CDD: a Conserved Domain Database for protein classification

The Conserved Domain Database (CDD) is the protein classification component of NCBI's Entrez query and retrieval system. CDD is linked to other Entrez databases such as Proteins, Taxonomy and PubMed®, and can be accessed at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=cdd. CD-Search, which is available at http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, is a fast, interactive tool to identify conserved domains in new protein sequences. CD-Search results for protein sequences in Entrez are pre-computed to provide links between proteins and domain models, and computational annotation visible upon request. Protein–protein queries submitted to NCBI's BLAST search service at http://www.ncbi.nlm.nih.gov/BLAST are scanned for the presence of conserved domains by default. While CDD started out as essentially a mirror of publicly available domain alignment collections, such as SMART, Pfam and COG, we have continued an effort to update, and in some cases replace these models with domain hierarchies curated at the NCBI. Here, we report on the progress of the curation effort and associated improvements in the functionality of the CDD information retrieval system

Whole-Exome Sequencing Identifies Homozygous AFG3L2 Mutations in a Spastic Ataxia-Neuropathy Syndrome Linked to Mitochondrial m-AAA Proteases

We report an early onset spastic ataxia-neuropathy syndrome in two brothers of a consanguineous family characterized clinically by lower extremity spasticity, peripheral neuropathy, ptosis, oculomotor apraxia, dystonia, cerebellar atrophy, and progressive myoclonic epilepsy. Whole-exome sequencing identified a homozygous missense mutation (c.1847G>A; p.Y616C) in AFG3L2, encoding a subunit of an m-AAA protease. m-AAA proteases reside in the mitochondrial inner membrane and are responsible for removal of damaged or misfolded proteins and proteolytic activation of essential mitochondrial proteins. AFG3L2 forms either a homo-oligomeric isoenzyme or a hetero-oligomeric complex with paraplegin, a homologous protein mutated in hereditary spastic paraplegia type 7 (SPG7). Heterozygous loss-of-function mutations in AFG3L2 cause autosomal-dominant spinocerebellar ataxia type 28 (SCA28), a disorder whose phenotype is strikingly different from that of our patients. As defined in yeast complementation assays, the AFG3L2Y616C gene product is a hypomorphic variant that exhibited oligomerization defects in yeast as well as in patient fibroblasts. Specifically, the formation of AFG3L2Y616C complexes was impaired, both with itself and to a greater extent with paraplegin. This produced an early-onset clinical syndrome that combines the severe phenotypes of SPG7 and SCA28, in additional to other “mitochondrial” features such as oculomotor apraxia, extrapyramidal dysfunction, and myoclonic epilepsy. These findings expand the phenotype associated with AFG3L2 mutations and suggest that AFG3L2-related disease should be considered in the differential diagnosis of spastic ataxias

Predisposition to Cancer Caused by Genetic and Functional Defects of Mammalian Atad5

ATAD5, the human ortholog of yeast Elg1, plays a role in PCNA deubiquitination. Since PCNA modification is important to regulate DNA damage bypass, ATAD5 may be important for suppression of genomic instability in mammals in vivo. To test this hypothesis, we generated heterozygous (Atad5+/m) mice that were haploinsuffficient for Atad5. Atad5+/m mice displayed high levels of genomic instability in vivo, and Atad5+/m mouse embryonic fibroblasts (MEFs) exhibited molecular defects in PCNA deubiquitination in response to DNA damage, as well as DNA damage hypersensitivity and high levels of genomic instability, apoptosis, and aneuploidy. Importantly, 90% of haploinsufficient Atad5+/m mice developed tumors, including sarcomas, carcinomas, and adenocarcinomas, between 11 and 20 months of age. High levels of genomic alterations were evident in tumors that arose in the Atad5+/m mice. Consistent with a role for Atad5 in suppressing tumorigenesis, we also identified somatic mutations of ATAD5 in 4.6% of sporadic human endometrial tumors, including two nonsense mutations that resulted in loss of proper ATAD5 function. Taken together, our findings indicate that loss-of-function mutations in mammalian Atad5 are sufficient to cause genomic instability and tumorigenesis

PAtENT: a student-centered entrepreneurial pathway to the engineering doctorate

AbstractCurrent structures of STEM graduate programs raise questions about addressing graduates’ interest in multiple career paths, and how programs prepare graduates for positions increasingly available in varied occupations. This problem is addressed through an innovative doctoral program in engineering, Pathways to Entrepreneurship (PAtENT), which works to develop a scalable alternative student-centered framework. This research explores how this program responds to calls for graduate STEM education to address changes in science and engineering, the nature of the workforce, career goals, and how program components build an entrepreneurial mindset. A mixed-methods design includes a curriculum analysis showing alignment of program components to recommendations for Ph.D. STEM programs from the National Academy of Sciences, Engineering, and Medicine. Direct measures include surveys and interviews developed for current doctoral students and faculty to describe students’ and faculty perspectives about program components, particularly entrepreneurship and the patent process. The curriculum analysis shows strong alignment of the PAtENT program components and activities to the ten elements of the National Academies’ recommendations. A survey of graduate students in engineering, computing, and business show strong measures in engineering and entrepreneurial self-efficacy. Interviews of program participants and faculty demonstrate strong interest in patents and developing entrepreneurship. This innovative program in engineering focusing on obtaining a patent as a capstone shows potential to reform doctoral studies, so candidates are prepared not only for academic careers but a range of industry and government work environments. This work will lead to development of a model for other graduate STEM programs

A New Ken-Ken Puzzle Pattern Based Reconfiguration Technique for Maximum Power Extraction in Partial Shaded Solar PV Array

Solar Photovoltaic array may often be subjected to partial shading, which may lead to uneven row current and creates local maximum power point on the power-voltage characteristics. One of the effective approaches to dilute the concentration of partial shading is the array reconfiguration technique. This study proposes a ken-ken puzzle-based reconfiguration technique for $4\times 4$ total-cross-tied configuration to rearrange the position of modules within the array and to improve the maximum power under partial shading conditions. Further, the performance of the ken-ken puzzle arrangement is compared with the total-cross-tied configuration and existing reconfiguration techniques namely odd-even, Latin Square, and Sudoku reported in the literature. The performance of all these configurations is evaluated in terms of fill factor, mismatch loss, power loss, execution ratio, and performance enhancement ratio. The proposed ken-ken puzzle-based reconfiguration technique mitigates the occurrence of local maximum power point and eliminates the need for a complex algorithm to track the global maximum power point. The simulation result shows that the KK puzzle-based reconfiguration technique has obtained an improved PE of 10.85 % compared to TCT configuration, followed by LS, Sudoku, and OE. Also, the experimental result shows the effectiveness of the ken-ken in diluting the effects of partial shading when the rows of the photovoltaic array are shaded. The ken-ken puzzle-based reconfiguration technique reduces the complexity, maintenance and increases reliability, scalability of the PV array

Transforming growth factor-β3 (TGF-β3) knock-in ameliorates inflammation due to TGF-β1 deficiency while promoting glucose tolerance.

Three homologues of TGF-β exist in mammals as follows: TGF-β1, TGF-β2, and TGF-β3. All three proteins share high homology in their amino acid sequence, yet each TGF-β isoform has unique heterologous motifs that are highly conserved during evolution. Although these TGF-β proteins share similar properties in vitro, isoform-specific properties have been suggested through in vivo studies and by the unique phenotypes for each TGF-β knock-out mouse. To test our hypothesis that each of these homologues has nonredundant functions, and to identify such isoform-specific roles, we genetically exchanged the coding sequence of the mature TGF-β1 ligand with a sequence from TGF-β3 using targeted recombination to create chimeric TGF-β1/3 knock-in mice (TGF-β1(Lβ3/Lβ3)). In the TGF-β1(Lβ3/Lβ3) mouse, localization and activation still occur through the TGF-β1 latent associated peptide, but cell signaling is triggered through the TGF-β3 ligand that binds to TGF-β receptors. Unlike TGF-β1(−/−) mice, the TGF-β1(Lβ3/Lβ3) mice show neither embryonic lethality nor signs of multifocal inflammation, demonstrating that knock-in of the TGF-β3 ligand can prevent the vasculogenesis defects and autoimmunity associated with TGF-β1 deficiency. However, the TGF-β1(Lβ3/Lβ3) mice have a shortened life span and display tooth and bone defects, indicating that the TGF-β homologues are not completely interchangeable. Remarkably, the TGF-β1(Lβ3/Lβ3) mice display an improved metabolic phenotype with reduced body weight gain and enhanced glucose tolerance by induction of beneficial changes to the white adipose tissue compartment. These findings reveal both redundant and unique nonoverlapping functional diversity in TGF-β isoform signaling that has relevance to the design of therapeutics aimed at targeting the TGF-β pathway in human disease

Reproduction and immunity-driven natural selection in the human WFDC locus

The whey acidic protein (WAP) four-disulfide core domain (WFDC) locus located on human chromosome 20q13 spans 19 genes with WAP and/or Kunitz domains. These genes participate in antimicrobial, immune, and tissue homoeostasis activities. Neighboring SEMG genes encode seminal proteins Semenogelin 1 and 2 (SEMG1 and SEMG2). WFDC and SEMG genes have a strikingly high rate of amino acid replacement (d(N)/d(S))(,) indicative of responses to adaptive pressures during vertebrate evolution. To better understand the selection pressures acting on WFDC genes in human populations, we resequenced 18 genes and 54 noncoding segments in 71 European (CEU), African (YRI), and Asian (CHB + JPT) individuals. Overall, we identified 484 single-nucleotide polymorphisms (SNPs), including 65 coding variants (of which 49 are nonsynonymous differences). Using classic neutrality tests, we confirmed the signature of short-term balancing selection on WFDC8 in Europeans and a signature of positive selection spanning genes PI3, SEMG1, SEMG2, and SLPI. Associated with the latter signal, we identified an unusually homogeneous-derived 100-kb haplotype with a frequency of 88% in Asian populations. A putative candidate variant targeted by selection is Thr56Ser in SEMG1, which may alter the proteolytic profile of SEMG1 and antimicrobial activities of semen. All the well-characterized genes residing in the WDFC locus encode proteins that appear to have a role in immunity and/or fertility, two processes that are often associated with adaptive evolution. This study provides further evidence that the WFDC and SEMG loci have been under strong adaptive pressure within the short timescale of modern humans

An analysis of exome sequencing for diagnostic testing of the genes associated with muscle disease and spastic paraplegia

In this study we assess exome sequencing (ES) as a diagnostic alternative for genetically heterogeneous disorders. Since ES readily identified a previously reported homozygous mutation in the CAPN3 gene for an individual with an undiagnosed limb girdle muscular dystrophy, we evaluated ES as a generalizable clinical diagnostic tool by assessing the targeting efficiency and sequencing-coverage of 88 genes associated with muscle disease (MD) and spastic paraplegia (SPG). We used three exome-capture kits on 125 individuals. Exons constituting each gene were defined using the UCSC and CCDS databases. The three exome-capture kits targeted 47–92% of bases within the UCSC-defined exons, and 97%–99% of bases within the CCDS-defined exons. An average of 61.2–99.5% and 19.1–99.5% of targeted bases per gene were sequenced to 20X coverage within the CCDS-defined MD and SPG coding exons, respectively. Greater than 95–99% of targeted known mutation positions were sequenced to ≥1X coverage and 55–87% to ≥20X coverage in every exome. We conclude therefore that ES is a rapid and efficient first tier method to screen for mutations, particularly within the CCDS annotated exons, although its application requires disclosure of the extent of coverage for each targeted gene and supplementation with second tier Sanger sequencing for full coverage

Additional file 1: Figure S1. of Replicate exome-sequencing in a multiple-generation family: improved interpretation of next-generation sequencing data

Systematic approach to study exome capture variability in exome-sequencing (A) Three-generation pedigree in which two individuals have an undiagnosed disease that segregated as an autosomal dominant disorder and a de novo variation arose in the second generation. (B) Model of individual subject sample blood DNA processing and sequencing. A sample of blood went through DNA isolation, and independent libraries (in triplicate) were sequenced to appropriate comparable depth and analyzed for various quality control parameters, target coverage, read depth and nucleotide variation detection. (C) Schematic illustration of sequencing read depth vs. targeted genomic region in relation to exome sequencing in replicate. Listed are also the main approaches taken in this study to analyzed exome replicate data. (D) Two main hypotheses tested using replicate exome data: (i) Biases in sequence capture resulting in poor coverage are addressable through repetition (ii) Library replication is beneficial to overall interpretation of sequence variation data. Figure S2. Titration of percentage targeted exome sequenced as a function of depth of sequencing thresholds in all three replicates per sample. Error bars show standard error for replicate sequencing. As expected, higher depth of sequencing thresholds (x-axis) result in higher-coverage (y-axis) variability in replicate exome data. Table S2.Titration of percentage target exome sequenced as a function of depth of sequencing thresholds (attached excel file). Table S3. Primers used for and results of Sanger sequencing analysis for resolution of replicate discordances in NGS data. Table S4. Primers used for and results of Sanger sequencing validation of de novo variants detected using NGS. (Concordant NGS and Sanger genotypes are highlighted in yellow). Figure S3. Box-plot of GC-content distribution in all first-exons (blue) and high-GC content (>70% GC; >=50 bp length). Table S5. Evaluation of coverage of targeted exons with high GC content (attached excel file). Table S6. Quote from Illumina for exome enrichment kits. Quotes in red indicate costs when the study was undertaken. Nextera prices, and other kit prices (in white) reflect current costs per sample (see last column). (DOC 1 mb

Additional file 2: Table S2. of Replicate exome-sequencing in a multiple-generation family: improved interpretation of next-generation sequencing data

Titration of percentage target exome sequenced as a function of depth of sequencing thresholds. (XLSX 59 kb