Identification of potential small molecule binding pockets on Rho family GTPases

Rho GTPases are conformational switches that control a wide variety of signaling pathways critical for eukaryotic cell development and proliferation. They represent attractive targets for drug design as their aberrant function and deregulated activity is associated with many human diseases including cancer. Extensive high-resolution structures (.100) and recent mutagenesis studies have laid the foundation for the design of new structure-based chemotherapeutic strategies. Although the inhibition of Rho signaling with drug-like compounds is an active area of current research, very little attention has been devoted to directly inhibiting Rho by targeting potential allosteric non-nucleotide binding sites. By avoiding the nucleotide binding site, compounds may minimize the potential for undesirable off-target interactions with other ubiquitous GTP and ATP binding proteins. Here we describe the application of molecular dynamics simulations, principal component analysis, sequence conservation analysis, and ensemble small-molecule fragment mapping to provide an extensive mapping of potential small-molecule binding pockets on Rho family members. Characterized sites include novel pockets in the vicinity of the conformationaly responsive switch regions as well as distal sites that appear to be related to the conformations of the nucleotide binding region. Furthermore the use of accelerated molecular dynamics simulation, an advanced sampling method that extends the accessible time-scale of conventional simulations, is found to enhance the characterization of novel binding sites when conformational changes are important for the protein mechanism.

HAMAP in 2015: updates to the protein family classification and annotation system.

HAMAP (High-quality Automated and Manual Annotation of Proteins-available at http://hamap.expasy.org/) is a system for the automatic classification and annotation of protein sequences. HAMAP provides annotation of the same quality and detail as UniProtKB/Swiss-Prot, using manually curated profiles for protein sequence family classification and expert curated rules for functional annotation of family members. HAMAP data and tools are made available through our website and as part of the UniRule pipeline of UniProt, providing annotation for millions of unreviewed sequences of UniProtKB/TrEMBL. Here we report on the growth of HAMAP and updates to the HAMAP system since our last report in the NAR Database Issue of 2013. We continue to augment HAMAP with new family profiles and annotation rules as new protein families are characterized and annotated in UniProtKB/Swiss-Prot; the latest version of HAMAP (as of 3 September 2014) contains 1983 family classification profiles and 1998 annotation rules (up from 1780 and 1720). We demonstrate how the complex logic of HAMAP rules allows for precise annotation of individual functional variants within large homologous protein families. We also describe improvements to our web-based tool HAMAP-Scan which simplify the classification and annotation of sequences, and the incorporation of an improved sequence-profile search algorithm.

The demands of family caregivers of elderly individuals with dementia

The purpose of this cross-sectional observational study was to identify characteristics of caregivers of elderly people with dementia, types of care demands and to relate demands to the stage of dementia. The study was carried out in 2004 with 104 older adults and 90 caregivers in Ribeirão Preto, state of São Paulo. The OARS instrument was utilized and a questionnaire answered by caregivers. Among older adults, 66.3% were female, aged 75.5 years in average and 86.5% had caregivers. Eighty percent of the caregivers were women family members, aged 52.3 years in average. They spent 15.10 hours/day with care, without help. An important relationship was observed between caregiver's burden, physical and emotional effort and stage of dementia. Emotional overburden was higher at dementia early and late stages, this difference was statistically non-significant. Results reveal the urgent need to plan formal and informal support strategies to caregivers of Brazilian elderly people with dementia.

Comfort of the patient's family in an Intensive Care Unit related to welcoming

This study aimed to identify the level of comfort of families of patients in a critical health condition related to the welcoming practices performed by the hospital staff. Interviews were conducted with 250 relatives in hospitals of the state Bahia, using a Likert scale. Data were analyzed as percentages and quartiles. For nine of the 12 statements of the scale, most relatives scored their comfort level between very and totally comfortable, median of 4,revealing kindness, tranquility and friendly communication with family members. More than half of the sample scored its level as not at all to more or less comfortable, median of 3, for statements about demonstration of interest towards the relative by the staff and flexible visiting of the patient. The necessity of greater interest of the team in the condition and needs of the family was observed. Promoting comfort from the dimension of welcoming demands interdisciplinary actions grounded in humanistic philosophy, in which the nurse has an important role to play.

Social network of family caregivers of disabled and dependent patients

Cross-sectional study that used the Social Network Index and the genogram to assess the social network of 110 family caregivers of dependent patients attended by a Home Care Service in São Paulo, Brazil. Data were analyzed using the test U of Mann-Whitney, Kruskal-Wallis and Spearman correlation. Results were considered statistically significant when p<0,05. Few caregivers participated in activities outside the home and the average number of people they had a bond was 4,4 relatives and 3,6 friends. Caregivers who reported pain and those who had a partner had higher average number of relatives who to trust. The average number of friends was higher in the group that reported use of medication for depression. Total and per capita incomes correlated with the social network. It was found that family members are the primary caregiver’s social network.





Adolescent support networks in a health care context: the interface between health, family and education

AbstractOBJECTIVEAnalyze adolescents' perceptions about support networks and their health needs.METHODAnalytical and interpretive study using focus groups conducted in municipal state schools in Fortaleza, in the State of Ceará during the first semester of 2012. The sample comprised 36 male and female adolescents aged between 13 and 16 years attending the ninth grade of the second phase of elementary school.RESULTSThematic analysis revealed that the health care support network and interaction between health professionals, education professionals and family members was insufficient, constituting a lack of an integrated network to enable and provide support for health promotion.CONCLUSIONCoordination between education, health and family services has the potential to act as a support network to help meet adolescents' healthcare needs and demands.

Identification of a new cancer/testis gene family, CT47, among expressed multicopy genes on the human X chromosome.

Cancer/testis (CT) genes are normally expressed in germ cells only, yet are reactivated and expressed in some tumors. Of the approximately 40 CT genes or gene families identified to date, 20 are on the X chromosome and are present as multigene families, many with highly conserved members. This indicates that novel CT gene families may be identified by detecting duplicated expressed genes on chromosome X. By searching for transcript clusters that map to multiple locations on the chromosome, followed by in silico analysis of their gene expression profiles, we identified five novel gene families with testis-specific expression and >98% sequence identity among family members. The expression of these genes in normal tissues and various tumor cell lines and specimens was evaluated by qualitative and quantitative RT-PCR, and a novel CT gene family with at least 13 copies was identified on Xq24, designated as CT47. mRNA expression of CT47 was found mainly in the testes, with weak expression in the placenta. Brain tissue was the only positive somatic tissue tested, with an estimated CT47 transcript level 0.09% of that found in testis. Among the tumor specimens tested, CT47 expression was found in approximately 15% of lung cancer and esophageal cancer specimens, but not in colorectal cancer or breast cancer. The putative CT47 protein consists of 288 amino acid residues, with a C-terminus rich in alanine and glutamic acid. The only species other than human in which a gene homologous to CT47 has been detected is the chimpanzee, with the predicted protein showing approximately 80% identity in its carboxy terminal region.

Study of a Swiss dopa-responsive dystonia family with a deletion in GCH1: redefining DYT14 as DYT5.

OBJECTIVE: To report the study of a multigenerational Swiss family with dopa-responsive dystonia (DRD). METHODS: Clinical investigation was made of available family members, including historical and chart reviews. Subject examinations were video recorded. Genetic analysis included a genome-wide linkage study with microsatellite markers (STR), GTP cyclohydrolase I (GCH1) gene sequencing, and dosage analysis. RESULTS: We evaluated 32 individuals, of whom 6 were clinically diagnosed with DRD, with childhood-onset progressive foot dystonia, later generalizing, followed by parkinsonism in the two older patients. The response to levodopa was very good. Two additional patients had late onset dopa-responsive parkinsonism. Three other subjects had DRD symptoms on historical grounds. We found suggestive linkage to the previously reported DYT14 locus, which excluded GCH1. However, further study with more stringent criteria for disease status attribution showed linkage to a larger region, which included GCH1. No mutation was found in GCH1 by gene sequencing but dosage methods identified a novel heterozygous deletion of exons 3 to 6 of GCH1. The mutation was found in seven subjects. One of the patients with dystonia represented a phenocopy. CONCLUSIONS: This study rules out the previously reported DYT14 locus as a cause of disease, as a novel multiexonic deletion was identified in GCH1. This work highlights the necessity of an accurate clinical diagnosis in linkage studies as well as the need for appropriate allele frequencies, penetrance, and phenocopy estimates. Comprehensive sequencing and dosage analysis of known genes is recommended prior to genome-wide linkage analysis.

Cohesion, satisfaction with family bonds, and emotional well-being in families with adolescents

The present paper investigated whether higher cohesion and satisfaction with family bonds were associated with the daily experience of emotional well-being in varying social circumstances. Using a sample of school-age adolescents (N = 95) and both their parents, data were gathered daily over 1 week using a diary approach in addition to self-report instruments. Multilevel analyses revealed higher cohesion to be associated with well-being in fathers and adolescents, but not in mothers. Parents also reported higher well-being when with friends or colleagues than when alone. Moreover, fathers who scored higher on cohesion reported higher well-being when with family members than when alone, whereas adolescents who scored higher on satisfaction with bonds reported lower well-being when with peers or siblings than when alone.

The development of family alliance from pregnancy to toddlerhood and child outcomes at 5 years.

This article presents a longitudinal study of the development of "family alliance" from pregnancy to toddlerhood in a community sample, as well as its links with the emotional and cognitive development of the child at age 5 years. Family alliance is defined as the quality of the interactive coordination between family members. We consider that the alliance constitutes a context for the child to learn emotion regulation and to develop an understanding of inner states. Family interactions (N = 38) were observed at the 5th month of pregnancy and at 3, 9, and 18 months after birth in a standardized situation of observation (Lausanne Trilogue Play). Marital satisfaction and child temperament were assessed through self-reported questionnaires. Several outcomes of the child at age 5 years were measured: theory of mind performances, predominant emotional themes in pretend play, internalized and externalized symptoms. Results show that (a) three patterns of evolution of family alliance occur: "high stable" (n = 19), "high to low" (n = 10), and "low stable" (n = 9); (b) a high stable alliance is predictive of better outcomes in children at age 5 years, especially regarding theory of mind; (c) the temperament of the child is predictive of child outcomes; and (d) an interaction effect occurs between family alliance and temperament. These results highlight the importance of both family-level and individual-level variables for understanding individual differences in the social and cognitive development of children.

The SLC2 (GLUT) family of membrane transporters.

GLUT proteins are encoded by the SLC2 genes and are members of the major facilitator superfamily of membrane transporters. Fourteen GLUT proteins are expressed in the human and they are categorized into three classes based on sequence similarity. All GLUTs appear to transport hexoses or polyols when expressed ectopically, but the primary physiological substrates for several of the GLUTs remain uncertain. GLUTs 1-5 are the most thoroughly studied and all have well established roles as glucose and/or fructose transporters in various tissues and cell types. The GLUT proteins are comprised of ∼500 amino acid residues, possess a single N-linked oligosaccharide, and have 12 membrane-spanning domains. In this review we briefly describe the major characteristics of the 14 GLUT family members.

Nomenclature of the GLUT/SLC2A family of sugar/polyol transport facilitators.

The recent identification of several additional members of the family of sugar transport facilitators (gene symbol SLC2A, protein symbol GLUT) has created a heterogeneous and, in part, confusing nomenclature. Therefore, this letter provides a summary of the family members and suggests a systematic nomenclature for SLC2A and GLUT symbols.

Epithelial sodium channel/degenerin family of ion channels: a variety of functions for a shared structure.

The recently discovered epithelial sodium channel (ENaC)/degenerin (DEG) gene family encodes sodium channels involved in various cell functions in metazoans. Subfamilies found in invertebrates or mammals are functionally distinct. The degenerins in Caenorhabditis elegans participate in mechanotransduction in neuronal cells, FaNaC in snails is a ligand-gated channel activated by neuropeptides, and the Drosophila subfamily is expressed in gonads and neurons. In mammals, ENaC mediates Na+ transport in epithelia and is essential for sodium homeostasis. The ASIC genes encode proton-gated cation channels in both the central and peripheral nervous system that could be involved in pain transduction. This review summarizes the physiological roles of the different channels belonging to this family, their biophysical and pharmacological characteristics, and the emerging knowledge of their molecular structure. Although functionally different, the ENaC/DEG family members share functional domains that are involved in the control of channel activity and in the formation of the pore. The functional heterogeneity among the members of the ENaC/DEG channel family provides a unique opportunity to address the molecular basis of basic channel functions such as activation by ligands, mechanotransduction, ionic selectivity, or block by pharmacological ligands.

A Novel Homozygous R764H Mutation in CRB1 Causes Autosomal Recessive Retinitis Pigmentosa in a Consanguineous Family

Purpose: Retinitis pigmentosa (RP; MIM 268000) is a hereditary disease characterized by poor night vision and progressive loss of photoreceptors, eventually leading to blindness. This degenerative process primarily affects peripheral vision due to the loss of rods. Autosomal recessive RP (arRP) is clinically and genetically heterogeneous. It has been associated with mutations in different genes, including CRB1 (Crumbs homolog 1). The aim of this study was to determine the causative gene in a Tunisian patient with arRP born to non consanguineous parents.Methods: Four accessible family members were included. They underwent full ophthalmic examination with best corrected Snellen visual acuity, fundus photography and fluoroangiography. Haplotype analyses were used to test linkage in the family to 20 arRP loci, including ABCA4, LRAT, USH2A, RP29, CERKL, CNGA1, CNGB1, CRB1, EYS, RP28, MERTK, NR2E3, PDE6A, PDE6B, RGR, RHO, RLBP1, TULP1. All exons and intron-exon junctions of candidate genes not excluded by haplotype analysis were PCR amplified and directly sequenced.Results: A 39 aged affected member was individualized. Best corrected visual acuity was OR: 20/63, OS: 20/80. Visual loss began at the third decade. Funduscopic examination and FA revealed typical advanced RP changes with bone spicule-shaped pigment deposits in the posterior pole and the mild periphery along with retinal atrophy, narrowing of the vessels and waxy optic discs. Haplotypes analysis revealed homozygosity with microsatellites markers D1S412 and D1S413 on chromosome 1q31.3. These markers flanked the CRB1 gene. Our results excluded linkage of all the other arRP loci/ genes tested. Sequencing of the 12 coding exons and splice sites of CRB1 gene disclosed a homozygous missense mutation in exon 7 at nucleotide c.(2291 G>A), resulting in an Arg to Hist substitution (p.R764H).Conclusions: R764H is a novel mutation associated with CRB1-related arRP. Previously, an R764C mutation was observed. Extending the mutation spectrum of CRB1 with additional families is important for genotype-phenotype correlations.

Formation of virus-like clusters is an intrinsic property of the tumor necrosis factor family member BAFF (B cell activating factor).

The oligomeric state of BAFF (B cell activing factor), a tumor necrosis factor (TNF) family cytokine that plays a critical role in B cell development and survival, has been the subject of recent debate. Myc-tagged BAFF starting at residue Gln136 was previously reported to crystallize as trimers at pH 4.5, whereas a histidine-tagged construct of BAFF, starting at residue Ala134, formed a virus-like cluster containing 60 monomers when crystallized at pH 9.0. The formation of the BAFF 60-mer was pH dependent, requiring pH &gt;or= 7.0. More recently, 60-mer formation was suggested to be artificially induced by the histidine tag, and it was proposed that BAFF, like all other TNF family members, is trimeric. We report here that a construct of BAFF with no amino-terminal tag (Ala134-BAFF) can form a 60-mer in solution. Using size exclusion chromatography and static light scattering to monitor trimer to 60-mer ratios in BAFF preparations, we find that 60-mer formation is pH-dependent and requires histidine 218 within the DE loop of BAFF. Biacore measurements established that the affinity of Ala134-BAFF for the BAFF receptor BAFFR/BR3 is similar to that of myc-Gln136-BAFF, which is exclusively trimeric in solution. However, Ala134-BAFF is more efficacious than myc-Gln136-BAFF in inducing B cell proliferation in vitro. We additionally show that BAFF that is processed and secreted by 293T cells transfected with full-length BAFF, or by a histiocytic lymphoma cell line (U937) that expresses BAFF endogenously, forms a pH-dependent 60-mer in solution. Our results indicate that the formation of the 60-mer in solution by the BAFF extracellular domain is an intrinsic property of the protein, and therefore that this more active form of BAFF may be physiologically relevant.