Salmonella enterica Typhimurium fljBA operon stability: implications regarding the origin of Salmonella enterica I 4,[5],12:i:.

Salmonella enterica subsp enterica serovar 4,5,12:i:- has been responsible for many recent Salmonella outbreaks worldwide. Several studies indicate that this serovar originated from S. enterica subsp enterica serovar Typhimurium, by the loss of the flagellar phase II gene (fljB) and adjacent sequences. However, at least two different clones of S. enterica 4,5,12:i:- exist that differs in the molecular events responsible for fljB deletion. The aim of this study was to test the stability of the fljBA operon responsible for the flagellar phase variation under different growth conditions in order to verify if its deletion is a frequent event that could explain the origin and dissemination of this serovar. In fact, coding sequences for transposons are present near this operon and in some strains, such as S. enterica Typhimurium LT2, the Fels-2 prophage gene is inserted near this operon. The presence of mobile DNA could confer instability to this region. In order to examine this, the cat (chloramphenicol acetyltransferase) gene was inserted adjacent to the fljBA operon so that deletions involving this genomic region could be identified. After growing S. enterica chloramphenicol-resistant strains under different conditions, more than 104 colonies were tested for the loss of chloramphenicol resistance. However, none of the colonies were sensitive to chloramphenicol. These data suggest that the origin of S. enterica serovar 4,5,12:i:- from Typhimurium by fljBA deletion is not a frequent event. The origin and dissemination of 4,5,12:i:- raise several questions about the role of flagellar phase variation in virulence.

Case report: Equine metacarpophalangeal joint partial and full thickness defects treated with allogenic equine synovial membrane mesenchymal stem/stromal cell combined with umbilical cord mesenchymal stem/stromal cell conditioned medium.

Here, we describe a case of a 5-year-old show-jumping stallion presented with severe lameness, swelling, and pain on palpation of the left metacarpophalangeal joint (MCj). Diagnostic imaging revealed full and partial-thickness articular defects over the lateral condyle of the third metacarpus (MC3) and the dorsolateral aspect of the first phalanx (P1). After the lesion's arthroscopic curettage, the patient was subjected to an innovative regenerative treatment consisting of two intra-articular injections of equine synovial membrane mesenchymal stem/stromal cells (eSM-MSCs) combined with umbilical cord mesenchymal stem/stromal cells conditioned medium (UC-MSC CM), 15 days apart. A 12-week rehabilitation program was accomplished, and lameness, pain, and joint effusion were remarkably reduced; however, magnetic resonance imaging (MRI) and computed tomography (CT) scan presented incomplete healing of the MC3's lesion, prompting a second round of treatment. Subsequently, the horse achieved clinical soundness and returned to a higher level of athletic performance, and imaging exams revealed the absence of lesions at P1, fulfillment of the osteochondral lesion, and cartilage-like tissue formation at MC3's lesion site. The positive outcomes suggest the effectiveness of this combination for treating full and partial cartilage defects in horses. Multipotent mesenchymal stem/stromal cells (MSCs) and their bioactive factors compose a novel therapeutic approach for tissue regeneration and organ function restoration with anti-inflammatory and pro-regenerative impact through paracrine mechanisms.

Preparedness and satisfaction of European medical students: development and validation of the EUSPSQ.

Context: In Europe, during an era of globalization in which traveling and studying abroad is becoming easier and more affordable, an open question remains on whether different European medical schools educate students with a dissimilar level of preparedness and/or satisfaction. Objective: To develop and validate the EUropean Students' Preparedness and Satisfaction Questionnaire (EUSPSQ), a standardized European tool to assess preparedness and satisfaction in medical education. Materials and methods: 30 participants belonging to the 5th and 6th year of two different European medical schools (Sapienza University of Rome and Universidad Complutense de Madrid) participated in the validation of the study. A standard validation protocol that included item development, test-retesting, and a series of statistical validation analyses, was used. Feasibility, test-retest reliability, inter-item reliability were assessed quantitatively. Face validity was evaluated qualitatively by an expert professor in the field. Results: Test-retest and inter-item reliability were considered satisfactory for all the parts of the questionnaire. Part III and IV presented lower Cohen's kappa values, probably as a consequence of their inherent subjectivity. When one item in Part III was removed, the Cronbach's alpha values of both initial and 48h answers consider-ably increased (-0.355 to 0.713 and -0.202 to 0.808 respectively). We therefore decided to eliminate the question from the EUSPSQ. Internal and external validity were considered appropriate. The questionnaire was also feasible and viable to complete. Discussion: Preliminary findings indicate that the EUSPSQ can be a valid, viable and reliable questionnaire to measure preparedness and satisfaction of 5th and 6th year medical students across different European Universities.

Liver depurative techniques: a single liver transplantation center experience.

BACKGROUND: In a liver transplant (LT) center, treatments with Prometheus were evaluated. The main outcome considered was 1 and 6 months survival. METHODS: During the study period, 74 patients underwent treatment with Prometheus; 64 were enrolled, with a mean age of 51 ± 13 years; 47 men underwent 212 treatments (mean, 3.02 per patient). The parameters evaluated were age, sex, laboratorial (liver enzymes, ammonia) and clinical (model for end-stage liver disease and Child-Turcotte-Pugh score) data. RESULTS: Death was verified in 23 patients (35.9%) during the hospitalization period, 20 patients (31.3%) were submitted to liver transplantation, and 21 were discharged. LT was performed in 4 patients with acute liver failure (ALF, 23.7%), in 7 patients with acute on chronic liver failure (AoCLF, 43.7%), and in 6 patients with liver disease after LT (30%). Seven patients who underwent LT died (35%). In the multivariate analysis, older age (P = .015), higher international normalized ratio (INR) (P = .019), and acute liver failure (P = .039) were independently associated with an adverse 1-month clinical outcome. On the other hand, older age (P = .011) and acute kidney injury (P = .031) at presentation were both related to worse 6-month outcome. For patients with ALF and AoCLF we did not observe the same differences. CONCLUSIONS: In this cohort, older age was the most important parameter defining 1- and 6-month survival, although higher INR and presence of ALF were important for 1-month survival and AKI for 6-month survival. No difference was observed between patients who underwent LT or did not have LT.

Infections After Liver Transplantation: A Retrospective, Single-center Study.

OBJECTIVE: To access the incidence of infectious problems after liver transplantation (LT). DESIGN: A retrospective, single-center study. MATERIALS AND METHODS: Patients undergoing LT from January 2008 to December 2011 were considered. Exclusion criterion was death occurring in the first 48 hours after LT. We determined the site of infection and the bacterial isolates and collected and compared recipient's variables, graft variables, surgical data, post-LT clinical data. RESULTS: Of the 492 patients who underwent LT and the 463 considered for this study, 190 (Group 1, 41%) developed at least 1 infection, with 298 infections detected. Of these, 189 microorganisms were isolated, 81 (51%) gram-positive bacteria (most frequently Staphylococcus spp). Biliary infections were more frequent (mean time of 160.4 ± 167.7 days after LT); from 3 months after LT, gram-negative bacteria were observed (57%). Patients with infections after LT presented lower aminotransferase levels, but higher requirements in blood transfusions, intraoperative vasopressors, hemodialysis, and hospital stay. Operative and cold ischemia times were similar. CONCLUSION: We found a 41% incidence of all infections in a 2-year follow-up after LT. Gram-positive bacteria were more frequent isolated; however, negative bacteria were commonly isolated later. Clinical data after LT were more relevant for the development of infections. Donors' variables should be considered in future analyses.

Field trials with oxamniquine in a Schistosomiasis mansoni-endemic area.

In Peri-Peri (Minas Gerais), an area endemic for Schistosoma mansoni, 591 inhabitants were examined. A coprological survey showed a prevalence of infection of 43.7%. After clinical examination 220 patients were distributed into three groups for treatment with oxamniquine; in Group 1, 19 children (2 to 15 years) were treated with a single oral dose of drug suspension around 20 mg/kg body weight; in Group 2, 47 children were treated orally with two 10 mg/kg doses with a 6- to 8-hour interval between them; in Group 3, 154 adults were treated with a single dose of about 15 mg/kg (capsules). The most frequent side effects were dizziness, drowsiness, and headache. No statistical difference in frequency of side effects was observed between Groups 1 and 2. Nevertheless, after treatment 32% of the patients in Group 1 complained of dizziness and 13% of headache, whereas in Group 2 the frequencies of these symptoms were 2% and 0%, respectively. Adults (Group 3) had a higher frequency of side effects, their chief complaints being dizziness and drowsiness. In Groups 1, 2, and 3, respectively, 73.7%, 62.2%, and 82.4% of the patients were cured. Statistical analysis did not show any difference in cure rates between children in Groups 1 and 2; however a significance was found when compared with the cure rate observed in adults, showing the drug to be more active in the latter group. In 61.8% of the 40 patients not cured a decrease of over 90% in the number of S. mansoni eggs/g feces was observed. From the data above oxamniquine seems to be an effective schistosomicidal drug suitable for use in endemic areas, although further studies are still needed.

The prolonged use of niclosamide as a molluscicide for the control of Schistosoma mansoni.

Applications of niclosamide at three-monthly intervals were undertaken for 14 years in foci of Biomphalaria glabrata in the water sources of Peri-Peri (Capim Branco, MG). All the residents of the area were submitted to an annual fecal examination (Kato/Katz) and those individuals eliminating Schistosoma mansoni eggs were treated with oxamniquine. A malacological survey was undertaken at three-monthly intervals by means of ten scoops with a perforated ladle each ten metres along the two banks of the ditches and streams of the region. Where snails were found, molluscicide was applied by means of dripping or aspersion using a 3 ppm aqueous suspension of niclosamide. Initially, a mean of 14.3% of snails in the region were found to be eliminating cercariae. Following the first four applications of molluscicide, this was reduced to 0.0% and maintained at about 1.5% throughout the program. Thus, there was a continued possibility of schistosomiasis transmission in the area and it was observed that the population of snails reestablished itself within three months of molluscicide application. The results obtained in this study do not encourage the continual use of niclosamide as the only method of control of schistosomiasis.

[The use of the shell of the cashew nut, Anacardium occidentale, as an alternative molluscacide]. / O uso da casca da castanha do caju, Anacardium occidentale, como moluscicida alternativo.

Bioassays using hexanolic extracts of cashew nut shells, of Anacardium occidentale, collected in Ceará in 1972 (Sample 1) and 1987 (Sample 2) were undertaken with adult snails and egg masses of Biomphalaria glabrata, B. tenagophila and B. straminea both in the laboratory and in the field. Non extracted shells, 18.5 g, sample 1, were also tested with adult snails and egg masses of the three species. The toxicity of extract was tested with fish (Poecilia reticulata) and tadpoles. The lethal concentration, CL90, of sample 1 was from 2.0 to 2.2 ppm for adult snails of the three species. With sample 2, the CL90 was 2.0, 0.5 and 30.0 ppm for B. glabrata adults, newly hatched snails and egg mass respectively. Non extracted shells caused 40-80% mortality of adult snails, 22-35% mortality of embryos and 40-55% reduction of egg production. The hexanolic extract, sample 2, were innocuous for tadpole and fish at 2 ppm. In the field, in pools of still water treatment with 20 ppm of extract, sample 1, caused a 97.1% mortality of B. straminea and 100% mortality of B. glabrata and B. tenagophila. Using Niclosamide, at 3 ppm, 100% mortality of the three species occurred.

[Molluscacide and cercariacide activity of different species of Eucalyptus]. / Atividade moluscicida e cercaricida de diferentes espécies de Eucalyptus.

Hydrolates and essential oils of several Eucalyptus species were tested on adult snails and egg masses of Biomphalaria glabrata, and cercaria of Schistosoma mansoni. These products were obtained by vapor dragging. Eight out of 21 hydrolates presented activity on snails, nine on egg masses and three on cercaria at 1:4 dilutions. Eleven oils were active on both snails and egg masses at 20 ppm concentrations. The hydrolate of E. deanei was active both on snails, egg masses and cercaria at 1:4 dilution and its essential oil at 20 ppm.

[Molluscacide activity of a mixture of 6-n-alkyl salicylic acids (anacardic acid) and 2 of its complexes with copper (II) and lead (II)]. / Atividade moluscicida da mistura de ácidos 6-n-alquil salicílicos (ácido anacárdico) e dos seus complexos com cobre (II) e chumbo (II).

The molluscicide activity of hexanic extract from Anacardium occidentale L. (cashew) nut shell, of copper (II) complex, of lead (II) complex and anacardic acid has been compared in the laboratory in an attempt to obtain better stability than anacardic acid. This was obtained from the hexanic extract of the cashew nut shell by precipitation with lead (II) hydroxide or cupric sulfate plus sodium hydroxide or (II) cupric hydroxide followed by treatment of lead (II) complex with a diluted solution of sulfuric acid. Ten products of the mixture obtained were tested on adults snails of Biomphalaria glabrata at 1 to 10 ppm. The most active products were copper (II) complex, obtained by cupric sulfate plus sodium hydroxide, and anacardic acid (sodium hydroxide) which presented activity at 4 ppm. The anacardic acid's lead content was above the limits accepted by the United States standards.

In vitro modulation of Bcl-2 levels in small cell lung cancer cells: effects on cell viability.

Small cell lung cancer (SCLC) is an aggressive disease, representing 15% of all cases of lung cancer, has high metastatic potential and low prognosis that urgently demands the development of novel therapeutic approaches. One of the proposed approaches has been the down-regulation of BCL2, with poorly clarified and controversial therapeutic value regarding SCLC. The use of anti-BCL2 small interfering RNA (siRNA) in SCLC has never been reported. The aim of the present study was to select and test the in vitro efficacy of anti-BCL2 siRNA sequences against the protein and mRNA levels of SCLC cells, and their effects on cytotoxicity and chemosensitization. Two anti-BCL2 siRNAs and the anti-BCL2 G3139 oligodeoxynucleotide (ODN) were evaluated in SCLC cells by the simultaneous determination of Bcl-2 and viability using a flow cytometry method recently developed by us in addition to Western blot, real-time reverse-transcription PCR, and cell growth after single and combined treatment with cisplatin. In contrast to previous reports about the use of ODN, a heterogeneous and up to 80% sequence-specific Bcl-2 protein knockdown was observed in the SW2, H2171 and H69 SCLC cell lines, although without significant sequence-specific reduction of cell viability, cell growth, or sensitization to cisplatin. Our results question previous data generated with antisense ODN and supporting the present concept of the therapeutic interest in BCL2 silencing per se in SCLC, and support the growing notion of the necessity of a multitargeting molecular approach for the treatment of cancer.

In vitro modulation of Bcl-2 levels in small cell lung cancer cells: effects on cell viability

Small cell lung cancer (SCLC) is an aggressive disease, representing 15 percent of all cases of lung cancer, has high metastatic potential and low prognosis that urgently demands the development of novel therapeutic approaches. One of the proposed approaches has been the down-regulation of BCL2, with poorly clarified and controversial therapeutic value regarding SCLC. The use of anti-BCL2 small interfering RNA (siRNA) in SCLC has never been reported. The aim of the present study was to select and test the in vitro efficacy of anti-BCL2 siRNA sequences against the protein and mRNA levels of SCLC cells, and their effects on cytotoxicity and chemosensitization. Two anti-BCL2 siRNAs and the anti-BCL2 G3139 oligodeoxynucleotide (ODN) were evaluated in SCLC cells by the simultaneous determination of Bcl-2 and viability using a flow cytometry method recently developed by us in addition to Western blot, real-time reverse-transcription PCR, and cell growth after single and combined treatment with cisplatin. In contrast to previous reports about the use of ODN, a heterogeneous and up to 80 percent sequence-specific Bcl-2 protein knockdown was observed in the SW2, H2171 and H69 SCLC cell lines, although without significant sequence-specific reduction of cell viability, cell growth, or sensitization to cisplatin. Our results question previous data generated with antisense ODN and supporting the present concept of the therapeutic interest in BCL2 silencing per se in SCLC, and support the growing notion of the necessity of a multitargeting molecular approach for the treatment of cancer.

[Molluscicide activity of some natural products on Biomphalaria glabrata]. / Atividade moluscicida de alguns produtos naturais sobre Biomphalaria glabrata.

The molluscicide activity of aqueous (macerated and boiled), hexanic and ethylic extracts of Aristolochia brasiliensis, Caesalpinia peltophoroides, Caesalpinia pulcherrima, Delonix regia, Spathodea campanulata and Tibouchina scrobiculata was evaluated in the laboratory. The solutions obtained from those extracts were tested on adults and egg masses of Biomphalaria glabrata reared in the laboratory at 1, 10, 20, 100 and 1000 ppm concentrations. The most active of the extracts studied was D. regia flowers' (flamboyant) ethylic extracts which presented molluscicidal activity on adult snails at 20 ppm.

[Molluscicidal properties of the Euphorbia cotinifolia L (author's transl)]. / Propriedades moluscicidas da Euphorbia cotinifolia L.

The hexanic extract from the leaves of Euphorbia cotinifolia L. has been experimentally used as molluscicide against Biomphalaria glabrata. The extract was fractionated and the most actives fractions were those of numbers 6 and 7. The lethal concentrations (LC50 and LC90 obtained using fraction 6 against adult snails were 1,2 and 2,4 ppm; for the eggs they were 25,0 and 48,0 ppm. With fraction 7 the lethal concentrations were 1,4 and 3,4 ppm for adult snails; for newly hatched snails they were 4,8 and 8,0 ppm; for the eggs they were 13,0 and 31,0 ppm. In the field the hexanic extract was lethal for the snails in concentrations of 20 ppm in stagnant water. Fractions 6 and 7 were lethal for S. mansoni cercariae and to fish (Lebistes reticulatus) in concentrations below 10 ppm. For mice the extract did not display any toxity in concentrations of 600 mg/kg.

[Effect of several extracts derived from plants on the survival of larvae of Aedes fluviatilis (Lutz) (Diptera: Culicidae) in the laboratory]. / Influência de diversos derivados de vegetais na sobrevida das larvas de Aedes fluviatilis (Lutz) (Diptera: Culicidae) em laboratório.

The larvicidal properties of 34 plant extracts were tested against Aedes fluviatilis (Lutz) (Diptera: Culicidae) larvae, at 100, 10 and 1 ppm concentrations; 26.6% of the extracts enhanced larval mortality (alpha = 0.05) at 100 ppm (Anacardium occidentale, Agave americana, Allium sativum, Coriandrum sativum, Nerium oleander, Spatodea campanulata, Tibouchina scrobiculata and Vernonia salzmanni). Anacardic acid (A. occidentale) was effective at 10 ppm and A. sativum (crude extract) at 1 ppm.

[Influence of EDTA on growth and reproduction of Biomphalaria glabrata (author's transl)]. / Influência do EDTA sobre crescimento e reprodução de Biomphalaria glabrata.

The influence of EDTA on B. glabrata has been investigated. Newly hatched snails were exposed to concentrations of EDTA from 1 to 64 ppm, and young (diameter of 6 mm) snails to concentrations of 50, 80 and 100 ppm, for periods of 90 days. Fifty percent reduction of egg productivity has been caused by concentration of 16 ppm of EDTA while 50% of mortality has occurred at about 70 ppm. The calcium and iron content both in treated and non-treated young snails have been estimated by atomic absorption photometry. The uptake of calcium was 40, 83 and 90% less for calcium and 37, 77 and 81% less for iron as compared with the untreated group. The calcium content of the shell was 5--15 times greater than that of the soft body, while the iron content of those two parts was in the proportion of 1:1. These proportions were maintained constant in the treated and non-treated groups. The interference of increasing concentrations of EDTA has resulted in the proportional reduction of growth-rate, reproduction rate and of longevity of the exposed snails.