Enzymes involved in hepatic acylglycerol metabolism in the chicken

In laying hens, massive hepatic mobilization of fatty acids is required for the synthesis of oocyte-targeted very-low density lipoproteins (VLDL). The current study aims at identification of enzymes that hydrolyze hepatic acylglycerol stores regulated in a fashion compatible with supporting enhanced VLDL synthesis. We show that unlike mammals, chickens express adipose triglyceride lipase (ATGL) also in liver, where it is upregulated by fasting, while the enzyme patatin-like phospholipase domain-containing lipase 3 (PNPLA3) is suppressed. For the first time in any system, we show that hepatic arylacetamide deacetylase (AADA) is upregulated by fasting, and that its affinity for an insoluble carboxylester substrate is compatible with an in-vivo function similar to that of ATGL. Unknown heretofore, hepatic expression of chicken AADA is estrogen-responsive, and is induced to the same degree as the stimulation of VLDL-production by estrogen. These observations support roles of chicken ATGL, PNPLA3, and AADA in acylglycerol metabolism related to the high rates of VLDL synthesis that are essential for reproduction.     

DNA condensation by TmHU studied by optical tweezers,AFM and molecular dynamics simulations

The compaction of DNA by the HU protein from Thermotoga maritima (TmHU) is analysed on a single-molecule level by the usage of an optical tweezers-assisted force clamp. The condensation reaction is investigated at forces between 2 and 40 pN applied to the ends of the DNA as well as in dependence on the TmHU concentration. At 2 and 5 pN, the DNA compaction down to 30% of the initial end-to-end distance takes place in two regimes. Increasing the force changes the progression of the reaction until almost nothing is observed at 40 pN. Based on the results of steered molecular dynamics simulations, the first regime of the length reduction is assigned to a primary level of DNA compaction by TmHU. The second one is supposed to correspond to the formation of higher levels of structural organisation. These findings are supported by results obtained by atomic force microscopy.     

The biological and structural characterization of Mycobacterium tuberculosis UvrA provides novel insights into its mechanism of action

Mycobacterium tuberculosis is an extremely well adapted intracellular human pathogen that is exposed to multiple DNA damaging chemical assaults originating from the host defence mechanisms. As a consequence, this bacterium is thought to possess highly efficient DNA repair machineries, the nucleotide excision repair (NER) system amongst these. Although NER is of central importance to DNA repair in M. tuberculosis, our understanding of the processes in this species is limited. The conserved UvrABC endonuclease represents the multi-enzymatic core in bacterial NER, where the UvrA ATPase provides the DNA lesion-sensing function. The herein reported genetic analysis demonstrates that M. tuberculosis UvrA is important for the repair of nitrosative and oxidative DNA damage. Moreover, our biochemical and structural characterization of recombinant M. tuberculosis UvrA contributes new insights into its mechanism of action. In particular, the structural investigation reveals an unprecedented conformation of the UvrB-binding domain that we propose to be of functional relevance. Taken together, our data suggest UvrA as a potential target for the development of novel anti-tubercular agents and provide a biochemical framework for the identification of small-molecule inhibitors interfering with the NER activity in M. tuberculosis.     

Distribution of ergot alkaloids and ricinoleic acid in different milling fractions

The sclerotia of the fungus Claviceps sp. are still a challenge for the milling industry. Ergot sclerotia are a constant contamination of the rye crop and have to be removed by modern milling technologies. Changing sizes and coloration of the sclerotia make it difficult to separate them from the grain. Ergot sclerotia are a problem when cleaning is insufficient and non-separated specimens or sclerotia fragments get into the milling stream and thus ergot alkaloids are distributed into the different cereal fractions. In model milling experiments, the residues of ergot in rye flour and the distribution of ergot into different milling fractions were investigated. Rye grains were mixed with whole ergot sclerotia and in another experiment with ergot powder and cleaned afterwards before milling. The ergot alkaloids ergometrine, ergosine, ergotamine, ergocornine, ergocryptine, ergocristineand their related isomeric forms (-inine-forms), and additionally ricinoleic acid as a characteristic component of ergot, were quantified in the different milling fractions. From the first experiment, it can be shown that after harvesting even simple contact of sclerotia with bulk grains during ordinary handling or movement of bulk grain in the granary is sufficient to contaminate all the healthy or sound rye grains with ergot alkaloids. Thereby, the amount of ergot residue correlates with the amount of peripheral layers of rye grains in the flour. In an additional experiment without sclerotia specimens, bulk rye grains were loaded with powder of sclerotia. After subsequent cleaning, aconcentration of ergot alkaloids was detected, which was tenfold higher than the ergot alkaloidconcentration of the experiment with intact ergot sclerotia.     

Developmental refinement of hair cell synapses tightens the coupling of Ca2+ influx to exocytosis

Cochlear inner hair cells (IHCs) develop from pre‐sensory pacemaker to sound transducer. Here, we report that this involves changes in structure and function of the ribbon synapses between IHCs and spiral ganglion neurons (SGNs) around hearing onset in mice. As synapses matured they changed from holding several small presynaptic active zones (AZs) and apposed postsynaptic densities (PSDs) to one large AZ/PSD complex per SGN bouton. After the onset of hearing (i) IHCs had fewer and larger ribbons; (ii) Ca_V1.3 channels formed stripe‐like clusters rather than the smaller and round clusters at immature AZs; (iii) extrasynaptic Ca_V1.3‐channels were selectively reduced, (iv) the intrinsic Ca²⁺ dependence of fast exocytosis probed by Ca²⁺ uncaging remained unchanged but (v) the apparent Ca²⁺ dependence of exocytosis linearized, when assessed by progressive dihydropyridine block of Ca²⁺ influx. Biophysical modeling of exocytosis at mature and immature AZ topographies suggests that Ca²⁺ influx through an individual channel dominates the [Ca²⁺] driving exocytosis at each mature release site. We conclude that IHC synapses undergo major developmental refinements, resulting in tighter spatial coupling between Ca²⁺ influx and exocytosis.     

Nectar robbing improves male reproductive success of the endangered Aconitum napellus ssp. lusitanicum

Nectar robbery is usually thought to impact negatively on the reproductive success of plants, but also neutral or even positive effects have been reported. Very few studies have investigated the effects of nectar robbing on the behaviour of legitimate pollinators so far. Such behavioural changes may lead to the reduction of geitonogamy or to increased pollen movement. We simulated nectar robbing in experimental sites as well as in natural populations of Aconitum napellus ssp. lusitanicum, a rare plant pollinated by long-tongued bumblebees. In an experimental setup, we removed the nectaries of 40 % of the flowers, which is similar to rates of robbing observed in wild populations. Patches of plants with experimentally robbed flowers were compared with control patches containing plants with untreated flowers. We observed pollinator behaviour, mimicked male reproductive success (pollen dispersal) using fluorescent dye, and measured female reproductive success (seed set). The main legitimate visitors were bumblebees while honeybees were often observed robbing nectar. They did so by “base working”, i.e. sliding between tepals. Bumblebees tended to visit fewer flowers per plant and spent less time per single flower when these had been experimentally robbed. This change in behaviour consequently increased the proportion of flowers visited by bumblebees in patches with robbed flowers. Fluorescent dye mimicking pollen flow was dispersed larger distances after pollinators had visited patches with robbed flowers compared to control patches. Average seed set per plant was not affected by nectar robbing. Our results demonstrated that A. napellus does not suffer from nectar robbery but may rather benefit via improved pollen dispersal and thus, male reproductive success. Knowledge on such combined effects of behavioural changes of pollinators due to nectar robbery is important to understand the evolutionary significance of exploiters of such mutualistic relationships between plants and their pollinators.     

Facets of diazotrophy in the oxygen minimum zone waters off Peru

Nitrogen fixation, the biological reduction of dinitrogen gas (N₂) to ammonium (NH₄⁺), is quantitatively the most important external source of new nitrogen (N) to the open ocean. Classically, the ecological niche of oceanic N₂ fixers (diazotrophs) is ascribed to tropical oligotrophic surface waters, often depleted in fixed N, with a diazotrophic community dominated by cyanobacteria. Although this applies for large areas of the ocean, biogeochemical models and phylogenetic studies suggest that the oceanic diazotrophic niche may be much broader than previously considered, resulting in major implications for the global N-budget. Here, we report on the composition, distribution and abundance of nifH, the functional gene marker for N₂ fixation. Our results show the presence of eight clades of diazotrophs in the oxygen minimum zone (OMZ) off Peru. Although proteobacterial clades dominated overall, two clusters affiliated to spirochaeta and archaea were identified. N₂ fixation was detected within OMZ waters and was stimulated by the addition of organic carbon sources supporting the view that non-phototrophic diazotrophs were actively fixing dinitrogen. The observed co-occurrence of key functional genes for N₂ fixation, nitrification, anammox and denitrification suggests that a close spatial coupling of N-input and N-loss processes exists in the OMZ off Peru. The wide distribution of diazotrophs throughout the water column adds to the emerging view that the habitat of marine diazotrophs can be extended to low oxygen/high nitrate areas. Furthermore, our statistical analysis suggests that NO₂⁻ and PO₄³⁻ are the major factors affecting diazotrophic distribution throughout the OMZ. In view of the predicted increase in ocean deoxygenation resulting from global warming, our findings indicate that the importance of OMZs as niches for N₂ fixation may increase in the future.     

A functional high‐content miRNA screen identifies miR‐30 family to boost recombinant protein production in CHO cells

The steady improvement of mammalian cell factories for the production of biopharmaceuticals is a key challenge for the biotechnology community. Recently, small regulatory microRNAs (miRNAs) were identified as novel targets for optimizing Chinese hamster ovary (CHO) production cells as they do not add any translational burden to the cell while being capable of regulating entire physiological pathways. The aim of the present study was to elucidate miRNA function in a recombinant CHO‐SEAP cell line by means of a genome‐wide high‐content miRNA screen. This screen revealed that out of the 1, 139 miRNAs examined, 21% of the miRNAs enhanced cell‐specific SEAP productivity mainly resulting in elevated volumetric yields, while cell proliferation was accelerated by 5% of the miRNAs. Conversely, cell death was diminished by 13% (apoptosis) or 4% (necrosis) of all transfected miRNAs. Besides these large number of identified target miRNAs, the outcome of our studies suggest that the entire miR‐30 family substantially improves bioprocess performance of CHO cells. Stable miR‐30 over expressing cells outperformed parental cells by increasing SEAP productivity or maximum cell density of approximately twofold. Our results highlight the application of miRNAs as powerful tools for CHO cell engineering, identified the miR‐30 family as a critical component of cell proliferation, and support the notion that miRNAs are powerful determinants of cell viability.