The thiazide-sensitive NaCl cotransporter is targeted for chaperone-dependent endoplasmic reticulum-associated degradation

The thiazide-sensitive NaCl cotransporter (NCC, SLC12A3) mediates salt reabsorption in the distal nephron of the kidney and is the target of thiazide diuretics, which are commonly prescribed to treat hypertension. Mutations in NCC also give rise to Gitelman syndrome, a hereditary salt-wasting disorder thought in most cases to arise from impaired NCC biogenesis through enhanced endoplasmic reticulum-associated degradation (ERAD). Because the machinery that mediates NCC quality control is completely undefined, we employed yeast as a model heterologous expression system to identify factors involved in NCC degradation. We confirmed that NCC was a bona fide ERAD substrate in yeast, as the majority of NCC polypeptide was integrated into ER membranes, and its turnover rate was sensitive to proteasome inhibition. NCC degradation was primarily dependent on the ER membrane-associated E3 ubiquitin ligase Hrd1. Whereas several ER luminal chaperones were dispensable for NCC ERAD, NCC ubiquitination and degradation required the activity of Ssa1, a cytoplasmic Hsp70 chaperone. Compatible findings were observed when NCC was expressed in mammalian kidney cells, as the cotransporter was polyubiquitinated and degraded by the proteasome, and mammalian cytoplasmic Hsp70 (Hsp72) coexpression stimulated the degradation of newly synthesized NCC. Hsp70 also preferentially associated with the ER-localized NCC glycosylated species, indicating that cytoplasmic Hsp70 plays a critical role in selecting immature forms of NCC for ERAD. Together, these results provide the first survey of components involved in the ERAD of a mammalian SLC12 cation chloride cotransporter and provide a framework for future studies on NCC ER quality control.     

Enrichment of omnivorous cercozoan nanoflagellates from coastal Baltic Sea waters

Free-living nano-sized flagellates are important bacterivores in aquatic habitats. However, some slightly larger forms can also be omnivorous, i.e., forage upon both bacterial and eukaryotic resources. This hitherto largely ignored feeding mode may have pronounced implications for the interpretation of experiments about protistan bacterivory. We followed the response of an uncultured group of omnivorous cercozoan nanoflagellates from the Novel Clade 2 (Cerc_BAL02) to experimental food web manipulation in samples from the Gulf of Gdańsk (Southern Baltic Sea). Seawater was either prefiltered through 5 μm filters to exclude larger predators of nanoflagellates (F-treatment), or prefiltered and subsequently 1∶10 diluted with sterile seawater (F+D-treatment) to stimulate the growth of both, flagellates and bacteria. Initially, Cerc_BAL02 were rapidly enriched under both conditions. They foraged on both, eukaryotic prey and bacteria, and were highly competitive at low concentrations of food. However, these omnivores were later only successful in the F+D treatment, where they eventually represented almost one fifth of all aplastidic nanoflagellates. By contrast, their numbers stagnated in the F-treatment, possibly due to top-down control by a concomitant bloom of other, unidentified flagellates. In analogy with observations about the enrichment of opportunistically growing bacteria in comparable experimental setups we suggest that the low numbers of omnivorous Cerc_Bal02 flagellates in waters of the Gulf of Gdańsk might also be related to their vulnerability to grazing pressure.     

Retinoids for treatment of Alzheimer's disease

Retinoids are Vitamin A derivatives involved in cellular regulatory processes including cell differentiation, neurite outgrowth and defense against oxidative stress. Retinoids may also influence Amyloid beta processing upregulation of alpha secretase via ADAM10. Vitamin A and other retinoids also directly inhibit formation of Amyloid fibrils in vivo. These properties of retinoids are relevant to theories of Alzheimer's disease pathogenesis. Retinoids are already used in treatment of acne vulgaris, psoriasis, neuroblastoma and acute promyelocytic leukemia. Clinical studies involving in cognitively impaired older adults with Alzheimer's disease are beginning with a variety of retinoids. These studies need to address safety issues of retinoids in older populations, and hold hope for demonstrating efficacy in translating these basic mechanisms to treatment of a widespread dementing illness.     

Comparability of tympanic and oral mercury thermometers at high ambient temperatures

ABSTRACT: BACKGROUND: Body temperature can be measured in seconds with tympanic thermometers as opposed to minutes with mercury ones. The aim of this study was to compare tympanic and oral mercury thermometer measurements under high ambient field temperatures. RESULTS: Tympanic temperature (measured thrice by 3 operators) was compared to oral temperature measured once with a mercury-in-glass thermometer in 201 patients (aged [greater than or equal to]5 years), on the Thai-Myanmar border. Ambient temperature was measured with an electronic thermo-hygrometer. Participants had a mean [min-max] age of 27 [5-60] years and 42% (84) were febrile by oral thermometer. The mean difference in the mercury and tympanic temperature measurement for all observers/devices was 0.09 (95%CI 0.07-0.12)degreesC and intra-class correlation for repeat tympanic measurements was high ([greater than or equal to]0.97) for each observer. Deviations in tympanic temperatures were not related to ambient temperature. CONCLUSION: Clinically significant differences were not observed between oral and tympanic temperature measurements at high ambient temperatures in a rural tropical setting.     

Modeling within-host dynamics of influenza virus infection including immune responses

Influenza virus infection remains a public health problem worldwide. The mechanisms underlying viral control during an uncomplicated influenza virus infection are not fully understood. Here, we developed a mathematical model including both innate and adaptive immune responses to study the within-host dynamics of equine influenza virus infection in horses. By comparing modeling predictions with both interferon and viral kinetic data, we examined the relative roles of target cell availability, and innate and adaptive immune responses in controlling the virus. Our results show that the rapid and substantial viral decline (about 2 to 4 logs within 1 day) after the peak can be explained by the killing of infected cells mediated by interferon activated cells, such as natural killer cells, during the innate immune response. After the viral load declines to a lower level, the loss of interferon-induced antiviral effect and an increased availability of target cells due to loss of the antiviral state can explain the observed short phase of viral plateau in which the viral level remains unchanged or even experiences a minor second peak in some animals. An adaptive immune response is needed in our model to explain the eventual viral clearance. This study provides a quantitative understanding of the biological factors that can explain the viral and interferon kinetics during a typical influenza virus infection.     

Characterization of Encapsulated Flavor Systems by NIR and Low-field TD-NMR: A Chemometric Approach

A quantitative method for measuring simultaneously the flavor and water contents in model spray-dried flavor delivery systems was developed using spectroscopic techniques and chemometrics. Nine encapsulated systems were prepared, consisting of a solid carrier (maltodextrin and gum arabic) and varying the amounts of water and flavor. The model flavors used in this work were a hydrophobic (limonene) and a more hydrophilic (2,5-dimethylpyrazine) single components. Near-infrared (NIR) and low-field time-domain nuclear magnetic resonance (low field TD-NMR) data were acquired on each system and analyzed using multivariate chemometric techniques to develop optimal prediction models. Partial least squares regression models showed good predictive ability, with coefficients of determination (R²) between 0.81 and 1.00 and low root mean square error of cross-validation values compared to the range of concentrations. The predictive ability of the chemometric models computed using the NIR spectra improved significantly when data were pre-processed using multiplicative signal correction. The development of good prediction models (i.e., robust models resulting in accurate predictions for water and flavor content) from the NMR relaxation data spectra was successful only for the hydrophobic limonene systems, yielding prediction models whose performance was better than the models obtained using the NIR data. Overall, NIR spectroscopy and NMR relaxometry were identified as complementary techniques rather than competitive methods in the characterization of encapsulated flavor systems.     

Prostaglandin I2 analogs inhibit proinflammatory cytokine production and T cell stimulatory function of dendritic cells

Signaling through the PGI(2) receptor (IP) has been shown to inhibit inflammatory responses in mouse models of respiratory syncytial viral infection and OVA-induced allergic responses. However, little is known about the cell types that mediate the anti-inflammatory function of PGI(2.) In this study, we determined that PGI(2) analogs modulate dendritic cell (DC) cytokine production, maturation, and function. We report that PGI(2) analogs (iloprost, cicaprost, treprostinil) differentially modulate the response of murine bone marrow-derived DC (BMDC) to LPS in an IP-dependent manner. The PGI(2) analogs decreased BMDC production of proinflammatory cytokines (IL-12, TNF-alpha, IL-1alpha, IL-6) and chemokines (MIP-1alpha, MCP-1) and increased the production of the anti-inflammatory cytokine IL-10 by BMDCs. The modulatory effect was associated with IP-dependent up-regulation of intracellular cAMP and down-regulation of NF-kappaB activity. Iloprost and cicaprost also suppressed LPS-induced expression of CD86, CD40, and MHC class II molecules by BMDCs and inhibited the ability of BMDCs to stimulate Ag-specific CD4 T cell proliferation and production of IL-5 and IL-13. These findings suggest that PGI(2) signaling through the IP may exert anti-inflammatory effects by acting on DC.     

Functional analysis of replication and stability regions of broad-host-range conjugative plasmid CTX-M3 from the IncL/M incompatibility group

Plasmid CTX-M3 (89 kb) isolated from Citrobacter freundii from a Warsaw hospital is a mosaic plasmid with replication functions 100% identical with those of pMU407.1 of the IncL/M group, conjugative operons with up to 60% homology to ColIb-P9 (IncI) and stability functions originating either from NR1(R100) (IncFII) or ColIb-P9 /R1/NR1 plasmids. We established the broad-host-range for pCTX-M3 and defined its minireplicon in Escherichia coli. We analyzed the role of stability cassettes and showed that the par operon consists of three orfs parA (stbA), parB (stbB) and nuc with a centromere-like region located upstream of the operon. Deletion of the par operon strongly destabilized pCTX-M3 despite the presence of the pemIK toxin-antidote system identical to that on NR1(R100) plasmids. Deletion of the pemIK operon had no effect on plasmid stability.     

Mass marking of Leuciscus idus larvae using Artemia salina as a vector of fluorescent dyes

A method for the mass marking of ide Leuciscus idus larvae by feeding them Artemia salina nauplii that were immersed in different solutions of alizarin red S, tetracycline hydrochloride and calcein was tested. The best quality marks were obtained after feeding fish for 4 days with nauplii that had been immersed in 200 mg l^?1 alizarin red S.