Investigation of non-precious metal cathode catalysts for direct borohydride fuel cells.

Borohydride crossover in anion exchange membrane (AEM) based direct borohydride fuel cells (DBFCs) impairs their performance and induces cathode catalyst poisoning. This study evaluates three non-precious metal catalysts, namely LaMn0.5Co0.5O3 (LMCO) perovskite, MnCo2O4 (MCS) spinel, and Fe-N-C, for their application as cathode catalysts in DBFCs. The rotating disk electrode (RDE) testing shows significant borohydride tolerance of MCS. Moreover, MCS has exhibited exceptional stability in accelerated durability tests (ADTs), with a minimal reduction of 10 mV in half-wave potential. DFT calculations further reveal that these catalysts predominantly adsorb over , unlike commercial Pt/C which preferentially adsorbs . In DBFCs, MCS can deliver a peak power density of 1.5 W cm-2, and a 3% voltage loss after a 5 hours durability test. In contrast, LMCO and Fe-N-C have exhibited significantly lower peak power density and stability. The analysis of the TEM, XRD, and XPS results before and after the single-cell stability tests suggests that the diminished stability of LMCO and Fe-N-C catalysts is due to catalyst detachment from carbon supports, resulting from the nanoparticle aggregation during the high-temperature preparation process. Such findings suggest that MCS can effectively mitigate the fuel crossover challenge inherent in DBFCs, thus enhancing its viability for practical application.

High Prevalence, Genetic Diversity, and Recombination of Porcine Sapelovirus in Pig Farms in Fujian, Southern China.

Porcine sapelovirus (PSV) is a ubiquitous virus in farmed pigs that is associated with SMEDI syndrome, polioencephalomyelitis, and diarrhea. However, there are few reports on the prevalence and molecular characterization of PSV in Fujian Province, Southern China. In this study, the prevalence of PSV and a poetical combinative strain PSV2020 were characterized using real-time PCR, sequencing, and bioinformatics analysis. As a result, an overall sample prevalence of 30.8% was detected in 260 fecal samples, and a farm prevalence of 76.7% was observed in 30 Fujian pig farms, from 2020 to 2022. Noteably, a high rate of PSV was found in sucking pigs. Bioinformatics analysis showed that the full-length genome of PSV2020 was 7550 bp, and the genetic evolution of its ORF region was closest to the G1 subgroup, which was isolated from Asia and America; the similarity of nucleotides and amino acids to other PSVs was 59.5~88.7% and 51.7~97.0%, respectively. However, VP1 genetic evolution analysis showed a distinct phylogenetic topology from the ORF region; PSV2020 VP1 was closer to the DIAPD5469-10 strain isolated from Italy than strains isolated from Asia and America, which comprise the G1 subgroup based on the ORF region. Amino acid discrepancy analysis illustrated that the PSV2020 VP1 gene inserted twelve additional nucleotides, corresponding to four additional amino acids (STAE) at positions 898-902 AAs. Moreover, a potential recombination signal was observed in the 2A coding region, near the 3' end of VP1, owing to recombination analysis. Additionally, 3D genetic evolutionary analysis showed that all reference strains demonstrated, to some degree, regional conservation. These results suggested that PSV was highly prevalent in Fujian pig farms, and PSV2020, a PSV-1 genotype strain, showed gene diversity and recombination in evolutionary progress. This study also laid a scientific foundation for the investigation of PSV epidemiology, molecular genetic characteristics, and vaccine development.

Effects of Toxin-Antitoxin System HicAB on Biofilm Formation by Extraintestinal Pathogenic E. coli.

The type II toxin-antitoxin (T-A) HicAB system is abundant in several bacteria and archaea, such as Escherichia coli, Burkholderia Pseudomallei, Yersinia pestis, Pseudomonas aeruginosa, and Streptococcus pneumoniae. This system engages in stress response, virulence, and bacterial persistence. This study showed that the biofilm-forming ability of the hicAB deletion mutant was significantly decreased to moderate ability compared to the extra-intestinal pathogenic Escherichia coli (ExPEC) parent strain and the complemented strain, which are strong biofilm producers. Congo red assay showed that the hicAB mutant maintained the ability to form curli fimbriae. Using RNA-seq and comparative real-time quantitative RT-PCR, we observed the difference in gene expression between the hicAB mutant and the parent strain, which was associated with biofilm formation. Our data indicate that the HicAB type II T-A system has a key role in biofilm formation by ExPEC, which may be associated with outer membrane protein (OMP) gene expression. Collectively, our results indicate that the hicAB type II T-A system is involved in ExPEC biofilm formation.

The Chemoreceptor Sensory Adaptation System Produces Coordinated Reversals of the Flagellar Motors on an Escherichia coli Cell.

In isotropic environments, an Escherichia coli cell exhibits coordinated rotational switching of its flagellar motors, produced by fluctuations in the intracellular concentration of phosphorylated CheY (CheY-P) emanating from chemoreceptor signaling arrays. In this study, we show that these CheY-P fluctuations arise through modifications of chemoreceptors by two sensory adaptation enzymes: the methyltransferase CheR and the methylesterase CheB. A cell containing CheR, CheB, and the serine chemoreceptor Tsr exhibited motor synchrony, whereas a cell lacking CheR and CheB or containing enzymatically inactive forms did not. Tsr variants with different combinations of methylation-mimicking Q residues at the adaptation sites also failed to show coordinated motor switching in cells lacking CheR and CheB. Cells containing CheR, CheB, and Tsr [NDND], a variant in which the adaptation site residues are not substrates for CheR or CheB modifications, also lacked motor synchrony. TsrΔNWETF, which lacks a C-terminal pentapeptide-binding site for CheR and CheB, and the ribose-galactose receptor Trg, which natively lacks this motif, failed to produce coordinated motor switching, despite the presence of CheR and CheB. However, addition of the NWETF sequence to Trg enabled Trg-NWETF to produce motor synchrony, as the sole receptor type in cells containing CheR and CheB. Finally, CheBc, the catalytic domain of CheB, supported motor coordination in combination with CheR and Tsr. These results indicate that the coordination of motor switching requires CheR/CheB-mediated changes in receptor modification state. We conclude that the opposing receptor substrate-site preferences of CheR and CheB produce spontaneous blinking of the chemoreceptor array's output activity. IMPORTANCE Under steady-state conditions with no external stimuli, an Escherichia coli cell coordinately switches the rotational direction of its flagellar motors. Here, we demonstrate that the CheR and CheB enzymes of the chemoreceptor sensory adaptation system mediate this coordination. Stochastic fluctuations in receptor adaptation states trigger changes in signal output from the receptor array, and this array blinking generates fluctuations in CheY-P concentration that coordinate directional switching of the flagellar motors. Thus, in the absence of chemoeffector gradients, the sensory adaptation system controls run-tumble swimming of the cell, its optimal foraging strategy.

The Immune Efficacy of Inactivated Pseudorabies Vaccine Prepared from FJ-2012ΔgE/gI Strain.

An emerging pseudorabies virus (PRV) variant has been reported on Bartha-K61-vaccinated farms since 2011, causing great economic losses to China's swine-feeding industry. In this study, two vaccines, FJ-2012ΔgE/gI-GEL02 and FJ-2012ΔgE/gI-206VG, were administered to piglets for immune efficacy investigation. Humoral immunity response, clinical signs, survival rate, tissue viral load, and pathology were assessed in piglets. The results showed that both vaccines were effective against the PRV FJ-2012 challenge, the piglets all survived while developing a high level of gB-specific antibody and neutralizing antibody, the virus load in tissue was alleviated, and no clinical PR signs or pathological lesions were displayed. In the unimmunized challenged group, typical clinical signs of pseudorabies were observed, and the piglets all died at 7 days post-challenge. Compared with commercial vaccines, the Bartha-K61 vaccine group could not provide full protection, which might be due to a lower vaccine dose; the inactivated vaccine vPRV* group piglets survived, displaying mild clinical signs. The asterisk denotes inactivation. These results indicate that FJ-2012ΔgE/gI-GEL02 and FJ-2012ΔgE/gI-206VG were effective and could be promising vaccines to control or eradicate the new PRV epidemic in China.

Application of Decision Tree in PE Teaching Analysis and Management under the Background of Big Data.

With the continuous development of physical education reform, the defects and deficiencies of physical education teaching in colleges and universities are increasingly exposed. The reform of the original physical education teaching thought, education system, teaching mode, and method has achieved little. At present, the research on the prediction of physical education teaching achievements is mainly aimed at the prediction of athletes and physical education teaching achievements or the prediction of the past data of college students. This paper studies the physical education under the decision tree under the background of big data and constructs the physical education management system. When the number of tests reaches 40, the qualified rate of long-distance running is 65.2%, that of basketball is 68.1%, and that of volleyball is 68.2%. The quality of physical education teaching determines the lifeline of the development of school physical education teaching. In the process of collecting and selecting teaching materials, this paper enriches teaching materials and teaching reflection, cultivates one's own understanding, and improves the artistic appeal and creativity of teaching.

Prebiotic-like cyclodextrin assisted silybin on NAFLD through restoring liver and gut homeostasis.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease with no currently approved treatment. The natural compound silybin (SLN) has versatile hepatoprotective efficacy with negligible adverse effects; however, poor absorption limits its clinical applications. Gut microbiota has been proposed to play a crucial role in the pathophysiology of NAFLD and targeted for disease control. Cyclodextrins, the cyclic oligosaccharides, were documented to have various health benefits with potential prebiotic properties. This study aimed to develop a silybin-2-hydroxypropyl-ß-cyclodextrin inclusion (SHßCD) to improve the therapeutic efficacy of SLN and elucidate the mechanisms of improvement. The results showed that SLN formed a 1:1 stoichiometric inclusion complex with HP-ß-CD. The solubility of SLN was increased by generating SHßCD, resulting in improved drug permeability and bioavailability. In high-fat diet (HFD)-fed hamsters, SHßCD modulated gut health by restoring the gut microbiota and intestinal integrity. SHßCD showed superior anti-lipid accumulation, antioxidant, and anti-inflammatory effects compared with SLN alone. Transcriptome analysis in the liver tissue implied that the improved inflammation and/or energy homeostasis was the potential mechanism. Therefore, SHßCD may be a promising alternative for the treatment of NAFLD, attributing to the dual functions of HßCD on drug absorption and gut microbial homeostasis.

Optical Properties of Thomson Scattering Diagnostics Lower Window Glass under Laser Irradiation.

Tokamak diagnostic window glass is an indispensable optical medium in fusion research. The transmittance of the device affects the optical performance and accuracy of the diagnostic system. Especially, the window glass serves as the entrance of the light source while performing the sealing function for the active diagnosis method represented by Thomson scattering diagnostics. In this work, we studied the influence of the laser irradiation and tokamak discharge on the EAST (Experimental Advanced Superconducting Tokamak) Thomson scattering diagnostic borosilicate glass window. Using X-ray photoelectron spectroscopy (XPS) and Raman scattering, we found that carbon-based impurities in the device aggravated the film damage due to laser irradiation, reducing the performance of the coating of the glass. Besides, the laser and the various rays of tokamak discharge generated many point defects in the glass, increasing the light absorption of the glass. These two factors caused the glass transmittance to drop significantly (from 99.99% to 77.62%). In addition, the long-term laser irradiation primarily reduced the transmittance, while environmental rays had a minor impact on the same. This work provides valuable insights into the selection and effective use of glass in optics-based diagnostics.

The sole rupture of flexor digitorum tendons to the index finger following volar locking plate fixation of distal radius fracture: A case report.

Distal radius fracture is a common injury, especially in elderly people, and internal fixation with volar locking plate (VLP) is becoming an increasingly popular technique for the management of displaced and/or unstable distal radius fractures. One of the most common complications of this treatment is the flexor tendon rupture, mostly of the flexor pollicis longus (FPL). While the rupture of flexor digitorum tendons to the index (FDI) mostly occurs concomitantly with the rupture of FPL after the treatment using volar plating for distal radial fracture, sole rupture of the FDI without FPL rupture is very rare. Here, we report a case of the sole rupture of FDI after volar locking plating and analyze its pathogenesis indicating that the lift-up of the distal ulnar edge of the plate related to the malcorrection of the fracture site is the culprit for this specific complication.

Fluctuations in Intracellular CheY-P Concentration Coordinate Reversals of Flagellar Motors in E. coli.

Signal transduction utilizing membrane-spanning receptors and cytoplasmic regulator proteins is a fundamental process for all living organisms, but quantitative studies of the behavior of signaling proteins, such as their diffusion within a cell, are limited. In this study, we show that fluctuations in the concentration of the signaling molecule, phosphorylated CheY, constitute the basis of chemotaxis signaling. To analyze the propagation of the CheY-P signal quantitatively, we measured the coordination of directional switching between flagellar motors on the same cell. We analyzed the time lags of the switching of two motors in both CCW-to-CW and CW-to-CCW switching (∆tCCW-CW and ∆tCW-CCW). In wild-type cells, both time lags increased as a function of the relative distance of two motors from the polar receptor array. The apparent diffusion coefficient estimated for ∆t values was ~9 µm2/s. The distance-dependency of ∆tCW-CCW disappeared upon loss of polar localization of the CheY-P phosphatase, CheZ. The distance-dependency of the response time for an instantaneously applied serine attractant signal also disappeared with the loss of polar localization of CheZ. These results were modeled by calculating the diffusion of CheY and CheY-P in cells in which phosphorylation and dephosphorylation occur in different subcellular regions. We conclude that diffusion of signaling molecules and their production and destruction through spontaneous activity of the receptor array generates fluctuations in CheY-P concentration over timescales of several hundred milliseconds. Signal fluctuation coordinates rotation among flagella and regulates steady-state run-and-tumble swimming of cells to facilitate efficient responses to environmental chemical signals.

[Effects of arbuscular mycorrhizal fungi on metabolism of aroma substances in tobacco]. / ä¸›æžèŒæ ¹çœŸèŒå¯¹çƒŸè‰é¦™æ°”ç›¸å ³ç‰©è´¨ä»£è°¢çš„å½±å“.

Arbuscular mycorrhizal fungi (AMF) can promote nutrient absorption and improve stress resistance of host plants. The effects of AMF on aroma substance metabolism were rarely examined. In this study, we investigated the effects of AMF (Glomus mosseae) on glands and metabolism of aroma substances in tobacco leaves. The results showed that the density of gland hair and the relative expression of the glandular-specific lipid transporter gene NtLTP1, which was necessary to induce lipid secretion, were higher in the leaves of tobacco inoculated with AMF. The content of main aroma substances in tobacco leaves, such as carotenoids, chlorogenic acid and solanesol, were increased. Moreover, AMF inoculation increased the activities of phenylalanine ammonia-lyase (PAL) and polyphenol oxidase (PPO), the key aroma substance synthesis enzymes, and caused upregulation in the relative expression of phenylalanine transaminase, polyphenoloxidase, flavonoids alcoholase and squalene synthase encoding genes. In all, the symbiosis with G. mosseae could increase the abundance and secretory activity of glandular hairs and promote the synthesis of aroma substances in tobacco leaves.

Isolation of a Reassortant H1N2 Swine Flu Strain of Type "Swine-Human-Avian" and Its Genetic Variability Analysis.

We isolated an influenza strain named A/Swine/Fujian/F1/2010 (H1N2) from a pig suspected to be infected with swine flu. The results of electron microscopy, hemagglutination (HA) assay, hemagglutination inhibition (HI) assay, and whole genome sequencing analysis suggest that it was a reassortant virus of swine (H1N1 subtype), human (H3N2 subtype), and avian influenza viruses. To further study the genetic evolution of A/Swine/Fujian/F1/2010 (H1N2), we cloned its whole genome fragments using RT-PCR and performed phylogenetic analysis on the eight genes. As a result, the nucleotide sequences of HA, NA, PB1, PA, PB2, NP, M, and NS gene are similar to those of A/Swine/Shanghai/1/2007(H1N2) with identity of 98.9%, 98.9%, 99.0%, 98.6%, 99.0%, 98.9%, 99.3%, and 99.3%, respectively. Similar to A/Swine/Shanghai/1/2007(H1N2), we inferred that the HA, NP, M, and NS gene fragments of A/Swine/Fujian/F1/2010 (H1N2) strain were derived from classical swine influenza H3N2 subtype, NA and PB1 were derived from human swine influenza H3N2 subtype, and PB2 and PA genes were derived from avian influenza virus. This further validates the role of swine as a "mixer" for influenza viruses.

Pathogenicity and Whole Genome Sequence Analysis of a Pseudorabies Virus Strain FJ-2012 Isolated from Fujian, Southern China.

The outbreaks of pseudorabies have been frequently reported in Bartha-K61-vaccinated farms in China since 2011. To study the pathogenicity and evolution of the circulating pseudorabies viruses in Fujian Province, mainland China, we isolated and sequenced the whole genome of a wild-type pseudorabies virus strain named "FJ-2012." We then conducted a few downstream bioinformatics analyses including phylogenetic analysis and pathogenic analysis and used the virus to infect 6 pseudorabies virus-free piglets. FJ-2012-infected piglets developed symptoms like high body temperature and central nervous system disorders and had high mortality rate. In addition, we identified typical micropathological changes such as multiple gross lesions in infected piglets through pathological analysis and conclude that the FJ-2012 genome is significantly different from known pseudorabies viruses, in which insertions, deletions, and substitutions are observed in multiple immune and virulence genes. In summary, this study shed lights on the molecular basis of the prevalence and pathology of the pseudorabies virus strain FJ-2012. The genome of FJ-2012 could be used as a reference to study the evolution of pseudorabies viruses, which is critical to the vaccine development of new emerging pseudorabies viruses.

[Vacuum sealing drainage combined with discontinuous windowing technique for repairing large area exposed wound of Achilles tendon].

OBJECTIVE: To explore clinical effect of vacuum sealing drainage (VSD) combined with discontinuous windowing technique for repairing large area exposed wounds of Achilles tendon. METHODS: From July 2009 to May 2014, 11 patients with large exposed wounds of Achilles tendon were treated, including 5 males and 6 females with an average age of 43 years old (aged from 7 to 65 years old). Among them, 4 cases were skin necrosis caused by heavy objects abrasion and contusion; 3 cases were caused by distal tibiofibula fractures; 3 cases were caused by bicycle-spoke injuries; 1 case was caused by diabetes. Areas of exposed Achilles tendon were from 6 cmx3 cm to 14 cmx5 cm without tendon rupture or bone exposed. After debridement, discontinuous fenestration on Achilles tendon was made by knife blade parallel with longitudinal axis of Achilles tendon, combined with Vacuum Sealing Drainage (VSD) treatment. RESULTS: After drainage treatment with one VSD cycle (5 to 7 days), abundant fresh granulation tissues were growing on all wounds and survived well after the second phase dermatoplasty. All patients were followed up for 12 to 24 months, the color of skin flap was good, the texture was soft without burst. At 3 to 4 months after operation, subcutaneous fat was appeared under the flap, the skin was sliding, movement of ankle joints was good. No delayed Achilles tendon rupture were occurred. CONCLUSION: Vacuum sealing drainage (VSD) combined with discontinuous fenestration is a simple, safe and effective method for repairing large area exposed wounds of Achilles tendon,which could minimize the secondary damage caused by wounds of skin flap grafting.

G226, a new epipolythiodioxopiperazine derivative, triggers DNA damage and apoptosis in human cancer cells in vitro via ROS generation.

AIM: G226 is a novel derivative of epipolythiodioxopiperazines with potent inhibitory activity against cancer cells. Here, we sought to identify potential targets involved in the anti-cancer activity of G226. METHODS: Cell proliferation assay was conducted in a panel of 12 human cancer cell lines. The activities of topoisomerase I (Topo I) and Topo II were studied using supercoiled pBR322 DNA relaxation and kDNA decatenation assays. ROS production was assessed with probes DCFH-DA and H&E. Western blot analysis and flow cytometry were used to examine DNA damage, apoptosis and cell cycle changes. RESULTS: G226 displayed potent cytotoxicity in the 12 human cancer cell lines with a mean IC50 value of 92.7 nmol/L. This compound (1-100 µmol/L) selectively inhibited the activity of Topo II, and elevated the expression of phosphorylated-H2AX in a dose-dependent manner. In Topo II-deficient HL60/MX2 cells, however, G226-induced DNA damage, apoptosis and cytotoxicity were only partially reduced, suggesting that Topo II was not essential for the anti-tumor effects of G226. Furthermore, G226 (0.125-2 µmol/L) dose-dependently elevated the intracellular levels of H2O2 and in the cancer cells, and pretreatment with GSH, NAC or DTT not only blocked G226-induced intracellular accumulation of ROS, but also abrogated G226-mediated phosphorylation of H2AX, apoptosis and cytotoxicity. CONCLUSION: G226-mediated ROS production contributes to the anti-cancer activity of this compound.

G226, a novel epipolythiodioxopiperazine derivative, induces autophagy and caspase-dependent apoptosis in human breast cancer cells in vitro.

AIM: To investigate the effects of G226, a novel epipolythiodioxopiperazine derivative, on human breast cancer cells in vitro, and to explore its anticancer mechanisms. METHODS: A panel of human breast cancer cell lines (MDA-MB-231, MDA-MB-468, MCF-7, ZR-75-30, BT474, BT549, SK-BR-3, T47D and HBL100) was examined. Cell proliferation was measured using sulforhodamine B assay, and cell apoptosis was detected with flow cytometry and caspase activity assay. Western blotting, immunofluorescence and targeted gene knockdowns were used to study autophagy in the cells. RESULTS: G226 suppressed proliferation of the 9 breast cancer cell lines with a mean IC50 value of 48.5 nmol/L (the mean IC50 value of adriamycin, a reference compound, was 170.6 nmol/L). G226 induced dose-dependent apoptosis of MDA-MB-231 and MCF-7 cells, accompanied by markedly increased activities of caspase-8 and caspase-3/7, which were abolished by caspase inhibitors zVAD or zIETD. G226 also induced mitochondrial outer membrane permeabilization, resulted in the caspase-9 activation. Moreover, G226 dose-dependently enhanced the autophagy marker LC3-II and autophagy substrate p62 accumulation in the cells, which were co-localized with caspase-8. Silencing of p62 or LC3 partially diminished caspase-8 and subsequent caspase-3 activation. LC3 silencing partially reversed G226-induced apoptosis, but p62 silencing elicited a subtle effect on G226-induced apoptosis. CONCLUSION: The novel epipolythiodioxopiperazine derivative G226 exerts potent anticancer action against human breast cancer cells in vitro, via triggering autophagy and caspase-dependent apoptosis.

Thiol-reactive amphiphilic block copolymer for coating gold nanoparticles with neutral and functionable surfaces.

Nanoparticles designed for biomedical applications are often coated with polymers containing reactive functional groups, such as -COOH and -NH2, to conjugate targeting ligands or drugs. However, introducing highly charged surfaces promotes binding of the nanoparticles to biomolecules in biological systems through ionic interactions, causing the nanoparticles to aggregate in biological environments and consequently undergo strong non-specific binding to off-target cells and tissues. Developing a unique polymer with neutral surfaces that can be further functionalized directly would be critical to develop suitable nanomaterials for nanomedicine. Here, we report a thiol-reactive amphiphilic block copolymer poly(ethylene oxide)-block-poly(pyridyldisulfide ethylmeth acrylate) (PEO-b-PPDSM) for coating gold nanoparticles (AuNPs). The resultant polymer-coated AuNPs have almost neutral surfaces with slightly negative zeta potentials from -10 to 0 mV over a wide pH range from 2 to 12. Although the zeta potential is close to zero we show that the PEO-b-PPDSM copolymer-coated AuNPs have both good stability in various physiological conditions and reduced non-specific adsorption of proteins/biomolecules. Because of the multiple pyridyldisulfide groups on the PPDSM block, these individually dispersed nanocomplexes with an overall hydrodynamic size around 43.8 nm can be directly functionalized via disulfide-thiol exchange chemistry.

Assembly and stoichiometry of FliF and FlhA in Salmonella flagellar basal body.

The bacterial flagellar export apparatus is required for the construction of the bacterial flagella beyond the cytoplasmic membrane. The membrane-embedded part of the export apparatus, which consists of FlhA, FlhB, FliO, FliP, FliQ and FliR, is located in the central pore of the MS ring formed by 26 copies of FliF. The C-terminal cytoplasmic domain of FlhA is located in the centre of the cavity within the C ring made of FliG, FliM and FliN. FlhA interacts with FliF, but its assembly mechanism remains unclear. Here, we fused yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) to the C-termini of FliF and FlhA and investigated their subcellular localization by fluorescence microscopy. The punctate pattern of FliF-YFP localization required FliG but neither FliM, FliN, FlhA, FlhB, FliO, FliP, FliQ nor FliR. In contrast, FlhA-CFP localization required FliF, FliG, FliO, FliP, FliQ and FliR. The number of FlhA-YFP molecules associated with the MS ring was estimated to be about nine. We suggest that FlhA assembles into the export gate along with other membrane components during the MS ring complex formation in a co-ordinated manner.

Load-sensitive coupling of proton translocation and torque generation in the bacterial flagellar motor.

The Salmonella flagellar motor consists of a rotor and about a dozen stator elements. Each stator element, consisting of MotA and MotB, acts as a proton channel to couple proton flow with torque generation. A highly conserved Asp33 residue of MotB is directly involved in the energy coupling mechanism, but it remains unknown how it carries out this function. Here, we show that the MotB(D33E) mutation dramatically alters motor performance in response to changes in external load. Rotation speeds of the MotA/B(D33E) and MotA(V35F)/B(D33E) motors were markedly slower than the wild-type motor and fluctuated considerably at low load but not at high load, whereas the rotation rate of the wild-type motor was stable at any load. At low load, pausing events were frequently observed in both mutant motors. The proton conductivities of these mutant stator channels in their 'unplugged' forms were only half of the conductivity of the wild-type channel. These results suggest that the D33E mutation induces a load-dependent inactivation of the MotA/B complex. We propose that the stator element is a load-sensitive proton channel that efficiently couples proton translocation with torque generation and that Asp33 of MotB is critical for this co-ordinated proton translocation.

Populational heterogeneity vs. temporal fluctuation in Escherichia coli flagellar motor switching.

The dynamic switching of the bacterial flagellar motor regulates cell motility in bacterial chemotaxis. It has been reported under physiological conditions that the switching bias of the flagellar motor undergoes large temporal fluctuations, which reflects noise propagating in the chemotactic signaling network. On the other hand, nongenetic heterogeneity is also observed in flagellar motor switching, as a large group of switching motors show different switching bias and frequency under the same physiological condition. In this work, we present simultaneous measurement of groups of Escherichia coli flagellar motor switching and compare them to long time recording of single switching motors. Consistent with previous studies, we observed temporal fluctuations in switching bias in long time recording experiments. However, the variability in switching bias at the populational level showed much higher volatility than its temporal fluctuation. These results suggested stable individuality in E. coli motor switching. We speculate that uneven expression of key regulatory proteins with amplification by the ultrasensitive response of the motor can account for the observed populational heterogeneity and temporal fluctuations.