Discovery of a non-canonical prototype long-chain monoacylglycerol lipase through a structure-based endogenous reaction intermediate complex.

The identification and characterization of enzyme function is largely lacking behind the rapidly increasing availability of large numbers of sequences and associated high-resolution structures. This is often hampered by lack of knowledge on in vivo relevant substrates. Here, we present a case study of a high-resolution structure of an unusual orphan lipase in complex with an endogenous C18 monoacylglycerol ester reaction intermediate from the expression host, which is insoluble under aqueous conditions and thus not accessible for studies in solution. The data allowed its functional characterization as a prototypic long-chain monoacylglycerol lipase, which uses a minimal lid domain to position the substrate through a hydrophobic tunnel directly to the enzyme's active site. Knowledge about the molecular details of the substrate binding site allowed us to modulate the enzymatic activity by adjusting protein/substrate interactions, demonstrating the potential of our findings for future biotechnology applications.

High-resolution analysis of individual Drosophila melanogaster larvae uncovers individual variability in locomotion and its neurogenetic modulation.

Neuronally orchestrated muscular movement and locomotion are defining faculties of multicellular animals. Due to its simple brain and genetic accessibility, the larva of the fruit fly Drosophila melanogaster allows one to study these processes at tractable levels of complexity. However, although the faculty of locomotion clearly pertains to the individual, most studies of locomotion in larvae use measurements aggregated across animals, or animals tested one by one, an extravagance for larger-scale analyses. This prevents grasping the inter- and intra-individual variability in locomotion and its neurogenetic determinants. Here, we present the IMBA (individual maggot behaviour analyser) for analysing the behaviour of individual larvae within groups, reliably resolving individual identity across collisions. We use the IMBA to systematically describe the inter- and intra-individual variability in locomotion of wild-type animals, and how the variability is reduced by associative learning. We then report a novel locomotion phenotype of an adhesion GPCR mutant. We further investigated the modulation of locomotion across repeated activations of dopamine neurons in individual animals, and the transient backward locomotion induced by brief optogenetic activation of the brain-descending 'mooncrawler' neurons. In summary, the IMBA is an easy-to-use toolbox allowing an unprecedentedly rich view of the behaviour and its variability of individual larvae, with utility in multiple biomedical research contexts.

Epstein-Barr Virus-Induced Genes and Endogenous Retroviruses in Immortalized B Cells from Patients with Multiple Sclerosis.

The immune pathogenesis of multiple sclerosis (MS) is thought to be triggered by environmental factors in individuals with an unfavorable genetic predisposition. Epstein-Barr virus (EBV) infection is a major risk factor for subsequent development of MS. Human endogenous retroviruses (HERVs) can be activated by EBV, and might be a missing link between an initial EBV infection and the later onset of MS. In this study, we investigated differential gene expression patterns in EBV-immortalized lymphoblastoid B cell lines (LCL) from MS-affected individuals (MSLCL) and controls by using RNAseq and qRT-PCR. RNAseq data from LCL mapped to the human genome and a virtual virus metagenome were used to identify possible biomarkers for MS or disease-relevant risk factors, e.g., the relapse rate. We observed that lytic EBNA-1 transcripts seemed to be negatively correlated with age leading to an increased expression in LCL from younger PBMC donors. Further, HERV-K (HML-2) GAG was increased upon EBV-triggered immortalization. Besides the well-known transactivation of HERV-K18, our results suggest that another six HERV loci are up-regulated upon stimulation with EBV. We identified differentially expressed genes in MSLCL, e.g., several HERV-K loci, ERVMER61-1 and ERV3-1, as well as genes associated with relapses. In summary, EBV induces genes and HERV in LCL that might be suitable as biomarkers for MS or the relapse risk.

A host-vector toolbox for improved secretory protein overproduction in Bacillus subtilis.

Target proteins in biotechnological applications are highly diverse. Therefore, versatile flexible expression systems for their functional overproduction are required. In order to find the right heterologous gene expression strategy, suitable host-vector systems, which combine different genetic circuits, are useful. In this study, we designed a novel Bacillus subtilis expression toolbox, which allows the overproduction and secretion of potentially toxic enzymes. This toolbox comprises a set of 60 expression vectors, which combine two promoter variants, four strong secretion signals, a translation-enhancing downstream box, and three plasmid backbones. This B. subtilis toolbox is based on a tailor-made, clean deletion mutant strain, which is protease and sporulation deficient and exhibits reduced autolysis and secondary metabolism. The appropriateness of this alternative expression platform was tested for the overproduction of two difficult-to-produce eukaryotic model proteins. These included the sulfhydryl oxidase Sox from Saccharomyces cerevisiae, which forms reactive hydrogen peroxide and undesired cross-linking of functional proteins, and the human interleukin-1ß, a pro-inflammatory cytokine. For the best performing Sox and interleukin, overproducing and secreting variants of these new B. subtilis toolbox fermentation strategies were developed and tested. This study demonstrates the suitability of the prokaryotic B. subtilis host-vector system for the extracellular production of two eukaryotic proteins with biotechnological relevance. KEY POINTS: • Construction of a versatile Bacillus subtilis gene expression toolbox. • Verification of the toolbox by the secretory overproduction of two difficult-to-express proteins. • Fermentation strategy for an acetoin-controlled overproduction of heterologous proteins.

The effect of tree shade on ambient conditions and heat stress indicator traits of new-born South African Mutton Merino and Dormer lambs: Preliminary results.

This preliminary study investigated the provision of shade on heat stress indicators of South African Mutton Merino (SAMM) and Dormer lambs shortly after birth, during the autumn 2017 and 2018 lambing seasons. Newborn lambs were assessed to determine whether welfare, as assessed by respiratory response and rectal temperature as heat stress indicators, survival and early growth benefitted from the provision of shade. Groups consisting of 4-17 pregnant SAMM and Dormer ewes were randomly allocated to 5-10 paddocks with natural shade from trees and 5-9 paddocks that were directly in the sun with no shade available. The lambs were recorded within 24 h of birth at noon. Climate data were obtained from a nearby weather station. The lambs were also weighed at 12 (SD = 2) days of age at tail-docking. Tree shade had a moderating effect on temperature, resulting in lower maximum daytime and higher minimum night-time temperatures. There was an interaction between a temperature-humidity index (THI) and the treatments (access to shade or no access to shade) for respiration and rectal temperature (P < 0.01). Both traits were relatively unaffected by the THI at values below 77. Unshaded lambs exhibited a pronounced upwards trend following a THI-threshold of 77-78. Tailing weight tended to be higher while lamb survival of live-born lambs to tail-docking was lower in lambs born in shaded paddocks but these trends did not persist to weaning. Shade is needed to enhance animal welfare by alleviating the effect of high THI-values on hot days in an autumn lambing season.

Overexpression of Endogenous Retroviruses and Malignancy Markers in Neuroblastoma Cell Lines by Medium-Induced Microenvironmental Changes.

Neuroblastoma (NB) is the commonest solid tumor outside the central nervous system in infancy and childhood with a unique biological heterogeneity. In patients with advanced, metastasizing neuroblastoma, treatment failure and poor prognosis is often marked by resistance to chemo- or immunotherapy. Thus, identification of robust biomarkers seems essential for understanding tumor progression and developing effective therapy. Here, we have studied the expression of human endogenous retroviruses (HERV) as potential targets in NB cell lines during stem-cell medium-induced microenvironmental change. Quantitative PCR revealed that relative expression of the HERV-K family and HERV-W1 ENV were increased in all three NB cell lines after incubation in stem-cell medium. Virus transcriptome analyses revealed the transcriptional activation of three endogenous retrovirus elements: HERV-R ENV (ERV3-1), HERV-E1 and HERV-Fc2 ENV (ERVFC1-1). Known malignancy markers in NB, e.g. proto-oncogenic MYC or MYCN were expressed highly heterogeneously in the three investigated NB cell lines with up-regulation of MYC and MYCN upon medium-induced microenvironmental change. In addition, SiMa cells exclusively showed a phenotype switching from loosely-adherent monolayers to low proliferating grape-like cellular aggregates, which was accompanied by an enhanced CD133 expression. Interestingly, the overexpression of HERV was associated with a significant elevation of immune checkpoint molecule CD200 in both quantitative PCR and RNA-seq analysis suggesting tumor escape mechanism in NB cell lines after incubation in serum-free stem cell medium.

Identification of Differentially Expressed Human Endogenous Retrovirus Families in Human Leukemia and Lymphoma Cell Lines and Stem Cells.

Endogenous retroviruses (ERVs) are becoming more and more relevant in cancer research and might be potential targets. The oncogenic potential of human ERVs (HERVs) has been recognized and includes immunosuppression, cell fusion, antigenicity of viral proteins, and regulation of neighboring genes. To decipher the role of HERVs in human cancers, we used a bioinformatics approach and analyzed RNA sequencing data from the LL-100 panel, covering 22 entities of hematopoietic neoplasias including T cell, B cell and myeloid malignancies. We compared HERV expression in this panel with hematopoietic stem cells (HSCs), embryonic stem cells (ESCs) and normal blood cells. RNA sequencing data were mapped against a comprehensive synthetic viral metagenome with 116 HERV sequences from 14 different HERV families. Of these, 13 HERV families and elements were differently expressed in malignant hematopoietic cells and stem cells. We found transcriptional upregulation of HERVE family in acute megakaryocytic and erythroid leukemia and of HERVFc family in multiple myeloma/plasma cell leukemia (PCL). The HERVFc member HERVFc-1 was found transcriptionally active in the multiple myeloma cell line OPM-2 and also in the Hodgkin lymphoma cell line L-428. The expression of HERVFc-1 in L-428 cells was validated by qRT-PCR. We also confirm transcriptional downregulation of ERV3 in acute megakaryocytic and erythroid leukemia, and HERVK in acute monocytic and myelocytic leukemia and a depression of HERVF in all malignant entities. Most of the higher expressed HERV families could be detected in stem cells including HERVK (HML-2), HERV-like, HERVV, HERVT, ERV9, HERVW, HERVF, HERVMER, ERV3, HERVH and HERVPABLB.

Biomass-degrading glycoside hydrolases of archaeal origin.

During the last decades, the impact of hyperthermophiles and their enzymes has been intensively investigated for implementation in various high-temperature biotechnological processes. Biocatalysts of hyperthermophiles have proven to show extremely high thermo-activities and thermo-stabilities and are identified as suitable candidates for numerous industrial processes with harsh conditions, including the process of an efficient plant biomass pretreatment and conversion. Already-characterized archaea-originated glycoside hydrolases (GHs) have shown highly impressive features and numerous enzyme characterizations indicated that these biocatalysts show maximum activities at a higher temperature range compared to bacterial ones. However, compared to bacterial biomass-degrading enzymes, the number of characterized archaeal ones remains low. To discover new promising archaeal GH candidates, it is necessary to study in detail the microbiology and enzymology of extremely high-temperature habitats, ranging from terrestrial to marine hydrothermal systems. State-of-the art technologies such as sequencing of genomes and metagenomes and automated binning of genomes out of metagenomes, combined with classical microbiological culture-dependent approaches, have been successfully performed to detect novel promising biomass-degrading hyperthermozymes. In this review, we will focus on the detection, characterization and similarities of archaeal GHs and their unique characteristics. The potential of hyperthermozymes and their impact on high-temperature industrial applications have not yet been exhausted.

Digitalization in microbiology - Paving the path to sustainable circular bioeconomy.

The transition to a sustainable bio-based circular economy requires cutting edge technologies that ensure economic growth with environmentally responsible action. This transition will only be feasible when the opportunities of digitalization are also exploited. Digital methods and big data handling have already found their way into life sciences and generally offer huge potential in various research areas. While computational analyses of microbial metagenome data have become state of the art, the true potential of bioinformatics remains mostly untapped so far. In this article we present challenges and opportunities of digitalization including multi-omics approaches in discovering and exploiting the microbial diversity of the planet with the aim to identify robust biocatalysts for application in sustainable bioprocesses as part of the transition from a fossil-based to a bio-based circular economy. This will contribute to solving global challenges, including utilization of natural resources, food supply, health, energy and the environment.

Extremely thermoactive archaeal endoglucanase from a shallow marine hydrothermal vent from Vulcano Island.

Already-characterized microbial cellulases have proven to be highly useful for industrial processes, since they can withstand harsh industrial conditions with characteristics such as high thermo- and acid stability. These properties provide promising features for the process of plant biomass degradation and biofuel generation. Nevertheless, the number of known extremely thermoactive archaeal cellulases is low. Hence, the discovery of archaeal cellulases with different characteristics is crucial for the development of efficient and sustainable biorefinery. In this work, the metagenome of a high-temperature enrichment culture from marine environment of Vulcano Island was screened for the presence of novel endoglucanase-encoding genes of archaeal origin. The ORF vul_cel5A was detected, and the deduced protein was characterized as the most thermoactive endoglucanase described to date. Vul_Cel5A was identified as a thermoactive glycoside hydrolase family 5 endoglucanase, with the highest sequence identity (72-75%) to putative endoglucanases from archaeal genera. Vul_Cel5A showed the highest activity at notable 115 °C towards barley ß-glucan (210.7 U/mg), and lichenan (209.9 U/mg), and further towards carboxymethyl cellulose (38.6 U/mg) and locust bean gum (83.0 U/mg). The endoglucanase exhibited a half-life time of 46 min at 100 °C and did not show any loss of activity after incubation for 48 h at 75 °C. Furthermore, Vul_Cel5A showed high affinity to barley ß-glucan with a Km of 0.52 mg/mL and showed tolerance against various chemical reagents. Due to the outstanding high thermoactivity and thermostability and tolerance to acidic conditions, Vul_Cel5A represents a promising novel archaeal endo-ß-glucanase for application in biorefineries for an efficient biomass pre-treatment.

The ß-Ketoacyl-CoA Synthase HvKCS1, Encoded by Cer-zh, Plays a Key Role in Synthesis of Barley Leaf Wax and Germination of Barley Powdery Mildew.

The cuticle coats the primary aerial surfaces of land plants. It consists of cutin and waxes, which provide protection against desiccation, pathogens and herbivores. Acyl cuticular waxes are synthesized via elongase complexes that extend fatty acyl precursors up to 38 carbons for downstream modification pathways. The leaves of 21 barley eceriferum (cer) mutants appear to have less or no epicuticular wax crystals, making these mutants excellent tools for identifying elongase and modification pathway biosynthetic genes. Positional cloning of the gene mutated in cer-zh identified an elongase component, ß-ketoacyl-CoA synthase (CER-ZH/HvKCS1) that is one of 34 homologous KCSs encoded by the barley genome. The biochemical function of CER-ZH was deduced from wax and cutin analyses and by heterologous expression in yeast. Combined, these experiments revealed that CER-ZH/HvKCS1 has a substrate specificity for C16-C20, especially unsaturated, acyl chains, thus playing a major role in total acyl chain elongation for wax biosynthesis. The contribution of CER-ZH to water barrier properties of the cuticle and its influence on the germination of barley powdery mildew fungus were also assessed.

Health-related quality of life in patients with poststroke epilepsy.

BACKGROUND: Lesional epilepsy is an important long-term sequela of stroke. Data on health-related quality of life (HrQoL) in patients with poststroke epilepsy are limited. We investigated HrQoL in patients with epilepsy after ischemic stroke and identified independent HrQoL-determinants. METHODS AND PATIENTS: All patients with acute ischemic stroke, who were permanent residents in the district Marburg-Biedenkopf (Hessia, Germany, reference population 240,000 inhabitants) were recruited within 12months in the population-based Marburg Stroke Register (MARSTREG). Follow-up visits were performed after 6, 12, and 24months, and patients who developed poststroke epilepsy were identified. Data on demographics, antiepileptic drugs (AEDs), stroke severity (National Institute of Heath Stroke Scale (NIHSS), Barthel-Index, modified Rankin Scale), depression (Geriatric Depression Scale), and HrQoL (EQ-5D and EQ VAS) were collected. A multiple regression analysis was performed to identify HrQoL-determinants. RESULTS: Among the study participants (n=374), 23 (6.1%) developed poststroke epilepsy. The HrQoL of patients with poststroke epilepsy was reduced in comparison with patients without seizures (24-month follow-up: EuroQol Visual Analogue Scale (EuroQol-VAS): 55.3±10.7 versus 64.2±11.4, p=0.03). Seizure frequency, depression, and functional impairment (Barthel-Index) were identified as independent determinants of HrQoL. The adjustment of AEDs between 6-month and 24-month follow-ups resulted in decrease of seizure frequency by 40% and reduction of complications (dizziness by 27.8%, nausea by 52.2%, fatigue by 84.2%). CONCLUSION: Lesional epilepsy is associated with decreased HrQoL in patients with stroke. We identified HrQoL-determinants, which would improve the management of patients with poststroke epilepsy. These determinants include proper adjustment of AEDs with reduction of seizure frequency, treatment of depression, and focused rehabilitation programs for poststroke epilepsy.

Towards a sustainable biobased industry - Highlighting the impact of extremophiles.

The transition of the oil-based economy towards a sustainable economy completely relying on biomass as renewable feedstock requires the concerted action of academia, industry, politics and civil society. An interdisciplinary approach of various fields such as microbiology, molecular biology, chemistry, genetics, chemical engineering and agriculture in addition to cross-sectional technologies such as economy, logistics and digitalization is necessary to meet the future global challenges. The genomic era has contributed significantly to the exploitation of naturés biodiversity also from extreme habitats. By applying modern technologies it is now feasible to deliver robust enzymes (extremozymes) and robust microbial systems that are active at temperatures up to 120°C, at pH 0 and 12 and at 1000bar. In the post-genomic era, different sophisticated "omics" analyses will allow the identification of countless novel enzymes regardless of the lack of cultivability of most microorganisms. Furthermore, elaborate protein-engineering methods are clearing the way towards tailor-made robust biocatalysts. Applying environmentally friendly and efficient biological processes, terrestrial and marine biomass can be converted to high value products e.g. chemicals, building blocks, biomaterials, pharmaceuticals, food, feed and biofuels. Thus, further application of extremophiles has the potential to improve sustainability of existing biotechnological processes towards a greener biobased industry.

[Suicide in children, youths and young adults]. / Suizide von Kindern, Jugendlichen und Heranwachsenden.

Suicides among children and youths are tragic events. The suicide cases of children, youths and young adults examined between 1995 and 2009 at the Institute of Legal Medicine of the Hanover Medical School were retrospectively analyzed. Altogether, 54 suicides among children, youths and young adults (37 males, 17 females; median age 17.5 years) were evaluated. The suicide victims were predominantly male (68.5%). The vast majority used "hard" methods such as hanging, jumping from a height or being run over by a train. In seven cases (13.0%) psychiatric disorders were known. Seven victims (13.0%) left a suicide note. Suicide was most frequently (40.7%) committed in the open air (40.7%). In addition, the evaluation shows that at the time of committing suicide more than 40% of the youths and adolescents, in whom a chemical toxicological examination was performed, were under the influence of alcohol, medical and/or illegal drugs.

[Combined edema reducing therapy in the treatment of advanced lower limb lymphedema]. / Kompleksowa terapia przeciwobrzekowa w leczeniu zaawansowanych postaci pierwotnego obrzeku limfatycznego konczyn dolnych.

UNLABELLED: Combined edema reducing therapy is a recognized method of lymphedema treatment. However such therapy can be difficult to implement from methodological and logistic point of view in cases of advanced forms of lymphedema. The aim of the study was the presentation and discussion of intensive phase of combined treatment in patient with advanced primary lymphedema. MATERIAL AND METHODS: Therapy was conducted on 19 patients (27 limbs) with edema reducing therapy program. Procedures were conducted daily for 4-6 weeks in out-patient and in-wards conditions. RESULTS: Intensive phase of treatment succeeded in 3870-15 330 ml edema reduction, consisting of 48-65% of initial status. Chronic leg ulcers were healed completely in 2 patients. Ten patients underwent minor adverse events (AE), such as superficial skin ulceration (n = 2), popliteal fossa skin maceration (n = 2), neuropathic foot pain (n = 3) and skin scratches (n = 3). Modification of the treatment allowed the complete healing of AEs within 2-7 days, but it produced significant delay in achievement of desired therapeutic result, In 2 cases it prolonged hospital stay for 7 days. CONCLUSIONS: 1. Combined edema reducing therapy is very efficient form of treatment in advanced primary lymphedema. 2. Intensive, 4-6 week, phase of the treatment allows 3.8 to 15.3 1 edema reduction. 3. In our opinion this phase should be conducted only in specialized centers for proper final results achievement with adverse events minimization. 4. The main point of the therapy is a combination of appropriate forms of available treatment.