Pathogen Gene Expression Research Articles

Antibacterial Activity of Ferulic Acid Ester against Ralstonia solanacearum and Its Synergy with Essential Oils

Ralstonia solanacearum is one of the ten most harmful plant bacteria worldwide, and traditional agrochemicals are not very effective in controlling this pathogen. Moreover, excessive pesticides always bring organic residues and resistant strains, which cause the unsustainability of the environment. In this paper, ferulic acid and essential oils are used as antibacterial materials. These compounds are natural substances with low toxicity and environmental safety. Through the structural optimization and the analysis of binary combined bacteriostatic efficiency, the MIC values of chlorobutyl ferulate (2e) and peppermint essential oil (EO1) were 0.64 mg/mL and 2.02 mg/mL, respectively, and the MIC value of 2e-EO1 (mass ratio 1:1.5) was 0.40 mg/mL. The growth rate of bacteria treated with 2e-EO1 was inhibited, the OD590nm value of cell membrane decreased by 57.83%, and the expression levels of hrpB, pehC, pilT, polA, aceE, egl, and phcA were downregulated to 18.81%, 30.50%, 14.00%, 44.90%, 86.79%, 23.90%, and 27.56%, respectively. The results showed that 2e-EO1 had a synergistic inhibitory effect against R. solanacearum. It significantly affected the formation of the bacterial cell membrane and the expression of pathogenic genes. Consequently, 2e-EO1 provides the potential to become a green pesticide and can promote the sustainability of the agricultural ecological environment.

TRNA modification enzyme MiaB connects environmental cues to activation of Pseudomonas aeruginosa type III secretion system.

Pseudomonas aeruginosa, a major inhabitant of numerous environmental reservoirs, is a momentous opportunistic human pathogen associated with severe infections even death in the patients suffering from immune deficiencies or metabolic diseases. Type III secretion system (T3SS) employed by P. aeruginosa to inject effector proteins into host cells is one of the pivotal virulence factors pertaining to acute infections caused by this pathogen. Previous studies showed that P. aeruginosa T3SS is regulated by various environmental cues such as calcium concentration and the host signal spermidine. However, how T3SS is regulated and expressed particularly under the ever-changing environmental conditions remains largely elusive. In this study, we reported that a tRNA modification enzyme PA3980, designated as MiaB, positively regulated T3SS gene expression in P. aeruginosa and was essential for the induced cytotoxicity of human lung epithelial cells. Further genetic assays revealed that MiaB promoted T3SS gene expression by repressing the LadS-Gac/Rsm signaling pathway and through the T3SS master regulator ExsA. Interestingly, ladS, gacA, rsmY and rsmZ in the LadS-Gac/Rsm signaling pathway seemed potential targets under the independent regulation of MiaB. Moreover, expression of MiaB was found to be induced by the cAMP-dependent global regulator Vfr as well as the spermidine transporter-dependent signaling pathway and thereafter functioned to mediate their regulation on the T3SS gene expression. Together, these results revealed a novel regulatory mechanism for MiaB, with which it integrates different environmental cues to modulate T3SS gene expression in this important bacterial pathogen.

The BRCAness Landscape of Cancer

BRCAness refers to the damaged homologous recombination (HR) function due to the defects in HR-involved non-BRCA1/2 genes. BRCAness is the important marker for the use of synthetic lethal-based PARP inhibitor therapy in breast and ovarian cancer treatment. The success provides an opportunity of applying PARP inhibitor therapy to treat other cancer types with BRCAness features. However, systematic knowledge is lack for BRCAness in different cancer types beyond breast and ovarian cancer. We performed a comprehensive characterization for 40 BRCAness-related genes in 33 cancer types with over 10,000 cancer cases, including pathogenic variation, homozygotic deletion, promoter hypermethylation, gene expression, and clinical correlation of BRCAness in each cancer type. Using BRCA1/BRCA2 mutated breast and ovarian cancer as the control, we observed that BRCAness is widely present in multiple cancer types. Based on the sum of the BRCAneass features in each cancer type, we identified the following 21 cancer types as the potential targets for PARPi therapy: adrenocortical carcinoma, bladder urothelial carcinoma, brain lower grade glioma, colon adenocarcinoma, esophageal carcinoma, head and neck squamous carcinoma, kidney chromophobe, kidney renal clear cell carcinoma, kidney renal papillary cell carcinoma, liver hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, mesothelioma, rectum adenocarcinoma, pancreatic adenocarcinoma, prostate adenocarcinoma, sarcoma, skin cutaneous melanoma, stomach adenocarcinoma, uterine carcinosarcoma, and uterine corpus endometrial carcinoma.

Myricetin Derivative LP11 Targets Cucumber Mosaic Virus 2b Protein to Achieve In Vivo Antiviral Activity in Plants.

Cucumber mosaic virus (CMV) 2b protein plays a key role in the process of CMV infecting plants and symptom formation and is a potential molecular target for the control of this important plant virus. The exploitation of antiviral compounds is one of the strategies with the highest input: output ratio in plant protection. In this study, the CMV 2b recombinant protein was cloned, purified, and identified as the target protein by mass spectrometry. Subsequently, we carried out preliminary functional screening of the LP series of myricetin derivatives designed and synthesized in our laboratory and commercial antiviral compounds by microscale thermophoresis (MST), which showed that LP compounds LP4, LP11, LP13, and LP20 interacted well with CMV 2b, with dissociation constant (Kd) values of 1.39, 0.88, 1.52, and 1.77 μM, respectively. Among the commercially available antiviral compounds, ningnanmycin (NNM) was the most active, with a Kd value of 4.09 μM. Then, the strongest binding force to CMV 2b was identified to be from LP11 by isothermal titration calorimetry (ITC) experiments, with a Kd of 1.19 μM. Among the commercial compounds, NNM had the strongest binding force with CMV 2b, with a Kd of 4.62 μM. Through the screening of commercial compounds and LP series compounds by MST and ITC, LP11, NNM (positive control), LP16 (negative control), and the blank control group were selected to test the in vivo impact of LP11 on CMV. Specifically, the screened compounds were sprayed onto CMV-inoculated Nicotiana benthamiana plants to determine their impact on the regulation of CMV pathogenic gene expression, symptoms, and virus titer. The results showed that LP11 had a strong ability to inhibit CMV infection of tobacco at the transcriptional and translational levels. By mutating the CMV 2b protein, the 15th amino acid leucine and the 18th amino acid methionine at the N-terminal region were shown to be potential sites for binding to compound LP11. This finding provided a theoretical basis for screening and developing anti-CMV agents.

Inhibitory effect and possible mechanism of oregano and clove essential oils against Pectobacterium carotovorum subsp. carotovorum as onion soft rot in storage

Soft rot caused by Pectobacterium carotovorum subsp. carotovorum (Pcc) is the most common bacterial disease of postharvest onion. This study aimed to determine the antibacterial activity of oregano and clove oils against Pcc and the control of stored soft rot decay in onion. Oregano oil had a minimum inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC) of 200 μL L−1, whereas clove oil had a MIC and MBC of 400 and 500 μL L−1, respectively. Treatment with oregano and clove oil could significantly reduce Pcc cell viability and coincided with a gradient change with time. The inhibitory degree on biofilm synthesis was correlated with oregano and clove oil concentrations. Consequently, 1 × and 2 ×MIC treatments could distinctly reduce biofilm adhesion. With high concentrations of oregano and clove oil, abnormal cell morphology along with cell membrane rupture and cellular leakage were observed by scanning electron microscope (SEM), resulting in a substantial increase in extracellular conductivity. Meanwhile, secretion of extracellular cell wall degrading enzymes (CWDEs) of Pcc was declined in varying degrees, with pectinase (Pel) being significantly inhibited (29.82–100 %). In comparison to the control group, RT-qPCR data indicated that oregano and clove oil downregulated the expression of pathogenic genes, resulting in a decrease in bacterial pathogenicity. Under in vivo culturing, the soft rot decay of onion was reduced by 34.07–100 % in a concentration-dependent manner. In short, essential oils (EOs) may mitigate the severity of onion soft rot by inhibiting the Pcc virulence-associated factors such as activity, motility, toxicity, biofilm formation, and enzyme secretion to various degrees. Thus, oregano and clove oil may be used as alternative bio-fungicides to control and treat the severe disease of soft rot caused by Pcc in onions after harvest.

Evaluating EcxR for Its Possible Role in Ehrlichia chaffeensis Gene Regulation.

Ehrlichia chaffeensis, a tick-transmitted intraphagosomal bacterium, is the causative agent of human monocytic ehrlichiosis. The pathogen also infects several other vertebrate hosts. E. chaffeensis has a biphasic developmental cycle during its growth in vertebrate monocytes/macrophages and invertebrate tick cells. Host- and vector-specific differences in the gene expression from many genes of E. chaffeensis are well documented. It is unclear how the organism regulates gene expression during its developmental cycle and for its adaptation to vertebrate and tick host cell environments. We previously mapped promoters of several E. chaffeensis genes which are recognized by its only two sigma factors: σ32 and σ70. In the current study, we investigated in assessing five predicted E. chaffeensis transcription regulators; EcxR, CtrA, MerR, HU and Tr1 for their possible roles in regulating the pathogen gene expression. Promoter segments of three genes each transcribed with the RNA polymerase containing σ70 (HU, P28-Omp14 and P28-Omp19) and σ32 (ClpB, DnaK and GroES/L) were evaluated by employing multiple independent molecular methods. We report that EcxR binds to all six promoters tested. Promoter-specific binding of EcxR to several gene promoters results in varying levels of gene expression enhancement. This is the first detailed molecular characterization of transcription regulators where we identified EcxR as a gene regulator having multiple promoter-specific interactions.

A Ribosomal Protein Homolog Governs Gene Expression and Virulence in a Bacterial Pathogen.

ABSTRACTThe molecular machine necessary for protein synthesis, the ribosome, is generally considered constitutively functioning and lacking any inherent regulatory capacity. Yet ribosomes are commonly heterogeneous in composition and the impact of ribosome heterogeneity on translation is not well understood. Here, we determined that changes in ribosome protein composition govern gene expression in the intracellular bacterial pathogen Francisella tularensis. F. tularensis encodes three distinct homologs for bS21, a ribosomal protein involved in translation initiation, and analysis of purified F. tularensis ribosomes revealed they are heterogeneous with respect to bS21. The loss of one homolog, bS21-2, resulted in significant changes to the cellular proteome unlinked to changes in the transcriptome. Among the reduced proteins were components of the type VI secretion system (T6SS), an essential virulence factor encoded by the Francisella Pathogenicity Island. Furthermore, loss of bS21-2 led to an intramacrophage growth defect. Although multiple bS21 homologs complemented the loss of bS21-2 with respect to T6SS protein abundance, bS21-2 was uniquely necessary for robust intramacrophage growth, suggesting bS21-2 modulates additional virulence gene(s) distinct from the T6SS. Our results indicate that ribosome composition in F. tularensis, either directly or indirectly, posttranscriptionally modulates gene expression and virulence. Our findings are consistent with a model in which bS21 homologs function as posttranscriptional regulators, allowing preferential translation of specific subsets of mRNAs, likely at the stage of translation initiation. This work also raises the possibility that bS21 in other organisms may function similarly and that ribosome heterogeneity may permit many bacteria to posttranscriptionally regulate gene expression.IMPORTANCE While bacterial ribosomes are commonly heterogeneous in composition (e.g., incorporating different homologs for a ribosomal protein), how heterogeneity impacts translation is unclear. We found that the intracellular human pathogen Francisella tularensis has heterogeneous ribosomes, incorporating one of three homologs for ribosomal protein bS21. Furthermore, one bS21 homolog posttranscriptionally governs the expression of the F. tularensis type VI secretion system, an essential virulence factor. This bS21 homolog is also uniquely important for robust intracellular growth. Our data support a model in which bS21 heterogeneity leads to modulation of translation, providing another source of posttranscriptional gene regulation. Regulation of translation by bS21, or other sources of ribosomal heterogeneity, may be a conserved mechanism to control gene expression across the bacterial phylogeny.

Indole Sensing Regulator (IsrR) Promotes Virulence Gene Expression in Enteric Pathogens.

ABSTRACTEnteric pathogens such as enterohemorrhagic E. coli (EHEC) and its surrogate murine model Citrobacter rodentium sense indole levels within the gut to navigate its biogeography and modulate virulence gene expression. Indole is a microbiota-derived signal that is more abundant in the intestinal lumen, with its concentration decreasing at the epithelial lining where it is absorbed. E. coli, but not C. rodentium, produces endogenous indole because it harbors the tnaA gene. Microbiota-derived exogenous indole is sensed by the CpxAR two-component system, where CpxA is a membrane-bound histidine-sensor-kinase (HK) and CpxR is a response regulator (RR). Indole inhibits CpxAR function leading to decreased expression of the locus of enterocyte effacement (LEE) pathogenicity island, which is essential for these pathogens to form lesions on enterocytes. In our transcriptome studies comparing wild-type (WT) EHEC and ΔtnaA ± indole, one of the most upregulated genes by indole is ygeV, which is a predicted orphan RR. Because of the role YgeV plays in the indole signaling cascade, we renamed this gene indole sensing regulator (isrR). In the absence of endogenous indole, IsrR activates LEE gene expression. IsrR only responds to endogenous indole, with exogenous indole still blocking virulence gene expression independently from IsrR. Notably, a C. rodentium isrR mutant is attenuated for murine infection, depicting delayed death, lower intestinal colonization, and LEE gene expression. IsrR aids in discriminating between microbiota-derived (exogenous) and endogenous self-produced indole in fine-tuning virulence gene expression by enteric pathogens in the intestine.

Host Respiratory Transcriptome Signature Associated with Poor Outcome in Children with Influenza-Staphylococcus aureus Pneumonia.

Respiratory coinfection of influenza with Staphylococcus aureus often causes severe disease; methicillin-resistant S. aureus (MRSA) coinfection is frequently fatal. Understanding disease pathogenesis may inform therapies. We aimed to identify host and pathogen transcriptomic (messenger RNA) signatures from the respiratory compartment of pediatric patients critically ill with influenza-S. aureus coinfection (ISAC), signatures that predict worse outcomes. Messenger RNA extracted from endotracheal aspirate samples was evaluated for S. aureus and host transcriptomic biosignatures. Influenza-MRSA outcomes were worse, but of 190 S. aureus virulence-associated genes, 6 were differentially expressed between MRSA-coinfected versus methicillin-susceptible S. aureus-coinfected patients, and none discriminated outcome. Host gene expression in patients with ISAC was compared with that in patients with influenza infection alone. Patients with poor clinical outcomes (death or prolonged multiorgan dysfunction) had relatively reduced expression of interferons and down-regulation of interferon γ-induced immune cell chemoattractants CXCL10 and CXCL11. In ISAC, airway host but not pathogen gene expression profiles predicted worse clinical outcomes.

Synthesis of Mucin O-Glycans Associated with Attenuation of Pathogen Virulence.

With the concerning rise in antibiotic-resistant infections, novel treatment options against pathogens are urgently sought. Several recent studies have identified mucin O-glycan mixtures as potent down-regulators of virulence-related gene expression in diverse pathogens. As individual mucin glycans cannot be isolated in sufficient purity and quantity for biological evaluation of discrete structures, we have developed an optimized synthetic approach to generate a small library of mucin glycans which were identified as most likely to display activity. The glycans have been prepared in sufficient quantity to assess biological function, studies of which are currently ongoing.

Transcriptome profile of spleen tissues from locally-adapted Kenyan pigs (Sus scrofa) experimentally infected with three varying doses of a highly virulent African swine fever virus genotype IX isolate: Ken12/busia.1 (ken-1033)

BackgroundAfrican swine fever (ASF) is a lethal hemorrhagic disease affecting domestic pigs resulting in up to 100% mortality rates caused by the ASF virus (ASFV). The locally-adapted pigs in South-western Kenya have been reported to be resilient to disease and harsh climatic conditions and tolerate ASF; however, the mechanisms by which this tolerance is sustained remain largely unknown. We evaluated the gene expression patterns in spleen tissues of these locally-adapted pigs in response to varying infective doses of ASFV to elucidate the virus-host interaction dynamics.MethodsLocally adapted pigs (n = 14) were experimentally infected with a high dose (1x106HAD50), medium dose (1x104HAD50), and low dose (1x102HAD50) of the highly virulent genotype IX ASFV Ken12/busia.1 (Ken-1033) isolate diluted in PBS and followed through the course of infection for 29 days. The in vivo pig host and ASFV pathogen gene expression in spleen tissues from 10 pigs (including three from each infective group and one uninfected control) were analyzed in a dual-RNASeq fashion. We compared gene expression between three varying doses in the host and pathogen by contrasting experiment groups against the naïve control.ResultsA total of 4954 differentially expressed genes (DEGs) were detected after ASFV Ken12/1 infection, including 3055, 1771, and 128 DEGs in the high, medium, and low doses, respectively. Gene ontology and KEGG pathway analysis showed that the DEGs were enriched for genes involved in the innate immune response, inflammatory response, autophagy, and apoptosis in lethal dose groups. The surviving low dose group suppressed genes in pathways of physiopathological importance. We found a strong association between severe ASF pathogenesis in the high and medium dose groups with upregulation of proinflammatory cytokines and immunomodulation of cytokine expression possibly induced by overproduction of prostaglandin E synthase (4-fold; p < 0.05) or through downregulation of expression of M1-activating receptors, signal transductors, and transcription factors. The host-pathogen interaction resulted in induction of expression of immune-suppressive cytokines (IL-27), inactivation of autophagy and apoptosis through up-regulation of NUPR1 [5.7-fold (high dose) and 5.1-fold (medium dose) [p < 0.05] and IL7R expression. We detected repression of genes involved in MHC class II antigen processing and presentation, such as cathepsins, SLA-DQB1, SLA-DOB, SLA-DMB, SLA-DRA, and SLA-DQA in the medium and high dose groups. Additionally, the host-pathogen interaction activated the CD8+ cytotoxicity and neutrophil machinery by increasing the expression of neutrophils/CD8+ T effector cell-recruiting chemokines (CCL2, CXCL2, CXCL10, CCL23, CCL4, CXCL8, and CXCL13) in the lethal high and medium dose groups. The recovered pigs infected with ASFV at a low dose significantly repressed the expression of CXCL10, averting induction of T lymphocyte apoptosis and FUNDC1 that suppressed neutrophilia.ConclusionsWe provide the first in vivo gene expression profile data from locally-adapted pigs from south-western Kenya following experimental infection with a highly virulent ASFV genotype IX isolate at varying doses that mimic acute and mild disease. Our study showed that the locally-adapted pigs induced the expression of genes associated with tolerance to infection and repression of genes involved in inflammation at varying levels depending upon the ASFV dose administered.

Metagenome Analysis of the Bacterial Characteristics in Invasive Klebsiella Pneumoniae Liver Abscesses.

Background Klebsiella pneumoniae liver abscess (KPLA) combined with extrahepatic migratory infection (EMI) is defined as invasive KPLA (IKPLA) and is associated with a poor prognosis. The mechanism of IKPLA formation is yet to be elucidated. In this study, metagenomic sequencing was used to compare the bacterial characteristics between IKPLA and KPLA to explore the underlying mechanism of invasiveness.MethodsClinical details, imaging, and microbial features were retrospectively evaluated by medical record review. Metagenomic sequencing was performed on the pus samples of liver abscesses whose culture results were indicative of monomicrobial Klebsiella pneumoniae (K. pneumoniae). Bacterial diversity and composition in IKPLA and KPLA were comparatively analyzed, and the key pathways and genes that may affect invasiveness were further explored.ResultsSixteen patients were included in this study. Five patients with EMI were included in the IKPLA group, and the other eleven patients without EMI were assigned to the KPLA group. There was no statistical difference in the hypermucoviscous phenotype and serotype of K. pneumoniae between the two groups. The bacterial diversity of IKPLA was lower than that of KPLA. The abundant taxa in the IKPLA group were primarily species of unclassified Enterobacteriaceae and K. pneumoniae. The KPLA group had a high abundance of the genera Tetrasphaera and Leuconostoc. Metabolic pathway genes represented most of the enriched genes in IKPLA. Fourteen pathogenic genes with significant differences in abundance were identified between the two groups, including ybtS, fepC, phoQ, acrB, fimK, magA, entC, arnT, iucA, fepG, oqxB, entA, tonB, and entF (p < 0.001).ConclusionThe diversity and bacterial composition of IKPLA were significantly different from those of KPLA. Microbiological changes in the abscess, activation of the related metabolic pathways, and the pathogenic gene expression may constitute a novel mechanism that regulates the invasiveness of KPLA.

The AraC/XylS Protein MxiE and Its Coregulator IpgC Control a Negative Feedback Loop in the Transcriptional Cascade That Regulates Type III Secretion in Shigella flexneri.

Members of the AraC family of transcriptional regulators (AFTRs) control the expression of many genes important to cellular processes, including virulence. In Shigella species, the type III secretion system (T3SS), a key determinant for host cell invasion, is regulated by the three-tiered VirF/VirB/MxiE transcriptional cascade. Both VirF and MxiE belong to the AFTRs and are characterized as positive transcriptional regulators. Here, we identify a novel regulatory activity for MxiE and its coregulator IpgC, which manifests as a negative feedback loop in the VirF/VirB/MxiE transcriptional cascade. Our findings show that MxiE and IpgC downregulate the virB promoter and, hence, VirB protein production, thus decreasing VirB-dependent promoter activity at ospD1, one of the nearly 50 VirB-dependent genes. At the virB promoter, regions required for negative MxiE- and IpgC-dependent regulation were mapped and found to be coincident with regions required for positive VirF-dependent regulation. In tandem, negative MxiE- and IpgC-dependent regulation of the virB promoter only occurred in the presence of VirF, suggesting that MxiE and IpgC can function to counter VirF activation of the virB promoter. Lastly, MxiE and IpgC do not downregulate another VirF-activated promoter, icsA, demonstrating that this negative feedback loop targets the virB promoter. Our study provides insight into a mechanism that may reprogram Shigella virulence gene expression following type III secretion and provides the impetus to examine if MxiE and IpgC homologs in other important bacterial pathogens, such as Burkholderia pseudomallei and Salmonella enterica serovars Typhimurium and Typhi, coordinate similar negative feedback loops. IMPORTANCE The large AraC family of transcriptional regulators (AFTRs) control virulence gene expression in many bacterial pathogens. In Shigella species, the AraC/XylS protein MxiE and its coregulator IpgC positively regulate the expression of type III secretion system genes within the three-tiered VirF/VirB/MxiE transcriptional cascade. Our findings suggest a negative feedback loop in the VirF/VirB/MxiE cascade, in which MxiE and IpgC counter VirF-dependent activation of the virB promoter, thus making this the first characterization of negative MxiE- and IpgC-dependent regulation. Our study provides insight into a mechanism that likely reprograms Shigella virulence gene expression following type III secretion, which has implications for other important bacterial pathogens with functional homologs of MxiE and IpgC.

Multifunctional peptide carrier-modified polymer micelle accelerates oral siRNA-delivery to the colon and improves gene silencing-mediated therapeutic effects in ulcerative colitis

In the treatment of inflammatory bowel diseases such as ulcerative colitis (UC), siRNAs are expected to enable fundamental suppression of the expression of pathogenic genes. Oral administration is considered the most desirable route of drug-administration as it can act directly on the affected area and improve the patient's quality of life. However, when orally administered, naked siRNA is easily degraded in the gastrointestinal tract; therefore, it is necessary to use a carrier to protect the siRNA from degradation. We have previously shown that systemic administration of the multifunctional peptide carrier (CH2R4H2C)-modified methoxy polyethylene glycol-polycaprolactone polymer micelles (MPEG-PCL-CH2R4H2C)/siRNA has high therapeutic efficacy against inflammatory diseases. In the present study, we aimed to apply MPEG-PCL-CH2R4H2C nano-micelles to apply MPEG-PCL-CH2R4H2C nano-micelles for oral delivery of siRNA to treat dextran sodium sulfate-induced UC model mice. Agarose gel electrophoresis and cell viability and cytotoxicity assays were performed. The therapeutic effects of siRelA in UC mice were assessed. MPEG-PCL-CH2R4H2C/siRNA complexes with an N/P ratio of more than 50 were not degraded at the gastrointestinal pH environment of 1.2 and 6.8, and showed high cellular uptake ability without showing cytotoxicity in macrophages. In addition, oral administration of the MPEG-PCL-CH2R4H2C/siRNA to mice showed improved delivery to the colon compared to naked siRNA. Finally, RelA-targeted siRNA suppressed the RelA mRNA expression and deterioration of clinical symptoms, such as bloody stool, diarrhea, weight loss, and colonic shortening, by complexation with MPEG-PCL-CH2R4H2C micelles. These results suggest that MPEG-PCL-CH2R4H2C micelles are useful as novel siRNA carriers for oral administration.

Bacterial Pathogens in Food: Modern Safety Criteria and Methods of Molecular Genetic Analysis.

The review is devoted to the issues of improving the food biosafety system by developing and implementing modern methods of microbiological and molecular genetic analysis and new safety standards for the main groups of food products. The results of fundamental and applied microbiological research in the field of food safety conducted by the Federal Research Center for Nutrition and Biotechnology are presented. We demonstrated the necessity of a detailed study of the ecology and features of survival of new types of pathogenic microorganisms and evaluation of the role of technological factors in the formation of altered properties of these pathogens in food production environment. It is noted that the most effective basis for establishing criteria for biosafety of food products is the use of a structural model of microbiological risk analysis (MRA), which implies assessment and integration of risks throughout the food chain and step-by-step study of microbial hazards, predicts the degree of danger to the health of certain foods contaminated by them and the risk and severity of adverse effects. Consideration of the variability of pathogens and the ability of MRA to forecast allows rationalizing resources in the most relevant areas when developing measures to protect the population. It is shown how, as a result of studying the biology of pathogenic bacteria of the genera Campylobacter, Salmonella, Cronobacter, and Listeria and mechanisms for regulating the expression of pathogenicity and resistance factors of these pathogens, improved schemes and modified methods have been developed over a number of years, allowing accelerated indication of pathogens in food products using combined schemes of bacteriological and molecular genetic analysis based on the selection of the most informative biochemical and immunological tests, and their genotyping. Accumulated scientific data in the field of biosafety indicate the need for further research in the prediction of the pathogen behavior in food, studying the mechanisms of regulation of the expression of pathogenicity genes in food-borne pathogens, the transmission of antibiotic resistance genes, and studying the interaction of the human with pathogens to calculate the biological response to microbial agents coming from food.

Pat- and Pta-mediated protein acetylation is required for horizontally-acquired virulence gene expression in Salmonella Typhimurium.

Salmonella Typhimurium is a Gram-negative facultative pathogen that causes a range of diseases, from mild gastroenteritis to severe systemic infection in a variety of animal hosts. S. Typhimurium regulates virulence gene expression by a silencing mechanism using nucleoid-associated proteins such as Histone-like Nucleoid Structuring protein (H-NS) silencing. We hypothesize that the posttranslational modification, specifically protein acetylation, of proteins in gene silencing systems could affect the pathogenic gene expression of S. Typhimurium. Therefore, we created acetylation-deficient mutant by deleting two genes, pat and pta, which are involved in the protein acetylation pathway. We observed that the pat and pta deletion attenuates mouse virulence and also decreases Salmonella's replication within macrophages. In addition, the Δpat Δpta strain showed a decreased expression of the horizontally-acquired virulence genes, mgtC, pagC, and ugtL, which are highly expressed in low Mg2+. The decreased virulence gene expression is possibly due to higher H-NS occupancy to those promoters because the pat and pta deletion increases H-NS occupancy whereas the same mutation decreases occupancy of RNA polymerase. Our results suggest that Pat- and Pta-mediated protein acetylation system promotes the expression of virulence genes by regulating the binding affinity of H-NS in S. Typhimurium.

Study of intestinal microbial flora of local chickens to investigate the effect of probiotics Bacillus subtilis and Bacillus coagulans on the expression of ctxM and luxS pathogenic genes in isolates of Escherichia coli

Background and objective: Antibiotics are widely used worldwide. However, due to the emergence of antibiotic resistance in a wide range of microorganisms, their use worldwide has failed. Probiotics are suggested as complementary and alternative medicine. The present study aimed to investigate the effects of probiotics isolated from local chickens on the expression of luxS and ctxM genes in resistant Escherichia coli. Materials and methods: 300 fecal samples were taken from patients referring to Imam Khomeini Hospital in Tehran during May–September 2016 and Escherichia coli samples were isolated using specific culture media and biochemical tests and then the presence of luxS and ctxM genes were identified using PCR with specific primers. In order to extract the probiotics forming spores, the intestinal contents of 10 poultry that had not used any antibiotics and probiotics were cultured, isolated, and identified using biochemical and PCR methods. Commercial strains of Bacillus subtilis and Bacillus coagulans were purchased to compare their effects with native bacteria. These strains were then co-cultured with resistant Escherichia coli strains containing ctxM and luxS genes. Real-time PCR was used to evaluate the effect of these probiotics on gene expression Results: The results indicated that 40 isolates (7.5%) of Escherichia coli were obtained from the 300 fecal samples. Thirteen samples (32.5%) were outpatients and 27 (67.5%) were inpatients. All isolates were isolated from men and women aged 21–62. Four Escherichia coli strains were isolated from patients carrying ctxM and luxS genes. Isolation of Bacillus coagulans and Bacillus subtilis from samples was confirmed by biochemical and molecular experiments. The commercial and native strains of Bacillus coagulans reduced the expression of the luxS and ctxM genes by 3.60, 3.30, 1.58, and 2.70 times respectively. Also, the commercial and native strains of Bacillus subtilis decreased the expression of the luxS and ctxM genes by 1.37, 1.10, 2.20, and 2.80 times respectively. The results of statistical analysis showed a significant relationship between the presence of native and commercial probiotics in culture and reduced expression of ctxM and luxS genes. Conclusion: According to the results, supplements of Bacillus coagulans and Bacillus subtilis increase the effect of antibiotics resistance in Escherichia coli by reducing the expression of resistance genes.

Analyzing Association Between Expression Quantitative Trait and CNV for Breast Cancer Based on Gene Interaction Network Clustering and Group Sparse Learning

Aims: Aims: The occurrence and development of tumor is accompanied by the change of pathogenic gene expression. Tumor cells avoid the damage of immune cells by regulating the expression of immune related genes. Background: Background: Tracing the causes of gene expression variation is helpful to understand tumor evolution and metastasis. Objective: Objective: Current gene expression variation explanation methods are confronted with several main challenges: low explanation power, insufficient prediction accuracy, and lack of biological meaning. Method: Method: In this study, we propose a novel method to analyze the mRNA expression variations of breast cancers risk genes. Firstly, we collected some high-confidence risk genes related to breast cancer and then designed a rank-based method to preprocess the breast cancers copy number variation (CNV) and mRNA data. Secondly, to elevate the biological meaning and narrow down the combinatorial space, we introduced a prior gene interaction network and applied a network clustering algorithm to generate high density subnetworks. Lastly, to describe the interlinked structure within and between subnetworks and target genes mRNA expression, we proposed a group sparse learning model to identify CNVs for pathogenic genes expression variations. Result: Result: The performance of the proposed method is evaluated by both significantly improved predication accuracy and biological meaning of pathway enrichment analysis. Conclusion: Conclusion: The experimental results show that our method has practical significance

Pseudomonas aeruginosa post‐translational responses to elevated c‐di‐GMP levels

C-di-GMP signaling can directly influence bacterial behavior by affecting the functionality of c-di-GMP-binding proteins. In addition, c-di-GMP can exert a global effect on gene transcription or translation, for example, via riboswitches or by binding to transcription factors. In this study, we investigated the effects of changes in intracellular c-di-GMP levels on gene expression and protein production in the opportunistic pathogen Pseudomonas aeruginosa. We induced c-di-GMP production via an ectopically introduced diguanylate cyclase and recorded the transcriptional, translational as well as proteomic profile of the cells. We demonstrate that rising levels of c-di-GMP under growth conditions otherwise characterized by low c-di-GMP levels caused a switch to a non-motile, auto-aggregative P. aeruginosa phenotype. This phenotypic switch became apparent before any c-di-GMP-dependent role on transcription, translation, or protein abundance was observed. Our results suggest that rising global c-di-GMP pools first affects the motility phenotype of P. aeruginosa by altering protein functionality and only then global gene transcription.

The Controversial Role of HCY and Vitamin B Deficiency in Cardiovascular Diseases

Plasma homocysteine (HCY) is an established risk factor for cardiovascular disease CVD and stroke. However, more than two decades of intensive research activities has failed to demonstrate that Hcy lowering through B-vitamin supplementation results in a reduction in CVD risk. Therefore, doubts about a causal involvement of hyperhomocysteinemia (HHcy) and B-vitamin deficiencies in atherosclerosis persist. Existing evidence indicates that HHcy increases oxidative stress, causes endoplasmatic reticulum (ER) stress, alters DNA methylation and, thus, modulates the expression of numerous pathogenic and protective genes. Moreover, Hcy can bind directly to proteins, which can change protein function and impact the intracellular redox state. As most mechanistic evidence is derived from experimental studies with rather artificial settings, the relevance of these results in humans remains a matter of debate. Recently, it has also been proposed that HHcy and B-vitamin deficiencies may promote CVD through accelerated telomere shortening and telomere dysfunction. This review provides a critical overview of the existing literature regarding the role of HHcy and B-vitamin deficiencies in CVD. At present, the CVD risk associated with HHcy and B vitamins is not effectively actionable. Therefore, routine screening for HHcy in CVD patients is of limited value. However, B-vitamin depletion is rather common among the elderly, and in such cases existing deficiencies should be corrected. While Hcy-lowering with high doses of B vitamins has no beneficial effects in secondary CVD prevention, the role of Hcy in primary disease prevention is insufficiently studied. Therefore, more intervention and experimental studies are needed to address existing gaps in knowledge.