Human intestinal epithelial cell line SK-CO15 is a new model system to study Na(+)/H(+) exchanger 3

The Caco-2 cell line represents absorptive polarized intestinal epithelial cells that express multiple forms of Na(+)/H(+) exchanger (NHE) in their plasma membranes. Caco-2 cells express the major apical NHE isoform NHE3, but low NHE3 expression together with inefficient transfection often hamper intended studies. In this study, we examined whether SK-CO15 cells could be used to study NHE3 regulation. SK-CO15 cells grown on Transwell inserts developed polarized epithelial cells with microvilli. The transfection efficiency of SK-CO15 cells was markedly higher compared with Caco-2 cells, an advantage in gene transfer and knockout. SK-CO15 cells expressed NHE1, NHE2, and NHE3. NHE3 expression was significantly greater in these cells than Caco-2, and NHE3 comprised more than half of total NHE activity. Apical expression of NHE3 in SK-CO15 cells was confirmed by confocal immunofluorescence and surface biotinylation. NHE regulatory factors NHERF1 and NHERF2, which are important for regulation of NHE3 activity, were expressed in these cells. Stimulatory response of NHE3 in SK-CO15 cells was assessed by dexamethasone and lysophosphatidic acid (LPA). Treatment with dexamethasone for 24-48 h increased NHE3 expression and activity. Similarly to Caco-2 cells, SK-CO15 cells lacked the expression of the LPA receptor LPA(5,) but exogenous expression of LPA(5) resulted in acute stimulation of NHE3. Forskolin acutely inhibited NHE3 activity in SK-CO15 cells, further attesting the validity of these cells. We conclude that SK-CO15 cells with the amenity for transfection and high endogenous NHE3 expression are a new and better cell model for NHE3 regulatory investigation than widely used Caco-2 cells.     

Invertebrate lysozymes: Diversity and distribution, molecular mechanism and in vivo function

Lysozymes are antibacterial enzymes widely distributed among organisms. Within the animal kingdom, mainly three major lysozyme types occur. Chicken (c)-type lysozyme and goose (g)-type lysozyme are predominantly, but not exclusively, found in vertebrate animals, while the invertebrate (i)-type lysozyme is typical for invertebrate organisms, and hence its name. Since their discovery in 1975, numerous research articles report on the identification of i-type lysozymes in a variety of invertebrate phyla. This review describes the current knowledge on i-type lysozymes, outlining their distribution, molecular mechanism and in vivo function taking the representative from Venerupis philippinarum (formerly Tapes japonica) (Vp-ilys) as a model. In addition, invertebrate g-type and ch-type (chalaropsis) lysozymes, which have been described in molluscs and nematodes, respectively, are also briefly discussed.     

'Because we see them naked' - nurses' experiences in caring for hospitalized patients with dementia: considering artificial nutrition or hydration (ANH)

The aim of this study was to explore and describe how Flemish nurses experience their involvement in the care of hospitalized patients with dementia, particularly in relation to artificial nutrition or hydration (ANH). We interviewed 21 hospital nurses who were carefully selected from nine hospitals in different regions of Flanders. 'Being touched by the vulnerability of the demented patient' was the central experience of the nurses, having great impact on them professionally as well as personally. This feeling can be described as encompassing the various stages of the care process: the nurses' initial meeting with the vulnerable patient; the intense decision-making process, during which the nurses experienced several intense emotions influenced by supporting or hindering contextual factors; and the final coping process, a time when nurses came to terms with this challenging experience. From our examination of this care process, it is obvious that nurses' involvement in ANH decision-making processes that concern patients with dementia is a difficult and ethically sensitive experience. On the one hand, the feeling of 'being touched' can imply strength, as it demonstrates that nurses are willing to provide good care. On the other hand, the feeling of 'being touched' can also imply weakness, as it makes nurses vulnerable to moral distress stemming from contextual influences. Therefore, nurses have to be supported as they carry out this ethically sensitive assignment. Practical implications are given.     

A role for neuronal piRNAs in the epigenetic control of memory-related synaptic plasticity

Small RNA-mediated gene regulation during development causes long-lasting changes in cellular phenotypes. To determine whether small RNAs of the adult brain can regulate memory storage, a process that requires stable and long-lasting changes in the functional state of neurons, we generated small RNA libraries from the Aplysia CNS. In these libraries, we discovered an unexpectedly abundant expression of a 28 nucleotide sized class of piRNAs in brain, which had been thought to be germline specific. These piRNAs have unique biogenesis patterns, predominant nuclear localization, and robust sensitivity to serotonin, a modulatory transmitter that is important for memory. We find that the Piwi/piRNA complex facilitates serotonin-dependent methylation of a conserved CpG island in the promoter of CREB2, the major inhibitory constraint of memory in Aplysia, leading to enhanced long-term synaptic facilitation. These findings provide a small RNA-mediated gene regulatory mechanism for establishing stable long-term changes in neurons for the persistence of memory.     

Ultra-fast pg/ml anthrax toxin (protective antigen) detection assay based on microwave-accelerated metal-enhanced fluorescence

Rapid presymptomatic diagnosis of Bacillus anthracis at early stages of infection plays a crucial role in prompt medical intervention to prevent rapid disease progression and accumulation of lethal levels of toxin. To detect low levels of the anthrax protective antigen (PA) exotoxin in biological fluids, we have developed a metal-enhanced fluorescence (MEF)-PA assay using a combination of the MEF effect and microwave-accelerated PA protein surface absorption. The assay is based on a modified version of our "rapid catch and signal" (RCS) technology previously designed for the ultra-fast and sensitive analysis of genomic DNA sequences. Technologically, the proposed MEF-PA assay uses standard 96-well plastic plates modified with silver island films (SiFs) grown within the wells. It is shown that the fluorescent probe, covalently attached to the secondary antibody, plays a crucial role of indicating complex formation (i.e., shows a strong MEF response to the recognition event). Microwave irradiation rapidly accelerates PA deposition onto the surface ("rapid catch"), significantly speeding up the MEF-PA assay and resulting in a total assay run time of less than 40 min with an analytical sensitivity of less than 1 pg/ml PA.     

The relevance of the non-canonical PTS1 of peroxisomal catalase

Catalase is sorted to peroxisomes via a C-terminal peroxisomal targeting signal 1 (PTS1), which binds to the receptor protein Pex5. Analysis of the C-terminal sequences of peroxisomal catalases from various species indicated that catalase never contains the typical C-terminal PTS1 tripeptide-SKL, but invariably is sorted to peroxisomes via a non-canonical sorting sequence. We analyzed the relevance of the non-canonical PTS1 of catalase of the yeast Hansenula polymorpha (-SKI). Using isothermal titration microcalorimetry, we show that the affinity of H. polymorpha Pex5 for a peptide containing -SKI at the C-terminus is 8-fold lower relative to a peptide that has a C-terminal -SKL. Fluorescence microscopy indicated that green fluorescent protein containing the -SKI tripeptide (GFP-SKI) has a prolonged residence time in the cytosol compared to GFP containing -SKL. Replacing the -SKI sequence of catalase into -SKL resulted in reduced levels of enzymatically active catalase in whole cell lysates together with the occurrence of catalase protein aggregates in the peroxisomal matrix. Moreover, the cultures showed a reduced growth yield in methanol-limited chemostats. Finally, we show that a mutant catalase variant that is unable to properly fold mislocalizes in protein aggregates in the cytosol. However, by replacing the PTS1 into -SKL the mutant variant accumulates in protein aggregates inside peroxisomes. Based on our findings we propose that the relatively weak PTS1 of catalase is important to allow proper folding of the enzyme prior to import into peroxisomes, thereby preventing the accumulation of catalase protein aggregates in the organelle matrix.     

Chronic ramelteon treatment in a mouse model of Alzheimer's disease

Prior research has reported beneficial effects of melatonin in rodent models of Alzheimer's disease (AD). This study evaluated the effect of ramelteon (Rozerem, a melatonin receptor agonist) on spatial learning & memory and neuropathological markers in a transgenic murine model of AD (the B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J transgenic mouse strain; hereafter 'AD mice'). Three months of daily ramelteon treatment (~3mg/kg/day), starting at 3 months of age, did not produce an improvement in the cognitive performance of AD mice (water maze). In contrast to wild-type control mice, AD mice did not show any evidence of having learned the location of the escape platform. The cortex and hippocampus of AD mice contained significant quantities of beta-amyloid plaques and PARP-positive (poly ADP ribose polymerase) cells, indicating apoptosis. Six months of ramelteon treatment, starting at 3 months of age, did not produce any change in these neuropathological markers. The ability of long term melatonin treatment to improve cognition and attenuate neuropathology in AD mice did not generalize to this dosage of ramelteon.     

Impact of restricted marital practices on genetic variation in an endogamous Gujarati group

Recent studies have examined the influence on patterns of human genetic variation of a variety of cultural practices. In India, centuries‐old marriage customs have introduced extensive social structuring into the contemporary population, potentially with significant consequences for genetic variation. Social stratification in India is evident as social classes that are defined by endogamous groups known as castes. Within a caste, there exist endogamous groups known as gols (marriage circles), each of which comprises a small number of exogamous gotra (lineages). Thus, while consanguinity is strictly avoided and some randomness in mate selection occurs within the gol, gene flow is limited with groups outside the gol. Gujarati Patels practice this form of “exogamic endogamy.” We have analyzed genetic variation in one such group of Gujarati Patels, the Chha Gaam Patels (CGP), who comprise individuals from six villages. Population structure analysis of 1,200 autosomal loci offers support for the existence of distinctive multilocus genotypes in the CGP with respect to both non‐Gujaratis and other Gujaratis, and indicates that CGP individuals are genetically very similar. Analysis of Y‐chromosomal and mitochondrial haplotypes provides support for both patrilocal and patrilineal practices within the gol, and a low‐level of female gene flow into the gol. Our study illustrates how the practice of gol endogamy has introduced fine‐scale genetic structure into the population of India, and contributes more generally to an understanding of the way in which marriage practices affect patterns of genetic variation. Am J PhyAnthropol 2012. © Wiley Periodicals, Inc.     

The dynamics of forest tent caterpillar outbreaks across east‐central Canada

An analysis of forest tent caterpillar Malacosoma disstria defoliation records from Ontario and Quebec over the period 1938–2002 indicates that outbreaks recur periodically and somewhat synchronously among regions of the two provinces. Cluster analysis revealed that the most strongly periodic, large‐scale, synchronized fluctuations occurred within three regions: northwestern Ontario, eastern Ontario/western Quebec and southeastern Quebec. Defoliation in the vast surrounding hinterlands tended to be infrequent and sporadic, loosely tracking defoliation in the core outbreak regions. One small cluster in northeastern Ontario stood out as anomalous, as a result of an increasing trend in the duration of periodic defoliation episodes, marked by an unprecedented double‐wave of defoliation that persisted from 1992 to 1999. This is the precise area where, in the early 2000s, trembling aspen Populus tremuloides stems were mapped as being in a state of decline of unprecedented severity and extent. Our results suggest forest tent caterpillar has the potential to cause significant impacts on forest health and, hence, carbon budgets in east‐central Canada and that the forest tent caterpillar deserves more attention as a model system of forest insect disturbance ecology.