To provide an increased understanding of avian influenza A(H7N9) activity in live-poultry market in space and time and hence improve H7N9 epidemic control, an ongoing environmental sampling program in multiple live-poultry markets across Guangdong, China was conducted during March 2013–June 2014.A total of 625 live-poultry markets throughout 21 prefecture areas took part in the study. A total of 10 environmental sites in markets for sampling were identified to represent 4 different poultry-related activity areas. At least 10 environmental samples were collected from each market every month. The real time RT-PCR was performed to detect the avian influenza A(H7N9) virus. Field survey was conducted to investigate the sanitation status of live-poultry markets.There were 109 human infections with H7N9 avian influenza in Guangdong, of which 37 (34%) died. A total of 18741 environmental swabs were collected and subjected to real-time RT-PCR test, of which 905(4.83%) were found positive for H7N9 virus. There were 201 (32.16%) markets affected by H7N9 in 16 prefecture areas. The detection of H7N9 virus in markets spiked in winter months. 63.33% markets (38/60) had no physical segregation for poultry holding, slaughter or sale zones. Closing live-poultry market significantly decreased the H7N9 detection rate from 14.83% (112/755) to 1.67% (5/300).This study indicates the importance of live-poultry market surveillance based on environmental sampling for H7N9 Avian Influenza control. Improving live-poultry market management and sanitation and changing consumer practices are critical to reduce the risk of H7N9 infection. In 2013 and 2014 two epidemic waves of an avian influenza A (H7N9) virus (hereafter "H7N9") affected China. The emerging infectious disease first started in Shanghai and spread to eastern and northern China in spring 2013 [Most human cases of H7N9 had direct or indirect poultry exposure at live-poultry markets (hereafter “LPMs”), where many environmental samples were found to be positive for H7N9 –6. Case-In addition to outbreak investigations and virus surveillance in humans, poultry and farms, environmental surveillance in LPMs can improve avian influenza control. Previous studies on H5N1 and H9N2 avian influenza have shown that regular environmental sampling from LPMs can help trace the virus activity and evaluate the risk of human outbreaks –16. To rGuangdong Province, located in southern China with a population over 100 million, was the epicenter of the second wave of the H7N9 outbreak in 2013–2014 where 109 human cases were reported till June 2014 . To provHuman infection with H7N9 is one of notifiable infectious diseases in China. Case definitions, surveillance for identification of infection, and laboratory test assays have been described previously . All labGuangdong CDC has conducted LPM surveillance since 2009. 4 prefectures out of 21 prefectures in the province were selected as sentinel sites where environmental specimens were collected and tested monthly from a total of 17 live poultry markets.Following the first detection of human infection with H7N9 in Shanghai March 2013, Guangdong CDC retrospectively tested environmental specimens collected from sentinel sites for the January to March 2013 period. Then the environmental sampling program for H7N9 expanded to the entire province, such that for each of the 21 prefectures, at least one local LPM was selected on the basis of their poultry sales and market coverage. There were two types of LPM included. One is the wholesale market where live poultry are assembled and held for sale and slaughter. Another is “wet market” retailing live poultry and fresh meat. A total of 625 LPMs, including 21 wholesale markets and 604 wet markets, throughout 21 prefecture cities took part in the study.By field observation and literature review, we identified 10 locations across 4 key activity areas within LMP for swab sampling in this study. The sampling locations include poultry feces, poultry cage, poultry-feeding trough, floor, poultry de-feather machine, Tub, Chopping board, display table, sewage and waste bin. The market activity areas included the poultry-holding zone, the slaughter zone, the sale zone and the waste-disposal zone. Guangdong CDC and 21 prefecture-level CDCs did environmental sampling by collecting at least 10 wet-swab specimens from sampling locations at each prefectures per month. In addition to the planned monitoring across the province, two-week emergence surveillance initiated to collected environmental swabs weekly from LPMs, which were suspected to be epidemiologically associated with human H7N9 infection. As infrastructure and processes differ between LPMs, not all locations were sampled for every market.A total of 22 national influenza collaborative laboratories located in 21 prefectures participated in this study. All swab samples were sent to the nearest laboratory for detection of H7N9 viral RNA by real-time RT-PCR method . All posTo access the poultry-related infrastructure and sanitation status in LPMs, we conducted a field survey in January 2014. 60 markets were randomly selected from the surveillance LPMs in Guangzhou, Shenzhen, Dongguan, Foshan and Yangjiang City. We developed a structured questionnaire to investigate each participating market by visual inspection and interviews with managers and vendors. The questionnaire included information about the demarcation of workflow including poultry holding, slaughter, sale, waste-disposal and workplace cleaning practices.http://api.map.baidu.com/lbsapi/getpoint/index.html). We analyzed the dataset and described the temporal and spatial trends by mapping the distribution of H7N9 incidence and affected markets. Chi-squared test (α = 0.05) and Fisher exact test were used to compare the H7N9-positive rate before and after LPM closure.Our study period is from January 2013 to June 2014. A dataset was created containing information on human H7N9 cases and LPMs sampled. The addresses of human H7N9 cases and LPMs were located and geocoded through an Internet geocoding service  had serious disease with pneumonia and 37 (33.94%) died. There were 69 male and 40 female H7N9 cases. The median age of the cases was 56 years (range 2.5–88 years) and 65 (59.63%) occurred in persons aged over 50 years old. The first H7N9 case had an onset of symptoms on July 28 and was confirmed on August 10 2013. Most cases  appeared in winter months since December 2013 and the incidence peaked in February 2014. The last H7N9 case in the study period was confirmed at the end of May 2014 . ConfirmOf 625 LPMs involved, 600 (96.00%) were located in urban areas and 25(4.00%) in rural areas. There were 201 (32.16%) LPMs affected by H7N9 in 16 prefecture areas . Of themThe first two environmental swabs positive for H7N9 virus were found in April and May 2013. They were collected respectively from two LPMs both in Meizhou, a prefecture located in the east of Guangdong. Then a neighboring prefecture Huizhou reported the first human H7N9 case in August 2013. After a comparatively “silent” period, Shenzhen identified a LPM with H7N9 contamination in late October, followed by a H7N9 case reported one week later in a neighboring city, Dongguan. More and more H7N9-positive LPMs and human infection had been identified since December 2013. The affected areas expanded to southern and western cities. Overall, 20 LPMs had been contaminated by H7N9 in 2013. The weekly detection of H7N9-positive LPMs increased sharply at the beginning of 2014 and reached its peak in March. Then it started to decline since April. Between January and June 2014, a total of 195 LPMs had been contaminated by H7N9.To response to outbreaks of human infection with H7N9, we took emergence surveillance by collecting environmental samples from epidemiologically associated LPMs. Case investigation determined that 87 local H7N9 cases had ever work or visit LPMs before their onset. Additionally, two cases reported in Hong Kong, China, which is closed to Guangdong, had a history of exposure to poultry in LPMs of Guangdong . A total2 = 92.45χ, P<0.01). The most common affected sites were sewage (10.07%), poultry-feeding trough (9.88%), and chopping board and knife (8.13%). We did not find any H7N9-positive samples in the holding tubs or waste bins  were found positive for H7N9 virus. In 2013, the H7N9-positive rate of environmental samples was 0.71 (44/6189). Between January and June 2014, the positive rate was up to 6.86 (861/12552). All four working zones and 8 of 10 sampling sites in LPMs had evidence of H7N9 contamination. The H7N9-positive rates differed significantly in working zones . All LPMs operated daily, the same vendors operated in the same stalls and the markets had segregated zones for waste disposal. It is shown that 38 markets (63.33%) have no physical segregation for poultry holding, slaughter or sale zones. Live poultry were generally kept in the market for a few days until sold, housing them overnight in stalls. Of 37 LPMs investigated, 24 (64.86%) markets had daily stall washing and 13 (35.14%) disinfected weekly.χ2 = 37.75, P<0.01) , 755 environmental swabs were sampled in the week before the market closure. 112 samples from 17 markets were H7N9-positive. The H7N9-positive rate was 14.83%. When these LPMs reopened, 300 environmental swabs were collected from the same 31 LPMs. Only 5 samples from 2 markets were found positive for H7N9 and the positive-rate significantly decreased to 1.67% ( P<0.01) .Our study provides baseline information for H7N9 contamination in live poultry markets of Guangdong, south China. With continuing environmental sampling throughout the H7N9 epidemic period, we have detected the importation of H7N9 and traced its time-space changes in LPMs. Our results indicate the H7N9 virus spread across Guangdong with a seasonal peak in winter months.Unlike other affected areas, Guangdong identified the H7N9 virus in local LPMs environment prior to the detection of human infection. Sequence analyses showed that the Guangdong H7N9 virus isolated from LPM shared a high sequence similarity (98.2% to 99.7%) with other strains from eastern China . First dEven though Guangdong enhanced human and environmental surveillance, further LPM contamination or human infection was not identified until three months later. It is unclear why H7N9 did not spread quickly in Guangdong following its initial detection. One explanation is that the imported viruses had limited viability within Guangdong poultry and they needed time to adapt to the local conditions. The introduction of H7N9 virus into Guangdong occurred at the end of spring 2013 when the first epidemic wave in China was ending. Before the second wave occurred in winter months, the H7N9 virus may have been circulating in poultries and markets at a low level that was difficult to detect. Another explanation could be the seasonality of H7N9 activity. The sharp increases in winter observed for human H7N9 cases and positive environmental specimens suggest that H7N9 avian influenza follows a similar seasonal pattern as H5N1 avian influenza . Higher We use the number of affected markets, rather than data on H7N9-positive samples, as an indicator to provide market-level analysis on the H7N9 contamination level in LPM environment and its temporal and geographical changes. Our figures show a close time-space association between human infection and LPM contamination of H7N9. Contaminated markets were found in most areas where human cases were reported. Our data provide ecological evidence that LPMs are the main source of H7N9 infection. The H7N9 epidemic started in eastern areas of the province and then expanded to southern and western areas. With H7N9 spreading, more and more human cases and contaminated LPMs appeared in the Pearl River delta region, the central part of the province. Given that H7N9 infection has not yet shown continuing person-to-person transmission, poultry trades and consumption contributed to the geographical distribution of H7N9 outbreaks. Because of high density of LPM and more connection between human and poultry, the Pearl River delta has always been referred as a high-risk region for avian influenza epidemics . A studyBirds infected with H7N9 normally have mild symptoms, but they can shed the virus for 7 to 10 days . We obseOur study shows that closing LPMs reduce H7N9 contamination. It has been already proved that closure of LPMs had indeed decreased the incidence of H7N9 human infection . HoweverThis study clearly indicates the importance of environmental sampling in LPMs for H7N9 prevention and control as it can be used to timely monitor the circulation of H7N9 virus and get a better understanding of any H7N9 epidemic. Improving LPM management and sanitation and changing consumer practices are critical to reduce the risk of H7N9 infection.S1 Table(DOCX)Click here for additional data file.


The TRAF-interacting protein (TRAIP) is an E3 ubiquitin ligase required for cell proliferation. TRAIP mRNA is downregulated in human keratinocytes after inhibition of the PI3K/AKT/mTOR signaling. Since E2F transcription factors are downstream of PI3K/AKT/mTOR we investigated whether they regulate TRAIP expression. E2F1 expression significantly increased the TRAIP mRNA level in HeLa cells. Reporter assays with the 1400bp 5′-upstream promoter in HeLa cells and human keratinocytes showed that E2F1-, E2F2- and E2F4-induced upregulation of TRAIP expression is mediated by 168bp upstream of the translation start site. Mutating the E2F binding site within this fragment reduced the E2F1- and E2F2-dependent promoter activities and protein-DNA complex formation in gel shift assays. Abundance of TRAIP mRNA and protein was regulated by the cell cycle with a peak in G2/M. Expression of GFP and TRAIP-GFP demonstrated that TRAIP-GFP protein has a lower steady-state concentration than GFP despite similar mRNA levels. Cycloheximide inhibition experiments indicated that the TRAIP protein has a half-life of around four hours. Therefore, the combination of cell cycle-dependent transcription of the TRAIP gene by E2F and rapid protein degradation leads to cell cycle-dependent expression with a maximum in G2/M. These findings suggest that TRAIP has important functions in mitosis and tumorigenesis. The TRAF-interacting protein  is a 53-kDa protein that contains at its N-terminal end a 55 amino acids long RING domain, followed by a putative coiled-coil domain and a leucine zipper region . TRAIP uTRAIP is required for early mice development since homozygous TRAIP knock-out mice die at embryonic day E6.5/7.5 caused by decreased number of proliferative cells and excessive apoptosis . This geRecently, it has been reported that NOPO/TRAIP interacts with DNA polymerase η which facilitates translesional synthesis after DNA damage . FurtherThe TRAIP mRNA level is strongly decreased in primary human epidermal keratinocytes (NHEK) undergoing differentiation induced by high calcium concentration, high cell density or phorbol ester , indicatE2F transcription factors are involved in many ways in the regulation of genes for proliferation, DNA replication, apoptosis and differentiation . E2F DNAIn this report we show that TRAIP gene expression is induced by E2F1, E2F2 and E2F4 through an E2F binding site located in the 5′upstream promoter region close to the transcription start site. TRAIP mRNA and protein expression correlated with a peak in the G2/M phase suggesting that TRAIP has physiological function(s) in G2/M.Previously, we have shown that inhibitors targeting PI3-Kinase LY294002) or mTOR (rapamycin) decreased TRAIP gene expression in proliferating keratinocytes . These f4002 or mPreviously, it has been shown that the hypo-phosphorylated form of the retinoblastoma (pRB1) protein binds to E2F1 leading to the inhibition of its transcriptional activity , 32. To In silico analysis of the 1.4kbp 5′-upstream region of the human and mouse TRAIP gene detected two conserved regions with similarity to the consensus E2F binding sequence [sequence . In the Reporter constructs driven by different deletion fragments A-F Fig.  from thein silico identified conserved E2F transcription factor binding site located at position +16 to +25  cultured in low-calcium medium. The results demonstrated that the fragments A-D had very similar reporter activities which were significantly higher than the basic vector whereas the activity with fragment F was not increased Fig. . Taken tPreviously, we have reported that addition of TPA to NHEK cultured in low calcium medium repressed TRAIP expression within 24 hours . TherefoTo further prove the binding of E2F transcription factors to the E2F site at +16 to +25 Fig. . In elecSince the TRAIP gene is a target of the cell cycle-dependent transcriptional activity of E2Fs we addressed the question whether endogenous TRAIP gene expression is modulated by the cell cycle. HeLa cells were synchronized either in the G0/G1, S or G2/M phase by serum starvation, double thymidine block or nocodazole treatment, respectively. Analysis of the cell cycle distributions by fluorescence-activated flow cytometry analysis of propidium iodide stained cells revealed that a high percentage of cells was arrested in the desired cell cycle phase: G0/G1, 62.8 ± 2.2%; S, 72.6 ± 5.3%; G2/M, 80.3 ± 3.0%; mean ± SEM, n=4. Endogenous TRAIP mRNA levels in the three cell cycle phases were measured by quantitative RT-PCR analysis using four house-keeping genes  for normalization. Previous experiments reported that their gene expression levels are minimally regulated during the cell cycle . The resTo investigate whether TRAIP protein expression was regulated post-transcriptionally, vectors driving expression of TRAIP-GFP and GFP from a CMV promoter were constructed. TRAIP-GFP was undergoing auto-ubiquitination  and locain silico analysis of the 5′ upstream region of the human and mouse TRAIP genes revealed two highly conserved putative E2F sites , and published data [Cell cycle progression is a complex physiological process coordinated by a multitude of signaling pathways that are regulated by internal and external cues. Experiments using shRNAs, knockout mice and inhibitors showed that the E3 ubiquitin ligase TRAIP is necessary for cellular proliferation , 19. Sintes Fig. , consisttes Fig.  which mites Fig.  requiredtes Fig. . Inspecthed data , 44–46 phed data  were fouThe distal E2F site (TTGGAGG) at the position −628/−621 . In addition, no good PEST [http://emboss.sourceforge.net/) were detected in the TRAIP amino acid sequence.The expression of E2F transcription factors and their target genes are modulated during the cell cycle. E2F transcription factors not only play an important role in controlling genes in G1/S phase to coordinate DNA replication, but also regulate genes in G2/M phase encoding proteins important for mitosis . Consistturnover , 63. Recturnover . Indeed,turnover  indicatiturnover , 67 predood PEST  motives The two findings that the TRAIP protein is unstable and expressed in a cell cycle-dependent manner as E2F target gene with a peak in the G2/M phase, confer upon TRAIP two properties commonly found for proteins involved in mitosis or cell cycle regulation . During In summary, we report novel biological properties of TRAIP which is an E3 ubiquitin ligase essential for cellular proliferation. We show that the cellular TRAIP level is tightly regulated by E2F-mediated transactivation and rapid protein turnover to precisely deliver high amounts of TRAIP in the G2/M phase, which is correlating well with its reported functions during mitosis. To understand fully the biological functions of TRAIP, the identification of its physiological substrates will be necessary. Since overexpression of TRAIP has been reported in basal cell carcinomas , breast HeLa, 293T, and 293FT cells were cultured in 3T3 medium . NHEK frFull-length human TRAIP cDNA was isolated from human keratinocytes by RT-PCR and inserted into the pGEM-T-Easy vector (Promega). All vectors used for the expression of TRAIP and GFP proteins were constructed by PCR amplification and cloning either into pEGFP-N3 (Clontech), pCI (Promega) or pCDH-CMV-MCS-EF1-Hygro (BioCat). Promoter fragments from the 5′-upstream region of the human TRAIP gene were isolated by PCR and cloned into the pGL3-Basic vector (Promega). Mutations in the E2F binding site were introduced by overlapping PCR amplification using Platinum Pfx DNA Polymerase (Invitrogen) and primers containing the desired mutations. All constructs were verified by sequence analysis. Expression plasmids were purchased from Open Biosystems  or Addgene .Lentiviral particles were produced in 293FT cells using the packaging vectors pMD2.G (Addgene plasmid 12259) and psPAX2 (Addgene plasmid 12260), and titrated as described previously . HeLa ceHeLa cells were synchronized in G0 phase by 3 days of serum starvation, in S phase by double thymidine block , and in M phase by 15h treatment with 400ng/ml nocodazole. Synchronized cells were stained with propidium iodide and analyzed for cell cycle distribution by flow cytometry as described .RNA was extracted from cultured cells using the RNeasy Mini Kit (Qiagen) and cDNA was produced using the Primescript RT reagent kit (TakaRa). Quantitative PCR analysis was performed with Power SYBRGreen PCR Mastermix (Applied Biosystems) using Quantitect Primer Assay (Qiagen) primers. Relative gene expression was calculated as described .4 cells per well) in 3T3 medium or EpiLife medium with growth supplements, respectively [HeLa cells or normal human epidermal keratinocytes (NHEK) were seeded in 24-well plates (2-3×10ectively . Cells wNuclear extracts from HeLa cells were prepared using the Nuclear and Cytoplasmic Extraction Kit (ThermoScientific). Electrophoretic mobility shift assays were carried out using the LightShift chemiluminescence EMSA kit (ThermoScientific) following the manufacturer's protocol. The following 5′-biotinylated double-stranded oligonucleotides were used as probes: WT, 5′-CCCGCTCCAGGAAGTCGTGCTGCGGAGC CAAATTTG-3′ (the E2F binding site is underlined) and Mut, 5′-CCCGCTCCAGGAAGTCGTGCTGCGAT CATGCGTTTG-3′ (the mutated E2F binding site is underlined). The same oligonucleotides without the biotin were used at 200-fold molar excess for competition experiments. After incubation, the reaction products were resolved on a 5% polyacrylamide gel in 0.5xTBE and transferred to HyBond N+ membrane (Amersham).Proteins were extracted from cells by lysis in 1% SDS/phosphate-buffered saline. The following antibodies were used for immunoblots: GFP (Clontech), actin (Sigma), FLAG (Sigma), and cyclin B1 . Signals from immunoblots were captured by LAS4000 and quantified by ImageJ.Statistical analysis was performed with GraphPad Prism 6 and results with p<0.05 were considered significant.The 5′upstream region of the human and mouse TRAIP genes were analysed by the rVISTA 2.0 program to find transcription factor binding sites that are phylogenetically conserved .


To the Editor: Since the first case of avian-origin influenza (H7N9) virus infection was reported in March 2013 in Shanghai, China, this virus has caused ≈400 confirmed cases . According to the Chinese Influenza Weekly Report (http://www.cnic.org.cn/eng/show.php?contentid=699), during the winter of 2013–14, multiple influenza viruses that infect humans co-circulated in southern China; these viruses were influenza A(H1N1)pdm09, seasonal influenza A(H3N2), and influenza B viruses, together with avian influenza (H7N9) virus,. Co-infection of humans with avian virus subtype H7N9 and human virus subtypes were reported in Jiangsu Province in June 2013 (http://www.who.int/influenza/vaccines/virus/recommendations/2013_14_north/en/). On December 26, 2013, the patient experienced influenza-like symptoms; the next day, she was hospitalized. Despite oral administration of oseltamivir, a rapidly progressive pneumonia developed, and the patient died of multiple organ failure 9 days after admission. On December 31, 2013 (day 5), 1 serum and 2 throat swab samples were collected; both throat swab samples were positive for influenza A and A(H1N1)pdm09 virus, according to the universal and subtype-specific primers provided by the Chinese Center for Disease Control and Prevention (http://www.who.int/influenza/resources/documents/diagnostic_recommendations/en/). On January 3, 2014 (day 8), 1 bronchial secretion and 1 serum sample were also collected.In a retrospective study, we identified co-infection with A(H1N1)pdm09 and novel avian-origin H7N9 virus in a 61-year-old female patient from the urban area of Shanghai, China. She had a 3-year history of thrombocythemia. In October 2013, she received an annual vaccine that included an A/California/7/2009(H1N1)pdm09–like virus, an A/Texas/50/2012(H3N2)–like virus, and an influenza B virus, as recommended by the World Health Organization  or embryonated eggs (H7N9) through several passages. The hemagglutination titers of the isolated H7N9 (A/Shanghai/Mix1/2014[H7N9]) and A(H1N1)pdm09 viruses (A/Shanghai/Mix1/2014[H1N1]) in allantoic fluid were 32 and 8, respectively. Cultures of the 2 isolates were subjected to RT-PCR for detection of 8 genomic segments. The RT-PCR products were sequenced on an ABI 3730XL Automatic DNA Analyzer by using an ABI Prism BigDye Terminator v3.1 Cycle Sequencing Kit . The full-genome sequences were submitted to the National Center for Biotechnology Information Influenza Virus Resource .To determine whether the reassortment had occurred between the 2 subtypes of influenza A viruses during the co-infection, we isolated the virus strains from the bronchial secretion on either MDCK cells (A[H1N1]pdm09) (http://www.megasoftware.net) to estimate relationships with selected influenza A virus reference sequences obtained from GenBank and the Global Initiative on Sharing Avian Influenza Data database. DNA sequence analysis showed that no genetic reassortment had occurred between the 2 subtypes because only H7N9 or A(H1N1)pdm09 genomic fragments were found in the culture of the respective isolate (Phylogenetic trees were constructed by using the neighbor-joining method in MEGA 5.1 ( isolate . Phyloge isolate .Co-infections with H7N9 and other influenza subtypes have been reported from Jiangsu (Phylogenetic analysis of 12 internal genes of the co-infection viruses in Shanghai, China.


Dermatophagoides pteronissinus (DP) inhibited neutrophil apoptosis, but Dermatophagoides farinae extract had no effect. Anti-apoptotic signaling mediated by DP involves in TLR4, Lyn, PI3K, Akt, ERK, and NF-κB in normal neutrophils. DP delayed cleavage of procaspase 9 and procaspase 3 and the decrease in Mcl-1 expression. Supernatant collected from DP-treated normal neutrophils inhibited the constitutive apoptosis of normal neutrophils, and S100A8 and S100A9 were identified as anti-apoptotic proteins in the supernatant. S100A8 and S100A9 transduced the anti-apoptotic signal via TLR4, Lyn, PI3K, Akt, ERK, and NF-κB. DP also suppressed asthmatic neutrophil apoptosis and induced secretion of S100A8 and S100A9, which delayed the constitutive apoptosis. The anti-apoptotic effects of DP, S100A8 and S100A9 in asthmatic neutrophils are associated with TLR4, Lyn, PI3K, Akt, ERK, and NF-κB. The concentrations of S100A8 and S100A9 were significantly elevated in asthmatic bronchoalveolar lavage fluid (BALF) when compared to normal BALF (p<0.01), but not in serum. S100A8 concentration in BALF was positively correlated with the number of BALF neutrophils and negatively correlated with FEV1(%). These findings improve our understanding of the role of HDM in regulation of neutrophil apoptosis in normal individuals and asthmatics and will enable elucidation of asthma pathogenesis.House dust mites (HDMs) induce allergic diseases such as asthma. Neutrophil apoptosis is an important process of innate immunity, and its dysregulation is associated with asthma. In this study, we examined the effects of HDM on constitutive apoptosis of normal and asthmatic neutrophils. Extract of  Dermatophagoides pteronissinus (DP) and Dermatophagoides farinae (DF), are heavily involved in asthma pathogenesis .Data were expressed as the means ± SD. Statistical differences were analyzed using a paired t-test for a two-group comparison and one-way ANOVA for comparison of more than two groups. The SPSS statistical software package  was used for statistical analysis. A To investigate the effects of HDM on neutrophil apoptosis, we first evaluated whether major allergens of HDM, DP and DF alter the regulation of neutrophil apoptosis. DP blocked neutrophil apoptosis in a dose-dependent manner, and DF had no effect on apoptosis Fig  and 1B. As shown in Fig As shown in Because S100A8 and S100A9 is secreted by neutrophils under the influence of DP and has an inhibitory effect on neutrophil apoptosis, we investigated how they act as anti-apoptotic proteins comparable to the DP-mediated signaling mechanism. TLR4i inhibited the anti-apoptotic effects of S100A8 and S100A9 in a dose-dependent manner . DP, S10p<0.01)  and FEV1/FVC(%), indicating that neutrophils are associated with asthma pathogenesis and severity (p<0.01) . As showp<0.01) . Supernap<0.01) . S100A8 p<0.01) . To furtp<0.01) . DP, S10p<0.01) .in vitro, we investigated the clinical relevance of the above findings in asthma subjects. Both S100A8 and S100A9 proteins were significantly elevated in BALF (p<0.01), but not in serum (p<0.01), whereas there was no correlation between neutrophil number and S100A9 expression  and western blotting (B).(TIF)Click here for additional data file.S2 FigLinear regression represents the correlation between neutrophils in BALF and serum and FEV1(%) or FEV1/FVC(%).(TIF)Click here for additional data file.S3 Figp < 0.05 and **p < 0.01 indicate a significant difference between the control and DP-treated groups.Normal, (A) and asthmatic (B) neutrophils (8<n<29), were incubated for 24 h with and without DP (10 μg/ml), Der p 1 (10 μg/ml) and Der p 2 (10 μg/ml) (left panel) or were pretreated in the absence and presence of E64 (50 μg/ml) and aprotinin (Ap) (50 μg/ml) for 1 h, after which the cells were incubated for 24 h in the absence and presence of DP (10 μg/ml) (right panel). Apoptosis was analyzed by measuring the binding of annexin V-FITC and PI. Data are expressed as the means ± SD and are presented relative to the control, which was set at 100%. *(TIF)Click here for additional data file.S4 Figp < 0.01 indicates a significant difference between the control and DP-treated groups.Normal neutrophils (n = 3) were pretreated in the absence and presence of anti-TLR2 blocking antibodies in the indicated concentration for 1 h, after which the cells were incubated for 24 h in the absence and presence of DP (10 μg/ml). Apoptosis was analyzed by measuring the binding of annexin V-FITC and PI. Data are expressed as the means ± SD and are presented relative to the control, which was set at 100%. **(TIF)Click here for additional data file.S5 Fig(A-B) Data are presented by classifying the results from (TIF)Click here for additional data file.S6 FigNormal neutrophils (n = 7) were incubated for 24 h with and without Der p 7 (10 μg/ml). Apoptosis was analyzed by measuring the binding of annexin V-FITC and PI. Data are expressed as the means ± SD and are presented relative to the control, which was set at 100%.(TIF)Click here for additional data file.


Drosophila melanogaster, dual oxidase (Duox) kills pathogenic microbes by generating the microbicidal reactive oxygen species (ROS), hypochlorous acid (HOCl) in response to bacterially excreted uracil. The physiological function of enzymatically generated HOCl in the gut is, however, unknown aside from its anti-microbial activity. Drosophila TRPA1 is an evolutionarily conserved receptor for reactive chemicals like HOCl, but a role for this molecule in mediating responses to gut microbial content has not been described. Here we identify a molecular mechanism through which bacteria-produced uracil facilitates pathogen-clearing defecation. Ingestion of uracil increases defecation frequency, requiring the Duox pathway and TrpA1. The TrpA1(A) transcript spliced with exon10b (TrpA1(A)10b) that is present in a subset of midgut enteroendocrine cells (EECs) is critical for uracil-dependent defecation. TRPA1(A)10b heterologously expressed in Xenopus oocytes is an excellent HOCl receptor characterized with elevated sensitivity and fast activation kinetics of macroscopic HOCl-evoked currents compared to those of the alternative TRPA1(A)10a isoform. Consistent with TrpA1’s role in defecation, uracil-excreting Erwinia carotovora showed higher persistence in TrpA1-deficient guts. Taken together, our results propose that the uracil/Duox pathway promotes bacteria expulsion from the gut through the HOCl-sensitive receptor, TRPA1(A)10b, thereby minimizing the chances that bacteria adapt to survive host defense systems.Pathogen expulsion from the gut is an important defense strategy against infection, but little is known about how interaction between the intestinal microbiome and host immunity modulates defecation. In  Duox pathway is known to kill ill-causing bacteria in the gut by producing chlorine bleach. Our study reveals that the bleach does not stop there, working further to promote expulsion of the bacteria from the gut through a sensitive bleach detector. Moreover, in the gut deficient for the bleach detector, the bacteria not only stayed longer, but also opportunistically survived the bleach that they encounter afterwards. This is the first identification of a mechanism illustrating how important it is that gut defense systems control defecation, and helps explain why troubles in the gut cause changes in defecation.The amount and pattern of defecation are often determined by the bacterial composition in the gut, and can have a significant impact on human health. It is however unknown how changes in the bacterial community affect defecation. A chemical defense system called the  Drosophila gut is under the control of two distinct innate immune mechanisms, the imd and Duox pathways [imd pathway [2+ pathway increases the enzymatic activity of Duox exploiting the intracellular Ca2+ -binding domain of Duox [Duox gene [Duox dramatically increases the concentration of highly microbicidal HOCl [phox-like oxidase domain of Duox extracts electrons from NADPH, and the electrons are delivered to the extracellular space through two heme structures in the transmembrane domains of Duox, sequentially forming superoxide and hydrogen peroxide (H2O2). The extracellularly generated H2O2 is then subjected to peroxidation by the peroxidase homology domain (PHD) of Duox to generate microbicidal HOCl. Thus, the Duox pathway plays a critical role for the control of the gut microbiome homeostasis by responding to bacterially excreted uracil and producing the reactive chlorine oxidant, HOCl.Encountering other organisms in nature offers opportunities of benefits or dangers depending on the relationship of the organisms facing each other. We intimately interact with bacteria in the gut, and the control of such interaction is the key to the health of animals –3. Althopathways –7. In thpathways –10, whic pathway ,8. The u of Duox , while tdal HOCl  in the gdal HOCl . The intDrosophila larvae was reported to be modulated by diuretic hormone 31 (DH31) [Drosophila provide the intestinal model system to study functional implication of the enteroendocrine system in various contexts including host/microbe interaction in the intestine.Gastrointestinal motility is a key determinant of defecation in mammals, and is controlled by enteroendocrine cells as well as enteric and central nervous systems. The majority of endocrine cells in the small and large intestine consists of enterochromaffin cells (EC cells) which contain >90% of intestinal serotonin. Serotonin in EC cells is closely associated with gastrointestinal motility ,14. Rece1 (DH31)  which is1 (DH31) ,17. ThusDrosophila melanogaster [As a reactive chemical, HOCl activates the mammalian ortholog of the evolutionarily conserved reactive chemical receptor TRPA1 . TRPA1s nogaster , which wnogaster . HoweverTrpA1 transcripts are present in the Drosophila gut [Drosophila TrpA1 in the gut has yet to be examined. In this study, we find that the TRPA1(A) isoform coupled with exon10b (referred to as TRPA1(A)10b) is the key receptor for the detection of the major reactive chlorine HOCl generated from activation of the Duox pathway in the gut and that such functional interaction between TRPA1 and the Duox pathway is critical for expulsion of the opportunistic pathogen Erwinia carotovora from the gut. These results indicate that the Duox innate immune system regulates defecation via TRPA1, thus maintaining the microbiome homeostasis in the Drosophila gut.Recently two independent transcriptome analyses showed that hila gut ,27, but wcs) 10a or TRPA1(A)10b expressed in Xenopus oocytes   or “foldwcs) , while tetection ,28,29, l animals . Note thc rescue ) restore animals . To exam animals . Spectra animals  or inges animals . Applica oocytes , indicatDuox activation is important for TrpA1-dependent defecation, N-methylmaleimide (NMM), a robust sulfhydryl-reactive chemical which activates TRPA1 [wcs with the extent similar to that of 20 μM uracil with TrpA1 required  fed with uracil abolished uracil-dependent defecation increase in wcs without significantly affecting defecation of a TrpA1 mutant  [TrpA1-deficient guts and TrpA1 RNAi-knockdown by EEC-restricted TrpA1(A)-Gal4 phenocopies TrpA1ins. TrpA1(A)-Gal4 contains the genomic fragment of the TrpA1(A) isoform-specific promoter upstream of the Gal4 coding sequence [TrpA1(A)-Gal4 comprised a subset of Prospero-positive cells (Bottom) throughout the anti-TRPA1-stained regions. In order to functionally demonstrate the role of TrpA1-positive cells in the defecation, TrpA1 RNAi knockdown was conducted with TrpA1(A)-Gal4, and showed significant reduction of TRPA1 expression in EECs -Gal4 did (Lower). These observations together with the Duox-dependent HOCl production in the gut lumen [TrpA1-positive neurons so far identified by immunostaining [TrpA1-expressing EECs in uracil-dependent defecation.Highly reactive HOCl produced by Duox was previously shown to accumulate in the lumen of the gut  where Tr present ,27. Indeand Top) , but notsequence . GFP expve cells  as in TRve cells , Bottom  in EECs  and urac in EECs . On the Gal4 did , Lower. ut lumen ,11 suggestaining ,20,22,37TrpA1 to conform to multiple sensory needs by modulating its temperature sensitivity [TrpA1(A), the chemosensory isoform expressed in taste neurons with much reduced thermal sensitivity [Lower). Interestingly, TrpA1(A)10b but not TrpA1(A)10a cDNA restored the uracil-dependent defecation when expressed in TrpA1(A)-Gal4 cells of TrpA1ins mutants (UAS-TrpA1(A)10a transgene in restoring the defecation is unlikely due to lack of functional expression based on two lines of evidence. First, functional expression of UAS-TrpA1(A)10a was comparable to that of UAS-TrpA1(A)10b in gustatory neurons (TrpA1 mutants by expressing either TrpA1(A)10a or TrpA1(A)10b in the TrpA1(A)-Gal4 cells (TrpA1(A)10a-expressing TrpA1–deficient animals in response to NMM ingestion demonstrates the functional expression of the UAS-TrpA1(A)10a transgene in the TrpA1(A)-Gal4 cells where expression of TrpA1(A)10a was unable to restore uracil-evoked defecation in TrpA1-deficient animals, while the former result (UAS-TrpA1(A)10a. The isoform dependence in uracil-evoked defecation might result from further functional diversification of TRPA1(A) via alternative exon10s altering response parameters to HOCl of the channel isoforms. To examine for any differential HOCl responsiveness of the two isoforms, TRPA1 isoforms were heterologously expressed in Xenopus laevis oocytes, and HOCl responses of the ion channel isoforms were characterized by the two-electrode voltage clamping approach. Interestingly, TRPA1(A)10b showed >3 times faster activation kinetics at 1 and 10 ppm of the HOCl-donating NaOCl at the membrane potential of -60 mV 10b, but not those expressing TrpA1(A)10a, showed spiking frequency increases in response to NaOCl 100 ppm (Duox-dependent defecation by rapidly reacting to low amounts of reactive chlorine species that are probably short-lived in the gut mucus due to their high reactivity. The enhanced sensitivity and response kinetics of TRPA1(A)10b might not originate from its intrinsic receptivity to reactive chemicals, because the transcript spliced with Exon10b loses a cysteine that is known to be important for electrophile sensitivity [In sensory systems, it has been reported that alternative use of exons  allows Tsitivity ,25. Our sitivity , is pred mutants A. The in neurons . Second,l4 cells , as was l4 cells . TRPA1, the well-characterized microbe excreting uracil, is sensitive to the Duox immune response [pyrE, was incapable of eliciting reactive chlorine production in the fly gut [pyrE, a trait requiring TrpA1 but independent of the imd pathway in flies (pyrE or WT ECC15 showed defecation spots per ingested volume (output/input) of 0.98+/-0.08 or 1.45+/-0.11, while ingestion of sucrose only or sucrose+uracil was led to output/input of 0.41+/-0.04 or 0.77+/-0.05  5 hrs after the start of the 2-hr Ecc15 ingestion session, and CFUs at the 8-hr time point were similar to those after the 2-hr feeding session . However, inability of uracil excretion was not led to apparent increases of ECC15 pyrE gut persistence. Given the roles of uracil and its derivative in a wide array of enzymatic events as coenzyme and regulator [pyrE with uracil raised CFU in the TrpA1ins not wcs guts albeit at a delayed time point, 8 hrs (right). Uracil supplementation might aid ECC15 pyrE to grow faster in the gut, but at the same time trigger the Duox pathway to drive the TrpA1-dependent expulsion. Because residing time or growth of ECC15 in the gut can be greatly influenced by the commensal microbiome that can differ in the genotypes of interest, germ-free animals were generated and tested for defecation induction by uracil  were least resistant to orally ingested ECC15, revealing that uracil-induced defecation is important for the survival of immune-compromised animals. This enhanced mortality in TrpA1ins, rele20 was not observed when ECC15 pyrE was ingested, suggesting the role of uracil excreted from ECC15 in resisting to the bacteria 10b. Thus, the exceptional activation kinetics and enhanced sensitivity to HOCl of the TRPA1(A)10b isoform reduce the risk of inflammation which may otherwise result from the need of prolonged exposure to excessive reactive chlorine. Our comparative analysis of human TRPA1 with Drosophila TRPA1 isoforms showed that human TRPA1 is inferior to TRPA1(A)10b in terms of NaOCl response kinetics and sensitivity (Drosophila TRPA1. A recent study proposed that TRPA1(A)10b is a potential receptor for H2O2 [2O2 responsiveness appeared to be similar to that of TRPA1(A)10a. Considering that TrpA1(A)10b but not TrpA1(A)10a is required for Duox-dependent defecation in EECs, HOCl rather than H2O2 is the key player resulting from uracil-induced Duox upregulation.Reactive chlorine species are not only highly bactericidal but also harmful to host cells. Although the gut contains a host-protecting antioxidant system , sustainsitivity , suggestfor H2O2  which isDrosophila TRPA1 functions in chemical nociception [How bacteria affect host defecation in mammals is not entirely understood. Mammals express Duox in mucosal epithelia, where bacteria densely colonize . At the iception ,22 and dUAS-TrpA1(A)10b transgene into the genome at the same site as UAS-TrpA1(A)10a [UAS-TrpA1(B) [Drosophila Stock Center, IN, USA. The da-Gal4 line is a kind gift from Won-Jae Lee at Seoul National University, TrpA1(A)-Gal4 [TrpA11 and TrpA1Gal4 [UAS-DuoxRNAi and UAS-TrpA1RNAi used in this study were the fly stocks of #33975 and #31384, respectively, from the Bloomington Drosophila Research Stock Center. These RNAi transgenic lines were generated by the Transgenic RNAi Resource Project (TRiP) [Site-specific transgenesis  was usedA1(A)10a  and UAS-TrpA1(B) , attp16 TrpA1(B)  in order(A)-Gal4  from DanrpA1Gal4  from Crat (TRiP) . Axenic In order to measure defecation frequencies, 12 to 15 male flies were used 4 to 6 days after eclosion. Female defecation is affected by reproduction status , which m2HPO4, 1.8 mM KH2PO4, 140 mM NaCl, 2.7 mM KCl, pH 7.3) and resuspended in 500 mM sucrose solution to the optical density (OD) of 10 at 600 nm to be offered via the capillaries to flies.To assess the defecation responses to Ecc15 strains, each strain was precultured for 8 hrs at 30°C in 4 ml of LB broth with the appropriate antibiotics. The preculture was transferred to a larger volume of LB at a ratio of 1:100 for the overnight main culture. The harvested bacteria were washed once in PBS . NaOCl  was freshly diluted immediately before experiments. TRPA1-expressing oocytes exhibited resting membrane potentials between -10 and -50 mV. Voltage was initially held at -60 mV, and a 300-ms voltage ramp from -60 to +60 mV was applied every second by the GeneClamp 500B amplifier  during data acquisition . To determine NaOCl sensitivities of the TRPA1 isoforms, currents were recorded till reaching steady state after evoked by a concentration of NaOCl, rather than fixing the application time across the experiments. Data points from dose dependence experiments were normalized with the respect to the current amplitude achieved by 100 ppm NaOCl, and fitted to the Hill equation by Sigmaplot12. Since current traces were often unable to be fitted to the single exponential equation, Time to the 70% maximum current at each NaOCl concentration was determined.TRPA1 currents in escribed ,22. BrieFive hours after start of ECC15-GFP at OD 10 in 500 mM sucrose, five guts per genotype were dissected out and ground in 0.2 ml of PBS. Each aliquot of 0.04 ml of ground guts was incubated with either 0, 0.1, 1, 5 or 10 ppm of NaOCl for 30 min at room temperature in 1 ml of PBS. Subsequently, the surviving bacteria were spot-titrated on spectinomycin LB agar plates with serial dilution in PBS.Extracellular recordings of gustatory neurons in L-bristles were conducted as previously described in detail .Student’s t-test and ANOVA Tukey multiple comparison and ANOVA repeated measures tests were performed with Sigmaplot12.Complementary DNA was prepared from dissected fly guts using the RETROscript kit . Primers used to determine exons encoding the N-terminus were described previously . RegardiE10com-F: 5’-GTGGACAAGGATGGGAAC-3’10a-R: 5’-CTCTCCGGTTTTCTCATCA-3’10b-R: 5’-GGTAGGGCCAAAACGAA-3’The response profile of R19S to the concentration range of NaOCl used in the study was determined with Deltascan . For confocal scanning of the R19S fluorescence signal , sucrose solutions containing 10 μM R19S with or without 20 nM uracil or with uracil and DTT or with NMM were fed to flies in Café configuration for 1 hr. Subsequently, the intestines of the flies were dissected out and fixed in 2% paraformaldehyde for 15 min. The mean pixel intensity was measured by Zen Pro  in a middle part of the anterior midgut captured with Plan-Apochromat 20x/0.8 M27 .S1 FigA-E) Defecation spot numbers normalized to ingestion amounts  are presented in addition to fold change of defecation results. The ingestion amount was assessed as the length change of food filling 5 microliter glass capillary tubes (15 mm/microliter). n = 6–14 (A-E). (D) Duox RNAi knockdown by pan-neuronal appl-Gal4 did not abolish uracil-dependent defecation (n = 7–11). Left: The results presented as “Output/Input”, Right: fold change of defection derived from the Left panel (n = 6–14). (F) The ingestion amount per animal was analyzed across genotypes (n = 6–18). Ingestion with uracil or DTT did not significantly alter the ingestion amounts. Only statistically significant comparisons were marked: *, p<0.05, **, p<0.01, ***, p<0.001. Student’s t-test or Tukey.((PDF)Click here for additional data file.S2 FigB) Indicated fly alleles were fed with 500 mM sucrose solution with 0.5% brilliant blue FCF containing indicated ECC15 strains, and allowed to defecate in fly vials for 8 hrs in total. Fecal spots were washed with 2.5 ml PBS. (C) Fecal spectral absorbances at 628 nm normalized with respect to input. Uracil (upper) or ECC15 WT ingestion (lower) resulted in increased spectral absorbance, compared to sucrose only or ECC15 pyrE ingestion, respectively. *: p<0.05 and ***:p<0.001, Student t-test. The number of experiments is indicated at the base of the graphs in grey.(A) Fecal spot sizes are not significantly changed by uracil ingestion. Sucrose solution with (+uracil) or without uracil (-uracil) was fed to indicated genotypes which were at the same time allowed to defecate on the inside wall of a cuvette for providing flat surface. The fecal spots were scanned and the area sizes were assessed by Image J. ((PDF)Click here for additional data file.S3 FigA and B) Prolonged application (6 min) of 0.02 mM uracil did not evoke TRPA1 currents in Xenopus oocytes expressing either TRPA1(A)10a or TRPA1(A)10b, while the well known TRPA1 activator, NMM, did. The currents were recorded with 300-ms voltage ramps between -60 and +60 mV every one second. (C) Summary of uracil responses appraised at +60 mV in the oocytes (n = 4–5). (D) Gustatory choice test results (n = 4–7). TrpA1 in the taste neurons mediates avoidance behavior to TRPA1 agonists [E-G) Uracil at 0.1 mM was mixed with 10 mM DTT, and incubated for 2 hrs at room temperature, after which the spectral absorbance was determined. Single chemical conditions were also subjected to the test as controls. (E) Spectral absorbance of indicated chemicals. Different colors indicate independent experiments (n = 3). Arrows indicate the absorbance peak of uracil at 260 nm. Inset (Top and Bottom panels): magnification for uracil peak absorbance. (F) The peak uracil absorbance at 260 nm of three different conditions. (G) The peak DTT absorbance at 205 nm.(agonists . Twenty (PDF)Click here for additional data file.S4 FigA) Spectral evaluation of R19S in the range of NaOCl concentrations from 0.1 to 100 ppm. Left, A representative NaOCl dose-dependent change of R19S fluorescence. Right, The averaged graph of R19S fluorescence at indicated NaOCl concentrations (n = 4). (B) Typical confocal images of intestines with R19S. Experimental conditions were indicated at the left side of images. A part of the anterior midgut indicated as a red box in (C) is shown for each experiment. Field of view: 420 microns. (C) An R19S image of the midgut and hindgut from a fly that ingested uracil. R19S fluorescence was observed throughout the intestine, suggesting that HOCl production is not spatially limited. The red box indicates approximate location of the part shown in (B). (D) Ingestion of the R19S and 0.1 ppm NaOCl premixture yields the intensity and pattern of R19S fluorescence similar to the guts from animals that ingested 20 nM uracil. Right, A composite image illustrating the fluorescence distribution in the mid- and hindgut. (E) Averaged data are presented as bar graphs. Letters indicate significantly distinct groups. ANOVA Tukey, p<0.001. The number of experiments is indicated at the base of the graphs in grey.((PDF)Click here for additional data file.S5 FigA) Schematic diagram of the temporal control of Gal4/Gal80ts-dependent transcription. (B-D) Genetic interventions such as Duox (B) or TrpA1 (D) RNAi knockdown and Jafrac1 overexpression (C) by indicated Gal4 lines. Non-permissive temperature at 30°C disrupts transcriptional suppression of GAL80ts and allows GAL4 to drive transcription, while GAL80ts restricts transcription at permissive temperatures such as 18°C. **: p<0.01, ANOVA Tukey.((PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.S7 FigA) Schematic illustration of the gut regions stained with anti-TRPA1 antibody. (B) TrpA1(A)-Gal4 cells are enteroendocrine cells marked with anti-Prospero, except for large cells in the middle midgut that appear to be enterocytes (not shown). (C) TrpA1 RNAi knockdown in TrpA1(A)-Gal4 cells presented as “Output/Input” (n = 5–8).((PDF)Click here for additional data file.S8 FigC).TRPA1 staining results for RNAi knockdowned animals are summarized in the table ((PDF)Click here for additional data file.S9 FigA-B) Dose dependence (Left) and current voltage relationship (Right) of the temperature-sensitive Drosophila TRPA1 isoform, TRPA1(B)10a (A), and human TRPA1 . (C) Summarized dose dependence of hsTRPA1 (n = 4–10) and TRPA1(B)10a (n = 4–6). hsTRPA1 has a dose dependence profile very similar to TRPA1(B)10a. (D) Time constants from dose dependence experiments reveal that hsTRPA1 exhibits faster activation than TRPA1(B)10a but slower activation thanTRPA1(A) at 10 ppm NaOCl (n = 4–10). (E) Time constants of Drosophila TRPA1 isoforms and human TRPA1 (hsTRPA1) at -60 (Upper) and +60 mV (Lower) (n = 4–9). Shaded boxes indicate the concentrations that failed to generate sufficient currents for time constant determination. Note that only TRPA1(A)10b had time constants measured for 0.1 ppm. (F) EC50s from various TRPA1s at -60 and +60 mV (n = 4–10). EC50 of hsTRPA1 is comparable to that measured by a previous report [D). ND: not determined. (G) Current amplitudes evoked by 100 ppm HOCl for estimation of expression in oocytes (n = 4–13). (H) L-bristles of animals with the indicated genotypes were examined for their NaOCl responsiveness. Note that Gr5a>TrpA1(A)10a taste neurons hardly showed action potentials to NaOCl 100 ppm, while responding to the immediately following NMM contact. In contrast, Gr5a cells expressing TRPA1(A)10b showed robust spikes from both sweet and water cells. The noisy low level spikes frequently appeared when the sensilla contact with NaOCl, probably due to its reactivity. (I) Summary of 30 sec-averaged spike frequencies evoked by NaOCl in L-bristles of flies used in (H) (n = 4–8). ***: p≤0.001, Tukey test.(s report . *: p<0.(PDF)Click here for additional data file.S10 FigA) and TRPA1(A)10b (B). (C) Citronellal dose dependences of the two isoforms. (D) Time to 70% amplitude at each concentration was plotted as function of citronellal concentrations.The non-covalent agonist citronellal opens TRPA1(A)10b faster and more sensitively than TRPA1(A)10a. Typical current recording with increasing concentrations of citronellal for TRPA1(A)10a ((PDF)Click here for additional data file.S11 FigUpper, Output/input results were presented for indicated genotypes. Defecation frequencies were normalized to the feeding amounts after ingestion of either ECC15 WT or pyrE. Lower, The results in the Upper panel were displayed as fold change of defecation for estimation of fold increase of defecation by uracil from ECC15. **: p<0.01, ANOVA Tukey or Student t-test.(PDF)Click here for additional data file.S12 FigA) NaOCl resistance was assessed with ECC15-GFP that previously resided in wcs or TrpA1ins guts in the range of indicated concentrations and is not significantly different between the two genotypes. (B) ECC15-GFP from TrpA1ins guts opportunistically survive the NaOCl concentration that eliminated the growth of the bacteria from wcs guts. (C) Enhanced survival to ingested ECC15 pyrE of TrpA1ins is not rescued by reintroduction of the TrpA1 genomic DNA, suggesting that the resistance TrpA1ins exhibited might have originated from its genetic background.((PDF)Click here for additional data file.


Auto-inflammatory syndromes now include polygenic diseases, such as Behcet's syndrome and Still's disease; however, the best characterized auto-inflammatory diseases are relatively rare, but florid conditions arising from mutations in single genes. The prevalence of each auto-inflammatory disease varies depending on ethnic background.Auto-inflammatory syndromes are defined as conditions caused by an exaggerated innate immune system response, resulting in episodes of spontaneous inflammation affecting multiple organs. The prototypical auto-inflammatory disorders are associated with periodic febrile episodes. To analyze the prevalence of polymorphisms of the genes responsible for auto-inflammatory diseases managed in a single rheumatology institute in Japan.® Terminator v3.1 kit and analyzed with an ABI 3130xl Prism Genetic Analyzer. For the most frequently reported 4 genes, genetic polymorphisms within MEFV (exons 1-10), TNFRSF1A (exons 2-4), MVK (exons 9-11), and NLRP3 (exon 3) were examined. With respect to the other 6 genes, the existence of polymorphisms was also determined within NOD2 (from L248R to P727L), LI1RN (from N52KfsX25 to C91F), IL36RN (from R10X to G141Mfs*29), PSMB8 (from T75M to G201V), NALP12 (from T260M to F402L and R1016X), and PSTPIP1 (from A230T to E277D), with reference to the INFEVERS database, an evolving mutation database for auto-inflammatory syndromes (http://fmf.igh.cnrs.fr/ISSAID/infevers/index.php).A total of 202 individuals < 40 years of age with recurrent febrile episodes were enrolled in this study. Recurrent fever was defined as > 2 episodes of fever > 38.5 degrees Celsius lasting > 3 days in a year. Infections, autoimmune disorders, and malignancies were excluded as causes of fever prior to enrollment. Genomic DNA was isolated from the patients' peripheral blood and a polymerase chain reaction (PCR) was used to amplify the indicated exons of 10 genes , which have been reported as the genes responsible for auto-inflammatory diseases. After cleaning the PCR products, cycle sequencing was carried out using the Big DyeGene polymorphisms in the targeted genes were identified in 137 of the 236 patients (58.1%) based on INFEVERS. One hundred thirty-five of the 137 (98.5%) were associated with MEFV genes. Other polymorphisms were identified in TNFRSF1A (n=7), NLRP3 (n=5), NOD2 (n=4), MVK (n=2), and PSTPIP1 (n=1).Polymorphisms in MEFV were most frequently identified among Japanese patients with recurrent fevers. Further evaluation with clinical features is warranted.


Schistosoma mansoni (S. mansoni) infection. We found that adenosine 5’-diphosphate (ADP) induced human eosinophils to secrete eosinophil peroxidase (EPO) in a P2Y12R dependent manner. However, ADP did not interfere with human eosinophil apoptosis or chemotaxis in vitro. In vivo, C57Bl/6 mice were infected with cercariae of the Belo Horizonte strain of S. mansoni. Analyses performed 55 days post infection revealed that P2Y12R blockade reduced the granulomatous hepatic area and the eosinophilic infiltrate, collagen deposition and IL-13/IL-4 production in the liver without affecting the parasite oviposition. As found for humans, murine eosinophils also express the P2Y12R. P2Y12R inhibition increased blood eosinophilia, whereas it decreased the bone marrow eosinophil count. Our results suggest that P2Y12R has an important role in eosinophil EPO secretion and in establishing the inflammatory response in the course of a S. mansoni infection.Identifying new target molecules through which eosinophils secrete their stored proteins may reveal new therapeutic approaches for the control of eosinophilic disorders such as host immune responses to parasites. We have recently reported the expression of the purinergic P2Y12 receptor (P2Y12R) in human eosinophils; however, its functional role in this cell type and its involvement in eosinophilic inflammation remain unknown. Here, we investigated functional roles of P2Y12R in isolated human eosinophils and in a murine model of eosinophilic inflammation induced by  Schistosoma mansoni (S. mansoni) ) in vitro [Because liver granulomas from rophages , 27, 28.rophages  and enhain vitro . As showIn vivo, we found that P2Y12R blockade reduces the hepatic granulomatous inflammatory response in S. mansoni infection without affecting the Th2 response. P2Y12R blockade modulated hepatic tissue inflammation but did not impair S. mansoni egg oviposition, indicating that the blockage of P2Y12R does not affect the infection regarding parasite load. In this regard, clopidogrel-treated mice had reduced granuloma areas with less eosinophils during S. mansoni infection. However, the plasma levels of IL-13 and IL-4, as well as the levels of IL-13 produced by splenocytes in response to SEA, were not importantly affected by P2Y12R blockage, indicating that P2Y12R is not essential to Th2 polarization. As found for humans, murine eosinophils also express the P2Y12R. Because liver granulomas from S. mansoni-infected, clopidogrel-treated mice had fewer eosinophils compared with those from untreated mice, we tested whether ADP could also be a chemoattractant to eosinophils or prevent eosinophil apoptosis. We found that this is unlikely based on our results in vitro, in which ADP did not interfere with eosinophil chemotaxis or survival. Our results demonstrate a previously unrecognized role for P2Y12R in as a modulator of eosinophil activation and in schistosomal inflammation.Identifying new target molecules that account for eosinophil activation may be highly important for our understanding of the pathophysiology of host immune responses to parasites, allergic inflammation and related eosinophilic diseases. We have recently reported the expression of P2Y12R on human eosinophils and isolated human eosinophil granules [S. mansoni and other worm infections in vitro [in vivo [S. mansoni infection in clopidogrel-treated mice is efficiently controlled concerning the parasite load, despite the reduced numbers of eosinophils. In our findings we noticed a prominent reduction in the area size of granulomas possible due to the reduction of infiltrating inflammatory cells. Despite we identified less eosinophils in the granulomas and found that mouse eosinophils also express the P2Y12R, whether they or other cells are responsible to the observations related to granuloma size is not possible to define. In addition, if mouse eosinophils are capable to secrete cationic proteins in vivo via the P2Y12R activation and contribute to the inflammatory host response is still not known. The granuloma formation around eggs also depends on Th2 responses, as demonstrated in IL-4/IL-13- [S. mansoni-infected, clopidogrel-treated mice does not seem to be the consequence of a weakened Th2 response because infected clopidogrel-treated mice do not have strongly reduced plasma concentrations of IL-4 and IL-13 and do not have lower levels of IL-13 production by spleen cells in response to SEA. These results indicate that the control exerted by the P2Y12R blockade on the area size of egg granulomas is likely not related to a disorder of the Th2 response.Using flow cytometry and immunofluorescence techniques, our results confirmed previous findings that demonstrate that peripheral human eosinophils express P2Y12R. Notably, immunofluorescence microscopy images indicate an intracellular clustered labeling for P2Y12R. In fact, our group previously described P2Y12R expression in intact, non-permeabilized eosinophils and on the membranes of intracellular eosinophil granules . Notablyin vitro  and in v[in vivo . Neverth[in vivo  or IL-5 [in vivo  control 4/IL-13- , IL-10/I4/IL-13- , and IL-4/IL-13- , in whicS. mansoni-infected mice showed that P2Y12R blockade is relevant for liver fibrosis induced by Th2 inflammation. One hypothesis is that the reduced inflammatory cells infiltration might be contributing to the impaired collagen deposition in clopidogrel-treated mice compared with the non-treated group. The role of eosinophils in promoting fibrosis and collagen deposition remains controversial. Although a previous study demonstrated an essential role for eosinophils on the collagen deposition and tissue remodeling induced by OVA immunization and challenge using eosinophil-ablated mice [S. mansoni [The production of IL-13  and tissted mice , a colla mansoni . Because mansoni , 26, we S. mansoni-infected sites might be associated with impaired leukocyte transmigration from the vascular compartment. S. mansoni infection causes robust eosinophilopoiesis and blood eosinophilia [S. mansoni is still unknown. P2Y12R may also be expressed in other cells of the immune system involved in the inflammatory host responses to S. mansoni. High amounts of P2Y12R mRNA have been found previously in both lymphocytes and CD34+ progenitor cells [The reduced granuloma sizes at nophilia . In thisnophilia . Beyond nophilia . Platelenophilia . Thus, anophilia , 37. Planophilia . In addinophilia . Althougor cells . In addior cells . In thesor cells . Howeveror cells .50 values of 8 μM, 0.07 μM and 0.06 μM, respectively [Because we demonstrated that P2Y12R is important for modulating the schistosomal inflammatory response and tissue eosinophilia, we wondered whether ADP would be capable of attracting human eosinophils in transwell assays as well as acting as a survival factor for these cells. ADP selectively activates the receptors for P2Y1, P2Y12 and P2Y13. ADP is known to activate P2Y1R, P2Y12R and P2Y13R, with ECectively . Human eectively . Thus faectively . Becauseectively . Augmentectively . Howeverectively . Once reectively . In our ectively –46 and oectively , 47. Burectively . These sectively , 45, 48.ectively . Howeverin vitro. However, even after IL-5 priming, ADP (100 and 10 nM) was not capable of promoting human eosinophil chemotaxis. We also verified possible autocrine/paracrine roles of P2Y12R in the chemotaxis induced by known eosinophil physiological activators such as eotaxin. A study published by Kronlage and colleagues revealed that C5a-induced macrophage chemotaxis is dependent on the paracrine/autocrine activation of the P2Y12 and P2Y2 receptors [IL-5 priming induces the random, chemokinetic migration of eosinophils  and enhaeceptors . Additioeceptors . In our S. mansoni infection occurred irrespective of robust alterations in the antigen-driven immune response, thus indicating that the response mediated by this receptor is downstream of these events. Our data also implicate P2Y12R as a mediator for ADP-induced EPO secretion in human eosinophils, but not for eosinophil chemotaxis or survival. Together, these results demonstrate a formerly unrecognized role for P2Y12R in eosinophil activation and schistosomal inflammation.In conclusion, our studies demonstrate that P2Y12R modulation plays a role in eosinophil biology and in the host response to schistosomal infection. The reduced granulomatous area in clopidogrel-treated mice during S1 Figt test. . n = 5–8 mice/group and represents animals from at least 2 independent infection studies. OD = optical density.The concentration of hemoglobin was measured spectrophotometrically using a microplate spectrophotometer at 540 nm. Data represent the mean ± SE and were analyzed by Student’s (PDF)Click here for additional data file.


Previous research has reported reduced serum 25-hydroxyvitamin D (25(OH)D) levels is associated with acute infectious illness. The relationship between vitamin D status, measured prior to acute infectious illness, with risk of community-acquired pneumonia (CAP) and sepsis has not been examined. Community-living individuals hospitalized with CAP or sepsis were age-, sex-, race-, and season-matched with controls. ICD-9 codes identified CAP and sepsis; chest radiograph confirmed CAP. Serum 25(OH)D levels were measured up to 15 months prior to hospitalization. Regression models adjusted for diabetes, renal disease, and peripheral vascular disease evaluated the association of 25(OH)D levels with CAP or sepsis risk. A total of 132 CAP patients and controls were 60 ± 17 years, 71% female, and 86% Caucasian. The 25(OH)D levels <37 nmol/L (adjusted odds ratio (OR) 2.57, 95% CI 1.08–6.08) were strongly associated with increased odds of CAP hospitalization. A total of 422 sepsis patients and controls were 65 ± 14 years, 59% female, and 91% Caucasian. The 25(OH)D levels <37 nmol/L  were associated with increased odds of sepsis hospitalization. Vitamin D status was inversely associated with risk of CAP and sepsis hospitalization in a community-living adult population. Further clinical trials are needed to evaluate whether vitamin D supplementation can reduce risk of infections, including CAP and sepsis. Relatively little progress has been made in improving mortality associated with community-acquired pneumonia (CAP)  which is2D), were originally recognized as important endocrine hormones in calcium homeostasis and bone health. However, studies over the past twenty years suggest a broader role of 25(OH)D in endothelial function, cell proliferation, and immunity. The vitamin D receptor (VDR) is essentially ubiquitous, including immune cells. It responds to 1,25(OH)2D [The major circulating form of vitamin D, 25-hydroxyvitamin D (25(OH)D), and its active form, 1,25-dihydroxyvitamin D 25(OH)2D ,5 and re25(OH)2D . Large e25(OH)2D  includin25(OH)2D , renal d25(OH)2D , and per25(OH)2D .Lower serum 25(OH)D levels are associated with increased risk of upper respiratory tract infection ,12,13. WA retrospective matched cohort study was performed using the Intermountain Healthcare Enterprise Data Warehouse, which incorporates comprehensive electronic health and administrative data for over 10 years . IntermoCase and control selection occurred between 1 January 2008 and 31 December 2010. CAP cases were identified through ICD-9 codes (480–488) and confirmed with chest radiograph. There were 187,132 CAP admissions of which 43,460 had discharge ICD-9 codes indicating pneumonia. Of these, 11,455 had chest radiography confirming pneumonia. 4352 Sepsis cases were identified through ICD-9 codes . Controls were randomly selected from a pool of 62,757 adult patients without a CAP or sepsis diagnosis admitted within the same time period and matched 1:1 with cases by age, sex, race, and season of 25(OH)D measurement. Cases and control had to have a serum 25(OH) level in the electronic medical record 3–15 months prior to admission; therefore, only 132 and 422 patients were included in the final CAP and sepsis analyses, respectively. These time points were chosen arbitrarily to assess the relationship between pre-infection serum 25(OH)D levels and infectious episodes.Serum 25(OH)D level were measured in all participants using an INCSTAR 25(OH)D two step assay procedure with a coefficient of variation of less than 10%. The first step in the procedure involves the rapid extraction of 25(OH)D from the serum using acetonitrile. Following extraction, the treated sample is assayed by using an equilibrium radioimmunoassay procedure. This method is based on an antibody with specificity to 25(OH)D. The sample, antibody, and tracer are incubated at 20–25 °C for ninety minutes. A second antibody-precipitating complex is used to achieve phase separation. The radioimmunoassay method tends to overestimate the level of 25(OH)D because the antibody recognizes all forms of dihydroxy-vitamin D and D steroids.2 Test of Independence tested the distribution of categorical variables and the Wilcoxon Rank Sum tested for differences in 25(OH)D levels among cases and controls. Cox logistic regression was performed using log-transformed 25(OH)D as a continuous variable and non-transformed 25(OH)D as a categorical variable . These thresholds were chosen using established definitions of 25(OH)D deficiency/insufficiency [p < 0.05 were considered statistically significant.The associations of serum 25(OH)D levels with CAP or sepsis admission were evaluated separately. The χficiency ,19. Modeficiency ,21 and ovs. cases . Renal disease was more prevalent in cases than controls . Serum 25(OH)D levels were recorded in 28 cases 3–5 months prior, in 18 cases 6–11 months prior, in 10 cases each 9–11 and 12–15 months prior to admission.The demographic and clinical characteristics of the 66 cases and 66 controls for the CAP cohort are described in p = 0.84). A lack of association remained after adjustment for diabetes, renal disease, and peripheral vascular disease . Likewise, 25(OH)D <75 nmol/L vs. ≥75 nmol/L and <50 nmol/L vs. ≥50 nmol/L were not associated with CAP in adjusted analyses D as a continuous variable was not associated with CAP . Comorbid conditions were more prevalent in sepsis cases than in controls: diabetes  and renal disease . Serum 25(OH)D levels were recorded in 93 cases 3–5 months prior, in 50 cases 6–11 months prior, in 47 cases 9–11 months prior, and 21 cases 12–15 months prior to admission.The demographic and clinical characteristics of the 211 cases and 211 controls for the sepsis cohort are described in p = 0.70). A lack of association remained after adjustment for diabetes, renal disease, and peripheral vascular disease . 25(OH)D <75 nmol/L vs. ≥75 nmol/L was not associated with sepsis in adjusted analyses  in adjusted analyses.In unadjusted logistic regression, log-transformed 25(OH)D as a continuous variable was not associated with sepsis D levels <37 nmol/L and for sepsis with serum 25(OH)D levels <50 nmol/L. This association was not observed for 25(OH)D <75 nmol/L, suggesting that 25(OH)D <37 nmol/L confers a greater risk of infection than vitamin D insufficiency.vs. 24%, p = 0.006) at initial evaluation and at 24 h  [These findings are consistent with other epidemiologic studies linking vitamin D deficiency with increased risk of infection and infection-associated complications. A large observational study using National Health and Nutrition Examination Survey data showed that in a diverse cohort of 18,883 individuals greater than 12 years of age, those with a serum 25(OH)D level <25 nmol/L and a serum 25(OH)D 25–75 nmol/L had a 36% and 24% increased risk of upper respiratory tract infection, respectively, compared to those with a serum 25(OH)D ≥75 nmol/L . Likewis= 0.005) . While o2D, are expressed in immune cells, suggesting that 25(OH)D has paracrine or autocrine function. Furthermore, 1-α-hydroxylase in macrophages is not regulated by parathyroid hormone (PTH) [4 resulting in local conversion of 25(OH)D to 1,25(OH)2D, which in turn increases the expression of cathelicidin and beta-defensing, bactericidal proteins. There is evidence that cathelicidin transcription is particularly dependent on sufficient levels of 25(OH)D [Both the VDR and CYP27B1, the gene encoding 1-α-hydroxylase, which converts 25(OH)D to its active form 1,25(OH)ne (PTH)  but depene (PTH) . When to 25(OH)D .2D may have other effects beyond those associated with immunity such as endothelial function, coagulation, and hemodynamic stability. In rats with induced sepsis, pretreatment with 1,25(OH)2D resulted in a more normal coagulation profile compared to placebo [2D has been shown to enhance the effect of inotropes [2D in sepsis [In sepsis, 25(OH)D and 1,25(OH) placebo  and 1,25notropes  suggestiThe findings of this study are consistent with other epidemiologic reports linking serum 25(OH)D <75 nmol/L with increased risk of infection and associated complications ,14,15,16This study has several limitations. First, it is retrospective and observational so causation cannot be assumed. Second, the use of ICD-9 codes to identify admissions for sepsis limits details of cause and severity. Third, while cases and controls were well-matched and regression models adjusted for comorbidities, there may be other unmeasured confounders that could potentially affect the results. Fourth, ascertainment bias may exist because only cases and controls with a serum 25(OH)D level available prior to admission were considered. Additionally, no information is available on outcomes during the hospitalization.In conclusion, serum 25(OH)D level <37 nmol/L in a community-living cohort was associated with increased risk of hospital admission for CAP and sepsis. Large randomized controlled trials are needed to establish whether or not 25(OH)D repletion will decrease CAP and sepsis incidence in community-living adult populations.


N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic messenger RNA (mRNA). Recent discoveries of demethylases and specific binding proteins of m6A as well as m6A methylomes obtained in mammals, yeast and plants have revealed regulatory functions of this RNA modification. Although m6A is present in the ribosomal RNA of bacteria, its occurrence in mRNA still remains elusive. Here, we have employed ultra-high pressure liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-QQQ-MS/MS) to calculate the m6A/A ratio in mRNA from a wide range of bacterial species, which demonstrates that m6A is an abundant mRNA modification in tested bacteria. Subsequent transcriptome-wide m6A profiling in Escherichia coli and Pseudomonas aeruginosa revealed a conserved m6A pattern that is distinct from those in eukaryotes. Most m6A peaks are located inside open reading frames and carry a unique consensus motif of GCCAU. Functional enrichment analysis of bacterial m6A peaks indicates that the majority of m6A-modified genes are associated with respiration, amino acids metabolism, stress response and small RNAs, suggesting potential functional roles of m6A in these pathways. N6-methyladenosine (m6A) is the most frequent internal mRNA modification in a wide range of eukaryotes and certain viral RNAs (6A is mediated by an N6-adenosine methyltransferase complex including a 70-kD SAM (S-adenosylmethionine)-binding subunit methyltransferase like 3 , methyltransferase like 14 (METTL14) and Wilms tumor 1 associated protein (WTAP)  are m6A RNA demethylases, which are involved in mammalian development, RNA metabolism and fertility .Transcriptome-wide profiling of mart site ,15,16. mrt codon . The maj6A has been well documented in the rRNA in bacteria, its presence on mRNA is still elusive. In Escherichia coli, A1618 and A2030 of 23S rRNA are methylated by methyltransferases RlmF and RlmJ, respectively . The primers used were 5′-CTCCTACGGGAGGCAGCAG-3′ and 5′-GTATTACCGCGGCGGCTG-3′. The P. aeruginosa MPAO1 strain were cultured overnight at different temperatures  and then subjected to the mRNA purification protocol described above in the temperature variation studies.The strains and culture conditions used in this study are listed in Table 2) at 37°C for 2 h, followed by the addition of NH4HCO3  and alkaline phosphatase (0.5 U) at 37°C for 2 h. The nucleosides were separated by reverse phase ultra-performance liquid chromatography by a C18 column on an Agilent 6410 QQQ triple-quadrupole LC mass spectrometer in positive electrospray ionization mode. The nucleosides were quantified using the nucleoside-to-base ion mass transitions of 282 to 150 (m6A), 294 to 164 (m62A) and 268 to 136 (A). Quantification was performed by comparison with the standard curve obtained from pure nucleoside standards. Three biological repeats have been performed for all bacterial strains.The highly purified bacterial mRNA was subjected to an UHPLC-QQQ-MS/MS (Agilent) analysis. Two hundred ng of mRNA or rRNA (on the beads of the mRNA Enrichment Kit) were digested by nuclease P1 (2 U) in 40 μl of nuclease buffer  were incubated for 2 h at 4°C in IPP buffer . The mixture was exposed to UV irradiation at 254 nm 3× (90 s each time), before RNase T1 (0.1 U/μl) digestion for 15 min at 22°C. After the digestion reaction was quenched on ice for 5 min, 200 μl pre-blocked protein A bead slurry was added into reaction for 1 h at 4°C. After washing thrice with IP wash buffer , the beads were treated by a second round of RNase T1 digestion (15 U/μl) at 22°C for 15 min. The beads were cooled down on ice for 5 min and then thrice washed with high salt wash buffer . The beads were then treated with Antarctic phosphatase (0.5 U/μl) for 20 min at 37°C. After dephosphorylation, beads were washed twice with phosphatase wash buffer  and twice with PNK buffer without DTT . Polynucleotide kinase (1 U/μl) and 200 μM adenosine triphosphate was then added to the beads at 37°C for 15 min. The RNA fragments were further purified by proteinase K digestion and TRIzol extraction. For IP samples, small RNA libraries were made by using NEBNext® Small RNA Library Prep Set for Illumina® (NEB). The input samples followed the above procedures without anti-m6A antibody pull-down and RNase T1 digestion. Libraries for input samples were made by using TruSeq RNA Sample Preparation Kits . Six libraries were constructed, containing one control sample and two duplicate IP samples for each strain.Procedures were slightly modified from previously described protocols . In a 0.E. coli IP libraries obtained 1,718,364 mapped reads, and two P. aeruginosa libraries obtained 3,720,658 mapped reads. Sequence data were analyzed by following the procedures described previously  and P. aeruginosa PAO1  genomes and annotation files (http://david.abcc.ncifcrf.gov/) (6A peaks were divided into three categories based on their relative positions in their corresponding genes: Overlap Start (±100 nucleotides around the start codon), Overlap End (±100 nucleotides around the stop codon) and Inside (other locations inside a coding region). The functional association with each gene was determined by NCBI annotation.All samples were sequenced using the HiSeq 2000 system  at the Genomics Core Facility at the University of Chicago. FastQC was done to check the quality of each dataset; all datasets were obtained in high quality and can afford further reliable analyses. Two eviously . Brieflyon files ,23. In Ton files . Consensrf.gov/) . The m6A6A is the most abundant internal mRNA modification in eukaryotes, its potential presence in the kingdom of bacteria has yet to be investigated. To this end, we selected seven diverse model bacterial species  to grow in a common laboratory environment and measured their m6A/A ratios in purified mRNA. Unlike eukaryotes, bacterial mRNA lacks a poly(A) tail, which makes it challenging to purify mRNA. By following the protocols described in the Method section, we were able to remove >90% rRNA in the purified mRNA sample .Although m6A/A level in the bacterial mRNA samples that contain residual rRNA (<10%). Given that two m6A  and two N6,N6-dimethladenosine  are known to be present in rRNA of E. coli and other related bacterial species . Based on the m62A/m6A ratio in rRNA, we were able to accurately calculate the real m6A/A level as ([m6A-m62A/1.30]/A) in the purified mRNA samples.We then tried to use an UHPLC-QQQ-MS/MS approach in order to quantify the m species ,19,27, t6A in all of the tested bacterial species, whose m6A/A ratio varied within the range of 0.02–0.28%  (>0.2%) and from two Gram-positive bacteria (S. aureus and B. subtilis) (<0.08%). Unlike E. coli and Pseudomonas spp., two other Gram-negative cyanobacteria (Anabaena sp. PCC 7120 and Synechocystis sp. PCC 6803) showed low m6A/A ratios (<0.04%). In order to test if m6A/A ratios also vary among different strains in the same species, three strains of E. coli , two strains of P. aeruginosa (MPAO1 and PA14) and six strains of S. aureus  were tested, all of which revealed a constant ratio in the same species  in mRNA from E. coli and Pseudomonas spp. is comparable to that from eukaryotes , which significantly improves the m6A peak resolution from ∼200 nt to around 23 nt (6A peaks representing the transcripts of 213 genes in E. coli (Supplementary Table S1).To obtain the transcriptome-wide mquencing ,16. In ond 23 nt . In tota6A in the whole transcriptome of E. coli. We determined the distribution of m6A reads along transcripts in both the m6A-IP and non-IP (input) samples. Intriguingly, we found that reads from m6A-IP tend to be equally distributed throughout a gene, with a peak in the middle of open reading frames (ORFs) , followed by regions at the start of gene (15%) and then the end of gene (13%)  Figure . The pre) Figure ,16. To f) Figure .6A consensus sequence , which could be found in more than 41.2% of all m6A peaks  found in eukaryotes. The unique feature of the m6A distribution suggests a likely unique role of m6A in perhaps bacteria-specific pathways.We then used the HOMER tool to identify a leading ms Figure . This mo6A-containing genes, which include metabolism (52%), transportation (12%), gene regulation (11%), cell envelope (5%), ribosome (3%), nucleic acids (1%), stress response (5%), genes with unknown function/annotation (6%) and pseudogenes (2%)  terms using the DAVID tool (26). We found that these genes are highly enriched in aerobic respiration, amino acids biosynthesis, response to abiotic stresses, cell wall biosynthesis and anaerobic respiration  genes of the six-gene-operon encoding hydrogenase 1 contain multiple m6A peaks inside the transcript, suggesting a clustering of m6A in this operon  Figure . To furtn Figure . The firn Figure  28). We. We6A-com Figure  29)..6A-conta6A position patterns are related to bacteria-specific GO categories. As aforementioned, we classified genes into three subgroups according to the location of m6A peaks on a gene: Overlap Start (m6A peaks within 100-bp from the start codon), Overlap End (m6A peaks within 100-bp from the stop codon) and Inside (m6A peaks inside the coding region)  were enriched in the Overlap Start subgroup, while amino acids biosynthesis and response to stimulus were enriched in the Overlap End subgroup .We next sought to determine if the unique m) Figure . We then6A peaks in a group of functionally important genes, such as lacZ and lacI  that is required for transport and metabolism of lactose (6A modification (Supplementary Table S1). These newly found m6A marks in these transcripts could open a new angle to study novel regulatory roles in well-established pathways.Beside these five GO categories, we identified high mI Figure . LacI ne lactose . InteresP. aeruginosa, a widely-spread human opportunistic pathogen, also possesses a high m6A/A ratio in mRNA, we applied the same modified photo-crosslinking-assisted m6A-seq approach to obtain a high-resolution map of its m6A methylome. We identified 109 m6A peaks representing the transcripts of 68 genes in P. aeruginosa (Supplementary Table S2). The m6A-modified transcripts identified are around half of those in E. coli.Given that 6A reads along transcripts in both the m6A-IP and non-IP (input) samples. Like in E. coli, we found that reads from m6A-IP are equally distributed throughout a gene, with two peaks in the middle of ORFs as well as in the beginning of genes , followed by the start of gene regions (15%) and end of gene regions (8%) , which were found in >70% of all m6A peaks . A similar feature of the m6A distribution in both E. coli and P. aeruginosa indicates that m6A possesses functions unique to these bacteria.We next determined the distribution of ms Figure . m6A pea) Figure . We were6A-containing genes cover different gene categories in P. aeruginosa, including metabolism (40%), gene regulation (8%), transportation (1%), virulence (4%), ribosome (12%), stress response (4%) and genes with unknown function/annotation (31%)  involved in glycolysis and TCA carry numerous m6A peaks  Figure . DAVID a) Figure , all of s Figure  31). We. We6A-com Figure . Beside B Figure .6A peaks have been observed under different stress conditions, indicating a link between m6A and stress response  for both E. coli and P. aeruginosa. For most tested conditions, we did not observe a significant difference in the m6A/A level compared to the normal condition  for both bacteria. Interestingly, we noticed that increasing the culture temperature (from 37–45°C) led to a clear decrease in the m6A/A ratio in P. aeruginosa , binders (m6A-specific binding protein) and writers (methyltransferase) as well as advances in profiling the m6A methylomes in eukaryotic systems reveal that m6A is a reversible and dynamic modification with important regulatory functions -binding domain in E. coli K-12, but only 24 in S. aureus USA300. We further present the high-resolution, transcriptome-wide m6A distributions in E. coli and human pathogen P. aeruginosa, which contain 265 and 109 peaks, respectively.Recent discoveries and characterization of munctions . On the 6A patterns in the bacterial kingdom, we compared these two newly identified methylomes. E. coli and P. aeruginosa share many similarities in their m6A distributions that are distinct from those of mammals: (i) a similar motif GCCAG instead of RRACU motif in mammals; (ii) most peaks are in the middle of coding region, while mammalian m6A peaks enrich around the stop codon and at 3′ UTRs; (iii) enrichment of GO categories of energy and amino acids metabolism; (iv) many small noncoding RNAs were found to carry m6A for both organisms. These shared characteristics suggest that other bacterial species, especially Gram-negative bacteria, may have similar m6A characteristics in mRNA. The new consensus sequence (GCCAG) is different from known rRNA methylation sites, including the two m6A sites on rRNA (CACA*GGU for RlmF and GUGA*AGA for RlmJ) and one on tRNAval (UACA*AGG for YfiC). Given that our recent study demonstrates that m6A and its specific binding protein, YTHDF2, affect the translation status and lifetime of mRNA in eukaryotes , we observed a very low rate of overlapping genes. Besides rRNA genes that are previously known to carry m6A modification, only one gene (aceA encoding isocitrate lyase) was shared between E. coli and P. aeruginosa. Each bacterium has distinct functional categories of mRNAs that carry m6A. For example, genes involved in cell wall biosynthesis and anaerobic respiration are enriched in the E. coli methylome only, while a group of virulence genes (RsmYZ and rhlAB) are enriched in the P. aeruginosa methylome . As a negative control, the ksgA mutant lost the m62A modification and showed a higher m6A/A ratio than the other strains, suggesting a small content of rRNA in the mRNA sample. We also tried to look for bacterial homologs of mammalian m6A methyltransferases , but could not identify one, reminiscent of a distinct bacterial m6A motif that is different from that of mammals. These results suggest that bacterial m6A modification in mRNA is possessing of a mechanism that differs from eukaryotes.Given that multiple known rRNA or tRNA adenine methylases have been characterized in 6A profiling in yeast revealed dynamic changes in methylation during meiosis, which led us to test if bacterial m6A patterns also vary in different growth stage (6A/A ratios during lag phase, log phase, stationary phase and death phase for both E. coli and P. aeruginosa. No significant difference was recorded throughout the bacterial growth curve, which indicates that m6A is a stable modification during bacterial growth.Recent mth stage . To this6A maps presented here provide a starting roadmap for uncovering bacterial distinct m6A functions in the future. Recent breakthroughs in the characterization of m6A-associated proteins as well as in the development of high-throughput assays in mammals present a very useful toolbox for us to study m6A in bacteria. Given the high abundance of m6A in numerous bacterial species, we foresee unique functions of m6A modification in mRNA in the wide bacteria kingdom.The first bacterial mSupplementary Data are available at NAR Online.