Lipid Composition Analysis Reveals Mechanisms of Ethanol Tolerance in the Model Yeast Saccharomyces cerevisiae.

Saccharomyces cerevisiae is an important unicellular yeast species within the biotechnological and the food and beverage industries. A significant application of this species is the production of ethanol, where concentrations are limited by cellular toxicity, often at the level of the cell membrane. Here, we characterize 61 S. cerevisiae strains for ethanol tolerance and further analyze five representatives with various ethanol tolerances. The most tolerant strain, AJ4, was dominant in coculture at 0 and 10% ethanol. Unexpectedly, although it does not have the highest noninhibitory concentration or MIC, MY29 was the dominant strain in coculture at 6% ethanol, which may be linked to differences in its basal lipidome. Although relatively few lipidomic differences were observed between strains, a significantly higher phosphatidylethanolamine concentration was observed in the least tolerant strain, MY26, at 0 and 6% ethanol compared to the other strains that became more similar at 10%, indicating potential involvement of this lipid with ethanol sensitivity. Our findings reveal that AJ4 is best able to adapt its membrane to become more fluid in the presence of ethanol and that lipid extracts from AJ4 also form the most permeable membranes. Furthermore, MY26 is least able to modulate fluidity in response to ethanol, and membranes formed from extracted lipids are least leaky at physiological ethanol concentrations. Overall, these results reveal a potential mechanism of ethanol tolerance and suggest a limited set of membrane compositions that diverse yeast species use to achieve this. IMPORTANCE Many microbial processes are not implemented at the industrial level because the product yield is poorer and more expensive than can be achieved by chemical synthesis. It is well established that microbes show stress responses during bioprocessing, and one reason for poor product output from cell factories is production conditions that are ultimately toxic to the cells. During fermentative processes, yeast cells encounter culture media with a high sugar content, which is later transformed into high ethanol concentrations. Thus, ethanol toxicity is one of the major stresses in traditional and more recent biotechnological processes. We have performed a multilayer phenotypic and lipidomic characterization of a large number of industrial and environmental strains of Saccharomyces to identify key resistant and nonresistant isolates for future applications.

A comparison of two clinical scores for bronchiolitis. A multicentre and prospective study conducted in hospitalised infants.

BACKGROUND: There are a number of clinical scores for bronchiolitis but none of them are firmly recommended in the guidelines. METHOD: We designed a study to compare two scales of bronchiolitis (ESBA and Wood Downes Ferres) and determine which of them better predicts the severity. A multicentre prospective study with patients <12 months with acute bronchiolitis was conducted. Each patient was assessed with the two scales when admission was decided. We created a new variable "severe condition" to determine whether one scale afforded better discrimination of severity. A diagnostic test analysis of sensitivity and specificity was made, with a comparison of the AUC. Based on the optimum cut-off points of the ROC curves for classifying bronchiolitis as severe we calculated new Se, Sp, LR+ and LR- for each scale in our sample. RESULTS: 201 patients were included, 66.7% males and median age 2.3 months (IQR=1.3-4.4). Thirteen patients suffered bronchiolitis considered to be severe, according to the variable severe condition. ESBA showed a Se=3.6%, Sp=98.1%, and WDF showed Se=46.2% and Sp=91.5%. The difference between the two AUC for each scale was 0.02 (95%CI: 0.01-0.15), p=0.72. With new cut-off points we could increase Se and Sp for ESBA: Se=84.6%, Sp=78.7%, and WDF showed Se=92.3% and Sp=54.8%; with higher LR. CONCLUSIONS: None of the scales studied was considered optimum for assessing our patients. With new cut-off points, the scales increased the ability to classify severe infants. New validation studies are needed to prove these new cut-off points.

Road rage in Sri Lanka: prevalence and psychiatric distress.

INTRODUCTION: Road traffic accidents are a major public health concern in Sri Lanka. Aggressive and reckless driving is an important contributor to the high rate of road traffic accidents. OBJECTIVE: We studied prevalence, nature, determinants and associated psychiatric morbidity of road rage among motorists in Sri Lanka. Methods Data were gathered from 238 randomly selected motorists in Sri Lanka using a modified questionnaire regarding road rage and the 6-item version of Kessler's psychological distress scale. RESULTS: While 98.7% participants reported being victims of road rage, 85.3% were involved in offending behaviour. However actual physical assault (0.8%) and damage to vehicles (2.5%) were rare. Male gender, young age, increased traffic density and driving a three-wheeler or bus were associated with daily road rage victimisation and perpetration. Psychiatric distress was associated with being a victim of road rage. CONCLUSIONS: High prevalence of road rage in Sri Lanka and significant psychiatric distress associated with it indicate the necessity of interventions at least for target groups.

Morphological and genetic variations of the freshwater leech, Hirudinaria spp., in Peninsular Malaysia.

In this study the genetic diversity of local freshwater leeches (Hirudinaria spp.) was inferred using mtDNA COI gene analysis and compared with the gross external variations of 26 freshwater leech specimens obtained from the wild and leech farms. Based on a neighbor-joining tree generated from 516 COI base sequences, four distinct clades of Hirudinaria were seen with interspecific genetic divergence in the range of 7.6-14.5%. The external morphological variations based on the presence of stripes, location of gonopores, and anus separated the samples into four morphologically distinct groups matching the four clades obtained from the molecular data. Two black stripes at the ventral region were observed only in specimens found clustered with clades that contained the GenBank-reported H. manillensis, whereas the brown or dark green coloration without stripes on the ventral region was seen in samples that clustered with H. javanica and H. bpling clades.

Characteristics and treatment of pyoderma gangrenosum in inflammatory bowel disease.

BACKGROUND: Pyoderma gangrenosum is a serious cutaneous complication seen in approximately 1 % of patients with inflammatory bowel disease (IBD). Oral corticosteroids are the mainstay treatment, although the evidence supporting their use is weak. AIMS: The purpose of this study was to investigate the characteristics of pyoderma gangrenosum associated with Crohn's disease or ulcerative colitis and which treatments are prescribed in Spanish clinical practice. METHODS: In this retrospective, observational study, the medical records from all patients with IBD and a diagnosis of pyoderma gangrenosum attended by the gastroenterology departments of 12 Spanish hospitals were reviewed. Data on patient demographics and characteristics, underlying IBD and treatment, and pyoderma gangrenosum characteristics, treatment, and outcome were collected and analyzed. RESULTS: The data from 67 patients were analyzed (41 [61.2 %] women, 41 [61.2 %] with Crohn's disease, 25 [37.3 %] with ulcerative colitis, and 1 [1.5 %] with indeterminate disease). The underlying disease was in remission in approximately one-third of patients at the time of presentation of pyoderma gangrenosum. Healing was achieved in all patients (in 3 without any systemic therapy). Oral corticosteroids were taken by 51 patients (76.1 %), almost always as first-line treatment, although definitive healing was attained in 19 (28.4 %). Biologic agents such as infliximab and adalimumab were taken by 31 patients (46.3 %) at some point (first-line in 6 patients [9.0 %]), with definitive healing in 29 patients (93.5 %). CONCLUSIONS: Oral corticosteroid therapy remains the most common treatment for pyoderma gangrenosum associated with inflammatory bowel disease. Biologic therapies such as infliximab and adalimumab should also be considered.

Evaluating clinical effectiveness and impact of anti-pneumococcal vaccination in adults after universal childhood PCV13 implementation in Catalonia, 2017-2018.

Background: At present, because of indirect effects derived from routine childhood immunisation, clinical benefits vaccinating adults with the 23-valent pneumococcal polysaccharide vaccine (PPsV23) and/or the 13-valent pneumococcal conjugate vaccine (PCV13) are uncertain. This study investigated clinical effectiveness for both PPsV23/PCV13 in preventing pneumonia among Catalonian adults during an earlier 2-year period post-PCV13 free (publicly funded) approval for infants. Methods: We conducted a Population-based cohort study involving 2,059,645 adults ≥ 50 years in Catalonia, Spain, who were followed between 01/01/2017-31/12/2018. Primary outcomes were hospitalisation from pneumococcal pneumonia (PP) or all-cause pneumonia (ACP) and main explanatory variable was PCV13/PPsV23 vaccination status. Cox regression models were used to estimate vaccination effectiveness adjusted by age/sex and underlying-risk conditions. Results: Cohort members were followed for 3,958,528 person-years (32,328 PCV13-vaccinated, 1,532,186 PPsV23-vaccinated), observing 3592 PP (131 in PCV13-vaccinated vs 2476 in PPsV23-vaccinated) and 24,136 ACP (876 in PCV13-vaccinated vs 17,550 in PPsV23-vaccinated). Incidence rates (per 100,000 person-years) were 90.7 for PP (394.2 in PCV13-vaccinated vs 161.6 in PPsV23-vaccinated) and 609.7 for ACP (2636.3 in PCV13-vaccinated vs 1145.4 in PPsV23-vaccinated). The PCV13 was associated with an increased risk of PP (hazard ratio [HR]: 1.24; 95% CI: 1.00-1.52; p = 0.046) and ACP (HR: 1.38; 95% CI: 1.28-1.49; p < 0.001) whereas the PPsV23 did not alter the risk of PP (HR: 1.07; 95% CI: 0.98-1.18; p = 0.153) and slightly increased the risk of ACP (HR: 1.14; 95% CI: 1.10-1.18; p < 0.001). In supplementary analyses focused on at-risk individuals (i.e., elderly persons, immunocompromissing and other chronic illnesses) protective effects of vaccination did not emerge either. Conclusions: Data does not support clinical benefits from pneumococcal vaccination (nor PCV13 neither PPsV23) against pneumonia among Catalonian middle-aged and older adults in the current era of universal PCV13 childhood immunisation in our setting. New extended valency PCVs are greatly needed.

Prevotella in Pigs: The Positive and Negative Associations with Production and Health.

A diverse and dynamic microbial community (known as microbiota) resides within the pig gastrointestinal tract (GIT). The microbiota contributes to host health and performance by mediating nutrient metabolism, stimulating the immune system, and providing colonization resistance against pathogens. Manipulation of gut microbiota to enhance growth performance and disease resilience in pigs has recently become an active area of research in an era defined by increasing scrutiny of antimicrobial use in swine production. In order to develop microbiota-targeted strategies, or to identify potential next-generation probiotic strains originating from the endogenous members of GIT microbiota in pigs, it is necessary to understand the role of key commensal members in host health. Many, though not all, correlative studies have associated members of the genus Prevotella with positive outcomes in pig production, including growth performance and immune response; therefore, a comprehensive review of the genus in the context of pig production is needed. In the present review, we summarize the current state of knowledge about the genus Prevotella in the intestinal microbial community of pigs, including relevant information from other animal species that provide mechanistic insights, and identify gaps in knowledge that must be addressed before development of Prevotella species as next-generation probiotics can be supported.

[Multicentre longitudinal study of the nutritional status and swallowing difficulties in children with severe neurological diseases]. / Estudio multicéntrico y longitudinal del estado nutricional y problemas de deglución en niños con enfermedad neurológica grave.

INTRODUCTION: Nutritional problems are common in children with neurological diseases, especially if they have significant motor impairment. Oropharyngeal dysphagia is very prevalent in these patients, and can contribute to worsening nutritional status and produce pulmonary aspirations. AIM: Longitudinal assessment of the nutritional status of a sample of pediatric patients with moderate-severe neurological disease and establish the prevalence of oropharyngeal dysphagia in that sample. PATIENTS AND METHODS: An observational multicenter prospective study was conducted. We included children under 16 years of age with moderate-to-severe neurological impairment from four hospitals, with clinical and anthropometric monitoring for one year. Questions were asked to conduct oropharyngeal dysphagia screening. RESULTS: Sixty-eight children were included, the main diagnosis obtained was cerebral palsy. In the anthropometric assessment, 42 patients (62%) showed weight z scores below -2, and 29 (43%) height z scores below -2, while body mass index, mid upper arm circumference and triceps and subscapular skinfolds remained less affected. We found an oropharyngeal dysphagia prevalence of 73.5% in our sample, increasing with greater motor impairment. CONCLUSIONS: These patients showed lower weight and height than children without neurological impairment. However, with a correct follow-up they remain stable with an adequate body composition. It is important to proactively investigate the presence of oropharyngeal dysphagia, especially in those with greater motor impairment, as it occurs very frequently and an adequate diagnosis can improve clinical evolution and prevent complications.

TITLE: Estudio multicéntrico y longitudinal del estado nutricional y problemas de deglución en niños con enfermedad neurológica grave.Introducción. Los problemas nutricionales son frecuentes en niños con enfermedades neurológicas, sobre todo si tienen una importante afectación motora. La disfagia orofaríngea es muy prevalente en estos pacientes y puede contribuir a empeorar el estado nutricional y a que se produzcan aspiraciones pulmonares. Objetivo. Evaluar de forma longitudinal el estado nutricional de una muestra de pacientes pediátricos con enfermedad neurológica moderada-grave y establecer la prevalencia de disfagia orofaríngea en dicha muestra. Pacientes y métodos. Estudio multicéntrico prospectivo observacional. Se incluyó a niños menores de 16 años controlados en cuatro centros hospitalarios. Se recogieron datos clínicos y antropométricos durante un año. Se realizaron preguntas dirigidas a realizar un cribado sistemático de disfagia orofaríngea. Resultados. Se seleccionó a 68 pacientes, y el diagnóstico más frecuente fue la parálisis cerebral infantil. En la valoración antropométrica, 42 pacientes (62%) presentaron puntuaciones z de peso inferiores a ­2, y 29 (43%), talla con puntuaciones z inferiores a ­2, pero con un índice de masa corporal, un perímetro braquial y unos pliegues cutáneos mucho menos alterados. La prevalencia de disfagia orofaríngea fue del 73,5%, que aumentó a mayor afectación motora. Conclusiones. Estos pacientes presentan un tamaño corporal menor que la población de su misma edad y sexo sin patología. Sin embargo, con un correcto seguimiento nutricional, mantienen estable su composición corporal. Es importante investigar de forma proactiva la presencia de disfagia orofaríngea, sobre todo en los que tienen mayor afectación motora, pues se presenta con mucha frecuencia y un adecuado diagnóstico puede mejorar la evolución clínica y prevenir complicaciones.

Denosumab Regulates Gut Microbiota Composition and Cytokines in Dinitrobenzene Sulfonic Acid (DNBS)-Experimental Colitis.

The pro-inflammatory mediator receptor activator of nuclear factor-kappa B ligand (RANKL) plays a significant role in the development of rheumatoid arthritis; however, its role in inflammatory bowel disease is unknown. Genome-wide association meta-analysis for Crohn's disease (CD) identified a variant near the TNFSF11 gene that encodes RANKL and CD risk allele increased expression of RANKL in specific cell lines. This study aims to elucidate if the RANKL inhibitor denosumab can reduce the severity of experimental colitis and modify the gut microbiota composition using murine dinitrobenzenesulfonic acid (DNBS)-experimental model of colitis mimicking CD. In colitic conditions, denosumab treatment significantly decreased the pro-inflammatory cytokines IL-6, IL-1ß, and TNF-α within the colonic mucosa. Moreover, colitis was accompanied by disruption of gut microbiota, and preventative treatment with denosumab modulated this disruption. Denosumab treatment also modified the alpha- and beta diversity of colonic mucosa and fecal microbiota. These results provide a rationale for considering denosumab as a future potential therapy in CD; however, more detailed experimental and clinical studies are warranted.

Discovery of Predictors of Mycoplasma hyopneumoniae Vaccine Response Efficiency in Pigs: 16S rRNA Gene Fecal Microbiota Analysis.

The gut microbiota comprises a large and diverse community of bacteria that play a significant role in swine health. Indeed, there is a tight association between the enteric immune system and the overall composition and richness of the microbiota, which is key in the induction, training and function of the host immunity, and may therefore, influence the immune response to vaccination. Using vaccination against Mycoplasma hyopneumoniae (M. hyo) as a model, we investigated the potential of early-life gut microbiota in predicting vaccine response and explored the post-vaccination dynamics of fecal microbiota at later time points. At 28 days of age (0 days post-vaccination; dpv), healthy piglets were vaccinated, and a booster vaccine was administered at 21 dpv. Blood samples were collected at 0, 21, 28, 35, and 118 dpv to measure M. hyo-specific IgG levels. Fecal samples for 16S rRNA gene amplicon sequencing were collected at 0, 21, 35, and 118 dpv. The results showed variability in antibody response among individual pigs, whilst pre-vaccination operational taxonomic units (OTUs) primarily belonging to Prevotella, [Prevotella], Anaerovibrio, and Sutterella appeared to best-predict vaccine response. Microbiota composition did not differ between the vaccinated and non-vaccinated pigs at post-vaccination time points, but the time effect was significant irrespective of the animals' vaccination status. Our study provides insight into the role of pre-vaccination gut microbiota composition in vaccine response and emphasizes the importance of studies on full metagenomes and microbial metabolites aimed at deciphering the role of specific bacteria and bacterial genes in the modulation of vaccine response.

Hospital economic impact of an outbreak of Pseudomonas aeruginosa infections.

A total of 67 patients involved in an outbreak of Pseudomonas aeruginosa in the intensive care unit (ICU) were retrospectively followed to determine whether case patients experienced differences in cost, length of stay and survival rates when compared with non-affected patients. The method of microcosting, a technique that involves detailed identification and measurement of all care items and services offered by the hospital, was used to identify attributable costs related to diagnostic procedures, pharmacy and ICU stay of each patient. Seventeen patients developed nosocomial P. aeruginosa infection. On average, these patients incurred adjusted hospital costs of euro 27,917, 66% higher than non-case patients (P=0.002). The extra length of ICU stay attributable to P. aeruginosa infection was 70 days (P=0.0001). In multiple linear regression analysis, we found that P. aeruginosa infection was an independent predictor of increased hospital costs and length of hospital stay. On the basis of these findings, a conservative estimate of the extra cost attributable to P. aeruginosa infection in our ICU was euro 312,936 (95% confidence interval: 305,676-320,196).

Colorectal cancer screening: strategies to select populations with moderate risk for disease.

OBJECTIVE: to analyse the association between rectal bleeding or a family history of colorectal cancer (CRC) and the results obtained in two rounds of a CRC screening pilot programme performed in L Hospitalet, Barcelona, Spain. SUBJECTS: males and females (50-69 years) were the target population. Together with the invitation letter, they received a questionnaire in which they were asked about rectal bleeding, family history of CRC and related neoplasms. The screening test was a guaiac-based faecal occult blood test (FOBT), and colonoscopy for positive tests. RESULTS: 25,829 FOBT were performed in 18,405 individuals. Information on rectal bleeding and a family history of CRC were obtained for 9,849 and 9,865 cases, respectively. Male sex (OR = 1.32), 60-69 years of age (OR = 1.48), rectal bleeding (OR = 1.84) and history of CRC (OR = 1.54) were independent predictors of positive FOBT. With regard to colonoscopy, a greater risk of diagnosing advanced neoplasm was observed among men (OR = 2.47) and subjects with a family history of CRC (OR = 1.98). CONCLUSIONS: CRC screening programmes must have instruments that make it possible to select the candidate population and the possibility of offering a study suited to the risk of individuals who are not susceptible to population screening by means of FOBT.

Characterization of whole blood transcriptome and early-life fecal microbiota in high and low responder pigs before, and after vaccination for Mycoplasma hyopneumoniae.

We investigated gene expression patterns in whole blood and fecal microbiota profile as potential predictors of immune response to vaccination, using healthy M. hyopneumoniae infection free piglets (n = 120). Eighty piglets received a dose of prophylactic antibiotics during the first two days of life, whereas the remaining 40 did not. Blood samples for RNA-Seq analysis were collected on experimental Day 0 (D0; 28 days of age) just prior to vaccination, D2, and D6 post-vaccination. A booster vaccine was given at D24. Fecal samples for microbial 16SrRNA sequencing were collected at 7 days of age, and at D0 and D35 post-vaccination. Pigs were ranked based on the levels of M. hyopneumoniae-specific antibodies in serum samples collected at D35, and groups of 'high' (HR) and 'low' (LR) responder pigs (n = 15 each) were selected. Prophylactic antibiotics did not influence antibody titer levels and differential expression analysis did not reveal differences between HR and LR at any time-point (FDR > 0.05); however, based on functional annotation with Ingenuity Pathway Analysis, D2 post-vaccination, HR pigs were enriched for biological terms relating to increased activation of immune cells. In contrast, the immune activation decreased in HR, 6 days post-vaccination. No significant differences were observed prior to vaccination (D0). Two days post-vaccination, multivariate analysis revealed that ADAM8, PROSER3, B4GALNT1, MAP7D1, SPP1, HTRA4, and ENO3 genes were the most promising potential biomarkers. At D0, OTUs annotated to Prevotella, CF21, Bacteroidales and S24-7 were more abundant in HR, whereas Fibrobacter, Paraprevotella, Anaerovibrio, [Prevotella], YRC22, and Helicobacter positively correlated with the antibody titer as well as MYL1, SPP1, and ENO3 genes. Our study integrates gene differential expression and gut microbiota to predict vaccine response in pigs. The results indicate that post-vaccination gene-expression and early-life gut microbiota profile could potentially predict vaccine response in pigs, and inform a direction for future research.

[Colonoscopic findings from a pilot screening study for colorectal cancer in Catalonia]. / Hallazgos colonoscópicos del estudio piloto de cribado de cáncer colorrectal realizado en Cataluña.

OBJECTIVE: to evaluate lesions detected in two screening rounds performed in a pilot screening programme for colorectal cancer in Catalonia, Spain. MATERIAL AND METHODS: a colorectal cancer screening programme was initiated in 2000. The target population included men and women aged 50-69 years. Screening consisted of biennial guaiac-based fecal occult blood testing (FOBT), and colonoscopy for participants with a positive FOBT. Any polyps found were removed, and biopsies were performed for any masses. RESULTS: colonoscopies were performed in 442 of 495 people with positive FOBT. In 213 (48.2%), 36 invasive cancers, 121 high-risk adenomas, 29 low-risk adenomas, and 27 hyperplastic polyps were diagnosed. Lesion size was smaller than 10 mm in 25.8% of cases. Most detected lesions (37.2%) were located in the distal colon, followed by the proximal colon (5.7%) and both locations (5.2%). Advanced neoplasm was significantly associated with male gender and distal location. The prevalence of advanced proximal neoplasms among patients with no distal polyps was 5.1%. CONCLUSIONS: the most common lesions detected by colonoscopy were high-risk adenomas located in the distal colon. FOBT is a suitable method for detecting small precancer lesions during population screening, and is thus a key factor in reducing the incidence of colorectal cancer.

Purtscher-like retinopathy associated with dermatomyositis and hemophagocytic lymphohistiocytosis. / Retinopatía Purtscher-like asociada a dermatomiositis y linfohistiocitosis hemofagocítica.

CASE REPORT: An 11 year-old girl with progressive muscle weakness due to dermatomyositis and hemophagocytic lymphohistiocytosis in which multiple cotton exudates, venous tortuosity and flame haemorrhages are detected in the funduscopic examination, leading to the diagnosis of Purtscher-like retinopathy. DISCUSSION: Purstcher's retinopathy is a microvascular disorder with clinical signs of probable thrombotic origin. Its treatment is controversial, with systemic corticosteroids being the most widespread choice.

Nutritional support and parenteral nutrition in cancer patients: an expert consensus report.

BACKGROUND: Malnutrition is a frequent medical problem of cancer patients that negatively impacts their quality of life. METHODS: A multidisciplinary group of experts in Medical Oncology, Pharmacy, and Nutrition convened to discuss the management of the nutritional support in cancer patients. RESULTS: Of the 18 questions addressed, 9 focused on nutritional support, 5 were related to parenteral nutrition (PN) and 4 about home PN (HPN). The panel of experts recommends using nutritional screening routinely, at diagnosis and throughout the disease course, for detecting the risk of malnutrition and, if it is positive, to perform a complete nutritional assessment, to diagnose malnutrition. Currently, there are different screening tools and methods that allow us to detect nutritional risk. Based on the evidence and experience, the panel stated that PN is indicated mainly when it is not possible to use the digestive tract and/or oral feeding and/or enteral nutrition is not sufficient or possible. The nutritional needs of the cancer patients, except in those cases where individualized measures are required, should be considered similar to healthy individuals (25-30 kcal/kg/day). The panel considers that the nutritional monitoring of the cancer patient should be multidisciplinary and adapted to the characteristics of each center. Additionally, the objective of the HPN is to improve or maintain the nutritional status of a patient at home. CONCLUSIONS: This document seeks to lay down a set of recommendations and to identify key issues that may be useful for the nutritional management of cancer patients.

Role of the 3' splice site in U12-dependent intron splicing.

U12-dependent introns containing alterations of the 3' splice site AC dinucleotide or alterations in the spacing between the branch site and the 3' splice site were examined for their effects on splice site selection in vivo and in vitro. Using an intron with a 5' splice site AU dinucleotide, any nucleotide could serve as the 3'-terminal nucleotide, although a C residue was most active, while a U residue was least active. The penultimate A residue, by contrast, was essential for 3' splice site function. A branch site-to-3' splice site spacing of less than 10 or more than 20 nucleotides strongly activated alternative 3' splice sites. A strong preference for a spacing of about 12 nucleotides was observed. The combined in vivo and in vitro results suggest that the branch site is recognized in the absence of an active 3' splice site but that formation of the prespliceosomal complex A requires an active 3' splice site. Furthermore, the U12-type spliceosome appears to be unable to scan for a distal 3' splice site.

An intergenic G-rich region in Leishmania tarentolae kinetoplast maxicircle DNA is a pan-edited cryptogene encoding ribosomal protein S12.

Six short G-rich intergenic regions in the maxicircle of Leishmania tarentolae are conserved in location and polarity in two other kinetoplastid species. We show here that G-rich region 6 (G6) represents a pan-edited cryptogene which contains at least two domains edited independently in a 3'-to-5' manner connected by short unedited regions. In the completely edited RNA, 117 uridines are added at 49 sites and 32 uridines are deleted at 13 sites, creating a translated 85-amino-acid polypeptide. Similar polypeptides are probably encoded by pan-edited G6 transcripts in two other species. The G6 polypeptide has significant sequence similarity to the family of S12 ribosomal proteins. A minicircle-encoded gRNA overlaps 12 editing sites in G6 mRNA, and chimeric gRNA/mRNA molecules were shown to exist, in agreement with the transesterification model for editing.

Epidemiological patterns of bovine besnoitiosis in an endemic beef cattle herd reared under extensive conditions.

Bovine besnoitiosis is a parasitic disease caused by the protozoan Besnoitia besnoiti. Described many decades ago, recent epidemiological studies reveal its important spread within Europe in the last years. To date, many epidemiological aspects related to life cycle, routes of transmission, incidence rates and associated risk factors are lacking; hence, the establishment of appropriate disease control programmes poses an important challenge. Thus, the aim of the present study was to determine the epidemiological pattern of the disease in an endemic herd reared under extensive conditions (Spanish Pyrenees) by identifying main factors associated with infection and clinical disease dynamics. The study population consisted of 276 Brown Swiss and Pirenaica adult animals and 145 calves born and weaned at the farm during the study. Three sampling time frames were used: January 2010, September 2010 and February 2011, which allowed us to differentiate two periods designated as mountain and valley periods. The data related to animals (breed, sex and age) and herd management (animal grouping and time in housing) were recorded. The data collection methodology was mainly based on clinical examinations and defining the serological status against bovine besnoitiosis by the immunofluorescent antibody testing of blood samples. The total prevalence among adult animals was 38.34% (CI95%: 34.53-42.07), with 18.54% of seropositive animals showing clinical signs. In regard to the cumulative incidence, 34.57% of new infections were detected during the mountain period, in contrast to the 24.59% observed in the valley period. The incidence density was 0.058 and 0.061 new infections per animal-month for the mountain and valley periods, respectively. According to the seroepidemiological study, the seroconversion probability of B. besnoiti infection was directly associated with the number of seropositive cows with whom an animal had been stabled as well as the housing period duration, supporting horizontal transmission by close contact as one of the most important methods of disease spread. In addition, the risk of developing the clinical course increased with age, and the presence of clinical signs was related to higher antibody responses. Among calves (from 3.1 to 7.1 months old) sampled once at weaning, the total seroprevalence was 15.17% (CI95%: 9.36-21.04), and the chronic stage was observed in three animals, supporting the ability of B. besnoiti to infect and even cause disease in animals less than 6 months old. Finally, the risk of calf seroconversion was positively related to the serological status of the cows, suggesting postnatal transmission between dams and offspring by contact during the suckling period.

Reactivation of Intestinal Inflammation Is Suppressed by Catestatin in a Murine Model of Colitis via M1 Macrophages and Not the Gut Microbiota.

While there is growing awareness of a relationship between chromogranin-A (CHGA) and susceptibility to inflammatory conditions, the role of human catestatin [(hCTS); CHGA352-67] in the natural history of established inflammatory bowel disease is not known. Recently, using two different experimental models, we demonstrated that hCTS-treated mice develop less severe acute colitis. We have also shown the implication of the macrophages in this effect. The aims of this study were to determine (1) whether hCTS treatment could attenuate the reactivation of inflammation in adult mice with previously established chronic colitis; (2) whether this effect is mediated through macrophages or the gut microbiota. Quiescent colitis was induced in 7-8-week-old C57BL6 mice using four cycles (2-4%) of dextran sulfate sodium. hCTS (1.5 mg/kg/day) treatment or vehicle started 2 days before the last induction of colitis and continuing for 7 days. At sacrifice, macro- and microscopic scores were determined. Colonic pro-inflammatory cytokines [interleukin (IL)-6, IL-1ß, and TNF- α], anti-inflammatory cytokines (IL-10, TGF- ß), classically activated (M1) (iNOS, Mcp1), and alternatively activated (M2) (Ym1, Arg1) macrophages markers were studied using ELISA and/or RT-qPCR. In vitro, peritoneal macrophages isolated from naïve mice and treated with hCTS (10-5 M, 12 h) were exposed to either lipopolysaccharide (100 ng/ml, 12 h) to polarize M1 macrophages or to IL-4/IL-13 (20 ng/ml) to polarize M2 macrophages. M1/M2 macrophage markers along with cytokine gene expression were determined using RT-qPCR. Feces and mucosa-associated microbiota (MAM) samples were collected, and the V4 region of 16 s rRNA was sequenced. Micro- and macroscopic scores, colonic IL-6, IL-1ß, TNF- α, and M1 macrophages markers were significantly decreased in the hCTS-treated group. Treatment did not have any effect on colonic IL-10, TGF-ß, and M2 markers nor modified the bacterial richness, diversity, or the major phyla in colitic fecal and MAM samples. In vitro, pro-inflammatory cytokines levels, as well as their gene expression, were significantly reduced in hCTS-treated M1 macrophages. hCTS treatment did not affect M2 macrophage markers. These findings suggest that hCTS treatment attenuates the severity of inflammatory relapse through the modulation of the M1 macrophages and the release of pro-inflammatory cytokines.