scholarly journals Regulatory protein HilD stimulates Salmonella Typhimurium invasiveness by promoting smooth swimming via the methyl-accepting chemotaxis protein McpC

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Kendal G. Cooper ◽  
Audrey Chong ◽  
Laszlo Kari ◽  
Brendan Jeffrey ◽  
Tregei Starr ◽  
...  
Keyword(s):  
Regulatory Protein ◽  
In Vitro Assays ◽  
Flagellar Motor ◽  
Independent Mechanism ◽  
Serovar Typhimurium ◽  
Chemotaxis Proteins ◽  
Exogenous Ligands ◽  
System 1 ◽  
And Control

AbstractIn the enteric pathogen Salmonella enterica serovar Typhimurium, invasion and motility are coordinated by the master regulator HilD, which induces expression of the type III secretion system 1 (T3SS1) and motility genes. Methyl-accepting chemotaxis proteins (MCPs) detect specific ligands and control the direction of the flagellar motor, promoting tumbling and changes in direction (if a repellent is detected) or smooth swimming (in the presence of an attractant). Here, we show that HilD induces smooth swimming by upregulating an uncharacterized MCP (McpC), and this is important for invasion of epithelial cells. Remarkably, in vitro assays show that McpC can suppress tumbling and increase smooth swimming in the absence of exogenous ligands. Expression of mcpC is repressed by the universal regulator H-NS, which can be displaced by HilD. Our results highlight the importance of smooth swimming for Salmonella Typhimurium invasiveness and indicate that McpC can act via a ligand-independent mechanism when incorporated into the chemotactic receptor array.

scholarly journals Dual Role of Integrin Alpha-6 in Glioblastoma: Supporting Stemness in Proneural Stem-Like Cells While Inducing Radioresistance in Mesenchymal Stem-Like Cells

Cancers ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 3055
Author(s):  
Elisabetta Stanzani ◽  
Leire Pedrosa ◽  
Guillaume Bourmeau ◽  
Oceane Anezo ◽  
Aleix Noguera-Castells ◽  
...  
Keyword(s):  
Cellular Heterogeneity ◽  
In Vitro Assays ◽  
Rna Seq ◽  
Promising Target ◽  
Damage Response ◽  
Integrin Alpha 6 ◽  
And Control ◽  
Improve Patient

Therapeutic resistance after multimodal therapy is the most relevant cause of glioblastoma (GBM) recurrence. Extensive cellular heterogeneity, mainly driven by the presence of GBM stem-like cells (GSCs), strongly correlates with patients’ prognosis and limited response to therapies. Defining the mechanisms that drive stemness and control responsiveness to therapy in a GSC-specific manner is therefore essential. Here we investigated the role of integrin a6 (ITGA6) in controlling stemness and resistance to radiotherapy in proneural and mesenchymal GSCs subtypes. Using cell sorting, gene silencing, RNA-Seq, and in vitro assays, we verified that ITGA6 expression seems crucial for proliferation and stemness of proneural GSCs, while it appears not to be relevant in mesenchymal GSCs under basal conditions. However, when challenged with a fractionated protocol of radiation therapy, comparable to that used in the clinical setting, mesenchymal GSCs were dependent on integrin a6 for survival. Specifically, GSCs with reduced levels of ITGA6 displayed a clear reduction of DNA damage response and perturbation of cell cycle pathways. These data indicate that ITGA6 inhibition is able to overcome the radioresistance of mesenchymal GSCs, while it reduces proliferation and stemness in proneural GSCs. Therefore, integrin a6 controls crucial characteristics across GBM subtypes in GBM heterogeneous biology and thus may represent a promising target to improve patient outcomes.

132 The Developmental Competence of Bovine Oocytes Exposed to Progesterone and Luteotrophic Hormones During the Second Step of Two-Step Maturation

2018 ◽  
Vol 30 (1) ◽  
pp. 206
Author(s):  
G. Singina ◽  
I. Lebedeva ◽  
T. Taradajnic ◽  
E. Shedova ◽  
A. Lopukhov ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Calf Serum ◽  
Developmental Competence ◽  
Second Step ◽  
Russian Science ◽  
Cleavage Rate ◽  
System 1 ◽  
And Control ◽  
Bovine Oocytes

Data on effects of progesterone (P4) during in vitro maturation of bovine oocytes on their capacity for embryonic development are contradictory. Our study was aimed at characterising effects of P4 and 2 luteotropic hormones, prolactin (PRL) and LH, on bovine oocyte developmental competence during the second step of two-step maturation (from metaphase (M)I to MII). Slaughterhouse-derived cumulus-enclosed oocytes (CEO) were matured for 12 or 24 h [one-step (OS) Control] in TCM-199 containing 10% fetal calf serum (FCS), 10 μg mL−1 porcine FSH, and 10 μg mL−1 ovine LH at 38.5°C and 5% CO2. The CEO cultured for 12 h were transferred to the following culture systems: (1) TCM-199 containing 10% FCS (Control 1) or (2) a monolayer of granulosa cells (GC) precultured for 12 h in TCM-199 containing 10% FCS (Control 2); then, the oocytes were matured for next 12 h. In both systems, the medium of experimental groups was supplemented with either P4 (50 ng mL−1) or bovine PRL (25 and 50 ng mL−1) or ovine LH (5 μg mL−1). All treatments were repeated 5 to 6 times using 138 to 196 oocytes per group. Following IVM, all oocytes underwent IVF as described previously (Singina et al. 2014 Reprod. Fertil. Dev. 26, 154). Embryos were cultured in CR1aa medium until Day 5 post-insemination and then transferred to the same medium supplemented with 5% FCS and cultured to Day 7. Embryo development was evaluated at Days 2 and 7 for cleavage and blastocyst formation. Apoptosis was detected by the TUNEL method using 26 to 47 blastocysts per group (from 4 to 5 separate experiments). For each system, arcsine-transformed data were analysed by one-way ANOVA. In OS Control, the cleavage and blastocyst rates were 68.9 ± 4.4% and 22.0 ± 2.4%, respectively. Regardless of the system or medium of two-step culture, the cleavage rate did not differ from that for OS Control, varying between 57.6 and 68.4%. In the absence of GC (System 1), the blastocyst yield in the P4 group (30.4 ± 0.8%) was greater (P < 0.05) than in OS Control and Control 1 (20.2 ± 2.7%) as well as in the groups treated with LH (19.1 ± 3.0%) and 25 ng mL−1 PRL (20.1 ± 2.7%). In the presence of GC, P4 raised the yield from 16.7 ± 2.3% (Control 2) to 27.7 ± 2.4% (P < 0.05). Furthermore, in System 2, the blastocyst rate in groups treated with P4 and 50 ng mL−1 PRL (25.0 ± 2.8%) was higher (P < 0.05) than in the LH group (13.9 ± 2.6%). Meanwhile, the proportion of apoptotic nuclei (2.3-6.9%) was not associated with the system of oocyte maturation or effects of hormones studied. Our data indicate that P4 (50 ng mL−1) can enhance the developmental competence of bovine oocytes during the second step of two-step maturation regardless of the presence of granulosa cells, whereas the similar effect of PRL (50 ng mL−1) is less pronounced and depends on the granulosa-conditioned environment. This research was supported by the Russian Science Foundation (project 16-16-10069).

scholarly journals Host-Derived Metabolites Modulate Transcription ofSalmonellaGenes Involved in L-Lactate Utilization during Gut Colonization

2019 ◽  
Vol 87 (4) ◽  
Author(s):  
Caroline C. Gillis ◽  
Maria G. Winter ◽  
Rachael B. Chanin ◽  
Wenhan Zhu ◽  
Luisella Spiga ◽  
...  
Keyword(s):  
Cell Metabolism ◽  
Regulatory Protein ◽  
Transcriptional Level ◽  
Lactate Oxidation ◽  
Terminal Electron Acceptor ◽  
Content Type ◽  
Gut Colonization ◽  
Serovar Typhimurium ◽  
Lactate Utilization

ABSTRACTDuringSalmonella entericaserovar Typhimurium infection, host inflammation alters the metabolic environment of the gut lumen to favor the outgrowth of the pathogen at the expense of the microbiota. Inflammation-driven changes in host cell metabolism lead to the release ofl-lactate and molecular oxygen from the tissue into the gut lumen.Salmonellautilizes lactate as an electron donor in conjunction with oxygen as the terminal electron acceptor to support gut colonization. Here, we investigated transcriptional regulation of the respiratoryl-lactate dehydrogenase LldDin vitroand in mouse models ofSalmonellainfection. The two-component system ArcAB repressed transcription ofl-lactate utilization genes under anaerobic conditionsin vitro. The ArcAB-mediated repression oflldDtranscription was relieved under microaerobic conditions. Transcription oflldDwas induced byl-lactate but notd-lactate. A mutant lacking the regulatory protein LldR failed to inducelldDtranscription in response tol-lactate. Furthermore, thelldRmutant exhibited reduced transcription ofl-lactate utilization genes and impaired fitness in murine models of infection. These data provide evidence that the host-derived metabolites oxygen andl-lactate serve as cues forSalmonellato regulate lactate oxidation metabolism on a transcriptional level.

scholarly journals Relationship Between Fungicide Sensitivity and Control of Gummy Stem Blight of Watermelon Under Field Conditions

Plant Disease ◽  
2012 ◽  
Vol 96 (12) ◽  
pp. 1780-1784 ◽  
Author(s):  
A. Thomas ◽  
D. B. Langston ◽  
H. F. Sanders ◽  
K. L. Stevenson
Keyword(s):  
Fungicide Resistance ◽  
Field Experiments ◽  
In Vitro Assays ◽  
Didymella Bryoniae ◽  
Fungicide Sensitivity ◽  
Thiophanate Methyl ◽  
Stem Blight ◽  
Gummy Stem Blight ◽  
And Control

Gummy stem blight (GSB), caused by the fungus Didymella bryoniae, is the most destructive disease of watermelon and is managed primarily with fungicides. D. bryoniae has developed resistance to many fungicides that were once very effective, including azoxystrobin, boscalid, and thiophanate-methyl. Field experiments were conducted in Tifton (TN) and Reidsville (RV), GA in 2009 and 2010 to establish a relationship between frequency of resistance to a fungicide based on in vitro assays and its efficacy in the management of GSB. Frequency of resistance to boscalid, thiophanate-methyl, and azoxystrobin was >0.80 in isolates collected from nontreated plots in both locations and years. All isolates collected after six applications of boscalid, thiophanate-methyl, or azoxystrobin were resistant to the respective fungicide. All isolates collected from treated and nontreated plots were sensitive to tebuconazole and difenoconazole. GSB severity was assessed on a weekly basis from 63 days after planting. GSB severity in plots treated with boscalid, thiophanate-methyl, or azoxystrobin was not significantly different from that in the nontreated plots (39%, TN-2009; 45%, TN-2010; and 16%, RV-2010). GSB severity in tebuconazole-treated plots (27%, TN-2009; 14%, TN-2010; and 4%, RV-2010) was significantly lower than all other treatments and the nontreated control. There was a consistent negative association between frequency of fungicide resistance and disease control in the field. Thus, knowledge of the frequency of fungicide resistance in the pathogen population will be helpful in selecting the most effective fungicides for the management of GSB in watermelon fields.

A multi-factor trafficking site on the spliceosomal remodeling enzyme, BRR2, recruits C9ORF78 to regulate alternative splicing

2021 ◽  
Author(s):  
Alexandra Bergfort ◽  
Marco Preussner ◽  
Benno Kuropka ◽  
İbrahim Ilik ◽  
Tarek Hilal ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Large Scale ◽  
Affinity Purification ◽  
Regulatory Protein ◽  
In Vitro Assays ◽  
Home Base ◽  
Complex Protein ◽  
C Complex ◽  
Affinity Purification Mass Spectrometry

Abstract The complete inventory of regulatory factors in human spliceosomes remains unknown, and many flexibly bound components are not revealed in present spliceosome structures. The intrinsically unstructured C9ORF78 protein was detected in C complex spliceosomes but is not contained in present spliceosome structures. We found a tight interaction between C9ORF78 and the key spliceosome remodeling factor, BRR2, in a large-scale yeast two-hybrid screen, validated by targeted in vitro assays. Affinity purification/mass spectrometry and RNA UV-crosslinking analyses identified several additional C9ORF78 interactors in spliceosomes. High-resolution cryogenic electron microscopy structures revealed how C9ORF78 and the spliceosomal B complex protein FBP21 wrap around the C-terminal helicase cassette of BRR2 in a mutually exclusive manner. Knock-down of C9ORF78 led to global alternative splicing changes, including a substantial usage of alternative NAGNAG 3’-splice sites, at least in part dependent on BRR2. Comparison of our structure to C* complex spliceosomes shows that C9ORF78 could contact several detected interactors from its BRR2 “home base”, in particular the RNA helicase PRPF22, a suggested 3’-splice site regulator. Together our data firmly establish C9ORF78 as a novel, late-stage splicing regulatory protein that takes advantage of a multi-factor trafficking site on BRR2, providing one explanation for the suggested, but puzzling, roles of BRR2 during splicing catalysis and alternative splicing.

Activity of Dasatinib Against Novel KIT-N822I Mutation In Familial Cutaneous Mastocytosis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4088-4088
Author(s):  
Bartosz Wasag ◽  
Marek Niedoszytko ◽  
Anna Piskorz ◽  
Magdalena Lange ◽  
Joanna Renke ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
In Vitro Assays ◽  
Kit Mutation ◽  
Imatinib Resistant ◽  
Buccal Swabs ◽  
The Family ◽  
Cutaneous Mastocytosis ◽  
And Control ◽  
Kit D816v

Abstract Abstract 4088 Background: In contrast to mastocytosis associated with somatic KIT mutations, the accounts of familial forms of mastocytosis with KIT germline mutations are extremely rare. We report the family that met the WHO criteria for cutaneous mastocytosis, which was diagnosed in the father and two children. Patients and Methods: The clinical follow-up of mastocytosis in the father included bone marrow histopatological/cytological examinations and flow cytometry, and histopatological examination of the skin. In children tryptase measurement and skin histopatological examination was performed. The father and children presented with urticaria pigmentosa as the only manifestation of the disease. Blood, urine and buccal swabs specimens were collected from the family members. Molecular analysis of the KIT coding sequence revealed a novel missense mutation (p.N822I) in the affected members of the family. Ba/F3 cell lines expressing KIT-N822I, KIT-D816V and KIT-V559D mutants were treated with different concentrations of imatinib and dasatinib. The effect of treatment on proliferation, survival, and signaling was determined. In addition, Cos-7 cells were transiently transfected with plasmids expressing KIT-WT and KIT-N882I. Results: By in vitro assays, both imatinib and dasatinib exhibited a high efficacy toward the control, imatinib-sensitive KIT-V559D mutant. In contrast, only dasatinib potently inhibited the KIT-N822I and control, imatinib-resistant KIT-D816V isoform with an IC50 value of 68 nmol/L and 77nmol/L, respectively. Finally, experiments with Cos-7 cells showed that on the contrary to KIT-WT the KIT-N822I mutation constitutively activated KIT tyrosine phosphorylation. Conclusion: In summary, we proved that novel germline KIT mutation (p.N822I) has transforming potential and cause a constitutive activation of KIT. Moreover, we demonstrated that treatment with dasatinib may provide a therapeutic alternative for patients with mastocytosis whose carry imatinib-resistant KIT mutant isoforms. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Effect of Growth Temperature on Crl-Dependent Regulation of σS Activity in Salmonella enterica Serovar Typhimurium

2008 ◽  
Vol 190 (13) ◽  
pp. 4453-4459 ◽  
Author(s):  
Véronique Robbe-Saule ◽  
Ingrid Carreira ◽  
Annie Kolb ◽  
Françoise Norel
Keyword(s):  
Stationary Phase ◽  
Salmonella Enterica ◽  
Regulatory Protein ◽  
Effect Of Temperature ◽  
Resistance Level ◽  
Serovar Typhimurium ◽  
Negative Effect ◽  
Transcription In Vitro ◽  
Physiological Impact

ABSTRACT The small regulatory protein Crl favors association of the stationary-phase sigma factor σS (RpoS) with the core enzyme polymerase and thereby increases σS activity. Crl has a major physiological impact at low levels of σS. Here, we report that the Crl effects on σS-dependent gene expression, the H2O2 resistance of Salmonella enterica serovar Typhimurium, and the resistance of this organism to acidic pH are greater at 28°C than at 37°C. Immunoblot experiments revealed a negative correlation between σS and Crl levels; the production of Crl was slightly greater at 28°C than at 37°C, whereas the σS levels were about twofold lower at 28°C than at 37°C. At both temperatures, Crl was present in excess of σS, and increasing the Crl level further did not increase the H2O2 resistance level of Salmonella and the expression of the σS-dependent gene katE encoding the stationary-phase catalase. In contrast, increasing the σS level rendered Salmonella more resistant to H2O2 at 28°C, increased the expression of katE, and reduced the magnitude of Crl activation. In addition, the effect of Crl on katE transcription in vitro was not dependent on temperature. These results suggest that the effect of temperature on Crl-dependent regulation of the katE gene and H2O2 resistance are mediated mainly via an effect on σS levels. In addition, our results revealed that σS exerts a negative effect on the production of Crl in stationary phase when the cells contain high levels of σS.

scholarly journals Physiological Effects of Crl in Salmonella Are Modulated by σS Level and Promoter Specificity

2007 ◽  
Vol 189 (8) ◽  
pp. 2976-2987 ◽  
Author(s):  
Véronique Robbe-Saule ◽  
Miguel Dias Lopes ◽  
Annie Kolb ◽  
Françoise Norel
Keyword(s):  
Rna Polymerase ◽  
Stationary Phase ◽  
Sigma Factor ◽  
Transcription Initiation ◽  
Regulatory Protein ◽  
Acid Stress ◽  
Physiological Effects ◽  
Wild Type ◽  
Serovar Typhimurium

ABSTRACT The small regulatory protein Crl activates σS (RpoS), the stationary-phase and general stress response sigma factor. Crl has been reported to bind σS in vitro and to facilitate the formation of RNA polymerase holoenzyme. In Salmonella enterica serovar Typhimurium, Crl is required for the development of the rdar morphotype and transcription initiation of the σS-dependent genes csgD and adrA, involved in curli and cellulose production. Here, we examined the expression of other σS-dependent phenotypes and genes in a Δcrl mutant of Salmonella. Gene fusion analyses and in vitro transcription assays indicate that the magnitude of Crl activation differs between promoters and is highly dependent on σS levels. We replaced the wild-type rpoS allele in S. enterica serovar Typhimurium strain ATCC 14028 with the rpoS LT2 allele that shows reduced expression of σS; the result was an increased Crl activation ratio and larger physiological effects of Crl on oxidative, thermal, and acid stress resistance levels during stationary phase. We also found that crl, rpoS, and crl rpoS strains grew better on succinate than did the wild type and expressed the succinate dehydrogenase sdhCDBA operon more strongly. The crl and rpoS LT2 mutations also increased the competitive fitness of Salmonella in stationary phase. These results show that Crl contributes to negative regulation by σS, a finding consistent with a role for Crl in sigma factor competition via the facilitation of σS binding to core RNA polymerase.

scholarly journals A new KSRP-binding compound suppresses distant metastasis of colorectal cancer by targeting the oncogenic KITENIN complex

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Jeong A Bae ◽  
Woo Kyun Bae ◽  
Sung Jin Kim ◽  
Yoo-Seung Ko ◽  
Keon Young Kim ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Liver Metastasis ◽  
Distant Metastasis ◽  
Regulatory Protein ◽  
Binding Pocket ◽  
In Vitro Assays ◽  
Docking Model ◽  
Small Molecule Compound

Abstract Background Distant metastasis is the major cause of death in patients with colorectal cancer (CRC). Previously, we identified KITENIN as a metastasis-enhancing gene and suggested that the oncogenic KITENIN complex is involved in metastatic dissemination of KITENIN-overexpressing CRC cells. Here, we attempted to find substances targeting the KITENIN complex and test their ability to suppress distant metastasis of CRC. Methods We screened a small-molecule compound library to find candidate substances suppressing the KITENIN complex in CRC cells. We selected a candidate compound and examined its effects on the KITENIN complex and distant metastasis through in vitro assays, a molecular docking model, and in vivo tumor models. Results Among several compounds, we identified DKC1125 (Disintegrator of KITENIN Complex #1125) as the best candidate. DKC1125 specifically suppressed KITENIN gain of function. After binding KH-type splicing regulatory protein (KSRP), DKC1125 degraded KITENIN and Dvl2 by recruiting RACK1 and miRNA-124, leading to the disintegration of the functional KITENIN–KSRP–RACK1–Dvl2 complex. A computer docking model suggested that DKC1125 specifically interacted with the binding pocket of the fourth KH-domain of KSRP. KITENIN-overexpressing CRC cells deregulated certain microRNAs and were resistant to 5-fluorouracil, oxaliplatin, and cetuximab. DKC1125 restored sensitivity to these drugs by normalizing expression of the deregulated microRNAs, including miRNA-124. DKC1125 effectively suppressed colorectal liver metastasis in a mouse model. Interestingly, the combination of DKC1125 with 5-fluorouracil suppressed metastasis more effectively than either drug alone. Conclusion DKC1125 targets the KITENIN complex and could therefore be used as a novel therapeutic to suppress liver metastasis in CRC expressing high levels of KITENIN.

Sign in / Sign up

Export Citation Format

Share Document