scholarly journals yggS Encoding Pyridoxal 5′-Phosphate Binding Protein Is Required for Acidovorax citrulli Virulence

2022 ◽  
Vol 12 ◽  
Author(s):  
Yuanjie Wang ◽  
Yuqiang Zhao ◽  
Liming Xia ◽  
Lin Chen ◽  
Yajie Liao ◽  
...  
Keyword(s):  
Hypersensitive Response ◽  
Molecular Mechanisms ◽  
Pyridoxal Phosphate ◽  
Economic Losses ◽  
Insertion Mutant ◽  
Limited Information ◽  
Phosphate Binding ◽  
Acidovorax Citrulli ◽  
Phosphate Binding Protein

Bacterial fruit blotch, caused by seed-borne pathogen Acidovorax citrulli, poses a serious threat to the production of cucurbits globally. Although the disease can cause substantial economic losses, limited information is available about the molecular mechanisms of virulence. This study identified that, a random transposon insertion mutant impaired in the ability to elicit a hypersensitive response on tobacco. The disrupted gene in this mutant was determined to be Aave_0638, which is predicted to encode a YggS family pyridoxal phosphate-dependent enzyme. YggS is a highly conserved protein among multiple organisms, and is responsible for maintaining the homeostasis of pyridoxal 5′-phosphate and amino acids in cells. yggS deletion mutant of A. citrulli strain XjL12 displayed attenuated virulence, delayed hypersensitive response, less tolerance to H2O2 and pyridoxine, increased sensitivity to antibiotic β-chloro-D-alanine, and reduced swimming. In addition, RNA-Seq analysis demonstrated that yggS was involved in regulating the expression of certain pathogenicity-associated genes related to secretion, motility, quorum sensing and oxidative stress response. Importantly, YggS significantly affected type III secretion system and its effectors in vitro. Collectively, our results suggest that YggS is indispensable for A.citrulli virulence and expands the role of YggS in the biological processes.

scholarly journals Rv3722c governs aspartate-dependent nitrogen metabolism in Mycobacterium tuberculosis

2019 ◽  
Author(s):  
Robert Jansen ◽  
Lungelo Mandyoli ◽  
Ryan Hughes ◽  
Shoko Wakabayashi ◽  
Jessica Pinkham ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Unknown Function ◽  
Drug Targets ◽  
Pyridoxal Phosphate ◽  
Phosphate Binding ◽  
Nitrogen Distribution ◽  
Natural Reservoir ◽  
Phosphate Binding Protein ◽  
Distinguishing Features

AbstractOrganisms are defined by their genomes, yet many distinguishing features of a given organism are encoded by genes that are functionally unannotated. Mycobacterium tuberculosis (Mtb), the leading cause of death due to a single microbe, co-evolved with humans as its only known natural reservoir, yet the factors mediating Mtb’s pathogenicity remain incompletely defined. rv3722c is a gene of unknown function predicted to encode a pyridoxal phosphate binding protein and to be essential for in vitro growth of Mtb. Using metabolomic, genetic and structural approaches, we show that Rv3722c is the primary aspartate aminotransferase of Mtb and mediates an essential but underrecognized role in metabolism: nitrogen distribution. Together with the attenuation of Rv3722c-deficient Mtb in macrophages and mice, these results identify aspartate biosynthesis and nitrogen distribution as potential species-selective drug targets in Mtb.

scholarly journals Emerging role of extracellular vesicles in communication of preimplantation embryos in vitro

2017 ◽  
Vol 29 (1) ◽  
pp. 66 ◽  
Author(s):  
Krishna C. Pavani ◽  
Carmen Alminana ◽  
Eline Wydooghe ◽  
Maaike Catteeuw ◽  
Miguel A. Ramírez ◽  
...  
Keyword(s):  
Embryo Development ◽  
Extracellular Vesicles ◽  
Molecular Mechanisms ◽  
Culture Media ◽  
Somatic Cells ◽  
Indirect Evidence ◽  
Preimplantation Embryos ◽  
Limited Information ◽  
Paracrine Factors

In vitro, efficient communication between mammalian embryos in groups or between embryos and cocultured somatic cells implies that there is a sender, a message and a receiver that is able to decode the message. Embryos secrete a variety of autocrine and paracrine factors and, of these, extracellular vesicles have recently been implicated as putative messengers in embryo–embryo communication, as well as in communication of the embryo with the maternal tract. Extracellular vesicles (EVs) are membrane-bound vesicles that are found in biofluids and in culture media conditioned by the presence of embryos or cells. EVs carry and transfer regulatory molecules, such as microRNAs, mRNAs, lipids and proteins. We conducted a systematic search of the literature to review and present the currently available evidence regarding the possible roles of EVs in in vitro embryo communication and embryo development. It is important to note that there is limited information available on the molecular mechanisms and many of the biologically plausible functions of EVs in embryo communication have not yet been substantiated by conclusive experimental evidence. However, indirect evidence, such as the use of media conditioned by embryos or by somatic cells with improved embryo development as a result, may indicate that EVs can be an important asset for the development of tailor-made media, allowing better embryo development in vitro, even for single embryo culture.

scholarly journals Towards a structural characterization of the IFIT antiviral complex

2014 ◽  
Vol 70 (a1) ◽  
pp. C246-C246
Author(s):  
Christina Dimech ◽  
Bhushan Nagar
Keyword(s):  
Innate Immunity ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Gel Filtration ◽  
Innate Immune ◽  
Limited Information ◽  
Virus Elimination ◽  
Tetratricopeptide Repeats ◽  
Immune Effector

Our first line of defense against viral pathogens is the innate immune system. Interferon-induced proteins with tetratricopeptide repeats (IFITs) are innate immune effector molecules that are thought to confer antiviral defense through the formation of the IFIT `Interactome', a multiprotein complex made up of IFIT1, IFIT2, IFIT3 and several other host factors1. Through IFIT1, this complex has the ability to distinguish self from non-self nucleic acids such as virus-derived RNA bearing 5´-triphosphate or viral mRNA lacking 2´-O methylation on the first two nucleotides1,2. We have limited information on the architecture of this complex, its role in innate immunity, and its activity downstream of RNA binding remain unclear. To better understand the mechanisms of Interactome formation, we are investigating the structure of its core, namely the IFIT1-IFIT2-IFIT3 complex. Since it is challenging to crystallize the complex as a whole, likely due to its size and heterogeneity, we are also targeting the structure of individual components and co-crystals of interacting domains. A crystal structure of human IFIT2 is available, and our lab has solved the structure of N-terminal human IFIT1 and, more recently, N-terminal IFIT3. In this study, we aim to characterize the interaction between IFIT1 and IFIT2, and between IFIT3 and IFIT2, through gel-filtration binding assays, in vitro pull-downs and deletion mutations. Preliminary results on the expression and purification of IFIT2-deletion mutants will be presented, as well as purification of IFIT subcomplexes. Understanding the molecular mechanisms behind IFIT-mediated virus elimination will help us unravel the complexities of these interactions and significantly advance our fundamental knowledge of innate immunity, paving the way for designing novel immunotherapeutics, which could potentially complement anti-cancer strategies that rely on oncolytic RNA viruses.

scholarly journals ET-7 Roles for hENT1 and dCK in gemcitabine sensitivity and malignancy of meningioma

2021 ◽  
Vol 3 (Supplement_6) ◽  
pp. vi5-vi5
Author(s):  
Masahiro Yamamoto ◽  
Hiroyuki Uchida ◽  
Hajime Yonezawa ◽  
Nayuta Higa ◽  
Yuki Yamada ◽  
...  
Keyword(s):  
Proliferative Activity ◽  
Intracellular Transport ◽  
Molecular Mechanisms ◽  
High Morbidity ◽  
High Grade ◽  
Nucleoside Transporter ◽  
Limited Information ◽  
Ki 67 ◽  
Who Grade

Abstract Background: High-grade meningiomas are aggressive tumors with high morbidity and mortality rates that frequently recur even after surgery and adjuvant radiotherapy. However, limited information is currently available on the biology of these tumors, and no alternative adjuvant treatment options exist. Although we previously demonstrated that high-grade meningioma cells were highly sensitive to gemcitabine in vitro and in vivo, the underlying molecular mechanisms remain unknown. Methods: We examined the roles of hENT1 (human equilibrative nucleoside transporter 1) and dCK (deoxycytidine kinase) in the gemcitabine sensitivity and growth of meningioma cells in vitro. Tissue samples from meningiomas (26 WHO grade I and 21 WHO grade II/III meningiomas) were immunohistochemically analyzed for hENT1 and dCK as well as for Ki-67 as a marker of proliferative activity. Results: hENT1 and dCK, which play critical roles in the intracellular transport and activation of gemcitabine, respectively, were responsible for the high gemcitabine sensitivity of high-grade meningioma cells and were strongly expressed in high-grade meningiomas. hENT1 expression was required for the proliferation and survival of high-grade meningioma cells and dCK expression. Furthermore, high hENT1 and dCK expression levels correlated with stronger tumor cell proliferative activity and shorter survival in meningioma patients. Conclusions: The present results suggest that hENT1 is a key molecular factor influencing the growth capacity and gemcitabine sensitivity of meningioma cells and also that hENT1, together with dCK, may be a viable prognostic marker for meningioma patients as well as a predictive marker of their responses to gemcitabine.

scholarly journals Heparan Sulfate Proteoglycan Is an Important Attachment Factor for Cell Entry of Akabane and Schmallenberg Viruses

2017 ◽  
Vol 91 (15) ◽  
Author(s):  
Shin Murakami ◽  
Akiko Takenaka-Uema ◽  
Tomoya Kobayashi ◽  
Kentaro Kato ◽  
Masayuki Shimojima ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Molecular Mechanisms ◽  
Substantial Reduction ◽  
Heparan Sulfate Proteoglycan ◽  
Economic Losses ◽  
Sulfate Proteoglycan ◽  
Content Type ◽  
Cellular Attachment ◽  
Attachment Factor

ABSTRACT Akabane virus (AKAV) and Schmallenberg virus (SBV) are members of the genus Orthobunyavirus, which are transmitted by arthropod vectors with a broad cellular tropism in vitro as well as in vivo. Both AKAV and SBV cause arthrogryposis-hydranencephaly syndrome in ruminants. The main cellular receptor and attachment factor for entry of these orthobunyaviruses are unknown. Here, we found that AKAV and SBV infections were inhibited by the addition of heparin or enzymatic removal of cell surface heparan sulfates. To confirm this finding, we prepared heparan sulfate proteoglycan (HSPG)-knockout (KO) cells by using a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system and measured the quantities of binding of these viruses to cell surfaces. We observed a substantial reduction in AKAV and SBV binding to cells, limiting the infections by these viruses. These data demonstrate that HSPGs are important cellular attachment factors for AKAV and SBV, at least in vitro, to promote virus replication in susceptible cells. IMPORTANCE AKAV and SBV are the etiological agents of arthrogryposis-hydranencephaly syndrome in ruminants, which causes considerable economic losses in the livestock industry. Here, we identified heparan sulfate proteoglycan as a major cellular attachment factor for the entry of AKAV and SBV. Moreover, we found that heparin is a strong inhibitor of AKAV and SBV infections. Revealing the molecular mechanisms of virus-host interactions is critical in order to understand virus biology and develop novel live attenuated vaccines.

scholarly journals The E3 Ubiquitin Ligase TRIM25 Inhibits Tembusu Virus Replication in vitro

2021 ◽  
Vol 8 ◽  
Author(s):  
Han Kaikai ◽  
Dongmin Zhao ◽  
Yuzhuo Liu ◽  
Qingtao Liu ◽  
Xinmei Huang ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Egg Production ◽  
Molecular Mechanisms ◽  
E3 Ubiquitin Ligase ◽  
Control Measures ◽  
Neurological Dysfunction ◽  
Health Concern ◽  
Economic Losses ◽  
Tembusu Virus

Duck Tembusu virus (DTMUV) is a newly emerging pathogenic flavivirus that has caused significant economic losses to the duck industry in China since 2010 due to egg production losses and neurological dysfunction. DTMUV is a public health concern because the infection spreads rapidly among birds. Retinoic acid-inducible gene-I (RIG-I)serves as an innate immune sensor and plays a key role in host antiviral defenses. Tripartite motif-containing protein 25 (TRIM25), an E3 ubiquitin ligase, is pivotal for RIG-I ubiquitination and activation. In addition, TRIM25 acts as an interferon-stimulated gene and mediates the antiviral activity. However, the effect of duck TRIM25 on DTMUV has not been assessed. Herein, we reportthe antiviral function of TRIM25 against DTMUV. First, we constructed the pcDNA3.1-c-myc-duTRIM25 plasmid. TRIM25 has a 2052 bp open reading frame that encodes a predicted 684 amino acid protein consisting of a RING finger domain, a B-box domain, a coiled-coil domain, and a PRY/SPRY domain. The protein sequence identity with chicken, mouse, and human TRIM25 is 69.7, 47.8, and 48.3%, respectively. TRIM25 was upregulated in BHK-21 cells, duck embryo fibroblasts, and 293T cellsupon DTMUV infection. The expression of viral RNA and proteins was significantly lower in cells over expressing TRIM25 than in control cells. Furthermore, siRNA-mediated silencing of TRIM25 increased the production of viral progeny. These results help elucidate the molecular mechanisms underlying the host response to DTMUV infection and suggest potential control measures for DTMUV outbreaks.

scholarly journals Transcriptome-Based Network Analysis Reveals Hirudin Potentiates Anti-Renal Fibrosis Efficacy in UUO Rats

2021 ◽  
Vol 12 ◽  
Author(s):  
Hang-Xing Yu ◽  
Wei Lin ◽  
Kang Yang ◽  
Li-Juan Wei ◽  
Jun-Li Chen ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Pyridoxal Phosphate ◽  
Interstitial Fibrosis ◽  
Remission Rate ◽  
Enrichment Analysis ◽  
Functional Enrichment Analysis ◽  
Functional Enrichment ◽  
Phosphate Binding ◽  
Renal Protection

Background: Hirudin has been widely used in the treatment of antifibrosis. Previous studies have shown that hirudin can effectively improve the clinical remission rate of chronic kidney disease. However, the mechanism of its renal protection has not been systematically investigated.Methods: In this study, the reliability of UUO-induced renal interstitial fibrosis was evaluated by histopathological verification. High-throughput transcriptome sequencing was used to elucidate the molecular mechanism of hirudin, differentially expressed mRNAs were identified, and their functions were analyzed by GO analysis and GSEA. In addition, the RNA-seq results were validated by in vitro and vivo experiments.Results: We found 322 identical differential expressed genes (IDEs) in the UUO hirudin-treated group compared with the sham group. Functional enrichment analysis indicated that cellular amino acid metabolic processes were the most obvious enrichment pathways in biological processes. In terms of molecular functional enrichment analysis, IDEs were mainly enriched in coenzyme binding, pyridoxal phosphate binding and other pathways. In addition, microbody is the most obvious pathway for cellular components. A total of 115 signaling pathways were enriched, and AMPK, JAK-STAT, and PI3K-Akt signaling pathways were the important signaling pathways enriched. We found that PI3K, p-Akt, and mTOR expression were significantly reduced by hirudin treatment. In particular, our results showed that hirudin could induce a decrease in the expression of autophagy-related proteins such as P62, LC3, Beclin-1 in TGF-β1-induced NRK-52E cells.Conclusion: Our results suggest that hirudin may protect the kidney by ameliorating renal autophagy impairment through modulating the PI3K/Akt pathway.

scholarly journals Roles for hENT1 and dCK in gemcitabine sensitivity and malignancy of meningioma

2021 ◽  
Author(s):  
Masahiro Yamamoto ◽  
Tomomi Sanomachi ◽  
Shuhei Suzuki ◽  
Hiroyuki Uchida ◽  
Hajime Yonezawa ◽  
...  
Keyword(s):  
Proliferative Activity ◽  
Intracellular Transport ◽  
Molecular Mechanisms ◽  
High Morbidity ◽  
High Grade ◽  
Limited Information ◽  
Ki 67 ◽  
Who Grade

Abstract Background High-grade meningiomas are aggressive tumors with high morbidity and mortality rates that frequently recur even after surgery and adjuvant radiotherapy. However, limited information is currently available on the biology of these tumors and no alternative adjuvant treatment options exist. Although we previously demonstrated that high-grade meningioma cells were highly sensitive to gemcitabine in vitro and in vivo, the underlying molecular mechanisms remain unknown. Methods We examined the roles of hENT1 and dCK in the gemcitabine sensitivity and growth of meningioma cells in vitro. Tissue samples from meningiomas (26 WHO grade I and 21 WHO grade II/III meningiomas) were immunohistochemically analyzed for hENT1 and dCK as well as for Ki-67 as a marker of proliferative activity. Results hENT1 and dCK, which play critical roles in the intracellular transport and activation of gemcitabine, respectively, were responsible for the high gemcitabine sensitivity of high-grade meningioma cells and were strongly expressed in high-grade meningiomas. hENT1 expression was required for the proliferation and survival of high-grade meningioma cells and dCK expression. Furthermore, high hENT1 and dCK expression levels correlated with stronger tumor cell proliferative activity and shorter survival in meningioma patients. Conclusions The present results suggest that hENT1 is a key molecular factor influencing the growth capacity and gemcitabine sensitivity of meningioma cells and also that hENT1, together with dCK, may be a viable prognostic marker for meningioma patients as well as a predictive marker of their responses to gemcitabine.

scholarly journals Spatio-Temporal Profiling of Metarhizium anisopliae—Responsive microRNAs Involved in Modulation of Plutella xylostella Immunity and Development

2021 ◽  
Vol 7 (11) ◽  
pp. 942
Author(s):  
Junaid Zafar ◽  
Yuxin Zhang ◽  
Junlin Huang ◽  
Shoaib Freed ◽  
Rana Fartab Shoukat ◽  
...  
Keyword(s):  
Plutella Xylostella ◽  
Metarhizium Anisopliae ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Defense Mechanism ◽  
Pathogenic Fungus ◽  
Insect Pest ◽  
Luciferase Assay ◽  
Economic Losses

Metarhizium anisopliae, a ubiquitous pathogenic fungus, regulates a wide array of the insect pest population. The fungus has been employed to control Plutella xylostella, an insecticide-resistant destructive lepidopteran pest, which causes substantial economic losses in crops worldwide. Integration of modern gene-silencing technologies in pest control strategies has become more crucial to counter pesticide-resistant insects. MicroRNAs (miRNA) play essential roles in the various biological process via post-transcriptional gene regulation. In the present study, RNA-seq analysis of control (CK36h, CK72h) and fungal-infected (T36h, T72h) midguts was performed to reveal underlying molecular mechanisms occurring in larval midgut at different time courses. We aimed at exploring M. anisopliae-responsive miRNAs and their target genes involved in development and immunity. After data filtration, a combined set of 170 miRNAs were identified from all libraries. Interestingly, miR-281, miR-263, miR-1, miR-6094 and miR-8 were listed among the most abundantly expressed conserved miRNAs. Furthermore, we experimentally studied the role of differentially expressed miR-11912-5p in regulating corresponding target trypsin-like serine proteinase (Px_TLSP). The luciferase assay (in vitro) revealed that miRNA-11912-5p significantly downregulated its target gene, suggesting it might play a crucial role in defense mechanism of P. xylostella against M.+ anisopliae infection. We used synthetic miRNA mimic/inhibitor (in vivo), to overexpress/silence miRNA, which showed harmful effects on larval duration, survival and adult fecundity. Additionally, fungal application in the presence of mimics revealed enhanced sensitivity of P. xylostella to infection. Our finding provides an insight into the relatively obscure molecular mechanisms involved in insect midgut during the fungal infection.

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