Membrane Protein-coding Genes Research Articles

The two-component system CpxA/CpxR regulates pathogenesis and stress adaptability in the poplar canker bacterium Lonsdalea populi.

Bacteria employ two-component systems (TCSs) to rapidly sense and respond to their surroundings often and during plant infection. Poplar canker caused by Lonsdalea populi is an emerging woody bacterial disease that leads to high mortality and poplar plantation losses in China. Nonetheless, the information about the underlying mechanism of pathogenesis remains scarce. Therefore, in this study, we reported the role of a TCS pair CpxA/CpxR in regulating virulence and stress responses in L. populi. The CpxA/R system is essential during infection, flagellum formation, and oxidative stress response. Specifically, the Cpx system affected flagellum formation by controlling the expression of flagellum-related genes. CpxR, which was activated by phosphorylation in the presence of CpxA, participated in the transcriptional regulation of a chaperone sctU and the type III secretion system (T3SS)-related genes, thereby influencing T3SS functions during L. populi infection. Phosphorylated CpxR directly manipulated the transcription of a membrane protein-coding gene yccA and the deletion of yccA resulted in reduced virulence and increased sensitivity to H2O2. Furthermore, we mutated the conserved phosphorylation site of CpxR and found that CpxRD51A could no longer bind to the yccA promoter but could still bind to the sctU promoter. Together, our findings elucidate the roles of the Cpx system in regulating virulence and reactive oxygen species resistance and provide further evidence that the TCS is crucial during infection and stress response.

Prevalence of multidrug-resistant hypervirulent Klebsiella pneumoniae without defined hypervirulent biomarkers in Anhui, China: a new dimension of hypervirulence.

Klebsiella pneumoniae is an opportunistic pathogen that mainly causes nosocomial infections and hospital-associated pneumonia in elderly and immunocompromised people. However, multidrug-resistant hypervirulent K. pneumoniae (MDR-hvKp) has emerged recently as a serious threat to global health that can infect both immunocompromised and healthy individuals. It is scientifically established that plasmid-mediated regulator of mucoid phenotype genes (rmpA and rmpA2) and other virulence factors (aerobactin and salmochelin) are mainly responsible for this phenotype. In this study, we collected 23 MDR-hvKp isolates and performed molecular typing, whole genome sequencing, comparative genomic analysis, and phenotypic experiments, including the Galleria mellonella infection model, to reveal its genetic and phenotypic features. Meanwhile, we discovered two MDR-hvKp isolates (22122315 and 22091569) that showed a wide range of hypervirulence and hypermucoviscosity without rmpA and rmpA2 and any virulence factors. In phenotypic experiments, isolate 22122315 showed the highest hypervirulence (infection model) with significant mucoviscosity, and conversely, isolate 22091569 exhibited the highest mucoviscosity (string test) with higher virulence compared to control. These two isolates carried carbapenemase (blaKPC - 2), β-lactamase (blaOXA - 1, blaTEM - 1B), extended-spectrum β-lactamase (ESBL) genes (blaCTX - M - 15, blaSHV - 106), outer membrane protein-coding genes (ompA), fimbriae encoding genes (ecpABCDER), and enterobactin coding genes (entAB, fepC). In addition, single nucleotide polymorphism analysis indicated that both isolates, 22122315 and 22091569, were found to have novel mutations in loci FEBNDAKP_03184 (c. 2084A > C, p. Asn695Thr), and EOFMAFIB_02276 (c. 1930C > A, p. Pro644Thr), respectively. Finally, NCBI blast analysis suggested these mutations are located in the wzc of the capsule polysaccharide (cps) region and are responsible for putative tyrosine kinase. This study would be a strong reference for enhancing the current understanding of identifying the MDR-hvKp isolates that lacked both mucoid regulators and virulence factors.

Detection of Chlamydia sp. by fluorescence in situ hybridization (FISH) in histologic sections of the liver from exotic and native avian species

Chlamydiosis is a zoonotic disease that affects several animal species. Therefore, reliable detection techniques are essential for efficient control of the disease. Fluorescence in situ hybridization (FISH) for Chlamydia sp. was applied to 137 formalin-fixed, paraffin-embedded liver sections from native and exotic birds. The samples were divided into two groups: retrospective (n=57) and prospective (n=80). The probe was designed based on an annealing sequence that targets the Major Outer Membrane Protein coding gene. Livers previously confirmed for Chlamydia psittaci by polymerase chain reaction (PCR) and immunohistochemistry (IHC) were used as positive controls. Also, 47 randomly selected samples from the prospective group were submitted to PCR for Chlamydia psittaci for confirmation. From all 137 samples, 67% (92/137) were positive for Chlamydia sp. through FISH, from which 39% (36/57) and 61% (56/80) were from the retrospective and prospective groups, respectively. From the samples of the prospective group submitted for PCR confirmation, 83% (39/47) of them had a positive correlation with FISH results. When considering the number of targeted microorganisms per 400x field, 42,39% (39/92) of the samples had up to five microorganisms, 14,13% (13/92) had from six to ten, and 43,47% (40/92) of the samples had 11 or more microorganisms per field. FISH is a specific and reliable method to identify Chlamydia sp. in histologic sections of the liver, providing an additional tool for detecting avian chlamydiosis.

Pan-Cancer Analysis Identifies Tumor Cell Surface Targets for CAR-T Cell Therapies and Antibody Drug Conjugates.

Tumor cells can be recognized through tumor surface antigens by immune cells and antibodies, which therefore can be used as drug targets for chimeric antigen receptor-T (CAR-T) therapies and antibody drug conjugates (ADCs). In this study, we aimed to identify novel tumor-specific antigens as targets for more effective and safer CAR-T cell therapies and ADCs. Here, we performed differential expression analysis of pan-cancer data obtained from the Cancer Genome Atlas (TCGA), and then performed a series of conditional screenings including Cox regression analysis, Pearson correlation analysis, and risk-score calculation to find tumor-specific cell membrane genes. A tumor tissue-specific and highly expressed gene set containing 3919 genes from 17 cancer types was obtained. Moreover, the prognostic roles of these genes and the functions of these highly expressed membrane proteins were assessed. Notably, 427, 584, 431 and 578 genes were identified as risk factors for LIHC, KIRC, UCEC, and KIRP, respectively. Functional enrichment analysis indicated that these tumor-specific surface proteins might confer tumor cells the ability to invade and metastasize. Furthermore, correlation analysis displayed that most overexpressed membrane proteins were positively correlated to each other. In addition, 371 target membrane protein-coding genes were sifted out by excluding proteins expressed in normal tissues. Apart from the identification of well-validated genes such as GPC3, MSLN and EGFR in the literature, we further confirmed the differential protein expression of 23 proteins: ADD2, DEF6, DOK3, ENO2, FMNL1, MICALL2, PARVG, PSTPIP1, FERMT1, PLEK2, CD109, GNG4, MAPT, OSBPL3, PLXNA1, ROBO1, SLC16A3, SLC26A6, SRGAP2, and TMEM65 in four types of tumors. In summary, our findings reveal novel tumor-specific antigens, which could be potentially used for next-generation CAR-T cell therapies and ADC discovery.

Reduced sensitivity of lager brewing yeast to premature yeast flocculation via adaptive evolution

Malt-induced premature yeast flocculation (PYF) is a sporadic problem within the brewing industry. The use of PYF malts is concomitant with a number of negative impacts on beer quality, including incomplete fermentation and/or flavor defects. Although malt-induced PYF is widely acknowledged, actions taken so far have proved insufficient to solve the PYF-related issues. To limit the detrimental effects of PYF malts on beer production, an adaptive laboratory evolution (ALE) process was applied in this study to an industrial lager brewing yeast strain (TT02), in an attempt to generate variant strains with improved fermentation performance in PYF wort. Through a batch fermentation-based adaptation process, evolved variants were isolated and screened for their phenotypic and metabolic traits. The investigation focused mainly on the tendency to remain in suspension, fermentation capacity and final acetaldehyde concentration. We successfully obtained a variant (TT02-30 T) with improved fermentation properties. The improvement was seen in worts prepared from different types of PYF malt as well as normal malt. Furthermore, ALE of lager brewing yeast in PYF wort yielded a wide array of mutations. Several changes in the genomes (copy number variation in flocculin encoding gene FLO1 and a missense SNP in a putative mitochondrial membrane protein coding gene FMP10) of the variant strains relative to the original strain were observed. These could potentially contribute to the improved yeast suspension during fermentation. Importantly, mutational enrichment in genes related to ion binding in PYF-evolved strains suggests the involvement of the yeast ion transportation process in dealing with the PYF stress. Our study demonstrates the possibility of attenuating yeast sensitivity to PYF malts over time through adaptive laboratory evolution via spontaneous mutation.

Genetic Spectrum of Inherited/Congenital Hemolytic Anemias in Indian Patients

IntroductionHemolytic anemias are a group of disorders caused by the premature destruction of red blood cells with reticulocytosis. Common causes of inherited/congenital hemolysis are hemoglobinopathies and thalassemia syndromes, red blood cell membrane, and enzyme disorders. Most of the common causes (thalassemia, glucose-6-phosphate dehydrogenase (G6PD) deficiency, hereditary spherocytosis, etc.) are diagnosed based on laboratory testing; however, for remaining causes laboratory tests are either inaccessible or cumbersome. We follow a stepwise diagnostic pipeline and red cell morphology is helpful with membrane disorders. Phenotypes vary from severe hemolysis (transfusion-dependent) to mild/asymptomatic patients. Undiagnosed haemolytic anemias are taken up for multi-gene panel-based targeted resequencing which is rapid, accurate, and cost-effective. The use of these panels expedites the diagnoses of inherited hemolytic anemias and is eventually helpful for evidence-based genetic counseling.ObjectivesThis study aimed to determine the genetic defects in inherited/congenital hemolytic anemias which remained unexplained after routine laboratory tests.MethodsSeventy-five families were enrolled based on the clinical and laboratory features of inherited/congenital hemolytic anemias. Common causes of inherited hemolysis are G6PD deficiency, hemoglobinopathies and thalassemia syndromes, autoimmune hemolytic anemias, hereditary spherocytosis, and pyruvate kinase (PK) deficiency were excluded on the basis of biochemical and molecular tests. DNA extraction was done QIAamp DNA Blood Mini Kit. Quantity and quality of DNA were verified using NanoDrop and Qubit Fluorometer respectively. DNA libraries were prepared using Amplicon custom panels for genes implicated in hemolytic anemias and sequenced on Illumina MiSeq Sequencer. Alignment and variant calling were done in Illumina Local run Manager and Variant annotation was done in Basespace VariantInterpretor. Sanger sequencing was done as orthogonal validation in the index case. Predictive testing was performed for the family members.ResultsAfter targeted resequencing of the total 75 index cases, 19 patients were found to have red blood cell enzymopathies, 15 patients had stomatocytosis, 13 had membranopathies and three patients had unstable hemoglobins. In 8 patients cause was not established either only heterozygous variant was found for autosomal recessive or due to the lack of samples of family members for screening. Seventeen cases remained unexplained even after next-generation sequencing.Out of 19 patients, unexpected PK deficiency was found in 12 patients and G6PD deficiency was found in 3 patients; despite the enzyme assay being normal in these cases. We also found 2 patients with glucose-6-phosphate isomerase deficiency. One case each with hexokinase deficiency and glutathione synthetase deficiency was found.Among 15 patients with stomatocytosis, 8 had Mediterranean stomatocytosis/macrothrombocytopenia (ABCG5/ABCG8). These 8 patients showed the presence of stomatocytosis along with giant platelets on peripheral smear evaluation. Of the remaining 7 cases , 2 were found to have overhydrated hereditary stomatocytosis (RHAG) and dehydrated Stomatocytosis/xerocytosis was found in 5 (PIEZO1/KCNN4).We also found 13 cases of hemolytic anemia to have a genetic defect in red blood cell membrane protein-coding genes. Of these 5 had probably pathogenic variants in the ANK1 gene, 5 had a pathogenic variant in SPTA1, 2 had SPTB 2, and 1 patient SLC4A1.We also encountered 3 cases of unstable hemoglobins where no abnormality was noted in Hb-HPLC patterns.A total of seven patients underwent splenectomy and are transfusion free.ConclusionsOur cohort of 75 families of hemolytic anemia of unexplained etiology showed a highly heterogeneous genetic spectrum. Of the total cases, the confirmed diagnosis was achieved in 67% of the patients. This approach of using a multi-gene panel is cost-effective and can provide a rapid and accurate diagnosis. Unexpected PK deficiency, G6PD deficiency, and unstable hemoglobins suggest that such cases can be missed. Providing accurate diagnosis in such cases provides evidence-based counseling and saves the families from inappropriate treatments. DisclosuresNo relevant conflicts of interest to declare.

Aspergillus fumigatus versus Genus Aspergillus: Conservation, Adaptive Evolution and Specific Virulence Genes.

Aspergillus is an important fungal genus containing economically important species, as well as pathogenic species of animals and plants. Using eighteen fungal species of the genus Aspergillus, we conducted a comprehensive investigation of conserved genes and their evolution. This also allows us to investigate the selection pressure driving the adaptive evolution in the pathogenic species A. fumigatus. Among single-copy orthologs (SCOs) for A. fumigatus and the closely related species A. fischeri, we identified 122 versus 50 positively selected genes (PSGs), respectively. Moreover, twenty conserved genes of unknown function were established to be positively selected and thus important for adaption. A. fumigatus PSGs interacting with human host proteins show over-representation of adaptive, symbiosis-related, immunomodulatory and virulence-related pathways, such as the TGF-β pathway, insulin receptor signaling, IL1 pathway and interfering with phagosomal GTPase signaling. Additionally, among the virulence factor coding genes, secretory and membrane protein-coding genes in multi-copy gene families, 212 genes underwent positive selection and also suggest increased adaptation, such as fungal immune evasion mechanisms (aspf2), siderophore biosynthesis (sidD), fumarylalanine production (sidE), stress tolerance (atfA) and thermotolerance (sodA). These genes presumably contribute to host adaptation strategies. Genes for the biosynthesis of gliotoxin are shared among all the close relatives of A. fumigatus as an ancient defense mechanism. Positive selection plays a crucial role in the adaptive evolution of A. fumigatus. The genome-wide profile of PSGs provides valuable targets for further research on the mechanisms of immune evasion, antimycotic targeting and understanding fundamental virulence processes.

Data-Driven Discovery of Molecular Targets for Antibody-Drug Conjugates in Cancer Treatment.

Antibody-drug conjugate therapy has attracted considerable attention in recent years. Since the selection of appropriate targets is a critical aspect of antibody-drug conjugate research and development, a big data research for discovery of candidate targets per tumor type is outstanding and of high interest. Thus, the purpose of this study was to identify and prioritize candidate antibody-drug conjugate targets with translational potential across common types of cancer by mining the Human Protein Atlas, as a unique big data resource. To perform a multifaceted screening process, XML and TSV files including immunohistochemistry expression data for 45 normal tissues and 20 tumor types were downloaded from the Human Protein Atlas website. For genes without high protein expression across critical normal tissues, a quasi H-score (range, 0-300) was computed per tumor type. All genes with a quasi H − score ≥ 150 were extracted. Of these, genes with cell surface localization were selected and included in a multilevel validation process. Among 19670 genes that encode proteins, 5520 membrane protein-coding genes were included in this study. During a multistep data mining procedure, 332 potential targets were identified based on the level of the protein expression across critical normal tissues and 20 tumor types. After validation, 23 cell surface proteins were identified and prioritized as candidate antibody-drug conjugate targets of which two have interestingly been approved by the FDA for use in solid tumors, one has been approved for lymphoma, and four have currently been entered in clinical trials. In conclusion, we identified and prioritized several candidate targets with translational potential, which may yield new clinically effective and safe antibody-drug conjugates. This large-scale antibody-based proteomic study allows us to go beyond the RNA-seq studies, facilitates bench-to-clinic research of targeted anticancer therapeutics, and offers valuable insights into the development of new antibody-drug conjugates.

Molecular detection of Coxiella burnetii in raw meat intended for pet consumption.

The discovery of antibodies against Coxiella burnetii in cattery-confined breeding cats indicating prior or current exposure (Shapiro etal., 2015) prompted an investigation into possible sources of infection. One hypothesis was that raw meat diets containing reservoir species may provide a source of C. burnetii transmission. The aim of this pilot study was to determine whether C. burnetii DNA was present in raw meat sold exclusively for companion animal consumption. The sample population consisted of raw meat packages (n=58) of primarily kangaroo origin, with three to four aliquots (50-120mg) randomly selected from each package. Genomic DNA was extracted from whole tissue in each of these aliquots using a modified protocol. Three quantitative PCR assays were used for the detection of C.burnetii targeting the IS1111 gene, the heat shock operon htpAB and the C.burnetii outer membrane protein-coding gene, com1. Coxiella burnetii DNA was detected in 25/58 samples (43%) using the IS1111, htpAB and/or com1 PCR assays and confirmed by DNA sequencing. All samples amplifying a product in the com1 assay also amplified a product in the htpAB and IS1111 assays. A total of 17/58 (29%) packets were positive with all three genes, 4/58 (7%) were positive with two genes (IS1111 and htpAB) and 4/58 (7%) were positive with the IS1111 gene only. Coxiella burnetii DNA was five times more likely to be found in offal than skeletal muscle meat samples. All meat samples in which C.burnetii DNA was found were from kangaroo tissues, while samples labelled as non-kangaroo meat (n=4) were negative. Multi-locus variable number of tandem repeat analysis (MLVA) identified three different genotypes of C.burnetii that have all been identified previously from Australian human clinical Q fever cases. Further investigations are required to determine the potential role of certain raw meats in the transmission of C.burnetii to cats and humans.

Next-Generation Sequencing–Based Diagnosis of Unexplained Inherited Hemolytic Anemias Reveals Wide Genetic and Phenotypic Heterogeneity

Determination of the cause of inherited hemolysis is based on clinical and stepwise conventional laboratory tests. Patients with obscure etiology require genetic diagnosis, which is time-consuming, expensive, and laborious, mainly because of numerous causal genes. This study enrolled 43 patients with clinical and laboratory evidence of unexplained hemolytic anemia. Initially, 13 patients were tested using a commercial (TruSight One) panel, and remaining cases underwent targeted sequencing using a customized 55-gene panel. Pyruvate kinase deficiency was found in eight, glucose-6-phosphate dehydrogenase (G6PD) deficiency in three (G6PD Guadalajara in two and p.Tyr227Ser: novel, named as G6PD Chandigarh), and glucose-6-phosphate isomerase (GPI) deficiency in two (GPI:p.Arg347His and p.Phe304Leu: novel, named as GPI Chandigarh). Three patients had Mediterranean stomatocytosis/macrothrombocytopenia, and two had overhydrated stomatocytosis. Xerocytosis was found in three patients, whereas six had potentially pathogenic variants in membrane protein-coding genes. Overall, 63% cases received a definite diagnosis. Timely determination of etiology was helpful in diagnosis, genetic counseling, and offering a prenatal diagnosis. Therapeutic implications include performing or avoiding splenectomy that may ameliorate the anemia in many but also predispose to thrombosis in other groups of patients. This first study on the genetic spectrum of unexplained hemolytic anemia from the Indian subcontinent also represents, currently, one of the largest cohort worldwide of such patients.

The Spectrum of Genetic Defects in Indian Patients with Rare Congenital Anemias: Next Generation Sequencing Based Approach

IntroductionDiagnosis of inherited anemias is based on automated red cell indices, morphology and reticulocyte count in an appropriate clinical presentation and a stepwise diagnostic algorithm needs to be followed. Major inherited causes of hemolysis include the hemoglobinopathies, membranopathies and enzymopathies. In contrast, ineffective erythropoiesis, a term that refers to a disturbance in proliferation, differentiation and maturation of erythroblasts, includes megaloblastic anemia, myelodysplastic syndromes, thalassemias, and congenital dyserythropoietic anemias (CDA). Phenotypes vary from severe transfusion-dependent hemolytic anemia (HA) to fully compensated hemolysis. Patients are encountered where the entire spectrum of diagnostic tests cannot identify the etiology. Causal genes implicated are numerous, making a gene-by-gene approach time consuming, expensive and laborious. Hence, the use of targeted resequencing can expedite molecular diagnosis, genetic counseling and prenatal diagnosis if indicated.ObjectivesThis study aimed at determining the spectrum of mutations in uncommon HA and CDA.MethodsSixty-one patients (HA=44, CDA=17) with clinical and laboratory evidence suggestive of HA/CDA were enrolled after excluding common causes of anemias. Various biochemical and molecular tests were used to exclude glucose-6-phosphate dehydrogenase (G6PD) deficiency, hemoglobinopathies, autoimmune HA, hereditary spherocytosis and pyruvate kinase (PK) deficiency. Peripheral blood genomic DNA was extracted using QIAamp DNA Blood Midi Kit, quantified on NanoDrop 2000 spectrophotometer and Qubit® 2.0 Fluorometer. Common G6PD, PKLR variants, SEC23B mutation were excluded by molecular tests. Since the genetic defects in Indian patients with unexplained HA/CDA are not available, we first tested 16 patients with a comprehensive commercially available sequencing panel that covers 4,813 clinically-relevant genes. We next sought to streamline testing and designed a customized panel of 55 genes (Illumina). Variants were annotated and classified and the most likely disease-causing variants were validated by Sanger sequencing in the patient and available affected and unaffected family members.ResultsOut of 61 patient's sequenced, unexpected pyruvate kinase (PK) deficiency were found in 11 patients. PK enzyme activity assay was within normal limits in all these cases. Three patients with G6PD deficiency, 2 patients with glucose-6-phosphate isomerase deficiency and 1 case with hexokinase deficiency were found. Of 5 patients with stomatocytes on peripheral blood film, 3 had Mediterranean stomatocytosis/ macrothrombocytopenia (ABCG5/ABCG8) and 2 were found to have overhydrated hereditary stomatocytosis (RHAG).We also found 6 cases of HA to have a mutation in erythrocyte membrane protein-coding genes. Heterozygous nonsense mutation in the ANK1 gene was found in 2 cases and compound heterozygous missense mutations in ANK1 were seen in 1. Missense mutations were also found in SPTB gene in other 3 cases. Xerocytosis or dehydrated hereditary stomatocytosis (PEIZO1) was found in 3 patients.Seventeen cases for suspected CDA were studied and SEC23B mutations were present in 7. In one patient only heterozygous mutation was found. There could be the possibility of intronic mutation or long deletion/insertion in her. Notably, 6 patients with CDA showed mutations in PKLR (n=3), 1 each had mutations in MTRR, SPTB and PIEZO1 genes. We also found a mutation in GATA1 gene in a patient of CDA with thrombocytopenia.Conclusion(s)HA/CDA showed highly varied etiology. Our experience demonstrates the high diagnostic yield (>75%) of targeted resequencing for molecular diagnosis of unexplained anemias. It is cost effective and can expedite the diagnosis where conventional testing is not helpful. Timely detection of the etiology was helpful in genetic counseling. It not only offered therapeutic benefits to our patients but may help us in future antenatal diagnosis if required. Therapeutic implications include performing splenectomy which can ameliorate the anemia in selected subgroups of patients (HS, PK deficiency, CDA type II). DisclosuresNo relevant conflicts of interest to declare.

Clinical manifestations of erythrocyte membrane protein coding gene mutations in hereditary spherocytosis

目的探讨遗传性球形红细胞增多症（HS）红细胞膜蛋白基因突变与患者临床表现的关系。方法对25例HS患者的HS常见突变进行靶向测序，并根据突变与患者临床特征评估两者相关性。结果25例患者中男13例，女12例，中位年龄20（4~55）岁。中位HGB为105（65~150）g/L，其中代偿性溶血9例，轻度贫血9例，中度贫血3例，重度贫血4例。18例（72%）患者携带与HS致病相关的基因突变，包括6例ANK1突变、6例SLC4A1突变、5例SPTB突变和1例EPB41突变；7例（28%）无相关突变。SPTB突变与SLC4A1突变患者中以男性为主，携带基因突变的HS患者发病年龄（P=0.130）与HGB水平（P=0.585）与无突变患者差异无统计学意义，但EMA标记缺失的程度（P=0.015）、酸化甘油溶血试验（AGLT50）（P=0.032）以及红细胞渗透脆性试验（EOF）开始溶血浓度（P=0.027）显著高于无突变患者。结论HS红细胞膜蛋白基因突变筛查对明确诊断有指导意义，无突变患者临床溶血表现较轻。

A Comprehensive Computational Analysis of Mycobacterium Genomes Pinpoints the Genes Co-occurring with YczE, a Membrane Protein Coding Gene Under the Putative Control of a MocR, and Predicts its Function.

Bacterial proteins belonging to the YczE family are predicted to be membrane proteins of yet unknown function. In many bacterial species, the yczE gene coding for the YczE protein is divergently transcribed with respect to an adjacent transcriptional regulator of the MocR family. According to in silico predictions, proteins named YczR are supposed to regulate the expression of yczE genes. These regulators linked to the yczE genes are predicted to constitute a subfamily within the MocR family. To put forward hypotheses amenable to experimental testing about the possible function of the YczE proteins, a phylogenetic profile strategy was applied. This strategy consists in searching for those genes that, within a set of genomes, co-occur exclusively with a certain gene of interest. Co-occurrence can be suggestive of a functional link. A set of 30 mycobacterial complete proteomes were collected. Of these, only 16 contained YczE proteins. Interestingly, in all cases each yczE gene was divergently transcribed with respect to a yczR gene. Two orthology clustering procedures were applied to find proteins co-occurring exclusively with the YczE proteins. The reported results suggest that YczE may be involved in the membrane translocation and metabolism of sulfur-containing compounds mostly in rapidly growing, low pathogenicity mycobacterial species. These observations may hint at potential targets for therapies to treat the emerging opportunistic infections provoked by the widespread environmental mycobacterial species and may contribute to the delineation of the genomic and physiological differences between the pathogenic and non-pathogenic mycobacterial species.

Genomic signatures of adaptation to wine biological ageing conditions in biofilm-forming flor yeasts.

The molecular and evolutionary processes underlying fungal domestication remain largely unknown despite the importance of fungi to bioindustry and for comparative adaptation genomics in eukaryotes. Wine fermentation and biological ageing are performed by strains of S.cerevisiae with, respectively, pelagic fermentative growth on glucose and biofilm aerobic growth utilizing ethanol. Here, we use environmental samples of wine and flor yeasts to investigate the genomic basis of yeast adaptation to contrasted anthropogenic environments. Phylogenetic inference and population structure analysis based on single nucleotide polymorphisms revealed a group of flor yeasts separated from wine yeasts. A combination of methods revealed several highly differentiated regions between wine and flor yeasts, and analyses using codon-substitution models for detecting molecular adaptation identified sites under positive selection in the high-affinity transporter gene ZRT1. The cross-population composite likelihood ratio revealed selective sweeps at three regions, including in the hexose transporter gene HXT7, the yapsin gene YPS6 and the membrane protein coding gene MTS27. Our analyses also revealed that the biological ageing environment has led to the accumulation of numerous mutations in proteins from several networks, including Flo11 regulation and divalent metal transport. Together, our findings suggest that the tuning of FLO11 expression and zinc transport networks are a distinctive feature of the genetic changes underlying the domestication of flor yeasts. Our study highlights the multiplicity of genomic changes underlying yeast adaptation to man-made habitats and reveals that flor/wine yeast lineage can serve as a useful model for studying the genomics of adaptive divergence.

Single strain infection of adult and larval cardamom thrips (Sciothrips cardamomi) by Wolbachia subgroup Con belonging to supergroup B in India

The presence of Wolbachia in cardamom thrips (Sciothrips cardamomi), a major insect pest of cardamom (Eletteria cardamomum) was detected by amplification of the fast evolving outer membrane protein coding gene wsp of the bacterium. Studies on the identity of Wolbachia showed infection with only subgroup Con belonging to supergroup B, and both male and female thrips were infected with the same Wolbachia subgroup. The incidence of Wolbachia infection varied from 15.0 to 87.8% in thrips collected from seven cardamom growing districts of different States in India. The overall infection rate was 53.5% with 57.1% male and 50.6% female populations infected with the bacterium. DNA sequencing and phylogenetic analysis revealed that all the Wolbachia isolates from thrips clustered together indicating that all thrips were infected by the same Wolbachia strain. This is the first ever report of Wolbachia infection in cardamom thrips and the possibility of using the bacterium as a tool in biological control of this important insect pest is discussed.

Detection ofCoxiella burnetiiDNA in Wildlife and Ticks in Northern Queensland, Australia

Wild animals and the tick species that feed on them form the natural transmission cycle and reservoir of Coxiella burnetii. The objective of this study was to determine whether C. burnetii was present in the blood of host animals and their ticks in northern Queensland, Australia. Three genomic targets were detected using real-time PCR assays-the Coxiella-specific outer membrane protein coding gene (Com1), the multicopy insertion element (IS1111), and the isocitrate dehydrogenase gene (Icd). Quantification of the single-copy targets identified a range of 1.48×10(1) to 4.10×10(3) C. burnetii genome equivalents per microliter in the ticks tested. The detection of Coxiella based on the presence of the genomic targets indicated the occurrence of C. burnetii in both the ticks and whole blood of a variety of native Australian marsupials and confirms these animals are capable of acting as reservoirs of Q fever in northern Queensland.

H+-myo-Inositol Transporter SLC2A13 as a Potential Marker for Cancer Stem Cells in an Oral Squamous Cell Carcinoma

Cancer Stem Cells (CSCs) from tumors of different phenotypes possess a marked capacity for proliferation, self-renewal, and differentiation. They also play a critical role in cancer recurrence. Although CSC has been regarded as a new target for cancer therapy, the fundamental questions in the CSC study have not been resolved mainly due to the lack of proper CSC markers. To find new CSC markers for oral squamous cell carcinoma (OSCC), we cultured the primary tumor cells from OSCC patients the regular culture condition and the sphere-forming culture condition to enrich primary tumor cells and potential CSCs. We compared gene expression profiles between sphere-forming and non-forming cells, thus identifying that 23 membrane protein-coding genes were over-expressed in the sphere-forming cells. Among them, 8 belonged to the solute carrier (SLC) protein family. H⁺-myo-inositol transporter SLC2A13 and monocarbohydrate transporter SLC16A6 genes that were consistently increased in the sphere-forming cells in the primary cultures of OSCC samples. Confocal microscopy revealed that SLC2A13-expressing cells were embedded in the limited areas of tumor tissue as a cluster, while SLC16A6 was uniformly detected in hyperplastic epithelium. Moreover, SLC2A13 an expression was induced in human breast adenocarcinoma MCF7 cells after serum starvation. Taken together, our results suggest that SLC2A13 can be a potential markers for CSC in various tumors.

Unbiased Quantitation of Escherichia coli Membrane Proteome Using Phase Transfer Surfactants

We developed a sample preparation protocol for rapid and unbiased analysis of the membrane proteome using an alimentary canal-mimicking system in which proteases are activated in the presence of bile salts. In this rapid and unbiased protocol, immobilized trypsin is used in the presence of deoxycholate and lauroylsarcosine to increase digestion efficiency as well as to increase the solubility of the membrane proteins. Using 22.5 microg of Escherichia coli whole cell lysate, we quantitatively demonstrated that membrane proteins were extracted and digested at the same level as soluble proteins without any solubility-related bias. The recovery of membrane proteins was independent of the number of transmembrane domains per protein. In the analysis of the membrane-enriched fraction from 22.5 microg of E. coli cell lysate, the abundance distribution of the membrane proteins was in agreement with that of the membrane protein-coding genes when this protocol, coupled with strong cation exchange prefractionation prior to nano-LC-MS/MS analysis, was used. Because this protocol allows unbiased sample preparation, protein abundance estimation based on the number of observed peptides per protein was applied to both soluble and membrane proteins simultaneously, and the copy numbers per cell for 1,453 E. coli proteins, including 545 membrane proteins, were successfully obtained. Finally, this protocol was applied to quantitative analysis of guanosine tetra- and pentaphosphate-dependent signaling in E. coli wild-type and relA knock-out strains.