First-time characterization of viable but non-culturable Proteus mirabilis: Induction and resuscitation

Pathogenic bacteria can enter into a viable but non-culturable (VBNC) state under unfavourable conditions. Proteus mirabilis is responsible for dire clinical consequences including septicaemia, urinary tract infections and pneumonia, but is not a species previously known to enter VBNC state. We suggested that stress-induced P. mirabilis can enter a VBNC state in which it retains virulence. P. mirabilis isolates were incubated in extreme osmotic pressure, starvation, low temperature and low pH to induce a VBNC state. Resuscitation was induced by temperature upshift and inoculation in tryptone soy broth with Tween 20 and brain heart infusion broth. Cellular ultrastructure and gene expression were examined using transmission electron microscopy (TEM) and quantitative real-time polymerase chain reaction (qPCR), respectively. High osmotic pressure and low acidity caused rapid entry into VBNC state. Temperature upshift caused the highest percentage of resuscitation (93%) under different induction conditions. In the VBNC state, cells showed aberrant and dwarf morphology, virulence genes and stress response genes (envZ and rpoS) were expressed (levels varied depending on strain and inducing factors). This is the first-time characterization of VBNC P. mirabilis. The ability of P. mirabilis pathogenic strains to enter a stress-induced VBNC state can be a serious public health threat.     

Individual Participant Data Meta-Analysis for a Binary Outcome: One-Stage or Two-Stage?

Background

A fundamental aspect of epidemiological studies concerns the estimation of factor-outcome associations to identify risk factors, prognostic factors and potential causal factors. Because reliable estimates for these associations are important, there is a growing interest in methods for combining the results from multiple studies in individual participant data meta-analyses (IPD-MA). When there is substantial heterogeneity across studies, various random-effects meta-analysis models are possible that employ a one-stage or two-stage method. These are generally thought to produce similar results, but empirical comparisons are few.

Objective

We describe and compare several one- and two-stage random-effects IPD-MA methods for estimating factor-outcome associations from multiple risk-factor or predictor finding studies with a binary outcome. One-stage methods use the IPD of each study and meta-analyse using the exact binomial distribution, whereas two-stage methods reduce evidence to the aggregated level (e.g. odds ratios) and then meta-analyse assuming approximate normality. We compare the methods in an empirical dataset for unadjusted and adjusted risk-factor estimates.

Results

Though often similar, on occasion the one-stage and two-stage methods provide different parameter estimates and different conclusions. For example, the effect of erythema and its statistical significance was different for a one-stage (OR = 1.35, ) and univariate two-stage (OR = 1.55, ). Estimation issues can also arise: two-stage models suffer unstable estimates when zero cell counts occur and one-stage models do not always converge.

Conclusion

When planning an IPD-MA, the choice and implementation (e.g. univariate or multivariate) of a one-stage or two-stage method should be prespecified in the protocol as occasionally they lead to different conclusions about which factors are associated with outcome. Though both approaches can suffer from estimation challenges, we recommend employing the one-stage method, as it uses a more exact statistical approach and accounts for parameter correlation.     

Robust post-translocational N-glycosylation at the extreme C-terminus of membrane and secreted proteins in Xenopus laevis oocytes and HEK293 cells

N-Glycosylation is normally a co-translational process that occurs as soon as a nascent and unfolded polypeptide chain has emerged ~12 residues into the lumen of the endoplasmic reticulum (ER). Here, we describe the efficient utilization of an N-glycosylation site engineered within the luminal extreme C-terminal residues of distinct integral membrane glycoproteins, a native ER resident protein and an engineered secreted protein. This N-glycan addition required that the acceptor asparagine within an Asn-Trp-Ser sequon be located at least four residues away from the C-terminus of the polypeptide and, in the case of membrane proteins, at least 13 residues away from the lumenal side of the transmembrane segment. Pulse-chase assays revealed that the natural N-glycans of the proteins studied were attached co-translationally, whereas C-terminal N-glycosylation occurred post-translocationally within a time frame of hours in Xenopus laevis oocytes and minutes in human embryonic kidney 293 (HEK293) cells. In oocyte and HEK cell expression systems, affinity tag-driven C-terminal N-glycosylation may facilitate the determination of orientation of the C-terminal tail of membrane proteins relative to the membrane.     

TMEM16A(a)/anoctamin-1 shares a homodimeric architecture with CLC chloride channels

TMEM16A/anoctamin-1 has been identified as a protein with the classic properties of a Ca(2+)-activated chloride channel. Here, we used blue native polyacrylamide gel electrophoresis (BN-PAGE) and chemical cross-linking to assess the quaternary structure of the mouse TMEM16A(a) and TMEM16A(ac) splice variants as well as a genetically concatenated TMEM16A(a) homodimer. The constructs carried hexahistidyl (His) tags to allow for their purification using a nondenaturing metal affinity resin. Neither His-tagging nor head-to-tail concatenation of two copies of TMEM16A(a) noticeably affected Ca(2+)-induced measured macroscopic Cl(-) currents compared with the wild-type TMEM16A(a) channel. The digitonin-solubilized, nondenatured TMEM16A(a) protein migrated in the BN-PAGE gel as a homodimer, as judged by comparison with the concatenated TMEM16A(a) homodimer and channel proteins of known oligomeric structures (e.g. the voltage-gated Cl(-) channel CLC-1). Cross-linking with glutaraldehyde corroborated the homodimeric structure of TMEM16A(a). The TMEM16A(a) homodimer detected in Xenopus laevis oocytes and HEK 293 cells dissociated into monomers following denaturation with SDS, and reducing versus nonreducing SDS-PAGE provided no evidence for the presence of intersubunit disulfide bonds. Together, our data demonstrate that the Ca(2+)-activated chloride channel member TMEM16A shares an obligate homodimeric architecture with the hCLC-1 channel.     

A simple qPCR-based method to detect correct insertion of homologous targeting vectors in murine ES cells

The identification of correctly targeted embryonic stem (ES) cell clones from among the large number of random integrants that result from most selection paradigms remains an important hurdle in the generation of animals bearing homologously targeted transgenes. Given the limitations inherent to Southern blotting and standard PCR, we utilized quantitative real-time polymerase chain reaction (qPCR) to rapidly identify murine ES cell clones containing insertions at the correct genomic locus. Importantly, this approach is useful for screening ES clones from conditional/insertional “knock-in” strategies in which there is no loss of genetic material. Simple validation avoids the generation of assays prone to false negative results. In this method, probe and primer sets that span an insertion site detect and quantify the unperturbed gene relative to an irrelevant reference gene, allowing ES cell clones to be screened for loss of detection of one copy of the gene (functional loss of homozygousity (LOH)) that occurs when the normal DNA is disrupted by the insertion event. Simply stated, detected gene copy number falls from two to one in correctly targeted clones. We have utilized such easily designed and validated qPCR LOH assays to rapidly and accurately identify insertions in multiple target sites (including the Lepr and mTOR loci) in murine ES cells, in order to generate transgenic animals.     

Development of newly synthesised quinazolinone-based CDK2 inhibitors with potent efficacy against melanoma

Inhibiting Cyclin-dependent kinase 2 (CDK2) has been established as a therapeutic strategy for the treatment of many cancers. Accordingly, this study aimed at developing a new set of quinazolinone-based derivatives as CDK2 inhibitors. The new compounds were evaluated for their anticancer activity against sixty tumour cell lines. Compounds 5c and 8a showed excellent growth inhibition against the melanoma cell line MDA-MB-435 with GI% of 94.53 and 94.15, respectively. Cell cycle analysis showed that compound 5c led to cell cycle cessation at S phase and G2/M phase revealing that CDK2 could be the plausible biological target. Thus, the most cytotoxic candidates 5c and 8a were evaluated in vitro for their CDK2 inhibitory activity and were able to display significant inhibitory action. The molecular docking study confirmed the obtained results. ADME study predicted that 5c had appropriate drug-likeness properties. These findings highlight a rationale for further development and optimisation of novel CDK2 inhibitors.     

Biosynthesized nanosilver as anti-oxidant,anti-apoptotic and anti-inflammatory agent against Plasmodium chabaudi infection in the mouse liver

In recent years, the use of plant-mediated nanoparticle synthesis to combat infectious diseases has become increasingly significant. Malaria is one of the world's most infectious diseases caused by Plasmodium species. The antioxidant, anti-apoptotic, and anti-inflammatory properties of nanosilver biosynthesized from Indigofera oblongifolia leaf extracts (NS) against Plasmodium chabaudi infection of the mouse liver were investigated in this research. Male mice were infected with P. chabaudi infected erythrocytes then treated with NS for 7 days. The parasitemia was suppressed by approximately 24, 28, 47 and 75% on days 4, 5, 6 and 7 postinfection, respectively after treatment of mice with NS. Also, NS was able to regulate the leucocytes count and the IL1β and TNF-α-mRNA expression in mice. Ns could increase the antioxidant activity in liver of mice and was able to regulate the apoptotic genes, Bcl2 and Casp3. We showed that NS has antioxidant, anti-apoptotic, and anti-inflammatory properties when it was used to treat the livers of mice infected with P. chabaudi.