Comparative proteomic analysis of Salmonella enterica serovar Typhimurium ppGpp-deficient mutant to identify a novel virulence protein required for intracellular survival in macrophages

<p>Abstract</p> <p>Background</p> <p>The global ppGpp-mediated stringent response in pathogenic bacteria plays an important role in the pathogenesis of bacterial infections. In <it>Salmonella enterica </it>serovar Typhimurium (<it>S</it>. Typhimurium), several genes, including virulence genes, are regulated by ppGpp when bacteria are under the stringent response. To understand the control of virulence genes by ppGpp in <it>S</it>. Typhimurium, agarose 2-dimensional electrophoresis (2-DE) combined with mass spectrometry was used and a comprehensive 2-DE reference map of amino acid-starved <it>S</it>. Typhimurium strain SH100, a derivative of ATCC 14028, was established.</p> <p>Results</p> <p>Of the 366 examined spots, 269 proteins were successfully identified. The comparative analysis of the wild-type and ppGpp⁰mutant strains revealed 55 proteins, the expression patterns of which were affected by ppGpp. Using a mouse infection model, we further identified a novel virulence-associated factor, STM3169, from the ppGpp-regulated and <it>Salmonella</it>-specific proteins. In addition, <it>Salmonella </it>strains carrying mutations in the gene encoding STM3169 showed growth defects and impaired growth within macrophage-like RAW264.7 cells. Furthermore, we found that expression of <it>stm3169 </it>was controlled by ppGpp and SsrB, a response regulator of the two-component system located on <it>Salmonella </it>pathogenicity island 2.</p> <p>Conclusions</p> <p>A proteomic approach using a 2-DE reference map can prove a powerful tool for analyzing virulence factors and the regulatory network involved in <it>Salmonella </it>pathogenesis. Our results also provide evidence of a global response mediated by ppGpp in <it>S. enterica</it>.</p

Multiple transcripts of Ca 2ϩ channel ␣ 1 -subunits and a novel spliced variant of the ␣ 1C -subunit in rat ductus arteriosus

3 H]thymidine incorporation, suggesting that L-and T-type Ca 2ϩ channels are involved in smooth muscle cell proliferation in the DA. Third, we found that a novel alternatively spliced variant of the ␣ 1C-isoform was highly expressed in the neointimal cushion of the DA, where proliferating and migrating smooth muscle cells are abundant. The basic channel properties of the spliced variant did not differ from those of the conventional ␣1C-subunit. We conclude that multiple VDCC subunits were identified in the DA, and, in particular, ␣ 1C-and ␣1G-subunits were predominant in the DA. A novel spliced variant of the ␣1C-subunit gene may play a distinct role in neointimal cushion formation in the DA. alternative spliced; development; gene expression; fetal circulation THE DUCTUS ARTERIOSUS (DA) is a fetal arterial connection between the pulmonary artery and the descending aorta. After birth, the DA closes immediately, in accordance with its smooth muscle contraction. An increase in oxygen tension and a dramatic decline in circulating prostaglandins are the most important triggers of DA contraction (5). Generally, vascular smooth muscle contraction is induced by Ca 2ϩ / calmodulin-dependent phosphorylation of the regulatory myosin light chain, which is mediated by an increase in intracellular Ca 2ϩ . Ca 2ϩ influx through voltage-dependent Ca 2ϩ channels (VDCCs) and Ca 2ϩ release from intracellular stores are major sources of this increase (8, 26). Thus VDCCs must play an important role in vascular myogenic reactivity and tone of the DA. VDCCs are classified, according to their distinct electrophysiological and pharmacological properties, into low (Ttype) and high (L-, N-, P-, Q-, and R-type) VDCCs (20, In addition to their role in determining contractile state, a growing body of evidence has demonstrated that VDCCs play an important role in regulating differentiation and remodeling of vascular smooth muscle cells (SMCs) (14, In the present study, we identified multiple VDCC subunits in the DA by semiquantitative and quantitative RT-PCR and immunodetection. In particular, ␣ 1C -and ␣ 1G -subunits were predominant in the DA. Furthermore, we will demonstrate the identification of a novel spliced variant of the ␣ 1C -subunit gene that may play a role in neointimal cushion formation of the DA

Concomitant administration of radiation with eribulin improves the survival of mice harboring intracerebral glioblastoma

Glioblastoma is the most common and devastating type of malignant brain tumor. We recently found that eribulin suppresses glioma growth in vitro and in vivo and that eribulin is efficiently transferred into mouse brain tumors at a high concentration. Eribulin is a non‐taxane microtubule inhibitor approved for breast cancer and liposarcoma. Cells arrested in M‐phase by chemotherapeutic agents such as microtubule inhibitors are highly sensitive to radiation‐induced DNA damage. Several recent case reports have demonstrated the clinical benefits of eribulin combined with radiation therapy for metastatic brain tumors. In this study, we investigated the efficacy of a combined eribulin and radiation treatment on human glioblastoma cells. The glioblastoma cell lines U87MG, U251MG and U118MG, and SJ28 cells, a patient‐derived sphere culture cell line, were used to determine the radiosensitizing effect of eribulin using western blotting, flow cytometry and clonogenic assay. Subcutaneous and intracerebral glioma xenografts were generated in mice to assess the efficacy of the combined treatment. The combination of eribulin and radiation enhanced DNA damage in vitro. The clonogenic assay of U87MG demonstrated the radiosensitizing effect of eribulin. The concomitant eribulin and radiation treatment significantly prolonged the survival of mice harboring intracerebral glioma xenografts compared with eribulin or radiation alone (P < .0001). In addition, maintenance administration of eribulin after the concomitant treatment further controlled brain tumor growth. Aberrant microvasculature was decreased in these tumors. Concomitant treatment with eribulin and radiation followed by maintenance administration of eribulin may serve as a novel therapeutic strategy for glioblastomas

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target

Effects of hypergravity environments on amphibian development, gene expression and apoptosis

This study investigates how rearing under conditions of hypergravity affects amphibian development, Xotx2 and Xag1 gene expression and apoptosis. Uncleaved Xenopus laevis eggs 20 min after insemination, 2 cell stage embryos, and gastrula stage embryos were raised at 2G and 5G, while controls were raised in normal gravity. Apoptosis in brain and eye inner structures of hatching embryos was scored using the TUNEL staining method, and gene expression in tail-bud embryos was analyzed by whole-mount in situ hybridization. Results showed that: (1) 5G retarded the development of eggs and embryos and induced microcephaly and microphthalmia. (2) 5G suppressed the expression of the two genes, Xotx2 (involved in fore- and midbrain and eye development) and Xag1 (regulating cement gland formation). (3) Eggs and 2 cell stage embryos raised at 5G showed a greater extent of brain and eye apoptosis compared with controls, while those raised at 2G showed no significant difference. These findings suggest that high gravity suppresses certain gene functions and induces abnormal apoptosis in brain and eyes, resulting in developmental retardation and various morphological abnormalities

A New Era of Neuro-Oncology Research Pioneered by Multi-Omics Analysis and Machine Learning

Although the incidence of central nervous system (CNS) cancers is not high, it significantly reduces a patient’s quality of life and results in high mortality rates. A low incidence also means a low number of cases, which in turn means a low amount of information. To compensate, researchers have tried to increase the amount of information available from a single test using high-throughput technologies. This approach, referred to as single-omics analysis, has only been partially successful as one type of data may not be able to appropriately describe all the characteristics of a tumor. It is presently unclear what type of data can describe a particular clinical situation. One way to solve this problem is to use multi-omics data. When using many types of data, a selected data type or a combination of them may effectively resolve a clinical question. Hence, we conducted a comprehensive survey of papers in the field of neuro-oncology that used multi-omics data for analysis and found that most of the papers utilized machine learning techniques. This fact shows that it is useful to utilize machine learning techniques in multi-omics analysis. In this review, we discuss the current status of multi-omics analysis in the field of neuro-oncology and the importance of using machine learning techniques