Distinct thyroid hormone‐dependent expression of trkB and p75NGFR in nonneuronal cells during the critical TH‐dependent period of the cochlea

Analyzing the thyroid hromone (TH)‐dependent period of the inner ear, we observed that the presence of triiodothyronine (T3) between postnatal day 3 (P3) and P12 is sufficient for functional maturation of the auditory system. Within this short time period, an unusual transient TH‐dependent expression of nonneuronal neurotrophin receptors (NT‐R) trkB and p75^NGFR was observed in correlation with neuronal and morphogenetic processes. The availability of thyroid hormone was revealed to be invariably correlated with (a) a transient expression of full‐length trkB in TRα1‐, TRα2‐ and TRβ1‐expressing hair cells concomitant to the segregation of afferent fibers and the synaptogenesis of efferent fibers; and (b) a transient expression of p75^NGFR in TRα1‐ and TRβ1‐expressing great epithelia ridge cells in direct spatiotemporal correlation with the appearance of apoptotic cells and morphogenetic maturation of the organ. For the first time, these data suggest a TH dependency of the expression of neurotrophin receptors in nonneuronal cells. A potential role of these peculiar neurotrophin receptor expression for the conversion of the biological function of TH on innervation patterning and morphogenesis during the critical TH‐dependent period of the inner ear may be considered. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 338–356, 1999     

A database of mutants and effects of site-directed mutagenesis experiments on G protein-coupled receptors

A database system and computer programs for storage and retrieval of information about guanine nucleotide-binding protein (G protein) -coupled receptor mutants and associated biological effects have been developed. Mutation data on the receptors were collected from the literature and a database of mutants and effects of mutations was developed. The G protein-coupled receptor, family A, point mutation database (GRAP) provides detailed information on ligand-binding and signal transduction properties of more than 2130 receptor mutants. The amino acid sequences of receptors for which mutation experiments have been reported were aligned, and from this alignment mutation data may be retrieved. Alternatively, a search form allowing detailed specification of which mutants to retrieve may be used, for example, to search for specific amino acid substitutions, substitutions in specific protein domains or reported biological effects. Furthermore, ligand and bibliographic oriented queries may be performed. GRAP is available on the Internet (URL: http://www-grap.fagmed.uit.no/GRAP/homepage.html ) using the World-Wide Web system. © 1996 Wiley-Liss, Inc.     

Sleeping site ecology,but not sex,affect ecto- and hemoparasite risk,in sympatric,arboreal primates (<Emphasis Type="Italic">Avahi occidentalis</Emphasis> and <Emphasis Type="Italic">Lepilemur edwardsi</Emphasis>)

Background

A central question in evolutionary parasitology is to what extent ecology impacts patterns of parasitism in wild host populations. In this study, we aim to disentangle factors influencing the risk of parasite exposure by exploring the impact of sleeping site ecology on infection with ectoparasites and vector-borne hemoparasites in two sympatric primates endemic to Madagascar. Both species live in the same dry deciduous forest of northwestern Madagascar and cope with the same climatic constraints, they are arboreal, nocturnal, cat-sized and pair-living but differ prominently in sleeping site ecology. The Western woolly lemur (Avahi occidentalis) sleeps on open branches and frequently changes sleeping sites, whereas the Milne-Edward’s sportive lemur (Lepilemur edwardsi) uses tree holes, displaying strong sleeping site fidelity. Sleeping in tree holes should confer protection from mosquito-borne hemoparasites, but should enhance the risk for ectoparasite infestation with mites and nest-adapted ticks. Sex may affect parasite risk in both species comparably, with males bearing a higher risk than females due to an immunosuppressive effect of higher testosterone levels in males or to sex-specific behavior. To explore these hypotheses, ectoparasites and blood samples were collected from 22 individuals of A. occidentalis and 26 individuals of L. edwardsi during the dry and rainy season.

Results

L. edwardsi, but not A. occidentalis, harbored ectoparasites, namely ticks (Haemaphysalis lemuris [Ixodidae], Ornithodoros sp. [Argasidae]) and mites (Aetholaelaps trilyssa, [Laelapidae]), suggesting that sleeping in tree holes promotes infestation with ectoparasites. Interestingly, ectoparasites were found solely in the hot, rainy season with a prevalence of 75% (N = 16 animals). Blood smears were screened for the presence and infection intensity of hemoparasites. Microfilariae were detected in both species. Morphological characteristics suggested that each lemur species harbored two different filarial species. Prevalence of microfilarial infection was significantly lower in L. edwardsi than in A. occidentalis. No significant difference in infection intensity between the two host species, and no effect of season, daytime of sampling or sex on prevalence or infection intensity was found. In neither host species, parasite infection showed an influence on body weight as an indicator for body condition.

Conclusions

Our findings support that sleeping site ecology affects ectoparasite infestation in nocturnal, arboreal mammalian hosts in the tropics, whereas there is no significant effect of host sex. The influence of sleeping site ecology to vector-borne hemoparasite risk is less pronounced. The observed parasite infections did not affect body condition and thus may be of minor importance for shaping reproductive fitness. Findings provide first evidence for the specific relevance of sleeping site ecology on parasitism in arboreal and social mammals. Further, our results increase the sparse knowledge on ecological drivers of primate host-parasite interactions and transmission pathways in natural tropical environments.

     

Adenosine A<Subscript>2A</Subscript> receptor regulates expression of vascular endothelial growth factor in feto-placental endothelium from normal and late-onset pre-eclamptic pregnancies

We aim to investigate whether A_2A/nitric oxide-mediated regulation of vascular endothelial growth factor (VEGF) expression is impaired in feto-placental endothelial cells from late-onset pre-eclampsia. Cultures of human umbilical vein endothelial cells (HUVECs) and human placental microvascular endothelial cells (hPMECs) from normal and pre-eclamptic pregnancies were used. Assays by using small interference RNA (siRNA) for A_2A were performed, and transfected cells were used for estimation of messenger RNA (mRNA) levels of VEGF, as well as for cell proliferation and angiogenesis in vitro. CGS-21680 (A_2A agonist, 24 h) increases HUVEC and hPMEC proliferation in a dose response manner. Furthermore, similar to CGS-21680, the nitric oxide donor, S-nitroso-N-acetyl-penicillamine oxide (SNAP), increased cell proliferation in a dose response manner (logEC₅₀ 10^?9.2 M). In hPMEC, CGS-21680 increased VEGF protein levels in both normal (～1.5-fold) and pre-eclamptic pregnancies (～1.2-fold), an effect blocked by the A_2A antagonist, ZM-241385 (10^?5 M) and the inhibitor of NO synthase, N _ω-nitro-L-arginine methyl ester hydrochloride (L-NAME). Subsequently, SNAP partially recovered cell proliferation and in vitro angiogenesis capacity of cells from normal pregnancies exposed to siRNA for A_2A. CGS-21680 also increased (～1.5-fold) the level of VEGF mRNA in HUVEC from normal pregnancies, but not in pre-eclampsia. Additionally, transfection with siRNA for A_2A decrease (～30 %) the level of mRNA for VEGF in normal pregnancy compared to untransfected cells, an effect partially reversed by co-incubation with SNAP. The A_2A-NO-VEGF pathway is present in endothelium from microcirculation and macrocirculation in both normal and pre-eclamptic pregnancies. However, NO signaling pathway seems to be impaired in HUVEC from pre-eclampsia.     

Intra- and Intergenomic variation of Ploidy and Clonality characterize <Emphasis Type="Italic">Phytophthora capsici</Emphasis> on <Emphasis Type="Italic">Capsicum</Emphasis> sp. in Taiwan

Phytophthora capsici is a devastating disease of pepper (Capsicum sp.) in Taiwan causing complete loss of commercial fields. The objective of this study was to characterize genetic diversity for 38 newly collected isolates and three historical isolates. Analysis of data includes whole genome sequence for two new isolates and for two isolates collected previously in 1987 and 1995. In addition, 63 single nucleotide polymorphism loci were genotyped using targeted-sequencing, revealing 27 unique genotypes. Genotypes fell into three genetic groups: two of the groups contain 90% (n = 33) of the 2016 isolates, are triploid (or higher), are exclusively the A2 mating type and appear to be two distinct clonal lineages. The isolates from 2016 that grouped with the historical isolates are diploid and the A1 mating type. Whole genome sequence revealed that ploidy varies by linkage group, and it appears the A2 clonal lineages may have switched mating type due to increased ploidy. Most of the isolates were recently race-typed on a set of differential C. annuum, and although there was no direct correlation between virulence and ploidy, many of the triploid isolates were less virulent as compared to the historical diploid isolates. The implications for breeding resistant pepper and conducting population analyses are discussed.     

High‐throughput downstream process development for cell‐based products using aqueous two‐phase systems (ATPS) – A case study

The availability of preparative‐scale downstream processing strategies for cell‐based products presents a critical juncture between fundamental research and clinical development. Aqueous two‐phase systems (ATPS) present a gentle, scalable, label‐free, and cost‐effective method for cell purification, and are thus a promising tool for downstream processing of cell‐based therapeutics. Here, the application of a previously developed robotic screening platform that enables high‐throughput cell partitioning analysis in ATPS is reported. In the present case study a purification strategy for two model cell lines based on high‐throughput screening (HTS)‐data and countercurrent distribution (CCD)‐modeling, and validated the CCD‐model experimentally is designed. The obtained data are shown an excellent congruence between CCD‐model and experimental data, indicating that CCD‐models in combination with HTS‐data are a powerful tool in downstream process development. Finally, the authors are shown that while cell cycle phase significantly influences cell partitioning, cell type specific differences in surface properties are the main driving force in charge‐dependent separation of HL‐60 and L929 cells. In order to design a highly robust purification process it is, however, advisable to maintain constant growth conditions.     

Compensation masks trophic cascades in complex food webs

Ecological networks, or food webs, describe the feeding relationships between interacting species within an ecosystem. Understanding how the complexity of these networks influences their response to changing top-down control is a central challenge in ecology. Here, we provide a model-based investigation of trophic cascades — an oft-studied ecological phenomenon that occurs when changes in the biomass of top predators indirectly effect changes in the biomass of primary producers — in complex food webs that are representative of the structure of real ecosystems. Our results reveal that strong cascades occur primarily in low richness and weakly connected food webs, a result in agreement with some prior predictions. The primary mechanism underlying weak or absent cascades was a strong compensatory response; in most webs, predators induced large population level cascades that were masked by changes in the opposite direction by other species in the same trophic guild. Thus, the search for a general theory of trophic cascades in food webs should focus on uncovering features of real ecosystems that promote biomass compensation within functional guilds or trophic levels.     

Exploring the linkage between cell culture process parameters and downstream processing utilizing a plackett‐burman design for a model monoclonal antibody

Linkage of upstream cell culture with downstream processing and purification is an aspect of Quality by Design crucial for efficient and consistent production of high quality biopharmaceutical proteins. In a previous Plackett‐Burman screening study of parallel bioreactor cultures we evaluated main effects of 11 process variables, such as agitation, sparge rate, feeding regimens, dissolved oxygen set point, inoculation density, supplement addition, temperature, and pH shifts. In this follow‐up study, we observed linkages between cell culture process parameters and downstream capture chromatography performance and subsequent antibody attributes. In depth analysis of the capture chromatography purification of harvested cell culture fluid yielded significant effects of upstream process parameters on host cell protein abundance and behavior. A variety of methods were used to characterize the antibody both after purification and buffer formulation. This analysis provided insight in to the significant impacts of upstream process parameters on aggregate formation, impurities, and protein structure. This report highlights the utility of linkage studies in identifying how changes in upstream parameters can impact downstream critical quality attributes. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:163–170, 2017