Cryptic diversity within the rotifer Polyarthra dolichoptera along an altitudinal gradient

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Mercaptosuccinate metabolism in Variovorax paradoxus strain B4—a proteomic approach

Variovorax paradoxus B4 was isolated due to its ability to degrade the organic thiol compound mercaptosuccinate, which could be a promising precursor for novel polythioesters. The analysis of the proteome of this Gram-negative bacterium revealed several proteins with significantly increased expression during growth of cells with mercaptosuccinate as carbon source when compared to cells grown with gluconate or succinate. Among those, a large number of proteins involved in amino acid metabolism were identified, e.g., adenosylhomocysteinase and glutamate-ammonia ligase. Additionally, detection of superoxide dismutase strengthened the assumption of enhanced stress levels in mercaptosuccinate-grown cells. Several isoforms of a rhodanese domain-containing protein exhibited particularly increased expression during growth with mercaptosuccinate in comparison to gluconate (factor 14.2, stationary phase) or to succinate (factor 15.4, stationary phase). Besides this, augmented expression of the hypothetical protein VAPA_1c41240 raised attention. VAPA_1c41240 exhibited up to 13.3-fold (mercaptosuccinate vs gluconate) or 9.5-fold (mercaptosuccinate vs succinate) increased expression levels, and in silico searches revealed that this protein might be a thiol dioxygenase. Based on these results, a novel degradation pathway is proposed for mercaptosuccinate. The newly identified putative mercaptosuccinate dioxygenase could convert mercaptosuccinate to sulfinosuccinate by the introduction of two molecules of oxygen. Subsequently, sulfinosuccinate would be cleaved into succinate and sulfite either by a yet unknown enzyme, by spontaneous hydrolysis, or by the putative mercaptosuccinate dioxygenase itself. Succinate could then enter the central metabolism, while detoxification of sulfite could be achieved by the previously identified putative molybdopterin oxidoreductase. Biochemical studies will be done in the future to confirm this pathway.     

Shedding of glycan‐modifying enzymes by signal peptide peptidase‐like 3 (SPPL3) regulates cellular N‐glycosylation

Protein N‐glycosylation is involved in a variety of physiological and pathophysiological processes such as autoimmunity, tumour progression and metastasis. Signal peptide peptidase‐like 3 (SPPL3) is an intramembrane‐cleaving aspartyl protease of the GxGD type. Its physiological function, however, has remained enigmatic, since presently no physiological substrates have been identified. We demonstrate that SPPL3 alters the pattern of cellular N‐glycosylation by triggering the proteolytic release of active site‐containing ectodomains of glycosidases and glycosyltransferases such as N‐acetylglucosaminyltransferase V, β‐1,3 N‐acetylglucosaminyltransferase 1 and β‐1,4 galactosyltransferase 1. Cleavage of these enzymes leads to a reduction in their cellular activity. In line with that, reduced expression of SPPL3 results in a hyperglycosylation phenotype, whereas elevated SPPL3 expression causes hypoglycosylation. Thus, SPPL3 plays a central role in an evolutionary highly conserved post‐translational process in eukaryotes.     

Interleukin-2-stimulated natural killer cells are less susceptible to mycophenolate mofetil than non-activated NK cells: possible consequences for immunotherapy

In a clinical phase I/II trial, pediatric patients with high-risk malignancies were treated with ex vivo IL-2-stimulated donor natural killer (NK) cells after transplantation with haploidentical stem cells. To evaluate the potential negative effects of the immunosuppressive drug mycophenolate mofetil (MMF) used for immunotherapy, the functionality and signaling of ex vivo NK cells was investigated. Our results show that during NK cell expansion, long-term (9 days) incubation with mycophenolic acid (MPA), the active metabolite of MMF, in therapeutically relevant concentrations led to the severe inhibition of NK cell proliferation. This correlated with a significantly reduced cytokine/chemokine secretion and the inhibited acquisition of surface receptors regarding cytotoxicity (e.g., NKp30, NKp44, NKp46, NKG2D), adhesion/migration (e.g., ICAM-1/CD54, LFA-1/CD11a, CD62L, CXCR3) and activation (e.g., CD25). Moreover, MPA prevented phosphorylation of the central signaling molecules STAT-3/-4/-5, AKT and ERK1/2. In contrast, short-term (24 h) MPA incubation of IL-2-stimulated NK cells had no or only marginal effects on the activated NK cell phenotype, including receptor expression, cytokine/chemokine secretion and intracellular signaling. Further, short-term MPA incubation only moderately affected the highly cytotoxic activity of previously IL-2-stimulated NK cells. In conclusion, while long-term MPA incubation significantly compromised ex vivo NK cell functionality, previously IL-2-activated NK cells seemed to be rather resistant to short-term MPA treatment. This finding supports the use of IL-2-activated NK cells as immunotherapy, especially for patients treated with MMF after haploidentical stem cell transplantation.     

In cryptozoospermia or severe oligozoospermia is sperm freezing useful?

Background

Intracytoplasmic Sperm Injection (ICSI) is an Assisted Reproduction Technique (ART) which offers the chance to conceive to patients presenting very low sperm counts (cryptozoospermia/severe oligozoospermia). Sperm freezing before the oocyte pick-up, can prevent from a lack of spermatozoa on the day of the ICSI. It can avoid the cancellation of the ICSI or the use of TESE (Testicular sperm extraction). The objective of this study was to analyse the practice of sperm freezing for these patients in our center over 8 years and the rate of use of these frozen sperms. We also compared the outcome of ICSIs with frozen versus ejaculated sperm.

Material and methods

We performed a retrospective epidemiological study between 2004 and 2011. We recruited all the patients having a sperm count below 1 Million/mL and who were waiting for their first ICSI attempt.

Results

169 patients were recruited: 84 cryopreserved their sperm before the ICSI (secured ICSI) while 85 did not (non-secured ICSI). Both groups were split in cryptozoospermia (<10³ spermatozoa/ml): 19 and 17 patients respectively, very severe oligozoospermia (10³–10⁵/ml): 37 and 13 patients, and severe oligozoospermia (10⁵–10⁶/ml): 28 and 55 patients. The part of secured ICSI significantly increased from 29% during 2004–2007 to 74% during 2008–2011(p?=?0.0029) and the frozen sperm was used in 5.9% of the cases. Median age was significantly higher in the non secured ICSI group (33.57 vs 35.52 for men, p?=?0.0069 and 30.45 vs 32.26 for women, p?=?0.025) but no significant difference was found in the outcome of the ICSI between frozen-thawed sperm and fresh ejaculated sperm.

Conclusion

Sperm freezing before ICSI for severe oligozoospermic and cryptozoospermic patients significantly increased in our practice but the rate of use remain very low. This encourages to define more accurate criteria leading to sperm freezing.

     

Improved appreciation of the functioning and importance of biological soil crusts in Europe: the Soil Crust International Project (SCIN)

Here we report details of the European research initiative “Soil Crust International” (SCIN) focusing on the biodiversity of biological soil crusts (BSC, composed of bacteria, algae, lichens, and bryophytes) and on functional aspects in their specific environment. Known as the so-called “colored soil lichen community” (Bunte Erdflechtengesellschaft), these BSCs occur all over Europe, extending into subtropical and arid regions. Our goal is to study the uniqueness of these BSCs on the regional scale and investigate how this community can cope with large macroclimatic differences. One of the major aims of this project is to develop biodiversity conservation and sustainable management strategies for European BSCs. To achieve this, we established a latitudinal transect from the Great Alvar of Öland, Sweden in the north over Gössenheim, Central Germany and Hochtor in the Hohe Tauern National Park, Austria down to the badlands of Tabernas, Spain in the south. The transect stretches over 20° latitude and 2,300 m in altitude, including natural (Hochtor, Tabernas) and semi-natural sites that require maintenance such as by grazing activities (Öland, Gössenheim). At all four sites BSC coverage exceeded 30 % of the referring landscape, with the alpine site (Hochtor) reaching the highest cyanobacterial cover and the two semi-natural sites (Öland, Gössenheim) the highest bryophyte cover. Although BSCs of the four European sites share a common set of bacteria, algae (including cyanobacteria) lichens and bryophytes, first results indicate not only climate specific additions of species, but also genetic/phenotypic uniqueness of species between the four sites. While macroclimatic conditions are rather different, microclimatic conditions and partly soil properties seem fairly homogeneous between the four sites, with the exception of water availability. Continuous activity monitoring of photosystem II revealed the BSCs of the Spanish site as the least active in terms of photosynthetic active periods.     

Astroglial connexin 43 sustains glutamatergic synaptic efficacy

Astrocytes dynamic interactions with neurons play an active role in neurotransmission. The gap junction (GJ) subunits connexins 43 and 30 are strongly expressed in astrocytes and have recently been shown to regulate synaptic activity and plasticity. However, the specific role of connexin 43 in the morphological and electrophysiological properties of astrocytes in situ as well as in synaptic transmission remains unknown. Here, we show that connexin 43, a major determinant of astroglial GJ coupling, regulates astrocyte cell volume, but has no impact on astroglial passive membrane properties. Furthermore, we demonstrate that connexin 43 modulates glutamatergic synaptic activity of hippocampal CA1 pyramidal cells. This regulation involves changes in synaptically released glutamate, with no alteration in neuronal excitability or postsynaptic function. These results reveal connexin 43 as a critical player in neuroglial interactions by supporting synaptic efficacy.     

Shigella flexneri Infection in Caenorhabditis elegans: Cytopathological Examination and Identification of Host Responses

The Gram-negative bacterium Shigella flexneri is the causative agent of shigellosis, a diarrhoeal disease also known as bacillary dysentery. S. flexneri infects the colonic and rectal epithelia of its primate host and induces a cascade of inflammatory responses that culminates in the destruction of the host intestinal lining. Molecular characterization of host-pathogen interactions in this infection has been challenging due to the host specificity of S. flexneri strains, as it strictly infects humans and non-human primates. Recent studies have shown that S. flexneri infects the soil dwelling nematode Caenorhabditis elegans, however, the interactions between S. flexneri and C. elegans at the cellular level and the cause of nematode death are unknown. Here we attempt to gain insight into the complex host-pathogen interactions between S. flexneri and C. elegans. Using transmission electron microscopy, we show that live S. flexneri cells accumulate in the nematode intestinal lumen, produce outer membrane vesicles and invade nematode intestinal cells. Using two-dimensional differential in-gel electrophoresis we identified host proteins that are differentially expressed in response to S. flexneri infection. Four of the identified genes, aco-1, cct-2, daf-19 and hsp-60, were knocked down using RNAi and ACO-1, CCT-2 and DAF-19, which were identified as up-regulated in response to S. flexneri infection, were found to be involved in the infection process. aco-1 RNAi worms were more resistant to S. flexneri infection, suggesting S. flexneri-mediated disruption of host iron homeostasis. cct-2 and daf-19 RNAi worms were more susceptible to infection, suggesting that these genes are induced as a protective mechanism by C. elegans. These observations further our understanding of the processes involved in S. flexneri infection of C. elegans, which is immensely beneficial to the routine use of this new in vivo model to study S. flexneri pathogenesis.     

Usefulness of a Darwinian System in a Biotechnological Application: Evolution of Optical Window Fluorescent Protein Variants under Selective Pressure

With rare exceptions, natural evolution is an extremely slow process. One particularly striking exception in the case of protein evolution is in the natural production of antibodies. Developing B cells activate and diversify their immunoglobulin (Ig) genes by recombination, gene conversion (GC) and somatic hypermutation (SHM). Iterative cycles of hypermutation and selection continue until antibodies of high antigen binding specificity emerge (affinity maturation). The avian B cell line DT40, a cell line which is highly amenable to genetic manipulation and exhibits a high rate of targeted integration, utilizes both GC and SHM. Targeting the DT40''s diversification machinery onto transgenes of interest inserted into the Ig loci and coupling selective pressure based on the desired outcome mimics evolution. Here we further demonstrate the usefulness of this platform technology by selectively pressuring a large shift in the spectral properties of the fluorescent protein eqFP615 into the highly stable and advanced optical imaging expediting fluorescent protein Amrose. The method is advantageous as it is time and cost effective and no prior knowledge of the outcome protein''s structure is necessary. Amrose was evolved to have high excitation at 633 nm and excitation/emission into the far-red, which is optimal for whole-body and deep tissue imaging as we demonstrate in the zebrafish and mouse model.