Novel carbohydrate binding modules in the surface anchored α‐amylase of Eubacterium rectale provide a molecular rationale for the range of starches used by this organism in the human gut

Gut bacteria recognize accessible glycan substrates within a complex environment. Carbohydrate binding modules (CBMs) of cell surface glycoside hydrolases often drive binding to the target substrate. Eubacterium rectale, an important butyrate‐producing organism in the gut, consumes a limited range of substrates, including starch. Host consumption of resistant starch increases the abundance of E. rectale in the intestine, likely because it successfully captures the products of resistant starch degradation by other bacteria. Here, we demonstrate that the cell wall anchored starch‐degrading α‐amylase, Amy13K of E. rectale harbors five CBMs that all target starch with differing specificities. Intriguingly these CBMs efficiently bind to both regular and high amylose corn starch (a type of resistant starch), but have almost no affinity for potato starch (another type of resistant starch). Removal of these CBMs from Amy13K reduces the activity level of the enzyme toward corn starches by ～40‐fold, down to the level of activity toward potato starch, suggesting that the CBMs facilitate activity on corn starch and allow its utilization in vivo. The specificity of the Amy13K CBMs provides a molecular rationale for why E. rectale is able to only use certain starch types without the aid of other organisms.     

The Dwarf Phenotype in GH240B Mice,Haploinsufficient for the Autism Candidate Gene Neurobeachin,Is Caused by Ectopic Expression of Recombinant Human Growth Hormone

Two knockout mouse models for the autism candidate gene Neurobeachin (Nbea) have been generated independently. Although both models have similar phenotypes, one striking difference is the dwarf phenotype observed in the heterozygous configuration of the GH240B model that is generated by the serendipitous insertion of a promoterless human growth hormone (hGH) genomic fragment in the Nbea gene. In order to elucidate this discrepancy, the dwarfism present in this Nbea mouse model was investigated in detail. The growth deficiency in Nbea ^+/− mice coincided with an increased percentage of fat mass and a decrease in bone mineral density. Low but detectable levels of hGH were detected in the pituitary and hypothalamus of Nbea ^+/− mice but not in liver, hippocampus nor in serum. As a consequence, several members of the mouse growth hormone (mGH) signaling cascade showed altered mRNA levels, including a reduction in growth hormone-releasing hormone mRNA in the hypothalamus. Moreover, somatotrope cells were less numerous in the pituitary of Nbea ^+/− mice and both contained and secreted significantly less mGH resulting in reduced levels of circulating insulin-like growth factor 1. These findings demonstrate that the random integration of the hGH transgene in this mouse model has not only inactivated Nbea but has also resulted in the tissue-specific expression of hGH causing a negative feedback loop, mGH hyposecretion and dwarfism.     

The Ecology of Influenza A Viruses in Wild Birds in Southern Africa

Avian influenza viruses (AIVs) are pathogens of global concern, but there has been little previous research on avian influenza in southern Africa and almost nothing is known about the dynamics of AIVs in the region. We counted, captured and sampled birds regularly at five sites, two in South Africa (Barberspan and Strandfontein) and one in each of Botswana (Lake Ngami), Mozambique (Lake Chuali) and Zimbabwe (Lakes Manyame and Chivero) between March 2007 and May 2009. The South African and Zimbabwean sites were visited every 2 months and the sites in Botswana and Mozambique every 4 months. During each visit we undertook 5-7?days of standardised bird counts followed by 5-10?days of capturing and sampling water-associated birds. We sampled 4,977 birds of 165 different species and completed 2,503 half-hour point counts. We found 125 positive rRT-PCR cases of avian influenza across all sites. Two viruses (H1N8 and H3N8) were isolated and additional H5, H6 and H7 strains were identified. We did not positively identify any highly pathogenic H5N1. Overall viral prevalence (2.51%) was similar to the lower range of European values, considerable spatial and temporal variation occurred in viral prevalence, and there was no detectable influence of the annual influx of Palearctic migrants. Although waterbirds appear to be the primary viral carriers, passerines may link wild birds and poultry. While influenza cycles are probably driven by the bird movements that result from rainfall patterns, the epidemiology of avian influenza in wild birds in the subregion is complex and there appears to be the possibility for viral transmission throughout the year.     

Generic Raman‐based calibration models enabling real‐time monitoring of cell culture bioreactors

Raman‐based multivariate calibration models have been developed for real‐time in situ monitoring of multiple process parameters within cell culture bioreactors. Developed models are generic, in the sense that they are applicable to various products, media, and cell lines based on Chinese Hamster Ovarian (CHO) host cells, and are scalable to large pilot and manufacturing scales. Several batches using different CHO‐based cell lines and corresponding proprietary media and process conditions have been used to generate calibration datasets, and models have been validated using independent datasets from separate batch runs. All models have been validated to be generic and capable of predicting process parameters with acceptable accuracy. The developed models allow monitoring multiple key bioprocess metabolic variables, and hence can be utilized as an important enabling tool for Quality by Design approaches which are strongly supported by the U.S. Food and Drug Administration. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1004–1013, 2015     

A novel 3D heterotypic spheroid model for studying extracellular vesicle-mediated tumour and immune cell communication

Cancer-derived extracellular vesicles (EVs) have emerged as important mediators of tumour-host interactions, and they have been shown to exert various functional effects in immune cells. In most of the studies on human immune cells, EVs have been isolated from cancer cell culture medium or patients' body fluids and added to the immune cell cultures. In such a setting, the physiological relevance of the chosen EV concentration is unknown and the EV isolation method and the timing of EV administration may bias the results. In the current study we aimed to develop an experimental cell culture model to study EV-mediated effects in human T and B cells at conditions mimicking the tumour microenvironment. We constructed a human prostate cancer cell line PC3 producing GFP-tagged EVs (PC3-CD63-GFP cells) and developed a 3D heterotypic spheroid model composed of PC3-CD63-GFP cells and human peripheral blood mononuclear cells (PBMCs). The transfer of GFP-tagged EVs from PC3-CD63-GFP cells to the lymphocytes was analysed by flow cytometry and fluorescence imaging. The endocytic pathway was investigated using three endocytosis inhibitors. Our results showed that GFP-tagged EVs interacted with a large fraction of B cells, however, the majority of EVs were not internalised by B cells but rather remained bound at the cell surface. T cell subsets differed in their ability to interact with the EVs - 15.7–24.1% of the total CD3⁺ T cell population interacted with GFP-tagged EVs, while only 0.3–5.8% of CD8⁺ T were GFP positive. Furthermore, a fraction of EVs were internalised in CD3⁺ T cells via macropinocytosis. Taken together, the heterotypic PC3-CD63-GFP and PBMC spheroid model provides the opportunity to study the interactions and functional effects of cancer-derived EVs in human immune cells at conditions mimicking the tumour microenvironment.     

Genetic structure,phylogeography, and demography of Anadara tuberculosa (Bivalvia) from East Pacific as revealed by mtDNA: Implications to conservation

Wild populations of the pustulose ark, Anadara tuberculosa (Bivalvia), an emblematic species of the East Pacific mangrove ecosystem declined in South American countries (Colombia, Ecuador, and Peru) mainly due to overharvesting and habitat loss or degradation. Understanding the genetic aspects of geographic variations and population structure of A. tuberculosa, currently unknown, appears as a priority to fishery authorities in order to elaborate integrated and collaborative conservation policies for fishery management, aquaculture, and stock enhancement programs. We used mtDNA sequence data to investigate haplotype diversity, genetic structure, and demography of A. tuberculosa. Results indicate genetic homogeneity of populations distributed north and south of the equator, respectively. However, statistically significant differentiation emerged between northern and southern populations with pairwise ф_ST values ranging between 0.036 and 0.092. The oceanic current system acting in the area (Panama Current and Humboldt Current) might play a role in limiting the larval dispersal of the species, still poorly understood. Demography reconstruction supported recent population expansion, possibly started after last glacial maximum. Our results would suggest separate and independent management of populations north and south of the equator.