The superior soft-tissue contrast of 4D-T2w MRI motivates its use for delineation in radiotherapy treatment planning. We address current limitations of slice-selective implementations, including thick slices and artefacts originating from data incompleteness and variable breathing.

Everyday speech perception is challenged by external acoustic interferences that hinder verbal communication. Here, we directly compared how different levels of the auditory system (brainstem vs. cortex) code speech and how their neural representations are affected by two acoustic stressors: noise and reverberation. We recorded multichannel (64 ch) brainstem frequency-following responses (FFRs) and cortical event-related potentials (ERPs) simultaneously in normal hearing individuals to speech sounds presented in mild and moderate levels of noise and reverb. We matched signal-to-noise and direct-to-reverberant ratios to equate the severity between classes of interference. Electrode recordings were parsed into source waveforms to assess the relative contribution of region-specific brain areas [i.e., brainstem (BS), primary auditory cortex (A1), inferior frontal gyrus (IFG)]. Results showed that reverberation was less detrimental to (and in some cases facilitated) the neural encoding of speech compared to additive noise. Inter-regional correlations revealed associations between BS and A1 responses, suggesting subcortical speech representations influence higher auditory-cortical areas. Functional connectivity analyses further showed that directed signaling toward A1 in both feedforward cortico-collicular (BS→A1) and feedback cortico-cortical (IFG→A1) pathways were strong predictors of degraded speech perception and differentiated "good" vs. "poor" perceivers. Our findings demonstrate a functional interplay within the brain's speech network that depends on the form and severity of acoustic interference. We infer that in addition to the quality of neural representations within individual brain regions, listeners' success at the "cocktail party" is modulated based on how information is transferred among subcortical and cortical hubs of the auditory-linguistic network.

Unrestricted by time and place, electronic health (eHealth) provides solutions for patient empowerment and value-based health care. Women in the reproductive age are particularly frequent users of internet, social media, and smartphone apps. Therefore, the pregnant patient seems to be a prime candidate for eHealth-supported health care with telemedicine for fetal and maternal conditions.

Duchenne muscular dystrophy (DMD) is an X-linked muscle-wasting disorder caused by mutations in the dystrophin gene, with an incidence of 1 in 3500 in new male births. Mdx mice are widely used as an animal model for DMD. However, these mice do not faithfully recapitulate DMD patients in many aspects, rendering the preclinical findings in this model questionable. Although larger animal models of DMD, such as dogs and pigs, have been generated, usage of these animals is expensive and only limited to several facilities in the world. Here, we report the generation of a rabbit model of DMD by co-injection of Cas9 mRNA and sgRNA targeting exon 51 into rabbit zygotes. The DMD knockout (KO) rabbits exhibit the typical phenotypes of DMD, including severely impaired physical activity, elevated serum creatine kinase levels, and progressive muscle necrosis and fibrosis. Moreover, clear pathology was also observed in the diaphragm and heart at 5 months of age, similar to DMD patients. Echocardiography recording showed that the DMD KO rabbits had chamber dilation with decreased ejection fraction and fraction shortening. In conclusion, this novel rabbit DMD model generated with the CRISPR/Cas9 system mimics the histopathological and functional defects in DMD patients, and could be valuable for preclinical studies.This article has an associated First Person interview with the first author of the paper.

Members of the miR-200 family are critical gatekeepers of the epithelial state, restraining expression of pro-mesenchymal genes that drive epithelial-mesenchymal transition (EMT) and contribute to metastatic cancer progression. Here, we show that miR-200c and another epithelial-enriched miRNA, miR-375, exert widespread control of alternative splicing in cancer cells by suppressing the RNA-binding protein Quaking (QKI). During EMT, QKI-5 directly binds to and regulates hundreds of alternative splicing targets and exerts pleiotropic effects, such as increasing cell migration and invasion and restraining tumour growth, without appreciably affecting mRNA levels. QKI-5 is both necessary and sufficient to direct EMT-associated alternative splicing changes, and this splicing signature is broadly conserved across many epithelial-derived cancer types. Importantly, several actin cytoskeleton-associated genes are directly targeted by both QKI and miR-200c, revealing coordinated control of alternative splicing and mRNA abundance during EMT These findings demonstrate the existence of a miR-200/miR-375/QKI axis that impacts cancer-associated epithelial cell plasticity through widespread control of alternative splicing.

MicroRNA regulation of key biological and developmental pathways is a rapidly expanding area of research, accompanied by vast amounts of experimental data. This data, however, is not widely available in bioinformatic resources, making it difficult for researchers to find and analyze microRNA-related experimental data and define further research projects. We are addressing this problem by providing two new bioinformatics data sets that contain experimentally verified functional information for mammalian microRNAs involved in cardiovascular-relevant, and other, processes. To date, our resource provides over 4400 Gene Ontology annotations associated with over 500 microRNAs from human, mouse, and rat and over 2400 experimentally validated microRNA:target interactions. We illustrate how this resource can be used to create microRNA-focused interaction networks with a biological context using the known biological role of microRNAs and the mRNAs they regulate, enabling discovery of associations between gene products, biological pathways and, ultimately, diseases. This data will be crucial in advancing the field of microRNA bioinformatics and will establish consistent data sets for reproducible functional analysis of microRNAs across all biological research areas.

An increasing body of literature suggests that both individual and collections of bacteria are associated with the progression of colorectal cancer. As the number of studies investigating these associations increases and the number of subjects in each study increases, a meta-analysis to identify the associations that are the most predictive of disease progression is warranted. We analyzed previously published 16S rRNA gene sequencing data collected from feces and colon tissue. We quantified the odds ratios (ORs) for individual bacterial taxa that were associated with an individual having tumors relative to a normal colon. Among the fecal samples, there were no taxa that had significant ORs associated with adenoma and there were 8 taxa with significant ORs associated with carcinoma. Similarly, among the tissue samples, there were no taxa that had a significant OR associated with adenoma and there were 3 taxa with significant ORs associated with carcinoma. Among the significant ORs, the association between individual taxa and tumor diagnosis was equal to or below 7.11. Because individual taxa had limited association with tumor diagnosis, we trained Random Forest classification models using only the taxa that had significant ORs, using the entire collection of taxa found in each study, and using operational taxonomic units defined based on a 97% similarity threshold. All training approaches yielded similar classification success as measured using the area under the curve. The ability to correctly classify individuals with adenomas was poor, and the ability to classify individuals with carcinomas was considerably better using sequences from feces or tissue.IMPORTANCE Colorectal cancer is a significant and growing health problem in which animal models and epidemiological data suggest that the colonic microbiota have a role in tumorigenesis. These observations indicate that the colonic microbiota is a reservoir of biomarkers that may improve our ability to detect colonic tumors using noninvasive approaches. This meta-analysis identifies and validates a set of 8 bacterial taxa that can be used within a Random Forest modeling framework to differentiate individuals as having normal colons or carcinomas. When models trained using one data set were tested on other data sets, the models performed well. These results lend support to the use of fecal biomarkers for the detection of tumors. Furthermore, these biomarkers are plausible candidates for further mechanistic studies into the role of the gut microbiota in tumorigenesis.

Biological nitrogen fixation is catalyzed by nitrogenase, a complex metalloenzyme found only in prokaryotes. N2 fixation is energetically highly expensive, and an energy-generating process such as photosynthesis can meet the energy demand of N2 fixation. However, synthesis and expression of nitrogenase are exquisitely sensitive to the presence of oxygen. Thus, engineering nitrogen fixation activity in photosynthetic organisms that produce oxygen is challenging. Cyanobacteria are oxygenic photosynthetic prokaryotes, and some of them also fix N2 Here, we demonstrate a feasible way to engineer nitrogenase activity in the nondiazotrophic cyanobacterium Synechocystis sp. PCC 6803 through the transfer of 35 nitrogen fixation (nif) genes from the diazotrophic cyanobacterium Cyanothece sp. ATCC 51142. In addition, we have identified the minimal nif cluster required for such activity in Synechocystis 6803. Moreover, nitrogenase activity was significantly improved by increasing the expression levels of nif genes. Importantly, the O2 tolerance of nitrogenase was enhanced by introduction of uptake hydrogenase genes, showing this to be a functional way to improve nitrogenase enzyme activity under micro-oxic conditions. To date, our efforts have resulted in engineered Synechocystis 6803 strains that, remarkably, have more than 30% of the N2 fixation activity of Cyanothece 51142, the highest such activity established in any nondiazotrophic oxygenic photosynthetic organism. This report establishes a baseline for the ultimate goal of engineering nitrogen fixation ability in crop plants.IMPORTANCE Application of chemically synthesized nitrogen fertilizers has revolutionized agriculture. However, the energetic costs of such production processes and the widespread application of fertilizers have raised serious environmental issues. A sustainable alternative is to endow to crop plants the ability to fix atmospheric N2in situ One long-term approach is to transfer all nif genes from a prokaryote to plant cells and to express nitrogenase in an energy-producing organelle, chloroplast, or mitochondrion. In this context, Synechocystis 6803, the nondiazotrophic cyanobacterium utilized in this study, provides a model chassis for rapid investigation of the necessary requirements to establish diazotrophy in an oxygenic phototroph.

The long noncoding RNA nuclear-enriched abundant transcript 1 (NEAT1) has been shown to regulate multiple cancer-related cellular activities including cell proliferation, apoptosis, and migration. In this study, we confirm that repression of NEAT1 induces DNA damage, disturbs the cell cycle, and arrests the proliferation of prostate cancer cells. By taking advantage of the prostate cancer tumor transcriptome profiles from The Cancer Genome Atlas, our data-mining pipeline identified a series of transcription factors (TF) whose regulatory activities on target genes depended on the level of NEAT1. Among them was putative TF CDC5L, which bound directly to NEAT1. Silencing NEAT1 in prostate cancer cells repressed the transcriptional activity of CDC5L, and RNA-seq and ChIP-seq analyses further revealed a handful of potential targets of CDC5L regulated by NEAT1 expression. One target of CDC5L, ARGN, mediated the strong phenotypic consequences of NEAT1 reduction, including DNA damage, cell-cycle dysregulation, and proliferation arrest. In summary, we have established the requirement of the CDC5L-AGRN circuit for the essential oncogenic role of NEAT1 in prostate cancer cells.Significance: An integrative methodology uncovers CDC5L-AGRN signaling as critical to the tumor-promoting function of long noncoding RNA NEAT1 in prostate cancer cells. Cancer Res; 78(15); 4138-49. ©2018 AACR.

Nucleotide oligomerization domain 2 (NOD2) is a major cytoplasmic sensor for pathogens and is critical for the clearance of cytosolic bacteria in mammals. However, studies regarding NOD2, especially the initiated signaling pathways, are scarce in teleost species. In this study, we identified a NOD2 molecule (PaNOD2) from ayu (Plecoglossus altivelis). Bioinformatics analysis showed the structure of NOD2 to be highly conserved during vertebrate evolution. Dual-luciferase reporter assays examined the activation of NF-κB signaling and Western blotting analysis detected the phosphorylation of three MAP kinases (p-38, Erk1/2, and JNK1/2). Functional study revealed that, like its mammalian counterparts, PaNOD2 was the receptor of the bacterial cell wall component muramyl dipeptide (MDP), and the leucine-rich repeat motif was responsible for the recognition and binding of PaNOD2 with the ligand. Overexpression of PaNOD2 activated the NF-κB signaling pathway, leading to the upregulation of inflammatory cytokines, including TNF-α and IL-1β in HEK293T cells and ayu head kidney-derived monocytes/macrophages (MO/MΦ). Particularly, we found that PaNOD2 activated the MAPK signaling pathways, as indicated by the increased phosphorylation of p-38, Erk1/2, and JNK1/2, which have not been characterized in any teleost species previously. Our findings proved that the NOD2 molecule and initiated pathways are conserved between mammals and ayu. Therefore, ayu could be used as an animal model to investigate NOD2-based diseases and therapeutic applications.

Many environmental, host, and microbial characteristics have been recognized as risk factors for dissemination of extrapulmonary tuberculosis (EPTB). However, there are few population-based studies investigating the association between the primary sites of tuberculosis (TB) infection and mortality during TB treatment. De-identified population-based surveillance data of confirmed TB patients reported from 2009 to 2015 in Texas, USA, were analyzed. Regression analyses were used to determine the risk factors for EPTB, as well as its subsite distribution and mortality. We analyzed 7007 patients with exclusively pulmonary TB, 1259 patients with exclusively EPTB, and 894 EPTB patients with reported concomitant pulmonary involvement. Age ≥45 years, female gender, human immunodeficiency virus (HIV)-positive status, and end-stage renal disease (ESRD) were associated with EPTB. ESRD was associated with the most clinical presentations of EPTB other than meningeal and genitourinary TB. Patients age ≥45 years had a disproportionately high rate of bone TB, while foreign-born patients had increased pleural TB and HIV+ patients had increased meningeal TB. Age ≥45 years, HIV+ status, excessive alcohol use within the past 12 months, ESRD, and abnormal chest radiographs were independent risk factors for EPTB mortality during TB treatment. The epidemiologic risk factors identified by multivariate analyses provide new information that may be useful to health professionals in managing patients with EPTB.

A better understanding of the seroprevalence and specificity of influenza HA stem-directed broadly neutralizing antibodies (bNAbs) in the human population could significantly inform influenza vaccine design efforts. Here, we utilized probes comprising headless, HA stabilized stem (SS) to determine the prevalence, binding and neutralization breadth of antibodies directed to HA stem-epitope in a cross-sectional analysis of the general population. Five group-1 HA SS probes, representing five subtypes, were chosen for this analyses. Eighty-four percent of samples analyzed had specific reactivity to at least one probe, with approximately 60% of the samples reactive to H1 probes, and up to 45% reactive to each of the non-circulating subtypes. Thirty percent of analyzed sera had cross-reactivity to at least four of five probes and this reactivity could be blocked by competing with F10 bNAb. Binding cross-reactivity in sera samples significantly correlated with frequency of H1+H5+ cross-reactive B cells. Interestingly, only 33% of the cross-reactive sera neutralized both H1N1 and H5N1 pseudoviruses. Cross-reactive and neutralizing antibodies were more prevalent in individuals >50 years of age. Our data demonstrate the need to use multiple HA-stem probes to assess for broadly reactive antibodies. Further, a universal vaccine could be designed to boost pre-existing B-cells expressing stem-directed bNAbs.

Forty-seven percent of falling accidents in older adults are caused by tripping over obstacles. Understanding what strategies are involved in obstacle negotiation in older adults could reduce fall risks. There is a paucity of research investigating how healthy adults negotiate multiple obstacles, which may better reflect the complexity of the real environment. The presence of a second obstacle has induced mixed results in the obstacle negotiation of healthy older adults with the interval between obstacles two steps apart. This study extended the knowledge to understand what strategies healthy younger and older adults used to step over two obstacles placed at three-step-length apart. Twenty healthy subjects performed 2 tasks: level ground walking and stepping over two obstacles. The height of each obstacle was set at 10% of subjects' leg height. We found that aging significantly increased the toe clearance in leading and trailing legs when stepping over the obstacles at a three-step-length interval. Toe clearance was higher while stepping over the second obstacle than the first one in older adults. These results had two-fold meanings: the three-step-length interval was long enough to trigger the adjustment of the obstacle negotiation strategy, and aging led older adults to use conservative negotiation strategies.

MCC950 a potent, highly specific small molecule inhibitor of canonical and noncanonical activation of NLRP3 inflammasome has been evaluated in a multitude of NLRP3 driven inflammatory diseases. However, the effect of MCC950 on colonic inflammation has not yet been reported. In the present study we investigated the effect of MCC950 in a spontaneous chronic colitis mouse model Winnie, which mimics human ulcerative colitis. Oral administration of 40 mg/kg MCC950 commencing at Winnie week seven for three weeks significantly improved body weight gain, colon length, colon weight to body weight ratio, disease activity index and histopathological scores. MCC950 significantly suppressed release of proinflammatory cytokines IL-1β, IL-18, IL1-α, IFNγ, TNF-α, IL6, IL17, chemokine MIP1a and Nitric Oxide in colonic explants. Moreover, MCC950 resulted in a significant decrease of IL-1β release and activation of caspase-1 in colonic explants and macrophage cells isolated from Winnie. Complete inhibition with MCC950 in Winnie colonic explants shows, for the first time, the contribution of inflammatory effects resulting exclusively from canonical and noncanonical NLRP3 inflammasome activation in colitis. Taken together, our results illustrate the efficacy of MCC950 in the treatment of murine ulcerative colitis and provides avenue for a potential novel therapeutic agent for human inflammatory bowel diseases.

Compensatory mutations have been suggested to promote multidrug-resistant tuberculosis (MDR-TB) transmission, but their role in facilitating the recent transmission of MDR-TB is unclear. To investigate the epidemiological significance of compensatory mutations, we analyzed a four-year population-based collection of MDR-TB strains from Shanghai (the most populous city in China) and 1346 published global MDR-TB strains. We report that MDR-TB strains with compensatory mutations in the rpoA, rpoB, or rpoC genes were neither more frequently clustered nor found in larger clusters than those without compensatory mutations. Our results suggest that compensatory mutations are not a major contributor to the current epidemic of MDR-TB.

We previously reported that serum N1-methylnicotinamide (me-NAM), an indicator of nicotinamide N-methyltransferase (NNMT) activity, was associated with obesity, diabetes, and coronary artery disease in Chinese patients. However, whether NNMT might play a role in the development of heart failure remains to be elucidated. In this study, the associations between levels of serum me-NAM and left ventricular structure and function were investigated in Chinese patients. Serum me-NAM was measured by liquid chromatography-mass spectrometry in 265 subjects. M-mode, 2-dimensional and Doppler echocardiography were performed with the GE Vivid E9 system to assess left ventricular structure and function. Of note, the participants in the top tertile of me-NAM had the lowest left ventricular ejection fraction (LVEF), preload recruitable stroke work (PRSW), and highest prevalence of left ventricular systolic dysfunction (LVSD). Serum me-NAM was negatively correlated with LVEF and PRSW before and after adjusted for potential confounding variables (P ≤ 0.02). In multiple logistic regression analyses, the subjects in the top tertile of me-NAM had highest risks for LVSD (OR 6.80; 95% CI 1.26-36.72; P = 0.026), which was also observed in continuous analyses (OR 9.48; 95% CI 1.41-63.48; P = 0.02). In conclusion, serum me-NAM is negatively associated with LVEF and PRSW and accordingly positively associated with the prevalence of LVSD in Chinese patients.

The Wnt/β-Catenin signaling pathway is one of the most frequently altered pathways in adrenocortical carcinomas (ACCs). The aim of this study was to investigate the status of Wnt/β-Catenin signaling pathway by analyzing the expression level of β-Catenin and the mutational status of APC, AXIN2, CTNNB1, and ZNRF3 in ACCs. Mutations in APC, CTNNB1, ZNRF3 and homozygous deletions in ZNRF3 were observed in 3.8% (2/52), 11.5% (6/52), 1.9% (1/52) and 17.3% (9/52) of the cohort respectively. Novel interstitial deletions in CTNNB1 spanning intron 1 to exon 3/intron 3 were also found in 7.7% (4/52) of the tumours. All the observed alterations were mutually exclusive. Nuclear accumulation of β-Catenin, increased expression of Cyclin D1 and significantly higher expression of AXIN2 (p = 0.0039), ZNRF3 (p = 0.0032) and LEF1(p = 0.0090) observed in the tumours harbouring the deletion in comparison to tumours without CTNNB1 mutation demonstrates that the truncated β-Catenin is functionally active and erroneously activates the downstream targets. Significantly lower overall survival rate in patients with tumours harbouring alterations in APC/CTNNB1/ZNRF3 in comparison to those without mutation was observed. In conclusion, the discovery of novel large deletions in addition to the point mutations in CTNNB1 infers that activation of Wnt/β-Catenin pathway via alterations in CTNNB1 occurs frequently in ACCs. We also confirm that alterations in Wnt/β-Catenin signaling pathway members have a negative effect on overall survival of patients.

At times, it can be difficult to discern if a lack of overlap in reported interactions for a protein-of-interest reflects differences in methodology or biology. In such instances, systematic analyses of protein-protein networks across diverse paradigms can provide valuable insights. Here, we interrogated the interactome of the prion protein (PrP), best known for its central role in prion diseases, in four mouse cell lines. Analyses made use of identical affinity capture and sample processing workflows. Negative controls were generated from PrP knockout lines of the respective cell models, and the relative levels of peptides were quantified using isobaric labels. The study uncovered 26 proteins that reside in proximity to PrP. All of these proteins are predicted to have access to the outer face of the plasma membrane, and approximately half of them were not reported to interact with PrP before. Strikingly, although several proteins exhibited profound co-enrichment with PrP in a given model, except for the neural cell adhesion molecule 1, no protein was highly enriched in all PrP-specific interactomes. However, Gene Ontology analyses revealed a shared association of the majority of PrP candidate interactors with cellular events at the intersection of transforming growth factor β and integrin signaling.

Hepatitis E is the most common type of acute hepatitis prevalent worldwide. The open reading frame 3 protein of HEV (HEV ORF3) is proposed to create a favorable environment for viral replication and pathogenesis. However, the mechanisms by which HEV overcomes the effects of host immunity, particularly the role of ORF3, remain to be established. Expression of IFNα and IFNβ in supernatant and cell samples was examined via ELISA and quantitative RT-PCR. The protein levels of specific signaling factors in cells overexpressing HEV ORF3 were examined via western blot. Analyses of cells transfected with vectors expressing ORF3 demonstrated that HEV ORF3 significantly impairs the generation of endogenous type I interferon through downregulating TLR3 and TLR7 as well as their corresponding downstream signaling pathways. Moreover, inhibition of NFκB, JAK/STAT and JNK/MAPK signaling pathways contributed significantly to suppression of increased levels of TLR7. Levels of p-P65, p-STAT1 and p-JNK were markedly impaired in ORF3-expressing cells, even upon treatment with the respective agonists. HEV ORF3 inhibits the production of endogenous type I interferon through downregulation of TLR3 and TLR7. Furthermore, suppression of TLR7 is achieved through impairment of multiple signaling pathways, including NFκB, JAK/STAT and JNK/MAPK.

Insights into the molecular pathogenesis of breast cancer might come from molecular analysis of tissue from early stages of the disease.