Reliability of the In Silico Prediction Approach to In Vitro Evaluation of Bacterial Toxicity.

Several pathogens that spread through the air are highly contagious, and related infectious diseases are more easily transmitted through airborne transmission under indoor conditions, as observed during the COVID-19 pandemic. Indoor air contaminated by microorganisms, including viruses, bacteria, and fungi, or by derived pathogenic substances, can endanger human health. Thus, identifying and analyzing the potential pathogens residing in the air are crucial to preventing disease and maintaining indoor air quality. Here, we applied deep learning technology to analyze and predict the toxicity of bacteria in indoor air. We trained the ProtBert model on toxic bacterial and virulence factor proteins and applied them to predict the potential toxicity of some bacterial species by analyzing their protein sequences. The results reflect the results of the in vitro analysis of their toxicity in human cells. The in silico-based simulation and the obtained results demonstrated that it is plausible to find possible toxic sequences in unknown protein sequences.

The Small Protein CydX Is Required for Cytochrome bd Quinol Oxidase Stability and Function in Salmonella enterica Serovar Typhimurium: a Phenotypic Study.

Cytochrome bd quinol oxidases, which have a greater affinity for oxygen than heme-copper cytochrome oxidases (HCOs), promote bacterial respiration and fitness in low-oxygen environments, such as host tissues. Here, we show that, in addition to the CydA and CydB subunits, the small protein CydX is required for the assembly and function of the cytochrome bd complex in the enteric pathogen Salmonella enterica serovar Typhimurium. Mutant S Typhimurium lacking CydX showed a loss of proper heme arrangement and impaired oxidase activity comparable to that of a ΔcydABX mutant lacking all cytochrome bd subunits. Moreover, both the ΔcydX mutant and the ΔcydABX mutant showed increased sensitivity to ß-mercaptoethanol and nitric oxide (NO). Cytochrome bd-mediated protection from ß-mercaptoethanol was not a result of resistance to reducing damage but, rather, was due to cytochrome bd oxidase managing Salmonella respiration, while ß-mercaptoethanol interacted with the copper ions necessary for the HCO activity of the cytochrome bo-type quinol oxidase. Interactions between NO and hemes in cytochrome bd and cytochrome bd-dependent respiration during nitrosative stress indicated a direct role for cytochrome bd in mediating Salmonella resistance to NO. Additionally, CydX was required for S Typhimurium proliferation inside macrophages. Mutants deficient in cytochrome bd, however, showed a significant increase in resistance to antibiotics, including aminoglycosides, d-cycloserine, and ampicillin. The essential role of CydX in cytochrome bd assembly and function suggests that targeting this small protein could be a useful antimicrobial strategy, but potential drug tolerance responses should also be considered.IMPORTANCE Cytochrome bd quinol oxidases, which are found only in bacteria, govern the fitness of many facultative anaerobic pathogens by promoting respiration in low-oxygen environments and by conferring resistance to antimicrobial radicals. Thus, cytochrome bd complex assembly and activity are considered potential therapeutic targets. Here we report that the small protein CydX is required for the assembly and function of the cytochrome bd complex in S Typhimurium under stress conditions, including exposure to ß-mercaptoethanol, nitric oxide, or the phagocytic intracellular environment, demonstrating its crucial function for Salmonella fitness. However, cytochrome bd inactivation also leads to increased resistance to some antibiotics, so considerable caution should be taken when developing therapeutic strategies targeting the CydX-dependent cytochrome bd.

SPME-GC/MS Analysis of Methanol in Biospecimen by Derivatization with Pyran Compound.

Methanol is metabolized in the body to highly toxic formaldehyde and formate when consumed accidentally. Methanol has been typically analyzed with gas chromatography-flame ionization detector (GC-FID). However, its retention time may overlap with other volatile compounds and lead to confusion. Alternative analysis of methanol using gas chromatography/mass spectrometry (GC/MS) also has limitations due to its similar molecular weight with oxygen and low boiling point. In this study, methanol and internal standard of deuterium-substituted ethanol were derivatized with 3,4-dihydro-2H-pyran under acid catalysis using concentrated hydrochloric acid. The reaction products including 2-methoxytetrahydropyran were extracted with solid-phase microextraction followed by GC/MS analysis. This method was successfully applied to measure the lethal concentration of methanol in the blood of a victim with a standard addition method to overcome the complex matrix effect of the biospecimen. Identification of the metabolite formate by ion chromatography confirmed the death cause to be methanol poisoning. This new method was a much more convenient and reliable process to measure methanol in complex matrix samples by reducing sample pretreatment effort and cost.

Antimicrobial Mechanism of Oleanolic and Ursolic Acids on Streptococcus mutans UA159.

Triterpenoid saponin derivatives oleanolic acid (OA) and ursolic acid (UA), but not betulinic acid (BA), were previously found to have strong antimicrobial activity against Streptococcus mutans. OA and UA inhibited the transcription of genes related to peptidoglycan biosynthesis, thereby preventing bacterial growth. However, it is not clear whether this is the only pathway involved in the antimicrobial activity of these compounds against S. mutans. Therefore, we used quantitative real-time PCR (qPCR) and microarray analyses to examine the expression of genes related to essential metabolic pathways in S. mutans UA159 following incubation with OA, UA, or BA. An oligonucleotide array consisting of 5363 probes was designed to survey 1928 of the 1963 genes in the genome of S. mutans UA159. Genes that showed >2-fold changes in expression in response to the treatment conditions were annotated, and selected target genes involved in central metabolism were analyzed by qPCR. Microarray analysis confirmed that the gene expression patterns of the OA- and UA-treated cells differed from that of the BA-treated culture, indicating differences in the antimicrobial mechanism. In particular, the expression of pfk and pykF, coding for glycolysis regulatory proteins phosphofructokinase and pyruvate kinase, respectively, were significantly decreased in the OA and UA groups (P < 0.05), as were genes involved in fatty acid and amino acid synthesis. In addition, the microarray analysis confirmed previous qPCR results showing that peptidoglycan synthesis is down-regulated in the OA- and UA-treated groups. OA and UA also appear to decrease the generation of organic acids by S. mutans UA159, which would have an anticaries effect. Overall, these findings suggest that OA and UA affect multiple genes involved in the central metabolism of S. mutans, with inhibition of glycolysis, fatty acid synthesis, amino acid synthesis, and peptidoglycan synthesis, all contributing to their antimicrobial activity.

Branched-chain amino acid supplementation promotes aerobic growth of Salmonella Typhimurium under nitrosative stress conditions.

Nitric oxide (NO) inactivates iron-sulfur enzymes in bacterial amino acid biosynthetic pathways, causing amino acid auxotrophy. We demonstrate that exogenous supplementation with branched-chain amino acids (BCAA) can restore the NO resistance of hmp mutant Salmonella Typhimurium lacking principal NO-metabolizing enzyme flavohemoglobin, and of mutants further lacking iron-sulfur enzymes dihydroxy-acid dehydratase (IlvD) and isopropylmalate isomerase (LeuCD) that are essential for BCAA biosynthesis, in an oxygen-dependent manner. BCAA supplementation did not affect the NO consumption rate of S. Typhimurium, suggesting the BCAA-promoted NO resistance independent of NO metabolism. BCAA supplementation also induced intracellular survival of ilvD and leuCD mutants at wild-type levels inside RAW 264.7 macrophages that produce constant amounts of NO regardless of varied supplemental BCAA concentrations. Our results suggest that the NO-induced BCAA auxotrophy of Salmonella, due to inactivation of iron-sulfur enzymes for BCAA biosynthesis, could be rescued by bacterial taking up exogenous BCAA available in oxic environments.

Val43 residue of NsrR is crucial for the nitric oxide response of Salmonella Typhimurium.

ABSTRACT: In pathogenic bacteria, the flavohemoglobin Hmp is crucial in metabolizing the cytotoxic levels of nitric oxide (NO) produced in phagocytic cells, contributing to bacterial virulence. Hmp expression is predominantly regulated by the Rrf2 family transcription repressor NsrR in an NO-dependent manner; however, the underlying molecular mechanism in enterobacteria remains poorly understood. In this study, we identified Val43 of Salmonella Typhimurium NsrR (StNsrR) as a critical amino acid residue for regulating Hmp expression. The Val43-to-Ala-substituted mutant NsrR isolated through random and site-directed mutagenesis showed high binding affinity to the target DNA irrespective of NO exposure, resulting in a severe reduction in hmp transcription and slow NO metabolism in Salmonella under NO-producing conditions. Conversely, the Val43-to-Glu-substituted NsrR caused effects similar to nsrR null mutation, which directed hmp transcription and NO metabolism in a constitutive way. Comparative analysis of the primary sequences of NsrR and another NO-sensing Rrf2 family regulator, IscR, from diverse bacteria, revealed that Val43 of enterobacterial NsrR corresponds to Ala in Pseudomonas aeruginosa or Streptomyces coelicolor NsrR and Glu in enterobacterial IscR, all of which are located in the DNA recognition helix α3. The predicted structure of StNsrR in complex with the hmp DNA suggests dissimilar spatial stoichiometry in the interactions of Val43 and its substituted residues with the target DNA, consistent with the observed phenotypic changes in StNsrR Val43 mutants. Our findings highlight the discriminative roles of the NsrR recognition helix in regulating species-specific target gene expression, facilitating effective NO detoxification strategies in bacteria across diverse environments. IMPORTANCE: The precise regulation of flavohemoglobin Hmp expression by NsrR is critical for bacterial fitness, as excessive Hmp expression in the absence of NO can disturb bacterial redox homeostasis. While the molecular structure of Streptomyces coelicolor NsrR has been recently identified, the specific molecular structures of NsrR proteins in enterobacteria remain unknown. Our discovery of the crucial role of Val43 in the DNA recognition helix α3 of Salmonella NsrR offers valuable insights into the Hmp modulation under NO stress. Furthermore, the observed amino acid polymorphisms in the α3 helices of NsrR proteins across different bacterial species suggest the diverse evolution of NsrR structure and gene regulation in response to varying levels of NO pressure within their ecological niches.

The NsrR regulon in nitrosative stress resistance of Salmonella enterica serovar Typhimurium.

Nitric oxide (NO·) is an important mediator of innate immunity. The facultative intracellular pathogen Salmonella has evolved mechanisms to detoxify and evade the antimicrobial actions of host-derived NO· produced during infection. Expression of the NO·-detoxifying flavohaemoglobin Hmp is controlled by the NO·-sensing transcriptional repressor NsrR and is required for Salmonella virulence. In this study we show that NsrR responds to very low NO· concentrations, suggesting that it plays a primary role in the nitrosative stress response. Additionally, we have defined the NsrR regulon in Salmonella enterica sv. Typhimurium 14028s using transcriptional microarray, qRT-PCR and in silico methods. A novel NsrR-regulated gene designated STM1808 has been identified, along with hmp, hcp-hcr, yeaR-yoaG, ygbA and ytfE. STM1808 and ygbA are important for S. Typhimurium growth during nitrosative stress, and the hcp-hcr locus plays a supportive role in NO· detoxification. ICP-MS analysis of purified STM1808 suggests that it is a zinc metalloprotein, with histidine residues H32 and H82 required for NO· resistance and zinc binding. Moreover, STM1808 and ytfE promote Salmonella growth during systemic infection of mice. Collectively, these findings demonstrate that NsrR-regulated genes in addition to hmp are important for NO· detoxification, nitrosative stress resistance and Salmonella virulence.

Molecular characterization of the InvE regulator in the secretion of type III secretion translocases in Salmonella enterica serovar Typhimurium.

The type III secretion systems (T3SSs) are exploited by many Gram-negative pathogenic bacteria to deliver a set of effector proteins into the host cytosol during cell entry. The T3SS of Salmonella enterica serovar Typhimurium is composed of more than 20 proteins that constitute the membrane-associated base, the needle and the tip complex at the distal end of the T3SS needle. Membrane docking and piercing between the T3SS and host cells is followed by the secretion of effector proteins. Therefore, a secretion hierarchy among the substrates of the T3SS is required. The secretion of the pore-forming translocase proteins SipB, SipC and SipD is controlled by the T3SS regulator protein, InvE. During an attempt to identify the regions of InvE that are involved in T3SS regulation, it was observed that the secretion of SipB, SipC and SipD was inhibited when the C-terminal 52 amino acids were removed from InvE. In addition, InvE derivatives lacking the N-terminal 30 and 100 residues were unable to secrete translocases into the culture medium. Interestingly, in the absence of the N-terminal 180 residues of InvE, SipD is unstable, resulting in the hypersecretion of SipB. We also found that both the type III secretion signals of SipB and SptP were functionally interchangeable with the first 30 amino acids of InvE, which could allow the secretion of a reporter protein. These results indicate that InvE may have two functional domains responsible for regulating the secretion of translocases: an N-terminal secretion signal and a C-terminal regulatory domain.

Agr function is upregulated by photodynamic therapy for Staphylococcus aureus and is related to resistance to photodynamic therapy.

Photodynamic therapy (PDT) has been considered a feasible alternative for antimicrobial therapy of multidrug-resistant pathogens. However, bacterial response mechanisms against PDT-generated photo-oxidative stress remain largely unknown. Herein, it is shown that the accessory gene regulator Agr is involved in Staphylococcus aureus response to photo-oxidative stress generated by laser-induced PDT with the photosensitizer chlorin e6 . Transcriptional profiling revealed that sublethal PDT induces a general stress response and also activates Agr-dependent gene regulation. Moreover, mutant S. aureus lacking Agr function showed hypersusceptibility to two independent PDT conditions with higher energy densities, demonstrating Agr-dependent S. aureus resistance against PDT.

Effect of iacP mutation on flagellar phase variation in Salmonella enterica serovar typhimurium strain UK-1.

Flagella are surface appendages that are important for bacterial motility and invasion of host cells. Two flagellin subunits in Salmonella enterica serovar Typhimurium, FliC and FljB, are alternatively expressed by a site-specific DNA inversion mechanism called flagellar phase variation. Although this inversion mechanism is understood at the molecular level, the key factor controlling the expression of the two flagellin subunits has not been determined. In this study, we found that a putative acyl carrier protein, IacP, affects flagellar phase variation in S. Typhimurium strain UK-1 under Salmonella pathogenicity island 1 (SPI1)-inducing conditions. Liquid chromatography-mass spectrometry analysis of the secreted proteins from S. Typhimurium determined that the amount of FljB secreted was significantly higher in the iacP mutant strain, a finding confirmed by Western blot analysis. Northern blotting, quantitative PCR, and microarray data showed that the level of FljB in the iacP mutant strain was regulated at the transcriptional level, although the transcription and expression of the fliC gene were independent of IacP. FljB production was abolished by the deletion of the Hin DNA invertase but could be restored by the introduction of a plasmid carrying the hin gene. We also found that in the iacP mutant strain, the orientation of the invertible H segment is in the FljB-expressing phase. Furthermore, electron microscopy observations indicated that the iacP mutant strain had more flagella per cell than the wild-type strain. These results suggest that IacP is associated with flagellar phase switching under SPI1-inducing conditions.

Expression and delivery of tetanus toxin fragment C fused to the N-terminal domain of SipB enhances specific immune responses in mice.

Live attenuated bacteria can be used as a carrier for the delivery of foreign antigens to a host's immune system. The N-terminal domain of SipB, a translocon protein of the type III secretion system of Salmonella enterica serovar Typhimurium, is required for secretion and outer membrane localization. In the present study, vaccine plasmids for antigen delivery in which the non-toxic tetanus toxin fragment C (TTFC), which contains a T cell epitope, is fused to the N-terminal 160 amino acids of SipB were developed. It was found that the recombinant proteins are secreted into the culture media and localized to the bacterial surface. TTFC-specific antibody responses are significantly increased in mice orally immunized with attenuated S. Typhimurium BRD509 strains carrying TTFC delivery plasmids. When the TTFC delivery cassettes were introduced into a low copy vector, the plasmid was stably maintained in the BRD509 strain and induced an immune response to the TTFC antigen in mice. These results suggest that expression and delivery of heterologous antigens fused to the N-terminus of SipB enhance the induction of antigen-specific immune responses, and that the N-terminal domain of SipB can be used as a versatile delivery system for foreign antigens.

Development of an Oral Salmonella-Based Vaccine Platform against SARS-CoV-2.

Effective vaccine development for global outbreaks, such as the coronavirus disease 2019 (COVID-19), has been successful in the short run. However, the currently available vaccines have been associated with a higher frequency of adverse effects compared with other general vaccines. In this study, the possibility of an oral bacteria-based vaccine that can be safely used as a platform for large-scale, long-term immunization was evaluated. A well-known Salmonella strain that was previously considered as a vaccine delivery candidate was used. Recombinant Salmonella cells expressing engineered viral proteins related with COVID-19 pathogenesis were engineered, and the formulation of the oral vaccine candidate strain was evaluated by in vitro and in vivo experiments. First, engineered S proteins were synthesized and cloned into expression vectors, which were than transformed into Salmonella cells. In addition, when orally administrated to mice, the vaccine promoted antigen-specific antibody production and cellular immunity was induced with no significant toxicity effects. These results suggest that Salmonella strains may represent a valuable platform for the development of an oral vaccine for COVID-19 as an alternative to tackle the outbreak of various mutated coronavirus strains and new infectious diseases in the future.

Role of Salmonella Pathogenicity Island 1 protein IacP in Salmonella enterica serovar typhimurium pathogenesis.

Gram-negative bacteria, including Salmonella enterica serovar Typhimurium, exploit type III secretion systems (T3SSs) through which virulence proteins are delivered into the host cytosol to reinforce invasive and replicative niches in their host. Although many secreted effector proteins and membrane-bound structural proteins in the T3SS have been characterized, the functions of many cytoplasmic proteins still remain unknown. In this study, we found that IacP, encoded by Salmonella pathogenicity island 1, was important for nonphagocytic cell invasion and bacterial virulence. When the iacP gene was deleted from several Salmonella serovar Typhimurium strains, the invasion into INT-407 epithelial cells was significantly decreased compared to that of their parental strains, and retarded rearrangements of actin fibers were observed for the iacP mutant-infected cells. Although IacP had no effect on the secretion of type III translocon proteins, the levels of secretion of the effector proteins SopB, SopA, and SopD into the culture medium were decreased in the iacP mutant. In a mouse infection model, mice infected with the iacP mutant exhibited alleviated pathological signs in the intestine and survived longer than did wild-type-infected mice. Taken together, IacP plays a key role in Salmonella virulence by regulating the translocation of T3SS effector proteins.

Nitrosative stress causes amino acid auxotrophy in hmp mutant Salmonella Typhimurium.

Cytotoxic nitic oxide (NO) damages various bacterial macromolecules, resulting in abnormal metabolism by mechanisms largely unknown. We show that NO can cause amino acid auxotrophy in Salmonella Typhimurium lacking major NO-metabolizing enzyme, flavohemoglobin Hmp. In NO-producing cultures, supplementation with amino acid pool restores growth of Hmp-deficient Salmonella to normal growth phases, whereas excluding Cys or BCAA Leu, Ile, or Val from amino acid pool reduces growth recovery. Data suggest that, without detoxification, NO might inactivate key enzymes in the biosynthesis pathway of amino acids essential for Salmonella replication in amino acid-limiting host environments.

Acid stress activation of the sigma(E) stress response in Salmonella enterica serovar Typhimurium.

The alternative sigma factor sigma(E) is activated by unfolded outer membrane proteins (OMPs) and plays an essential role in Salmonella pathogenesis. The canonical pathway of sigma(E) activation in response to envelope stress involves sequential proteolysis of the anti-sigma factor RseA by the PDZ proteases DegS and RseP. Here we show that sigma(E) in Salmonella enterica sv. Typhimurium can also be activated by acid stress. A sigma(E)-deficient mutant exhibits increased susceptibility to acid pH and reduced survival in an acidified phagosomal vacuole. Acid activation of sigma(E)-dependent gene expression is independent of the unfolded OMP signal or the DegS protease but requires processing of RseA by RseP. The RseP PDZ domain is indispensable for acid induction, suggesting that acid stress may disrupt an inhibitory interaction between RseA and the RseP PDZ domain to allow RseA proteolysis in the absence of antecedent action of DegS. These observations demonstrate a novel environmental stimulus and activation pathway for the sigma(E) regulon that appear to be critically important during Salmonella-host cell interactions.

Antimicrobial effect of photodynamic therapy using a highly pure chlorin e6.

The aim of the present study is to evaluate the antimicrobial effect of photodynamic therapy (PDT) using a highly pure chlorin e(6) (Ce(6)), against various pathogenic bacteria. To examine the antimicrobial effect of Ce(6)-mediated PDT against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Salmonella enterica serovar Typhimurium, inhibition zone formation, CFU quantification, and bacterial viability were evaluated. Inhibition zone analysis showed that Ce(6)-mediated PDT is very effective to inhibit the growth of S. aureus and P. aeruginosa, but has only minor effect to E. coli and S. Typhimurium, which was dependent on the energy density of laser and dose of Ce(6). Ce(6)-mediated PDT also nearly inhibited the colony formation of S. aureus and P. aeruginosa, and partially inhibited that of E. coli and S. Typhimurium. In addition, the number of viable bacteria decreased greatly after PDT application with LS-chlorin e6 of 10 microM and laser and energy density of 20 J/cm(2). These results show that Ce(6)-mediated PDT can be an effective alternative for antimicrobial treatment.

Transcriptomic Identification and Biochemical Characterization of HmpA, a Nitric Oxide Dioxygenase, Essential for Pathogenesis of Vibrio vulnificus.

Nitric oxide (NO) and its derivatives are important effectors of host innate immunity, disrupting cellular function of infecting pathogens. Transcriptome analysis of Vibrio vulnificus, an opportunistic human pathogen, identified a set of genes induced upon exposure to NO. Among them, VvhmpA (V. vulnificus hmpA), encoding a multidomain NO dioxygenase, was the most greatly induced upon exposure to NO and was thus further characterized. Absorption spectra demonstrated that VvHmpA is a heme protein in which the heme iron can exist in either reduced, NO-bound, or oxidized state. Biochemical studies revealed that VvHmpA is a flavohemoglobin containing equimolar amounts of heme and FAD as cofactors. The K M and k cat values of VvHmpA for NO at 37°C, the temperature encountered by V. vulnificus in the host, were greater than those at 30°C, indicating that VvHmpA detoxifies high levels of NO effectively during infection. Compared with the wild type, the VvhmpA mutant exhibited a lower NO-decomposition activity and impaired growth in the presence of NO in vitro. Also, the cytotoxicity and survival of the VvhmpA mutant infecting the NO-producing murine macrophage cells were lower than those of the wild type. Furthermore, the mouse lethality of the VvhmpA mutant was reduced compared to that of the parental wild type. The combined results revealed that VvHmpA is a potent virulence factor that is induced upon exposure to NO and important for the survival and pathogenesis of V. vulnificus during infection.

The membrane-bound transcriptional regulator CadC is activated by proteolytic cleavage in response to acid stress.

Proteolytic processes often participate in signal transduction across bacterial membranes. In Salmonella enterica serovar Typhimurium, the transcriptional regulator CadC activates genes of lysine decarboxylase system in response to external acidification and exogenous lysine. However, the signaling mechanism of CadC activation remains unexplored. We report here that CadC is located on the inner membrane under normal growth conditions but rapidly cleaved under acid stress conditions, leading to the induction of target gene transcription. As full-length CadC is degraded, the N-terminal fragment containing the DNA-binding domain accumulates in the inner membrane. Moreover, we show that C-terminal truncations of CadC abolish its degradation, resulting in complete loss of activator function. Together, these observations suggest that site-specific proteolysis at the periplasmic domain of CadC generates a biologically active form of N-terminal DNA-binding domain to promote target gene activation.

A 60-Hz sinusoidal magnetic field induces apoptosis of prostate cancer cells through reactive oxygen species.

PURPOSE: To explore the effects of power frequency magnetic fields (MF) on cell growth in prostate cancer, DU145, PC3, and LNCaP cells were examined in vitro. MATERIALS AND METHODS: The cells were exposed to various intensities and durations of 60-Hz sinusoidal MF in combination with various serum concentrations in the media. To analyze MF effects on cell growth, cell counting, trypan blue exclusion assay, Western blot analysis, flow cytometry, enzyme-linked immunosorbent assay (ELISA), semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), fluorescence microscopy, and spectrofluorometry were used. RESULTS: MF exposure induced significant cell growth inhibition and apoptosis in an intensity- and time-dependent manner, in which cell cycle arrest, cleaved Caspase-3, and reactive oxygen species (ROS) increased. Pretreatment with a Caspase-3 inhibitor or antioxidant, N-acetyl-L-cysteine (NAC), significantly attenuated MF-induced cell growth inhibition and cell death. Media replacement experiments failed to show any notable change in the MF effects. CONCLUSIONS: These results demonstrate 60-Hz sinusoidal MF-activated cell growth inhibition of prostate cancer in vitro. Apoptosis together with cell cycle arrest were the dominant causes of the MF-elicited cell growth inhibition, mediated by MF-induced ROS. These results suggest that a possibility of using 60-Hz MF in radiation therapy of prostate cancer could usefully be investigated.

Inducible spy Transcription Acts as a Sensor for Envelope Stress of Salmonella typhimurium.

Salmonella enterica infects a broad range of host animals, and zoonostic infection threatens both public health and the livestock and meat processing industries. Many antimicrobials have been developed to target Salmonella envelope that performs essential bacterial functions; however, there are very few analytical methods that can be used to validate the efficacy of these antimicrobials. In this study, to develop a potential biosensor for Salmonella envelope stress, we examined the transcription of the S. enterica serovar typhimurium spy gene, the ortholog of which in Escherichia coli encodes Spy (spheroplast protein y). Spy is a chaperone protein expressed and localized in the periplasm of E. coli during spheroplast formation, or by exposure to protein denaturing conditions. spy expression in S. typhimurium was examined by constructing a spy-gfp transcriptional fusion. S. typhimurium spy transcription was strongly induced during spheroplast formation, and also when exposed to membrane-disrupting agents, including ethanol and the antimicrobial peptide polymyxin B. Moreover, spy induction required the activity of regulator proteins BaeR and CpxR, which are part of the major envelope stress response systems BaeS/BaeR and CpxA/CpxR, respectively. Results suggest that monitoring spy transcription may be useful to determine whether a molecule particularly cause envelope stress in Salmonella.