Virulent bacteriophage of Edwardsiella ictaluri isolated from kidney and liver of striped catfish Pangasianodon hypophthalmus in Vietnam.

Striped catfish Pangasianodon hypophthalmus farmed in the Mekong Delta, Vietnam, represents an important contribution to Vietnamese aquaculture exports. However, these fish are affected by frequent disease outbreaks across the entire region. One of the most common infections involves white spots in the internal organs, caused by the bacterium Edwardsiella ictaluri. In this study, a virulent phage specific to E. ictaluri, designated MK7, was isolated from striped catfish kidney and liver samples and characterized. Morphological analysis indicates probable placement in the family Myoviridae with a 65 nm icosahedral head and a 147 %%CONV_ERR%% 19 nm tail. A double-stranded DNA genome of approximately 34 kb was predicted by restriction fragment analysis following digestion with SmaI. The adsorption affinity (ka) of the MK7 phage was estimated as 1.6 %%CONV_ERR%% 10-8 ml CFU-1 min-1, and according to a 1-step growth curve, its latent period and burst size were ~45 min and ~55 phage particles per infected host cell, respectively. Of the 17 bacterial strains tested, MK7 only infected E. ictaluri, although other species of Edwardsiella were not tested. E. ictaluri was also challenged in vitro, in both broth and water from a striped catfish pond and was inactivated by MK7 for 15 h in broth and 51 h in pond water. Thus, initial characterization of phage MK7 indicates its potential utility as a biotherapeutic agent against E. ictaluri infection in striped catfish. This is the first report of a lytic phage specific to an important striped catfish pathogen.

Impact of sitting at work on musculoskeletal complaints of German workers - results from the study on mental health at work (S-MGA).

INTRODUCTION: Sedentary behavior (including prolonged sitting) is a form of physical inactivity that has a negative impact on health, possibly including musculoskeletal complaints (MSCs). The purpose of this study was to determine the extent to which time spent sitting at work is associated with the one-year prevalence of MSCs in the neck, shoulder, upper back/thoracic spine, and lower back among workers from the Study of Mental Health in the Workplace (S-MGA). In addition, the study also examined whether leisure time, physical activity, and sex modify the relationship between occupational sitting and MSCs. METHODS: For this analysis, we used the S-MGA, a 5-year prospective study in Germany. The S-MGA is a nationwide representative employee cohort study with a baseline survey in 2012 and a follow-up survey in 2017. Sitting at work was measured using a question asked at baseline. The Nordic Musculoskeletal Questionnaire was used to determine the one-year prevalence of MSCs in the neck, shoulder, upper back, and lower back pain (yes/no). The assessment of MSCs was only conducted at the 2017 follow-up. Adjusted Poisson regression models were used to determine the association of baseline level of weekly hours spent sitting at work with MSCs during follow-up. In addition to unadjusted models, models were adjusted for demographic (age, sex, body mass index and occupational skill level), occupational (heavy lifting at work), psychological disorders and lifestyle factors (smoking status and leisure time physical activity), as well as preexisting musculoskeletal conditions reported at baseline. To examine whether the relationship between sitting time and pain was modified by sex and leisure time physical activity, the models were stratified for both these variables. RESULTS: Among the participants analyzed (n = 2,082), 49.8% were male, while 50.2% were female, and more than 60% of the study population spent over half of their working hours in a sitting position. Exposure to increased sitting at work reported at baseline was not consistently associated with 12-month prevalence of MSCs in the upper body at follow-up. However, differences in the association between occupational sitting and MSCs were dependent on the intensity of leisure time physical activity. Prevalence ratios (PRs) indicated an increased prevalence of MSC in the neck (PR = 1.46; 95% CI = 1.18-1.80) and shoulder (PR = 1.30; 95% CI = 1.03-1.64) in workers without leisure time physical activity who spent 25 to < 35 weekly working hours sitting. DISCUSSION: These findings suggest that leisure time physical activity interacts with the relationship between sitting at work and MSCs. The relationship between sitting at work and musculoskeletal pain needs further investigation, but we found indications that leisure time physical activity may counter the effects of sitting at work.

Bacteriophage PVN06 protected catfish Pangasianodon hypophthalmus from Edwardsiella ictaluri infection.

Bacillary necrosis of pangasius (BNP) is a disease caused by Edwardsiella ictaluri bacteria in striped catfish Pangasianodon hypophthalmus that results in high mortality rates. To control this disease, bacteriophages have been considered as alternatives to antibiotics. In this study, we applied the lytic bacteriophage PVN06 in striped catfish fingerlings to prevent E. ictaluri infection. In an experimental trial, the phage was administered to fish by feeding phage-coated feed with doses of 7.17±0.09, 8.17±0.09 and 9.17±0.09 log PFU/g feed per day before bacterial infection. Fish were infected by bacteria once with concentrations ranging from 3.01 to 7.01 log CFU/ml tank water. A day after infection, phage treatment resumed at a rate of once per day until the end of the trial. The results of the trial show that bacterial infection caused typical symptoms of BNP in fish with the cumulative fish death rate of 36.7±2.9 to 75.0±5.0%, depending on the bacterial concentration used for infection. Phage treatment with 9.17±0.09 log PFU/g significantly reduced the mortality rate, while treatments with 8.17±0.09 and 7.17±0.09 log PFU/g did not. This phage dose resulted in a 61.7-fold reduction in the toxicity of the bacterial pathogen and the survival rate of 15-23.3% in fish. Our study has demonstrated that the bacteriophage PVN06 protected striped catfish from BNP.

Novel inhibitors of surface layer processing in Clostridium difficile.

Clostridium difficile, a leading cause of hospital-acquired bacterial infection, is coated in a dense surface layer (S-layer) that is thought to provide both physicochemical protection and a scaffold for host-pathogen interactions. The key structural components of the S-layer are two proteins derived from a polypeptide precursor, SlpA, via proteolytic cleavage by the protease Cwp84. Here, we report the design, synthesis and in vivo characterization of a panel of protease inhibitors and activity-based probes (ABPs) designed to target S-layer processing in live C. difficile cells. Inhibitors based on substrate-mimetic peptides bearing a C-terminal Michael acceptor warhead were found to be promising candidates for further development.

Indel-II region deletion sizes in the white spot syndrome virus genome correlate with shrimp disease outbreaks in southern Vietnam.

Sequence comparisons of the genomes of white spot syndrome virus (WSSV) strains have identified regions containing variable-length insertions/deletions (i.e. indels). Indel-I and Indel-II, positioned between open reading frames (ORFs) 14/15 and 23/24, respectively, are the largest and the most variable. Here we examined the nature of these 2 indel regions in 313 WSSV-infected Penaeus monodon shrimp collected between 2006 and 2009 from 76 aquaculture ponds in the Mekong Delta region of Vietnam. In the Indel-I region, 2 WSSV genotypes with deletions of either 5950 or 6031 bp in length compared with that of a reference strain from Thailand (WSSV-TH-96-II) were detected. In the Indel-II region, 4 WSSV genotypes with deletions of 8539, 10970, 11049 or 11866 bp in length compared with that of a reference strain from Taiwan (WSSV-TW) were detected, and the 8539 and 10970 bp genotypes predominated. Indel-II variants with longer deletions were found to correlate statistically with WSSV-diseased shrimp originating from more intensive farming systems. Like Indel-I lengths, Indel-II lengths also varied based on the Mekong Delta province from which farmed shrimp were collected.

Activity-based probes: discovering new biology and new drug targets.

The development and application of chemical technologies enabling direct analysis of enzyme activity in living systems has undergone explosive growth in recent years. Activity-based protein profiling (ABPP) is a key constituent of this broad field, and is among the most powerful and mature chemical proteomic technologies. This tutorial review introduces the essential features of ABPP and the design and application of activity-based probes (ABPs) from drug target elucidation and in vivo visualisation of enzyme activity to comprehensive profiling of the catalytic content of living systems, and the discovery of new biological pathways.

Pathogenicity of gill-associated virus and Mourilyan virus during mixed infections of black tiger shrimp (Penaeus monodon).

Gill-associated virus (GAV) and Mourilyan virus (MoV) can occur at very high prevalence in healthy black tiger shrimp (Penaeus monodon) in eastern Australia, and both have been detected in moribund shrimp collected from mid-crop mortality syndrome (MCMS) outbreaks. Experimental evidence presented here indicates that GAV, but not MoV, is the cause of MCMS. Firstly, in healthy P. monodon used for experimental infections, pre-existing MoV genetic loads were very high (mean >10(9) viral RNA copies µg(-1) total RNA) and did not increase significantly following lethal challenge with an inoculum containing both GAV and MoV. In contrast, GAV genetic loads prior to challenge were low (mean ∼10(5) RNA copies µg(-1) total RNA) and increased >10(4)-fold in moribund shrimp. Secondly, dsRNAs targeted to the GAV RNA-dependent RNA polymerase (RdRp) or helicase gene regions reduced GAV genetic loads, delayed the onset of mortalities and improved survival following challenge. In contrast, dsRNA targeted to the MoV RdRp gene (L RNA) was highly effective in reducing MoV genetic loads, but mortality rates were unaffected. Targeting of the MoV S2 RNA, encoding a small non-structural protein (NSs2), a putative supressor of RNA interference, did not reduce the MoV genetic loads or enhance knockdown of GAV when administered simultaneously with dsRNA targeted to the GAV helicase gene. Overall, the data show that P. monodon can tolerate a high-level MoV infection and that mortalities are associated with GAV infection.

Mixed-genotype white spot syndrome virus infections of shrimp are inversely correlated with disease outbreaks in ponds.

Outbreaks of white spot syndrome virus (WSSV) in shrimp culture and the relationship between the virus and virulence are not well understood. Here, we provide evidence showing that WSSV mixed-genotype infections correlate with lower outbreak incidence and that disease outbreaks correlate with single-genotype infections. We tested 573 shrimp samples from 81 shrimp ponds in the Mekong delta with outbreak or non-outbreak status. The variable number tandem repeat (VNTR) loci of WSSV were used as molecular markers for the characterization of single- and mixed-genotype infections. The overall prevalence of mixed-genotype WSSV infections was 25.7 %. Non-outbreak ponds had a significantly higher frequency of mixed-genotype infections than outbreak ponds for all VNTR loci, both at the individual shrimp as well as at the pond level. The genetic composition of WSSV populations appears to correlate with the health status of shrimp culture in ponds. The causal relationship between genotypic diversity and disease outbreaks can now be experimentally approached.

Selection of Phages to Control Aeromonas hydrophila - An Infectious Agent in Striped Catfish.

Striped catfish (Pangasianodon hypophthalmus) farming in the Mekong Delta Vietnam (MKDVN) importantly contributes to national aquaculture export. Currently, however, diseases occur more frequently across the entire MKDVN region. One of the most common types is hemorrhagic septicemia caused by Aeromonas hydrophila. In this study, isolation and selection of the phages for control in vitro Aeromonas hydrophila were conducted. 24 phages were isolated from 100 striped catfish pond water samples. Next, lytic activity of these phages was clarified. Four phages with short latent period (about 25 to 40 min) and/or high burst size (about 67 to 94 PFU/ cell) were selected to evaluate their infection activity to different phage-resistant A. hydrophila strains. Two phages termed as TG25P and CT45P were subjected to the phage cocktail to inactivate A. hydrophila. Re-growth of the host bacteria appeared about eight hours after treatment. Usage of the phage cocktail that attach different host bacterial receptors is not always much effective than usage of single phage. This is the first report about phage therapy to control A. hydrophila isolated from striped catfish. Some challenges in the phage cocktail were shown to achieve strategies in prospective studies in the context of high antibiotic resistance of A. hydrophila.

Regioselective synthesis of functionalized 3,5-diketoesters and 2,4-diketosulfones by uncatalyzed condensation of 1-methoxy-1,3-bis(trimethylsilyloxy)-1,3-butadienes with alpha,beta-unsaturated acid chlorides and sulfonyl chlorides.

The reaction of 1-methoxy-1,3-bis(trimethylsilyloxy)-1,3-butadienes with alpha,beta-unsaturated and functionalized acid chlorides afforded a variety of 3,5-diketoesters which are not readily available by other methods. The reaction of 1-methoxy-1,3-bis(trimethylsilyloxy)-1,3-butadiene with sulfonyl chlorides allows a direct synthesis of 2,4-diketosulfones.

MR1-restricted mucosal-associated invariant T (MAIT) cells respond to mycobacterial vaccination and infection in nonhuman primates.

Studies on mucosal-associated invariant T cells (MAITs) in nonhuman primates (NHP), a physiologically relevant model of human immunity, are handicapped due to a lack of macaque MAIT-specific reagents. Here we show that while MR1 ligand-contact residues are conserved between human and multiple NHP species, three T-cell receptor contact-residue mutations in NHP MR1 diminish binding of human MR1 tetramers to macaque MAITs. Construction of naturally loaded macaque MR1 tetramers facilitated identification and characterization of macaque MR1-binding ligands and MAITs, both of which mirrored their human counterparts. Using the macaque MR1 tetramer we show that NHP MAITs activated in vivo in response to both Bacillus Calmette-Guerin vaccination and Mycobacterium tuberculosis infection. These results demonstrate that NHP and human MR1 and MAITs function analogously, and establish a preclinical animal model to test MAIT-targeted vaccines and therapeutics for human infectious and autoimmune disease.

Chemical probes of surface layer biogenesis in Clostridium difficile.

Clostridium difficile, a leading cause of hospital-acquired infection, possesses a dense surface layer (S-layer) that mediates host-pathogen interactions. The key structural components of the S-layer result from proteolytic cleavage of a precursor protein, SlpA, into high- and low-molecular-weight components. Here we report the discovery and optimization of the first inhibitors of this process in live bacteria and their application for probing S-layer processing. We also describe the design and in vivo application of activity-based probes that identify the protein Cwp84 as the cysteine protease that mediates SlpA cleavage. This work provides novel chemical tools for the analysis of S-layer biogenesis and for the potential identification of novel drug targets within clostridia and related bacterial pathogens.

A key gene of the RNA interference pathway in the black tiger shrimp, Penaeus monodon: identification and functional characterisation of Dicer-1.

RNA interference (RNAi) is an evolutionarily conserved mechanism by which double-stranded RNA (dsRNA) initiates post-transcriptional silencing of homologous genes. Here we report the amplification and characterisation of a full length cDNA from black tiger shrimp (Penaeus monodon) that encodes the bidentate RNAase III Dicer, a key component of the RNAi pathway. The full length of the shrimp Dicer (Pm Dcr1) cDNA is 7629bp in length, including a 5' untranslated region (UTR) of 130bp, a 3' UTR of 77bp, and an open reading frame of 7422bp encoding a polypeptide of 2473 amino acids with an estimated molecular mass of 277.895kDa and a predicted isoelectric point of 4.86. Analysis of the deduced amino acid sequence indicated that the mature peptide contains all the seven recognised functional domains and is most similar to the mosquito (Aedes aegypti) Dicer-1 sequence with a similarity of 34.6%. Quantitative RT-PCR analysis showed that Pm Dcr1 mRNA is most highly expressed in haemolymph and lymphoid organ tissues (P<0.05). However, there was no correlation between Pm Dcr1 mRNA levels in lymphoid organ and the viral genetic loads in shrimp naturally infected with gill-associated virus (GAV) and Mourilyan virus (P>0.05). Treatment with synthetic dsRNA corresponding to Pm Dcr1 sequence resulted in knock-down of Pm Dcr1 mRNA expression in both uninfected shrimp and shrimp infected experimentally with GAV. Knock-down of Pm Dcr1 expression resulted in more rapid mortalities and higher viral loads. These data demonstrated that Dicer is involved in antiviral defence in shrimp.

Efficient synthesis of salicylates by catalytic [3 + 3] cyclizations of 1,3-bis(silyl enol ethers) with 1,1,3,3-tetramethoxypropane.

Salicylic acid derivatives were prepared by Me3SiOTf-catalyzed [3 + 3] cyclization of 1,3-bis(silyl enol ethers) with 1,1,3,3-tetramethoxypropane.

Synthesis of 1,3,5-tricarbonyl derivatives by condensation of 1,3-bis(silyl enol ethers) with acid chlorides.

A variety of 1,3,5-tricarbonyl derivatives were prepared by reaction of 1,3-bis(silyl enol ethers) with acid chlorides under mild conditions. This includes reactions of both aromatic and aliphatic acid chlorides and bis(acid chlorides). The yields vary depending on the type of acid chloride employed.

Nuclear forward scattering for high energy mössbauer transitions.

We have studied nuclear forward scattering of synchrotron radiation for the 67.41 keV resonance of 61Ni using a silicon crystal monochromator with low-index reflections and a multielement detector. This approach can be extended to other high-energy Mössbauer transitions and does not pose any restrictions on the sample environment. Under conditions of large sample thickness and short nuclear lifetime, typical for work with high-energy nuclear resonances, the nuclear decay follows a universal dependence where both thickness effects and hyperfine interactions are taken into account by time scaling.

Effect of inhaled preservatives on asthmatic subjects. II. Benzalkonium chloride.

Benzalkonium chloride has been used as a preservative in some antiasthma respirator solutions and is known to cause bronchoconstriction in asthmatic subjects. To increase understanding of how it causes bronchoconstriction, the characteristics of airway response in 28 asthmatic subjects were documented. Subjects inhaled histamine, in doses ranging from 0.03 to 7.8 mumol, or benzalkonium in doses ranging from 0.04 to 5.33 mumol on separate days. The dose of histamine or benzalkonium that caused a 20% fall in the 1-s forced expiratory volume (PD20FEV1) was measured. All subjects responded to histamine, with PD20FEV1 values in the range of 0.14 to 7.8 mumol and 17 responded to benzalkonium, with PD20FEV1 values in the range 0.35 to 5.55 mumol. Subjects who responded to benzalkonium were more sensitive to histamine (mean PD20FEV1 0.44 mumol) than subjects who did not respond (mean PD20FEV1 1.84 mumol) and, among the benzalkonium responders, there was a significant correlation between PD20FEV1 values for histamine and benzalkonium (r = 0.5, p less than 0.05). Inhalation of benzalkonium enhanced subsequent responses to histamine, causing a decrease in mean PD20FEV1 from 0.51 to 0.18 mumol histamine (p less than 0.001), but did not alter subsequent responses to benzalkonium. The response to benzalkonium reached a maximum 1 min after inhalation and was slow to recover, taking up to 60 min to return to baseline values. Response to benzalkonium was inhibited by 8 mg cromolyn sodium but not by 160 micrograms ipratropium bromide. The characteristics of the response to benzalkonium suggest a mechanism of action via release of mediators.