The Expression of Amino Acid and Fatty Acid Receptors Show an Age-Dependent Pattern Involving Oral Cavity, Jejunum and Lower Gut Sensing in Broiler Chickens.

This work aimed to evaluate the gene expression of amino acids (AA) and fatty acids (FA) sensors in the gastrointestinal tract (GIT) of chickens at two different ages (7 and 26 days post-hatch). Sixteen broilers (Ross 308) were selected, and ten sections of the GIT, including upper (tongue base, upper palate, crop, proventriculus), middle (gizzard, duodenum, jejunum, ileum), and lower GIT section (cecum, colon) were collected for analysis. Relative gene expression of AA (T1R1, T1R3, mGluR1, mGluR4, CaSR, GPR139, GPRC6A, GPR92) and FA (FFAR2, FFAR3, FFAR4) sensors were assessed using qPCR. The statistical model included age, GIT section, and gene. In addition, the correlations between gene expressions were calculated. At day 7, a significantly (p = 0.004) higher expression of AA sensors in the oral cavity and FA sensors in the lower GIT section (i.e., cecum and colon) compared to the middle section was recorded. A higher expression of AA compared to FA sensors was detected at the upper GIT section in 7 (p < 0.001) and 26-day-old chickens (p = 0.026). Thus, at day 7, AA sensors were predominantly (p < 0.05) expressed in the upper GIT section (mainly oral cavity), while FA sensors were mainly expressed in the lower GIT section, at cecum (FFR2 and 4) or colon (FFAR3). These results may indicate that in early life, both ends of the GIT are fundamental for feed intake (oral cavity) and development of the microbiota (cecum and colon). In contrast, at 26 days of age, the results showed the emergence of both AA and FA sensors in the jejunum, presumably indicating the essential role of the jejunum in the digestion absorption of nutrients and the signaling to the brain (gut-brain axis) through the enteroendocrine system. Significant positive correlations were observed between T1R1 and T1R3 (r = 0.85, p < 0.001), CaSR and T1R1 (r = 0.78, p < 0.001), CaSR and T1R3 (r = 0.45, p < 0.050), and mGluR1 and FFAR3 (r = 0.46, p < 0.050). It is concluded that the gene expression is greater in the oral cavity for AA sensors and the lower gut for FA sensors. On day 26, the role of jejunum regarding nutrient sensing is highlighted.

Genomic Distribution of Pro-Virulent cpdB-like Genes in Eubacteria and Comparison of the Enzyme Specificity of CpdB-like Proteins from Salmonella enterica, Escherichia coli and Streptococcus suis.

The cpdB gene is pro-virulent in avian pathogenic Escherichia coli and in Salmonella enterica, where it encodes a periplasmic protein named CpdB. It is structurally related to cell wall-anchored proteins, CdnP and SntA, encoded by the also pro-virulent cdnP and sntA genes of Streptococcus agalactiae and Streptococcus suis, respectively. CdnP and SntA effects are due to extrabacterial hydrolysis of cyclic-di-AMP, and to complement action interference. The mechanism of CpdB pro-virulence is unknown, although the protein from non-pathogenic E. coli hydrolyzes cyclic dinucleotides. Considering that the pro-virulence of streptococcal CpdB-like proteins is mediated by c-di-AMP hydrolysis, S. enterica CpdB activity was tested as a phosphohydrolase of 3'-nucleotides, 2',3'-cyclic mononucleotides, linear and cyclic dinucleotides, and cyclic tetra- and hexanucleotides. The results help to understand cpdB pro-virulence in S. enterica and are compared with E. coli CpdB and S. suis SntA, including the activity of the latter on cyclic-tetra- and hexanucleotides reported here for the first time. On the other hand, since CpdB-like proteins are relevant to host-pathogen interactions, the presence of cpdB-like genes was probed in eubacterial taxa by TblastN analysis. The non-homogeneous genomic distribution revealed taxa with cpdB-like genes present or absent, identifying eubacteria and plasmids where they can be relevant.

Overexpression of DAZL, STRA8, and BOULE Genes and Treatment With BMP4 or Retinoic Acid Modulate the Expression of MSC Overexpressing Germ Cell Genes.

In vitro gamete derivation from stem cells has potential applications in animal reproduction as an alternative method for the dissemination of elite animal genetics, production of transgenic animals, and conservation of endangered species. Mesenchymal stem cells (MSCs) may be suitable candidates for in vitro gamete derivation considering their differentiative capacity and their potential for cell therapy. Due to its relevance in gametogenesis, it has been reported that retinoic acid (RA) and bone morphogenetic protein (BMP) 4 are able to upregulate the expression of specific markers associated to the early stages of germ cell (GCs) differentiation in bovine fetal MSCs (bfMSCs). In the present study, we used polycistronic vectors containing combinations of GC genes DAZL, STRA8, and BOULE followed by exposure to BMP4 or RA to induce GC differentiation of bovine fetal adipose tissue-derived MSC (AT-MSCs). Cells samples at Day 14 were analyzed according to the expression of pluripotent genes NANOG and OCT4 and GC genes DAZL, STRA8, BOULE, PIWI, c-KIT, and FRAGILIS using Q-PCR. Fetal and adult testis and AT-MSCs samples were also analyzed for the expression of DAZL, STRA8, and NANOG using immunofluorescence. Increased gene expression levels in the adult testis and cell-specific distribution of DAZL, STRA8, and NANOG in the fetal testis suggest that these markers are important components of the regulatory network that control the in vivo differentiation of bovine GCs. Overexpression of DAZL and STRA8 in bi-cistronic and DAZL, STRA8, and BOULE in tri-cistronic vectors resulted in the upregulation of OCT4, NANOG, and PIWIL2 in bovine fetal AT-MSCs. While BMP4 repressed NANOG expression, this treatment increased DAZL and c-KIT and activated FRAGILIS expression in bovine fetal AT-MSCs. Treatment with RA for 14 days increased the expression of DAZL and FRAGILIS and maintained the mRNA levels of STRA8 in bovine fetal AT-MSCs transfected with bi-cistronic and tri-cistronic vectors. Moreover, RA treatment repressed the expression of OCT4 and NANOG in these cells. Thus, overexpression of DAZL, STRA8, and BOULE induced the upregulation of the pluripotent markers and PIWIL2 in transfected bovine fetal AT-MSCs. The partial activation of GC gene expression by BMP4 and RA suggests that both factors possess common targets but induce different gene expression effects during GC differentiation in overexpressing bovine fetal AT-MSCs.

Myogenic Differentiation Potential of Mesenchymal Stem Cells Derived from Fetal Bovine Bone Marrow.

The myogenic potential of bovine fetal MSC (bfMSC) derived from bone marrow (BM) remains unknown; despite its potential application for the study of myogenesis and its implications for livestock production. In the present study, three protocols for in vitro myogenic differentiation of bfMSC based on the use of DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza), myoblast-secreted factor Galectin-1 (Gal-1), and myoblast culture medium SkGM-2 BulletKit were used. Plastic-adherent bfMSC were isolated from fetal BM collected from abattoir-derived fetuses. Post-thaw viability analyses detected 85.6% bfMSC negative for propidium iodine (PI). Levels of muscle regulatory factors (MRF) MYF5, MYF6, MYOD, and DES mRNA were higher (P < 0.05) in bfMSC cultured under 100 µM of 5-Aza compared to 1 and 10 µM. Treatment of bfMSC with 10 µM of 5-Aza resulted in down-regulation of MYOD mRNA (Days 7 to 21) and up-regulation of MYF6 (Day 7), MYF5, and DES mRNA (Day 21). Gal-1 and SkGM-2 BulletKit induced sequential down-regulation of early MRF (MYF5) and up-regulation of intermediate (MYOD) and late MRF (DES) mRNA. Moreover, DES and MYF5 were immunodetected in differentiated bfMSC. In conclusion, protocols evaluated in bfMSC induced progress into myogenic differentiation until certain extent evidenced by changes in MRF gene expression.

Neumomediastino espontáneo / Spontaneous pneumomediastinum

No disponible

Sobre los métodos microbiológicos para la detección de la resistencia a oxacilina en Staphylococcus coagulasa negativos / About microbiological methods for detection of oxacillin resistance in coagulase-negative staphylococci

Introducción. Los Staphylococcus coagulasa negativos (SCN) forman parte de la microbiota humana y están implicados en infecciones de materiales protésicos, dispositivos intravasculares o bacteriemias relacionadas con el catéter. Presentan mayor resistencia que Staphylococcus aureus frente a las diferentes familias de antimicrobianos, y existe un aumento de la morbi-mortalidad de los pacientes cuando se instaura un tratamiento inadecuado. Material y métodos. Analizar los resultados obtenidos mediante diferentes técnicas comerciales: dos sistemas de microdilución automática en placa (MicroScan y Vitek2 Compact), aglutinación de la PBP2a con y sin inducción previa con disco de oxacilina de 1 μg, y detección del gen mecA mediante técnicas de amplificación de ácidos nucleicos, para realizar el diagnóstico de resistencia a meticilina en 170 aislados de SCN provenientes de hemocultivos. Resultados. Se detectó la resistencia a meticilina en las 170 cepas mediante MicroScan, en 167 por Vitek 2 Compact, en 115 mediante PBP2a sin inducción con oxacilina de 1μg y en 168 tras la inducción. Finalmente, se detectó la presencia del gen mecA en 167 cepas mediante amplificación de ácidos nucleicos. Conclusiones. Es necesario realizar una inducción con oxacilina 1μg antes de realizar la detección de PBP2a para evitar falsos negativos. Existe una gran variabilidad fenotípica en la expresión de la resistencia a meticilina en SCN (AU)

Introduction. Coagulase-negative staphylococci (CoNS) take part of the human skin and mucous membranes, but they are also involving in infections with the increasing use of prosthetic, in-dwelling devices or intravascular catheter-related bacteraemia. They are more resistance than Staphylococcus aureus against a wide range of antimicrobial agents, and it have been observed an increase in morbidity and mortality of patients with incorrect treatment. Material and methods. To analyze the results obtained by different commercial techniques: two automatic microdilution systems (MicroScan and Vitek2 Compact), PBP2a agglutination test, with and without 1 μg oxacillin disk induction, and detection of mecA gene by nucleic acids amplification techniques, for the diagnosis of methicillin resistance staphylococci in 170 strains of CoNS isolated from blood cultures. Results. One hundred and seventy methicillin resistance staphylococci were detected by MicroScan, 167 strains by Vitek 2 Compact, 115 strains were PBP2a positive without oxacillin induction and 168 after oxacillin induction. Finally, 167 strains were mecA gene positive detected by nucleic acids amplification techniques. Conclusions. It is necessary to do oxacillin induction before PBP2a test to avoid false negatives. There are a great variability in the phenotypic expression of methicillin resistance in CoNS (AU)

CGDEase, a Pseudomonas fluorescens protein of the PLC/APase superfamily with CDP-ethanolamine and (dihexanoyl)glycerophosphoethanolamine hydrolase activity induced by osmoprotectants under phosphate-deficient conditions.

A novel enzyme, induced by choline, ethanolamine, glycine betaine or dimethylglycine, was released at low temperature and phosphate from Pseudomonas fluorescens (CECT 7229) suspensions at low cell densities. It is a CDP-ethanolamine pyrophosphatase/(dihexanoyl)glycerophosphoethanolamine phosphodiesterase (CGDEase) less active on choline derivatives, and inactive on long-chain phospholipids, CDP-glycerol and other NDP-X compounds. The reaction pattern was typical of phospholipase C (PLC), as either phosphoethanolamine or phosphocholine was produced. Peptide-mass analyses, gene cloning and expression provided a molecular identity for CGDEase. Bioinformatic studies assigned it to the PLC branch of the phospholipase C/acid phosphatase (PLC/APase) superfamily, revealed an irregular phylogenetic distribution of close CGDEase relatives, and suggested their genes are not in operons or conserved contexts. A theoretical CGDEase structure was supported by mutagenesis of two predicted active-site residues, which yielded essentially inactive mutants. Biological relevance is supported by comparisons with CGDEase relatives, induction by osmoprotectants (not by osmotic stress itself) and repression by micromolar phosphate. The low bacterial density requirement was related to phosphate liberation from lysed bacteria in denser populations, rather than to a classical quorum-sensing effect. The results fit better a CGDEase role in phosphate scavenging than in osmoprotection.

Tuberculosis pancreática primaria en un paciente inmunocompetente: primer caso comunicado en España / Primary pancreatic tuberculosis in an immunocompetent patient: first case report in Spain

La tuberculosis pancreática primaria (TBPP) es una entidad excepcional definida por una lesión aislada del páncreas con confirmación microbiológica, en ausencia de TB conocida y sin afectación a ningún otro nivel. Se presenta el caso de un varón de 47 años con dolor abdominal y síndrome constitucional, diagnosticado mediante técnicas de imagen de masa sólida localmente avanzada en la cabeza del páncreas. La PAAF detectó granulomas necróticos. La prueba de la tuberculina intradérmica fue positiva. La tinción para bacilos ácido-alcohol resistentes del material de la 2.a PAAF fue negativa. No se encuentra TB tras un exhaustivo estudio diagnóstico. Se plantea laparotomía exploradora, pero a los 50 días crece Mycobacterium tuberculosis en el cultivo. La lesión pancreática desapareció tras 4 meses de tratamiento antituberculostático. Este es el primer caso descrito en España de TBPP en inmunocompetentes. Su diagnóstico exige una elevada sospecha y muestras óptimas microbiológicas para evitar cirugías innecesarias (AU)

Primary pancreatic tuberculosis (PPTB) is an extremely rare entity defined by an isolated pancreatic lesion with microbiological confirmation, in the absence of previously identified tuberculosis (TB) and involvement of any other organ. We report the case of a 47-year-old man referred for abdominal pain and weight loss, in whom several imaging techniques revealed a solid mass in the head of the pancreas. CT-guided fine-needle aspiration cytology was consistent with necrotic granuloma. Intradermic tuberculin reaction was positive, but acid fast bacilli staining was negative in repeat cytology. No additional evidence of TB was found after exhaustive diagnostic work-up. Exploratory laparotomy was proposed for a definitive diagnosis, but cultures grew Mycobacterium tuberculosis at 50 days. The pancreatic lesion disappeared after 4 months of antitubercular therapy. This is the first case report of PPTB in an immunocompetent person in Spain. A high index of suspicion and accurate samples for microbiology are mandatory to avoid unnecessary surgical procedures (AU)

[Primary pancreatic tuberculosis in an immunocompetent patient: first case report in Spain]. / Tuberculosis pancreática primaria en un paciente inmunocompetente: primer caso comunicado en España.

Primary pancreatic tuberculosis (PPTB) is an extremely rare entity defined by an isolated pancreatic lesion with microbiological confirmation, in the absence of previously identified tuberculosis (TB) and involvement of any other organ. We report the case of a 47-year-old man referred for abdominal pain and weight loss, in whom several imaging techniques revealed a solid mass in the head of the pancreas. CT-guided fine-needle aspiration cytology was consistent with necrotic granuloma. Intradermic tuberculin reaction was positive, but acid fast bacilli staining was negative in repeat cytology. No additional evidence of TB was found after exhaustive diagnostic work-up. Exploratory laparotomy was proposed for a definitive diagnosis, but cultures grew Mycobacterium tuberculosis at 50 days. The pancreatic lesion disappeared after 4 months of antitubercular therapy. This is the first case report of PPTB in an immunocompetent person in Spain. A high index of suspicion and accurate samples for microbiology are mandatory to avoid unnecessary surgical procedures.

CDP-alcohol hydrolase, a very efficient activity of the 5'-nucleotidase/UDP-sugar hydrolase encoded by the ushA gene of Yersinia intermedia and Escherichia coli.

Nucleoside 5'-diphosphate-X hydrolases are interesting enzymes to study due to their varied activities and structure-function relationships and the roles they play in the disposal, assimilation, and modulation of the effects of their substrates. Few of these enzymes with a preference for CDP-alcohols are known. In Yersinia intermedia suspensions prepared from cultures on Columbia agar with 5% sheep blood, we found a CDP-alcohol hydrolase liberated to Triton X-100-containing medium. Growth at 25 degrees C was deemed optimum in terms of the enzyme-activity yield. The purified enzyme also displayed 5'-nucleotidase, UDP-sugar hydrolase, and dinucleoside-polyphosphate hydrolase activities. It was identified as the protein product (UshA(Yi)) of the Y. intermedia ushA gene (ushA(Yi)) by its peptide mass fingerprint and by PCR cloning and expression to yield active enzyme. All those activities, except CDP-alcohol hydrolase, have been shown to be the properties of UshA of Escherichia coli (UshA(Ec)). Therefore, UshA(Ec) was expressed from an appropriate plasmid and tested for CDP-alcohol hydrolase activity. UshA(Ec) and UshA(Yi) behaved similarly. Besides being the first study of a UshA enzyme in the genus Yersinia, this work adds CDP-alcohol hydrolase to the spectrum of UshA activities and offers a novel perspective on these proteins, which are viewed here for the first time as highly efficient enzymes with k(cat)/K(m) ratios near the theoretical maximum level of catalytic activities. The results are discussed in the light of the known structures of UshA(Ec) conformers and the respective homology models constructed for UshA(Yi), and also in relation to possible biological functions. Interestingly, every Yersinia species with a sequenced genome contains an intact ushA gene, except Y. pestis, which in all its sequenced biovars contains a ushA gene inactivated by frameshift mutations.