Characterisation and expression of SPLUNC2, the human orthologue of rodent parotid secretory protein

We recently described the Palate Lung Nasal Clone (PLUNC) family of proteins as an extended group of proteins expressed in the upper airways, nose and mouth. Little is known about these proteins, but they are secreted into the airway and nasal lining fluids and saliva where, due to their structural similarity with lipopolysaccharide-binding protein and bactericidal/permeability-increasing protein, they may play a role in the innate immune defence. We now describe the generation and characterisation of novel affinity-purified antibodies to SPLUNC2, and use them to determine the expression of this, the major salivary gland PLUNC. Western blotting showed that the antibodies identified a number of distinct protein bands in saliva, whilst immunohistochemical analysis demonstrated protein expression in serous cells of the major salivary glands and in the ductal lumens as well as in cells of minor mucosal glands. Antibodies directed against distinct epitopes of the protein yielded different staining patterns in both minor and major salivary glands. Using RT-PCR of tissues from the oral cavity, coupled with EST analysis, we showed that the gene undergoes alternative splicing using two 5' non-coding exons, suggesting that the gene is regulated by alternative promoters. Comprehensive RACE analysis using salivary gland RNA as template failed to identify any additional exons. Analysis of saliva showed that SPLUNC2 is subject to N-glycosylation. Thus, our study shows that multiple SPLUNC2 isoforms are found in the oral cavity and suggest that these proteins may be differentially regulated in distinct tissues where they may function in the innate immune response

The karyotype of three Brazilian Terrarana frogs (Amphibia, Anura) with evidence of a new Barycholos species

A recent substantial rearrangement of the 882 described eleutherodactyline frog species has considerably improved the understanding of their systematics. Nevertheless, many taxonomic aspects of the South American eleutherodactyline species remain unknown and require further investigation using morphological, cytogenetic and molecular approaches. In this work, the karyotypes of the Brazilian species Ischnocnema juipoca (Atibaia and Campos do Jordão, SP), Barycholos cf. ternetzi (Uberlândia, MG, and Porto Nacional, TO), and Pristimantis crepitans (Chapada dos Guimarães and São Vicente, MT) were analyzed using Giemsa staining, Ag-NOR labeling, and C-banding techniques. All individuals had a diploid number of 22 chromosomes, but the Fundamental Numbers were different among species. The herein described low chromosome number of Pristimantis crepitans is unique within this genus, suggesting that cytogenetically this species is not closely related either to its congeneric species or to Ischnocnema. In addition, karyotype differences, mainly in the NOR position, clearly distinguished the two Barycholos populations, besides indicating the existence of a so far undescribed species in this genus. A taxonomic review could clarify the systematic position of P. crepitans and verify the hypothetic new Barycholos species

Nonlinear changes in the activity of the oxygen-dependent demethylase system in Rhodococcus erythropolis cells in the presence of low and very low doses of formaldehyde

The effect of exogenous, highly diluted formaldehyde on the rate of demethylation/re-methylation of veratric acid by the bacteria Rhodococcus erythropolis was studied using electrophoretic and microscopic techniques. The activity of 4-O-demethylase, responsible for accumulation of vanillic acid, and the levels of veratric and vanillic acids were determined using capillary electrophoresis. Formaldehyde was serially diluted at 1:100 ratios, and the total number of iterations was 20. After incubation of the successive dilutions of formaldehyde with the bacteria, demethylase activity oscillated in a sinusoidal manner. It was established using capillary electrophoresis that methylation of vanillic acid to veratric acid occurred at a double rate, as shown by the doubled fluctuation in the concentration of veratrate. There were also changes in the NADH oxidase activity, which is associated with methylation processes. Microscopic observations revealed the presence of numerous enlarged vacuoles in bacterial cells during the accumulation of large amounts of vanillic acid, and their disappearance together with a decrease in 4-O-demethylase activity. The presented results give evidence for the ability of living cells to detect the presence of submolecular concentrations of biological effectors in their environment and provide a basis for a scientific explanation of the law of hormesis and the therapeutic effect of homeopathic dilutions

Does a local Alcohol Health Champion programme have a measurable impact on health and crime outcomes? A natural experiment evaluation of Communities in Charge of Alcohol (CICA) based on triangulation of methods

Background and Aim: Drinking alcohol may cause harm to an individual's health and social relationships, while a drinking culture may harm societies as it may increase crime rates and make an area feel less safe. Local councils in Greater Manchester, UK, developed the Communities in Charge of Alcohol (CICA) intervention, in which volunteers were trained to give alcohol-related advice to the public and taught how to influence policies to restrict when, where and how alcohol is sold. As part of a larger study, the aim of the current project is to measure the impact of CICA on health and crime outcomes at the lower super output (LSOA) geographical aggregation. Design: Quantitative evaluation using four time series analytic methods (stepped-wedge design, and comparisons to local controls, national controls and synthetic controls) with findings triangulated across these methods. A cost–benefit analysis was carried out alongside the effectiveness analysis. Setting and Participants: The general public in Greater Manchester, UK, between 2010 and 2020. Measurements: The primary outcome of interest was alcohol-related hospital admissions. Secondary outcomes were accident and emergency (A&E) attendances, ambulance callouts, recorded crimes and anti-social behaviour incidents. Findings: Triangulation of the results did not indicate any consistent effect on area-level alcohol-related hospital admissions, A&E attendances, ambulance callouts, reported crimes or anti-social behaviour associated with the implementation of CICA. The primary stepped-wedge analysis indicated an increase in alcohol-related hospital admissions following the implementation of CICA of 13.4% (95% confidence interval −3.3%, +30.1%), which was consistent with analyses based on other methods with point estimates ranging from +3.4% to 16.4%. Conclusion: There is no evidence of a measurable impact of the Communities in Charge of Alcohol (CICA) programme on area-level health and crime outcomes in Greater Manchester, UK, within 3 years of the programme start. The increase in alcohol-related hospital admissions was likely the result of other temporal trends rather than the CICA programme. Possible explanations include insufficient follow-up time, too few volunteers trained, volunteers being unwilling to get involved in licensing decisions or that the intervention has no direct impact on the selected outcomes

Evaluation of cytotoxicity, antimicrobial activity and physicochemical properties of a calcium aluminate-based endodontic material

A calcium aluminate-based endodontic material, EndoBinder, has been developed in order to reduce MTA negative characteristics, preserving its biological properties and clinical applications. OBJECTIVES: The aim of this study was to evaluate the cytotoxicity, antimicrobial activity, pH, solubility and water sorption of EndoBinder and to compare them with those of white MTA (WMTA). MATERIAL AND METHODS: Cytotoxicity was assessed through a multiparametric analysis employing 3T3 cells. Antimicrobial activity against Enterococcus faecalis (ATCC 29212), Staphylococcus aureus. (ATCC 25923) and Candida albicans (ATCC 10556) was determined by the agar diffusion method. pH was measured at periods of 3, 24, 72 and 168 hours. Solubility and water sorption evaluation were performed following ISO requirements. Data were statistically analyzed by ANOVA and Tukey`s test with a significance level of 5%. RESULTS: EndoBinder and WMTA were non-cytotoxic in all tested periods and with the different cell viability parameters. There was no statistical differences between both materials (P>.05). All tested materials were inhibitory by direct contact against all microbial strains tested. EndoBinder and WMTA presented alkaline pH in all tested times with higher values of pH for WMTA (P<.05). Both materials showed values complying with the solubility minimum requirements. However, EndoBinder showed lower solubility than WMTA (P<.05). No statistical differences were observed regarding water sorption (P>.05). CONCLUSION: Under these experimental conditions, we concluded that the calcium aluminate-based endodontic material EndoBinder demonstrated suitable biological and physicochemical properties, so it can be suggested as a material of choice in root resorption, perforations and root-end filling

Substantial Alterations of the Cutaneous Bacterial Biota in Psoriatic Lesions

For psoriasis, an idiopathic inflammatory disorder of the skin, the microbial biota has not been defined using cultivation-independent methods. We used broad-range 16S rDNA PCR for archaea and bacteria to examine the microbiota of normal and psoriatic skin. From 6 patients, 19 cutaneous samples (13 from diseased skin and 6 from normal skin) were obtained. Extracted DNA was subjected to the broad range PCR, and 1,925 cloned products were compared with 2,038 products previously reported from healthy persons. Using 98% sequence identity as a species boundary, 1,841 (95.6%) clones were similar to known bacterial 16S rDNA, representing 6 phyla, 86 genera, or 189 species-level operational taxonomic unit (SLOTU); 84 (4.4%) clones with <98% identity probably represented novel species. The most abundant and diverse phylum populating the psoriatic lesions was Firmicutes (46.2%), significantly (P<0.001) overrepresented, compared to the samples from uninvolved skin of the patients (39.0%) and healthy persons (24.4%). In contrast, Actinobacteria, the most prevalent and diverse phylum in normal skin samples from both healthy persons (47.6%) and the patients (47.8%), was significantly (P<0.01) underrepresented in the psoriatic lesion samples (37.3%). Representation of Propionibacterium species were lower in the psoriatic lesions (2.9±5.5%) than from normal persons (21.1±18.2%; P<0.001), whereas normal skin from the psoriatic patients showed intermediate levels (12.3±21.6%). We conclude that psoriasis is associated with substantial alteration in the composition and representation of the cutaneous bacterial biota

Drug resistance, AmpC-β-lactamase and extended-spectrum β-lactamase-producing Enterobacteriaceae isolated from fish and shrimp

ABSTRACT The present study aims to detect the production of extended-spectrum beta-lactamases (ESBL) by enterobacteria isolated from samples of fresh shrimp and fish obtained from the retail trade of the city of Sobral, Ceará State, Brazil. All bacterial isolates were submitted to identification and antimicrobial susceptibility testing using aminopenicillin, beta-lactamase inhibitors, carbapenem, 1st, 2nd, 3rd and 4th generation cephalosporins, and monobactam. Three types of beta-lactamases - ESBL, AmpC and KPC - were investigated. 103 strains were identified, and the most frequent species in shrimp and fish samples was Enterobacter cloacae (n = 54). All the strains were resistant to penicillin and more than 50% of the isolates were resistant to ampicillin and cephalothin. Resistance to three 3rd generation cephalosporins (cefotaxime, ceftriaxone and ceftazidime) and one fourth generation cephalosporin (cefepime) was detected in two isolates of E. cloacae from shrimp samples. Phenotypic detection of AmpC was confirmed in seven strains. The ESBL was detected in two strains of E. cloacae from shrimp samples. No strain showed KPC production. These data can be considered alarming, since food (shrimp and fish) may be carriers of enterobacteria resistant to drugs of clinical interest

Community Analysis of Chronic Wound Bacteria Using 16S rRNA Gene-Based Pyrosequencing: Impact of Diabetes and Antibiotics on Chronic Wound Microbiota

Background: Bacterial colonization is hypothesized to play a pathogenic role in the non-healing state of chronic wounds. We characterized wound bacteria from a cohort of chronic wound patients using a 16S rRNA gene-based pyrosequencing approach and assessed the impact of diabetes and antibiotics on chronic wound microbiota. Methodology/Principal Findings: We prospectively enrolled 24 patients at a referral wound center in Baltimore, MD; sampled patients' wounds by curette; cultured samples under aerobic and anaerobic conditions; and pyrosequenced the 16S rRNA V3 hypervariable region. The 16S rRNA gene-based analyses revealed an average of 10 different bacterial families in wounds-approximately 4 times more than estimated by culture-based analyses. Fastidious anaerobic bacteria belonging to the Clostridiales family XI were among the most prevalent bacteria identified exclusively by 16S rRNA gene-based analyses. Community-scale analyses showed that wound microbiota from antibiotic treated patients were significantly different from untreated patients (p = 0.007) and were characterized by increased Pseudomonadaceae abundance. These analyses also revealed that antibiotic use was associated with decreased Streptococcaceae among diabetics and that Streptococcaceae was more abundant among diabetics as compared to non-diabetics. Conclusions/Significance: The 16S rRNA gene-based analyses revealed complex bacterial communities including anaerobic bacteria that may play causative roles in the non-healing state of some chronic wounds. Our data suggest that antimicrobial therapy alters community structure-reducing some bacteria while selecting for others