αV-Integrins Are Required for Mechanotransduction in MDCK Epithelial Cells

The properties of epithelial cells within tissues are regulated by their immediate microenvironment, which consists of neighboring cells and the extracellular matrix (ECM). Integrin heterodimers orchestrate dynamic assembly and disassembly of cell-ECM connections and thereby convey biochemical and mechanical information from the ECM into cells. However, the specific contributions and functional hierarchy between different integrin heterodimers in the regulation of focal adhesion dynamics in epithelial cells are incompletely understood. Here, we have studied the functions of RGD-binding αV-integrins in a Madin Darby Canine Kidney (MDCK) cell model and found that αV-integrins regulate the maturation of focal adhesions (FAs) and cell spreading. αV-integrin-deficient MDCK cells bound collagen I (Col I) substrate via α2β1-integrins but failed to efficiently recruit FA components such as talin, focal adhesion kinase (FAK), vinculin and integrin-linked kinase (ILK). The apparent inability to mature α2β1-integrin-mediated FAs and link them to cellular actin cytoskeleton led to disrupted mechanotransduction in αV-integrin deficient cells seeded onto Col I substrate.     

Quantifying Beetle-Mediated Effects on Gas Fluxes from Dung Pats

Agriculture is one of the largest contributors of the anthropogenic greenhouse gases (GHGs) responsible for global warming. Measurements of gas fluxes from dung pats suggest that dung is a source of GHGs, but whether these emissions are modified by arthropods has not been studied. A closed chamber system was used to measure the fluxes of carbon dioxide (CO₂), methane (CH₄) and nitrous oxide (N₂O) from dung pats with and without dung beetles on a grass sward. The presence of dung beetles significantly affected the fluxes of GHGs from dung pats. Most importantly, fresh dung pats emitted higher amounts of CO₂ and lower amounts of CH₄ per day in the presence than absence of beetles. Emissions of N₂O showed a distinct peak three weeks after the start of the experiment – a pattern detected only in the presence of beetles. When summed over the main grazing season (June–July), total emissions of CH₄ proved significantly lower, and total emissions of N₂O significantly higher in the presence than absence of beetles. While clearly conditional on the experimental conditions, the patterns observed here reveal a potential impact of dung beetles on gas fluxes realized at a small spatial scale, and thereby suggest that arthropods may have an overall effect on gas fluxes from agriculture. Dissecting the exact mechanisms behind these effects, mapping out the range of conditions under which they occur, and quantifying effect sizes under variable environmental conditions emerge as key priorities for further research.     

Quality along the Continuum: A Health Facility Assessment of Intrapartum and Postnatal Care in Ghana

Objective

To evaluate quality of routine and emergency intrapartum and postnatal care using a health facility assessment, and to estimate “effective coverage” of skilled attendance in Brong Ahafo, Ghana.

Methods

We conducted an assessment of all 86 health facilities in seven districts in Brong Ahafo. Using performance of key signal functions and the availability of relevant drugs, equipment and trained health professionals, we created composite quality categories in four dimensions: routine delivery care, emergency obstetric care (EmOC), emergency newborn care (EmNC) and non-medical quality. Linking the health facility assessment to surveillance data we estimated “effective coverage” of skilled attendance as the proportion of births in facilities of high quality.

Findings

Delivery care was offered in 64/86 facilities; only 3-13% fulfilled our requirements for the highest quality category in any dimension. Quality was lowest in the emergency care dimensions, with 63% and 58% of facilities categorized as “low” or “substandard” for EmOC and EmNC, respectively. This implies performing less than four EmOC or three EmNC signal functions, and/or employing less than two skilled health professionals, and/or that no health professionals were present during our visit. Routine delivery care was “low” or “substandard” in 39% of facilities, meaning 25/64 facilities performed less than six routine signal functions and/or had less than two skilled health professionals and/or less than one midwife. While 68% of births were in health facilities, only 18% were in facilities with “high” or “highest” quality in all dimensions.

Conclusion

Our comprehensive facility assessment showed that quality of routine and emergency intrapartum and postnatal care was generally low in the study region. While coverage with facility delivery was 68%, we estimated “effective coverage” of skilled attendance at 18%, thus revealing a large “quality gap.” Effective coverage could be a meaningful indicator of progress towards reducing maternal and newborn mortality.     

The U1 snRNP-associated factor Luc7p affects 5' splice site selection in yeast and human

yLuc7p is an essential subunit of the yeast U1 snRNP and contains two putative zinc fingers. Using RNA–protein cross-linking and directed site-specific proteolysis (DSSP), we have established that the N-terminal zinc finger of yLuc7p contacts the pre-mRNA in the 5′ exon in a region close to the cap. Modifying the pre-mRNA sequence in the region contacted by yLuc7p affects splicing in a yLuc7p-dependent manner indicating that yLuc7p stabilizes U1 snRNP–pre-mRNA interaction, thus reminding of the mode of action of another U1 snRNP component, Nam8p. Database searches identified three putative human yLuc7p homologs (hLuc7A, hLuc7B1 and hLuc7B2). These proteins have an extended C-terminal tail rich in RS and RE residues, a feature characteristic of splicing factors. Consistent with a role in pre-mRNA splicing, hLuc7A localizes in the nucleus and antibodies raised against hLuc7A specifically co-precipitate U1 snRNA from human cell extracts. Interestingly, hLuc7A overexpression affects splicing of a reporter in vivo. Taken together, our data suggest that the formation of a wide network of protein–RNA interactions around the 5′ splice site by U1 snRNP-associated factors contributes to alternative splicing regulation.     

Transgenerational effects of parental larval diet on offspring development time, adult body size and pathogen resistance in Drosophila melanogaster

Environmental conditions experienced by parents are increasingly recognized to affect offspring performance. We set out to investigate the effect of parental larval diet on offspring development time, adult body size and adult resistance to the bacterium Serratia marcescens in Drosophila melanogaster. Flies for the parental generation were raised on either poor or standard diet and then mated in the four possible sex-by-parental diet crosses. Females that were raised on poor food produced larger offspring than females that were raised on standard food. Furthermore, male progeny sired by fathers that were raised on poor food were larger than male progeny sired by males raised on standard food. Development times were shortest for offspring whose one parent (mother or the father) was raised on standard and the other parent on poor food and longest for offspring whose parents both were raised on poor food. No evidence for transgenerational effects of parental diet on offspring disease resistance was found. Although paternal effects have been previously demonstrated in D. melanogaster, no earlier studies have investigated male-mediated transgenerational effects of diet in this species. The results highlight the importance of not only considering the relative contribution each parental sex has on progeny performance but also the combined effects that the two sexes may have on offspring performance.     

Cloning, expression, and characterization of novel thermostable family 7 cellobiohydrolases

As part of the effort to find better cellulases for bioethanol production processes, we were looking for novel GH-7 family cellobiohydrolases, which would be particularly active on insoluble polymeric substrates and participate in the rate-limiting step in the hydrolysis of cellulose. The enzymatic properties were studied and are reported here for family 7 cellobiohydrolases from the thermophilic fungi Acremonium thermophilum, Thermoascus aurantiacus, and Chaetomium thermophilum. The Trichoderma reesei Cel7A enzyme was used as a reference in the experiments. As the native T. aurantiacus Cel7A has no carbohydrate-binding module (CBM), recombinant proteins having the CBM from either the C. thermophilum Cel7A or the T. reesei Cel7A were also constructed. All these novel acidic cellobiohydrolases were more thermostable (by 4-10 degrees C) and more active (two- to fourfold) in hydrolysis of microcrystalline cellulose (Avicel) at 45 degrees C than T. reesei Cel7A. The C. thermophilum Cel7A showed the highest specific activity and temperature optimum when measured on soluble substrates. The most effective enzyme for Avicel hydrolysis at 70 degrees C, however, was the 2-module version of the T. aurantiacus Cel7A, which was also relatively weakly inhibited by cellobiose. These results are discussed from the structural point of view based on the three-dimensional homology models of these enzymes.     

hiPSC-derived hepatocytes closely mimic the lipid profile of primary hepatocytes: A future personalised cell model for studying the lipid metabolism of the liver

Hepatocyte-like cells (HLCs) differentiated from human-induced pluripotent stem cells offer an alternative platform to primary human hepatocytes (PHHs) for studying the lipid metabolism of the liver. However, despite their great potential, the lipid profile of HLCs has not yet been characterized. Here, we comprehensively studied the lipid profile and fatty acid (FA) metabolism of HLCs and compared them with the current standard hepatocyte models: HepG2 cells and PHHs. We differentiated HLCs by five commonly used methods from three cell lines and thoroughly characterized them by gene and protein expression. HLCs generated by each method were assessed for their functionality and the ability to synthesize, elongate, and desaturate FAs. In addition, lipid and FA profiles of HLCs were investigated by both mass spectrometry and gas chromatography and then compared with the profiles of PHHs and HepG2 cells. HLCs resembled PHHs by expressing hepatic markers: secreting albumin, lipoprotein particles, and urea, and demonstrating similarities in their lipid and FA profile. Unlike HepG2 cells, HLCs contained low levels of lysophospholipids similar to the content of PHHs. Furthermore, HLCs were able to efficiently use the exogenous FAs available in their medium and simultaneously modify simple lipids into more complex ones to fulfill their needs. In addition, we propose that increasing the polyunsaturated FA supply of the culture medium may positively affect the lipid profile and functionality of HLCs. In conclusion, our data showed that HLCs provide a functional and relevant model to investigate human lipid homeostasis at both molecular and cellular levels.     

Thermotolerant and thermostable laccases

Laccases are phenol-oxidizing, usually four-copper containing metalloenzymes. For industrial and biotechnological purposes, laccases were among the first fungal oxidoreductases providing larger-scale applications such as removal of polyphenols in wine and beverages, conversion of toxic compounds and textile dyes in waste waters, and in bleaching and removal of lignin from wood and non-wood fibres. In order to facilitate novel and more efficient bio-catalytic process applications, there is a need for laccases with improved biochemical properties, such as thermostability and thermotolerance. This review gives a current overview on the sources and characteristics of such laccases, with particular emphasis on the fungal enzymes.