Knockdown of Sec16 causes early lethality and defective deposition of the protein Rp30 in the eggshell of the vector Rhodnius prolixus.

In nearly every species of insect, embryonic development takes place outside of the mother's body and is entirely dependent on the elements that the mother had previously stored within the eggs. It is well known that the follicle cells (FCs) synthesize the eggshell (chorion) components during the process of choriogenesis, the final step of oogenesis before fertilization. These cells have developed a specialization in the massive production of chorion proteins, which are essential for the protection and survival of the embryo. Here, we investigate the function of Sec16, a protein crucial for the endoplasmic reticulum (ER) to Golgi traffic, in the oocyte development in the insect Rhodnius prolixus. We discovered that Sec16 is strongly expressed in vitellogenic females' ovaries, particularly in the choriogenic oocyte and it is mainly associated with the FCs. Silencing of Sec16 by RNAi caused a sharp decline in oviposition rates, F1 viability, and longevity in adult females. In the FCs, genes involved in the unfolded protein response (UPR), the ubiquitin-proteasome system (UPS), and autophagy were massively upregulated, whereas the mRNAs of Rp30 and Rp45-which code for the two major chorion proteins - were downregulated as a result of Sec16 silencing, indicating general proteostasis disturbance. As a result, the outer surface ultrastructure of Sec16-silenced chorions was altered, with decreased thickness, dityrosine crosslinking, sulfur signals, and lower amounts of the chorion protein Rp30. These findings collectively demonstrate the critical role Sec16 plays in the proper functioning of the FCs, which impacts the synthesis and deposition of particular components of the chorion as well as the overall reproduction of this vector.

Biophysical tools to study the oligomerization dynamics of Prx1-class peroxiredoxins.

Peroxiredoxins (Prx) are ubiquitous, highly conserved peroxidases whose activity depends on catalytic cysteine residues. The Prx1-class of the peroxiredoxin family, also called typical 2-Cys Prx, organize as head-to-tail homodimers containing two active sites. The peroxidatic cysteine CP of one monomer reacts with the peroxide substrate to form sulfenic acid that reacts with the resolving cysteine (CR) of the adjacent subunit to form an intermolecular disulfide, that is reduced back by the thioredoxin/thioredoxin reductase/NADPH system. Although the minimal catalytic unit is the dimer, these Prx oligomerize into (do)decamers. In addition, these ring-shaped decamers can pile-up into high molecular weight structures. Prx not only display peroxidase activity reducing H2O2, peroxynitrous acid and lipid hydroperoxides (antioxidant enzymes), but also exhibit holdase activity protecting other proteins from unfolding (molecular chaperones). Highly relevant is their participation in redox cellular signaling that is currently under active investigation. The different activities attributed to Prx are strongly ligated to their quaternary structure. In this review, we will describe different biophysical approaches used to characterize the oligomerization dynamics of Prx that include the classical size-exclusion chromatography, analytical ultracentrifugation, calorimetry, and also fluorescence anisotropy and lifetime measurements, as well as mass photometry.

Apolipoprotein E4 heterologous expression, purification under non-denaturing conditions, and effects on neuronal clonal cell lines.

The ε4 allele of the apolipoprotein E gene (APOE4) constitutes the main genetic risk factor for late-onset Alzheimer disease (AD). High amounts of pure apolipoprotein E4 (ApoE4), in a rapid and reproducible fashion, could be of value for studying its pathophysiological roles in AD. The aim of the present work was to optimize a preparative method to obtain highly purified recombinant ApoE4 (rApoE4) with full biological activity. rApoE4 was expressed in the E. Coli BL21(D3) strain and a soluble form of the protein was purified by a combination of affinity and size-exclusion chromatography that precluded a denaturation step. The structural integrity and the biochemical activity of the purified rApoE4 were confirmed by circular dichroism and a lipid-binding assay. Several biological parameters affected by rApoE4, such as mitochondrial morphology, mitochondrial membrane potential and reactive oxygen species production were studied in CNh cells, a neuronal cell line, and neurodifferentiation and dendritogenesis were analyzed in the SH-SY5Y neuroblastoma cell line. The improved rApoE4 purification technique reported here enables the production of highly purified protein that retain the structural properties and functional activity of the native protein, as confirmed by tests in two different neuronal cell lines in culture.

Sec22b-dependent antigen cross-presentation is a significant contributor of T cell priming during infection with the parasite Trypanosoma cruzi.

Antigen cross-presentation is a vital mechanism of dendritic cells and other antigen presenting cells to orchestrate the priming of cytotoxic responses towards killing of infected or cancer cells. In this process, exogenous antigens are internalized by dendritic cells, processed, loaded onto MHC class I molecules and presented to CD8+ T cells to activate them. Sec22b is an ER-Golgi Intermediate Compartment resident SNARE protein that, in partnership with sintaxin4, coordinates the recruitment of the transporter associated with antigen processing protein and the peptide loading complex to phagosomes, where antigenic peptides that have been proteolyzed in the cytosol are loaded in MHC class I molecules and transported to the cell membrane. The silencing of Sec22b in dendritic cells primary cultures and conditionally in dendritic cells of C57BL/6 mice, critically impairs antigen cross-presentation, but neither affects other antigen presentation routes nor cytokine production and secretion. Mice with Sec22b conditionally silenced in dendritic cells (Sec22b-/-) show deficient priming of CD8+ T lymphocytes, fail to control tumor growth, and are resistant to anti-checkpoint immunotherapy. In this work, we show that Sec22b-/- mice elicit a deficient specific CD8+ T cell response when challenged with sublethal doses of Trypanosoma cruzi trypomastigotes that is associated with increased blood parasitemia and diminished survival.

A disordered region retains the full protease inhibitor activity and the capacity to induce CD8+ T cells in vivo of the oral vaccine adjuvant U-Omp19.

U-Omp19 is a bacterial protease inhibitor from Brucella abortus that inhibits gastrointestinal and lysosomal proteases, enhancing the half-life and immunogenicity of co-delivered antigens. U-Omp19 is a novel adjuvant that is in preclinical development with various vaccine candidates. However, the molecular mechanisms by which it exerts these functions and the structural elements responsible for these activities remain unknown. In this work, a structural, biochemical, and functional characterization of U-Omp19 is presented. Dynamic features of U-Omp19 in solution by NMR and the crystal structure of its C-terminal domain are described. The protein consists of a compact C-terminal beta-barrel domain and a flexible N-terminal domain. The latter domain behaves as an intrinsically disordered protein and retains the full protease inhibitor activity against pancreatic elastase, papain and pepsin. This domain also retains the capacity to induce CD8+ T cells in vivo of U-Omp19. This information may lead to future rationale vaccine designs using U-Omp19 as an adjuvant to deliver other proteins or peptides in oral formulations against infectious diseases, as well as to design strategies to incorporate modifications in its structure that may improve its adjuvanticity.

Rheological Behavior of Pectin Gels Obtained from Araçá (Psidium cattleianum Sabine) Fruits: Influence of DM, Pectin and Calcium Concentrations.

In this work, purified pectins from Araçá fruits (Psidium cattleianum Sabine) were obtained and characterized after partial demethylation. On each prepared sample, the carboxylic yield was obtained by titration, the degree of methylation (DM) by 1H-NMR, and the molecular weight distribution by steric exclusion chromatography (SEC). Then, the gelation ability in the presence of calcium counterions was investigated and related to DM (59-0%); the pectin concentration (2-10 g L-1); and the CaCl2 concentration (0.1-1 mol L-1) used for dialysis. The critical pectin concentration for homogeneous gelation was above 2 g L-1 when formed against 1 mol L-1 CaCl2. The elastic modulus (G') increased with pectin concentration following the relationship G'~C2.8 in agreement with rigid physical gel network predictions. The purified samples APP and APP-A with DM ≥ 40% in the same conditions released heterogeneous systems formed of large aggregates. Gels formed against lower concentrations of CaCl2 down to 0.1 mol L-1 had a higher degree of swelling, indicating electrostatic repulsions between charged chains, thus, counterbalancing the Ca2+ cross-linkage. Compression/traction experiments demonstrated that an irreversible change in the gel structure occurred during small compression with an enhancement of the G' modulus.

Molecular cloning, inducible expression and function analysis of Epinephelus coioides Sec6 response to SGIV infection.

Exocyst complex component 3 Sec6 of mammals, one of the components of the exocyst complex, participates in numerous cellular functions, such as promoting cell migration and inhibiting apoptosis. In this study, the Sec6 was obtained from Epinephelus coioides, an economically important cultured fish. The full length of E. coioides Sec6 was 2655 bp including a 245 bp 5' UTR, a 154 bp 3' UTR, and a 2256 bp open reading frame (ORF) encoding 751 amino acids, with a molecular mass of 86.76 kDa and a theoretical pI of 5.57. Sec6 mRNA was detected in all the tissues examined, but the expression level is different in these tissues. Using fluorescence microscopy, Sec6 were distributed in both the nucleus and the cytoplasm. After SGIV infection, the expression of E. coioides Sec6 was significantly up-regulated in both trunk kidney and spleen response to Singapore grouper iridovirus (SGIV), an important pathogens of E. coioides. Sec6 could increase the SGIV-induced cytopathic effects (CPE), the expression of the SGIV genes VP19, LITAF, MCP, ICP18 and MCP, and the viral titers. Besides, E. coioides Sec6 significantly downregulated the promoter of NF-κB and AP-1, and inhibited the SGIV-induced apoptosis. The results demonstrated that E. coioides Sec6 might play important roles in SGIV infection.

A gold revision of the Golgi Dynamics (GOLD) domain structure and associated cell functionalities.

The classical secretory pathway is the key membrane-based delivery system in eukaryotic cells. Several families of proteins involved in the secretory pathway, with functionalities going from cargo sorting receptors to the maintenance and dynamics of secretory organelles, share soluble globular domains predicted to mediate protein-protein interactions. One of them is the 'Golgi Dynamics' (GOLD) domain, named after its strong association with the Golgi apparatus. There are many GOLD-containing protein families, such as the transmembrane emp24 domain-containing proteins (TMED/p24 family), animal SEC14-like proteins, human Golgi resident protein ACBD3, a splice variant of TICAM2 called TRAM with GOLD domain, and FYCO1. Here, we critically review the state-of-the-art knowledge of the structures and functions of the main representatives of GOLD-containing proteins in vertebrates. We provide the first unified description of the GOLD domain structure across different families since the first high-resolution structure was determined. With a brand-new update on the definition of the GOLD domain, we also discuss how its tertiary structure fits the ß-sandwich-like fold map and give exciting new directions for forthcoming studies.

Sec3 exocyst component knockdown inhibits axonal formation and cortical neuronal migration during brain cortex development.

Neurons are the largest known cells, with complex and highly polarized morphologies and consist of a cell body (soma), several dendrites, and a single axon. The establishment of polarity necessitates initial axonal outgrowth in concomitance with the addition of new membrane to the axon's plasmalemma. Axolemmal expansion occurs by exocytosis of plasmalemmal precursor vesicles primarily at the neuronal growth cone membrane. The multiprotein exocyst complex drives spatial location and specificity of vesicle fusion at plasma membrane. However, the specific participation of its different proteins on neuronal differentiation has not been fully established. In the present work we analyzed the role of Sec3, a prominent exocyst complex protein on neuronal differentiation. Using mice hippocampal primary cultures, we determined that Sec3 is expressed in neurons at early stages prior to neuronal polarization. Furthermore, we determined that silencing of Sec3 in mice hippocampal neurons in culture precluded polarization. Moreover, using in utero electroporation experiments, we determined that Sec3 knockdown affected cortical neurons migration and morphology during neocortex formation. Our results demonstrate that the exocyst complex protein Sec3 plays an important role in axon formation in neuronal differentiation and the migration of neuronal progenitors during cortex development.

Biochemical and biophysical characterization of the RVB-1/RVB-2 protein complex, the RuvBL/RVB homologues in Neurospora crassa.

The RVB proteins, composed of the conservative paralogs, RVB1 and RVB2, belong to the AAA+ (ATPases Associated with various cellular Activities) protein superfamily and are present in archaea and eukaryotes. The most distinct structural features are their ability to interact with each other forming the RVB1/2 complex and their participation in several macromolecular protein complexes leading them to be involved in many biological processes. We report here the biochemical and biophysical characterization of the Neurospora crassa RVB-1/RVB-2 complex. Chromatographic analyses revealed that the complex (APO) predominantly exists as a dimer in solution although hexamers were also observed. Nucleotides influence the oligomerization state, while ATP favors hexamers formation, ADP favors the formation of multimeric states, likely dodecamers, and the Molecular Dynamics (MD) simulations revealed the contribution of certain amino acid residues in the nucleotide stabilization. The complex binds to dsDNA fragments and exhibits ATPase activity, which is strongly enhanced in the presence of DNA. In addition, both GFP-fused proteins are predominantly nuclear, and their nuclear localization signals (NLS) interact with importin-α (NcIMPα). Our findings show that some properties are specific of the fungus proteins despite of their high identity to orthologous proteins. They are essential proteins in N. crassa, and the phenotypic defects exhibited by the heterokaryotic strains, mainly related to growth and development, indicate N. crassa as a promising organism to investigate additional biological and structural aspects of these proteins.

Rapid size-exclusion high performance liquid chromatography method for the quality control of amyloid-ß oligomers.

Amyloid-ß (Aß) dysmetabolism is thought to be the main trigger for neurodegenerative events in Alzheimer's disease (AD). In particular, soluble Aß oligomers (AßOs) are proposed as key mediators of synaptic and cognitive dysfunction in AD. Over the past few decades, AßOs prepared from synthetic Aß have been widely applied in vitro and in vivo, the so-called chemical models of AD, uncovering their multiple neurotoxic mechanisms. However, the lack of a reliable quality control (QC) for synthetic AßOs may reflect poor experimental reproducibility. In keeping with this, we optimized and validated a rapid and reproducible SECHPLC method using fluorescence detection for the QC of synthetic AßOs. Our analytical method offers an unprecedent alternative to improve the reproducibility of AD chemical models.

Seryl-tRNA synthetase specificity for tRNASec in Bacterial Sec biosynthesis.

tRNA synthetases are responsible for decoding the molecular information, from codons to amino acids. Seryl-tRNA synthetase (SerRS), besides the five isoacceptors of tRNASer, recognizes tRNA[Ser]Sec for the incorporation of selenocysteine (Sec, U) into selenoproteins. The selenocysteine synthesis pathway is known and is dependent on several protein-protein and protein-RNA interactions. Those interactions are not fully described, in particular, involving tRNA[Ser]Sec and SerRS. Here we describe the molecular interactions between the Escherichia coli Seryl-tRNA synthetase (EcSerRS) and tRNA[Ser]Sec in order to determine their specificity, selectivity and binding order, leading to tRNA aminoacylation. The dissociation constant of EcSerRS and tRNA[Ser]Sec was determined as (126 ± 20) nM. We also demonstrate that EcSerRS binds initially to tRNA[Ser]Sec in the presence of ATP for further recognition by E. coli selenocysteine synthetase (EcSelA) for Ser to Sec conversion. The proposed studies clarify the mechanism of tRNA[Ser]Sec incorporation in Bacteria as well as of other domains of life.

Schistosoma mansoni cathepsin D1: Biochemical and biophysical characterization of the recombinant enzyme expressed in HEK293T cells.

Schistosomes express a variety of aspartyl proteases (APs) with distinct roles in the helminth pathophysiology, among which degradation of host haemoglobin is key, since it is the main amino acid source for these parasites. A cathepsin D-like AP from Schistosoma mansoni (SmCD1) has been used as a model enzyme for vaccine and drug development studies in schistosomes and yet a reliable expression system for readily producing the recombinant enzyme in high yield has not been reported. To contribute to further advancing the knowledge about this valuable antischistosomal target, we developed a transient expression system in HEK 293T mammalian cells and performed a biochemical and biophysical characterization of the recombinant enzyme (rSmCD1). It was possible to express a recombinant C-terminal truncated form of SmCD1 (rSmCD1ΔCT) and purify it with high yield (16 mg/L) from the culture supernatant. When analysed by Size-Exclusion Chromatography and multi-angle laser light scattering, rSmCD1ΔCT behaved as a dimer at neutral pH, which is unusual for cathepsins D, turning into a monomer after acidification of the medium. Through analytical ultrancentrifugation, the dimer was confirmed for free rSmCD1ΔCT in solution as well as stabilization of the monomer during interaction with pepstatin. The mammalian cell expression system used here was able to produce rSmCD1ΔCT with high yields allowing for the first time the characterization of important kinetic parameters as well as initial description of its biophysical properties.

Bacterial identification, somatic cell count, antimicrobial profile and toxigenic Staphylococcus strains search from mastitic cow milk samples on small farms properties / Identificação bacteriana, contagem de células somáticas, perfil antimicrobiano e pesquisa de linhagens toxigênicas de Staphylococcus em amostras de leite bovino de pequenas propriedades rurais

Bovine mastitis has a negative impact on milk production and can pose risks to public health. The present study aimed to evaluate the quality of bovine milk from small farms in the Botucatu/SP region. Somatic cell counts (SCC), identification of pathogens involved in mastitis, and sensitivity antimicrobial profile of staphylococci isolated were performed. The presence of enterotoxin encoding genes in isolates of staphylococci obtained from milk was investigated. Milk samples from individual mammary quarters of cows were submitted to the California mastitis test (CMT) and SCC. Of the 239 dairy cows from 21 dairy herds evaluated (mean = 11.4 animals/property), two cows (0.8%) presented clinical mastitis and 86 (35.9%) subclinical mastitis. Bacterial culture was performed in 177 quarter milk samples. Staphylococci were identified in 55 (31.1%), corynebacteria in 45 (25.4%), streptococci in 25 (14.1%) and coliforms in four (2.3%) milk samples. Average SCC from culture-positive samples was 1598x103 cells/mL, in case of staphylococci was 1362x103 cells/ml, streptococci was 2857x103 cells/mL, corynebacteria was 976x103 cells/mL and in the cases of coliforms 1161x103 cells/mL were obtained. Staphylococci showed a high sensitivity (>95%) to cephalothin, cotrimoxazole, enrofloxacin, and gentamicin, with a 41.2% resistance to penicillin and 11.8% to oxacillin. Both coagulase positive (CPS) and negative staphylococci (CNS) carried genes encoding enterotoxins in 21.6% of the first group and 41.9% in the second. The sea gene was the most detected 45.8% (n=24) between them, followed by seb with 29.2% and sec with 25.0%. The sed gene was not identified. We highlight the potential risk to public health in the possibility of strains of Staphylococcus spp. enterotoxin-producing genes that can cause staphylococcal food poisoning.(AU)

A mastite bovina impacta negativamente a produção leiteira e pode acarretar riscos à saúde pública. O presente estudo teve como objetivo a avaliação da qualidade do leite bovino proveniente de pequenas propriedades na região de Botucatu/SP. Foi realizada a contagem de células somáticas (CCS), identificação dos patógenos envolvidos nas mastites, e realizado o perfil de sensibilidade aos antimicrobianos dos estafilococos isolados. Pesquisou-se a presença de genes codificadores de enterotoxinas em isolados de estafilococos obtidos a partir do leite mastítico. Amostras de leite de quartos mamários individuais de vacas foram submetidas ao "California mastitis test" (CMT) e à CCS. Das 239 vacas em lactação provenientes de 21 rebanhos leiteiros avaliados (média = 11,4 animais/propriedade), dois (0,8%) animais apresentaram mastite clínica e, 86 (35,9%) mastite subclínica. 177 amostras de leite foram cultivadas em ágar sangue bovino 5% e ágar MacConckey e obteve-se 55 (31,1%) Staphylococcus spp., 25 (14,1%) Streptococcus spp., 45 (25,4%) Corynebacterium spp. e quatro (2,3%) coliformes. A média da CCS das amostras procedentes de todos os quartos mamários infectados avaliados foi de 1598x103 células/mL, enquanto que nos casos que foram isolados Staphylococcus spp. foi de 1362x103 células/mL, Streptococcus spp. 2857x103 células/mL, Corynebacterium spp. de 976x103 células/mL e nos casos de coliformes 1161x103 células/mL. Os estafilococos revelaram grande sensibilidade (>95%) à cefalotina, cotrimoxazol, enrofloxacina e gentamicina, com resistência de 41,2% à penicilina e 11,8% à oxacilina. Tanto estafilococos coagulase positivos (ECP) como negativos (ECN) revelaram genes codificadores de enterotoxinas em 21,6% do primeiro grupo e 41,9% no segundo. O gene sea foi o mais detectado 45,8% (n=24), seguido pelo seb com 29,2% e sec com 25,0%. O gene codificador da sed não foi identificado. Frente aos resultados, destaca-se o risco potencial à saúde pública pela possibilidade de veiculação de linhagens de Staphylococcus spp. carreadores de genes produtores de enterotoxinas, podendo ocasionar toxi-infecções alimentares.(AU)

Bacterial identification, somatic cell count, antimicrobial profile and toxigenic Staphylococcus strains search from mastitic cow milk samples on small farms properties / Identificação bacteriana, contagem de células somáticas, perfil antimicrobiano e pesquisa de linhagens toxigênicas de Staphylococcus em amostras de leite bovino de pequenas propriedades rurais

Bovine mastitis has a negative impact on milk production and can pose risks to public health. The present study aimed to evaluate the quality of bovine milk from small farms in the Botucatu/SP region. Somatic cell counts (SCC), identification of pathogens involved in mastitis, and sensitivity antimicrobial profile of staphylococci isolated were performed. The presence of enterotoxin encoding genes in isolates of staphylococci obtained from milk was investigated. Milk samples from individual mammary quarters of cows were submitted to the California mastitis test (CMT) and SCC. Of the 239 dairy cows from 21 dairy herds evaluated (mean = 11.4 animals/property), two cows (0.8%) presented clinical mastitis and 86 (35.9%) subclinical mastitis. Bacterial culture was performed in 177 quarter milk samples. Staphylococci were identified in 55 (31.1%), corynebacteria in 45 (25.4%), streptococci in 25 (14.1%) and coliforms in four (2.3%) milk samples. Average SCC from culture-positive samples was 1598x103 cells/mL, in case of staphylococci was 1362x103 cells/ml, streptococci was 2857x103 cells/mL, corynebacteria was 976x103 cells/mL and in the cases of coliforms 1161x103 cells/mL were obtained. Staphylococci showed a high sensitivity (>95%) to cephalothin, cotrimoxazole, enrofloxacin, and gentamicin, with a 41.2% resistance to penicillin and 11.8% to oxacillin. Both coagulase positive (CPS) and negative staphylococci (CNS) carried genes encoding enterotoxins in 21.6% of the first group and 41.9% in the second. The sea gene was the most detected 45.8% (n=24) between them, followed by seb with 29.2% and sec with 25.0%. The sed gene was not identified. We highlight the potential risk to public health in the possibility of strains of Staphylococcus spp. enterotoxin-producing genes that can cause staphylococcal food poisoning.(AU)

A mastite bovina impacta negativamente a produção leiteira e pode acarretar riscos à saúde pública. O presente estudo teve como objetivo a avaliação da qualidade do leite bovino proveniente de pequenas propriedades na região de Botucatu/SP. Foi realizada a contagem de células somáticas (CCS), identificação dos patógenos envolvidos nas mastites, e realizado o perfil de sensibilidade aos antimicrobianos dos estafilococos isolados. Pesquisou-se a presença de genes codificadores de enterotoxinas em isolados de estafilococos obtidos a partir do leite mastítico. Amostras de leite de quartos mamários individuais de vacas foram submetidas ao "California mastitis test" (CMT) e à CCS. Das 239 vacas em lactação provenientes de 21 rebanhos leiteiros avaliados (média = 11,4 animais/propriedade), dois (0,8%) animais apresentaram mastite clínica e, 86 (35,9%) mastite subclínica. 177 amostras de leite foram cultivadas em ágar sangue bovino 5% e ágar MacConckey e obteve-se 55 (31,1%) Staphylococcus spp., 25 (14,1%) Streptococcus spp., 45 (25,4%) Corynebacterium spp. e quatro (2,3%) coliformes. A média da CCS das amostras procedentes de todos os quartos mamários infectados avaliados foi de 1598x103 células/mL, enquanto que nos casos que foram isolados Staphylococcus spp. foi de 1362x103 células/mL, Streptococcus spp. 2857x103 células/mL, Corynebacterium spp. de 976x103 células/mL e nos casos de coliformes 1161x103 células/mL. Os estafilococos revelaram grande sensibilidade (>95%) à cefalotina, cotrimoxazol, enrofloxacina e gentamicina, com resistência de 41,2% à penicilina e 11,8% à oxacilina. Tanto estafilococos coagulase positivos (ECP) como negativos (ECN) revelaram genes codificadores de enterotoxinas em 21,6% do primeiro grupo e 41,9% no segundo. O gene sea foi o mais detectado 45,8% (n=24), seguido pelo seb com 29,2% e sec com 25,0%. O gene codificador da sed não foi identificado. Frente aos resultados, destaca-se o risco potencial à saúde pública pela possibilidade de veiculação de linhagens de Staphylococcus spp. carreadores de genes produtores de enterotoxinas, podendo ocasionar toxi-infecções alimentares.(AU)

Optimizing Periplasmic Expression in Escherichia coli for the Production of Recombinant Proteins Tagged with the Small Metal-Binding Protein SmbP.

We have previously shown that the small metal-binding protein (SmbP) extracted from the gram-negative bacterium Nitrosomonas europaea can be employed as a fusion protein for the expression and purification of recombinant proteins in Escherichia coli. With the goal of increasing the amounts of SmbP-tagged proteins produced in the E. coli periplasm, we replaced the native SmbP signal peptide with three different signal sequences: two were from the proteins CusF and PelB, for transport via the Sec pathway, and one was the signal peptide from TorA, for transport via the Tat pathway. Expression of SmbP-tagged Red Fluorescent Protein (RFP) using these three alternative signal peptides individually showed a considerable increase in protein levels in the periplasm of E. coli as compared to its level using the SmbP signal sequence. Therefore, for routine periplasmic expression and purification of recombinant proteins in E. coli, we highly recommend the use of the fusion proteins PelB-SmbP or CusF-SmbP, since these signal sequences increase periplasmic production considerably as compared to the wild-type. Our work, finally, demonstrates that periplasmic expression for SmbP-tagged proteins is not limited to the Sec pathway, in that the TorA-SmbP construct can export reasonable quantities of folded proteins to the periplasm. Although the Sec route has been the most widely used, sometimes, depending on the nature of the protein of interest, for example, if it contains cofactors, it is more appropriate to consider using the Tat route over the Sec. SmbP therefore can be recommended in terms of its particular versatility when combined with signal peptides for the two different routes.

The role of GYP-3 in cellular morphogenesis of Neurospora crassa: Analyzing its relationship with the polarisome.

In fungal hyphae multiple protein complexes assemble at sites of apical growth to maintain cell polarity. The polarisome, which in Saccharomyces cerevisiae consists of Spa2, Pea2, Bud6 and Bni1 is described as a small network of functionally related proteins that regulate polarized growth. In yeast Msb3 and Msb4 are considered polarisome components since both proteins interact directly with Spa2 and are involved in Bni1-nucleated actin assembly in vivo. Additionally they regulate exocytosis through their GAP activity towards Sec4 and perhaps other Rab GTPases. In filamentous fungi the role of these proteins has not been investigated, and in the genome of Neurospora crassa only the gene gyp-3 (NCU04514) was found to correlate with MSB3 and MSB4 of S. cerevisiae. Therefore in this work the role of GYP-3 and its relationship with the polarisome in N. crassa was analyzed. The results show that GYP-3 is required for normal colony development and cell morphology since the Δgyp-3 strain displayed a substantial reduction in colony diameter and hyphae showed a distorted morphology expressed as a general pattern of bulging areas in the distal region and hyphae were thinner at the active growing zone. The lack of GYP-3 had no effects on the localization of the polarisome components SPA-2 and BNI-1. Likewise, GYP-3 was not necessary for the normal localization of the F-actin population, however the dynamics of the Spitzenkörper (Spk) and the actin population at the apical region seemed to be destabilized. Additionally, the lack of GYP-3 strongly affects the localization and dynamics of SEC-4; which no longer accumulates at the tip of hyphae. The results presented here strongly suggest that GYP-3 is not part of the polarisome; however it requires the scaffold protein SPA-2 for arriving at the tip of hyphae. Although GYP-3 is not essential for cell survival, it has an important role in maintaining normal cell growth and morphology in N. crassa.

Interplay between early-life malnutrition, epigenetic modulation of the immune function and liver diseases.

Early-life nutrition plays a critical role in fetal growth and development. Food intake absence and excess are the two main types of energy malnutrition that predispose to the appearance of diseases in adulthood, according to the hypothesis of 'developmental origins of health and disease'. Epidemiological data have shown an association between early-life malnutrition and the metabolic syndrome in later life. Evidence has also demonstrated that nutrition during this period of life can affect the development of the immune system through epigenetic mechanisms. Thus, epigenetics has an essential role in the complex interplay between environmental factors and genetics. Altogether, this leads to the inflammatory response that is commonly seen in non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome. In conjunction, DNA methylation, covalent modification of histones and the expression of non-coding RNA are the epigenetic phenomena that affect inflammatory processes in the context of NAFLD. Here, we highlight current understanding of the mechanisms underlying developmental programming of NAFLD linked to epigenetic modulation of the immune system and environmental factors, such as malnutrition.

Optimization of the protein extraction method of goat meat using factorial design and response surface methodology.

Protein extraction from goat meat was carried out based on the combination of response surface methodology and factorial design to optimize the variables: temperature (25-50 °C), extraction time (8-20 min), volume (3-10 mL) and extractor concentration (0.05-0.1 mol L-1). The proposed model did not present a lack of fit, explaining 96% of the total data variance (R2 = 0.96). The optimum extraction conditions were: 0.05 mol L-1 for extractor concentration, extraction time of 10 min, temperature of 44 °C and extractor volume of 3.5 mL. The protein content (19.3 g/100 g) obtained by the optimized method was higher than some results reported in the literature. HPLC-SEC-DAD analysis revealed that the extraction conditions used did not significantly modify the protein structure. The proposed method proves to be simple, fast, robust, cheap and adequate for native protein extraction, being a potential approach for proteomic research focusing in goat meat.

Bacterial identification, somatic cell count, antimicrobial profile and toxigenic Staphylococcus strains search from mastitic cow milk samples on small farms properties

ABSTRACT: Bovine mastitis has a negative impact on milk production and can pose risks to public health. The present study aimed to evaluate the quality of bovine milk from small farms in the Botucatu/SP region. Somatic cell counts (SCC), identification of pathogens involved in mastitis, and sensitivity antimicrobial profile of staphylococci isolated were performed. The presence of enterotoxin encoding genes in isolates of staphylococci obtained from milk was investigated. Milk samples from individual mammary quarters of cows were submitted to the California mastitis test (CMT) and SCC. Of the 239 dairy cows from 21 dairy herds evaluated (mean = 11.4 animals/property), two cows (0.8%) presented clinical mastitis and 86 (35.9%) subclinical mastitis. Bacterial culture was performed in 177 quarter milk samples. Staphylococci were identified in 55 (31.1%), corynebacteria in 45 (25.4%), streptococci in 25 (14.1%) and coliforms in four (2.3%) milk samples. Average SCC from culture-positive samples was 1598x103 cells/mL, in case of staphylococci was 1362x103 cells/ml, streptococci was 2857x103 cells/mL, corynebacteria was 976x103 cells/mL and in the cases of coliforms 1161x103 cells/mL were obtained. Staphylococci showed a high sensitivity (>95%) to cephalothin, cotrimoxazole, enrofloxacin, and gentamicin, with a 41.2% resistance to penicillin and 11.8% to oxacillin. Both coagulase positive (CPS) and negative staphylococci (CNS) carried genes encoding enterotoxins in 21.6% of the first group and 41.9% in the second. The sea gene was the most detected 45.8% (n=24) between them, followed by seb with 29.2% and sec with 25.0%. The sed gene was not identified. We highlight the potential risk to public health in the possibility of strains of Staphylococcus spp. enterotoxin-producing genes that can cause staphylococcal food poisoning.

RESUMO: A mastite bovina impacta negativamente a produção leiteira e pode acarretar riscos à saúde pública. O presente estudo teve como objetivo a avaliação da qualidade do leite bovino proveniente de pequenas propriedades na região de Botucatu/SP. Foi realizada a contagem de células somáticas (CCS), identificação dos patógenos envolvidos nas mastites, e realizado o perfil de sensibilidade aos antimicrobianos dos estafilococos isolados. Pesquisou-se a presença de genes codificadores de enterotoxinas em isolados de estafilococos obtidos a partir do leite mastítico. Amostras de leite de quartos mamários individuais de vacas foram submetidas ao California mastitis test (CMT) e à CCS. Das 239 vacas em lactação provenientes de 21 rebanhos leiteiros avaliados (média = 11,4 animais/propriedade), dois (0,8%) animais apresentaram mastite clínica e, 86 (35,9%) mastite subclínica. 177 amostras de leite foram cultivadas em ágar sangue bovino 5% e ágar MacConckey e obteve-se 55 (31,1%) Staphylococcus spp., 25 (14,1%) Streptococcus spp., 45 (25,4%) Corynebacterium spp. e quatro (2,3%) coliformes. A média da CCS das amostras procedentes de todos os quartos mamários infectados avaliados foi de 1598x103 células/mL, enquanto que nos casos que foram isolados Staphylococcus spp. foi de 1362x103 células/mL, Streptococcus spp. 2857x103 células/mL, Corynebacterium spp. de 976x103 células/mL e nos casos de coliformes 1161x103 células/mL. Os estafilococos revelaram grande sensibilidade (>95%) à cefalotina, cotrimoxazol, enrofloxacina e gentamicina, com resistência de 41,2% à penicilina e 11,8% à oxacilina. Tanto estafilococos coagulase positivos (ECP) como negativos (ECN) revelaram genes codificadores de enterotoxinas em 21,6% do primeiro grupo e 41,9% no segundo. O gene sea foi o mais detectado 45,8% (n=24), seguido pelo seb com 29,2% e sec com 25,0%. O gene codificador da sed não foi identificado. Frente aos resultados, destaca-se o risco potencial à saúde pública pela possibilidade de veiculação de linhagens de Staphylococcus spp. carreadores de genes produtores de enterotoxinas, podendo ocasionar toxi-infecções alimentares.