Emerging role of the calcium-activated, small conductance, SK3 K ⁺ channel in distal tubule function: Regulation by TRPV4

The Ca2+-activated, maxi-K (BK) K+ channel, with low Ca2+-binding affinity, is expressed in the distal tubule of the nephron and contributes to flow-dependent K+ secretion. In the present study we demonstrate that the Ca2+-activated, SK3 (KCa2.3) K + channel, with high Ca2+-binding affinity, is also expressed in the mouse kidney (RT-PCR, immunoblots). Immunohistochemical evaluations using tubule specific markers demonstrate significant expression of SK3 in the distal tubule and the entire collecting duct system, including the connecting tubule (CNT) and cortical collecting duct (CCD). In CNT and CCD, main sites for K+ secretion, the highest levels of expression were along the apical (luminal) cell membranes, including for both principal cells (PCs) and intercalated cells (ICs), posturing the channel for Ca2+- dependent K+ secretion. Fluorescent assessment of cell membrane potential in native, split-opened CCD, demonstrated that selective activation of the Ca2+-permeable TRPV4 channel, thereby inducing Ca2+ influx and elevating intracellular Ca2+ levels, activated both the SK3 channel and the BK channel leading to hyperpolarization of the cell membrane. The hyperpolarization response was decreased to a similar extent by either inhibition of SK3 channel with the selective SK antagonist, apamin, or by inhibition of the BK channel with the selective antagonist, iberiotoxin (IbTX). Addition of both inhibitors produced a further depolarization, indicating cooperative effects of the two channels on Vm. It is concluded that SK3 is functionally expressed in the distal nephron and collecting ducts where induction of TRPV4-mediated Ca2+ influx, leading to elevated intracellular Ca2+ levels, activates this high Ca2+- affinity K+ channel. Further, with sites of expression localized to the apical cell membrane, especially in the CNT and CCD, SK3 is poised to be a key pathway for Ca2+-dependent regulation of membrane potential and K+ secretion. © 2014 Berrout et al

MicroRNA-21 dysregulates the expression of MEF2C in neurons in monkey and human SIV/HIV neurological disease

MicroRNAs (miRNAs) have important roles in regulating a plethora of physiological and pathophysiogical processes including neurodegeneration. In both human immunodeficiency virus (HIV)-associated dementia in humans and its monkey model simian immunodeficiency virus encephalitis (SIVE), we find miR-21, a miRNA largely known for its link to oncogenesis, to be significantly upregulated in the brain. In situ hybridization of the diseased brain sections revealed induction of miR-21 in neurons. miR-21 can be induced in neurons by prolonged N-methyl--aspartic acid receptor stimulation, an excitotoxic process active in HIV and other neurodegenerative diseases. Introduction of miR-21 into human neurons leads to pathological functional defects. Furthermore, we show that miR-21 specifically targets the mRNA of myocyte enhancer factor 2C (MEF2C), a transcription factor crucial for neuronal function, and reduces its expression. MEF2C is dramatically downregulated in neurons of HIV-associated dementia patients, as well as monkeys with SIVE. Together, this study elucidates a novel role for miR-21 in the brain, not only as a potential signature of neurological disease, but also as a crucial effector of HIV-induced neuronal dysfunction and neurodegeneration

Meeting the home-care needs of disabled older persons living in the community: does integrated services delivery make a difference?

<p>Abstract</p> <p>Background</p> <p>The PRISMA Model is an innovative coordination-type integrated-service-delivery (ISD) network designed to manage and better match resources to the complex and evolving needs of elders. The goal of this study was to examine the impact of this ISD network on unmet needs among disabled older persons living in the community.</p> <p>Methods</p> <p>Using data from the PRISMA study, we compared unmet needs of elders living in the community in areas with or without an ISD network. Disabilities and unmet needs were assessed with the Functional Autonomy Measurement System (SMAF). We used growth-curve analysis to examine changes in unmet needs over time and the variables associated with initial status and change. Sociodemographic characteristics, level of disability, self-perceived health status, cognitive functioning, level of empowerment, and the hours of care received were investigated as covariates. Lastly, we report the prevalence of needs and unmet needs for 29 activities in both areas at the end of the study.</p> <p>Results</p> <p>On average, participants were 83 years old; 62% were women. They had a moderate level of disability and mild cognitive problems. On average, they received 2.07 hours/day (SD = 1.08) of disability-related care, mostly provided by family. The findings from growth-curve analysis suggest that elders living in the area where ISD was implemented and those with higher levels of disability experience better fulfillment of their needs over time. Besides the area, being a woman, living alone, having a higher level of disability, more cognitive impairments, and a lower level of empowerment were linked to initial unmet needs (r²= 0.25; p < 0.001). At the end of the study, 35% (95% CI: 31% to 40%) of elders with needs living in the ISD area had at least one unmet need, compared to 67% (95% CI: 62% to 71%) in the other area. In general, unmet needs were highest for bathing, grooming, urinary incontinence, walking outside, seeing, hearing, preparing meals, and taking medications.</p> <p>Conclusions</p> <p>In spite of more than 30 years of home-care services in the province of Quebec, disabled older adults living in the community still have unmet needs. ISD networks such as the PRISMA Model, however, appear to offer an effective response to the long-term-care needs of the elderly.</p

Universal Primers Used for Species Identification of Foodstuff of Animal Origin: Effects of Oligonucleotide Tails on PCR Amplification and Sequencing Performance

M13 universal non-homologous oligonucleotide tails incorporated into universal primers have been shown to improve amplification and sequencing performance. However, a few protocols use these tails in the field of food inspection. In this study, two types of M13 tails (by Steffens and Messing) were selected to assess their benefits using universal cytochrome oxidase subunit I (COI) and 16S ribosomal RNA gene (16SrRNA) primers in standard procedures. The primer characteristics were tested in silico. Then, using 20 DNA samples of edible species (birds, fishes, and mammals), their performance during PCR amplification (band recovery and intensity) and sequencing (sequence recovery, length, and Phred score) was assessed and compared. While 16SrRNA tailed and non-tailed primers performed similarly, differences were found for COI primers. Messing’s tails negatively affected the reaction outputs, while Steffens’ tails significantly improved the band intensity and the length of the final contigs based on the individual bidirectional read sequence. This different performance could be related to a destabilization effect of certain tails on primers with unfavorable mismatches on the annealing region. Even though our results cannot be generalized because the tail performances are strictly dependent on laboratory conditions, they show that appropriate tails can improve the overall throughput of the analysis, supporting food traceabilit

A Pro-Cathepsin L Mutant Is a Luminal Substrate for Endoplasmic-Reticulum-Associated Degradation in C. elegans

Endoplasmic-reticulum associated degradation (ERAD) is a major cellular misfolded protein disposal pathway that is well conserved from yeast to mammals. In yeast, a mutant of carboxypeptidase Y (CPY*) was found to be a luminal ER substrate and has served as a useful marker to help identify modifiers of the ERAD pathway. Due to its ease of genetic manipulation and the ability to conduct a genome wide screen for modifiers of molecular pathways, C. elegans has become one of the preferred metazoans for studying cell biological processes, such as ERAD. However, a marker of ERAD activity comparable to CPY* has not been developed for this model system. We describe a mutant of pro-cathepsin L fused to YFP that no longer targets to the lysosome, but is efficiently eliminated by the ERAD pathway. Using this mutant pro-cathepsin L, we found that components of the mammalian ERAD system that participate in the degradation of ER luminal substrates were conserved in C. elegans. This transgenic line will facilitate high-throughput genetic or pharmacological screens for ERAD modifiers using widefield epifluorescence microscopy

A case report and brief review of the literature on bilateral retinal infarction following cardiopulmonary bypass for coronary artery bypass grafting

Postoperative visual loss is a devastating perioperative complication. The commonest aetiologies are anterior ischaemic optic neuropathy (AION), posterior ischaemic optic neuropathy (PION), and central retinal artery occlusion (CRAO). These appear to be related to certain types of operation, most commonly spinal and cardiac bypass procedures; with the rest divided between: major trauma causing excessive blood loss; head/neck and nasal or sinus surgery; major vascular procedures (aortic aneurysm repair, aorto-bifemoral bypass); general surgery; urology; gynaecology; liposuction; liver transplantation and duration of surgery. The non-surgical risk factors are multifactorial: advanced age, prolonged postoperative anaemia, positioning (supine v prone), alteration of venous drainage of the retina, hypertension, smoking, atherosclerosis, hyperlipidaemia, diabetes, hypercoagulability, hypotension, blood loss and large volume resuscitation. Other important cardiac causes are septic emboli from bacterial endocarditis and emboli caused by atrial myxomata. The majority of AION cases occur during CPB followed by head/neck surgery and prone spine surgery. CPB is used to allow coronary artery bypass grafting on a motionless heart. It has many side-effects and complications associated with its use and we report here a case of bilateral retinal infarction during routine coronary artery bypass grafting in a young male patient with multiple risk factors for developing this complication despite steps to minimise its occurrence

Ecological Niche Modelling and nDNA Sequencing Support a New, Morphologically Cryptic Beetle Species Unveiled by DNA Barcoding

DNA sequencing techniques used to estimate biodiversity, such as DNA barcoding, may reveal cryptic species. However, disagreements between barcoding and morphological data have already led to controversy. Species delimitation should therefore not be based on mtDNA alone. Here, we explore the use of nDNA and bioclimatic modelling in a new species of aquatic beetle revealed by mtDNA sequence data. The aquatic beetle fauna of Australia is characterised by high degrees of endemism, including local radiations such as the genus Antiporus. Antiporus femoralis was previously considered to exist in two disjunct, but morphologically indistinguishable populations in south-western and south-eastern Australia. We constructed a phylogeny of Antiporus and detected a deep split between these populations. Diagnostic characters from the highly variable nuclear protein encoding arginine kinase gene confirmed the presence of two isolated populations. We then used ecological niche modelling to examine the climatic niche characteristics of the two populations. All results support the status of the two populations as distinct species. We describe the south-western species as Antiporus occidentalis sp.n. In addition to nDNA sequence data and extended use of mitochondrial sequences, ecological niche modelling has great potential for delineating morphologically cryptic species

Group II Intron-Anchored Gene Deletion in Clostridium

Clostridium plays an important role in commercial and medical use, for which targeted gene deletion is difficult. We proposed an intron-anchored gene deletion approach for Clostridium, which combines the advantage of the group II intron “ClosTron” system and homologous recombination. In this approach, an intron carrying a fragment homologous to upstream or downstream of the target site was first inserted into the genome by retrotransposition, followed by homologous recombination, resulting in gene deletion. A functional unknown operon CAC1493–1494 located in the chromosome, and an operon ctfAB located in the megaplasmid of C. acetobutylicum DSM1731 were successfully deleted by using this approach, without leaving antibiotic marker in the genome. We therefore propose this approach can be used for targeted gene deletion in Clostridium. This approach might also be applicable for gene deletion in other bacterial species if group II intron retrotransposition system is established

Absence of Colony Stimulation Factor-1 Receptor Results in Loss of Microglia, Disrupted Brain Development and Olfactory Deficits

The brain contains numerous mononuclear phagocytes called microglia. These cells express the transmembrane tyrosine kinase receptor for the macrophage growth factor colony stimulating factor-1 (CSF-1R). Using a CSF-1R-GFP reporter mouse strain combined with lineage defining antibody staining we show in the postnatal mouse brain that CSF-1R is expressed only in microglia and not neurons, astrocytes or glial cells. To study CSF-1R function we used mice homozygous for a null mutation in the Csflr gene. In these mice microglia are >99% depleted at embryonic day 16 and day 1 post-partum brain. At three weeks of age this microglial depletion continues in most regions of the brain although some contain clusters of rounded microglia. Despite the loss of microglia, embryonic brain development appears normal but during the post-natal period the brain architecture becomes perturbed with enlarged ventricles and regionally compressed parenchyma, phenotypes most prominent in the olfactory bulb and cortex. In the cortex there is increased neuronal density, elevated numbers of astrocytes but reduced numbers of oligodendrocytes. Csf1r nulls rarely survive to adulthood and therefore to study the role of CSF-1R in olfaction we used the viable null mutants in the Csf1 (Csf1op) gene that encodes one of the two known CSF-1R ligands. Food-finding experiments indicate that olfactory capacity is significantly impaired in the absence of CSF-1. CSF-1R is therefore required for the development of microglia, for a fully functional olfactory system and the maintenance of normal brain structure