From mouth to gut: microfluidic in vitro simulation of human gastro-intestinal digestion and intestinal permeability.

Reproducible in vitro studies of bioaccessibility, intestinal absorption, and bioavailability are key to the successful development of novel food ingredients or drugs intended for oral administration. There is currently a lack of methods that offer the finesse required to study these parameters for valuable molecules typically found in small volumes - as is the case of nanomaterials, which are often used to carry and protect bioactives. Here, we describe a modular microfluidic-based platform for total simulation of the human gastro-intestinal tract. Digestion-chips and cell-based gut-chips were fabricated from PDMS by soft lithography. On-chip digestion was validated using a fluorescently labelled casein derivative, which followed typical Michaelis-Menten kinetics and showed temporal resolution and good agreement with well-established bench-top protocols. Irreversible inhibition of serine proteases using Pefabloc® SC and a 1 : 6 dilution was sufficient to mitigate the cytotoxicity of simulated digestion fluids. Caco-2/HT29-MTX co-cultures were grown on-chip under a continuous flow for 7 days to obtain a differentiated cell monolayer forming a 3D villi-like epithelium with clear tight junction formation, and with an apparent permeability (Papp) of Lucifer Yellow closely approximating values reported ex vivo (3.7 × 10-6 ± 1.4 × 10-6vs. 4.0 × 10-6 ± 2.2 × 10-6). Digesta from the digestion-chips were flowed through the gut-chip, demonstrating the capacity to study sample digestion and intestinal permeability in a single microfluidic platform holding great promise for use in pharmacokinetic studies.

UPLC-HDMSE to discover serum biomarkers in adults with type 1 diabetes.

Type 1 diabetes (T1D) is a complex disease with metabolic and functional changes that can alter an individual's proteome. An LC-MS/MS analytical method, in an HDMSE system, was used to identify differentially expressed proteins in the high abundance protein-depleted serum of T1D patients and healthy controls. Samples were processed in Progenesis QI for Proteomics software. A functional enrichment of the proteins was performed with Gene Ontology and ToppGene, and the interactions were visualized by STRING 11.5. As a result, 139 proteins were identified, 14 of which were downregulated in the serum of patients with T1D compared to controls. Most of the differentially expressed proteins were shown to be involved with the immune system, inflammation, and growth hormone stimulus response, and were associated with the progression of T1D. Differential protein expression data showed for the first-time changes in CPN2 expression levels in the serum of patients with T1D. Our findings indicate that these proteins are targets of interest for future investigations and for validation of protein biomarkers in T1D.

Neutralizing Effect of Synthetic Peptides toward SARS-CoV-2.

The outbreak caused by SARS-CoV-2 has taken many lives worldwide. Although vaccination has started, the development of drugs to either alleviate or abolish symptoms of COVID-19 is still necessary. Here, four synthetic peptides were assayed regarding their ability to protect Vero E6 cells from SARS-CoV-2 infection and their toxicity to human cells and zebrafish embryos. All peptides had some ability to protect cells from infection by SARS-CoV-2 with the D614G mutation. Molecular docking predicted the ability of all peptides to interact with and induce conformational alterations in the spike protein containing the D614G mutation. PepKAA was the most effective peptide, by having the highest docking score regarding the spike protein and reducing the SARS-CoV-2 plaque number by 50% (EC50) at a concentration of 0.15 mg mL-1. Additionally, all peptides had no toxicity to three lines of human cells as well as to zebrafish larvae and embryos. Thus, these peptides have potential activity against SARS-CoV-2, making them promising to develop new drugs to inhibit cell infection by SARS-CoV-2.

Potential protein markers in children with Autistic Spectrum Disorder (ASD) revealed by salivary proteomics.

The lack of specific pharmacological therapy for Autistic Spectrum Disorder (ASD) and its clinical heterogeneity demand efforts directed toward the identification of biomarkers to aid in diagnosis. Proteomics offers a new perspective for studying the altered proteins associated with autism spectrum disorders (ASD) and we have saliva as an easy-to-collect biological fluid with important biomolecules for investigating biomarkers in various diseases. In this sense, saliva could be used to identify potential biomarkers of ASD. In the current work, saliva samples were collected from children with different degrees of ASD and healthy children and proteomics approaches were applied to generate data on differentially expressed proteins between groups which will serve as a basis for future validation studies as protein markers. Data are available via ProteomeXchange with identifier PXD030065. As results, 132 proteins were present in 80% of the saliva pools of all analyzed groups. Twenty-five proteins were identified as overexpressed in the group of severe and mild/moderate ASD carriers, among which, eight were identified as potential biomarkers for ASD.

RcAlb-PepII, a synthetic small peptide bioinspired in the 2S albumin from the seed cake of Ricinus communis, is a potent antimicrobial agent against Klebsiella pneumoniae and Candida parapsilosis.

Antimicrobial peptides (AMPs) are important constituents of the innate immunity system of all living organisms. They participate in the first line of defense against invading pathogens such as viruses, bacteria, and fungi. In view of the increasing difficulties to treat infectious diseases due to the emergence of antibiotic-resistant bacterial strains, AMPs have great potential to control infectious diseases in humans and animals. In this study, two small peptides, RcAlb-PepI and RcAlb-PepII, were designed based on the primary structure of Rc-2S-Alb, a 2S albumin from the seed cake of Ricinus communis, and their antimicrobial activity assessed. RcAlb-PepII strongly inhibited the growth of Klebsiella pneumoniae and Candida parapsilosis, and induced morphological alterations in their cell surface. C. parapsilosis exposed to RcAlb-PepII presented higher cell membrane permeabilization and elevated content of reactive oxygen species. RcAlb-PepII also degraded and reduced the biofilm formation in C. parapsilosis and in K. pneumonia cells. Experimentally, RcAlb-PepII was not hemolytic and had low toxicity to mammalian cells. These are advantageous characteristics, which suggest that RcAlb-PepII is safe and apparently effective for its intended use and has great potential for the future development of an antimicrobial agent with the ability to kill or inhibit K. pneumoniae and C. parapsilosis cells.

Mo-CBP3-PepI, Mo-CBP3-PepII, and Mo-CBP3-PepIII are synthetic antimicrobial peptides active against human pathogens by stimulating ROS generation and increasing plasma membrane permeability.

The efficiency of current antimicrobial drugs is noticeably decreasing and thus the development of new treatments is necessary. Natural and synthetic antimicrobial peptides (AMPs) have attracted great attention as promising candidates. Inspired on Mo-CBP3, an antimicrobial protein from Moringa oleifera seeds, we designed and synthesized three AMPs named Mo-CBP3-PepI, Mo-CBP3-PepII, and Mo-CBP3-PepIII. All these three peptides inhibited the growth of Candida species and pathogenic bacteria, penetrate into microbial cells, but none is hemolytic or toxic to human cells. Mo-CBP3-PepIII, particularly, showed the strongest antimicrobial activity against Staphylococcus aureus and Candida species, important human pathogens. Additionally, Mo-CBP3-PepIII did not exhibit hemolytic or toxic activity to mammalian cells, but increased Staphylococcus aureus plasma membrane permeabilization. In Candida parapsilosis, Mo-CBP3-PepIII induced pore formation in the plasma membrane and overproduction of reactive oxygen species. Bioinformatics analysis suggested that Mo-CBP3-PepIII is resistant to pepsin digestion and other proteolytic enzymes present in the intestinal environment, which opens the possibility of oral delivery in future treatments. Together, these results suggest that Mo-CBP3-PepIII has great potential as an antimicrobial agent against the bacterium S. aureus and the fungi C. parapsilosis.

Antibacterial activity and antibiotic modulating potential of the essential oil obtained from Eugenia jambolana in association with led lights.

Bacterial resistance has risen as an important health problem with impact on the pharmaceutical industry because many antibiotics have become ineffective, which has affected their commercialization. The Brazilian biodiversity is marked by a vast variety of natural products with significant therapeutic potential, which could bring new perspectives in the treatment of infections caused by resistant microorganisms. The present study aimed to evaluate the antibacterial effect of the essential oil obtained from Eugenia jambolana (EjEO) using the method of microdilution method to determine the Minimum Inhibitory Concentration (MIC). The modulatory effect of this oil on antibiotic activity was determined using both the broth microdilution and gaseous contact methods. The antibacterial effect of the association of the gaseous contact and the use of a LED unit with red and blue lights was also determined. The chemical components of the EjEO were characterized by HPLC, which revealed the presence of α-pinene as a major constituent. The EjEO presented a MIC≥128µg/mL against S. aureus and ≥1024µg/mL against E. coli. The combination of the EjEO with antibiotics presented synergism against E. coli and antagonism against S. aureus. An antagonistic effect was obtained from the association of EjEO with amikacin and erythromycin by the method of gaseous contact. On the other hand, the association of EjEO with ciprofloxacin presented a synergistic effect against S. aureus and E. coli exposed to LED lights. A similar effect was observed in the association of the EjEO with norfloxacin presented synergism against S. aureus in the same conditions. In conclusion, our results demonstrated that the essential oil obtained from Eugenia jambolana interfere with the action of antibiotics against bacteria exposed to LED lights. Thus, further researches are required to elucidate the mechanisms underlying these effects, which could open new perspectives in the development of new antibacterial therapies.

In vitro evaluation of the antibacterial potential and modification of antibiotic activity of the Eugenia uniflora L. essential oil in association with led lights.

Due to the great biodiversity of its flora, Brazil provides combat tools against bacterial resistance with the utilization of natural products with vegetable origin. Therefore, the present study had as its objective to evaluate the antibacterial potential of the Eugenia uniflora essential oil (EuEO) in vitro, as well as to analyze the modulatory effect of the oil against antibiotics by gaseous contact and to compare them when associated with a LED apparatus. The chemical components were characterised by gas chromatography which revealed the presence of the isoflurane-germacrene, considered the major component (61.69%). The MIC obtained from the EuEO was ≥256 µg/mL for S. aureus and ≥1024 µg/mL for E. coli. When combined with antibiotics, the EuEO presented synergism reducing the MIC when associated, with the exception of gentamicin against E. coli, where an antagonistic effect was observed. The was an interference of the EuEO over the activity of ciprofloxacin when associated with red and blue LED lights, increasing the inhibition halos against S. aureus and E. coli. Norfloxacin presented similar results to ciprofloxacin against S. aureus bacteria. When combined, norfloxacin and the EuEO presented synergism against S. aureus, which did not occur in the combination with ciprofloxacin. Interference occurred only with blue light for E. coli. Thus, it was observed that the EuEO causes changes in the activity of antibiotics, the same occurring with the use of LED lights, without significant differences in the association of the oil and the lights with the antibiotics tested. Further research is needed to elucidate the modulatory effects of the EuEO, as well as its association with LED lights.

Chemical Characterization and Cytoprotective Effect of the Hydroethanol Extract from Annona coriacea Mart. (Araticum).

INTRODUCTION: Annona coriacea Mart. (araticum) is a widely distributed tree in the cerrado. Its value is attributed principally to the consumption of its fruit which possesses a large nutritive potential. The objective was to identify the chemical profile and evaluate the antimicrobial and cytoprotective activity of the hydroethanol extract of A. coriacea Mart. (HEAC) leaves against the toxicity of mercury chloride. MATERIALS AND METHODS: The characterization of components was carried out using high-performance liquid chromatography (HPLC). The minimum inhibitory concentration (MIC) was determined by microdilution method in broth with strains of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. For evaluation of the modulatory and cytoprotective activity of aminoglycoside antibiotics (gentamicin and amikacin) and mercury chloride (HgCl2), the substances were associated with the HEAC at subinhibitory concentrations (MIC/8). RESULTS AND DISCUSSION: The HPLC analysis revealed the presence of flavonoids such as Luteolin (1.84%) and Quercetin (1.19%) in elevated concentrations. The HEAC presented an MIC ≥512 µg/mL and significant antagonistic action in aminoglycosides modulation, and it also showed cytoprotective activity to S. aureus (significance P < 0.0001) and E. coli (significance P < 0.05) bacteria against the mercury chloride heavy metal with significance, this action being attributed to the chelating properties of the flavonoids found in the chemical identification. CONCLUSIONS: The results acquired in this study show that the HEAC presents cytoprotective activity over the tested strains in vitro and can also present antagonistic effect when associated with aminoglycosides, reinforcing the necessity of taking caution when combining natural and pharmaceutical products. SUMMARY: The hydroalcoholic extract of A. coriacea Mart. presents in vitro cytoprotective activity against the toxic effect of Hg. Abbreviations Used: HPLC-DAD: High-performance liquid chromatography with a diode array detector; MIC: Minimum inhibitory concentration; DMSO: Dimethyl sulfoxide.

Post-pandemic influenza A (H1N1) 2009 virus infection in pregnant women in Ceará, Brazil.

OBJECTIVE: The aim of this study was to present results of the post-pandemic phase of A(H1N1)pdm09 virus infection in pregnant women in Ceará, Brazil, during the January-June 2012 influenza season. RESULTS: One hundred and fifty-four nasopharyngeal swab samples were collected from pregnant women admitted to hospitals with suspected severe acute respiratory infection (SARI). Fifty-three (34·4%) had laboratory-confirmed A(H1N1)pdm09 virus infection with 15 (28·3%) outpatients and 38 (71·7%) hospitalized. Five (9·4%) women were in the first trimester of pregnancy, 20 (37·7%) in the second trimester of pregnancy, and 24 (45·2%) in the third trimester of pregnancy. Three had no information about the time of pregnancy. Six samples from newborns were also analyzed, of which three were nasopharyngeal swab positive for A(H1N1)pdm09. These swabs were collected immediately after birth, with the exception of one that was collected on the day after birth. CONCLUSION: Our findings suggest that transplacental transfer of influenza viruses could occur as a result of severe illness in pregnancy. It is therefore important to encourage women to be vaccinated against influenza in order to avoid pregnancy complications.

The ultrastructure of shelled and unshelled cashew nuts.

Cashew nuts have many attributes, including sensory, nutritional and health appeal, which contribute to their worldwide acceptance. We demonstrate details of the microstructure of shelled and unshelled cashew kernels with regard to pericarp and cotyledon organization. This study also provides evidence of the colonization of these kernels by filamentous fungi. Nuts were examined by scanning electron and confocal scanning laser microscopy. Staining with acridine orange was performed. A tight lignified palisade layer adjacent to the exocarp surface explains the hardness of the shell's pericarp. The mesocarp contains large secretory cavities that confer a spongy property to this tissue. Papillose cells, which are responsible for secreting CNSL (cashew nutshell liquid), were observed to cover the inner wall of these cavities. Lipid components are readily released from the parenchyma and appear as oil droplets. The outer surface of the shelled samples exhibited a dense Aspergillus infestation.

Characterization of a new Vaccinia virus isolate reveals the C23L gene as a putative genetic marker for autochthonous Group 1 Brazilian Vaccinia virus.

Since 1999, several Vaccinia virus (VACV) isolates, the etiological agents of bovine vaccinia (BV), have been frequently isolated and characterized with various biological and molecular methods. The results from these approaches have grouped these VACV isolates into two different clusters. This dichotomy has elicited debates surrounding the origin of the Brazilian VACV and its epidemiological significance. To ascertain vital information to settle these debates, we and other research groups have made efforts to identify molecular markers to discriminate VACV from other viruses of the genus Orthopoxvirus (OPV) and other VACV-BR groups. In this way, some genes have been identified as useful markers to discriminate between the VACV-BR groups. However, new markers are needed to infer ancestry and to correlate each sample or group with its unique epidemiological and biological features. The aims of this work were to characterize a new VACV isolate (VACV DMTV-2005) molecularly and biologically using conserved and non-conserved gene analyses for phylogenetic inference and to search for new genes that would elucidate the VACV-BR dichotomy. The VACV DMTV-2005 isolate reported in this study is biologically and phylogenetically clustered with other strains of Group 1 VACV-BR, the most prevalent VACV group that was isolated during the bovine vaccinia outbreaks in Brazil. Sequence analysis of C23L, the gene that encodes for the CC-chemokine-binding protein, revealed a ten-nucleotide deletion, which is a new Group 1 Brazilian VACV genetic marker. This deletion in the C23L open reading frame produces a premature stop-codon that is shared by all Group 1 VACV-BR strains and may also reflect the VACV-BR dichotomy; the deletion can also be considered to be a putative genetic marker for non-virulent Brazilian VACV isolates and may be used for the detection and molecular characterization of new isolates.

Vaccinia virus infection in monkeys, Brazilian Amazon.

To detect orthopoxvirus in the Brazilian Amazon, we conducted a serosurvey of 344 wild animals. Neutralizing antibodies against orthopoxvirus were detected by plaque-reduction neutralizing tests in 84 serum samples. Amplicons from 6 monkey samples were sequenced. These amplicons identified vaccinia virus genetically similar to strains from bovine vaccinia outbreaks in Brazil.

Human Vaccinia virus and Pseudocowpox virus co-infection: clinical description and phylogenetic characterization.

BACKGROUND: Occupational exanthematic diseases represent an important cause of public health impact and economical losses. Among the viral exanthematic diseases, two caused by poxviruses are noteworthy: the bovine vaccinia (BV), caused by the Vaccinia virus (VACV); and the milker's nodule, in which the agent is the Pseudocowpox virus (PCPV). Both agents are zoonotic and have been associated with several cases of bovine infection. In Brazilian rural areas BV has been highly prevalent, particularly in milk herds. Farmers, milkers and their close contacts developed lesions on the hands, forearms, legs and face accompanied by several systemic symptoms. Although VACV and PCPV present with similar epidemiological and transmission patterns, no VACV and PCPV co-infection cases have to date been described. OBJECTIVES: To describe the first case of zoonotic VACV and PCVP co-infection, based on serological and molecular methods. STUDY DESIGN AND RESULTS: In this work we report a case of a Brazilian rural worker who presented with a large severely ulcerated-pustule skin lesion, associated with fever, headache, malaise, myalgia and axillary, inguinal and cervical limphadenopathy. The worker declared occupational contact with cattle that had notable injuries on their teats. Human and bovine clinical samples were collected and submitted to serological and molecular tests. PCR and phylogenetic analysis revealed the presence of VACV DNA and PCPV DNA in the patient's lesion. Serological tests indicated anti-VACV neutralizing antibodies and molecular assays showed the presence of VACV and PCPV DNA in the patient sera. VACV and PCPV also were detected in dairy cattle. CONCLUSION: Together, these results indicate a case of zoonotic VACV/PCPV co-infection. Epidemiological surveillance and appropriate medical treatment are essential for the control of both diseases, especially in the most severe cases, as described in the present study.

Detection and phylogenetic analysis of Orf virus from sheep in Brazil: a case report.

BACKGROUND: Orf virus (ORFV), the prototype of the genus Parapoxvirus (PPV), is the etiological agent of contagious ecthyma, a severe exanthematic dermatitis that afflicts domestic and wild small ruminants. Although South American ORFV outbreaks have occurred and diagnosed there are no South American PPV major membrane glycoprotein B2L gene nucleotide sequences available. CASE PRESENTATION: an outbreak of ovine contagious ecthyma in Midwest Brazil was investigated. The diagnosis was based on clinical examinations and molecular biology techniques. The molecular characterization of the virus was done using PCR amplification, cloning and DNA sequencing of the B2L gene. The phylogenetic analysis demonstrated a high degree of identity with ORFV strains, and the isolate was closest to the ORFV-India 82/04 isolate. Another Brazilian ORFV isolate, NE1, was sequenced for comparative analysis and also showed a high degree of identity with an Asian ORFV strain. CONCLUSION: Distinct ORFV strains are circulating in Brazil. This is the first report on the phylogenetic analysis of an ORFV in South America.

Zoonotic vaccinia virus: clinical and immunological characteristics in a naturally infected patient.

Vaccinia virus was used as vaccine to eradicate smallpox. We report a zoonotic case of vaccinia virus infection in a 30-year-old patient who became infected after handling sick dairy cattle. The patient had inflamed lesions and systemic symptoms. Laboratory findings were indicative of down-modulated immune responses to the virus.

Short report: Isolation of two vaccinia virus strains from a single bovine vaccinia outbreak in rural area from Brazil: Implications on the emergence of zoonotic orthopoxviruses.

Outbreaks of bovine vaccinia disease caused by circulation of Vaccinia virus (VACV) strains have been a common occurrence in Brazil in the recent years, being an important emergent zoonosis. During a single outbreak that took place in 2001, two genetically different VACV strains were isolated and named Guarani P1 virus (GP1V) and Guarani P2 virus (GP2V). Molecular diagnosis was done through restriction fragment length polymorphism (RFLP) of ati gene (A26L) and by sequence analysis of a group of five VACV genes including the C11R, J2R, A56R, B18R, and E3L genes. These findings confirmed the co-circulation of two different Vaccinia virus strains during the same outbreak, raising important questions about the origin, emergence, and circulation of VACV strains in Brazil.

Passatempo virus, a vaccinia virus strain, Brazil.

Passatempo virus was isolated during a zoonotic outbreak. Biologic features and molecular characterization of hemagglutinin, thymidine kinase, and vaccinia growth factor genes suggested a vaccinia virus infection, which strengthens the idea of the reemergence and circulation of vaccinia virus in Brazil. Molecular polymorphisms indicated that Passatempo virus is a different isolate.