Proteomic Profiling of Burkholderia thailandensis During Host Infection Using Bio-Orthogonal Noncanonical Amino Acid Tagging (BONCAT)

Burkholderia pseudomallei and B. mallei are the causative agents of melioidosis and glanders, respectively, and are often fatal to humans and animals. Owing to the high fatality rate, potential for spread by aerosolization, and the lack of efficacious therapeutics, B. pseudomallei and B. mallei are considered biothreat agents of concern. In this study, we investigate the proteome of Burkholderia thailandensis, a closely related surrogate for the two more virulent Burkholderia species, during infection of host cells, and compare to that of B. thailandensis in culture. Studying the proteome of Burkholderia spp. during infection is expected to reveal molecular mechanisms of intracellular survival and host immune evasion; but proteomic profiling of Burkholderia during host infection is challenging. Proteomic analyses of host-associated bacteria are typically hindered by the overwhelming host protein content recovered from infected cultures. To address this problem, we have applied bio-orthogonal noncanonical amino acid tagging (BONCAT) to B. thailandensis, enabling the enrichment of newly expressed bacterial proteins from virtually any growth condition, including host cell infection. In this study, we show that B. thailandensis proteins were selectively labeled and efficiently enriched from infected host cells using BONCAT. We also demonstrate that this method can be used to label bacteria in situ by fluorescent tagging. Finally, we present a global proteomic profile of B. thailandensis as it infects host cells and a list of proteins that are differentially regulated in infection conditions as compared to bacterial monoculture. Among the identified proteins are quorum sensing regulated genes as well as homologs to previously identified virulence factors. This method provides a powerful tool to study the molecular processes during Burkholderia infection, a much-needed addition to the Burkholderia molecular toolbox

Genotype Dependent Somatic Embryogenesis from Egyptian Rice Mature Zygotic Embryos

Abs tract: Seven agronomically and genetically different rice (Oryza sativa L.) c u lt iv a rs were compared for their capacity for callus induction and plant regene ra t io n from mature zygotic embryos . W hite embryonic calli were formed within three weeks in the pres ence of 3 mgl 2,4,D . T h e s o matic Giza 178 s howed high regeneration fre q u e ncy (39%) followed by Giza 175 (38%) where the cultivar H1 s howed the lowes t record (24%). The data obtained fro m t h e random amplified polymorphic DNA analys is s howed that a total of 45 DNA markers were detected among the s e v e n ric e cultivars of wh ich, 29 bands were polymorphic (64.4%) and can be cons idered as us eful RA PD markers for th e rice cultivars us ed. Based on the data obtained by RA PD analys is , it was pos s ible to dis c riminate between the different genotyp e s u s e d . Sixteen out of the twenty nine polymorphic RAPD markers generated were found to be genotype-specific. Thes e markers can be verified as being RAPD markers ass ociated with the rege n e ra t io n c a pacity in the s even rice genotypes. Genetic s tability in tis s uecultured rice plants was examined by randomly amplified polymorphic DNA (RA PD) an a ly s is . T he genetic s imilarity between the mother and the regenerated plants was hig h fo r Giza 178 (91%) and it w a s 87.5 and 82 for Giza 175 and Sakha 102 respectively. The cultivar Giza 178 proved t o b e a us eful genetic res ource with unique regeneration ability and can be us ed for efficient generation of trans genic rice plants

Ionotropic Glutamate Receptor AMPA 1 Is Associated with Ovulation Rate

Ionotropic glutamate receptors mediate most excitatory neurotransmission in the central nervous system by opening ion channels upon the binding of glutamate. Despite the essential roles of glutamate in the control of reproduction and anterior pituitary hormone secretion, there is a limited understanding of how glutamate receptors control ovulation. Here we reveal the function of the ionotropic glutamate receptor AMPA-1 (GRIA1) in ovulation. Based on a genome-wide association study in Bos taurus, we found that ovulation rate is influenced by a variation in the N-terminal leucine/isoleucine/valine-binding protein (LIVBP) domain of GRIA1, in which serine is replaced by asparagine. GRIA1Asn has a weaker affinity to glutamate than GRIA1Ser, both in Xenopus oocytes and in the membrane fraction of bovine brain. This single amino acid substitution leads to the decreased release of gonadotropin-releasing hormone (GnRH) in immortalized hypothalamic GT1-7 cells. Cows with GRIA1Asn have a slower luteinizing hormone (LH) surge than cows with GRIA1Ser. In addition, cows with GRIA1Asn possess fewer immature ovarian follicles before superovulation and have a lower response to hormone treatment than cows with GRIA1Ser. Our work identified that GRIA1 is a critical mediator of ovulation and that GRIA1 might be a useful target for reproductive therapy

Variations in Helicobacter pylori Cytotoxin-Associated Genes and Their Influence in Progression to Gastric Cancer: Implications for Prevention

Helicobacter pylori (HP) is a bacterium that colonizes the human stomach and can establish a long-term infection of the gastric mucosa. Persistent Hp infection often induces gastritis and is associated with the development of peptic ulcer disease, atrophic gastritis, and gastric adenocarcinoma. Virulent HP isolates harbor the cag (cytotoxin-associated genes) pathogenicity island (cagPAI), a 40 kb stretch of DNA that encodes components of a type IV secretion system (T4SS). This T4SS forms a pilus for the injection of virulence factors into host target cells, such as the CagA oncoprotein. We analyzed the genetic variability in cagA and other selected genes of the HP cagPAI (cagC, cagE, cagL, cagT, cagV and cag Gamma) using DNA extracted from frozen gastric biopsies or from clinical isolates. Study subjects were 95 cagA+ patients that were histologically diagnosed with chronic gastritis or gastric cancer in Venezuela and Mexico, areas with high prevalence of Hp infection. Sequencing reactions were carried out by both Sanger and next-generation pyrosequencing (454 Roche) methods. We found a total of 381 variants with unambiguous calls observed in at least 10% of the originally tested samples and reference strains. We compared the frequencies of these genetic variants between gastric cancer and chronic gastritis cases. Twenty-six SNPs (11 non-synonymous and 14 synonymous) showed statistically significant differences (P<0.05), and two SNPs, in position 1039 and 1041 of cagE, showed a highly significant association with cancer (p-value = 2.07×10−6), and the variant codon was located in the VirB3 homology domain of Agrobacterium. The results of this study may provide preliminary information to target antibiotic treatment to high-risk individuals, if effects of these variants are confirmed in further investigations

Immunoproteasome LMP2 60HH Variant Alters MBP Epitope Generation and Reduces the Risk to Develop Multiple Sclerosis in Italian Female Population

Background: Albeit several studies pointed out the pivotal role that CD4+T cells have in Multiple Sclerosis, the CD8+ T cells involvement in the pathology is still in its early phases of investigation. Proteasome degradation is the key step in the production of MHC class I-restricted epitopes and therefore its activity could be an important element in the activation and regulation of autoreactive CD8+ T cells in Multiple Sclerosis. Methodology/Principal Findings: Immunoproteasomes and PA28-ab regulator are present in MS affected brain area and accumulated in plaques. They are expressed in cell types supposed to be involved in MS development such as neurons, endothelial cells, oligodendrocytes, macrophages/macroglia and lymphocytes. Furthermore, in a genetic study on 1262 Italian MS cases and 845 controls we observed that HLA-A*02+ female subjects carrying the immunoproteasome LMP2 codon 60HH variant have a reduced risk to develop MS. Accordingly, immunoproteasomes carrying the LMP2 60H allele produce in vitro a lower amount of the HLA-A*0201 restricted immunodominant epitope MBP111\u2013119. Conclusion/Significance: The immunoproteasome LMP2 60HH variant reduces the risk to develop MS amongst Italian HLAA* 02+ females. We propose that such an effect is mediated by the altered proteasome-dependent production of a specific MBP epitope presented on the MHC class I. Our observations thereby support the hypothesis of an involvement of immunoproteasome in the MS pathogenesis

The genetic basis and evolution of red blood cell sickling in deer

Crescent-shaped red blood cells, the hallmark of sickle-cell disease, present a striking departure from the biconcave disc shape normally found in mammals. Characterized by increased mechanical fragility, sickled cells promote haemolytic anaemia and vaso-occlusions and contribute directly to disease in humans. Remarkably, a similar sickle-shaped morphology has been observed in erythrocytes from several deer species, without obvious pathological consequences. The genetic basis of erythrocyte sickling in deer, however, remains unknown. Here, we determine the sequences of human β-globin orthologues in 15 deer species and use protein structural modelling to identify a sickling mechanism distinct from the human disease, coordinated by a derived valine (E22V) that is unique to sickling deer. Evidence for long-term maintenance of a trans-species sickling/non-sickling polymorphism suggests that sickling in deer is adaptive. Our results have implications for understanding the ecological regimes and molecular architectures that have promoted convergent evolution of sickling erythrocytes across vertebrates

Neuroprotection by adenosine in the brain: From A1 receptor activation to A2A receptor blockade

Adenosine is a neuromodulator that operates via the most abundant inhibitory adenosine A1 receptors (A1Rs) and the less abundant, but widespread, facilitatory A2ARs. It is commonly assumed that A1Rs play a key role in neuroprotection since they decrease glutamate release and hyperpolarize neurons. In fact, A1R activation at the onset of neuronal injury attenuates brain damage, whereas its blockade exacerbates damage in adult animals. However, there is a down-regulation of central A1Rs in chronic noxious situations. In contrast, A2ARs are up-regulated in noxious brain conditions and their blockade confers robust brain neuroprotection in adult animals. The brain neuroprotective effect of A2AR antagonists is maintained in chronic noxious brain conditions without observable peripheral effects, thus justifying the interest of A2AR antagonists as novel protective agents in neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease, ischemic brain damage and epilepsy. The greater interest of A2AR blockade compared to A1R activation does not mean that A1R activation is irrelevant for a neuroprotective strategy. In fact, it is proposed that coupling A2AR antagonists with strategies aimed at bursting the levels of extracellular adenosine (by inhibiting adenosine kinase) to activate A1Rs might constitute the more robust brain neuroprotective strategy based on the adenosine neuromodulatory system. This strategy should be useful in adult animals and especially in the elderly (where brain pathologies are prevalent) but is not valid for fetus or newborns where the impact of adenosine receptors on brain damage is different

دراسة جيوفيزيقية لشدة المغناطيسية الكلية منطقة الفيوم بالصحراء الغربية - مصر

The present paper deals with the interpretation of the aeromagnetic survey executed over the Faiyum area. Western Desert, Egypt. Qualitative as well as quantitative interpretation of the aeromagnetic data were carried out to obtain more information about the crystalline basement structure and the local structure in the sedimentary section. The analysis of the constructed magnetic maps which include the total intensity map, the vertical map, the regional map, the residual map, the second vertical derivative map and the downward continuation maps serve as basis for revealing the structural pattern of the basement complex, and the shallower structures. In connection with a quantitative interpretation by 2-dimensional model bodies a structural map for the basement was constructed.تضمنت هذه الدراسة بمنطقة الفيوم تحليل نوعي وكمي لشدة المغناطيسية الكلية للتعرف على تفاصيل شكل سطح القاعدة والتعرف على تراكيبها والتراكيب الموجودة في القطاع الصخري الرسوبي الدي يعلوها . ولقد وجد أن العمق المحسوب لسطح صخور القاعدة في اجزاء منطقة البحث المختلفة متفق مع نتائج الحفر ووجد أن متوسط هذا العمق يتراوح بين 2.5 و 3 كليو متر

دراسة نوعية لاتجاهات الشواذ المغناطيسية بمنطقة الفيوم بالصحراء الغربية - مصر

The present paper contains brief results of the trend analysis of the aeromagnetic survey in the Fayum District, Western Desert, Egypt. The trend analysis was the constructed magnetic maps which included the total magnetic intensity anomalies, the residual magnetic anomalies, the second vertical derivative magnetic anomalies and the downward continuation magnetic anomalies in depth of two kms to 5 kms. The trend patterns of the anomalies and their distribution were plotted in azimuth-frequency diagrams. Results show that the trends of the anomalies are characterized by a degree of preference in orientation; specially the pattern is dominated by WNW and ENE orientations at both the Poisson's 95% and 99% significance levels.تقوم هذه الدراسة أساسا على استخدام الخرائط المغناطيسية لمنطقة البحث وتد اثبتت الدراسة النوعية لاتجاهات الشواذ المغناطيسية أن الاتجاهات التركيبية السائدة هي شمال شرق ، شمال غرب ، وشرق غرب - وأنها مرتبطة بتصدع كلى في صخور القاعدة وبالدراسة الكمية المبنية على استخدام التوزيع البواسيني الإحصائي لدرجة ثقة 95% و 99% فان الاتجاهات التي سادت هي غرب شمال غرب وشرق شرق شمال شرق