Fuzzle 2.0: Ligand Binding in Natural Protein Building Blocks

Modern proteins have been shown to share evolutionary relationships via subdomain-sized fragments. The assembly of such fragments through duplication and recombination events led to the complex structures and functions we observe today. We previously implemented a pipeline that identified more than 1,000 of these fragments that are shared by different protein folds and developed a web interface to analyze and search for them. This resource named Fuzzle helps structural and evolutionary biologists to identify and analyze conserved parts of a protein but it also provides protein engineers with building blocks for example to design proteins by fragment combination. Here, we describe a new version of this web resource that was extended to include ligand information. This addition is a significant asset to the database since now protein fragments that bind specific ligands can be identified and analyzed. Often the mode of ligand binding is conserved in proteins thereby supporting a common evolutionary origin. The same can now be explored for subdomain-sized fragments within this database. This ligand binding information can also be used in protein engineering to graft binding pockets into other protein scaffolds or to transfer functional sites via recombination of a specific fragment. Fuzzle 2.0 is freely available at https://fuzzle.uni-bayreuth.de/2.0.

Development of a SCAR Marker Based on SCoT Polymorphisms for Sugarcane Smut Resistance

Sugarcane smut caused by Sporisorium scitanmineumis the most severe sugarcane disease that causes major economic losses in sugarcane production in China, and disease resistance breeding is an important way of preventing and controlling this disease. In this study, BC3F1lines derived from the cross between YC 73-226 and YCE 06-111 were used to generate sugarcane smut-resistant and -susceptible gene pools using bulked segregant analysis (BSA). Eighty-nine random primers of start codon targeted (SCoT) polymorphisms were screened, whereas only primer SCoT44 could stably amplify the specific fragment (HE-Ss44) in the resistant pool. Then, several primer pairs of sequence characterized amplified regions (SCARs) were designed based on the sequence alignment of HE-Ss44 (920 bp), which was recovered after purification, and only one pair of SCAR primers (Ss44-F2/R2, forward: 5'-GGCGGGCACCGTCGAGTCCACAT-3'; reverse: 5'-CCGTCCGTCGG TCTCGTCCTTACG-3') could stably amplify a 400-bp specific band in resistant gene pool and its individuals. A validation test of SCAR marker Ss44-F2/R2 was performed using 34 sugarcane cultivars with known smut resistance, which revealed a selection accuracy of 82.35% between marker detection and known smut resistance. Moreover, Pearson’s correlation analysis also showed that the SCAR marker Ss44-F2/R2 was significantly correlated (r= 0.583, P= 0.0003 < 0.01) with the smut resistance trait in sugarcane. In addition, the nucleotide sequence of HE-Ss44 linked with smut-resistancewas not aligned to the homologous sequence in GenBank (NCBI), and the accession number was MG740763. The SCAR marker Ss44-F2/R2 developed in this study can be used for the rapid detection of smut resistance in sugarcane and may be utilized as reference for the improvement of sugarcane smut resistance based on molecular marker-assisted selection.© 2021 Friends Science Publishers

DEM modeling of the one-dimensional compression of sands incorporating a statistical particle fragmentation scheme

This paper presents a novel framework of modeling crushable granular materials under mechanical loadings based on the discrete element method (DEM). The framework is featured with the construction of the one-to-one model in which every particle in a physical experiment has its own numerical twin and allows the modeling of irregular shaped fragments during the continuous breakage process. First, image processing techniques and spherical harmonic (SH) analysis were adopted, respectively, to segment and label particles and to construct a one-to-one model mathematically in DEM. Then, a particle crushing criterion based on the maximum inter-particle contact force was used to predict the crushing events, showing fitting results that agreed very well with a large number of single particle crushing tests. Next, a statistical approach for the generation of particle fragmentation modes of a given type of sand particles based on the principal component analysis (PCA) was proposed. The aim of the PCA was to analyze the statistical trends of the coefficient matrix, which was composed of the SH coefficients of all the particles involved in the analysis. Finally, a successful modeling of a particle crushing event was achieved by replacing the particle, which was judged by the crushing criterion to undergo crushing, with a few sub-particles chosen randomly from a specific fragment template constructed using the micro-computed tomography (micro-CT) data.

Insight into Functional Membrane Proteins by Solution NMR: The Human Bcl-2 Protein—A Promising Cancer Drug Target

Evasion from programmed cell death (apoptosis) is the main hallmark of cancer and a major cause of resistance to therapy. Many tumors simply ensure survival by over-expressing the cell-protecting (anti-apoptotic) Bcl-2 membrane protein involved in apoptotic regulation. However, the molecular mechanism by which Bcl-2 protein in its mitochondrial outer membrane location protects cells remains elusive due to the absence of structural insight; and current strategies to therapeutically interfere with these Bcl-2 sensitive cancers are limited. Here, we present an NMR-based approach to enable structural insight into Bcl-2 function; an approach also ideal as a fragment-based drug discovery platform for further identification and development of promising molecular Bcl-2 inhibitors. By using solution NMR spectroscopy on fully functional intact human Bcl-2 protein in a membrane-mimicking micellar environment, and constructs with specific functions remaining, we present a strategy for structure determination and specific drug screening of functional subunits of the Bcl-2 protein as targets. Using 19F NMR and a specific fragment library (Bionet) with fluorinated compounds we can successfully identify various binders and validate our strategy in the hunt for novel Bcl-2 selective cancer drug strategies to treat currently incurable Bcl-2 sensitive tumors.

Endotrophin, a pro-peptide of Type VI collagen, is a biomarker of survival in cirrhotic patients with hepatocellular carcinoma

Aim: Type VI collagen, is emerging as a signaling collagen originating from different types of fibroblasts. A specific fragment of Type VI collagen, the pro-peptide, is also known as the hormone endotrophin. We hypothesized that this fibroblast hormone would be of particular relevance in cancer types with a high amount of fibrosis activity, namely for outcome in hepatocellular carcinoma (HCC) cirrhotic patients. Patients & methods: Plasma C6M, PRO-C6 and alphafeto-protein (AFP) were assessed in 309 patients with mixed etiologies (hepatitis C, hepatitis B, alcohol and nonalcoholic fatty liver) diagnosed as cirrhotics, cirrhotics with HCC, noncirrhotics and healthy controls. Progression-free survival and overall survival (OS) data were collected up to 6120 days after diagnosis. The ability of each marker to predict survival was investigated. Results & conclusion: The level of endotrophin assessed by PRO-C6 was able to separate healthy controls, noncirrhotics and cirrhotics from HCC (p < 0.05–0.0001). Both endotrophin and C6M provided value in the prediction of OS in cirrhotic patients with HCC. In the multivariate analysis for identifying HCC, in patients with high endotrophin (highest quartile) and that were positive for AFP (≥20 IU/ml), the hazard ratio for predicting OS was increased from 3.7 (p = 0.0006) to 14.4 (p = 0.0001) when comparing with AFP positive as a stand-alone marker. In conclusion, plasma levels for markers of Type VI collagen remodeling were associated with survival in cirrhotic patients with HCC. A combination of AFP with endotrophin improved the prognostic value compared with AFP alone for predicting OS in cirrhotic patients with HCC.

Exploring DNA Diversity in Leathers:

DNA based approaches have become widespread in recent times to identify the origin of samples when its phenotypic characteristics are not distinguishable. This, in particular, applies to the leather industry wherein with an increase in duplicated embossing of grain patterns; there is a need to detect the animal origin of commercial leather articles. Thus, the characterization of molecular markers that enables rapid detection of the leather source helps us in precise species identification. The present study aims to generate definite sequences between the four major species in the Bovidae family (Buffalo, Cow, Goat, and Sheep), which are the major players in the manufacture of leather products, especially in India. Based on specific mitochondrial sequences, a specific fragment of the mitochondrial 12SrRNA gene was amplified by PCR as a marker for species-level identification. By the maximum homogeneity, from the NCBI and BOLD database, the BLAST analysis of the sequences of amplicons from unknown sources, distinguish closely related species of the subfamilies Bovinae (buffalo and Cow) and Caprinae (sheep and goat) and this 12SrRNA based PCR-BLAST analysis is a good tool to identify the origin and control the quality of leathers that are being manufactured. The present study has optimized an approach for the extraction and amplification of DNA from the finished leather, which is one of the most significant challenges because of the vigorous processes encountered during their manufacture. The findings of the study have commercial value at large scale.

Energy sharing based on microscopic level densities

The transformation of a moderately excited heavy nucleus into two excited fission fragments is modeled as a strongly damped evolution of the nuclear shape. The resulting Brownian motion in the multi-dimensional deformation space is guided by the shape-dependent level density which has been calculated microscopically for each of nearly ten million shapes (given in the three-quadratic-surfaces parametrization) by using a previously developed combinatorial method that employs the same single-particle levels as those used for the calculation of the pairing and shell contributions to the five-dimensional macroscopic-microscopic potential-energy surface. The stochastic shape evolution is followed until a small critical neck radius is reached, at which point the mass, charge, and shape of the two proto-fragments are extracted. The available excitation energy is divided statistically on the basis of the microscopic level densities associated with the two distorted fragments. Specific fragment structure features may cause the distribution of the energy disvision to deviate significantly from expectations based on a Fermi-gas level density. After their formation at scission, the initially distorted fragments are being accelerated by their mutual Coulomb repulsion as their shapes relax to their equilibrium forms. The associated distortion energy is converted to additional excitation energy in the fully accelerated fragments. These subsequently undergo sequential neutron evaporation which is calculated using again the appropriate microscopic level densities. The resulting dependence of the mean neutron multiplicity on the fragment mass, as well as the dependence of on the initial excitation energy of the fissioning compound nucleus, exhibit features that are similar to the experimentally observed behavior, suggesting that the microscopic energy sharing mechanism plays an important role in low-energy fission.

The influence of author’s provocation upon interpretation of the fourth chapter of “Finnegans Wake” by James Joyce

This article is dedicated to the peculiarities of the last novel of the Irish writer James Joyce &ldquo;Finnegans Wake&rdquo;, written in 1939. James Joyce paid deliberate attention to the linguistic arrangement of his work, resulting in the novel becoming difficult to translate, as well as to read and comprehend. Analysis is conducted on the fourth chapter of &ldquo;Finnegans Wake&rdquo; for demonstrating a peculiar feature of James Joyce's style of writing. Provocation of the Irish novelist is consists in usage of various puns for confusing the reader. This instigates the reader to seek different meanings that correspond to the writer&rsquo;s concept throughout the text or a specific fragment. The article employs semantic and structural methods of analysis for interpretation of pun. The research also uses historical- cultural and biographical methods for analyzing the complex instances of interpretation. Field analysis is applied for allocating the acquired results into three zones: nuclear, close, and far peripheries. The main result of this research consists in analysis of the novel in the context of author's provocation. The fancy linguistic arrangement of the literary text should be viewed from the two perspectives: on the one hand, the reader analyzes the language and perceives pun as an intended concealment of the novel&rsquo;s message; on the other hand, interrelation of the meanings of one pun with forces the reader to ponder on a play of sorts between the author and the reader. It can be unequivocally claimed that the reader is constantly uncertain in their correctness.

Comparison of loop-mediated isothermal amplification and conventional PCR tests for diagnosis of common Brucella species

Objective Rapid, reliable, and affordable detection of Brucella species via the molecular methods remains a challenge. In recent years, loop-mediated isothermal amplification (LAMP) is a functional nucleic acid amplification technique offering a substitute to polymerase chain reaction (PCR). So, we compared the LAMP assay with the conventional PCR for the identification of common Brucella species in Iran. In this study, LAMP assay was comprehensively evaluated against the common PCR method. A group of specific LAMP primers were used to amplify a highly specific fragment from the sequence of the Brucella abortus, bcsp31 gene. Sensitivity and specificity values of tests were done with a set of 78 (50 Brucella and 28 non-Brucella) strains. Results A dilution series of B. abortus DNA indicated that the LAMP reaction could reliably detect 10 (fg/µl) DNA target copies per reaction within 36 min, which is 10 times greater than the PCR assay. In summary, we conclude that LAMP assay provide accurate and fast test results to identify of common Brucella species in low-complexity labs, mainly in low and lower middle income countries.

Comparison of Loop-Mediated Isothermal Amplification and conventional PCR for Diagnosis of common Brucella species

Objective: Rapid, reliable, and affordable detection of Brucella species via the molecular methods remains a challenge. In recent years, loop-mediated isothermal amplification (LAMP) is a functional nucleic acid amplification technique offering a substitute to polymerase chain reaction (PCR). So, we compared the LAMP assay with the conventional PCR for the identification of common Brucella species in Iran. In this study, LAMP assay was comprehensively evaluated against the common PCR method. A group of specific LAMP primers were used to amplify a highly specific fragment from the sequence of the Brucella abortus bcsp31 gene. Sensitivity and specificity values of tests were done with a set of 78 (50 Brucella and 28 non-Brucella) strains.Results: A dilution series of Brucella abortus DNA indicated that the LAMP reaction could reliably detect 10 (fg/µl) DNA target copies per reaction within 36 minute, which is 10 times greater than the PCR assay. In summary, we conclude that LAMP assay provide accurate and fast test results to identify of common Brucella species in low-complexity labs, mainly in low and lower middle income countries.