Putative Determinants Research Articles

In vitro and in vivo characterization of molecular determinants of virulence in reassortant betanodavirus.

We previously reported that betanodavirus reassortant strains [redspotted grouper nervous necrosis virus/striped jack nervous necrosis virus (SJNNV)] isolated from Senegalese sole (Solea senegalensis) exhibited a modified SJNNV capsid amino acid sequence, with changes at aa 247 and 270. In the current study, we investigated the possible role of both residues as putative virulence determinants. Three recombinant viruses harbouring site-specific mutations in the capsid protein sequence, rSs160.03247 (S247A), rSs160.03270 (S270N) and rSs160.03247+270 (S247A/S270N), were generated using a reverse genetics system. These recombinant viruses were studied in cell culture and in vivo in the natural fish host. The three mutant viruses were shown to be infectious and able to replicate in E-11 cells, reaching final titres similar to the WT virus, although with a somewhat slower kinetics of replication. When the effect of the amino acid substitutions on virus pathogenicity was evaluated in Senegalese sole, typical clinical signs of betanodavirus infection were observed in all groups. However, fish mortality induced by all three mutant viruses was clearly affected. Roughly 40 % of the fish survived in these three groups in contrast with the WT virus which killed 100 % of the fish. These data demonstrated that aa 247 and 270 play a major role in betanodavirus virulence although when both mutated aa 247 and 270 are present, corresponding recombinant virus was not further attenuated.

Defective hCNT1 transport contributes to gemcitabine chemoresistance in ovarian cancer subtypes: Overcoming transport defects using a nanoparticle approach

Nucleoside analogs are used as chemotherapeutic options for the treatment of platinum-resistant ovarian cancers. Human concentrative nucleoside transporter 1 (hCNT1) is implicated in sensitizing solid tumors to nucleoside analogs although its role in determining drug efficacy in ovarian cancers remains unclear. Here we examined the functional expression of hCNT1 and compared its contributions toward gemcitabine efficacy in histological subtypes of ovarian cancer. Radioactivity analysis identified hCNT1-mediated 3H-gemcitabine transport in ovarian cancer cells to be significantly reduced compared with that of normal ovarian surface epithelial cells. Biochemical and immunocytochemical analysis identified that unlike normal ovarian cells which expressed high levels of hCNT1 at the apical cell surface, the transporter was either diminished in expression and/or mislocalized in cell lines of various subtypes of ovarian cancer. Retroviral expression of hCNT1 selectively rescued gemcitabine transport in cell lines representing serous, teratocarcinoma, and endometrioid subtypes, but not clear cell carcinoma (CCC). In addition, exogenous hCNT1 predominantly accumulated in intracytoplasmic vesicles in CCC suggesting defective cellular trafficking of hCNT1 as a contributing factor to transport deficiency. Despite diminution of hCNT1 transport in the majority of ovarian cancers and apparent trafficking defects with CCC, the chemotherapeutic efficacy of gemcitabine was broadly enhanced in all subtypes when delivered via engineered nanoparticles (NPs). Additionally, by bypassing the transport requirement, the delivery of a gemcitabine-cisplatin combination in NP formulation increased their synergistic interactions. These findings uncover hCNT1 as a putative determinant for nucleoside analog chemoresistance in ovarian cancer and may help rationalize drug selection and delivery strategies for various histological subtypes of ovarian cancer.

Virulence and the presence of aminoglycoside resistance genes of Staphylococcus haemolyticus strains isolated from clinical specimens.

We examined thirty methicillin-resistant Staphylococcus haemolyticus isolates cultured from clinical specimens for antibiotic resistance, various important interactions of the bacteria with epithelial cells and putative virulence determinants. All strains were resistant to oxacillin and carried the mecA gene. Aminocyclitol-3′-phosphotransferase (aph(3′)-IIIa) gene encoding nucleotidyltransferases was detected in 43 %, aminocyclitol-6′-acetyltransferase-aminocyclitol-2″-phosphotransferase (aac(6′)/aph(2″)) gene encoding bifunctional acetyltransferases/phosphotransferases in 33 %, aminocyclitol-4′-adenylyltransferase (ant(4′)-Ia) gene encoding phosphotransferases in 20 %. The coexistence of resistance to methicillin and aminoglycosides was investigated in multi-resistant strains. Coexisting (aac(6′)/aph(2″)) and (aph(3′)-IIIa) genes were detected in 33 % of isolates, whereas 63 % of isolates had at least one of these genes. All strains revealed adherence ability and most of them (63 %) were invasive to epithelial cells. Electron microscopy revealed that the bacteria were found in vacuoles inside the cells. We observed that the contact of the bacteria with host epithelial cells is a prerequisite to their cytotoxicity at 5 h-incubation. Culture supernatant of the strains induced a low effect of cytotoxicity at the same time of incubation. Cell-free supernatant of all isolates expressed cytotoxic activity which caused destruction of HEp-2 cells at 24 h. None of the strains was cytotonic towards CHO cells. Among thirty strains, 27 % revealed lipolytic activity, 43 % produced lecithinase and 20 % were positive for proteinase activity. Analyses of cellular morphology and DNA fragmentation exhibited typical characteristic features of those undergoing apoptosis. The Pearson linear test revealed positive correlations between the apoptotic index at 24 h and percentage of cytotoxicity. Our results provided new insights into the mechanisms contributing to the development of S. haemolyticus-associated infections. The bacteria adhered and invaded to non-professional phagocytes. The invasion of epithelial cells by S. haemolyticus could be similar to phagocytosis that requires polymerization of the actin cytoskeleton. The process is inhibited by cytochalasin D. Moreover, they survived within the cells by residing in membrane bound compartments and induced apoptotic cell death.

Genome Sequences of Three Highly Copper-Resistant Salmonella enterica subsp. I Serovar Typhimurium Strains Isolated from Pigs in Denmark.

Salmonella typhimurium is the causative agent of typhoid fever, which causes nearly 21.7 million illnesses and 217,000 deaths around the world each year. Here, we describe the draft genome sequences of the Salmonella typhimurium strains S7, S15, and S23, isolated from copper-fed pigs in Denmark and containing additional putative determinants conferring resistances to copper and other metals and metalloids.

Comparative analysis of surface-exposed virulence factors of Acinetobacter baumannii.

BackgroundAcinetobacter baumannii is a significant hospital pathogen, particularly due to the dissemination of highly multidrug resistant isolates. Genome data have revealed that A. baumannii is highly genetically diverse, which correlates with major variations seen at the phenotypic level. Thus far, comparative genomic studies have been aimed at identifying resistance determinants in A. baumannii. In this study, we extend and expand on these analyses to gain greater insight into the virulence factors across eight A. baumannii strains which are clonally, temporally and geographically distinct, and includes an isolate considered non-pathogenic and a community-acquired A. baumannii.ResultsWe have identified a large number of genes in the A. baumannii genomes that are known to play a role in virulence in other pathogens, such as the recently studied proline-alanine-alanine-arginine (PAAR)-repeat domains of the type VI secretion systems. Not surprising, many virulence candidates appear to be part of the A. baumannii core genome of virulent isolates but were often found to be insertionally disrupted in the avirulent A. baumannii strain SDF. Our study also reveals that many known or putative virulence determinants are restricted to specific clonal lineages, which suggests that these virulence determinants may be crucial for the success of these widespread common clones. It has previously been suggested that the high level of intrinsic and adaptive resistance has enabled the widespread presence of A. baumannii in the hospital environment. This appears to have facilitated the expansion of its repertoire of virulence traits, as in general, the nosocomial strains in this study possess more virulence genes compared to the community-acquired isolate.ConclusionsMajor genetic variation in known or putative virulence factors was seen across the eight strains included in this study, suggesting that virulence mechanisms are complex and multifaceted in A. baumannii. Overall, these analyses increase our understanding of A. baumannii pathogenicity and will assist in future studies determining the significance of virulence factors within clonal lineages and/or across the species.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1020) contains supplementary material, which is available to authorized users.

Kidney disease in Aboriginal Australians: a perspective from the Northern Territory.

This article outlines the increasing awareness, service development and research in renal disease in Aboriginal people in Australia's Northern Territory, among whom the rates of renal replacement therapy (RRT) are among the highest in the world. Kidney failure and RRT dominate the intellectual landscape and consume the most professional energy, but the underlying kidney disease has recently swung into view, with increasing awareness of its connection to other chronic diseases and to health profiles and trajectories more broadly. Albuminuria is the marker of the underlying kidney disease and the best treatment target, and glomerulomegaly and focal glomerulosclerosis are the defining histologic features. Risk factors in its multideterminant genesis reflect nutritional and developmental disadvantage and inflammatory/infectious milieu, while the major putative genetic determinants still elude detection. A culture shift of “chronic disease prevention” has been catalyzed in part by the human pain, logistic problems and great costs associated with RRT. Nowadays chronic disease management is the central focus of indigenous primary care, with defined protocols for integrated testing and management of chronic diseases and with government reimbursed service items and free medicines for people in remote areas. Blood pressure, cardiovascular risk and chronic kidney disease (CKD) are all mitigated by good treatment, which centres on renin-angiotensin system blockade and good metabolic control. RRT incidence rates appear to be stabilizing in remote Aboriginal people, and chronic disease deaths rates are falling. However, the profound levels of disadvantage in many remote settings remain appalling, and there is still much to be done, mostly beyond the direct reach of health services.

Resistance to Clover yellow vein virus in Common Bean Germplasm

ABSTRACTClover yellow vein virus (ClYVV) is a potentially devastating viral pathogen of temperate common bean (Phaseolus vulgaris L.) production. Recent research demonstrated that resistance to ClYVV is conditioned by bc‐3 and bc‐32 and that a nonsynonymous mutation in P. vulgaris eukaryotic translation initiation factor 4E (PveIF4E) is the putative functional determinant of this resistance. The objective of this research was to expand the characterization of ClYVV resistance in common bean through greenhouse‐based phenotypic evaluation of 391 accessions of the USDA P. vulgaris Core Collection, a collection of 99 snap bean cultivars and breeding lines, and an extended host differential panel of 75 genotypes with known response to other viral pathogens and to determine if previously designed Kompetitive Allele Specific Polymerase Chain Reaction (KASP) assays could be validated for future bc‐3 resistance allele mining. As a result, one accession from the Core Collection, one additional snap bean breeding line, and 19 dry bean genotypes representing most major market classes of common bean were identified to be resistant. All genotypes that remained symptomless throughout the evaluation were demonstrated to possess mutations in PveIF4E that corresponded with either bc‐3 or bc‐32. In addition, the bc‐u bc‐22 allele combination and the By‐2 allele were also demonstrated to condition resistance phenotypes to ClYVV. These results synthesize a new model for genetic resistance to ClYVV and validate the use of the KASP assays for large‐scale bc‐3 allele mining.

Abstract 803: A vertical combination strategy hitting multiple steps along the MAPK cascade: Molecular mechanisms of action and putative genetic determinants of synergism

Abstract Preclinical evidence shows that hitting a single point along the RAF/MEK/ERK cascade disrupts intra-pathway negative feedback loop, may cause paradoxical pathway activation and may lead to functional resistance. Thus, the “vertical” combination of agents simultaneously inhibiting RAF and MEK has been proposed as a strategy to synergistically inhibit tumor growth and delay resistance. Experimental procedures: Molecular and functional effects of single and combined MEK (using trametinib, T), BRAF (using dabrafenib, D), and RAF (using the pan-RAF inhibitor, RAF265, R) inhibition were dissected by WB analysis and conservative isobologram analysis to assess functional synergism, using a fixed dose-ratio experimental design. Summary data: We examined the in vitro molecular and functional consequences of D and T, alone or in combination, in a panel of different human BRAFV600E melanoma cell lines; both drugs inhibited cell growth and inactivated the MAPK pathway, but little or no synergistic growth inhibition was observed with their combination (CI: 0.7 - 1.3x106). Conversely, combined D+T suppressed malignant growth with highly synergistic effects in KRAS-mutant lung (2/5 cell lines tested) and pancreatic (4/6 cell lines tested) cancer models (CI: 0.1 - 0.7), in which selective BRAF inhibition induced hyperphosphorylation of MEK, ERK, and p90RSK (paradox effect). At concentrations inhibiting both BRAF and CRAF, R did not induce paradox MAPK activation and did not result in growth inhibitory synergism when combined with T. To define the role of RAS gene status in determining sensitivity/resistance to single and combined RAF and MEK blockade, we analyzed two isogenic tumor cell line models: H1299 expressing individual codon 12 KRAS mutants and isogenic HCT116 clones differing for the presence of a homo or heterozygous G13D KRAS mutation. Conversely, in lung cancer models driven by either EGFR mutations (HCC827, H1650) or HER-2 overexpression (Calu-3), selective BRAF inhibition also induced a paradox MAPK activation, which could be blocked using a reversible EGFR/HER-2 inhibitor (Lapatinib); in this context, combination (D+T) resulted in a non-synergic growth inhibitory effects. Conclusions: Overall, our data indicate that, in appropriate cellular contexts, vertical RAF/MEK inhibition-based combination strategies exert highly synergistic antitumor effects across different cancer models. Citation Format: Anais Del Curatolo, Ursula Cesta Incani, Ludovica Ciuffreda, Italia Falcone, Senji Shirasawa, Massimo Broggini, Isabella Sperduti, Adriana Eramo, Ruggero De Maria, Francesco Cognetti, Michele Milella. A vertical combination strategy hitting multiple steps along the MAPK cascade: Molecular mechanisms of action and putative genetic determinants of synergism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 803. doi:10.1158/1538-7445.AM2014-803

Swimming motility in a longitudinal collection of clinical isolates of Burkholderia cepacia complex bacteria from people with cystic fibrosis.

Chronic bacterial lung infections in cystic fibrosis (CF) are the leading cause of morbidity and mortality. While a range of bacteria are known to be capable of establishing residence in the CF lung, only a small number have a clearly established link to deteriorating clinical status. The two bacteria with the clearest roles in CF lung disease are Pseudomonas aeruginosa and bacteria belonging to the Burkholderia cepacia complex (BCC). A number of common adaptations by P. aeruginosa strains to chronic lung infection in CF have been well described. Typically, initial isolates of P. aeruginosa are nonmucoid and display a range of putative virulence determinants. Upon establishment of chronic infection, subsequent isolates ultimately show a reduction in putative virulence determinants, including swimming motility, along with an acquisition of the mucoid phenotype and increased levels of antimicrobial resistance. Infections by BCC are marked by an unpredictable, but typically worse, clinical outcome. However, in contrast to P. aeruginosa infections in CF, studies describing adaptive changes in BCC bacterial phenotype during chronic lung infections are far more limited. To further enhance our understanding of chronic lung infections by BCC bacteria in CF, we assessed the swimming motility phenotype in 551 isolates of BCC bacteria from cystic fibrosis (CF) lung infections between 1981 and 2007. These data suggest that swimming motility is not typically lost by BCC during chronic infection, unlike as seen in P. aeruginosa infections. Furthermore, while we observed a statistically significant link between mucoidy and motility, we did not detect any link between motility phenotype and clinical outcome. These studies highlight the need for further work to understand the adaptive changes of BCC bacteria during chronic infection in the CF lung.

Campylobacter spp. isolation from infected poultry livers with and without necrotic lesions

This study was developed in order to understand the possible intervention of Campylobacter spp. as etiological agent of necrotic lesions in poultry livers. This way, Campylobacter spp. was isolated from poultry livers with and without necrotic lesions. Additionally, virulence factors (cadF and cdtB) and antimicrobial resistance profile of the isolated strains were analyzed. From a total of 39 liver samples analyzed, 21 presented lesions and 18 were clean. Campylobacter spp. was isolated from 80.9% of liver samples with necrotic lesions (17/21) and from 38.9% of liver samples without lesions (7/18). These results indicate poultry liver as a potential source of human Campylobacter infection, since this bacteria may remain viable in the internal liver tissue in undercooked conditions. A high resistance to nalidixic acid (100%), norfloxacin (100%), ciprofloxacin (95.8%), ampicillin (91.6%) and tetracycline (75%) was observed among Campylobacter spp. isolates. Also, PCR detection of cdtB and cadF virulence and toxin genes, revealed 75% and 68.8% of positive samples, respectively. Strains isolated from livers with and without lesions presented similar results with respect to virulence factors and to antimicrobial resistance profiles, evidencing that these putative pathogenic determinants are widespread among the isolates from poultry livers. Phi coefficient calculated in order to measure the degree of association, revealed a highly significant association (Phi ¼ 0.472; p-value < 0.01) between the presence of livers with hepatic necrosis lesions and Campylobacter isolation. This result indicates the possibility of using these macroscopic lesions as visible and reliable indicator of Campylobacter spp. presence in poultry flock, and, thus, becoming an important tool to support the implementation of corrective measures at poultry farms level. This methodology could contribute for an accurate time-efficient monitoring and the development of effective prevention and intervention measures for Campylobacter spp. infection with reduced cost.

Identification and functional analysis of Penicillium digitatum genes putatively involved in virulence towards citrus fruit.

The fungus Penicillium digitatum, the causal agent of green mould rot, is the most destructive post-harvest pathogen of citrus fruit in Mediterranean regions. In order to identify P. digitatum genes up-regulated during the infection of oranges that may constitute putative virulence factors, we followed a polymerase chain reaction (PCR)-based suppression subtractive hybridization and cDNA macroarray hybridization approach. The origin of expressed sequence tags (ESTs) was determined by comparison against the available genome sequences of both organisms. Genes coding for fungal proteases and plant cell wall-degrading enzymes represent the largest categories in the subtracted cDNA library. Northern blot analysis of a selection of P. digitatum genes, including those coding for proteases, cell wall-related enzymes, redox homoeostasis and detoxification processes, confirmed their up-regulation at varying time points during the infection process. Agrobacterium tumefaciens-mediated transformation was used to generate knockout mutants for two genes encoding a pectin lyase (Pnl1) and a naphthalene dioxygenase (Ndo1). Two independent P. digitatum Δndo1 mutants were as virulent as the wild-type. However, the two Δpnl1 mutants analysed were less virulent than the parental strain or an ectopic transformant. Together, these results provide a significant advance in our understanding of the putative determinants of the virulence mechanisms of P. digitatum.

'Stealth' lipid-based formulations: poly(ethylene glycol)-mediated digestion inhibition improves oral bioavailability of a model poorly water soluble drug.

For over 20years, stealth drug delivery has been synonymous with nanoparticulate formulations and intravenous dosing. The putative determinants of stealth in these applications are the molecular weight and packing density of a hydrophilic polymer (commonly poly(ethylene glycol) (PEG)) that forms a steric barrier at the surface of the nanoparticle. The current study examined the potential translation of the concepts learned from stealth technology after intravenous administration to oral drug delivery and specifically, to enhance drug exposure after administration of oral lipid-based formulations (LBFs) containing medium-chain triglycerides (MCT). MCT LBFs are rapidly digested in the gastrointestinal tract, typically resulting in losses in solubilisation capacity, supersaturation and drug precipitation. Here, non-ionic surfactants containing stealth PEG headgroups were incorporated into MCT LBFs in an attempt to attenuate digestion, reduce precipitation risk and enhance drug exposure. Stealth capabilities were assessed by measuring the degree of digestion inhibition that resulted from steric hindrance of enzyme access to the oil-water interface. Drug-loaded LBFs were assessed for maintenance of solubilising capacity during in vitro digestion and evaluated in vivo in rats. The data suggest that the structural determinants of stealth LBFs mirror those of parenteral formulations, i.e., the key factors are the molecular weight of the PEG in the surfactant headgroup and the packing density of the PEG chains at the interface. Interestingly, the data also show that the presence of labile ester bonds within a PEGylated surfactant also impact on the stealth properties of LBFs, with digestible surfactants requiring a PEG Mw of ~1800g/mol and non-digestible ether-based surfactants ~800g/mol to shield the lipidic cargo. In vitro evaluation of drug solubilisation during digestion showed stealth LBFs maintained drug solubilisation at or above 80% of drug load and reduced supersaturation in comparison to digestible counterparts. This trend was also reflected in vivo, where the relative bioavailability of drug after administration in two stealth LBFs increased to 120% and 182% in comparison to analogous digestible (non-stealth) formulations. The results of the current study indicate that self-assembled "stealth" LBFs have potential as a novel means of improving LBF performance.

Beyond the chromosome: the prevalence of unique extra-chromosomal bacteriophages with integrated virulence genes in pathogenic Staphylococcus aureus.

In Staphylococcus aureus, the disease impact of chromosomally integrated prophages on virulence is well described. However, the existence of extra-chromosomal prophages, both plasmidial and episomal, remains obscure. Despite the recent explosion in bacterial and bacteriophage genomic sequencing, studies have failed to specifically focus on extra-chromosomal elements. We selectively enriched and sequenced extra-chromosomal DNA from S. aureus isolates using Roche-454 technology and uncovered evidence for the widespread distribution of multiple extra-chromosomal prophages (ExPΦs) throughout both antibiotic-sensitive and -resistant strains. We completely sequenced one such element comprised of a 43.8 kbp, circular ExPΦ (designated ФBU01) from a vancomycin-intermediate S. aureus (VISA) strain. Assembly and annotation of ФBU01 revealed a number of putative virulence determinants encoded within a bacteriophage immune evasion cluster (IEC). Our identification of several potential ExPΦs and mobile genetic elements (MGEs) also revealed numerous putative virulence factors and antibiotic resistance genes. We describe here a previously unidentified level of genetic diversity of stealth extra-chromosomal elements in S. aureus, including phages with a larger presence outside the chromosome that likely play a prominent role in pathogenesis and strain diversity driven by horizontal gene transfer (HGT).

Correlates of low testosterone in men with chronic spinal cord injury.

Although high rates of serum testosterone deficiency have been reported in men with spinal cord injury (SCI), its determinants and attributes are not yet established. The aim of this study was to recognize, among putative determinants and attributes of androgen deficiency, those significantly associated to low testosterone after adjustment for confounders recognizable in men with chronic SCI. A biochemical androgen deficiency (total testosterone <300ng/dL) was exhibited by 18 of 51 patients (35.3%). Significant correlates of testosterone levels were as follows: weekly leisure time physical activity (LTPA) explored by the LTPA Questionnaire for people with SCI, body mass index (BMI), homeostatic model assessment of insulin resistance (HOMA-IR), triglycerides and sexual symptoms, explored by the aging males' symptom (AMS) questionnaire. At the multiple linear regression analysis, among putative determinants of low testosterone, only weekly LTPA and BMI exhibited a significant association with testosterone levels, explaining 54.2 and 9.0% of testosterone variability respectively. At the linear regression models, among various putative attributes of androgen deficiency, only lower sexual desire and, at a lesser extent, higher HOMA-IR, exhibited significant associations with lower testosterone levels, after adjustment for BMI, age, comorbidities and weekly LTPA. In conclusion, poor LTPA, high BMI and low sexual desire are independent predictors of low testosterone in men with chronic SCI. This is relevant to clinical practice, as all these features are routinely assessed in rehabilitation settings for SCI. As poor LTPA and high BMI are modifiable life-style related risk factors, prospective studies could clarify whether life-style modification could increase the level of testosterone and improve the low sexual desire, relevant clinical attribute of low testosterone in men with SCI.

De novo sequencing and transcriptome analysis of Ustilaginoidea virens by using Illumina paired-end sequencing and development of simple sequence repeat markers.

Ustilaginoidea virens is the causal agent of rice false smut, which is a rice disease of increasing importance worldwide that has caused with the quantitative and qualitative rice losses. However, research on the pathogenic mechanism of U. virens is limited. In this study, we reported a de novo assembling, annotation, and characterization of the transcriptome and developed simple sequence repeat (SSR) markers of U. virens. U. virens transcripts of the mycelia and conidia mixture were sequenced using Illumina RNA-seq technology. A total of 52,554,142 clean reads were assembled into 36,496 transcripts representing 18,534 unigenes. Assembled unigenes were annotated through sequence comparison with known protein databases, and 48.48% of the unigenes were without hits in any of these databases. Clusters of orthologous groups for eukaryotic complete genome analysis identified the largest set of genes associated with posttranslational modification, protein turnover and chaperones. Kyoto Encyclopedia of Genes and Genomes pathway analyses identified the number of genes associated with mitogen-activated protein kinase and calcium-calcineurin pathways. The study also identified several putative pathogenicity determinants and candidate effectors in U. virens by using the pathogen-host interaction database. In addition, bioinformatics analysis revealed the presence of 12,298 SSR markers. This study provides a better understanding of the biology of U. virens and is an excellent resource for candidate genes required for pathogenesis discovery.

0408 PubMed search strings for the study of putative environmental determinants of disease

ObjectivesSeveral optimised search strategies have been developed in Medicine, and more recently in Occupational Medicine. Aim of this study is to identify efficient PubMed search strategies for the study of...

Prediction of the determinants of thermal stability by linear discriminant analysis: The case of the glutamate dehydrogenase protein family

Little is known about the determinants of thermal stability in individual protein families. Most of the knowledge on thermostability comes, in fact, from comparative analyses between large, and heterogeneous, sets of thermo- and mesophilic proteins. Here, we present a multivariate statistical approach aimed to detect signature sequences for thermostability in a single protein family. It was applied to the glutamate dehydrogenase (GDH) family, which is a good model for investigating this peculiar process. The structure of GDH consists of six subunits, each of them organized into two domains. Formation of ion-pair networks on the surface of the protein subunits, or increase in the inter-subunit hydrophobic interactions, have been suggested as important factors for explaining stability at high temperatures. However, identification of the amino acid changes that are involved in this process still remains elusive. Our approach consisted of a linear discriminant analysis on a set of GDH sequences from Archaea and Bacteria (33 thermo- and 36 mesophilic GDHs). It led to detection of 3 amino acid clusters as the putative determinants of thermal stability. They were localized at the subunit interface or in close proximity to the binding site of the NAD(P)+ coenzyme. Analysis within the clusters led to prediction of 8 critical amino acid sites. This approach could have a wide utility, in the ligth of the notion that each protein family seems to adopt its own strategy for achieving thermostability.

A novel begomovirus isolated from sida contains putative cis- and trans-acting replication specificity determinants that have evolved independently in several geographical lineages.

A novel begomovirus isolated from a Sida rhombifolia plant collected in Sinaloa, Mexico, was characterized. The genomic components of sida mosaic Sinaloa virus (SiMSinV) shared highest sequence identity with DNA-A and DNA-B components of chino del tomate virus (CdTV), suggesting a vertical evolutionary relationship between these viruses. However, recombination analysis indicated that a short segment of SiMSinV DNA-A encompassing the plus-strand replication origin and the 5´-proximal 43 codons of the Rep gene was derived from tomato mottle Taino virus (ToMoTV). Accordingly, the putative cis- and trans-acting replication specificity determinants of SiMSinV were identical to those of ToMoTV but differed from those of CdTV. Modeling of the SiMSinV and CdTV Rep proteins revealed significant differences in the region comprising the small β1/β5 sheet element, where five putative DNA-binding specificity determinants (SPDs) of Rep (i.e., amino acid residues 5, 8, 10, 69 and 71) were previously identified. Computer-assisted searches of public databases led to identification of 33 begomoviruses from three continents encoding proteins with SPDs identical to those of the Rep encoded by SiMSinV. Sequence analysis of the replication origins demonstrated that all 33 begomoviruses harbor potential Rep-binding sites identical to those of SiMSinV. These data support the hypothesis that the Rep β1/β5 sheet region determines specificity of this protein for DNA replication origin sequences.

Differential changes in amygdala and frontal cortex Pde10a expression during acute and protracted withdrawal.

Alcohol use disorders are persistent problems with high recidivism rates despite repeated efforts to quit drinking. Neuroadaptations that result from alcohol exposure and that persist during periods of abstinence represent putative molecular determinants of the propensity to relapse. Previously we demonstrated a positive association between phosphodiesterase 10A (PDE10A) gene expression and elevations in relapse-like alcohol self-administration in rats with a history of stress exposure. Because alcohol withdrawal is characterized by heightened anxiety-like behavior, activation of stress-responsive brain regions and an elevated propensity to self-administer alcohol, we hypothesized that Pde10a expression also would be upregulated in reward- and stress-responsive brain regions during periods of acute (8–10 h) and protracted (6 weeks) alcohol withdrawal. During acute withdrawal, elevated Pde10a mRNA expression was found in the medial and basolateral amygdala (BLA), as well as the infralimbic and anterior cingulate subdivisions of the medial prefrontal cortex, relative to alcohol-naïve controls. The BLA was the only region with elevated Pde10a mRNA expression during both acute and protracted withdrawal. In contrast to the elevations, Pde10a mRNA levels tended to be reduced during protracted withdrawal in the dorsal striatum, prelimbic prefrontal cortex, and medial amygdala. Together these results implicate heightened PDE10A expression in the BLA as a lasting neuroadaptation associated with alcohol dependence.

Heterologous Expression of Thuricin CD Immunity Genes in Listeria monocytogenes

Bacteriocins are ribosomally synthesized peptides that can have a narrow or broad spectrum of antimicrobial activity. Bacteriocin producers typically possess dedicated immunity systems that often consist of an ATP-binding cassette (ABC) transporter system and/or a dedicated immunity protein. Here we investigated the genes responsible for immunity to thuricin CD, a narrow-spectrum two-peptide sactibiotic produced by Bacillus thuringiensis DPC6431. Heterologous expression of putative thuricin CD immunity determinants allowed us to identify and investigate the relative importance of the individual genes and gene products that contribute to thuricin CD immunity. We established that TrnF and TrnG are the individual components of an ABC transporter system that provides immunity to thuricin CD. We also identified a hitherto overlooked open reading frame located upstream of trnF predicted to encode a 79-amino-acid transmembrane protein. We designated this newly discovered gene trnI and established that TrnI alone can provide protection against thuricin CD.