Miracle-dz/UniFAR-FTUs huggingface dataset:
Big trouble with little inserts: boundaries in metagenomic screenings using lacZα based vectors

The vast biochemical repertoire found in microbial communities from a wide-range of environments allows screening and isolation of novel enzymes with improved catalytic features. In this sense, metagenomics approaches have been of high relevance for providing enzymes used in diverse industrial applications. For instance, glycosyl hydrolases, which catalyze the hydrolysis of carbohydrates to sugars, are essential for bioethanol production from renewable resources. In the current study, we have focused on the prospection of protease and glycosyl hydrolase activities from microbial communities inhabiting a soil sample by using the lacZα-based plasmid pSEVA232 in the generation of a screenable metagenomic library. For this, we used a functional screen based on skimmed milk agar and a pH indicator dye as previously reported in literature. Although we effectively identified nine positive clones in the screenings, subsequent experiments revealed that this phenotype was not because of the hydrolytic activity encoded in the metagenomic fragments, but rather due to the insertion of small metagenomic DNA fragments in frame within the coding region of the lacZα alpha gene present in the original vector. We concluded that the current method has a higher tendency for false positive recovery of clones, when used in combination with a lacZα-based vector. Finally, we discuss the molecular explanation for positive phenotype recovering and highlight the importance of reporting boundaries in metagenomic screenings methodologies.
A high-throughput analysis method of microdroplet PCR coupled with fluorescence spectrophotometry

Here we report a novel microdroplet PCR method combined with fluorescence spectrophotometry (MPFS), which allows for qualitative, quantitative and high -throughput detection of multiple DNA targets. In this study, each pair of primers was labeled with a specific fluorophore. Through microdroplet PCR, a target DNA was amplified and labeled with the same fluorophore. After products purification, the DNA products tagged with different fluorophores could be analyzed qualitatively by the fluorescent intensity determination. The relative fluorensence unit was also measured to construct the standard curve and to achieve quantitative analysis. In a reaction, the co -amplified products with different fluorophores could be simultaneously analyzed to achieve high -throughput detection. We used four kinds of GM maize as a model to confirm this theory. The qualitative results revealed high specificity and sensitivity of 0.5% (w / w). The quantitative results revealed that the limit of detection was 103copies and with good repeatability. Moreover, reproducibility assay were further performed using four foodborne pathogenic bacteria. Consequently, the same qualitative, quantitative and high-throughput results were confirmed as the four GM maize.
A Potent Anti-Inflammatory Response in Bat Macrophages May Be Linked to Extended Longevity and Viral Tolerance

Bats are unique among mammals given their ability to fly, apparent tolerance of deadly viruses and extraordinary longevity. We propose that these traits are linked and driven by adaptations of the innate immune system. To explore this hypothesis we challenged macrophages from the greater mouse-eared bat, Myotis myotis and the house mouse, Mus musculus with toll like receptors (TLRs) ligands, lipopolysaccharides, LPS and polyinosinic-polycytidylic acid, Poly(I:C). Macrophages from both species presented a high level of mRNA induction of inferon β (INF-β), tumor necrosis factor (TNF) and interleukin-1β (Il-1β). However, in bat macrophages, this antiviral, proinflammatory response was balanced by a sustained high-level transcription of the anti-inflammatory cytokine Il-10, which was not observed in mouse, potentially resulting from adaptive regulation in bats. Additionally, phylogenomic selection tests across the basal divergences in mammals (n = 39) uncovered bat-specific adaptations in six genes involved in antiviral and proinflammatory signalling. Based on this pilot study, we put forward a hypothesis that bats may have evolved unique anti-inflammatory responses to neutralize proinflammatory stimuli resulting from flight. This in turn may drive their extraordinary longevity and viral tolerance by limiting inflammation driven ageing and infection-induced immunopathology. Further data from other individuals and bat species are required to advance this intriguing hypothesis.
Spot blotch disease of wheat: the current status of research on genetics and breeding

The spot blotch disease of wheat is caused by Bipolaris sorokiniana, which is an anamorph (teleomorph Cochliobolus sativus). The disease mainly occurs in warm humid wheat growing regions, and the Eastern Gangetic Plains (EGP) of South Asia is a hotspot. Significant progress has been made in recent years in characterizing the host-pathogen interaction. The study of the pathogen's life cycle and diversity have been an active area of research. A number of resistance sources have also been identified, characterized and utilized for breeding. Although immunity has not been observed in any genotype, cultivars displaying a relatively high level of resistance have been developed and made available to farmers. Further progress will require a regular use of marker-assisted breeding, genomic selection gene editing and transgenic interventions. This review summarizes the current state of knowledge about genetic and breeding efforts on wheat-B. sorokiniana pathosystem and discusses ways in which emerging tools can be used for future research to understand the mechanism involved in infection and for developing cultivars exhibiting a high level of resistance. This article is protected by copyright. All rights reserved.
Recurrent diversification patterns and taxonomic complexity in morphologically conservative ancient lineages of Pimelia (Coleoptera: Tenebrionidae)

The genus Pimelia Fabricius is the most speciose in the tribe Pimeliini (Coleoptera: Tenebrionidae). It consists of approximately 320 species distributed mainly in xeric environments in the western Palaearctic region and northern deserts in the Afrotropical region. Pimelia is a very heterogenous group despite its conserved morphology. Based on two mitochondrial (cox1 and 16S) and five nuclear markers (ITS1, ITS2, 18S, Wg and H3), we carried out a phylogenetic and biogeographic study to: (i) provide a comprehensive phylogenetic hypothesis for the relationships within Pimelia; (ii) re‐evaluate the taxonomic status and organization of clades (subgenera) within the genus; and (iii) establish a temporal framework and historical scenario for the evolution and diversification of Pimelia in the western Mediterranean Region. Pimelia presents a high degree of morphological conservatism and possibly stasis, often resulting in complexes of cryptic species. Repetitive patterns of variation are found recurrently between and within groups, which resulted in a conundrum of nonmonophyletic taxonomic assemblages based on morphological homoplastic characters. Based on our results, we propose a new taxonomic arrangement for Pimelia with 14 clades, treated as subgenera, with the description of six new subgenera: Iberomelia subgen.n. , Italomelia subgen.n. , Magrebmelia subgen.n. , Hispanomelia subgen.n. , Massadraamelia subgen.n. and Amblypteraca subgen.n. Our results also indicate an Oligocene (Rupelian) origin for Pimelia, and North Africa as the most likely area of origin. The complex geological and climatic history in the western Mediterranean region has shaped the distribution and diversity of species in the area. The current faunal assemblages observed in Pimelia are the result of complex patterns of recurrent processes of dispersal and vicariance, including in situ diversification through time in different areas of the Mediterranean basin. Pimelia is a fascinating yet very challenging group, and the phylogenetic and systematic framework developed here will pave the way for further ecological, taxonomic, and evolutionary studies at local and regional scales.
Peptide Terminus Tilting: an Unusual conformational transition of MHC Class I Revealed by Molecular Dynamics Simulations

A conventional picture for major histocompatibility complex class I (MHCI) antigen presentation is that the terminal anchor residues of the antigenic peptide bind to the pockets at the bottom of the MHC cleft, leaving the central peptide residues exposed for T cell antigen receptor (TCR) recognition. However, in the present study, we show that in canonical or accelerated molecular dynamics (MD) simulations, the peptide terminus in some immunodominant peptide-MHCI (pMHCI) complexes can detach from their binding pockets and stretch outside the MHC cleft. These pMHCI complexes include the complex of the H-2Kb and the lymphocytic choriomeningitis virus (LCMV) gp33 peptide, and the complex of the HLA-A*0201 and the influenza A virus M1 peptide. The detached peptide terminus becomes the most prominent spot at the pMHC interface, and so can serves as a novel TCR recognition target. Thus, peptide terminus detaching may be a novel mechanism for MHC antigen presentation.
Genetic parameters of calving ease in dairy cattle using threshold and linear models

Abstract Genetic parameters of direct and maternal effects for calving ease (CE) in Portuguese dairy cattle were estimated applying a threshold model (TM) and three linear models (LM) using 320,953 CE records. In LM, CE scores were directly used as dependent variables (LM1) or were transformed into values on an underlying continuous liability scale (average and maximum values for LM2 and LM3, respectively). The estimate of heritability for direct effects was lower on linear models (0.04–0.05) than in TM (0.09). Furthermore, direct heritability was higher than maternal heritability, ranging between 0.04–0.09 and 0.01–0.02, respectively. The genetic correlation between direct and maternal effects was negative in all models, ranging between –0.75 and –0.82. We investigated the association between bulls estimated breeding values (EBV) attained by TM and three LM, for 2223 bulls. Results show that all correlations were higher than 0.95 and slightly greater correlations were observed for direct effects than for maternal effects. Particular emphasis was given to individual differences between EBV ranking lists. The average difference between rank positions of TM and the three LM ranged from 64 to 122. Therefore, the selection of breeding animals is influenced by the model’s choice. Even with clear theoretical advantages in analysing categorical data, perhaps due to its more recent implementation, TM has not yet achieved the popularity of LM. The results of this work show that TM ranking list is more in accordance with LM2 than with LM1 or LM3. Therefore, with linear models, CE should be considered as average liability value instead of usual numerical score or maximum liability.
Using underwater video to assess megabenthic community vulnerability to trawling in the Grande Vasière (Bay of Biscay)

SUMMARY Trawling activities are considered to be one of the main sources of disturbance to the seabed worldwide. We aimed to disentangle the dominance of environmental variations and trawling intensity in order to explain the distribution of diversity patterns over 152 sampling sites in the French trawl fishing-ground, the Grande Vasière. Using a towed underwater video device, we identified 39 taxa to the finest taxonomic level possible, which were clustered according to their vulnerability to trawling disturbance based on functional traits. Using generalized linear models, we investigated whether the density distribution of each vulnerability group was sensitive to trawling intensity and habitat characteristics. Our analyses revealed a structuring effect of depth and substratum on community structure. The distribution of the more vulnerable group was a negative function of trawling intensity, while the distributions of the less vulnerable groups were independent of trawling intensity. Video monitoring coupled with trait-based vulnerability assessment of macro-epibenthic communities might be more relevant than the traditional taxonomic approach to identifying the areas that are most vulnerable to fishing activities in conservation planning.
REGULATION OF THE BROAD-SPECTRUM DISEASE RESISTANCE PROTEIN RPW8.2 BY PHOSPHORYLATION AND 14-3-3 IN ARABIDOPSIS

Title of Dissertation: REGULATION OF THE BROADSPECTRUM DISEASE RESISTANCE PROTEIN RPW8.2 BY PHOSPHORYLATION AND 14-3-3 IN ARABIDOPSIS Harlan King, Doctor of Philosophy, 2017 Dissertation directed by: Professor Shunyuan Xiao, Institute for Bioscience and Biotechnology Research & Department of Plant Science and Landscape Architecture Ascomycete powdery mildew (PM) fungi belonging to the order of Erysiphales cause diseases on more than 10,000 plant species including economically important staple food crops and numerous horticultural plants. The Arabidopsis protein RPW8.2 confers broad-spectrum resistance against all infectious powdery mildew pathogens. RPW8.2 is unique among characterized plant R proteins in that it activates broad-spectrum resistance to PM fungi, and the protein is specifically targeted to the extra-haustorium membrane (EHM). However, how RPW8.2 is regulated to exert haustoriumtargeted defenses remains poorly characterized. To understand how RPW8.2 is regulated, I first performed a thorough sitespecific mutagenesis of potential serine or threonine residues in RPW8.2 and identified two residues, threonine at 64 and serine at 138 to be critical for RPW8.2’s function. While the T64A mutation makes RPW8.2 auto-active, the S138A mutation abolishes RPW8.2’s ability to activate cell death and defense, with S138A being dominant over T64A. This suggests that RPW8.2 is negatively and positively regulated by (de)phosphorylation at T64 and S138, respectively. One candidate phosphatase and two kinases were genetically and biochemically tested for a potential role in (de)phosphorylation of RPW8.2. I also investigated how an RPW8.2-interacting protein 14-3-3λ regulates RPW8.2’s function. To this end, I developed a novel, divalent 14-33-sequestering protein named RYC to circumvent likely functional redundancy among different 14-3-3 isoforms. Our results demonstrate that RYC can effectively sequester multiple 14-3-3 isoforms from plants and human. When expressed in guard cells of Arabidopsis, RYC sequestered 14-33s away from H+-ATPases, thereby inducing stomatal closure, which in turn increased drought tolerance of transgenic Arabidopsis. When expressed in powdery-mildew-invaded cells, RYC abrogated RPW8.2-mediared resistance to powdery mildew, yet did not grossly affect its EHM-specific localization, suggesting that the C-terminus of RPW8.2 may exert a self-inhibition function which can be relieved when 14-3-3λ binds to the C-terminus during fungal infection. Taken together, our results lead to a better understanding of the molecular mechanisms regulating RPW8.2. REGULATION OF THE BROAD-SPECTRUM DISEASE RESISTANCE PROTEIN RPW8.2 BY PHOSPHORYLATION AND 14-3-3 IN ARABIDOPSIS
Potential of ligninolytic enzymatic complex produced by white‐rot fungi from genus Trametes isolated from Bulgarian forest soil

Because of the crucial role of ligninolytic enzymes in a variety of industrial processes, the demand for a new effective producer has been constantly increasing. Furthermore, information on enzyme synthesis by autochthonous fungal strains is very seldom found. Two fungal strains producing ligninolytic enzymes were isolated from Bulgarian forest soil. They were identified as being Trametes trogii and T. hirsuta. These two strains were assessed for their enzyme activities, laccase (Lac), lignin peroxidase (LiP) and Mn‐dependent peroxidase (MnP) in culture filtrate depending on the temperature and the type of nutrient medium. T. trogii was selected as the better producer of ligninolytic enzymes. The production process was further improved by optimizing a number of parameters such as incubation time, type of cultivation, volume ratio of medium/air, inoculum size and the addition of inducers. The maximum activities of enzymes synthesized by T. trogii was detected as 11100 U/L for Lac, 2.5 U/L for LiP and 4.5 U/L for MnP after 14 days of incubation at 25°C under static conditions, volume ratio of medium/air 1:6, and 3 plugs as inoculum. Among the supplements tested, 5% glycerol increased Lac activity to a significant extent. The addition of 1% veratryl alcohol had a positive effect on MnP.
Protein folding - Preface

The ability of proteins to fold rapidly and efficiently into their intricate and highly specific structures following their synthesis on ribosomes is an essential part of the conversion of genetic information into cellular activity. But in contrast to our understanding of the transcription and translation events, little is understood in detail about how this occurs. In the cell folding is now recognized to be a highly controlled process, with a cascade of proteins involved in ensuring that it occurs in the right place at the right time, and that the newly formed polypeptide chains do not fall victim to unproductive and irreversible events. Many proteins, however, are able to fold in vitro in the absence of all of these factors, indicating that the information necessary for folding is encoded in the amino acid sequence. This also allows folding to be investigated in vitro , and enables physical and chemical methods to be used to probe the structural transitions involved. The role of individual cellular factors can then be explored by examining their influence on the in vitro process, and by comparison with studies of the in vivo events themselves.
Effect of the Growth Promoter VIUSID Agro on the Morphophysiological and Productive Performance of Tobacco Growth (Nicotiana tabacum L.)

With the objective of evaluating the effect of VIUSID agro on the morphophysiological and productive performance of tobacco seedlings, a randomised block experiment was designed with five treatments and three replications. The variants were: dosage of 0.2, 0.5, 0.7 and 1.0 L/ha and one control. The variables are recorded at three points of the cultivation cycle 10th, 20th and 30th days after planting (DAP). Among the indicators evaluated were the fresh and dry mass of the plants, the leaves per plant, foliar area, the length and diameter of the stalk as well as the characteristics of the root. The rates of physiological growth and agricultural yield were also evaluated. The results showed a significant increase (p ≤ 0.05) in the fresh and dry mass of the plants when VIUSID was used. Regarding the length of the stalk, the average increases of those treated in comparison to the control in the final evaluation were 28.42%, 30.51%, 41.17% and 38.43%. In terms of the diameter of the stalk and the foliar area in all evaluations, the treatments with VIUSID significantly exceeded the control. The absolute growth rate and net assimilation was significantly higher when the product was used. In the relative growth rate, the most favourable behaviour was the dosage of 0.5 L/ha, in final evaluation. With regard to yield, the best performance with significant differences (p ≤ 0.05) to the other treatments was the dosage of 0.5, 0.7 and 1.0 L/ha. Therefore, VIUSID agro had a positive influence on the morphophysiological and productive indicators of the tobacco seedlings.
Dominance of ST131 Escherichia coli carrying blaCTX-M in patients with bloodstream infections caused by cephalosporin-resistant strains in Australia, New Zealand and Singapore: whole genome analysis of isolates from a randomised trial

Objectives To characterise multi-drug resistant Escherichia coli isolated from patients in Australia, New Zealand and Singapore with bloodstream infection (BSI). Methods We prospectively collected third-generation cephalosporin resistant (3GC-R) E. coli from blood cultures obtained from patients enrolled in a randomised controlled trial. Whole genome sequencing was used to characterise antibiotic resistance genes, sequence types (STs), plasmids and phylogenetic relationships. Antibiotic susceptibility was determined using disk diffusion and Etest. Results A total of 70 E. coli were included, of which the majority were ST131 (61.4%). BSI was most frequently from a urinary source (69.6%), community-associated (62.9%) and in older patients (median age 71 years [IQR 64-81]). The median Pitt bacteraemia score at presentation was 1 (IQR 0-2, range 0-3) and ICU admission was infrequent (3.1%). ST131 possessed significantly more acquired resistance genes than non-ST131 (p=0.003). Clade C1/C2 ST131 predominated (30.2% and 53.5% of all ST131 respectively) and these were all resistant to ciprofloxacin. All clade A ST131 were community-associated. The predominant ESBL types were blaCTX-M (78.6% of isolates) and were strongly associated with ST131, with the majority blaCTX-M-15. Clade C1 was associated with blaCTX-M-14 and blaCTX-M-27, whereas blaCTX-M-15 predominated in clade C2. Plasmid-mediated AmpC (p-AmpC) genes (mainly blaCMY-2) were also frequent (17.1%) but were more common with non-ST131 strains (p< 0.001). The majority of plasmid replicon types were IncF. Conclusions In a prospective collection of 3GC-R E. coli causing BSI in the Australasian region, community-associated Clade C1/C2 ST131 predominate in association with blaCTX-M ESBLs, although a significant proportion of non-ST131 strains carried blaCMY-2.
Glucose monitoring in living cells with single fluorescent protein-based sensors

Glucose is the main source of energy and carbon in organisms and plays a central role in metabolism and cellular homeostasis. However, the sensitive fluctuation of glucose in living cells is difficult to monitor. Thus, we developed a series of ratiometric, highly responsive, single fluorescent protein-based glucose sensors of wide dynamic range by combining a circularly permuted yellow fluorescent protein with a bacterial periplasmic glucose/galactose-binding protein. We used these sensors to monitor glucose transport in living Escherichia coli cells, and found that the cells take up glucose within 10 min to maintain physiological glucose levels, and observed the differences in glucose uptake and glucose metabolism between wild-type and Mlc knockout cells. These sensors can be specific and simple tools for glucose detection in vitro and non-invasive tools for real-time monitoring of glucose metabolism in vivo.