Baseline susceptibility of Asian corn borer (Ostrinia furnacalis (Guenée)) populations in Vietnam to Cry1Ab insecticidal protein

Susceptibility of Asian corn borer (ACB), Ostrinia furnacalis (Guenée) to Bacillus thuringiensis (Bt) Cry1Ab protein was studied between 2015 and 2016 with 11 ACB populations, collected from various geographical regions in Vietnam. A concentration range of Cry1Ab from 0.20 to 26.10 ng/cm² of diet was evaluated against F₁ ACB neonates using diet surface-overlay bioassays. Mortality data was recorded daily until seven days after infestation. Growth inhibition was recorded at the end of seven days. The median lethal concentration (LC₅₀) varied ≈3-fold among the different populations, ranging from 0.58 to 1.83 ng/cm² of diet with an overall mean of 0.86 ng/cm² of diet. Even the lowest concentration of 0.20 ng/cm² caused 73.53% growth inhibition. >90% growth inhibition was achieved at 0.82 ng/cm² or higher concentrations. The results reflect natural variation in Bt susceptibility among ACB populations rather than variation caused by prior exposure to selection pressures. LC₉₉ value (17.26 ng/cm²) was generated by pooling mortality data across different populations. The upper fiducial limit of LC₉₉ (24.38 ng/cm²) could be a potential diagnostic dose for future resistance monitoring programs. The findings from this study suggest that ACB populations in Vietnam are highly susceptible to Cry1Ab protein. This is the first report of Cry1Ab susceptibility of different ACB populations in Vietnam and will serve as a baseline for future resistance monitoring work.     

Effects of isoleucine 135 side chain length on the cofactor donor-acceptor distance within F420H2:NADP+ oxidoreductase: A kinetic analysis

F₄₂₀H₂:NADP⁺ Oxidoreductase (Fno) catalyzes the reversible reduction of NADP⁺ to NADPH by transferring a hydride from the reduced F₄₂₀ cofactor. Here, we have employed binding studies, steady-state and pre steady-state kinetic methods upon wtFno and isoleucine 135 (I135) Fno variants in order to study the effects of side chain length on the donor-acceptor distance between NADP⁺ and the F₄₂₀ precursor, FO. The conserved I135 residue of Fno was converted to a valine, alanine and glycine, thereby shortening the side chain length. The steady-state kinetic analysis of wtFno and the variants showed classic Michaelis-Menten kinetics with varying FO concentrations. The data revealed a decreased k_cat as side chain length decreased, with varying FO concentrations. The steady-state plots revealed non-Michaelis-Menten kinetic behavior when NADPH was varied. The double reciprocal plot of the varying NADPH concentrations displays a downward concave shape, while the NADPH binding curves gave Hill coefficients of less than 1. These data suggest that negative cooperativity occurs between the two identical monomers. The pre steady-state Abs₄₂₀ versus time trace revealed biphasic kinetics, with a fast phase (hydride transfer) and a slow phase. The fast phase displayed an increased rate constant as side chain length decreased. The rate constant for the second phase, remained ~2 s^?1 for each variant. Our data suggest that I135 plays a key role in sustaining the donor-acceptor distance between the two cofactors, thereby regulating the rate at which the hydride is transferred from FOH₂ to NADP⁺. Therefore, Fno is a dynamic enzyme that regulates NADPH production.     

Pyruvate Fermentation by Oenococcus oeni and Leuconostoc mesenteroides and Role of Pyruvate Dehydrogenase in Anaerobic Fermentation

The heterofermentative lactic acid bacteria Oenococcus oeni and Leuconostoc mesenteroides are able to grow by fermentation of pyruvate as the carbon source (2 pyruvate → 1 lactate + 1 acetate + 1 CO₂). The growth yields amount to 4.0 and 5.3 g (dry weight)/mol of pyruvate, respectively, suggesting formation of 0.5 mol ATP/mol pyruvate. Pyruvate is oxidatively decarboxylated by pyruvate dehydrogenase to acetyl coenzyme A, which is then converted to acetate, yielding 1 mol of ATP. For NADH reoxidation, one further pyruvate molecule is reduced to lactate. The enzymes of the pathway were present after growth on pyruvate, and genome analysis showed the presence of the corresponding structural genes. The bacteria contain, in addition, pyruvate oxidase activity which is induced under microoxic conditions. Other homo- or heterofermentative lactic acid bacteria showed only low pyruvate fermentation activity.     

Empirical profile mixture models for phylogenetic reconstruction

MOTIVATION: Previous studies have shown that accounting for site-specific amino acid replacement patterns using mixtures of stationary probability profiles offers a promising approach for improving the robustness of phylogenetic reconstructions in the presence of saturation. However, such profile mixture models were introduced only in a Bayesian context, and are not yet available in a maximum likelihood (ML) framework. In addition, these mixture models only perform well on large alignments, from which they can reliably learn the shapes of profiles, and their associated weights. RESULTS: In this work, we introduce an expectation-maximization algorithm for estimating amino acid profile mixtures from alignment databases. We apply it, learning on the HSSP database, and observe that a set of 20 profiles is enough to provide a better statistical fit than currently available empirical matrices (WAG, JTT), in particular on saturated data.     

A Novel Glucosyltransferase Involved in O-Antigen Modification of Shigella flexneri Serotype 1c

The O antigen of serotype 1c differs from the unmodified O antigen of serotype Y by the addition of a disaccharide (two glucosyl groups) to the tetrasaccharide repeating unit. It was shown here that addition of the first glucosyl group is mediated by the previously characterized gtrI cluster, which is found within a cryptic prophage at the proA locus in the bacterial chromosome. Transposon mutagenesis was performed to disrupt the gene responsible for addition of the second glucosyl group, causing reversion to serotype 1a. Colony immunoblotting was used to identify the desired revertants, and subsequent sequencing, cloning, and functional expression successfully identified the gene encoding serotype 1c-specific O-antigen modification. This gene (designated gtrIC) was present as part of a three-gene cluster, similar to other S. flexneri glucosyltransferase genes. Relative to the other S. flexneri gtr clusters, the gtrIC cluster is more distantly related and appears to have arrived in S. flexneri from outside the species. Analysis of surrounding sequence suggests that the gtrIC cluster arrived via a novel bacteriophage that was subsequently rendered nonfunctional by a series of insertion events.Shigella flexneri is a pathovar of Escherichia coli that is the main causative agent of endemic bacillary dysentery (shigellosis). It is estimated that S. flexneri is responsible for approximately 100 million shigellosis cases annually, resulting in hundreds of thousands of deaths, predominantly in young children (11). Currently no vaccine is available, although there is evidence to suggest that serotype-specific immunity occurs following infection and that induction of immunity can be replicated with vaccines (9). Shigella serotype diversity arises due to differences in the chemical structure of the O-antigen repeating unit in the lipopolysaccharide, which is the main target of the adaptive host immune response following infection.Because immunity to S. flexneri can be conferred by the induction of antibodies directed against the O antigen, an understanding of the prevalence of different serotypes and the underlying basis of serotype diversity can inform appropriate vaccine design. All S. flexneri serotypes (with the exception of serotype 6) share a common O-antigen backbone, consisting of a repeating tetrasaccharide unit that is comprised of one N-acetylglucosamine residue (GlcNAc) and three rhamnose residues (RhaI, RhaII, and RhaIII) (14). The 12 traditionally recognized S. flexneri serotypes differ by the presence or absence of just six different chemical modifications (glucosylations or O acetylations) of the O antigen. The genes responsible for these O-antigen modifications are introduced into the bacterial genome via bacteriophages (3). Glucosylation of the S. flexneri O antigen is mediated by three genes [gtrA, gtrB, and gtr(type)] that are arranged in a single operon known as a gtr cluster. gtrA and gtrB are highly conserved between different gtr clusters and encode proteins involved in transferring the glucosyl group from the cytoplasm into the periplasm, where O-antigen modification is thought to take place. gtr(type) is unique to each gtr cluster and encodes a glucosyltransferase that is responsible for attaching the glucosyl group to a specific sugar unit of the O antigen via a specific linkage (3).Investigations of S. flexneri have typically focused on serotypes for which commercially available typing sera are available. More recently, it has become clear that other serotypes are also epidemiologically important. In Bangladesh in the late 1980s, two novel S. flexneri strains that did not agglutinate with antibodies specific for the traditionally recognized serotypes were isolated (4). Chemical analysis of the O antigen revealed that these strains belonged to a new serotype, which was named serotype 1c due to the similarity its O antigen shares with the O antigens of serotype 1a and 1b strains (19). Serotype 1c has since been isolated in Egypt, Indonesia, Pakistan, and Vietnam (6, 15, 18). Serotype 1c was shown to be the most prevalent S. flexneri serotype in a northern province of Vietnam, accounting for more than a third of all S. flexneri strains isolated from 1998 to 1999 (15). Identification of serotype 1c currently relies on agglutination testing using monoclonal antibody MASF Ic (19).The O antigen of serotype 1c is distinguished by the presence of a disaccharide (two glucosyl groups) linked to the GlcNAc in the tetrasaccharide repeating unit of the O antigen. The first glucosyl group is joined to GlcNAc via an α1→4 linkage, as occurs in the O antigen of serotype 1a and serotype 1b strains (type I modification). The O antigen of serotype 1c is distinguished by the presence of a second glucosyl group that is linked to the first via an α1→2 linkage (Fig. ​(Fig.1).1). Type Ia modification is prerequisite to type Ic modification.Open in a separate windowFIG. 1.Chemical structure of the tetrasaccharide repeat units in the O antigens of S. flexneri serotypes 1a and 1c. Note that the O antigen of serotype 1b (not shown) differs from that of serotype 1a by the O acetylation of l-RhaIII.In this study, the genetic basis of O-antigen modification in serotype 1c was elucidated. Serotype 1c strains isolated from different locations and times were compared to gain insight into the evolution of this serotype. This is the first report of the identification of a glucosyltransferase gene that is responsible for addition of the second glucosyl group, causing serotype conversion from serotype 1a to serotype 1c.     

Multilocus variable-number tandem repeat analysis for molecular typing and phylogenetic analysis of Shigella flexneri

Background

Enteropathogenic Escherichia coli (EPEC) produce attaching/effacing (A/E) lesions on eukaryotic cells mediated by the outer membrane adhesin intimin. EPEC are sub-grouped into typical (tEPEC) and atypical (aEPEC). We have recently demonstrated that aEPEC strain 1551-2 (serotype O non-typable, non-motile) invades HeLa cells by a process dependent on the expression of intimin sub-type omicron. In this study, we evaluated whether aEPEC strains expressing other intimin sub-types are also invasive using the quantitative gentamicin protection assay. We also evaluated whether aEPEC invade differentiated intestinal T84 cells.

Results

Five of six strains invaded HeLa and T84 cells in a range of 13.3%–20.9% and 5.8%–17.8%, respectively, of the total cell-associated bacteria. The strains studied were significantly more invasive than prototype tEPEC strain E2348/69 (1.4% and 0.5% in HeLa and T84 cells, respectively). Invasiveness was confirmed by transmission electron microscopy. We also showed that invasion of HeLa cells by aEPEC 1551-2 depended on actin filaments, but not on microtubules. In addition, disruption of tight junctions enhanced its invasion efficiency in T84 cells, suggesting preferential invasion via a non-differentiated surface.

Conclusion

Some aEPEC strains may invade intestinal cells in vitro with varying efficiencies and independently of the intimin sub-type.     

Stacking of G-quadruplexes: NMR structure of a G-rich oligonucleotide with potential anti-HIV and anticancer activity

G-rich oligonucleotides T30695 (or T30923), with the sequence of (GGGT)(4), and T40214, with the sequence of (GGGC)(4), have been reported to exhibit anti-HIV and anticancer activity. Here we report on the structure of a dimeric G-quadruplex adopted by a derivative of these sequences in K(+) solution. It comprises two identical propeller-type parallel-stranded G-quadruplex subunits each containing three G-tetrad layers that are stacked via the 5'-5' interface. We demonstrated control over the stacking of the two monomeric subunits by sequence modifications. Our analysis of possible structures at the stacking interface provides a general principle for stacking of G-quadruplexes, which could have implications for the assembly and recognition of higher-order G-quadruplex structures.     

Transient infection of the zebrafish notochord with E. coli induces chronic inflammation

Zebrafish embryos and larvae are now well-established models in which to study infectious diseases. Infections with non-pathogenic Gram-negative Escherichia coli induce a strong and reproducible inflammatory response. Here, we study the cellular response of zebrafish larvae when E. coli bacteria are injected into the notochord and describe the effects. First, we provide direct evidence that the notochord is a unique organ that is inaccessible to leukocytes (macrophages and neutrophils) during the early stages of inflammation. Second, we show that notochord infection induces a host response that is characterised by rapid clearance of the bacteria, strong leukocyte recruitment around the notochord and prolonged inflammation that lasts several days after bacteria clearance. During this inflammatory response, il1b is first expressed in macrophages and subsequently at high levels in neutrophils. Moreover, knock down of il1b alters the recruitment of neutrophils to the notochord, demonstrating the important role of this cytokine in the maintenance of inflammation in the notochord. Eventually, infection of the notochord induces severe defects of the notochord that correlate with neutrophil degranulation occurring around this tissue. This is the first in vivo evidence that neutrophils can degranulate in the absence of a direct encounter with a pathogen. Persistent inflammation, neutrophil infiltration and restructuring of the extracellular matrix are defects that resemble those seen in bone infection and in some chondropathies. As the notochord is a transient embryonic structure that is closely related to cartilage and bone and that contributes to vertebral column formation, we propose infection of the notochord in zebrafish larvae as a new model to study the cellular and molecular mechanisms underlying cartilage and bone inflammation.KEY WORDS: Zebrafish, Neutrophils, Inflammation, Interleukin-1β     

Dosage and Cell Line Dependent Inhibitory Effect of bFGF Supplement in Human Pluripotent Stem Cell Culture on Inactivated Human Mesenchymal Stem Cells

Many different culture systems have been developed for expanding human pluripotent stem cells (hESCs and hiPSCs). In general, 4–10 ng/ml of bFGF is supplemented in culture media in feeder-dependent systems regardless of feeder cell types, whereas in feeder-free systems, up to 100 ng/ml of bFGF is required for maintaining long-term culture on various substrates. The amount of bFGF required in native hESCs growth niche is unclear. Here we report using inactivated adipose-derived human mesenchymal stem cells as feeder cells to examine long-term parallel cultures of two hESCs lines (H1 and H9) and one hiPSCs line (DF19-9-7T) in media supplemented with 0, 0.4 or 4 ng/ml of bFGF for up to 23 passages, as well as parallel cultures of H9 and DF19 in media supplemented with 4, 20 or 100 ng/ml bFGF for up to 13 passages for comparison. Across all cell lines tested, bFGF supplement demonstrated inhibitory effect over growth expansion, single cell colonization and recovery from freezing in a dosage dependent manner. In addition, bFGF exerted differential effects on different cell lines, inducing H1 and DF19 differentiation at 4 ng/ml or higher, while permitting long-term culture of H9 at the same concentrations with no apparent dosage effect. Pluripotency was confirmed for all cell lines cultured in 0, 0.4 or 4 ng/ml bFGF excluding H1-4 ng, as well as H9 cultured in 4, 20 and 100 ng/ml bFGF. However, DF19 demonstrated similar karyotypic abnormality in both 0 and 4 ng/ml bFGF media while H1 and H9 were karyotypically normal in 0 ng/ml bFGF after long-term culture. Our results indicate that exogenous bFGF exerts dosage and cell line dependent effect on human pluripotent stem cells cultured on mesenchymal stem cells, and implies optimal use of bFGF in hESCs/hiPSCs culture should be based on specific cell line and its culture system.