratios per standard deviation increase (95% confidence interval) were 1.07 (0.84-1.36) and 1.40 (1.09-1.80) for the best performing MRS and ERS, respectively, and 1.48 (1.16-1.90) for the GRS. A full risk model combining the MRS, GRS, and ERS predicted BC incidence with the highest accuracy (AUC = 0.64) and might be useful for identifying high-risk populations for BC screening.

Free and bioavailable 25-hydroxyvitamin D (25OHD) are emerging measurements of vitamin D status. It remains unclear whether circulating free or bioavailable 25OHD are relevant to hepatocellular carcinoma (HCC) prognosis. Our aim was to test the hypothesis that bioavailable 25OHD may be a better serum biomarker of vitamin D status than total 25OHD on the association with HCC survival.

Anthropogenic organic inputs to freshwaters can exert detrimental effects on aquatic ecosystems, raising growing concern for both environmental conservation and water security. Current regulation by the EU water framework directive (European Union, 2000/60/EC) relates to organic pollution by monitoring selected micropollutants; however, aquatic ecosystem responses require a comprehensive understanding of dissolved organic matter (DOM) composition. The introduction of high-resolution mass spectrometry (HRMS) is set to greatly increase our understanding of the composition of DOM of both natural and anthropogenic origin derived from diffuse and point sources.

Genetic associations imply a role for CD8+ T cells and the interleukin-23 (IL-23)/IL-17 axis in psoriatic arthritis (PsA) and other spondyloarthritides (SpA). IL-17A+CD8+ (Tc17) T cells are enriched in the synovial fluid (SF) of patients with PsA, and IL-17A blockade is clinically efficacious in PsA/SpA. This study was undertaken to determine the immunophenotype, molecular profile, and function of synovial Tc17 cells in order to elucidate their role in PsA/SpA pathogenesis.