Polycystin-2 accelerates Ca2+ release from intracellular stores in Caenorhabditis elegans

Polycystin-2, a member of the TRP family of calcium channels, is encoded by the human PKD2 gene. Mutations in that gene can lead to swelling of nephrons into the fluid-filled cysts of polycystic kidney disease. In addition to expression in tubular epithelial cells, human polycystin-2 is found in muscle and neuronal cells, but its cell biological function has been unclear. A homologue in Caenorhabditis elegans is necessary for male mating behavior. We compared the behavior, calcium signaling mechanisms, and electrophysiology of wild-type and pkd-2 knockout C. elegans. In addition to characterizing PKD-2-mediated aggregation and mating behaviors, we found that polycystin-2 is an intracellular Ca(2+) release channel that is required for the normal pattern of Ca(2+) responses involving IP(3) and ryanodine receptor-mediated Ca(2+) release from intracellular stores. Activity of polycystin-2 creates brief cytosolic Ca(2+) transients with increased amplitude and decreased duration. Polycystin-2, along with the IP(3) and ryanodine receptors, acts as a major calcium-release channel in the endoplasmic reticulum in cells where rapid calcium signaling is required, and polycystin-2 activity is essential in those excitable cells for rapid responses to stimuli.     

The effect of human bile on Bacteroides fragilis in health and disease

Bacteroides fragilis is generally believed to be stimulated by bile. Although B. fragilis is rarely found in the human duodenum, it is relatively frequent in gall bladder infections. To investigate this paradox, the growth of B. fragilis in the human bile in both health and disease was studied, and compared with the effect of bovine and porcine bile. B. fragilis was stimulated by the bovine bile and inhibited by the porcine bile. The normal human bile was either bacteriostatic or inhibitory whereas bile from cholelithiatic patients, in 50% of cases, stimulated the growth of B. fragilis. This explains the relative prevalence of B. fragilis in cholelithiatic cholecystitis patients although it is not a resident flora of the duodenum.     

Radiation induces senescence and a bystander effect through metabolic alterations

Cellular senescence is a state of irreversible growth arrest; however, the metabolic processes of senescent cells remain active. Our previous studies have shown that radiation induces senescence of human breast cancer cells that display low expression of securin, a protein involved in control of the metaphase–anaphase transition and anaphase onset. In this study, the protein expression profile of senescent cells was resolved by two-dimensional gel electrophoresis to investigate associated metabolic alterations. We found that radiation induced the expression and activation of glyceraldehyde-3-phosphate dehydrogenase that has an important role in glycolysis. The activity of lactate dehydrogenase A, which is involved in the conversion of pyruvate to lactate, the release of lactate and the acidification of the extracellular environment, was also induced. Inhibition of glycolysis by dichloroacetate attenuated radiation-induced senescence. In addition, radiation also induced activation of the 5′-adenosine monophosphate-activated protein kinase (AMPK) and nuclear factor kappa B (NF-κB) pathways to promote senescence. We also found that radiation increased the expression of monocarboxylate transporter 1 (MCT1) that facilitates the export of lactate into the extracellular environment. Inhibition of glycolysis or the AMPK/NF-κB signalling pathways reduced MCT1 expression and rescued the acidification of the extracellular environment. Interestingly, these metabolic-altering signalling pathways were also involved in radiation-induced invasion of the surrounding, non-irradiated breast cancer and normal endothelial cells. Taken together, radiation can induce the senescence of human breast cancer cells through metabolic alterations.     

Recombinase mediated cassette exchange into genomic targets using an adenovirus vector

Recombinase mediated cassette exchange (RMCE) is a process in which site-specific recombinases exchange one gene cassette flanked by a pair of incompatible target sites for another cassette flanked by an identical pair of sites. Typically one cassette is present in the host genome, whereas the other gene cassette is introduced into the host cell by chemical or biological means. We show here that the frequency of cassette exchange is dependent on the relative and absolute quantities of the transgene cassette and the recombinase. We were able to successfully modify genomic targets not only by electroporation or chemically mediated gene transfer but also by using an adenovirus vector carrying both the transgene cassette to be inserted and the recombinase coding region. RMCE proceeds efficiently in cells in which the adenovirus vector is able to replicate. In contrast, insufficient quantities of the transgene cassette are produced in cells in which the virus cannot replicate. Additional transfection of the transgene cassette significantly enhances the RMCE frequency. This demonstrates that an RMCE system in the context of a viral vector allows the site directed insertion of a transgene into a defined genomic site.     

The Genetic Diversity of the Nguni Breed of African Cattle (Bos spp.): Complete Mitochondrial Genomes of Haplogroup T1

Domesticated cattle were commonplace in northern Africa by about 7,000 years ago. Archaeological evidence, however, suggests they were not established in southern Africa until much later, no earlier than 2,000 years ago. Genetic reconstructions have started to shed light on the movement of African cattle, but efforts have been frustrated by a lack of data south of Ethiopia and the nature of the mitochondrial haplogroup T1 which is almost fixed across the continent. We sequenced 35 complete mitochondrial genomes from a South African herd of Nguni cattle, a breed historically associated with Bantu speaking farmers who were among the first to bring cattle to southern Africa. As expected, all individuals in the study were found to be members of haplogroup T1. Only half of the sub-haplogroups of T1 (T1a-T1f) are represented in our sample and the overwhelming majority (94%) in this study belong to subhaplogroup T1b. A previous study of African cattle found frequencies of T1b of 27% in Egypt and 69% in Ethiopia. These results are consistent with serial multiple founder effects significantly shaping the gene pool as cattle were moved from north to south across the continent. Interestingly, these mitochondrial data give no indication that the impacts of the founder effects were ameliorated by gene flow from recently introduced Indian cattle breeds.     

Molecular characterisation of phaCAB from Comamonas sp. EB172 for functional expression in Escherichia coli JM109

In this study, PHA biosynthesis operon of Comamonas sp. EB172, an acid-tolerant strain, consisting of three genes encoding acetyl-CoA acetyltransferase (phaA(Co) gene, 1182bp), acetoacetyl-CoA reductase (phaB(Co) gene, 738bp) and PHA synthase, class I (phaC(Co) gene, 1694bp) were identified. Sequence analysis of the phaA(Co), phaB(Co) and phaC(Co) genes revealed that they shared more than 85%, 89% and 69% identity, respectively, with orthologues from Delftia acidovorans SPH-1 and Acidovorax ebreus TPSY. The PHA biosynthesis genes (phaC(Co) and phaAB(Co)) were successfully cloned in a heterologous host, Escherichia coli JM109. E. coli JM109 transformants harbouring pGEM'-phaC(Co)AB(Re) and pGEM'-phaC(Re)AB(Co) were shown to be functionally active synthesising 33wt.% and 17wt.% of poly(3-hydroxybutyrate) [P(3HB)]. E. coli JM109 transformant harbouring the three genes from the acid-tolerant Comamonas sp. EB172 (phaCAB(Co)) under the control of native promoter from Cupriavidus necator, in vivo polymerised P(3HB) when fed with glucose and volatile mixed organic acids (acetic acid:propionic acid:n-butyric acid) in ration of 3:1:1, respectively. The E. coli JM109 transformant harbouring phaCAB(Co) could accumulate P(3HB) at 2g/L of propionic acid. P(3HB) contents of 40.9% and 43.6% were achieved by using 1% of glucose and mixed organic acids, respectively.     

Microbial Analysis of Bite Marks by Sequence Comparison of Streptococcal DNA

Bite mark injuries often feature in violent crimes. Conventional morphometric methods for the forensic analysis of bite marks involve elements of subjective interpretation that threaten the credibility of this field. Human DNA recovered from bite marks has the highest evidentiary value, however recovery can be compromised by salivary components. This study assessed the feasibility of matching bacterial DNA sequences amplified from experimental bite marks to those obtained from the teeth responsible, with the aim of evaluating the capability of three genomic regions of streptococcal DNA to discriminate between participant samples. Bite mark and teeth swabs were collected from 16 participants. Bacterial DNA was extracted to provide the template for PCR primers specific for streptococcal 16S ribosomal RNA (16S rRNA) gene, 16S–23S intergenic spacer (ITS) and RNA polymerase beta subunit (rpoB). High throughput sequencing (GS FLX 454), followed by stringent quality filtering, generated reads from bite marks for comparison to those generated from teeth samples. For all three regions, the greatest overlaps of identical reads were between bite mark samples and the corresponding teeth samples. The average proportions of reads identical between bite mark and corresponding teeth samples were 0.31, 0.41 and 0.31, and for non-corresponding samples were 0.11, 0.20 and 0.016, for 16S rRNA, ITS and rpoB, respectively. The probabilities of correctly distinguishing matching and non-matching teeth samples were 0.92 for ITS, 0.99 for 16S rRNA and 1.0 for rpoB. These findings strongly support the tenet that bacterial DNA amplified from bite marks and teeth can provide corroborating information in the identification of assailants.     

Surface temperature patterns in seals and sea lions: A validation of temporal and spatial consistency

We assessed infrared thermography (IRT) as a tool for evaluating spatial and temporal surface temperature patterns in juvenile female harbor seals (Phoca vitulina, n=6) and adult female Steller sea lions (Eumetopias jubatus, n=2). Following a technical assessment of the influence of environmental parameters on the specific camera to be used, we identified regional and seasonal variability of surface temperatures. Variation was observed in several seasonal transitions (winter, reproductive, molt) in ten monitored body regions. Spatially and temporally consistent thermal patterns in the shoulder, axillae, foreflipper and hindflipper suggest thermal windows in both species.     

Thiol redox-sensitive seed proteome in dormant and non-dormant hybrid genotypes of wheat

The thiol redox-sensitive and the total proteome in harvest-ripe grains of closely related genotypes of wheat (Triticum aestivum L.), with either a dormant or a non-dormant phenotype, were investigated using hybrid lines of spring wheat double haploid population segregating transgressively, to gain further insight into seed dormancy controlling events. Redox signalling by reactive oxygen species has been shown to play a role in seed dormancy alleviation. Thiol-disulfide proteins are of particular importance in the context of redox-dependent regulation as a central and flexible mechanism to control metabolic and developmental activities of the cells. Here we describe functional proteomic profiling of reversible oxidoreductive changes and characterize in vivo intrinsic reactivity of cysteine residues using thiol-specific fluorescent labelling, solubility-based protein fractionation, two-dimensional electrophoresis, and mass spectrometry analysis in conjunction with wheat EST sequence libraries. Quantitative differences between genotypes were found for 106 spots containing 64 unique proteins. Forty seven unique proteins displayed distinctive abundance pattern, and among them 31 proteins contained 78 unique redox active cysteines. Seventeen unique proteins with 19 reactive modified cysteines were found to have differential post-translational thiol redox modification. The results provide an insight into the alteration of thiol-redox profiles in proteins that function in major processes in seeds and include groups of redox- and stress-responsive, genetic information processing and cell cycle control, transport and storage proteins, enzymes of carbohydrate metabolism, proteases and their inhibitors.