Expression and characterization of the Na+/H+ exchanger in the mammalian myocardium

We examined two expression systems for studying the Na⁺/H⁺ exchanger in the mammalian myocardium. Mammalian NHE1 with a hemagglutinin (HA) tag and was cloned behind the alpha myosin heavy chain promoter. Transgenic mice were made with wild type NHE1 protein or with a hyperactive NHE1 protein mutated at the calmodulin-binding domain. Three lines of transgenic mice were made of each cDNA with expression levels of each type varying from high to low. Higher levels and activity of the Na⁺/H⁺ exchanger were associated with decreased long-term survival of mice, and with dilated or hypertrophic cardiomyopathy. The exogenous NHE1 protein was present in freshly made cardiomyocytes from transgenic mice, however, expression from the alpha myosin heavy chain promoter declined rapidly and little exogenous NHE1 was apparent on the fourth day after cardiomyocyte isolation. To express NHE1 protein in isolated cardiomyocytes, we transferred a mutated form of the protein into an adenoviral expression system. Infection of neonatal rat cardiomyocytes resulted in robust expression of the exogenous NHE1 protein. The mutant form of the NHE1 protein could be distinguished from the endogenous Na⁺/H⁺ exchanger by its resistance to inhibition by amiloride analogs. Our results suggest that for in vivo studies on intact hearts and animals, expression in transgenic mice is an appropriate system, however for long-term studies on cardiomyocytes, this model is inappropriate due to waning expression from the alpha myosin heavy chain promoter. Therefore, infection by adenovirus is a superior system for long-term studies on cardiomyocytes in culture.     

The in vitro behaviour and patterns of colony formation of murine epithelial stem cells

OBJECTIVE: The mechanisms of renewal of skin and mucosal epithelia in vivo are associated with hierarchies of stem and amplifying cells organized in distinct spatial patterns. Stem and amplifying characteristics persist after isolation and growth of human keratinocytes in vitro but the pattern for murine keratinocytes has been less clear. MATERIALS AND METHODS: Murine keratinocytes were grown in low calcium media and examined for their patterns of colony morphologies. RESULTS: We consistently identified three types of colonies, one of which contains concentric zones of amplifying and differentiated cells surrounding a central zone of cells that have patterns of expression and behavioural characteristic of stem cells. This zonal organization facilitated analysis of stem cell formation and loss. Cells in the central stem cell zone undergo rapid symmetric divisions but expansion of this population is partially limited by their peripheral transition into amplifying cells. A striking feature of central zone cells is their enhanced apoptotic susceptibility and stem cell expansion limited by consistently high background rates of apoptosis. This occasionally reaches catastrophic levels with elimination of the entire central zone. CONCLUSION: In vitro amplification of stem cells for the generation of engineered tissue has tended to focus on control of asymmetric division but these findings suggest that development of mechanisms protecting stem cells from apoptotic changes are also likely to be of particular value.     

Plant mitochondrial recombination surveillance requires unusual RecA and MutS homologs

For >20 years, the enigmatic behavior of plant mitochondrial genomes has been well described but not well understood. Chimeric genes appear, and occasionally are differentially replicated or expressed, with significant effects on plant phenotype, most notably on male fertility, yet the mechanisms of DNA replication, chimera formation, and recombination have remained elusive. Using mutations in two important genes of mitochondrial DNA metabolism, we have observed reproducible asymmetric recombination events occurring at specific locations in the mitochondrial genome. Based on these experiments and existing models of double-strand break repair, we propose a model for plant mitochondrial DNA replication, chimeric gene formation, and the illegitimate recombination events that lead to stoichiometric changes. We also address the physiological and developmental effects of aberrant events in mitochondrial genome maintenance, showing that mitochondrial genome rearrangements, when controlled, influence plant reproduction, but when uncontrolled, lead to aberrant growth phenotypes and dramatic reduction of the cell cycle.     

Methicillin-Resistant Staphylococcus aureus in Commercial Swine Herds Is Associated with Disinfectant and Zinc Usage

Methicillin-resistant Staphylococcus aureus (MRSA) originating from swine is concerning for public health, but an understanding of the emergence and persistence of MRSA in nursery herds is lacking. The aim of this study was to determine whether MRSA in nursery pigs is associated with particular herd-level parameters, including the use of antimicrobials, disinfectants, and heavy metals, which may be driving the selection and persistence of antimicrobial resistance. Nasal cultures for MRSA were completed for 390 pigs from 26 farms at the end of the suckling phase and again at 3 weeks postweaning. Herd-level information was collected, and a random subset of MRSA isolates was screened for resistance to zinc and quaternary ammonium compounds (QACs). Multivariate analysis revealed that in-feed concentrations of zinc (P < 0.001) and frequent disinfection of nursery pens (P < 0.001) are associated with MRSA shedding in nursery pigs. Furthermore, 62.5% (25/40) of MRSA isolates carried the zinc resistance gene czrC and demonstrated decreased susceptibility to zinc. All MRSA isolates carried at least 1 QAC resistance gene. The most common genotype was qacG qacH smr, which occurred in 32.5% (13/40) of isolates. Seven isolates (17.5%) demonstrated a significant tolerance to benzalkonium chloride, indicating a potential to survive commercial QAC exposure in the presence of organic matter. Overall, these findings indicate that high levels of in-feed zinc and QAC-based disinfectants are important drivers in the selection and persistence of MRSA in commercial swine herds, and these agents may be coselecting for other antimicrobial resistance genes.     

Fish can show emotional fever: stress-induced hyperthermia in zebrafish

Whether fishes are sentient beings remains an unresolved and controversial question. Among characteristics thought to reflect a low level of sentience in fishes is an inability to show stress-induced hyperthermia (SIH), a transient rise in body temperature shown in response to a variety of stressors. This is a real fever response, so is often referred to as ‘emotional fever’. It has been suggested that the capacity for emotional fever evolved only in amniotes (mammals, birds and reptiles), in association with the evolution of consciousness in these groups. According to this view, lack of emotional fever in fishes reflects a lack of consciousness. We report here on a study in which six zebrafish groups with access to a temperature gradient were either left as undisturbed controls or subjected to a short period of confinement. The results were striking: compared to controls, stressed zebrafish spent significantly more time at higher temperatures, achieving an estimated rise in body temperature of about 2–4°C. Thus, zebrafish clearly have the capacity to show emotional fever. While the link between emotion and consciousness is still debated, this finding removes a key argument for lack of consciousness in fishes.     

East Weddell Sea echinoids from the JR275 expedition

Information regarding the echinoids in this dataset is based on the Agassiz Trawl (AGT) and epibenthic sledge (EBS) samples collected during the British Antarctic Survey cruise JR275 on the RRS James Clark Ross in the austral summer 2012. A total of 56 (1 at the South Orkneys and 55 in the Eastern Weddell Sea) Agassiz Trawl and 18 (2 at the South Orkneys and 16 in the Eastern Weddell Sea) epibenthic sledge deployments were performed at depths ranging from ~280 to ~2060 m. This presents a unique collection for the Antarctic benthic biodiversity assessment of an important group of benthic invertebrates. In total 487 specimens belonging to six families, 15 genera, and 22 morphospecies were collected. The species richness per station varied between one and six. Total species richness represents 27% of the 82 echinoid species ever recorded in the Southern Ocean (David et al. 2005b, Pierrat et al. 2012, Saucède et al. 2014). The Cidaridae (sub-family Ctenocidarinae) and Schizasteridae are the two most speciose families in the dataset. They comprise seven and nine species respectively. This is illustrative of the overall pattern of echinoid diversity in the Southern Ocean where 65% of Antarctic species belong to the families Schizasteridae and Cidaridae (Pierrat et al. 2012).