New pulsed EPR methods and their application to characterize mitochondrial complex I

Electron Paramagnetic Resonance (EPR) spectroscopy is the method of choice to study paramagnetic cofactors that often play an important role as active centers in electron transfer processes in biological systems. However, in many cases more than one paramagnetic species is contributing to the observed EPR spectrum, making the analysis of individual contributions difficult and in some cases impossible. With time-domain techniques it is possible to exploit differences in the relaxation behavior of different paramagnetic species to distinguish between them and separate their individual spectral contribution. Here we give an overview of the use of pulsed EPR spectroscopy to study the iron–sulfur clusters of NADH:ubiquinone oxidoreductase (complex I). While FeS cluster N1 can be studied individually at a temperature of 30 K, this is not possible for FeS cluster N2 due to its severe spectral overlap with cluster N1. In this case Relaxation Filtered Hyperfine (REFINE) spectroscopy can be used to separate the overlapping spectra based on differences in their relaxation behavior.Collaborative Research Centre 472 (Project P2)Collaborative Research Centre 472 (Project P15)Goethe University in Frankfurt/Main. Center for Biomolecular Magnetic Resonanc

Asynchronous Execution of Python Code on Task Based Runtime Systems

Despite advancements in the areas of parallel and distributed computing, the complexity of programming on High Performance Computing (HPC) resources has deterred many domain experts, especially in the areas of machine learning and artificial intelligence (AI), from utilizing performance benefits of such systems. Researchers and scientists favor high-productivity languages to avoid the inconvenience of programming in low-level languages and costs of acquiring the necessary skills required for programming at this level. In recent years, Python, with the support of linear algebra libraries like NumPy, has gained popularity despite facing limitations which prevent this code from distributed runs. Here we present a solution which maintains both high level programming abstractions as well as parallel and distributed efficiency. Phylanx, is an asynchronous array processing toolkit which transforms Python and NumPy operations into code which can be executed in parallel on HPC resources by mapping Python and NumPy functions and variables into a dependency tree executed by HPX, a general purpose, parallel, task-based runtime system written in C++. Phylanx additionally provides introspection and visualization capabilities for debugging and performance analysis. We have tested the foundations of our approach by comparing our implementation of widely used machine learning algorithms to accepted NumPy standards

Near Chromosome-Level Genome Assembly and Annotation of Rhodotorula babjevae Strains Reveals High Intraspecific Divergence

The genus Rhodotorula includes basidiomycetous oleaginous yeast species. Rhodotorula babjevae can produce compounds of biotechnological interest such as lipids, carotenoids, and biosurfactants from low value substrates such as lignocellulose hydrolysate. High-quality genome assemblies are needed to develop genetic tools and to understand fungal evolution and genetics. Here, we combined short- and long-read sequencing to resolve the genomes of two R. babjevae strains, CBS 7808 (type strain) and DBVPG 8058, at chromosomal level. Both genomes are 21 Mbp in size and have a GC content of 68.2%. Allele frequency analysis indicates that both strains are tetraploid. The genomes consist of a maximum of 21 chromosomes with a size of 0.4 to 2.4 Mbp. In both assemblies, the mitochondrial genome was recovered in a single contig, that shared 97% pairwise identity. Pairwise identity between most chromosomes ranges from 82 to 87%. We also found indications for strain-specific extrachromosomal endogenous DNA. A total of 7591 and 7481 protein-coding genes were annotated in CBS 7808 and DBVPG 8058, respectively. CBS 7808 accumulated a higher number of tandem duplications than DBVPG 8058. We identified large translocation events between putative chromosomes. Genome divergence values between the two strains indicate that they may belong to different species.Peer Reviewe

Appetite, food intake, and gut hormone responses to glycomacropeptide protein ingestion in older adults: : A feasibility, acceptability, and pilot study

We would like express huge gratitude to our participants for taking part, and for making data collection such an enjoyable experience for the research team. We would like to thank our students Vicky Catterall, Beth Minion, Joe Ashworth, Monty Hardcastle, and Anna Brooks for supporting data collection. We also thank Agropur Ingredients (Eden Prairie, MN, USA) for providing GMP.Peer reviewe

What the Phage: a scalable workflow for the identification and analysis of phage sequences

Phages are among the most abundant and diverse biological entities on earth. Phage prediction from sequence data is a crucial first step to understanding their impact on the environment. A variety of bacteriophage prediction tools have been developed over the years. They differ in algorithmic approach, results, and ease of use. We, therefore, developed "What the Phage"(WtP), an easy-to-use and parallel multitool approach for phage prediction combined with an annotation and classification downstream strategy, thus supporting the user's decision-making process by summarizing the results of the different prediction tools in charts and tables. WtP is reproducible and scales to thousands of datasets through a workflow manager (Nextflow). WtP is freely available under a GPL-3.0 license (https://github.com/replikation/What_the_Phage)

The Native 3D Organization of Bacterial Polysomes

SummaryRecent advances have led to insights into the structure of the bacterial ribosome, but little is known about the 3D organization of ribosomes in the context of translating polysomes. We employed cryoelectron tomography and a template-matching approach to map 70S ribosomes in vitrified bacterial translation extracts and in lysates of active E. coli spheroplasts. In these preparations, polysomal arrangements were observed in which neighboring ribosomes are densely packed and exhibit preferred orientations. Analysis of characteristic examples of polysomes reveals a staggered or pseudohelical organization of ribosomes along the mRNA trace, with the transcript being sequestered on the inside, the tRNA entrance sites being accessible, and the polypeptide exit sites facing the cytosol. Modeling of elongating nascent polypeptide chains suggests that this arrangement maximizes the distance between nascent chains on adjacent ribosomes, thereby reducing the probability of intermolecular interactions that would give rise to aggregation and limit productive folding

"Non-healing" claw horn lesions in dairy cows: Clinical, histopathological and molecular biological characterization of four cases.

The increasing prevalence of bovine digital dermatitis (BDD) contributes to a higher occurrence of secondary infections of exposed corium with Treponema spp. in bovine claws. "Non-healing" claw horn lesions (NHL) clinically resemble BDD lesions. They are severe, cause chronic lameness, and may persist for several months. They poorly respond to standard treatments of BDD and represent a serious welfare issue. In this study, four cases of NHL were classified clinically either as BDD-associated axial horn fissures (BDD-HFA; n = 3) or BDD-associated sole ulcer (BDD-SU; n = 1). In all four cases, pronounced multifocal keratinolysis of the stratum corneum, ulceration, and severe chronic lymphoplasmacytic perivascular to interstitial dermatitis were observed. All lesional samples tested positive for Treponema spp., Fusobacterium (F.) necrophorum, and Porphyromonas (P.) levii by PCRs. BDD-HFA lesions contained Treponema pedis as revealed by genetic identities of 93, 99, and 100%. Treponemes in the BDD-SU lesion were 94% homologous to Treponema phylotype PT3. Fluorescent in situ hybridization (FISH) revealed extensive epidermal infiltration by treponemes that made up > 90% of the total bacterial population in all four lesions. FISH also tested positive for P. levii and negative for F. necrophorum in all four cases, whilst only one BDD-HFA contained Dichelobacter nodosus. Our data point to BDD-associated treponemes and P. levii constituting potential etiological agents in the development of "non-healing" claw horn lesions in cattle

Proof of an optimized salicylic acid paste-based treatment concept of ulcerative M2-stage digital dermatitis lesions in 21 dairy cows.

The efficacy of salicylic acid paste (SA) in the treatment of ulcerative bovine digital dermatitis (BDD) was assessed by combining clinical and histopathological analyses with molecular biological techniques. The latter were conducted in a blinded manner to reach maximum objectivity. Prior to treatment, M2-stage BDD lesions (n = 26, diagnosed in 21 dairy cows) exhibited ulceration, with severe perivascular, chronic, lymphoplasmacytic dermatitis and extensive keratinolysis being noted in most cases. Pretreatment biopsy samples (n = 12) followed by povidone-iodine ointment under bandage for one week before administration of SA paste were tested positive for Treponema spp. by blinded PCR and fluorescent in situ hybridization (FISH). Subsequent treatment consisted of application of SA and bandaging at weekly intervals until lesions had completely resolved. The treatment duration ranged between 2 and 4 weeks. Complete healing was achieved in 100% of cases, with 2/21 animals requiring a second round of treatment upon disease reoccurrence. Importantly, only 3/26 biopsies taken from previously affected sites still tested positive by Treponema PCR, and in another biopsy, the outermost layers of the stratum corneum scored weakly positive by Treponema-specific FISH. None of these Treponema DNA-positive biopsies showed signs of ulceration. One case exhibited focal keratinolysis. Positive PCR or FISH in these cases may have arisen from DNA traces of dead bacteria or environmental contamination during biopsy harvesting. To our knowledge, this is the first study on blinded molecular biological monitoring of the therapeutic efficacy of SA with respect to treponemal infection, and on complete BDD M2-stage remission in all animals achieved by SA treatment according to an optimized protocol. Although the etiology of BDD is considered as multifactorial, our data further support the concept that treponemes have a decisive role in BDD pathogenesis

Enhanced glycerol assimilation and lipid production in Rhodotorula toruloides CBS14 upon addition of hemicellulose primarily correlates with early transcription of energy-metabolism-related genes

BackgroundLipid formation from glycerol was previously found to be activated in Rhodotorula toruloides when the yeast was cultivated in a mixture of crude glycerol (CG) and hemicellulose hydrolysate (CGHH) compared to CG as the only carbon source. RNA samples from R. toruloides CBS14 cell cultures grown on either CG or CGHH were collected at different timepoints of cultivation, and a differential gene expression analysis was performed between cells grown at a similar physiological situation.ResultsWe observed enhanced transcription of genes involved in oxidative phosphorylation and enzymes localized in mitochondria in CGHH compared to CG. Genes involved in protein turnover, including those encoding ribosomal proteins, translation elongation factors, and genes involved in building the proteasome also showed an enhanced transcription in CGHH compared to CG. At 10 h cultivation, another group of activated genes in CGHH was involved in beta-oxidation, handling oxidative stress and degradation of xylose and aromatic compounds. Potential bypasses of the standard GUT1 and GUT2-glycerol assimilation pathway were also expressed and upregulated in CGHH 10 h. When the additional carbon sources from HH were completely consumed, at CGHH 36 h, their transcription decreased and NAD(+)-dependent glycerol-3-phosphate dehydrogenase was upregulated compared to CG 60 h, generating NADH instead of NADPH with glycerol catabolism. TPI1 was upregulated in CGHH compared to cells grown on CG in all physiological situations, potentially channeling the DHAP formed through glycerol catabolism into glycolysis. The highest number of upregulated genes encoding glycolytic enzymes was found after 36 h in CGHH, when all additional carbon sources were already consumed.ConclusionsWe suspect that the physiological reason for the accelerated glycerol assimilation and faster lipid production, was primarily the activation of enzymes that provide energy

Assessing genetic diversity and similarity of 435 KPC-carrying plasmids.

Brandt C, Viehweger A, Singh A, et al. Assessing genetic diversity and similarity of 435 KPC-carrying plasmids. Scientific reports. 2019;9(1): 11223.The global spread and diversification of multidrug-resistant Gram-negative (MRGN) bacteria poses major challenges to healthcare. In particular, carbapenem-resistant Klebsiella pneumoniae strains have been frequently identified in infections and hospital-wide outbreaks. The most frequently underlying resistance gene (blaKPC) has been spreading over the last decade in the health care setting. blaKPC seems to have rapidly diversified and has been found in various species and on different plasmid types. To review the progress and dynamics of this diversification, all currently available KPC plasmids in the NCBI database were analysed in this work. Plasmids were grouped into 257 different representative KPC plasmids, of which 79.4% could be clearly assigned to incompatibility (Inc) group or groups. In almost half of all representative plasmids, the KPC gene is located on Tn4401 variants, emphasizing the importance of this transposon type for the transmission of KPC genes to other plasmids. The transposons also seem to be responsible for the occurrence of altered or uncommon fused plasmid types probably due to incomplete transposition. Moreover, many KPC plasmids contain genes that encode proteins promoting recombinant processes and mutagenesis; in consequence accelerating the diversification of KPC genes and other colocalized resistance genes