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The original CHESS database of human genes was assembled from nearly 10,000 RNA sequencing experiments in 53 human body sites produced by the Genotype-Tissue Expression (GTEx) project, and then augmented with genes from other databases to yield a comprehensive collection of protein-coding and noncoding transcripts. The construction of the new CHESS 3 database employed improved transcript assembly algorithms, a new machine learning classifier, and protein structure predictions to identify genes and transcripts likely to be functional and to eliminate those that appeared more likely to represent noise. The new catalog contains 41,356 genes on the GRCh38 reference human genome, of which 19,839 are protein-coding, and a total of 158,377 transcripts. These include 14,863 novel protein-coding transcripts. The total number of transcripts is substantially smaller than earlier versions due to improved transcriptome assembly methods and to a stricter protocol for filtering out noisy transcripts. Notably, CHESS 3 contains all of the transcripts in the MANE database, and at least one transcript corresponding to the vast majority of protein-coding genes in the RefSeq and GENCODE databases. CHESS 3 has also been mapped onto the complete CHM13 human genome, which gives a more-complete gene count of 43,773 genes and 19,968 protein-coding genes. The CHESS database is available athttp://ccb.jhu.edu/chess.

We explore a new hypothesis in genome annotation, namely whether computationally predicted protein structures can help to identify which of multiple possible gene isoforms represents a functional protein product. Guided by structure predictions, we evaluated over 140,000 isoforms of human protein-coding genes assembled from over 10,000 RNA sequencing experiments across many human tissues. We illustrate our new method with examples where structure provides a guide to function in combination with expression and evolutionary evidence. Additionally, we provide the complete set of structures as a resource to better understand the function of human genes and their isoforms. These results demonstrate the promise of protein structure prediction as a genome annotation tool, allowing us to refine even the most highly-curated catalog of human proteins.One-Sentence SummaryWe describe the use of 3D protein structures on a genome-wide scale to evaluate human protein isoforms for biological functionality.

A Síndrome de Williams-Beuren (SWB) é um distúrbio genético autossômico dominante caracterizado por dismorfologias faciais típicas e cardiopatias que acometem aproximadamente 1 em 10 mil pessoas; determinada pela deleção gênica contígua hemizigótica de aproximadamente 1,5 a 1,8 Mb no cromossomo 7q11-23, região que abrange 27 genes da elastina. A deficiência da elastina, responsável pela distensibilidade da aorta, acomete o sistema cardiovascular. Com isso, objetivou-se uma revisão literária para analisar a genética da estenose aórtica supravalvular (EAS) na SWB e sua intervenção cirúrgica, que é necessária em alguns casos. Foi realizada uma revisão literária com buscas no PubMed/MEDLINE e SciElo, utilizando os descritores (“williams syndrome” AND “chromosome 7”) pesquisados no MeSh e no DeCs. Como resultado, os efeitos típicos da SWB são EAS, que acomete cerca de 70% dos pacientes; estenose da artéria pulmonar e coarctação; e prolapso da válvula mitral. A haploinsuficiência e a deleção da elastina são responsáveis pela morbimortalidade mais significante. Outrossim, os principais acometimentos são explicados pelo crescimento circunferencial anormal das artérias. Dessarte, as intervenções cirúrgicas são indicadas nos casos de EAS por lesão progressiva, caracterizada pelo aumento no gradiente de pressão do ventrículo esquerdo devido à hipoplasia da aorta ascendente, podendo ocorrer risco de morte súbita. Reportou-se que 21 pacientes com EAS na SWB necessitaram de intervenção cirúrgica. A taxa de mortalidade variou entre 0-26% e sobrevida de 95-98% em 5 anos. Como conclusão, a EAS é a má formação mais encontrada na SWB e a cirurgia de reparo apresenta baixa taxa de mortalidade.

Metagenomic experiments expose the wide range of microscopic organisms in any microbial environment through high-throughput DNA sequencing. The computational analysis of the sequencing data is critical for the accurate and complete characterization of the microbial community. To facilitate efficient and reproducible metagenomic analysis, we introduce a step-by-step protocol for the Kraken suite, an end-to-end pipeline for the classification, quantification and visualization of metagenomic datasets. Our protocol describes the execution of the Kraken programs, via a sequence of easy-to-use scripts, in two scenarios: (1) quantification of the species in a given metagenomics sample; and (2) detection of a pathogenic agent from a clinical sample taken from a human patient. The protocol, which is executed within 1-2 h, is targeted to biologists and clinicians working in microbiome or metagenomics analysis who are familiar with the Unix command-line environment.

ObjectiveIntraductal papillary mucinous neoplasms (IPMNs) are non-invasive precursor lesions that can progress to invasive pancreatic cancer and are classified as low-grade or high-grade based on the morphology of the neoplastic epithelium. We aimed to compare genetic alterations in low-grade and high-grade regions of the same IPMN in order to identify molecular alterations underlying neoplastic progression.DesignWe performed multiregion whole exome sequencing on tissue samples from 17 IPMNs with both low-grade and high-grade dysplasia (76 IPMN regions, including 49 from low-grade dysplasia and 27 from high-grade dysplasia). We reconstructed the phylogeny for each case, and we assessed mutations in a novel driver gene in an independent cohort of 63 IPMN cyst fluid samples.ResultsOur multiregion whole exome sequencing identified KLF4, a previously unreported genetic driver of IPMN tumorigenesis, with hotspot mutations in one of two codons identified in >50% of the analyzed IPMNs. Mutations in KLF4 were significantly more prevalent in low-grade regions in our sequenced cases. Phylogenetic analyses of whole exome sequencing data demonstrated diverse patterns of IPMN initiation and progression. Hotspot mutations in KLF4 were also identified in an independent cohort of IPMN cyst fluid samples, again with a significantly higher prevalence in low-grade IPMNs.ConclusionHotspot mutations in KLF4 occur at high prevalence in IPMNs. Unique among pancreatic driver genes, KLF4 mutations are enriched in low-grade IPMNs. These data highlight distinct molecular features of low-grade and high-grade dysplasia and suggest diverse pathways to high-grade dysplasia via the IPMN pathway.

Epilepsy affects approximately 70 million people worldwide, and it is a significant contributor to the global burden of neurological disorders. Despite the advent of new AEDs, drug resistant-epilepsy continues to affect 30-40% of PWE. Once identified as having drug-resistant epilepsy, these patients should be referred to a comprehensive epilepsy center for evaluation to establish if they are candidates for potential curative surgeries. Unfortunately, a large proportion of patients with drug-resistant epilepsy are poor surgical candidates due to a seizure focus located in eloquent cortex, multifocal epilepsy or inability to identify the zone of ictal onset. An alternative treatment modality for these patients is neuromodulation. Here we present the evidence, indications and safety considerations for the neuromodulation therapies in vagal nerve stimulation (VNS), responsive neurostimulation (RNS), or deep brain stimulation (DBS).

Background: Metagenomic sequencing has the potential to identify a wide range of pathogens in human tissue samples. Sarcoidosis is a complex disorder whose etiology remains unknown and for which a variety of infectious causes have been hypothesized. We sought to conduct metagenomic sequencing on cases of ocular and periocular sarcoidosis, none of them with previously identified infectious causes. Methods: Archival tissue specimens of 16 subjects with biopsies of ocular and periocular tissues that were positive for non-caseating granulomas were used as cases. Four archival tissue specimens that did not demonstrate non-caseating granulomas were also included as controls. Genomic DNA was extracted from tissue sections. DNA libraries were generated from the extracted genomic DNA and the libraries underwent next-generation sequencing. Results: We generated between 4.8 and 20.7 million reads for each of the 16 cases plus four control samples. For eight of the cases, we identified microbial pathogens that were present well above the background, with one potential pathogen identified for seven of the cases and two possible pathogens for one of the cases. Five of the eight cases were associated with bacteria (Campylobacter concisus, Neisseria elongata, Streptococcus salivarius, Pseudopropionibacterium propionicum, and Paracoccus yeei), two cases with fungi (Exophiala oligosperma, Lomentospora prolificans and Aspergillus versicolor) and one case with a virus (Mupapillomavirus 1). Interestingly, four of the five bacterial species are also part of the human oral microbiome. Conclusions: Using a metagenomic sequencing we identified possible infectious causes in half of the ocular and periocular sarcoidosis cases analyzed. Our findings support the proposition that sarcoidosis could be an etiologically heterogenous disease. Because these are previously banked samples, direct follow-up in the respective patients is impossible, but these results suggest that sequencing may be a valuable tool in better understanding the etiopathogenesis of sarcoidosis and in diagnosing and treating this disease.

The worth of crayfish as a model system for studies of addiction was not previously recognized because a drug-reward phenomenon had not been documented in this model system. In our previous experiments, we demonstrate that the crayfish natural reward pathways are sensitive to human drugs of abuse. This finding supports crayfish as a suitable model to characterize specific behaviors that are relevant in drug addiction research, and the current study builds on our previous findings. The aim of the present study was to investigate unconditioned neurobehavioral effects of repeated treatment regimens using cocaine, morphine, and methamphetamine for three consecutive days. We analyzed mobility, immobility and characterized stereotypic behaviors following intracardial infusions of 2.0μg/g or 10.0μg/g doses of cocaine, morphine, and methamphetamine for three days. The results showed that systemic cocaine, morphine, and methamphetamine increased mobility at a low dose of 2.0μg/g more effectively than a high dose of 10.0μg/g, while simultaneously showing that the high dose exerted a more prominent effect in increasing immobility. Moreover, systemic cocaine, morphine, and methamphetamine injections have discerning effects towards a group of defined unconditioned stereotyped behavioral patterns associated with each drug, rather than a shared universal behavioral effect. These findings provide insight into the behavioral and pharmacological basis responsible for the unconditioned effects of these drugs in crayfish.

Objective. To study the safety and efficacy of a clopidogrel loading dose in patients with moderate and severe acute ischemic strokes.Background. The safety of clopidogrel loading has been extensively investigated in patients with minor strokes and transient ischemic attacks.Methods. Acute ischemic stroke patients presenting consecutively to our center from 07/01/08 to 07/31/13 were screened. Clopidogrel loading was defined as at least 300 mg dose (with or without aspirin) given within 6 hours of admission. We compared outcomes in patients with baseline NIHSS > 3 with and without clopidogrel loading.Results. Inclusion criteria were met for 1011 patients (43.6% females, 69.1% black, median age 63). Patients with clopidogrel loading had lower baseline NIHSS than patients who were not loaded (8 versus 9,p=0.005). The two groups had similar risk for hemorrhagic transformation (p=0.918) and symptomatic hemorrhage (p=0.599). Patients who were loaded had a lower rate of neurological worsening (38.9% versus 48.3%,p=0.031) and less in-hospital mortality (4.3% versus 13.4%,p=0.001) compared to those who were not loaded. The likelihood of having a poor functional outcome did not differ between the two groups after adjusting for NIHSS on admission (OR = 0.71, 95% CI 0.4633–1.0906,p=0.118).Conclusion. Clopidogrel loading dose was not associated with increased risk for hemorrhagic transformation or symptomatic intracranial hemorrhage in our retrospective study and was associated with reduced rates of neuroworsening following moderate and severe stroke.

Objective: To study the safety and efficacy of clopidogrel loading in patients with moderate and severe strokes. Background: The safety of clopidogrel loading has been extensively investigated in patients with minor strokes and transient ischemic attacks. Methods: AIS patients presenting consecutively to our center from 06/07/07-07/31/13 were screened. Clopidogrel loading was defined as at least 300 mg dose (with or without aspirin) given within 6 hours of admission. We compared outcomes in patients with baseline NIHSS >3 with and without clopidogrel loading. Results: Inclusion criteria were met for 1011 patients (43.6% females, 69.1% black, median age 63). Patients with clopidogrel loading had lower baseline NIHSS than patients not loaded (8 vs 9, p=0.005). The two groups had similar risk of hemorrhagic transformation (p=0.918) and symptomatic hemorrhage (p=0.599). Patients who were loaded had a lower rate of neuroworsening (defined as an increase in NIHSS score by 2 points within 24 hours, 38.9% vs 48.3%, p=0.031) and less in-hospital mortality (4.3% vs 13.4%, p=0.001) compared to those who were not loaded. The likelihood of having a poor functional outcome (defined as mRS > 2 on discharge) did not differ between the two groups after adjusting for NIHSS on admission (OR=0.71, 95% CI 0.4633-1.0906, p=0.118). Conclusion: Clopidogrel loading dose is safe and was associated with reduced rates of neuroworsening and mortality following AIS in patients with more than minor stroke. A prospective study of safety and efficacy at preventing stroke progression more than minor strokes is warranted.

Objective: To determine if an acute loading dose of clopidogrel is safe in acute ischemic stroke (AIS) patients with chronic intracerebral hemorrhage (ICH). Background: Clopidogrel loading is a promising therapy for AIS patients not eligible for tissue plasminogen activator (tPA) who are at risk for progressive stroke. Previous ICH is a risk factor for developing a new ICH. However, the acute risk of these events in this population after loading with clopidogrel has not been studied. Methods: We examined 1,011 AIS patients presenting to our center from 06/07/07-07/31/13. Only those loaded with at least 300mg of clopidogrel (with or without aspirin) within 6 hours of admission were analyzed. We compared new onset hemorrhagic complications in patients with and without chronic ICH, defined as areas of parenchymal hypodensity on gradient recall echo (GRE) sequencing on MRI. Repeat CT or MRI during admission was evaluated by a vascular neurologist for evidence of new ICH, hemorrhagic infarct using ECASS II criteria, or new ischemic infarct. Results: Of 365 AIS patients loaded with clopidogrel, 67 had chronic ICH on GRE. Patients with chronic ICH were more likely to be African American (80.0% vs. 65.9%, p=0.028) and male (69.2% vs. 50.9%, p=0.008). These patients were more likely to have existing comorbidities: history of stroke (67.7% vs 37.5%, p 2 on discharge, p=0.889), or unfavorable discharge disposition (defined as disposition other than home or inpatient rehabilitation, p=0.166). Conclusion: The presence of chronic ICH on GRE did not increase the risk of new ICH, hemorrhagic infarct, ischemic event, or neurologic deterioration after administration of an acute loading dose of clopidogrel for AIS.

In January 2013, Diabetes retracted the above-listed article at the authors’ request. The original retraction statement appears below: The authors have formally requested to retract the above-titled paper, which was published online on 19 November 2012. The authors cite concerns that portions of Fig. 4 were submitted without knowledge of inherent errors or abnormalities that they recognized in retrospect after submission. Therefore, the article has been retracted so the authors can readdress the work and submit it for publication at a later time. The corresponding author requests to update …

Diabetes mellitus types 1 and 2 (DM1 and DM2) and/or hypertension (HTN) can contribute to cognitive decline, cerebral atrophy and white matter abnormalities in humans. Adult rat models of streptozotocin-induced DM1 and genetic strains of DM2 and HTN were used to investigate relative contributions of DM and HTN for alterations in cerebral structure and function as well as insulin receptor biology using cognitive testing, magnetic resonance imaging (MRI), and histological and molecular methods. The effects of DM1 or DM2 were generally similar. DM was associated with earlier onset of cognitive impairment than with HTN alone. DM was independently correlated with brain atrophy, whereas HTN had minimal effects on brain volume. The combination of DM and HTN led to identifiable mild hippocampal neuronal loss while either DM or HTN led to synaptic loss. Only DM led to downregulation of the insulin receptor pathways' activation. In contrast, only HTN was associated with vascular luminal reduction and restricted cerebral perfusion on MRI. The impacts of DM and HTN in the brain differ, while their separate contributions can lead to some additive adverse effects within rodent brain grey matter.

Humans with type 1 diabetes mellitus (DM) are subject to the development of diabetic leukoencephalopathy (DLE) with cognitive decline, brain atrophy, and white matter abnormalities. We examined advanced glycation end products (AGE) and upregulation of their receptor (RAGE) stimulated by persistent hyperglycemia as a causative pathway, intervening with RAGE blockade in a murine DLE model of type 1 (streptozotocin) DM. Over 8 months, DM mice received intraperitoneal injections of soluble RAGE or placebo alongside non-DM mice and RAGE 2/2 mice. Cognitive testing and magnetic resonance imaging (T2, magnetization transfer, diffusion tensor imaging), neuronal and oligodendroglial counts, synaptic and myelin measures, and RAGE–nuclear factor (NF)-kB pathway assessment was performed serially. DM mice developed cognitive deficits after 8 to 20 weeks of DM, oligodendroglial loss after 3 months, brain atrophy after 5 months, loss of synaptic machinery and fractional anisotropy at 8 months, and RAGE–NF-kB pathway activation. DM mice receiving soluble RAGE and RAGE 2/2 DM mice were protected from most of these abnormalities. Although DM led to NF-k Bu pregulation, NF-kB–mediated gene transcription was downregulated at 8 months, possibly due to later upregulation of transcription repressors B-cell lymphoma 3-encoded protein and NF-k Bi nhibitor Ikbz. Blockade of RAGE has therapeutic importance in type 1D M–mediated DLE within the complex regulation of NF-kB.