Maria Silvia Viccari Gatti

Molecular characterization of picobirnaviruses from new hosts.

Picobirnaviruses (PBVs) have recently been classified into the Picobirnaviridae family. They are small, non-enveloped viruses with bisegmented, double-stranded (ds) RNA genomes. Although they are found in the feces of a broad range of hosts, information regarding their genomes is limited to viruses detected from humans, rabbits, and porcine. Identification of PBVs has been done using PAGE and reverse transcription PCR (RT-PCR). In this study, we present a phylogenetic analysis of PBVs detected in the feces of dogs, snakes, and rats. In addition, we compare these strains to those from human and porcine hosts. To do so, 487 fecal specimens from dogs, snakes and rats were analyzed by PAGE. The positive specimens for PBV were tested by RT-PCR using primers for genogroup I of the PBVs. From the 11 genogroup I PBV samples, at least one from each host was sequenced and submitted for phylogenetic analysis. All of the sequences showed high homology with the human and porcine genogroup I PBV sequences. In this study we report the first detection of PBVs in snakes (8.5%). We also report a phylogenetic analysis that goes beyond humans and pigs to include dogs, rats, and snakes. However, more hosts must be included in the analysis so that we may reach better conclusions regarding the spread of these viruses.

Different types of aqueous two‐phase systems for biomolecule and bioparticle extraction and purification

Upstream improvements have led to significant advances in the productivity of biomolecules and bioparticles. Today, downstream processes are the bottleneck in the production of some biopharmaceuticals, a change from previous years. Current purification platforms will reach their physical limits at some point, indicating the need for new approaches. This article reviews an alternative method to extract and purify biomolecules/bioparticles named aqueous two‐phase system (ATPS). Biocompatibility and readiness to scale up are some of the ATPS characteristics. We also discuss some of ATPS applications in the biotechnology field.

Avian anticoccidial activity of a novel membrane-interactive peptide selected from phage display libraries

In the present work, we describe the discovery of PW2, a novel peptide presenting in vitro activity against Eimeria acervulina and E. tenella sporozoites. PW2 was selected from phage display (Ph.D.) peptide libraries by an alternative method of panning using living purified E. acervulina sporozoites as targets. Our results showed that the peptide disrupts the sporozoite pellicle, resembling the effect caused by most natural antimicrobial peptides. PW2 peptide was also effective against fungi and showed low activity against Toxoplasma gondii tachyzoites, but no activity against Trypanosoma cruzi, Crithidia fasciculata epimastigotes, and bacteria. Additionally, the parasiticidal concentrations of PW2 produced a very low lytic effect on mammalian and avian cells. The effectiveness against Eimeria sporozoites and the absence of adverse effects to host cells indicates that PW2 may be used as a model to generate new drugs for the control of avian coccidiosis.

Changes in Chromatin Structure in NIH 3T3 Cells Induced by Valproic Acid and Trichostatin A

Valproic acid (VPA) and trichostatin A (TSA) are known histone deacetylase inhibitors (HDACIs) with epigenetic activity that affect chromatin supra‐organization, nuclear architecture, and cellular proliferation, particularly in tumor cells. In this study, chromatin remodeling with effects extending to heterochromatic areas was investigated by image analysis in non‐transformed NIH 3T3 cells treated for different periods with different doses of VPA and TSA under conditions that indicated no loss of cell viability. Image analysis revealed chromatin decondensation that affected not only euchromatin but also heterochromatin, concomitant with a decreased activity of histone deacetylases and a general increase in histone H3 acetylation. Heterochromatin protein 1‐α (HP1‐α), identified immunocytochemically, was depleted from the pericentromeric heterochromatin following exposure to both HDACIs. Drastic changes affecting cell proliferation and micronucleation but not alteration in CCND2 expression and in ratios of Bcl‐2/Bax expression and cell death occurred following a 48‐h exposure of the NIH 3T3 cells particularly in response to higher doses of VPA. Our results demonstrated that even low doses of VPA (0.05 mM) and TSA (10 ng/ml) treatments for 1 h can affect chromatin structure, including that of the heterochromatin areas, in non‐transformed cells. HP1‐α depletion, probably related to histone demethylation at H3K9me3, in addition to the effect of VPA and TSA on histone H3 acetylation, is induced on NIH 3T3 cells. Despite these facts, alterations in cell proliferation and micronucleation, possibly depending on mitotic spindle defects, require a longer exposure to higher doses of VPA and TSA. J. Cell. Biochem. 115: 1937–1947, 2014.

Nomenclature proposal for picobirnavirus

Picobirnaviruses have been identified in the feces of a broad range of hosts by several international research groups. Because there is no standard nomenclature for these viruses, we propose a clear and unique name for each strain.

Changing distribution of human rotavirus serotypes during two epidemic outbreaks of gastroenteritis in Campinas, São Paulo, Brazil, 2003-2004: Detection of G6 strains

BACKGROUND Rotavirus serotypes G1-G4 and G9 are the most important agents of severe diarrhea in children worldwide. OBJECTIVE To characterize rotavirus serotypes/genotypes causing two large outbreaks of diarrhea in Campinas, São Paulo, during 2003-2004. STUDY Rotavirus infection was investigated in 328 stool specimens collected from children and adults with diarrhea by PAGE and RT-PCR and further characterized by semi-nested PCR-typing assays. RESULTS G3P[8] (26.1%), G9P[8] (18.7%) and G1P[8] (17.9%) were the most frequently detected serotypes/genotypes. G1P[8] was predominant in 2003, but significantly decreased the following year when G3P[8] and G9P[8] prevailed. G5P[8] was identified in about 9% of the typed specimens from each year consistent with its endemic nature in Brazil for over two decades. The other globally common serotypes (G4P[8] and G2P[4]), uncommon G-P combinations, and multiple G serotypes were also found. Rarely found in humans, and not previously reported in Brazil, serotype G6 was identified in three specimens obtained from children in 2004. CONCLUSION Multiple rotavirus serotypes were observed co-circulating in the city with serotype predominance changing between the two-year study. This study provides pre-vaccine baseline information on locally endemic strains that might help analysis of post-vaccine data.

Rotavirus excretion in naturally infected pigs with and without diarrhoea

Seven hundred and fifty faecal samples from piglets ranging from 1 to 60 days old were studied for the presence of group A rotavirus by polyacrylamide gel electrophoresis (PAGE) and by enzyme immunoassay (EIA). From 451 diarrhoeic pigs, 117 (25.94%) were positive for rotavirus and only 45 (15.05%) of 299 pigs without diarrhoea excreted the virus (P < 0.005). When these animals were separated into four age groups with regard to the presence or absence of diarrhoea, it was observed that the excretion of rotavirus was associated with diarrhoea in piglets, both before and after weaning.

Presence of group A and non-A rotaviruses in neonatal piglets in Campinas, SP, Brazil

Rotaviruses were detected by polyacrylamide gel electrophoresis (PAGE) in 11 (84,6%) of 13 faecal specimens from neonatal piglets with acute diarrhoea in a piggery near the city of Campinas, State of São Paulo, Brazil. An immunoenzimatic assay for group A rotavirus (IEA-A) was positive in ten of the samples, all of which showed a PAGE profile typical of that group. Another sample was showed a group B profile in PAGE. An immunoenzimatic assay specific for group B (IEA-B) for this faecal sample was positive, confirming the PAGE results.

DNA Methylation Changes in Valproic Acid-Treated HeLa Cells as Assessed by Image Analysis, Immunofluorescence and Vibrational Microspectroscopy.

Valproic acid (VPA), a well-known histone deacetylase inhibitor, has been reported to affect the DNA methylation status in addition to inducing histone hyperacetylation in several cell types. In HeLa cells, VPA promotes histone acetylation and chromatin remodeling. However, DNA demethylation was not checked in this cell model for standing effects longer than those provided by histone acetylation, which is a rapid and transient phenomenon. Demonstration of VPA-induced DNA demethylation in HeLa cells would contribute to understanding the effect of VPA on an aggressive tumor cell line. In the present work, DNA demethylation in VPA-treated HeLa cells was assessed by image analysis of chromatin texture, the abundance of 5-methylcytosine (5mC) immunofluorescence signals and Fourier transform-infrared (FT-IR) microspectroscopy centered on spectral regions related to the vibration of–CH3 groups. Image analysis indicated that increased chromatin unpacking promoted by a 4-h-treatment with 1.0 mM VPA persisted for 24 h in the absence of the drug, suggesting the occurrence of DNA demethylation that was confirmed by decreased 5mC immunofluorescence signals. FT-IR spectra of DNA samples from 1 mM or 20 mM VPA-treated cells subjected to a peak fitting analysis of the spectral window for–CH3 stretching vibrations showed decreased vibrations and energy of these groups as a function of the decreased abundance of 5mC induced by increased VPA concentrations. Only the 20 mM-VPA treatment caused an increase in the ratio of -CH3 bending vibrations evaluated at 1375 cm-1 in relation to in-plane vibrations of overall cytosines evaluated at 1492 cm-1. CH3 stretching vibrations showed to be more sensitive than–CH3 bending vibrations, as detected with FT-IR microspectroscopy, for studies aiming to associate vibrational spectroscopy and changes in DNA 5mC abundance.

Differential Response of Human Hepatocyte Chromatin to HDAC Inhibitors as a Function of Microenvironmental Glucose Level

Diabetes is a complex multifactorial disorder characterized by chronic hyperglycemia due to impaired insulin secretion. Recent observations suggest that the complexity of the disease cannot be entirely accounted for genetic predisposition and a compelling argument for an epigenetic component is rapidly emerging. The use of histone deacetylase inhibitor (HDACi) in clinical setting is an emerging area of investigation. In this study, we have aimed to understand and compare the response of hepatocyte chromatin to valproic acid (VPA) and trichostatin A (TSA) treatments under normoglycemic or hyperglycemic conditions to expand our knowledge about the consequences of HDACi treatment in a diabetes cell model. Under normoglycemic conditions, these treatments promoted chromatin remodeling, as assessed by image analysis and H3K9ac and H3K9me2 abundance. Simultaneously, H3K9ac marks shifted to the nuclear periphery accompanied by HP1 dissociation from the heterochromatin and a G1 cell cycle arrest. More striking changes in the cell cycle progression and mitotic ratios required drastic treatment. Under hyperglycemic conditions, high glucose per se promoted chromatin changes similar to those promoted by VPA and TSA. Nonetheless, these results were not intensified in cells treated with HDACis under hyperglycemic conditions. Despite the absence of morphological changes being promoted, HDACi treatment seems to confer a physiological meaning, ameliorating the cellular hyperglycemic state through reduction of glucose production. These observations allow us to conclude that the glucose level to which the hepatocytes are subjected affects how chromatin responds to HDACi and their action under high‐glucose environment might not reflect on chromatin remodeling. J. Cell. Physiol. 231: 2257–2265, 2016.