Highly Specific Culture-Independent Detection of YGNGV Motif-Containing Pediocin-Producing Strains

A novel method based on (1) initial microbiological screening and (2) a highly specific PCR is described for selection of strains expressing YGNGV motif-containing pediocin. Initial screening is carried out using spot on the lawn assay for selection of acid-free, hydrogen peroxide (H₂O₂)-free and secreted heat-stable inhibitory activity producing strains. This is followed by highly specific PCR for amplification of 406-bp fragment using forward primer: 5′-tggccaatatcattggtggt-3′ targeting signal peptide sequence of pediocin structural gene and reverse primer: 5′-ctactaacgcttggctggca-3′ encoding N-terminus of immunity gene. The assay was validated with Pediococcus pentosaceus NCDC273 and Pediococcus acidilactici NCDC252 using (1) digestion of amplified 406-bp fragment with HindIII restriction enzyme-producing two restriction fragments of expected sizes (227 and 179 bp), (2) nucleotide sequencing of 406-bp fragment from both strains found these pediocins identical to pediocin PA-1/AcH and (3) identification of both pediocins as pediocin PA-1 at protein level using RP-HPLC. The assay was used for screening six strains (3 pediococci, 2 lactobacilli and an Enterococcus faecium) producing acid-free, hydrogen peroxide (H₂O₂)-free and secreted heat-stable inhibitory activity. This resulted in the detection of three new strains (P. pentosaceus NCDC35, E. faecium NCDC124 and Lactobacillus plantarum NCDC20) producing YGNGV motif-containing pediocins.     

AUDIT,AUDIT-C,and AUDIT-3: Drinking Patterns and Screening for Harmful,Hazardous and Dependent Drinking in Katutura,Namibia

Objectives

To describe alcohol drinking patterns among participants in Katutura, Namibia, and to evaluate brief versions of the AUDIT against the full AUDIT to determine their effectiveness in detecting harmful drinking.

Methods

A cross-sectional survey was conducted in four constituencies and 639 participants, 18 years or older, completed a sociodemographic survey and the AUDIT. The effectiveness of the AUDIT-C (first three questions) and the AUDIT-3 (third question) was compared to the full AUDIT.

Results

Approximately 40% were identified as harmful, hazardous or likely dependent drinkers, with men having a higher likelihood than women (57.2% vs. 31.0%, p<.0001). Approximately 32% reported making and/or selling alcohol from home. The AUDIT-C performed best at a cutoff ≥ 3, better in men (sensitivity: 99.3%, specificity: 77.8%) than women (sensitivity: 91.7%, specificity: 77.4%). The AUDIT-3 performed poorly (maximum sensitivity: < 90%, maximum specificity: <51%). According to AUROC, the AUDIT-C performed better than the AUDIT-3.

Conclusions

A large proportion of participants met criteria for alcohol misuse, indicating a need for screening and referral for further evaluation and intervention. The AUDIT-C was almost as effective as the full AUDIT and may be easier to implement in clinical settings as a routine screening tool in resource-limited settings because of its brevity.     

Apo and Calcium-Bound Crystal Structures of Cytoskeletal Protein Alpha-14 Giardin (Annexin E1) from the Intestinal Protozoan Parasite Giardia lamblia

Alpha-14 giardin (annexin E1), a member of the alpha giardin family of annexins, has been shown to localize to the flagella of the intestinal protozoan parasite Giardia lamblia. Alpha giardins show a common ancestry with the annexins, a family of proteins most of which bind to phospholipids and cellular membranes in a Ca²⁺-dependent manner and are implicated in numerous membrane-related processes including cytoskeletal rearrangements and membrane organization. It has been proposed that alpha-14 giardin may play a significant role during the cytoskeletal rearrangement during differentiation of Giardia. To gain a better understanding of alpha-14 giardin's mode of action and its biological role, we have determined the three-dimensional structure of alpha-14 giardin and its phospholipid-binding properties. Here, we report the apo crystal structure of alpha-14 giardin determined in two different crystal forms as well as the Ca²⁺-bound crystal structure of alpha-14 giardin, refined to 1.9, 1.6 and 1.65 Å, respectively. Although the overall fold of alpha-14 giardin is similar to that of alpha-11 giardin, multiwavelength anomalous dispersion phasing was required to solve the alpha-14 giardin structure, indicating significant structural differences between these two members of the alpha giardin family. Unlike most annexin structures, which typically possess N-terminal domains, alpha-14 giardin is composed of only a core domain, followed by a C-terminal extension that may serve as a ligand for binding to cytoskeletal protein partners in Giardia. In the Ca²⁺-bound structure we detected five bound calcium ions, one of which is a novel, highly coordinated calcium-binding site not previously observed in annexin structures. This novel high-affinity calcium-binding site is composed of seven protein donor groups, a feature rarely observed in crystal structures. In addition, phospholipid-binding assays suggest that alpha-14 giardin exhibits calcium-dependent binding to phospholipids that coordinate cytoskeletal disassembly/assembly during differentiation of the parasite.     

The alternative splice variant of DAPK-1, s-DAPK-1, induces proteasome-independent DAPK-1 destabilization

Death-associated protein kinase 1 (DAPK-1) is a Ca²⁺/CaM-regulated kinase involved in multiple cellular signalling pathways that trigger cell survival, apoptosis, and autophagy. An alternatively spliced product expressed from the dapk1 locus, named s-DAPK-1, does not contain the kinase domain but has part of the DAPK-1 ankyrin-repeat and a novel polypeptide tail extension which is processed proteolytically in vivo. Cleavage of this polypeptide tail from s-DAPK-1 can regulate the ability of the protein to mimic one of the biological functions of DAPK-1 in promoting membrane blebbing. The full-length DAPK-1 protein is a relatively long-lived protein whose half-life is regulated by stress-activated signals from TNFR1 or HSP90 that can promote DAPK-1 protein degradation. Transfection of s-DAPK-1 into cells can also have a direct effect on DAPK-1 protein itself by promoting DAPK-1 de-stabilization. This effect does not require the novel polypeptide tail-extension of s-DAPK-1, as the core ankyrin-repeat containing region of s-DAPK-1 is sufficient to promote DAPK-1 protein de-stabilization. Conversely, the minimal domain on full-length DAPK-1 that responds to the effect of s-DAPK-1 is not the ankyrin-repeat domain but the core kinase domain of DAPK-1. The de-stabilization of DAPK-1 by s-DAPK-1 is not dependent upon the proteasome. However, s-DAPK-1 itself is a very short-lived protein which is regulated by a proteasomal-dependent pathway. Together, these data identify a novel function of s-DAPK-1 in controlling the half-life of DAPK-1 protein itself and indicate that the degradation of each gene product is controlled by two distinct degradation pathways.     

Evolving possible link between PI3K and NO pathways in neuroprotective mechanism of ischemic postconditioning in mice

The present study was conducted to pharmacologically investigate the influence of NO signaling pathway in PI3K mediated neuroprotective mechanism of ischemic postconditioning (iPoCo). Bilateral carotid artery occlusion (BCAO) of 12 min followed by reperfusion for 24 h was employed to produce ischemia/reperfusion-induced cerebral injury in male Swiss mice. Memory was assessed using Morris water maze test. Degree of motor incoordination was evaluated using inclined beam walk test, rotarod test, and lateral push test. Cerebral infarct size was measured using triphenyltetrazolium chloride staining. Brain acetylcholinesterase activity, thiobarbituric acid reactive species (TBARS), nitrite/nitrate and reduced glutathione (GSH) levels were also estimated. BCAO followed by reperfusion produced significant rise in cerebral infarct size, acetylcholinesterase activity, and TBARS levels along with fall in nitrite/nitrate and GSH levels. A significant impairment of memory and motor coordination was also noted. iPoCo consisting of three episodes of 10 s carotid artery occlusion and reperfusion significantly attenuated infarct size, memory impairment, motor incoordination, and altered biochemicals. iPoCo-induced neuroprotective effects were significantly abolished by wortmannin (a selective PI3K inhibitor). However, administration of l-Arginine (a NO precursor) in the presence of wortmannin restored the protective effect of ischemic postconditioning. It may be concluded that neuroprotective mechanism of iPoCo involves PI3K-mediated pathway with NO signaling as an important downstream step.     

Safety and Immunogenicity of a Live Oral Recombinant Cholera Vaccine VA1.4: A Randomized,Placebo Controlled Trial in Healthy Adults in a Cholera Endemic Area in Kolkata,India

Background

A live oral cholera vaccine VA 1.4 developed from a non-toxigenic Vibrio cholerae O1 El Tor strain using ctxB gene insertion was further developed into a clinical product following cGMP and was evaluated in a double-blind randomized placebo controlled parallel group two arm trial with allocation ratio of 1∶1 for safety and immunogenicity in men and women aged 18–60 years from Kolkata, India.

Method

A lyophilized dose of 1.9×10⁹ CFU (n = 44) or a placebo (n = 43) reconstituted with a diluent was administered within 5 minutes of drinking 100 ml of a buffer solution made of sodium bicarbonate and ascorbic acid and a second dose on day 14.

Result

The vaccine did not elicit any diarrhea related adverse events. Other adverse events were rare, mild and similar in two groups. One subject in the vaccine group excreted the vaccine strain on the second day after first dose. The proportion of participants who seroconverted (i.e. had 4-folds or higher rise in reciprocal titre) in the vaccine group were 65.9% (95% CI: 50.1%–79.5%) at both 7 days (i.e. after 1^st dose) and 21 days (i.e. after 2^nd dose). None of the placebo recipients seroconverted. Anti-cholera toxin antibody was detected in very few recipients of the vaccine.

Conclusion

This study demonstrates that VA 1.4 at a single dose of 1.9×10⁹ is safe and immunogenic in adults from a cholera endemic region. No additional benefit after two doses was seen.

Trial Registration

Clinical Trials Registry-India, National Institute of Medical Statistics (Indian Council of Medical Research) CTRI/2012/04/002582     

HS‐SPME‐GC‐FID method for detection and quantification of Bacillus cereus ATCC 10702 mediated 2‐acetyl‐1‐pyrroline

A rapid micro‐scale solid‐phase micro‐extraction (SPME) procedure coupled with gas‐chromatography with flame ionized detector (GC‐FID) was used to extract parts per billion levels of a principle basmati aroma compound “2‐acetyl‐1‐pyrroline” (2‐AP) from bacterial samples. In present investigation, optimization parameters of bacterial incubation period, sample weight, pre‐incubation time, adsorption time, and temperature, precursors and their concentrations has been studied. In the optimized conditions, detection of 2‐AP produced by Bacillus cereus ATCC10702 using only 0.5 g of sample volume was 85 μg/kg. Along with 2‐AP, 15 other compounds produced by B. cereus were also reported out of which 14 were reported for the first time consisting mainly of (E)?2‐hexenal, pentadecanal, 4‐hydroxy‐2‐butanone, n‐hexanal, 2–6‐nonadienal, 3‐methoxy‐2(5H) furanone and 2‐acetyl‐1‐pyridine and octanal. High recovery of 2‐AP (87 %) from very less amount of B. cereus samples was observed. The method is reproducible fast and can be used for detection of 2‐AP production by B. cereus. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1356–1363, 2014     

Age-related differences in polyfunctional T cell responses

Background

A reduced number of naïve T cells along with an accumulation of differentiated cell types in aging have been described but little is known about the polyfunctionality of the T cell responses. In this study we compared the individual and polyfunctional expression of IFN-γ, MIP-1α, TNF-α, perforin, and IL-2 by T cell subsets, including the newly described stem cell like memory T cells (T_SCM), in response to stimulation with superantigen staphylococcal enterotoxin B (SEB) in older (median age 80, n?=?23) versus younger (median age 27; n?=?23) adults.

Results

Older age was associated with a markedly lower frequency of CD8+ naïve T cells (11% vs. 47%; p?<?0.0001) and an expansion in memory T cell subsets including central memory (p?<?0.05), effector memory and effector T cells (p?<?0.001 for both). There was also a decline in CD4+ naïve T cells in older subjects (33% vs. 45%; p?=?0.02). There were no differences in frequencies or polyfunctional profiles of T_SCM between groups. CD8+ naïve cells in the older group had increased expression of all functional parameters measured compared to the younger subjects and exhibited greater polyfunctionality (p?=?0.04). CD4+ naïve T cells in the older group also showed greater polyfunctionality with a TNF-α and IL-2 predominance (p?=?0.005). CD8+ effector memory and effector T cells exhibited increased polyfunctionality in the older group compared with younger (p?=?0.01 and p?=?0.003).

Conclusions

These data suggest that aging does not have a negative effect on polyfunctionality and therefore this is likely not a major contributor to the immunesenescence described with aging.