Conserved Endonuclease Function of Hantavirus L Polymerase.

Hantaviruses are important emerging pathogens belonging to the Bunyaviridae family. Like other segmented negative strand RNA viruses, the RNA-dependent RNA polymerase (RdRp) also known as L protein of hantaviruses lacks an intrinsic "capping activity". Hantaviruses therefore employ a "cap snatching" strategy acquiring short 5' RNA sequences bearing 5'cap structures by endonucleolytic cleavage from host cell transcripts. The viral endonuclease activity implicated in cap snatching of hantaviruses has been mapped to the N-terminal domain of the L protein. Using a combination of molecular modeling and structure-function analysis we confirm and extend these findings providing evidence for high conservation of the L endonuclease between Old and New World hantaviruses. Recombinant hantavirus L endonuclease showed catalytic activity and a defined cation preference shared by other viral endonucleases. Based on the previously reported remarkably high activity of hantavirus L endonuclease, we established a cell-based assay for the hantavirus endonuclase function. The robustness of the assay and its high-throughput compatible format makes it suitable for small molecule drug screens to identify novel inhibitors of hantavirus endonuclease. Based on the high degree of similarity to RdRp endonucleases, some candidate inhibitors may be broadly active against hantaviruses and other emerging human pathogenic Bunyaviruses

Aseptic Barriers Allow a Clean Contact for Contaminated Stethoscope Diaphragms.

Objective:To determine whether a single-use stethoscope diaphragm barrier surface remains aseptic when placed on pathogen-contaminated stethoscopes. Methods:From May 31 to August 5, 2019, we tested 2 separate barriers using 3 different strains of 7 human pathogens, including extended-spectrum β-lactamase-producing Escherichia coli, methicillin-resistant Staphylococcus aureus, and vancomycin resistant Enterococcus faecium. Results:For all diaphragms with either of the 2 barriers tested, no growth was recorded for any of the pathogens. Stethoscopes with aseptic barriers remained sterile for up to 24 hours. These single-use barriers also provided aseptic surfaces when stethoscope diaphragms were inoculated with human specimens, including saliva, stool, urine, and sputum. Conclusion:Disposable aseptic diaphragm barriers may provide robust and efficient solutions to reduce transmission of pathogens via stethoscopes

Interactions between Saccharomyces and Oenococcus oeni strains from Amarone wine affect malolactic fermentation and wine composition

Research Note

Suitability of the nisin Z-producer lactococcus lactis subsp. lactis CBM 21 to be used as an adjunct culture for squacquerone cheese production

This research investigated the technological and safety effects of the nisin Z producer Lactococcus lactis subsp. lactis CBM 21, tested as an adjunct culture for the making of Squacquerone cheese in a pilot-scale plant. The biocontrol agent remained at a high level throughout the cheese refrigerated storage, without having a negative influence on the viability of the conventional Streptococcus thermophilus starter. The inclusion of CBM 21 in Squacquerone cheesemaking proved to be more effective compared to the traditional one, to reduce total coliforms and Pseudomonas spp. Moreover, the novel/innovative adjunct culture tested did not negatively modify the proteolytic patterns of Squacquerone cheese, but it gave rise to products with specific volatile and texture profiles. The cheese produced with CBM 21 was more appreciated by the panelists with respect to the traditional one

Low-resolution structural studies of human Stanniocalcin-1

Background: Stanniocalcins (STCs) represent small glycoprotein hormones, found in all vertebrates, which have been functionally implicated in Calcium homeostasis. However, recent data from mammalian systems indicated that they may be also involved in embryogenesis, tumorigenesis and in the context of the latter especially in angiogenesis. Human STCI is a 247 amino acids protein with a predicted molecular mass of 27 kDa, but preliminary data suggested its di- or multimerization. The latter in conjunction with alternative splicing and/or post-translational modification gives rise to forms described as STC(50) and "big STC", which molecular weights range from 56 to 135 kDa. Results: In this study we performed a biochemical and structural analysis of STCI with the aim of obtaining low resolution structural information about the human STCI, since structural information in this protein family is scarce. We expressed STCI in both E. coli and insect cells using the baculo virus system with a C-terminal 6 x His fusion tag. From the latter we obtained reasonable amounts of soluble protein. Circular dichroism analysis showed STCI as a well structured protein with 52% of alpha-helical content. Mass spectroscopy analysis of the recombinant protein allowed to assign the five intramolecular disulfide bridges as well as the dimerization Cys202, thereby confirming the conservation of the disulfide pattern previously described for fish STCI. SAXS data also clearly demonstrated that STCI adopts a dimeric, slightly elongated structure in solution. Conclusion: Our data reveal the first low resolution, structural information for human STCI. Theoretical predictions and circular dichroism spectroscopy both suggested that STCI has a high content of alpha-helices and SAXS experiments revealed that STCI is a dimer of slightly elongated shape in solution. The dimerization was confirmed by mass spectrometry as was the highly conserved disulfide pattern, which is identical to that found in fish STCI

Differences in the Volatile Profile of Apple Cider Fermented with Schizosaccharomyces pombe and Schizosaccharomyces japonicus

In this study, two strains of Schizosaccharomyces pombe (NCAIM Y01474(T) and SBPS) and two strains of Schizosaccharomyces japonicus (DBVPG 6274(T), M23B) were investigated for their capacity to ferment apple juice and influence the volatile compounds of cider compared to Saccharomyces cerevisiae EC1118. The ethanol tolerance and deacidification capacity of Schizosaccharomyces yeasts could make them potential substitutes for the commonly used S. cerevisiae starter cultures. Despite different time courses (10-30 d), all strains could complete the fermentation process, and Schizosaccharomyces strains reduced the concentration of malic acid in the apple juice. Results indicated that each yeast exerted a distinctive impact on the volatile profile of the apple cider, giving final products separated using a principal component analysis. The volatile composition of the cider exhibited significant differences in the concentration of alcohols, esters, and fatty acids. Particularly, the flocculant strain S. japonicus M23B increased the levels of ethyl acetate (315.44 +/- 73.07 mg/L), isoamyl acetate (5.99 +/- 0.13 mg/L), and isoamyl alcohol (24.77 +/- 15.19 mg/L), while DBVPG 6274(T) incremented the levels of phenyl ethyl alcohol and methionol up to 6.19 +/- 0.51 mg/L and 3.72 +/- 0.71 mg/L, respectively. A large production of terpenes and ethyl esters (e.g., ethyl octanoate) was detected in the cider fermented by S. cerevisiae EC1118. This study demonstrates, for the first time, the possible application of S. japonicus in cider-making to provide products with distinctive aromatic notes"

Combined pangenomics and transcriptomics reveals core and redundant virulence processes in a rapidly evolving fungal plant pathogen

Background Studying genomic variation in rapidly evolving pathogens potentially enables identification of genes supporting their “core biology”, being present, functional and expressed by all strains or “flexible biology”, varying between strains. Genes supporting flexible biology may be considered to be “accessory”, whilst the “core” gene set is likely to be important for common features of a pathogen species biology, including virulence on all host genotypes. The wheat-pathogenic fungus Zymoseptoria tritici represents one of the most rapidly evolving threats to global food security and was the focus of this study. Results We constructed a pangenome of 18 European field isolates, with 12 also subjected to RNAseq transcription profiling during infection. Combining this data, we predicted a “core” gene set comprising 9807 sequences which were; (1) present in all isolates; (2) lacking inactivating polymorphisms; and (3) expressed by all isolates. A large accessory genome, consisting of 45% of the total genes was also defined. We classified genetic and genomic polymorphism at both chromosomal and individual gene scales. Proteins required for essential functions including virulence, had lower-than average sequence variability amongst core genes. Both core and accessory genomes encoded many small, secreted candidate effector proteins that likely interact with plant immunity. Viral vector-mediated transient in planta overexpression of 88 candidates failed to identify any which induced leaf necrosis characteristic of disease. However, functional complementation of a non-pathogenic deletion mutant lacking five core genes, demonstrated that full virulence was restored by re-introduction of the single gene exhibiting least sequence polymorphism and highest expression. Conclusions These data support the combined use of pangenomics and transcriptomics for defining genes which represent core, and potentially exploitable, weaknesses in rapidly evolving pathogens

Mapping contacts between regulatory domains of skeletal muscle TnC and Tnl by analyses of a single-chain chimeras.

The troponin (Tn) complex is formed by TnC, TnI and TnT and is responsible for the calcium-dependent inhibition of muscle contraction. TnC and TnI interact in an antiparallel fashion in which the N domain of TnC binds in a calcium-dependent manner to the C domain of TnI, releasing the inhibitory effect of the latter on the actomyosin interaction. While the crystal structure of the core cardiac muscle troponin complex has been determined, very little high resolution information is available regarding the skeletal muscle TnITnC complex. With the aim of obtaining structural information regarding specific contacts between skeletal muscle TnC and TnI regulatory domains, we have constructed two recombinant chimeric proteins composed of the residues 191 of TnC linked to residues 98182 or 98147 of TnI. The polypeptides were capable of binding to the thin filament in a calcium-dependent manner and to regulate the ATPase reaction of actomyosin. Small angle X-ray scattering results showed that these chimeras fold into compact structures in which the inhibitory plus the C domain of TnI, with the exception of residues 148182, were in close contact with the N-terminal domain of TnC. CD and fluorescence analysis were consistent with the view that the last residues of TnI (148182) are not well folded in the complex. MS analysis of fragments produced by limited trypsinolysis showed that the whole TnC N domain was resistant to proteolysis, both in the presence and in the absence of calcium. On the other hand the TnI inhibitory and C-terminal domains were completely digested by trypsin in the absence of calcium while the addition of calcium results in the protection of only residues 114137

Simultaneous and time resolved X-ray scattering and differential Scanning calorimetry experiments (SAXS/WAXD/DSC) using synchrotron radiation

New instrumentation designed to perform simultaneous time-resolved X-ray scattering experiments at small and wide angles (SAXS/WAXD) as well as differential scanning calorimetry (DSC) has recently been installed at the SAXS beamline of the Laboratório Nacional de Luz Síncrotron. The DSC device proved to be comparable with conventional equipment, allowing temperature variation with rates of up to 60 °C/min with precision of 0.1 °C. The use of a synchrotron radiation source and position sensitive X-ray detectors allows data collection in real time with 30 s resolution. The application of this experimental set-up in the isothermal crystallization and fusion of polymeric materials is given as an example. We present results of experiments with polycaprolactone (PCL) and its blends with chlorinated polyethylene (PCL/PECl), in which the simultaneous appearance of a crystalline structure and lamellar formation can be observed and the rate of process change for different compositions and thermal treatments can be determined. As a concluding remark, we mention that the SAXS/WAXD/DSC simultaneous experiments can also be performed with great advantage in the study of colloids and gel formation, as well as phase transitions in a variety of samples.Neste trabalho apresentamos uma nova instrumentação instalada na linha de SAXS do LNLS. Este equipamento permite a realização de experimentos simultâneos e resolvidos no tempo de espalhamento de raios X a baixos e altos ângulos (SAXS/WAXD) e calorimetria diferencial de varredura (DSC). O dispositivo de DSC mostrou-se comparável a equipamentos convencionais, com taxas de variação de temperatura de até 60 °C/min e uma precisão de 0.1 °C. O uso de uma fonte de radiação síncrotron e de detetores de raios X sensíveis à posição permitiu a obtenção de dados com uma resolução temporal de 30 s. A aplicação deste arranjo experimental no estudo da cristalização isotérmica e da fusão em materiais poliméricos é mostrada para o caso da policaprolactona (PCL) e suas blendas com polietileno clorado (PCL/PECl). As experiências mostraram a formação simultânea da estrutura cristalina e da morfologia lamelar nos diferentes estágios da cristalização assim como mudanças na cinética do processo com o tratamento isotérmico e a composição da blenda. Finalmente cabe destacar que experimentos simultâneos de SAXS/WAXD/DSC permitem o estudo de distintos processos abrangendo não apenas os de cristalização, mas também a formação de colóides e géis ou as transições de fase estruturais em diversos materiais.199206Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq