North American triple reassortant swine (TRS) influenza A viruses have caused sporadic human infections since 2005, but human-to-human transmission has not been documented. These viruses have six gene segments  closely related to those of the 2009 H1N1 pandemic viruses. Therefore, understanding of these viruses' pathogenicity and transmissibility may help to identify determinants of virulence of the 2009 H1N1 pandemic viruses and to elucidate potential human health threats posed by the TRS viruses. Here we evaluated in a ferret model the pathogenicity and transmissibility of three groups of North American TRS viruses containing swine-like and/or human-like HA and NA gene segments. The study was designed only to detect informative and significant patterns in the transmissibility and pathogenicity of these three groups of viruses. We observed that irrespective of their HA and NA lineages, the TRS viruses were moderately pathogenic in ferrets and grew efficiently in both the upper and lower respiratory tracts. All North American TRS viruses studied were transmitted between ferrets via direct contact. However, their transmissibility by respiratory droplets was related to their HA and NA lineages: TRS viruses with human-like HA and NA were transmitted most efficiently, those with swine-like HA and NA were transmitted minimally or not transmitted, and those with swine-like HA and human-like NA (N2) showed intermediate transmissibility. We conclude that the lineages of HA and NA may play a crucial role in the respiratory droplet transmissibility of these viruses. These findings have important implications for pandemic planning and warrant confirmation. North American triple reassortant swine (TRS) influenza A viruses have caused sporadic human infections, but human-to-human transmission has not been established. We wished to elucidate potential human health threats posed by the TRS viruses and to identify determinants of virulence in the TRS and closely related 2009 H1N1 pandemic viruses. We used a ferret model to evaluate the pathogenicity and transmissibility of North American TRS viruses with the HA and NA antigenic proteins of swine viruses and of human viruses. We observed that the North American TRS viruses grew efficiently in both the upper and lower respiratory tracts and caused moderate pathogenicity in ferrets. The viruses were readily transmissible via direct contact, irrespective of their HA and NA lineages. However, transmissibility via respiratory droplets was substantially greater when the viruses carried the HA and NA of human influenza A viruses rather than of swine influenza A viruses. Because ferrets are a useful model of human influenza infection, this finding helps to predict features that increase the risk to human health. For nearly 70 years, swine influenza virus in North America was relatively stable, dominated by the classical-swine H1N1 (cH1N1) subtype Sporadic infections with TRS H1N1 (swine-like HA and NA) and H1N2  viruses have been reported in humans exposed to swine in North America Despite extensive recent studies of the pathogenicity and transmissibility of pH1N1 viruses in different animal models Most of the North American TRS viruses belong to three subtypes: H1N1, H1N2, and H3N2; H1 and N1 are of the classical swine or seasonal human lineages, while H3 and N2 are of seasonal human lineage only. TRS viruses with human-like HA and NA have recently become the predominant influenza viruses isolated from swine in the US , but their transmissibility has not been tested in the ferret model. Ferrets are an established small-animal model that appears to recapitulate the pathogenicity and transmissibility of human seasonal influenza A viruses In the present study, we used the ferret model to evaluate the pathogenicity and transmissibility of three distinct groups of North American TRS viruses  and of the Eurasian avian-like swine virus A/sw/Italy/1369-7/1994 (H1N1) . Because a limited number of ferrets could be used, the study was designed to detect patterns in the transmissibility and pathogenicity of TRS viruses that, once confirmed, will have important implications for pandemic preparedness. Italy/94 virus was less efficiently transmissible than the North American TRS viruses. The North American TRS viruses, regardless of their HA and NA lineages, were readily transmissible to co-housed  ferrets, while viruses with human-like HA and NA or with human-like NA alone showed enhanced transmission via respiratory droplets.6 pfu of each virus (a dose previously found to result in consistent infection 4 pfu/ml vs. 106 pfu/ml for the other two viruses in this group) (Ferrets inoculated with 10s group) .6 pfu/ml) substantially higher than that caused by the other two viruses in this group . TRS isolates containing human-like HA and NA caused the highest peak nasal wash titers . Infectious virus particles were identified at various titers in the lungs of all ferrets inoculated with the studied TRS viruses (5 pfu/ml) and exhibited almost no clinical signs. However, infectious virus particles were obtained from the lungs of both inoculated ferrets.One of the TRS isolates (A/sw/IN/9K035/1999 [H1N2]) with swine-like HA but human-like NA caused peak nasal virus shedding , was transmitted only by direct contact  (6 pfu/ml) in donor ferrets, transmission was observed in only one of two DC ferrets and in neither RD ferret. Infectious A/sw/MN/6998/2003 was detected on day 7 post-exposure (p.e.) (day 8 p.i.) in the nasal wash of one of the two RD ferrets. Overall, only one of the three viruses in this group was transmitted via RD, to one of two ferrets; the remaining two viruses were not transmitted via RD. Therefore, TRS viruses with swine-like HA and NA were poorly transmitted via RD. Poor RD transmission of TRS viruses containing swine-like HA and NA has been reported previously Among the TRS viruses with swine-like HA and NA, A/sw/NC/47438/2000 (H1N1), which had the lowest mean peak nasal wash titer (≤10 0/2 RD) . Althoug6 pfu/ml) and were efficiently transmitted  in ferrets  transmissibility of Italy/94 (Eurasian avian-like swine) virus is poor, 2) North American TRS viruses are readily transmissible by direct contact irrespective of their HA and NA lineages, and 3) transmissibility via RD of TRS viruses with swine-like HA and NA, swine-like HA but human-like NA, and human-like HA and NA is poor, moderate, and efficient, respectively.8 pfu/ml. TRS viruses with human-like HA and NA grew to substantially higher titers (∼109 pfu/ml), while TRS viruses with swine-like HA and human-like NA grew to titers similar to those of seasonal H1N1 or 2009 pH1N1 viruses .Our results show that unlike seasonal H1N1 viruses, whose replication is reported to occur primarily in the upper respiratory tract At all three temperatures, TRS viruses with human-like HA and NA grew to the highest titers, whereas those with swine-like HA and NA grew to the lowest titers. These replication characteristics somewhat paralleled the viruses' overall respiratory droplet transmission efficiency.Despite sporadic human infections with North American TRS influenza A viruses, their human-to-human transmission has not been established, and pathogenicity and transmission studies in animal models have been very limited. This study of the pathogenicity and transmissibility of North American TRS viruses containing both swine- and human-like HA and NA found that the viruses grow efficiently in both the upper and lower respiratory tracts and cause moderate pathogenicity similar to that reported for the 2009 pandemic H1N1 viruses 6 pfu/ml) and were efficiently transmitted in ferrets (6 pfu/ml) similar to those of viruses with human HA and/or NA but was least transmissible  in ferrets. In contrast, the TRS virus A/sw/MN/1192/2001(H1N2), with swine-like HA and human-like NA, had a significantly lower average peak nasal wash virus titer (2×105 pfu/ml) than A/sw/NC/18161/2002 (106 pfu/ml) but was transmitted efficiently in ferrets.Multiple viral factors, including HA receptor specificity, human-specific amino acid residues  in PB2, and balance between HA and NA, are known to influence transmission and pathogenicity in humans  ferrets , transmiH1 HA of North American swine isolates comprises four distinct phylogenetic groups, H1α (cH1N1), H1β (TRS H1N1-like), H1γ (TRS H1N2-like), and H1δ (human-like) 6 to 2×108 pfu/ml at 39.5°C, while seasonal human H1N1 A/Brisbane/59/07 yielded only 6×103 pfu/ml under identical growth conditions (8 pfu/ml) and TRS viruses was similar. Interestingly, at all three temperatures the growth kinetics of the TRS viruses with swine-like HA was very similar to that of the 2009 pandemic A/Mexico/4482/2009 (H1N1) virus (in vitro growth of the TRS and pH1N1 viruses at 39.5°C and the relation of this growth to their efficient replication in ferret lungs are of interest for future studies.We found that the North American TRS viruses grew well at 39.5°C. The TRS viruses yielded 2×10nditions . This fi1) virus . It was in vitro and in vivo), efficient release of progeny virions in the presence of human-like NA, and efficient re-infection in the presence of human-like HA. In contrast, this group of TRS viruses causes only sporadic human infection, indicating a possible limitation of the ferret model. Importantly, however, ferrets used in this study  were farm-raised and sero-negative for influenza A viruses. Acquired immunity within the human population, in addition to viral and environmental factors, plays a critical role in human-to-human transmission of influenza A viruses TRS viruses containing human-like HA and NA showed the highest RD transmissibility in the ferret model, likely reflecting a higher rate of replication  in the World Health Organization's recommended trivalent vaccine. TRS viruses are reported to cause severe lower respiratory tract disease and diarrhea in humans Four- to six-month-old male ferrets  that were serologically negative for currently circulating influenza viruses by hemagglutination inhibition (HI) assay were used. All animal experiments were conducted in an Animal Biosafety Level 2+ (level 2 with enhanced biocontainment for pandemic H1N1 influenza A virus) facility at St. Jude Children's Research Hospital, in compliance with the policies of the National Institutes of Health and the Animal Welfare Act and with the approval of the St. Jude Children's Research Hospital Animal Care and Use Committee.MDCK cells were maintained in Dulbecco modified Eagle's medium  supplemented with 10% fetal bovine serum and antibiotics-antimycotic . Stock viruses were propagated in embryonated chicken eggs at 37°C for 48 h. All isolates underwent a limited number of passages in eggs to maintain their original properties. The genome sequences of A/sw/OK/011521-5/2008 (H1N2), A/sw/OK/011506/2007 (H3N2), A/sw/IN/9K035/99 (H1N2), and A/Mexico/4482/2009 (H1N1) have been described previously 3 [Invitrogen], antibiotics-antimycotic [Sigma], and 1.0 µg/ml of tosylsulfonyl-phenylalanyl-chloromethyl-ketone [TPCK]-treated trypsin [Sigma]) (1.6 ml per well in 6-well plates) at different temperatures. Plaque assays were done in MDCK cells in the presence of 1.0 µg/ml TPCK-treated trypsin in agarose overlay medium , as reported previously MDCK cell monolayers were inoculated with viruses at an MOI of 0.001 and maintained in virus growth medium . The next day (day 1 p.i.), two donor ferrets were moved into separate cages, each containing one naïve direct contact (co-housed) ferret (Baseline body weight and temperature were documented before inoculation or contact exposure. Four donor ferrets per virus were housed in the lower cages of isolators (configured as shown in ) ferret . For res) ferret  were eutLung tissue was collected from control (un-inoculated) and virus-inoculated ferrets on day 5 p.i., fixed in 10% neutral buffered formalin, and embedded in paraffin. 5-µm sections were stained with hematoxylin and eosin and examined by microscopy in a blinded fashion. Histopathology was examined separately for the bronchi, bronchioles, alveoli and alveolar interstitial walls of each lung lobe.Serum samples were collected from ferrets at day 20 p.i., treated for 18 h at 37°C with receptor-destroying enzyme, heat-inactivated at 56°C for 30 min, and tested by HI assay with 0.5% packed chicken red blood cells as described previously CY058484-91, CY098465-72, CY098473-80, CY098481-88, CY098489-96, CY098497-504, CY098505, CY098506-12, and CY098513-20 for A/Brisbane/59/2007 (H1N1), A/swine/MN/1192/2001 [H1N2], A/swine/NC/47834/2000 [H1N1], A/swine/MN/6998/2003 [H1N1], A/swine/MN/5763/2003 [H1N2], A/sw/Italy/1369-7/1994 [H1N1], A/Mexico/4482/2009 (H1N1), A/swine/NC/38448-1/2005 [H1N1], and A/swine/NC/18161/2002 [H1N1], respectively.Figure S1Phylogenetic tree of the A) HA and B) NA gene segments based on nucleotide sequences from GenBank. Evolutionary history was inferred by using the neighbor-joining method. The percentage of replicate trees in which the associated taxa clustered in the bootstrap test (500 replicates) are shown next to the branches. Only bootstrap values >60 are shown. The tree is drawn to scale, with branch lengths indicating the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances (the number of base substitutions per site) were computed by using the Kimura 2-parameter method. All ambiguous positions were removed for each sequence pair (pairwise deletion option). Evolutionary analyses were conducted by using the MEGA5 program. sw, swine; Eu sw, Eurasian avian-like swine; hu, human; pan, pandemic. •, Viruses used in this study.(EPS)Click here for additional data file.


Diet contributes to colorectal cancer development and may be potentially modified. We wanted to identify the biological mechanisms underlying colorectal carcinogenesis by assessment of diet-gene interactions.IL10 C-592A (rs1800872), C-rs3024505-T, IL1b C-3737T (rs4848306), G-1464C (rs1143623), T-31C (rs1143627) and PTGS2 (encoding COX-2) A-1195G (rs689466), G-765C (rs20417), and T8473C (rs5275) were assessed in relation to risk of colorectal cancer (CRC) and interaction with diet  and lifestyle (non-steroid-anti-inflammatory drug use and smoking status) was assessed in a nested case-cohort study of nine hundred and seventy CRC cases and 1789 randomly selected participants from a prospective study of 57,053 persons.The polymorphisms IL1b C-3737T, G-1464C and PTGS2 T8473C variant genotypes were associated with risk of CRC compared to the homozygous wildtype genotype . Interactions were found between diet and IL10 rs3024505 ; meat=0.04, fish=0.007, fibre=0.0008, vegetables=0.0005), C-592A , IL1b C-3737T  and PTGS2 genotypes G-765C , and T8473C  and A-1195G .IL10, IL1b, and PTGS2 and diet and lifestyle factors in relation to CRC. The present study demonstrates that gene-environment interactions may identify genes and environmental factors involved in colorectal carcinogenesis.Genetically determined low COX-2 and high IL-1β activity were associated with increased risk of CRC in this northern Caucasian cohort. Furthermore, interactions were found between  Colorectal cancer (CRC) is one of the most common cancers in the Western World . IncreasChronic intestinal inflammation is a well-known risk factor for CRC . Diet anIL10, IL1B, and PTGS2, respectively), are important mediators of intestinal inflammation. Both SCFA and TLR activation have been found to affect production of IL-10 and IL-1β and thereby COX-2 activation [IL1B has been associated with risk of lung cancer and multiple myeloma [IL-10, IL-1β and COX-2 , IL1B C-3737T (rs4848306), G-1464C (rs1143623), T-31C (rs1143627) and PTGS2 (encoding COX-2) A-1195G (rs689466), G-765C (rs20417), T8473C (rs5275) and the IL10 marker polymorphism C-rs3024505-T in relation to diet  and lifestyle (non-steroid-anti-inflammatory drug use and smoking status) in a nested case-cohort study of nine hundred and seventy CRC cases and 1789 randomly selected participants from the prospective Diet, Cancer and Health study encompassing 57,053 persons. Therefore, we assessed the functional polymorphisms The Diet, Cancer and Health Study is an ongoing Danish cohort study designed to investigate the relation between diet, lifestyle and cancer risk . The cohFollow-up was based on population-based cancer registries. Between 1994 and 31th December 2009, nine hundred and seventy CRC cases were diagnosed. A subcohort of 1897 persons was randomly selected within the cohort. Of these, 108 with missing genotype data were excluded. All information on genotypes and diet and lifestyle factors was available for nine hundred and seventy CRC cases and 1789 subcohort members. Information on diet, lifestyle, weight, height, medical treatment, environmental exposures, and other socio-economic factors were collected at enrolment using questionnaires and interviews. In the food-frequency questionnaire, diet consumption was assessed in 12 categories of predefined responses, ranking from ’never’ to ’eight times or more per day’. The daily intake was then calculated by using FoodCalc ; this prContributing food items to the food group ‘cereals’ included wholegrain foods  and refined grain foods  and was measured in grams per day . Intake Smoking status was classified as never, past or current. Persons smoking at least 1 cigarette daily during the last year were classified as smokers.The lifestyle questionnaire included this question regarding use of NSAID: “Have you taken more than one pain relieving pill per month during the last year?” If the answer was yes, the participant was asked to record how frequently they took each of the following medications: “Aspirin”, “Paracetamol”, “Ibuprofen”, or “Other pain relievers”. The latter category included NSAID preparations other than aspirin and ibuprofen. Based on all records, we classified study subjects according to use of “any NSAID” (≥ 2 pills per month during one year) at baseline. KASP™ genotyping assay. (http://www.lgcgenomics.com/). One marker polymorphism, the IL10 C-rs3024505-T, and 8 functional polymorphisms were selected; IL10 C-592A (rs1800872), IL1B C-3737T (rs4848306), G-1464C (rs1143623), T-31C (rs1143627) and PTGS2 (encoding COX-2) A-1195G (rs689466), G-765C (rs20417), and T8473C (rs5275). IL1B T-31C (rs1143627) [PTGS2 (encoding COX-2) A-1195G (rs689466), G-765C (rs20417), and T8473C (rs5275) [Buffy coat preparations were stored at minus 150°C until use. DNA was extracted as described . The DNAC-592A rs00872, IL(rs5275) . To confDeviation from Hardy-Weinberg equilibrium was assessed using a Chi square test.Incidence rate ratios (IRR) and 95% Confidence Interval (95%CI) were calculated according to the principles for analysis of case-cohort studies using an un-weighted approach . Age was2, continuous), NSAID (yes/no), use of hormone replacement therapy (HRT) , smoking status (never/past/current), intake of dietary fibre , and red meat and processed meat . Cereals, fibre, fruit and vegetables were also entered linearly. All analyses were stratified by gender, so that the basic (underlying) hazards were gender specific. For all the polymorphisms, IRR was calculated separately for heterozygous and homozygous variant allele carriers. For all the SNPs except for PTGS2 A-1195G, all variant allele carriers were subsequently grouped for interaction analyses since no recessive effects were observed. For PTGS2 A-1195G, a recessive mode was used in the subsequent analyses.All models were adjusted for baseline values of suspected risk factors for colorectal cancer such as body mass index (BMI)  . A p<0.0All participants gave verbal and written informed consent. The Diet, Cancer and Health study was approved by the National Committee on Health Research Ethics  01-345/93) and the Danish Data Protection Agency.IL10 C-592A 0.22, rs3024505 0.17, IL1B C-3737T 0.43, G-1464C 0.27, T-31C 0.33 and PTGS2 A-1195G 0.19, G-765C 0.15, and T8473C 0.34, respectively.Characteristics of the study population and risk factors for CRC are shown in IL1B C-3737T variant allele carriers were at lowered risk of CRC and the G-1464C variant allele carriers were at higher risk of CRC compared to the homozygous wildtype genotype carriers   were associated with increased risk of CRC compared to the reference TGT/TGT haplotype . Carriers of one copy of the haplotype CCC had an IRR of 1.20 (p=0.04) and carriers of two CCC haplotypes had an IRR of 1.29 (p=0.12) (reference group: no CCC haplotype) (ctively) . Haplotyaplotype . Howeverplotype) . Carrierplotype) .PTGS2 T8473C variant allele were at lower risk of CRC and homozygous carriers of the low COX-2 activity PTGS2 A-1195G variant G-allele were at marginally higher risk of CRC than the homozygous wildtype genotype  (GGT/GGT), were at increased risk of CRC (p=0.09) compared to the reference PTGS2AGT/AGT  haplotype combination (Carriers of the high COX-2 activity ctively) . Furtherbination . In a sebination . PTGS2 A-1195G and consequently, AA and AG carriers were grouped versus GG carriers. Where no recessive effects were observed, variant genotypes were combined in the interaction analysis to maximize the statistical power. A recessive effect was found for IL10 rs3024505 variant carriers were at 6% increased risk of CRC per 25 g red and processed meat per day (95%CI: 1.00-1.11) whereas homozygous wildtype carriers were at no risk by meat intake =0.04) .t)=0.04) . These ft)=0.04) . IL10 rsPTGS2 G-765C variant allele carriers were at 8% increased risk of CRC per 25 g red and processed meat per day (95% CI: 1.00-1.15) whereas homozygous wildtype carriers were at no risk by meat intake (Pint=0.006)  (t=0.006) . Also, Pt=0.005) . IL10 rs3024505 homozygous wildtype carriers were at 10 % reduced risk of CRC per 25 g fish per day whereas variant carriers had no risk reduction by similar intake (Pint=0.007).IL10 rs3024505 homozygous wildtype carriers were at 23 and 6 % reduced risk of CRC per 10 g fibre and 50 g vegetables per day whereas variant carriers had no risk reduction by similar intake . Furthermore, IL10 rs3024505 homozygous wildtype carriers were at lowered risk of CRC among study participants with the highest intake of fibre  and vegetables . PTGS2 G-765C homozygous wildtype carriers were at 21% and 5% reduced risk of CRC per 10 g fibre and 50 g fruit per day whereas variant carriers had no risk reduction by similar intake . In the tertile analysis, PTGS2 G-765C homozygous wildtype carriers were at low risk of CRC by high intake of fibre , and fruit .No interaction between any genotypes and cereal in relation to risk of CRC was found  whereas the risk estimates in the highest tertile was similar for the two alleles (Pint=0.03) (as found . In the nt=0.03) . IL1B C-3737T and use of NSAID was found  but not among NSAID users  compared to the homozygous wildtype carriers (reference) (Pint=0.04).A statistically significant association between as found . Low risPTGS2 A-1195G and smoking was found  compared to homozygous wildtype carriers who had never smoked (reference group). A statistically significant association between as found . Among cIL1B and PTGS2 polymorphisms were associated with risk of CRC  haplotype was associated with increased risk of CRC. This haplotype gives high transcription levels in studies of the promoter using transient transfections [IL1B transcription and encompasses the variant allele of C-3737T. The IL1B C-3737T polymorphism abolishes a binding site for the anti-inflammatory NF-κB subunit p50 [IL1B polymorphisms and any of the studied diet variables. However, in the tertile analysis of vegetable intake  and 1.47 (95%CI: 1.11-1.95) fold increased risk of CRC, respectively, whereas carriers of the variant allele of C-3737T were at reduced risk of CRC . This may suggest that the association between IL1B polymorphisms and risk of CRC can only be detected in populations with relatively low vegetable intake such as the Danish population [In the present study, the fections . Conversunit p50 . Thus, oe intake  the assoPTGS2 A-1195G variant allele leads to low transcription levels of COX-2 [PTGS2 T8473C gives high mRNA levels [PTGS2 GGT  haplotype was associated with increased risk of CRC (P=0.024). The PTGS2 A-1195G homozygous variant genotype was marginally associated with increased risk of CRC (P=0.07) and PTGS2 T8473C variant carriers with genetically determined high COX-2 activity were at lowered risk of CRC (P=0.02). In accordance with the present study, genetically low COX-2 activity was found to predispose to inflammatory bowel disease, a risk factor for CRC [The of COX-2  whereas A levels . In DaneA levels . In the  for CRC .PTGS2 G-765C. Thus, among variant allele carriers, daily intake of meat was associated with 8% increased risk of CRC pr 25g meat, whereas homozygous wildtype allele carriers were not at increased risk (Pint=0.006). The result is in accordance with the finding of a statistically significant association between PTGS2 G-765C variant genotypes and CRC among subjects with high n-6 PUFA intake in a prospective, population-based cohort of 310 Singapore Chinese cases [IL10 rs3024505. A similar interaction was found for fish intake. However, it is difficult to interpret the functional implications as IL10 rs3024505 is a marker SNP with no known function. The lack of interaction with the functional promoter polymorphism C-592A may suggest that the detected interaction may be related to other genes than IL10, but on the other hand, IL10 rs3024505 is located very far away from other genes [PTGS2 and IL10 on the other hand, suggest that inflammation plays a role in meat related carcinogenesis. In support of this, we have also found interaction between the functional promoter polymorphism NFKB1 -94ins/del and meat intake in relation to CRC [The intake of meat in the Danish population is among the highest intakes world-wide and we have previously identified interactions between meat and genes ,5. We fose cases . N-6 PUFer genes . In summIL10 rs3024505 and intake of fibre and vegetables. In both cases, homozygous wildtype allele carriers benefited from high intake, whereas variant allele carriers had no risk reduction when eating fibre or vegetables in relation to CRC risk. However, since variant allele carriers in the tertile with the lowest fibre intake were at marginally lowered risk of CRC  the results suggest that wildtype allele carriers experience a risk reduction by fibre intake that variant allele carriers already have. The results support and extend our previously finding of interaction between fibre and IL10 in a subcohort of the present study cohort [PTGS2 on the other. The interactions are quite consistent and suggest that subjects with genetically low PTGS2 activity benefit the most from high intake of fibres, fruit, and cereals. Furthermore, tertile analyses showed that those with the genetically determined lowest COX-2 activity, namely homozygous carriers of the variant allele of PTGS2 A-1195G, were at high risk of CRC in the group with the lowest intake of fibres (IRR=3.08 (95%CI:1.51-6.28), and fruits , whereas those with the genetically determined high COX-2 activity, carriers of the variant allele of PTGS2 G-765C, were at low CRC risk even in the tertile with the lowest fibre intake . Thus, COX-2 seems intimately implicated in the biological mechanism underlying the protective effect of fibres in relation to CRC.We observed strong interaction between the marker y cohort . SimilarIL1B C-3737T in relation to development of CRC suggesting that NSAID intake reduce the risk of CRC among those with high risk of CRC due to genetically determined high IL1B level. We found no statistically significant interaction between NSAID and COX-2 in relation to CRC. A non-statistically significant tendency towards protection by NSAID use among those with genetically low COX-2 activity was found. Long-term intake of aspirin (a COX-1 inhibitor) has been found to confer protection against CRC including the presently used Diet, Cancer and Health cohort [We found interaction between NSAID use and h cohort . It was h cohort ,48. cox-2 expression is high in il-10 deficient mice [The biological interpretation of our results is supported by other findings. IL-1B, IL-10, and COX-2 are part of the same inflammatory pathways. IL-1 has been found to induce the synthesis of COX-2 through activation of the pro-inflammatory p65 unit of nuclear factor κB (NF-κB) . Furtherent mice . TherefoPTGS2 polymorphisms may have differential impact on CRC risk dependent on environmental factors. However, once carcinogenesis has been initiated, a high COX-2 enzyme activity seems to be a risk factor for further progression [Furthermore, our results suggest that those with genetically determined low COX-2 activity are at high risk of CRC by smoking and meat intake and, furthermore, protected by fibre intake. Thus gression ,50,51. Taken together, our interaction analyses suggest that diet modify intestinal carcinogenesis through impact on inflammatory response and furthermore suggest that the effect may differ among various populations depending on gene-environment interactions. Our findings should be explored in other well-characterized prospective cohorts.IL10, fibre and PTGS2, vegetables and IL10, and fruit and PTGS2 withstood correction for multiple analyses. However, as our hypothesis was biologically based we did not correct for multiple analyses [This study used a nested prospective case-cohort design and has the major advantage that data and samples were collected before diagnosis thus minimizing the risk of differential misclassification between cases and comparison group. The risk estimates were adjusted for known confounding factors affecting risk of CRC in this cohort including dietary factors, body mass index (BMI), alcohol, smoking status and NSAID use. A main strength of the study is the large sample size. The genes were carefully selected based on their role in the inflammatory pathway and the polymorphisms were mainly selected based on their functional effects in order to allow interpretation of the involved biological pathways in colorectal carcinogenesis. Only the interactions between fibre and analyses . In the We found evidence that genetically determined variation in IL-1β and COX-2 levels is associated with risk of CRC. Moreover, gene-environment interactions suggest that COX-2 and IL10 are implicated in both meat-related carcinogenesis and in the protective effects of fibre in relation to CRC. This study demonstrates that gene-environment interactions provide an efficient tool for identifying factors involved in colorectal carcinogenesis. Our findings should be replicated in other well-characterized prospective cohorts.Table S1Combinations of genotypes/haplotypes and risk of colorectal cancer.(DOCX)Click here for additional data file.Table S2Incidence rate ratio (IRR) for colorectal cancer for tertiles of intake of diet for the studied polymorphisms.(DOCX)Click here for additional data file.Table S3Interactions between NSAID use  and studied polymorphisms in relation to risk of colorectal cancer.(DOCX)Click here for additional data file.Table S4Interaction between smoking status  and the studied polymorphisms in relation to risk of colorectal cancer.(DOCX)Click here for additional data file.


Recent reports show that R70Q and L/C91M amino acid substitutions in the core from different hepatitis C virus (HCV) genotypes have been associated with variable responses to interferon (IFN) and ribavirin (RBV) therapy, as well to an increase of hepatocellular carcinoma (HCC) risk, liver steatosis and insulin resistance (IR). Mutations in NS5B have also been associated to IFN, RBV, nucleoside and non-nucleoside inhibitors drug resistance. The prevalence of these mutations was studied in HCV RNA samples from chronically HCV-infected drug-naïve patients.After amplification of core and NS5B region by nested-PCR, 12 substitutions were analyzed in 266 Venezuelan HCV isolates subtype 1a, 1b, 2a, 2c, 2b, 2j (a subtype frequently found in Venezuela) and 3a (n = 127 and n = 228 for core and NS5B respectively), and compared to isolates from other countries (n = 355 and n = 646 for core and NS5B respectively).R70Q and L/C91M core substitutions were present exclusively in HCV G1b. Both substitutions were more frequent in American isolates compared to Asian ones . In Venezuelan isolates NS5B D310N substitution was detected mainly in G3a (100%) and G1a (13%), this later with a significantly higher prevalence than in Brazilian isolates (p = 0.03). The NS5B mutations related to IFN/RBV treatment D244N was mainly found in G3a, and Q309R was present in all genotypes, except G2. Resistance to new NS5B inhibitors (C316N) was only detected in 18% of G1b, with a significantly lower prevalence than in Asian isolates, where this polymorphism was surprisingly frequent (p < 0.001).Genotypical, geographical and regional differences were found in the prevalence of substitutions in HCV core and NS5B proteins. The substitutions found in the Venezuelan G2j type were similar to that found in G2a and G2c isolates. Our results suggest a high prevalence of the R70Q and L/C91M mutations of core protein for G1b and D310N substitution of NS5B protein for the G3a. C316N polymorphism related with resistance to new NS5B inhibitors was only found in G1b. Some of these mutations could be associated with a worse prognosis of the disease in HCV infected patients. Around 170 million persons (3% of the world population) are infected with the hepatitis C virus (HCV). More than 350,000 people die from HCV-related liver diseases, including hepatocellular carcinoma (HCC), each year-4. AboutSeven HCV genotypes, and a large number of subtypes in each genotype, have been described. Genotypes 1, 2, and 3 have a worldwide distribution, and their prevalence varies according to the region of the world. In Venezuela, genotype 1 is the most frequently detected, followed by genotypes 2 and 3, however a significant reduction of HCV G1b circulation was observed in the last decade, with the increase in circulation of genotype G2j, a subtype quite rare in other countries, including the Americas,7.While a vaccine for HCV is not available, many new antiviral agents are being tested to treat HCV infection, such as nucleoside (NIs) and non-nucleoside (NNIs) polymerase and protease inhibitors. The combination of pegylated interferon alpha (IFN) and ribavirin (RBV), plus new HCV NS3/4A serine protease inhibitors (boceprevir or telaprevir) is the current treatment for patients chronically infected with HCV. HCV genWithin the HCV genome, core is thought to be the most conserved protein; results of nucleotide and deduced amino acid sequence analysis across diverse strains of HCV reveal 81–88% nucleotide and 96% amino acid sequence homology,11. The In the present study, we analyzed core and NS5B polymorphisms/mutations which have been associated with non-responsiveness or a better response to IFN/RBV combination therapy, and/or resistance to polymerase inhibitor drugs, HCC, liver steatosis and IR, in chronically infected drug-naïve patients from Venezuela, and these polymorphisms/mutations were compared with worldwide HCV isolates.Some relevant core and NS5B substitutions associated with treatment response, as well as, with a worse prognosis of the disease were analyzed. Four core substitutions-30 and eCore mutations R70Q and L/C91M, associated with IR, increasIn NS5B region, D310N, D244N, T329I and S326G substitutions have been described to be induced by RBV treatment. D310N wQ309R and A333E NS5B mutations, frequents in patients with SVR and end-of-treatment response (ETR) after INF/RB therapy, were al′-C-methyl modified ribonucleosides and HCV-796, respectively) and core sequences [GenBank accession numbers HM777360 -HM777430 and JQ924868-JQ924946] of Venezuelan HCV-infected naïve patients were analyzed. The sequences of each genotype from other countries were obtained from GenBank and HCV database (http:VHC.lanl.gov/content/VHC-db/index).Sequence alignment performed by the global alignment algorithm, using DNAman 5.2.2 . Phylogenetic analysis was performed by the Neighbor Joining method .Statistical differences were evaluated by the Chi-Squares test with Yates correction, or Fisher Exact test, according to a computerized Epi Info program, version 3.5.3 .ETR: End-of-treatment response; G: Genotype; HCC: Hepatocellular carcinoma; HCV: Hepatitis C virus; IFN: Pegylated Interferon alpha; IR: Insulin resistance; NIs: Nucleoside inhibitors; NNIs: Non- nucleoside inhibitors; RBV: Ribavirin; SVR: Sustained virological response.The authors manifest non competing interests.RCJ, YFS, MZS, CLL, HRR, and FHP carried out the molecular genetic studies. RCJ, YFS and FHP participated in the sequence alignment and phylogenetic analysis. RCJ and FHP drafted the manuscript. All authors read and approved the final manuscript.


In the past twenty years, protective actions of the RAS, not only in the cardiovascular, but also in the nervous system, have been demonstrated. The so-called protective arm of the RAS includes AT2-receptors and Mas receptors (AT2R and MasR) and is characterized by effects different from and often opposing those of the AT1R. These include anti-inflammation, anti-fibrosis, anti-apoptosis and neuroregeneration that can counterbalance pathological processes and enable recovery from disease. The recent development of novel, small-molecule AT2R agonists offers a therapeutic potential in humans with a variety of clinical indications.The renin-angiotensin system (RAS) plays an important role in the initiation and progression of tissue injuries in the cardiovascular and nervous systems. The detrimental actions of the AT The AT2R was finally cloned in 1993 by two independent groups [2R was described as an “enigmatic” seven transmembrane receptor [2R had to cope with several problems including a very selective tissue expression pattern in adult life and unusual, sometimes contradictory, physiological properties. Thus, the AT2R became “a matter of love and hate” [2R forms part of the “protective arm of RAS” with a great potential in tissue protection and regeneration [2R, while being only sparsely expressed in most healthy tissues, is strongly upregulated following tissue damage [2R with a recently available small-molecule ligand, compound 21, has not only greatly helped to elucidate the major molecular pathways involved in AT2R-mediated tissue protection, such as anti-inflammation, anti-fibrosis and anti-apoptosis [2R stimulation with restriction to cardiac, vascular and neuronal disease.Research on the ATneration . The AT2e damage  and neure damage , myocarde damage –10 and be damage . The selpoptosis . This re2R stimulation activates at least three different classical signaling pathways: the cGMP/nitric oxide pathway [2R are characterized mainly by regulation of inflammation, fibrosis and apoptosis [AT pathway , 13, pro pathway , 15 and  pathway . Since t2R-mediated anti-inflammatory actions are the inhibition of NF-κB activity [Key molecular mechanisms involved in the ATactivity , 18 and activity , 20 . In this study, the authors showed that C21 caused a dose-dependent reduction of TNFα-induced interleukin-6 (Il-6) levels in primary human and murine dermal fibroblasts. Moreover, this study elucidated the anti-inflammatory AT2R-coupled signaling demonstrating that this pathway involves activation of protein phosphatases, CYP-dependent epoxidation of arachidonic acid to EETs, and inhibition of NF-κB activity. With this mechanism, AT2R counteracts not only the pro-inflammatory effects of TNFα but also those mediated by the AT1R, which involve CYP-dependent hydroxylation of arachidonic acid to 20-HETE and induces NF-κB activation [In 2010, Rompe and colleagues demonstrated anti-inflammatory effects of ATtivation .2R-mediated anti-inflammation can be achieved through an inhibition of oxidative stress. In fact, McCarthy et al., found that stimulation of the AT2R caused a reduction of stroke-induced superoxide production. They showed an inverse relationship between superoxide production and AT2R expression and suggested that AT2R reduces oxidative stress related to ischemia [As mentioned above, ATischemia .1R activation. Pendergrass et al., showed that AT1R-induced oxidative stress involves NADPH oxidase activation to generate reactive oxygen species (ROS) [2R is anti-inflammatory via reduction of pro-oxidant signals by inhibiting NADPH oxidase expression and ROS generation leading to a downregulation of p38 and p44/42 MAP kinase phosphorylation [It is well known that Ang II induces oxidative stress via ATes (ROS) . On the rylation .2R via increased interleukin-10 (IL-10) production in an NO-dependent manner [It is also known that, during oxidative stress, the production of ROS exceeds the available antioxidant defense systems. As a consequence, increased ROS concentrations reduce the amount of bioactive NO . Moreovet manner .2R via cellular mechanisms [In addition to the above-mentioned effects, increasing evidence demonstrates new protective anti-inflammatory actions of the ATchanisms –29.2R in the regulation of the cellular immune response in the context of ischemic heart injury. The authors identified a cardioprotective T cell population, CD8+AT2R+, characterized by upregulated IL-10 and downregulated IL-2 and INF-γ expression compared with CD8+AT2R- T cells, which increased in response to ischemic cardiac injury. The authors demonstrated an immune-regulatory, cardioprotective action of the AT2R involving downregulation of the expression of proinflammatory cytokines and sustained IL-10 production, mediated, at least in part, via CD8+AT2R+ T cells [Curato and colleagues studied the role of the AT T cells .2R. Valero-Esquitino et al., evaluated the effects of AT2R stimulation on T cell differentiation in vitro. The authors concluded that AT2R stimulation induces an inhibition of T cells recruitment and modulation of the differentiation of naïve T cells into pro-inflammatory T helper (Th)1 and Th17 subsets while promoting differentiation into anti-inflammatory T regulatory cells [Another recent study supports the immune regulatory role of the ATry cells .2R concomitantly with anti-fibrosis suggesting a possible cross-talk between the two mechanisms [Several investigators have observed anti-inflammatory actions of the ATchanisms , 30.2R seems to be due to a regulation of matrix metalloproteinases (MMP) and their inhibitors (TIMP) [Moreover, different studies demonstrate that the anti-fibrotic activity of the ATs (TIMP) , 31–33 t2R-mediated anti-fibrosis appears to be an increased expression and activity of TIMP1 and TIMP2 with consequent inhibition of MMP9 and MMP2; however, the exact signaling pathway is still unknown.An important mechanism of AT2R in a tetracyclin-regulated system, that the AT2R counteracted the effects elicited by AT1R signaling and caused a marked reduction in MMP2 levels [2R-mediated decrease in MMP2, counteracting the AT1R-mediated increase in MMP2 activity and decrease in TIMP2 activity [Jing et al., showed in rat vascular smooth muscle cells (VSMCs) expressing the AT2 levels . In agreactivity .2R in atherosclerotic plaque, Dandapat et al., found that the AT2R reduced the expression and activity of MMP2 and MMP9, and collagen accumulation. The authors hypothesized that, like the anti-inflammatory pathway, this mechanism involves downregulation of NADPH oxidase and subsequent ROS generation leading to an inhibition of p38 MAPKS and p44/42 MAPKs phosphorylation [In a study investigating the role of the ATrylation .2R-mediated regulation of MMPs has been recently confirmed in a setting of atherosclerosis by Kljajic and colleagues. In this study, stimulation of AT2R, with the peptide AT2R agonist, CGP42112, caused a reduction of MMP2 and MMP9 [In accordance with previous studies, an ATand MMP9 .2R in the TIMP/MMPs axis, Lauer et al., recently reported that in a rat model of infarct-induced heart failure, stimulation of the AT2R with the selective, non-peptide AT2R agonist, C21 induced an activation of TIMP1 and subsequent inhibition of MMP9-mediated proteolysis. Moreover, AT2R stimulation led to a downregulation of TGF-β1 followed by decreased collagen accumulation [Supporting the regulatory role of the ATmulation .2R stimulation with C21 reduced fibrosis in stroke-prone spontaneously hypertensive rats without any difference in MMP2 and MMP9 expression [Conversely, Rehman et al., found that ATpression . A possi2R in apoptosis is controversial and seems to differ heavily depending on the experimental conditions .The role of the AT2R regulates apoptosis via different pathways. AT2R stimulation can activate tyrosine phosphatases, such as mitogen-activated protein (MAP) kinase-phosphatase-1 (MKP-1) and inactivate MAP kinase extracellular signal-regulated kinase (ERK)1/2, resulting in dephosphorylation of Bcl-2 and upregulation of bax-induced pro-apoptotic effects [The AT effects , 35, 36 2R stimulation by C21 on neuroprotection and neurite outgrowth and plasticity both in vitro and in vivo in a model of spinal cord injury in mice. This study reported improved neuronal survival with elevated expression of the neurotrophin, brain-derived neurotrophic factor (BDNF), and the neurotrophin receptors, TrkA and TrkB, as well as Bcl-2 that may link AT2R to anti-apoptosis [Recently, we investigated the effect of ATpoptosis .2R-mediated regulation of apoptosis includes activation of caspases. Long-term stimulation of AT2R has been reported to cause ceramide generation leading to the activation of stress kinases and caspases and finally to apoptosis [Another pathway describing the ATpoptosis –40.2R mediates inducible transcriptional regulatory protein GATA-6 expression via activation of mitogen-activated protein kinase kinase (MEK)– ERK1/2 and c-Jun N-terminal kinase (JNK). GATA-6 in turn activates FasL promoter, FasL expression and consequently apoptosis via caspase 8 [In fact, it has been reported in SMC that the ATaspase 8 .2R especially in pathological conditions. For instance, Kaschina et al., demonstrated that direct AT2R stimulation with C21 exerts beneficial effects after MI by anti-apoptotic and anti-inflammatory mechanisms. They showed that, stimulation of the AT2R engendered anti-apoptosis after MI by rescuing p38 MAPK and p44/42 MAPK expression and decreasing Fas-ligand and caspase-3 expression [Conversely, different studies reported anti-apoptotic properties of the ATpression  Fig. 1)2R especi2R stimulation is anti-hypertrophic via two main mechanisms: activation of protein tyrosine phosphatase SHP-1, which blocks growth factor signals [2R binding protein (ATBP = ATIP) [2R after the discovery of the promyelocytic leukemia zinc finger protein (PLZF) as an interacting protein of the AT2R [It has been demonstrated that AT signals –44 and i = ATIP) , 46. A hthe AT2R , see bel2R-related pathways in neuronal regeneration have been extensively studied in vitro in PC12W and NG108-15 cell-lines [2R-mediated neurite outgrowth seems to be a complex process that involves several pathways necessary for cytoskeleton rearrangements. In contrast to nerve growth factor, stimulation of the AT2R with Ang II leads to upregulation of beta-tubulin and MAP2 but downregulation of MAP1B [2R leads to a decreased activity of PKCα followed by a decreased p21RAS activity [mapk mediated by Rap1/B-Raf [2R [2R-mediated neurite outgrowth in primary cortical neurons [The ATll-lines , 49. AT2of MAP1B  and neurof MAP1B  as shownactivity . The secp1/B-Raf . However-Raf [2R . This is neurons . However neurons . Finally neurons , 46 and  neurons , 57. Oth neurons .1 receptor antagonists (ARBs) are well recognized and commonly used in the treatment of many cardiovascular disorders, the potential cardiovascular protection offered by the AT2R per se is less known. Direct AT2R stimulation has no impact on blood pressure; however, it seems to afford tissue/organ protection via anti-inflammatory and anti-fibrotic actions [2R is increased in different cardiovascular disorders such as left ventricular hypertrophy (LVH), heart failure, cardiac fibrosis and atherosclerosis [2R.While angiotensin AT actions . As reviclerosis , 59. Her1R stimulation in various kinds of heart disease is unanimously accepted. In particular, after myocardial infarction (MI), the AT1R contributes to tissue injury via inflammation and tissue remodeling. The AT2R, on the other hand, is widely recognized to induce actions counteracting those of the AT1R and this seems particularly true during cardiac injury [The pathophysiological role of ATc injury .2R stimulation post MI using the selective AT2R agonist, C21 [2R stimulation can also afford long-term cardioprotection as highlighted recently in the late stage of MI six weeks after the acute event, when heart failure begins to develop [Kaschina et al., performed the first study looking at the impact of direct ATist, C21 . In a moist, C21 . The und develop . C21 als develop .2R also protected cardiac function and attenuated MI-induced cardiac hypertrophy [2R. Moreover, the MI-induced increase of collagen I was also attenuated [2R-mediated cardioprotective mechanisms, implying cardiac stem cells as well as CD8-positive T cells, have been identified [2R expression in cardiac stem cells has been reported. Treatment of these cells with C21 attenuated the apoptosis of cardiomyocytes [+ AT2R+ T cells did not induce cytotoxicity to cardiomyocytes in opposition to the CD8+ AT2R- T cells [+ AT2R+ T cells after MI reduced the infarct size [In a model of MI with permanent ligature of the left anterior descending coronary artery, moderate cardio-selective overexpression of the ATertrophy . The uprtenuated . In addientified . Indeed  T cells . They alrct size .2R stimulation [2R to cardiac remodeling, which is commonly observed with chronic ARB treatment in the aging hypertensive heart [1R blockade, and this cardioprotective effect was reversed by blockade of the AT2R, thus, highlighting the anti-fibrotic effects of the AT2R in the heart.In a hypertension-induced model of left ventricular hypertrophy (SHR-SP), administration of C21, alone or in combination with an ARB, led to anti-fibrotic and anti-hypertrophic effects on the heart independently of any blood pressure change . In partmulation . Anotherve heart . In this2R antagonist, PD123319, was administered concomitantly with C21. Moreover, C21 also attenuated the increase in pro-inflammatory cytokines contributing to the protective mechanisms of the AT2R stimulation in pulmonary hypertension [In a model of pulmonary hypertension, a disease that often leads to right ventricular failure due to the increased vasculature resistance in the lung, C21 treatment slowed the progression of the associated pulmonary and cardiac disease , 64, as rtension , 64.2R, thus, seems to be mainly the result of anti-inflammatory and anti-fibrotic actions. AT2R-mediated anti-inflammation is controlled by mechanisms initiated at two levels: within cardiac as well as within immune cells. This may offer new therapeutic perspectives against cardiac injuries.Cardioprotection afforded by the AT2R has also been considered as a mediator of cardiac hypertrophy. Infusion of Ang II in mice induces cardiac hypertrophy. However, in AT2R knock-out mice, cardiac hypertrophy is absent suggesting that the AT2R mediates this effect in response to increased blood pressure [2R may involve the transcription factor PLZF [2R and recent in vivo studies confirmed its involvement in AT2R-mediated cardiac hypertrophy [2R antagonist [2R by itself does not induce cardiac hypertrophy, but that the recruitment of PLZF to the AT2R may, under appropriate conditions, switch the AT2R-mediated cardiac actions from antihypertrophic to hypertrophic ones [In contrast to the results summarized above, the ATpressure . Furthertor PLZF , 66. PLZertrophy . Followitagonist . However2R stimulation with C21 alone or in combination with the ARB olmesartan [1R blockade with olmesartan completely prevented hypertension as well as the increase in PWV, aortic wall thickness and elastic modulus. Hydroxyproline accumulation in the aorta was slightly reduced by olmesartan, and the combination with C21 further reduced and even restored this parameter. Thus, the C21 + olmesartan combination led to a more pronounced stiffness reduction than each component alone, independently of any effect on blood pressure [Using the L-NAME hypertension model that exhibits vascular remodeling with increased stiffness, Paulis et al., investigated the effect of chronic ATmesartan . L-NAME pressure .2R stimulation have also been reported in different vascular beds of SHR-SP  [Similar mechanical/structural improvement of the vasculature after ATrteries) . Chronicrteries) .2R has also been investigated in the pathological context of the Marfan syndrome, a disease that predisposes for aortic root aneurysm and aortic rupture. The authors crossed AT2R knock-out mice with a genetic mouse model of Marfan syndrome [2R compared to mice with Marfan syndrome expressing the AT2R. This effect was maintained in one-year-old mice. In addition, they observed an increased death rate in AT2R KO mice with Marfan syndrome [2R in vascular remodeling since its absence accelerates the process of aortic aneurysm. The authors also investigated the contribution of indirect AT2R stimulation by using losartan in Marfan syndrome mice that express or not express the AT2R. They showed that AT2R signaling is needed to observe the protection afforded by losartan in this model [The role of the ATsyndrome . They obsyndrome . This hiis model .2R may play a role, since its absence increased the extent of the vascular lesions [2R [2R-mediated anti-atherogenic actions were kinin / NO-dependent. Another recent study investigated the effect of direct AT2R stimulation using CGP42112 [2R stimulation showed vaso-protective and athero-protective effects with increased plaque stability. These effects were observed without any change on blood pressure and were associated with an improvement of the endothelial function and an increased NO bioavailability [In the pathological context of atherosclerosis, it has been previously suggested that the AT lesions , 71. Thiions [2R . The autCGP42112 . ApoE knlability .2R-mediated vasoprotection, there are still some controversies. Opposed to the findings described above, a recent study showed that AT2R deficiency has no effect on either aortic aneurysms or atherosclerosis [2R during tissue injuries should not be studied indirectly using AT2R blockade and/or AT2R knock-out animals but rather using direct stimulation of the AT2R.Although many studies have highlighted an ATclerosis . However2R-mediated neuroprotection and neuroregeneration. Neuroprotection can be defined as a process that directly prevents necrotic or apoptotic neuronal cell death (primary neuroprotection) or affords protection of myelin, axons, and neurons by, e.g., anti-inflammation (secondary neuroprotection). Neuroregeneration can be defined as a complex process restoring the interrupted neuronal connectivity and resulting in functional recovery. The underlying mechanisms may involve cell renewal, synaptic plasticity, regrowth of severed axons and sprouting of non-damaged neurons compensating the loss of a neighboring tract [This chapter focuses on the ATng tract .2R in neuronal injury. The expression of AT2R is massively upregulated in neuronal damage as demonstrated in animal models of stroke and of sciatic or optic nerve crush [2R subjected to cerebral ischemia have larger infarcts as compared to the wild-type animals, mainly due to exacerbated inflammation and generation of ROS [2R exerts neuroprotection either directly, or by the anti-inflammatory activity (secondary neuroprotection).Several studies have suggested a protective role of the ATve crush , 11, 75.n of ROS . Subsequ2R-stimulation was demonstrated in vitro in N-methyl-d-aspartate (NMDA)-mediated cell death [-7 M Ang II inhibited neuronal apoptosis and suppressed the NMDA-mediated reduction of anti-apoptotic Bcl-2. This effect was inhibited by the co-treatment with PD123319, but not with losartan, suggesting an AT2R-mediated primary neuroprotection [2R stimulation with CGP42112 significantly reduced cell death during glucose deprivation [2R specificity.The neuro-protective potential of ATll death . The surotection . In primrivation . This ef2R stimulation inhibits inflammatory response in vitro in CNS-derived cells. As shown in primary rat astrocytes exposed to LPS, the elevated expression of IL-6 and TNF-α was significantly reduced by treatment with C21 as compared to vehicle [2R stimulation also inhibited microglial activation as shown by reduced nitric oxide release, and abolished cell migration [Postischemic inflammation significantly contributes to ischemic brain injury. Therefore, anti-inflammatory therapeutic strategies can afford secondary neuroprotection in ischemic stroke . Previou vehicle . AT2R stigration .2R. Previously, we had shown that the AT1R blocker telmisartan, but not the ACE inhibitor ramipril, reduced infarct volume, neuronal apoptosis and inflammation in normotensive rats subjected to middle cerebral artery occlusion (MCAO) [1R blockade, endogenous Ang II stimulates the unopposed AT2R and by this exerts neuroprotective actions.In animal models of neuronal injury, both primary and secondary neuroprotection may significantly contribute to the beneficial effects mediated by the ATn (MCAO) . This ob2R-mediated neuroprotection in cerebral ischemia was extensively studied by another group [2R even six hours after stroke induction was still effective, showing for the first time a neuroprotective effect of delayed AT2R stimulation after cerebral ischemia [2R agonist, CGP42112, during the reperfusion reduced infarct and edema volume as well as improved functional outcome [The ATer group , 78, 83.er group . Importa2R-mediated neuroprotective mechanisms in MCAO in mice [Our group studied the AT in mice . Systemi in mice . These f2R [2R was stimulated directly; either with CGP42112 [Neurite outgrowth has been attributed to the AT2R , 86 indi2R . In prim2R . SimilarCGP42112  or with CGP42112 , 87.2R stimulation engendered neurotrophic-like actions in the nervous system of adult animals [2R-dependent since it could be inhibited by an AT2R-antagonist [2R was also demonstrated in a sciatic nerve crush model in rats [2R but also accelerated functional recovery as shown by increased toe spread distance (parameter for motor-function) and improved the foot reflex withdrawal reaction (a parameter of sensomotoric function) [2R immunoreactivity was associated with intense neurite outgrowth in ischemic striatum [Lucius et al., demonstrated already in 1998 that AT animals . Rats sutagonist . The neu in rats . Ang II unction) . Finallystriatum .2R may be implied in neuro-regenerative processes beyond the well-described neurite outgrowth. It has been shown that in AT2R-KO mice subjected to MCAO, impaired spatial memory was associated with decreased hippocampal neurogenesis as compared to wild-type animals [2R-mediated neuroregeneration. In a related manner, the impact of AT2R-stimulation on renal function also differs between sexes in that direct AT2R-stimulation with C21 increased renal blood flow to a greater extent in female rats than in males [The AT animals . This efin males .2R stimulation with C21 on neuroregeneration in an animal model of spinal cord injury (SCI) [2R-mediated neuroregeneration [We have recently evaluated the impact of the direct ATry (SCI) . In miceneration .2R agonists, such as C21, via the activation of neutrophic pathways, may provide a therapeutic option in the above-mentioned diseases.Apart from neuronal injury, the exogenous delivery of BDNF to the CNS may exert therapeutic effects in a variety of other neurological diseases including Alzheimer’s, Parkinson’s and Huntington’s disease, amyotrophic lateral sclerosis, Rett syndrome and vascular dementia , 92. How2R, our understanding of its protective role in tissue injury has greatly improved, but it is still far from complete. Development of pharmacological tools such as small-molecule ligands and the generation of the AT2R-KO animals have facilitated the elucidation of the major molecular pathways engaged by the AT2R. Results from various experimental disease models revealed the protective and regenerative potential of AT2R stimulation in cardiovascular and neuronal injury. Future research on the AT2R should concentrate on the complexity of molecular AT2R-related pathways and the cross-talk with other receptors and pathways in the context of tissue-protection. In addition to basic science, translational research will have to address the therapeutic potential of AT2R stimulation with available AT2R agonists in humans and define therapeutic cardiovascular indications and non-cardiovascular indications.During the twenty-five years following the discovery of the AT


Pantoea ananatis. Previous in silico analyses described three different T6SS loci present in the pathogenic strain of P. ananatis LMG 20103. This initial investigation has been extended to include an additional seven sequenced strains of P. ananatis together with 39 strains from different ecological niches. Comparative and phylogenetic analyses were used to investigate the distribution, evolution, intra-strain variability and operon structure of the T6SS in the sequenced strains.The Type VI secretion system (T6SS) has been identified in several different bacteria, including the plant pathogenP. ananatis strain LMG 20103 and designated PA T6SS 1-3. PA T6SS-1 was present in all sequenced strains of P. ananatis and in all 39 additional strains examined in this study. In addition, PA T6SS-1 included all 13 core T6SS genes required for synthesis of a functional T6SS. The plasmid-borne PA T6SS-2 also included all 13 core T6SS genes but was restricted to only 33% (15/46) of the strains examined. In addition, PA T6SS-2 was restricted to strains of P. ananatis isolated from symptomatic plant material. This finding raises the possibility of an association between PA T6SS-2 and either pathogenicity or host specificity. The third cluster PA T6SS-3 was present in all strains analyzed in this study but lacked 11 of the 13 core T6SS genes suggesting it may not encoded a functional T6SS. Inter-strain variability was also associated with hcp and vgrG islands, which are associated with the T6SS and encode a variable number of proteins usually of unknown function. These proteins may play a role in the fitness of different strains in a variety of ecological niches or as candidate T6SS effectors. Phylogenetic analysis indicated that PA T6SS-1 and PA T6SS-2 are evolutionarily distinct.Three different T6SS loci were identified in P. ananatis appear to have been independently acquired and may play different roles relating to pathogenicity, host range determination and/or niche adaptation. Future work will be directed toward understanding the roles that these T6SSs play in the biology of P. ananatis.Our analysis indicates that the three T6SSs of  Pantoea ananatis is a Gram-negative, motile, facultative anaerobe belonging to the gamma Proteobacteria. This bacterium can survive and multiply in a variety of ecological niches as a saprophyte, endophyte, epiphyte and pathogen [P. ananatis infects a wide range of economically important plants. In South Africa the pathogen causes disease on maize, onion and Eucalyptus spp. [P. ananatis between host plants is largely unknown. However, Walcot et al. [P. ananatis from onion seeds, which went on to produce disease symptoms on susceptible onion plants [et al. [Frankliniella fusca) were able to transmit P. ananatis into 52% of onion plants analysed [P. ananatis to different geographical regions. Current control measures are limited to cultivation of resistant plant cultivars, eradication of infected plant material and/or the use of biocontrol in the form of lytic phages [P. ananatis, there is limited information on the genetic determinants of virulence and ecological fitness of the species.pathogen . In its tus spp. -5 but hatus spp. , rice [7tus spp. , sudan gtus spp. ,10. The t et al.  isolatedn plants . In addi [et al.  demonstranalysed . These sc phages ,14. Despet al. [Vibrio cholerae and was shown to be required for virulence against amoeba and macrophages [Pseudomonas aeruginosa, Vibrio cholerae, Pseudomonas fluorescens, Pseudomonas protegens, Burkholderia thailandensis and Serratia marcescens have been shown to secrete bactericidal effectors which inhibit growth of bacterial species that lack the cognate immunity protein [Pseudomonas syringae pv. tomato DC3000, Acinetobacter baumannii, Acinetobacter baylyi, Vibrio parahaemolyticus and Citrobacter rodentium have also been to play a role in inter-bacterial competition [To date, seven different secretion systems have been described in bacteria; namely type I-VII ,16. Theset al.  in Vibrirophages . This serophages -20. The rophages -24. The rophages -30. In a protein -40. Simipetition -44. Somepetition . Multiplpetition ,24,45.Pantoea ananatis. To date, the genome sequences for eight strains of P. ananatis are available, representing both pathogenic and non-pathogenic strains. Type II, type III and type IV secretion systems are well documented virulence determinants of several human and plant pathogens [P. ananatis strain LMG 20103 [P. ananatis could play a role in either virulence or ecological fitness of the species. Previous comparative studies identified three distinct T6SS loci on the genome of a single Eucalyptus pathogenic P. ananatis strain LMG 20103 [P. ananatis.This study focused on the T6SS of the pathogen athogens ,16, althMG 20103 . These fMG 20103 -48. ThusP. ananatis strain LMG 20103 [P. ananatis. BLASTN and BLASTP searches were done using all eight currently available genomes of P. ananatis, which included the LPP-1 megaplasmid found in all sequenced strains [et al. [tss A-M/ (Type Six Secretion A-M), while the accessory or non-conserved T6SS genes were designated tag A-P (Type Six Associated Genes A-P) [The 13 conserved gene components of the T6SS from MG 20103  were use strains . In this [et al. . Based ones A-P) .P. ananatis. These regions were designated PA T6SS-1 and PA T6SS-2 . The PA T6SS-1 gene cluster was located on the genome of all eight sequenced strains of P. ananatis while PA T6SS-2 gene cluster was located on a ~30 kb region of the LPP-1 megaplasmid of strains AJ13355, LMG 20103 and PA-4. This 30 kb region was missing from the LPP-1 plasmid of P. ananatis strains LMG 2665T, LMG 5342, BD442, B1-9 and PA-13. We also found that PA T6SS-1 and PA T6SS-2 gene clusters contained all 13 core gene components of the T6SS [icmF and dotU in all eight sequenced strains of P. ananatis. This gene cluster, was designated T6SS-3 and was found to be missing the remaining 11 core gene components of the T6SS. Whether or not PA T6SS-3 gene cluster encodes a functional T6SS or represents a truncated T4SS is currently unknown. However, this putative PA T6SS-3 gene cluster was included in this study for purposes of comparative analysis. The overall genetic organisation of each T6SS of P. ananatis is presented in Figure P. ananatis, including their putative functions and COG classification is presented in Table Homologs of the T6SS genes were clustered in two distinct genomic regions in all sequenced strains of the T6SS ,24. In aP. ananatis T6SS. The core genes of PA T6SS-1 were clustered in three highly conserved operons; group 1 (tssJ-tssK-dotU-icmF) group 2 (tssB-tssC-hcp) and group 3 (tssE-tssF-tssG-tssH). PA T6SS-2 showed a considerable level of gene shuffling compared to PA T6SS-1, with gene order being highly variable between each of the different groupings. The consensus grouping in PA T6SS-2 included tssB-tssC-tssK-dotU and tssF-tssG-tssJ-tssE, while tssH-vgrG and icmF-tssA were stand-alone operons linked to non-conserved T6SS genes. The start and stop codons of all 9 genes located in PA T6SS-3 gene cluster overlapped with each other, suggesting that PA T6SS-3 represents a single transcriptional unit. These different operon structures suggested the independent acquisition of the T6SS clusters, each of which may play a different role in the biology of P. ananatis.The T6SS encoded by most bacteria is organized in discreet transcriptional units or operons ,52, suggP. ananatis strains, we analyzed the distribution of each T6SS cluster in 46 different strains of the pathogen. BlastP analysis showed that the gene products of PA T6SS-1 and 2 shared less than 50% amino acid similarity  tested . Our results showed that PA T6SS-2 was present in strains of P. ananatis isolated from symptomatic maize, onion, pineapple fruit and Eucalyptus spp. However, not all strains of P. ananatis isolated from the same host plant contained PA T6SS-2. The cluster was only found in 3 of the 6 strains of P. ananatis (50%) isolated from either maize, onion, pineapple and Eucalyptus spp., while the remaining 3 strains, isolated from the same host but different plants, did not have the cluster. In addition, PA T6SS-2 was absent in all strains of P. ananatis isolated from symptomatic wheat (2 isolates), honeydew melon (4 isolates), rice (7 isolates) and sugarcane (1 isolate). These results suggest that PA T6SS-2 could be a host range or virulence determinant of P. ananatis. Future studies will undertake pathogenicity and cross inoculation trials to validate this correlation. In addition, the cluster was also found in P. ananatis strains AJ13355 and Yomogi-101 which have been shown not to cause disease on a range of host plants  PA T6SS-1P. ananatis. The tssD (hcp) and tssI (vgrG) genes found in this cluster encoded Hcp and VgrG proteins that do not have C-terminal extensions as found in “evolved” VgrG and Hcp proteins [V. cholerae and Aeromonas hydrophila, have been associated with actin cross-linking and actin ADP ribosylation activity in mammalian host cells, respectively [P. ananatis strains PA-4, BD442, B1-9 and LMG 20103 had a single vgrG gene, while strains AJ13355, PA-13, LMG 5342 and LMG 2665T had an additional copy of vgrG. These vgrG genes appear to encode VgrG proteins with different domain architectures, characterized by the presence or absence of a C-terminal Beta-N-acetylglucosaminidase domain  repeat or pentapeptide_4 domains. PAAR repeat proteins of bacteria have categorised into different classes (Class 1-7) based on their domain architectures [V. cholerae (4jiv_D–Hhpred score 105.4). A recent study showed that the PAAR repeat proteins of E. coli and V. cholerae bind to the Gp5-VgrG complex by means of non-covalent interactions [V. cholerae and Acinetobacter baylyi were shown to be bactericidal effectors associated with T6SS-mediated killing of E. coli[vgrG island of P. ananatis T6SS-1 gene cluster may either be T6SS effectors associated with inter-bacterial competition or may mediate secretion of other effectors.Regions associated with tectures . PAAR retectures . Some oftectures . The genractions . In addif E. coli. These ff E. coli,60. It if E. coli. It is, rhsD gene of PA T6SS-1. A recent study, aimed at identifying polymorphic toxins in bacterial genomes using comparative analysis, sequence and structural analysis, identified RhsD as putative T6SS effectors of P. ananatis, based on the presence of PAAR repeats on this protein [Serratia marcescens was recently identified in a T6SS secretome analysis, suggesting that this protein is secreted by the T6SS [rhsA and rhsB genes of Dickeya dadantii encode endonuclease toxins which have been associated with contact-dependent killing of other bacteria species [D. dadantii also express the cognate immunity factors from the rhsI gene located downstream of the rhsA and rhsB genes [rhsD locus of P. ananatis has a genetic organization similar to that of D. dadantii consisting of the rhsD gene which is followed by the rhsI homolog  [rhsD loci associated with the T6SS-1 gene clusters of P. ananatis may encode different toxin/immunity factors which play different roles as either bactericidal or insecticidal toxins. Alternatively, RhsD proteins of P. ananatis may mediate secretion of other T6SS effectors which are non-covalently associated with either their N or C-terminal extension, as predicted for other PAAR proteins [PAAR-repeat domains are also found in the  protein . Similarthe T6SS . Bacterithe T6SS -69. In a species ,71. ToxisB genes ,71. The g Figure . In additagAB and tagB, which encode pentapeptide repeat proteins were also identified in the vgrG islands of P. ananatis strains PA-13, AJ13355 and LMG 5342. In strain PA 13, the pentapeptide repeat proteins are encoded by PAGR_g1680 and PAGR_g1681 which correspond to tagAB and tagB genes, respectively. Sequence analysis performed on PAGR_g1680 and PAGR_g1681 predicted no signal sequence and no transmembrane helices [tagAB and tagB genes have also been in identified in the T6SS gene clusters of Serratia marcescens, Azotobacter vinelandii, Burkolderia bronchiseptica, including the T6SS-5 gene clusters of B. mallei, B. pseudomallei and B. thailandensis, however, these genes have not been functionally characterised and the biological function of their gene products is currently unknown [Homologs of  helices ,76. Psor helices . Homolog unknown ,38,50.P. ananatis T6SS-1 gene cluster is located between hcp and tagH and contains a gene encoding a protein with a peptidoglycan binding domain (PG_binding). In strain LMG 5342, the protein is encoded by PANA5342_1731 and showed structural homology to the lytic transglycosylase enzyme of Pseudomonas aeruginosa bacteriophage phiKZ (3bkh_A Hhpred–score 146) while the corresponding homologs found in strains LMG 20103, PA-4, B1-9 and AJ13355 showed structural homology to the pesticin toxin of Yersinia pestis . Pesticin and lytic transglycosylase enzymes are bactericidal effectors which degrade peptidoglycan by cleaving the β-1,4 glycosidic bond between N-acetylmuramic acid and the N-acetylglucosamine moieties [hcp island of P. ananatis T6SS-1 cluster may encode bactericidal effectors which are functionally related to the Type VI secretion glycoside hydrolase effectors 1-3 (Tge1-3) [hcp island of P. ananatis strain LMG 2665T. However, the gene product of LMG 2665T N454_00628 showed weak structural homology to Colicin S4 of Escherichia coli (3few_X–Hhpred score 55.8). Colicin S4 is a pore-forming bacteriocin which kills bacteria species not expressing the immunity factor [T N454_00628 may be a bacteriocidal effector which plays a role in inter-bacterial competition. We did not identify homologs of Type VI amidase 1-4 (Tae 1-4), Type VI lipase effectors 1-5 (Tle 1-5), Small secreted proteins 1-6 (Ssp 1-6) and Type VI secretion exported 1, 2  in the T6SS-1 gene cluster of P. ananatis, based on sequence and structural analysis [The second variable region found in moieties -82. Thes(Tge1-3) ,83. Geney factor ,85. It ianalysis ,39,40.2) PA T6SS-2vgrG gene that encodes a VgrG protein with an additional C-terminal DUF2345 (COG4253) domain of unknown function. Conserved Domain architecture analysis showed that this domain was conserved in the vgrG genes of several different bacteria. All strains of P. ananatis that harbor this cluster encode two copies of the tssA gene within the cluster showing 22% amino acid similarity. Duplication of the tssA gene has also been reported in the T6SS gene clusters of Vibrio cholerae, Escherichia coli O157:H7, Yersinia pseudotuberculosis and Salmonella enterica serotype Gallinarum [The genetic architecture of PA T6SS-2 is highly conserved in all strains that harbor the cluster. PA T6SS-2 was found to contain a single llinarum ,45,50. TtssA and tssF and encodes several strain specific hypothetical proteins including proteins with a PAAR_motif. The genes products of LMG 20103 PANA_4136, AJ13355 PAJ_p0154 and PA-4 N455_00706 contain 424 amino acids, sharing 99% amino acid similarity and belong to the Class 2 PAAR domain architecture proteins. Furthermore, these PAAR proteins all contained identical C-terminal extensions with no putative effector domains. The gene products of PANA_4136, PAJ_p0154 and PA-4 N455_00706 may either be T6SS effectors or mediate secretion of other effectors bound to their C-terminal extensions [icmF and vgrG. This region in LMG20103 contains four genes (PANA_4140 to 4143) encoding hypothetical proteins with homologs present in strain AJ13355 but is missing from strain PA-4. PANA_4143 encodes a protein with a peptidase_M23 domain. This protein is a putative endopeptidase and is predicted to be a T6SS effector based on its high structural homology to the secreted chitinase G of Streptomyces coelicolor[P. ananatis strain PA-4 contains a gene with an esterase_lipase domain and belongs to the family lecithin: cholesterol acyltransferase (PF02450). This protein is predicted to be involved in extracellular metabolism of plasma lipoproteins, including cholesterol [vgrG genes outside the major T6SS of Pseudomonas spp. In P. aeroginosa these genes form part of the “cargo” genes predicted to have been acquired by recent horizontal gene transfer [vgrG island of strain PA-4 T6SS-2 cluster, encodes a Class 1 PAAR domain architecture protein containing 98 amino acids with no N or C-terminal extensions. This PAAR protein may play a role as a T6SS effector associated with inter-bacterial competition [P. ananatis T6SS-2 gene cluster, using sequence analysis and structural homology search tools.Two variable regions were identified in PA T6SS-2, which contained genes encoding non-conserved T6SS components and hypothetical proteins. The first variable region is located between tensions ,60. The oelicolor,86. The lesterol . Genes wtransfer . In addipetition ,60. Homo3) PA T6SS-3P. ananatis. The genetic architecture, gene order and gene content of PA T6SS-3 was conserved in all sequenced strains of P. ananatis. Interestingly, all genes found in PA T6SS-3 showed high sequence similarity to genes found in Pantoea sp. At-9b, Pantoea sp. aB-valens, P. vagans C9-1, P. agglomerans E325 and Erwinia billingiae Eb661. These Pantoea and Erwinia spp. have a homologous cluster highly similar to PA T6SS-3 in terms of gene content and operon structure [Comparative analysis of PA T6SS-3 showed that there was no variability of this cluster between the different strains of tructure . The highcp and vgrG are present outside the T6SS they are often referred to as “orphan” vgrG and hcp genes. Our analysis did not identify “orphan” vgrG genes in any of the sequenced genomes of P. ananatis. However, we identified three different “orphan” hcp genes in P. ananatis strain PA-13 . These “orphan” hcp genes were designated hcp-1, hcp-2 and hcp-3 to correspond to PAGR_g1583, PAGR_g1584 and PAGR_g3636, respectively. Genes hcp-1 and hcp-2 are adjacent to each other and have homologs present in all sequenced strains of P. ananatis, while hcp-3 is restricted to P. ananatis PA-13. Multiple alignments of the amino acid sequences of representative P. ananatis Hcp proteins showed that Hcp-3 protein is highly divergent from Hcp-1, Hcp-2 and T6SS-associated Hcp proteins , while PA T6SS-2 was closest to T6SS loci 2 of Erwinia amylovora CFBP1430 (a plant pathogen). Our phylogenetic analysis resembled the analysis performed by Bingle et al. [P. aeruginosa which is known secreted Tse1 to 3 antimicrobial effectors was found in Group A while the T6SSs of Vibrio cholerae and Peudomonas. syringae pv tomato with a similar role in inter-bacterial competition belonged to Group D [Phylogenetic analysis was used to infer the evolutionary history of the T6SS using the Maximum Likelihood Method base on the Le and Gascuel (LG) + G + F amino acid substitution model, as determined by ProtTest -92. Repre et al.  which she et al. . Similar Group D ,32,41. I Group D ,24.P. ananatis T6SS-1 and T6SS-2 clustered away from the T6SS of several important plant pathogens such as Xanthomonas citri, X. campestris, X. oryzae, Pseudomonas syringae pv. syringae, Pseudomonas syringae pv. phaseolicola, Pectobacterium atrosepticum SCRI1043, Pectobacterium carotovorum subsp. carotovorum and Pectobacterium carotovorum subsp. brasiliensis. Functional studies have shown that the T6SS gene clusters of P. atrosepticum and P. syringae are only partially required for disease development. The T6SS mutants of these bacteria were either only slightly reduced in the ability to cause disease or caused disease symptoms on susceptible host plants similar to the wild type strains [P. ananatis in susceptible host plants.Our phylogenetic tree also showed that  strains ,93-96. Thcp genes  in P. ananatis we constructed a second Maximum Likelihood tree using the General Reverse Transcriptase (rtRev) + I + G + F amino acid substitution models, as determined by ProtTest [hcp genes and “orphan” hcp genes from other bacteria  whether the T6SSs of P. ananatis are functionally active and what roles they play in host-pathogen interactions and fitness; 2) which in vitro and in vivo conditions activate the T6SSs; 3) the presence of different potential effectors secreted by the T6SSs of P. ananatis and their physiological relevance to fitness and host-pathogen interactions; and 4) how T6SSs are regulated in these strains.Comparative analyses of the T6SS in the genomes of sequenced strains of nal T6SS ,24,30. Paptation ,23,27,39P. ananatis strains were downloaded from NCBI with the accession numbers LMG 5342 ; AJ13355 ; PA-13 ; LMG 20103 (chromosome CP001875.2) and the draft genome of B1-9 (CAEJ00000000.1). The draft genomes of P. ananatis BD 442, PA-4 and LMG 2665T were sequenced and partially assembled in our laboratory. The sequences and fully annotated contigs representing the different type VI secretion system gene clusters have been deposited in GenBank for strain BD 442 , PA-4  and LMG 2665T . All eight sequenced strains of P. ananatis were obtained from different geographical regions and were isolated from different diseased plants or other environmental samples. Detailed information on the strains is presented in Additional file The annotated genome sequences of different P. ananatis strain LMG 20103 by De Maayer et al. [P. ananatis genomes and plasmids using BLASTN and BLASTP [de novo gene prediction using F-GenesB (http://www.Softberry.com). All predicted genes were searched against non-redundant protein databases at NCBI to identify homologs. SignalP 4.0 and TMHMM Server v.2.0 were used to predict signal peptides and trans-membrane helices [Genes associated with the T6SS, including flanking regions identified in r et al. , were usd BLASTP ,99. Nucl helices ,76. Prot helices , InterPr helices , Conserv helices . Protein helices . ProteinP. ananatis. BLASTP analysis showed that T6SS gene products from homologous T6SS cluster found in all sequenced strains of P. ananatis were over 98% similar [Additional file P. ananatis. Genomic DNA was isolated from 46 different P. ananatis strains using the Quick-gDNA™MiniPrepkit  following the manufacturer’s instructions. PCR amplification using SuperTherm DNA polymerase  was performed with a VeritiR Thermal Cycler . DNA sequencing was done using the ABI PRISM3100 Genetic Analyzer (Applied Biosystems) at the DNA Sequencing Facility (University of Pretoria-RSA). Colony hybridisation was used to validate the PCR results. Probes for hybridisation were labeled with Digoxigenin-11-dUTP using DIG PCR labeling Kit  according to the manufacturer’s instructions. Membrane hybridization, washing and detection were done using DIG DNA labeling and detection kit  as directed by the manufacturer.A minimum of four primer pairs were designed per T6SS cluster to amplifying locus specific genes. For primer design, the nucleotide sequences for each gene of interest were extracted from all eight sequenced strains of P. ananatis, while a further phylogenetic grouping examined the relationship between T6SS-associated Hcp and orphan Hcp proteins. Amino acid sequences of TssB, TssC, Hcp and “orphan” Hcp proteins representing bacteria from T6SS phylogenetic Groups A-D were downloaded from NCBI [Two phylogenetic analyses were carried out, one using concatenated amino acid sequences of TssB and TssC and the second using amino acid sequences of Hcp and “orphan” Hcp. The TssBC analysis showed the phylogenetic grouping of the different T6SSs of rom NCBI ,24. Aminrom NCBI . The amirom NCBI . The evorom NCBI  for TssBrom NCBI  for Hcp The data sets supporting the results of this article are included within the article and its additional files. Alignments and Phylogenetic trees which support the findings presented in this research article are available online in the Dryad Digital Repository (doi: 10.5061/dryad.vd7k7).The authors declare that they have no competing interests.DYS, LNM, SNV, IKT and TAC conceived the study. DYS performed experiments and analysis. DYS, LNM, SNV, IKT and TAC wrote the original manuscript. All authors read and approved the final version.Pantoea ananatis strain LMG 20103. This table shows the percentage amino acid identify between the T6SS gene products of P. ananatis T6SS-1 to 3. The gene products of LMG 20103 T6SS-1 were used as bait for BlastP analysis.BLASTP analysis of type VI secretion system (T6SS) homologs found in Click here for filePantoea ananatis. This file contains PCR primer sequences and the T6SS genes of P. ananatis which were targeted for Polymerase Chain Reaction (PCR) amplification.List of primers used for PCR amplification of type VI secretion system genes from different strains of Click here for filePantoea ananatis tested for the presence of T6SS gene clusters. This file contains the list of all strains of Pantoea ananatis screened for the presence or absence type VI secretion system gene homologs. The file also contains information on the host of isolation and country from which the different strains of P. ananatis were isolated from.Strains of Click here for filePantoea ananatis. This file contains results of the PCR screen, which clearly shows the distribution of type VI secretion system genes in all strains of Pantoea ananatis analysed in this study.Distribution of type VI secretion system gene clusters in 46 strains of Click here for filePantoea ananatis type VI secretion system 1, 2 and 3 . This file contains pairwise alignment of the homologous type VI secretion system gene clusters found in sequenced strains of P. ananatis. Alignments were generated using Mauve v.2.3.1 and show variable and conserved regions between the homologous T6SS gene clusters from different strains of P. ananatis.Synteny between Click here for filePantoea ananatis. This file contains all genes found within the contiguous region representing type VI secretion system 1 (PA T6SS-1) in all eight sequenced strains of Pantoea ananatis analysed in this study. For each strain of P. ananatis, the genes found in T6SS-1 are shown, including the product sizes, cluster of orthologous groups of proteins (COG) classification, conserved domain, subcellular localization, and the gene locus tags.Gene content of type VI secretion system 1 (T6SS-1) found in all sequenced strains of Click here for filePantoea ananatis. This file contains all genes found within the contiguous region representing type VI secretion system 2 (PA T6SS-2) in different strains of Pantoea ananatis. For each strain of P. ananatis, the genes found in T6SS-2 are shown, including the product sizes, COG classification, conserved domain, subcellular localization, and the gene locus tags.Gene content of type VI secretion system 2 (T6SS-2) in sequenced strains of Click here for filePantoea ananatis type VI secretion system 1. A) Shows alignment of the variable C-terminal domain of RhsD proteins from eight different strains of P. ananatis. This variable C-terminal domain is demarcated from the conserved N-terminal domain by a PxxxxxxDPxGL peptide motif indicated in the figure by blue stars. B) Shows alignment of the full length RhI proteins encoded by rhsI genes located downstream of the rhsD gene. Conserved residues are indicated by the different shadings. P. ananatis strains B1-9, LMG 2665T and LMG 5342 have identical C-terminal extensions and encode identical RhsI homologs.Alignment of the RhsD and RhsI proteins associated with Click here for filePantoea ananatis. Representative Hcp amino acid sequences were aligned in BioEdit using ClustalW2 with default settings. The orphan Hcp proteins of P. ananatis strain LMG 20103 encoded by PANA_2446 and PANA_2447 are highly similar to the T6SS-associated Hcp protein encoded by PANA_2364. P. ananatis strain PA-13 encodes an additional orphan Hcp protein (PAGR_g3636) which is unique to this strain and is highly divergent from all other Hcp proteins.Alignment of all representative Hcp proteins encoded by strains of Click here for filetssB and tssD gene locus tags are provided.List of bacterial strains used for TssBC phylogenetic analysis. This file contains the list of TssB and TssC homologs from different bacterial species which were used for phylogenetic analysis. The accession numbers of all bacterial strains used in this study including the Click here for filetssD gene locus tags are provided.List of bacterial strains used for Hcp phylogenetic analysis. This file contains all TssD homologs used for phylogenetic analysis. The accession numbers of all bacterial strains used in this study including the Click here for filePantoea ananatis. Phylogenetic analysis were conducted in MEGA6 [Hcp phylogenetic tree. The figure shows the evolutionary history of Hcp and “orphan” Hcp proteins of in MEGA6 . The evoin MEGA6 ,97. The Click here for file


Presence of drug-resistance mutations may get in the way of the efficacy of antiviral therapy. Our study was aimed at defining the prevalence of HBV drug-resistance in HBVrt region in a population of 147 HBsAg positive patients.Antiviral therapy using nucleos(t)ide analogues (NAs) is an effective control measure of chronic hepatitis B virus (HBV) infection; however they need long term treatmentHBV/D has shown multiple types of HBVrt mutations both among treatment naïve  and treated patients . In additional, several mutations, with a suggested role in drug resistance, were detected among the treatment naïve as well as the treated patients. The mutations reported to be involved in reduction of drug effectiveness, was common among non-responders to therapy as well as among the naïve patients. Notably, classical antiviral resistance mutations (rtL80I/V-rtI169T-rtV173L-rtL180M-rtA181T/V/S-rtT184A/S/G/C-rtA194T-rtS202C /G/I -rtM204V/I-rtN236T-rtM250V) were not detected.The prevalence of putative NAr mutations among non responders to therapy suggests that they might have role in reduced efficacy of currently available antivirals and requires further investigations. Chronic hepatitis B virus (HBV) infection remains a serious public health problem in India. Antiviral therapy using nucleos(t)ide analogues (NAs) is an effective control measure; however they need long term treatment. The limited numbers of NAs available and development of drug resistance conferred by viral mutations in the HBV reverse transcriptase (rt) domain during long term treatment remains the major concern leading to treatment failure . Recent Mutations reported in response to available NA therapy for HBV is defined as primary mutations, which reduces HBV replication fitness . SecondaEight HBV genotypes (A–H) and several subgenotypes within certain HBV genotypes have been identified , with diBlood samples obtained from 147 HBV surface antigen (HBsAg) positive patients referred to our unit were included in this retrospective study. The inclusion criteria were hepatitis B surface antigen (HBsAg) positive; exclusion criteria were hepatitis C virus or human immunodeficiency virus co-infection. Among them HBVrt region could be successfully amplified and sequenced from 85 samples . The low amplification of the samples may be due to the low copy number of HBV DNA in the plasma. The treatment drug selection was lamivudine and combinational therapy (adefovir and lamivudine). The serology of the patients was done as described previously . The sel′-AAGCTCTGCTAGATCCCAGAGT-3′ (sense 18 to 40); HB6R 5′-AACAGACCAATTTATGCCTA-3′ (antisense 1809 to 1790) in 30 μl reaction volume and 4 μl from the first round product was amplified using primers B2 5′- GGCTCACAGTTCACGGAACAGT-3′(sense 65 to 86) and HS4R 5′- CATACTTTCCAATCAATAGG-3′(antisense 992 to 973) and a standard thermal cycling profile, with annealing 55°C for 45 s, 45 cycles (40 cycles for the second round).Nested polymerase chain reaction was used to amplify HBV RT region using primers (HB1F-5http://www.ncbi.nlm.nih.gov/projects/genotyping/formpage.cgi) and phylogenetic analysis with MEGA 4.0 software. Multiple clonal analyses of the samples were done as described previously from our laboratory [The amplicons were subjected to direct sequencing and the data were analyzed as previously described . HBV genboratory . For staAmong the 85 DNA positive subjects whose HBV RT sequences were determined, 70 (82.4%) were male. Of the treated patients the 12/30 was found to be HBeAg positive as compared to 28/55 HBeAg positive among the naive patients. Prevalent HBV genotypes were similar to that previously reported from eastern India. The main characteristics of the treated and naïve patients were compared in Table Whole genome sequences of a particular genotype retrieved from GenBank were used to produce a consensus sequence of the whole polymerase gene and were aligned with the study samples and different mutations and substitutions were analyzed. Three HBV genotypes were found, with a prevalence of HBV/A , HBV/C  and HBV/D . Compared to HBV/A and HBV/C genotypes, HBV/D has shown multiple different types of HBVrt mutation patterns (Putative NAr mutation and Pretreatment mutation) both among naïve and treated patients as 65.0% (13/20) and 56.2% (9/16) respectively  and 5 interdomains  ,13. AnalAmong the treated patients no primary antiviral drug resistant mutations, confirmed by in vitro studies as associated with resistance to NA (rtL80I/V-rtI169T-rtV173LrtL180M-rtA181T/V/S-rtT184A/S/G/C-rtA194T-rtS202C/G/I-rtM204V/I-rtN236T-rtM250V) were found. The prevalence of rtI91L was 80% (24/30) among them. Several other pretreatment mutations were also found in the treated patients  were detected neither among the therapy naïve patients nor among the non-responders to NA therapy. Although putative mutations widely found in treatment naive patients would suggest that they may not be wholly responsible for non-response to NA therapy; however, this needs confirmation by invitro and follow-up studies. Thus, routine use of resistance testing in patients before initiating antiviral therapy is not necessary at present.In conclusion, in our study classical antiviral resistance mutations (rtL80I/V-rtI169T-rtV173L-rtL180M-rtA181T/V/S-rtT184A/S/G/C-rtA194T-rtS202C/G/I-rtM204V/I-rtN236T-rtM250V) were not detected both in naïve patients and NA therapy non responders infected with any of the three HBV genotypes present. the prevalence of putative NAr mutations among the study subjects suggest that they might have role in reduced efficacy of currently available therapy, but their impact on antiviral efficacy requires further The authors declare that they have no competing interests.RP performed the majority of experiments; AB and RC were involved in experiment design; BKD, SC and RC participated in designing the study, preparation and editing the manuscript. All authors have read and approved the final manuscript.