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Much evidence suggests a role for human milk oligosaccharides (HMOs) in establishing the infant microbiota in the large intestine, but the response of particular bacteria to individual HMOs is not well known. Here twelve bacterial strains belonging to the genera Bifidobacterium, Enterococcus, Limosilactobacillus, Lactobacillus, Lacticaseibacillus, Staphylococcus and Streptococcus were isolated from infant faeces and their growth was analyzed in the presence of the major HMOs, 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 2',3-difucosyllactose (DFL), lacto-N-tetraose (LNT) and lacto-N-neo-tetraose (LNnT), present in human milk. Only the isolated Bifidobacterium strains demonstrated the capability to utilize these HMOs as carbon sources. Bifidobacterium infantis Y538 efficiently consumed all tested HMOs. Contrarily, Bifidobacterium dentium strains Y510 and Y521 just metabolized LNT and LNnT. Both tetra-saccharides are hydrolyzed into galactose and lacto-N-triose (LNTII) by B. dentium. Interestingly, this species consumed only the galactose moiety during growth on LNT or LNnT, and excreted the LNTII moiety. Two β-galactosidases were characterized from B. dentium Y510, Bdg42A showed the highest activity towards LNT, hydrolyzing it into galactose and LNTII, and Bdg2A towards lactose, degrading efficiently also 6'-galactopyranosyl-N-acetylglucosamine, N-acetyl-lactosamine and LNnT. The work presented here supports the hypothesis that HMOs are mainly metabolized by Bifidobacterium species in the infant gut., This work is part of the Grants AGL2017-84165-C2 (1-R and 2-R) and PID2020-115403RB (C21 and C22) funded by the Spanish Ministry of Science and Innovation (MICIN)/Spanish State Research Agency (AEI)/10.13039/501100011033, and by "ERDF A way of making Europe".

The gastrointestinal microbiota members produce α-l-fucosidases that play key roles in mucosal, human milk, and dietary oligosaccharide assimilation. Here, 36 open reading frames (ORFs) coding for putative α-l-fucosidases belonging to glycosyl hydrolase family 29 (GH29) were identified through metagenome analysis of breast-fed infant fecal microbiome. Twenty-two of those ORFs showed a complete coding sequence with deduced amino acid sequences displaying the highest degree of identity with α-l-fucosidases from Bacteroides thetaiotaomicron, Bacteroides caccae, Phocaeicola vulgatus, Phocaeicola dorei, Ruminococcus gnavus, and Streptococcus parasanguinis. Based on sequence homology, 10 α-l-fucosidase genes were selected for substrate specificity characterization. The α-l-fucosidases Fuc18, Fuc19A, Fuc35B, Fuc39, and Fuc1584 showed hydrolytic activity on α1,3/4-linked fucose present in Lewis blood antigens and the human milk oligosaccharide (HMO) 3-fucosyllactose. In addition, Fuc1584 also hydrolyzed fucosyl-α-1,6-N-acetylglucosamine (6FN), a component of the core fucosylation of N-glycans. Fuc35A and Fuc193 showed activity on α1,2/3/4/6 linkages from H type-2, Lewis blood antigens, HMOs and 6FN. Fuc30 displayed activity only on α1,6-linked l-fucose, and Fuc5372 showed a preference for α1,2 linkages. Fuc2358 exhibited a broad substrate specificity releasing l-fucose from all the tested free histo-blood group antigens, HMOs, and 6FN. This latest enzyme also displayed activity in glycoconjugates carrying lacto-N-fucopentaose II (Lea) and lacto-N-fucopentaose III (Lex) and in the glycoprotein mucin. Fuc18, Fuc19A, and Fuc39 also removed l-fucose from neoglycoproteins and human α-1 acid glycoprotein. These results give insight into the great diversity of α-l-fucosidases from the infant gut microbiota, thus supporting the hypothesis that fucosylated glycans are crucial for shaping the newborn microbiota composition. IMPORTANCE α-l-Fucosyl residues are frequently present in many relevant glycans, such as human milk oligosaccharides (HMOs), histo-blood group antigens (HBGAs), and epitopes on cell surface glycoconjugate receptors. These fucosylated glycans are involved in a number of mammalian physiological processes, including adhesion of pathogens and immune responses. The modulation of l-fucose content in such processes may provide new insights and knowledge regarding molecular interactions and may help to devise new therapeutic strategies. Microbial α-l-fucosidases are exoglycosidases that remove α-l-fucosyl residues from free oligosaccharides and glycoconjugates and can be also used in transglycosylation reactions to synthesize oligosaccharides. In this work, α-l-fucosidases from the GH29 family were identified and characterized from the metagenome of fecal samples of breastfed infants. These enzymes showed different substrate specificities toward HMOs, HBGAs, naturally occurring glycoproteins, and neoglycoproteins. These novel glycosidase enzymes from the breast-fed infant gut microbiota, which resulted in a good source of α-l-fucosidases, have great biotechnological potential., This work was supported by grant PID2020-115403RB (C21 and C22) from the Spanish Ministry of Science and Innovation (MICIN)/Spanish State Research Agency (AEI) (10.13039/501100011033).

Intestinal microbiota-virus-host interaction has emerged as a key factor in mediating enteric virus pathogenicity. With the aim of analyzing whether human gut bacteria improve the inefficient replication of human rotavirus in mice, we performed fecal microbiota transplant (FMT) with healthy infants as donors in antibiotic-treated mice. We showed that a simple antibiotic treatment, irrespective of FMT, resulted in viral shedding for 6 days after challenge with the human rotavirus G1P[8] genotype Wa strain (RVwa). Rotavirus titers in feces were also significantly higher in antibiotic-treated animals with or without FMT but they were decreased in animals subject to self-FMT, where a partial re-establishment of specific bacterial taxons was evidenced. Microbial composition analysis revealed profound changes in the intestinal microbiota of antibiotic-treated animals, whereas some bacterial groups, including members of Lactobacillus, Bilophila, Mucispirillum, and Oscillospira, recovered after self-FMT. In antibiotic-treated and FMT animals where the virus replicated more efficiently, differences were observed in gene expression of immune mediators, such as IL1β and CXCL15, as well as in the fucosyltransferase FUT2, responsible for H-type antigen synthesis in the small intestine. Collectively, our results suggest that antibiotic-induced microbiota depletion eradicates the microbial taxa that restrict human rotavirus infectivity in mice., This research was funded by Spanish Government (Ministerio de Economía y Competitividad) grants AGL2017-84165-C2-1-R to MJY, AGL2017-84165-C2-2-R to JRD. The study was also supported by Valencian Government grant IDIFEDER/2018/056 and by European FEDER founds. RGR is the recipient of a postdoctoral grant from the Valencian Government (APOST/2017/037). CSB is the recipient of a FPI predoctoral grant from the Spanish Government (RE2018-083315). SVV is the recipient of a predoctoral grant from the Valencian Government (ACIF/2016/437).

The gut microbiota has emerged as a key factor in the pathogenesis of intestinal viruses, including enteroviruses, noroviruses and rotaviruses (RVs), where stimulatory and inhibitory effects on infectivity have been reported. With the aim of determining whether members of the microbiota interact with RVs during infection, a combination of anti-RV antibody labeling, fluorescence-activated cell sorting and 16S rRNA amplicon sequencing was used to characterize the interaction between specific bacteria and RV in stool samples of children suffering from diarrhea produced by G1P[8] RV. The genera Ruminococcus and Oxalobacter were identified as RV binders in stools, displaying enrichments between 4.8- and 5.4-fold compared to samples nonlabeled with anti-RV antibodies. In vitro binding of the G1P[8] Wa human RV strain to two Ruminococcus gauvreauii human isolates was confirmed by fluorescence microscopy. Analysis in R. gauvreauii with antibodies directed to several histo-blood group antigens (HBGAs) indicated that these bacteria express HBGA-like substances on their surfaces, which can be the target for RV binding. Furthermore, in vitro infection of the Wa strain in differentiated Caco-2 cells was significantly reduced by incubation with R. gauvreauii. These data, together with previous findings showing a negative correlation between Ruminococcus levels and antibody titers to RV in healthy individuals, suggest a pivotal interaction between this bacterial group and human RV. These results reveal likely mechanisms of how specific bacterial taxa of the intestinal microbiota could negatively affect RV infection and open new possibilities for antiviral strategies., This research was supported by Spanish Government (Ministerio de Economía y Competitividad) grants AGL2017-84165-C2-1-R to M.J.Y. and AGL2017-84165-C2-2-R and RYC-2013-12442 to J.R.-D. This work was also supported by Valencian Government grant IDIFEDER/2018/056. R.G.-R. is the recipient of a postdoctoral grant from the Valencian Government APOST/2017/037. C.S.-B. is the recipient of a predoctoral grant FPI from the Spanish Government RE2018-083315. S.V.-V. is the recipient of a predoctoral grant from the Valencian Government ACIF/2016/437.

Human milk glycans present a unique diversity of structures that suggest different mechanisms by which they may affect the infant microbiome development. A humanized mouse model generated by infant fecal transplantation was utilized here to evaluate the impact of fucosyl-α1,3-GlcNAc (3FN), fucosyl-α1,6-GlcNAc, lacto-N-biose (LNB) and galacto-N-biose on the fecal microbiota and host–microbiota interactions. 16S rRNA amplicon sequencing showed that certain bacterial genera significantly increased (Ruminococcus and Oscillospira) or decreased (Eubacterium and Clostridium) in all disaccharide-supplemented groups. Interestingly, cluster analysis differentiates the consumption of fucosyl-oligosaccharides from galactosyl-oligosaccharides, highlighting the disappearance of Akkermansia genus in both fucosyl-oligosaccharides. An increment of the relative abundance of Coprococcus genus was only observed with 3FN. As well, LNB significantly increased the relative abundance of Bifidobacterium, whereas the absolute levels of this genus, as measured by quantitative real-time PCR, did not significantly increase. OTUs corresponding to the species Bifidobacterium longum, Bifidobacterium adolescentis and Ruminococcus gnavus were not present in the control after the 3-week intervention, but were shared among the donor and specific disaccharide groups, indicating that their survival is dependent on disaccharide supplementation. The 3FN-feeding group showed increased levels of butyrate and acetate in the colon, and decreased levels of serum HDL-cholesterol. 3FN also down-regulated the pro-inflammatory cytokine TNF-α and up-regulated the anti-inflammatory cytokines IL-10 and IL-13, and the Toll-like receptor 2 in the large intestine tissue. The present study revealed that the four disaccharides show efficacy in producing beneficial compositional shifts of the gut microbiota and in addition, the 3FN demonstrated physiological and immunomodulatory roles., This work was supported by the Spanish Ministry for Economy and Competitiveness (MINECO)/FEDER under Grant AGL2017-84165-C2 (1-R and 2-R). JRD was supported by a Ramon y Cajal Contract RYC-2013-12442 by the Spanish Ministry for Economy and Competitiveness and by Valencian Government/FEDER grant IDIFEDER/2018/056. EMMG was supported by a pre-doctoral Contract PRE2018-085768 by the Spanish Ministry of Science, Innovation and Universities.

The aims of the present work were to determine the prevalence and titer of serum antibodies against several rotavirus VP8* proteins from different P genotypes in children and adults in Valencia, Spain; and to determine the role of the secretor status (FUT2G428A polymorphism) in the antibody response. The VP8* protein from the P[4], P[6], P[8], P[9], P[11], P[14] and P[25] genotypes were produced in E. coli. These proteins were tested with 88 serum samples from children (n = 41, 3.5 years old in average) and from adults (n = 47, 58 years old in average) by ELISA. A subset of 55 samples were genotyped for the FUT2G428A polymorphism and the antibody titers compared. The same subset of samples was also analysed by ELISA using whole rotavirus Wa particles (G1P[8]) as antigen. Ninety-three per cent of the samples were positive for at least one of the VP8* antigens. Differences in the IgG seroprevalence were found between children and adults for the P[4], P[8] and P[11] genotypes. Similarly, significant differences were found between adults and children in their antibody titers against the P[4], P[8], and P[11] VP8* genotypes, having the children higher antibody titers than adults. Interestingly, positive samples against rare genotypes such as P[11] (only in children), P[14] and P[25] were found. While no statistical differences in the antibody titers between secretors and non-secretors were found for any of the tested P genotypes studied, a higher statistic significant prevalence for the P[25] genotype was found in secretors compared to non-secretors. Significant differences in the antibody titers between secretors and non-secretors were found when the whole viral particles from the Wa rotavirus strain (G1P[8]) were used as the antigen.

Human milk oligosaccharides (HMOs) are a mixture of structurally diverse carbohydrates that contribute to shape a healthy gut microbiota composition. The great diversity of the HMOs structures does not allow the attribution of specific prebiotic characteristics to single milk oligosaccharides. We analyze here the utilization of four disaccharides, lacto-N-biose (LNB), galacto-N-biose (GNB), fucosyl-α1,3-GlcNAc (3FN) and fucosyl-α1,6-GlcNAc (6FN), that form part of HMOs and glycoprotein structures, by the infant fecal microbiota. LNB significantly increased the total levels of bifidobacteria and the species Bifidobacterium breve and Bifidobacterium bifidum. The Lactobacillus genus levels were increased by 3FN fermentation and B. breve by GNB and 3FN. There was a significant reduction of Blautia coccoides group with LNB and 3FN. In addition, 6FN significantly reduced the levels of Enterobacteriaceae family members. Significantly higher concentrations of lactate, formate and acetate were produced in cultures containing either LNB or GNB in comparison with control cultures. Additionally, after fermentation of the oligosaccharides by the fecal microbiota, several Bifidobacterium strains were isolated and identified. The results presented here indicated that each, LNB, GNB and 3FN disaccharide, might have a specific beneficial effect in the infant gut microbiota and they are potential prebiotics for application in infant foods., This work was supported by the Spanish Ministry for Economy and Competitiveness (MINECO)/FEDER through the Project AGL2017-84165-C2 (1-R and 2-R). M.C.C. would like to acknowledge the support from European Research Council under the European Union's Horizon 2020 research and innovation programme (ERC starting grant, n° 639226)

Purpose To estimate the prevalence and importance of GSTT1, GSTM1, and CYP1B1 genotypes in renal cell carcinoma (RCC), and to identify their value as a prognostic factor. Materials and methods Cross-sectional study of a group of patients diagnosed with RCC (n = 133) and a control group (n = 208) with benign conditions and no history of tumor. Controls were selected by cumulative samples and mixed pairing. All subjects pertained to the catchment area for our hospital. Sociodemographic variables, anatomical pathology features, and presence of GSTT1, GSTM1, and CYP1B1 polymorphisms by multiplex PCR and sequencing techniques. Results There were no differences in the genotype distribution of the GSTT1 and GSTM1 genes between cases and controls. In the case of CYP1B1, the GG genotype (Ala119) was more prevalent in patients with RCC (OR = 2.08; 95% CI: 1.32–2.28) and may be implicated in 34.3% (95% CI: 16.3–52.2) of RCCs. In patients with GSTT1 deletion, TNM stages III to IV were more common (39.1%); whereas in Val432 homozygous patients in CYP1B1, Fuhrman grades 3 to 4 (54.6%) were more common. Because this was a cross-sectional study, longitudinal studies are needed in the future to confirm these data. Conclusions No relationship between GSTT1 and GSTM1 genotypes and RCC risk was observed. Homozygous subjects with Ala119 in CYP1B1 had twice the risk of RCC as homozygous for Ser119 or heterozygotes. Patients with GSTT1 deletion had tumors of more advanced stages, and those with Val432 polymorphism in CYP1B1 had tumors of higher Fuhrman grade.

We have previously characterized from Lactobacillus casei BL23 three α- l -fucosidases, AlfA, AlfB, and AlfC, which hydrolyze in vitro natural fucosyl-oligosaccharides. In this work, we have shown that L. casei is able to grow in the presence of fucosyl-α-1,3- N -acetylglucosamine (Fuc-α-1,3-GlcNAc) as a carbon source. Interestingly, L. casei excretes the l -fucose moiety during growth on Fuc-α-1,3-GlcNAc, indicating that only the N -acetylglucosamine moiety is being metabolized. Analysis of the genomic sequence of L. casei BL23 shows that downstream from alfB , which encodes the α- l -fucosidase AlfB, a gene, alfR , that encodes a transcriptional regulator is present. Divergently from alfB , three genes, alfEFG , that encode proteins with homology to the enzyme IIAB (EIIAB), EIIC, and EIID components of a mannose-class phosphoenolpyruvate:sugar phosphotransferase system (PTS) are present. Inactivation of either alfB or alfF abolishes the growth of L. casei on Fuc-α-1,3-GlcNAc. This proves that AlfB is involved in Fuc-α-1,3-GlcNAc metabolism and that the transporter encoded by alfEFG participates in the uptake of this disaccharide. A mutation in the PTS general component enzyme I does not eliminate the utilization of Fuc-α-1,3-GlcNAc, suggesting that the transport via the PTS encoded by alfEFG is not coupled to phosphorylation of the disaccharide. Transcriptional analysis with alfR and ccpA mutants shows that the two gene clusters alfBR and alfEFG are regulated by substrate-specific induction mediated by the inactivation of the transcriptional repressor AlfR and by carbon catabolite repression mediated by the catabolite control protein A (CcpA). This work reports for the first time the characterization of the physiological role of an α- l -fucosidase in lactic acid bacteria and the utilization of Fuc-α-1,3-GlcNAc as a carbon source for bacteria.

UDP-sugars are used as glycosyl donors in many enzymatic glycosylation processes. In bacteria UDP-N-acetylglucosamine (UDP-GlcNAc) is synthesized from fructose-6-phosphate by four successive reactions catalyzed by three enzymes: Glucosamine-6-phosphate synthase (GlmS), phosphoglucosamine mutase (GlmM), and the bi-functional enzyme glucosamine-1-phosphate acetyltransferase/N-acetylglucosamine-1-phosphate uridyltransferase (GlmU). In this work several metabolic engineering strategies, aimed to increment UDP-GlcNAc biosynthesis, were applied in the probiotic bacterium Lactobacillus casei strain BL23. This strain does not produce exopolysaccharides, therefore it could be a suitable host for the production of oligosaccharides. The genes glmS, glmM, and glmU coding for GlmS, GlmM, and GlmU activities in L. casei BL23, respectively, were identified, cloned and shown to be functional by homologous over-expression. The recombinant L. casei strain over-expressing simultaneously the genes glmM and glmS showed a 3.47 times increase in GlmS activity and 6.43 times increase in GlmM activity with respect to the control strain. Remarkably, these incremented activities resulted in about fourfold increase of the UDP-GlcNAc pool. In L. casei BL23 wild type strain transcriptional analyses showed that glmM and glmU are constitutively transcribed. By contrast, glmS transcription is down-regulated with a 21-fold decrease of glmS mRNA in cells cultured with N-acetylglucosamine as the sole carbon source compared to cells cultured with glucose. Our results revealed for the first time that GlmS, GlmM, and GlmU are responsible for UDP-GlcNAc biosynthesis in lactobacilli.

What’s known on the subject? and What does the study add? Bladder cancer susceptibility may be determined by genetic differences in the activity of glutathione S-transferases, enzymes that regulate the conversion of exogenous carcinogens to excretable hydrophilic metabolites by glutathione conjugation. The discrepancy of results regarding the association of common genetic polymorphisms and complex diseases such as cancer has raised scepticism in this area of research. Although the evidence generally supports the implication of GSTM1 and GSTT1 polymorphisms in bladder cancer, there is still some debate, with some studies in favour and some against. This study shows a greater risk of bladder cancer in individuals with GSTM1 null genotype, particularly women. This relationship is less evident with GSTT1 null genotypes. Null genotypes in both genes appear to be synergistic, particularly among smokers, and to increase the predisposition to more aggressive tumours. Nevertheless, the role of GSTM1 and GSTT1 polymorphisms in predisposition to bladder cancer should be viewed with caution, due to the multifactorial genetic origin of this condition and the need for long-term longitudinal studies to confirm these results. OBJECTIVE • To estimate the prevalence and importance of GSTT1 and GSTM1 genotypes (implicated in glutathione S-transferase activity) in bladder cancer, to determine whether smoking and occupational factors influence this relationship, and to identify the value of GSTT1 and GSTM1 genotypes as prognostic factors. PATIENTS AND METHODS • A cross-sectional study was conducted with a group of patients with bladder carcinoma and a control group with benign conditions and no history of tumours. The controls were selected and paired as subjects were recruited. • Sociodemographic variables, smoking, professional occupation, histological features and the presence of GSTT1 and GSTM1 polymorphisms by multiplex PCR techniques were assessed. RESULTS • GSTM1 genotypes were investigated in 201 patients and 193 controls and GSTT1 genotypes in 190 patients and 163 controls. • In the patients group, GSTT1 null genotype was observed in 22.1% (not significant) and GSTM1 null genotype in 54.2% (P= 0.008) (odds ratio, OR, 1.7); when considered together, 15.5% (P < 0.05; OR, 3.5) of patients had both null genotypes. In the multivariate analysis, the presence of GSTM1 null genotype remained in the model (OR, 2.1) in addition to smoking and age. • Subjects with bladder tumour and GSTM1 null genotype were younger than patients without gene deletion (P= 0.049). • Women with GSTM1 null genotype presented a higher OR than men (P= 0.024). When stratified by smoking habit, smokers with both null genotypes showed an OR of 4.7. • The percentage of patients with G3 tumours was higher in patients with GSTT1 null genotype (P= 0.013) and in patients with both null genotypes (P= 0.002). • A higher percentage of infiltrating tumours was also observed in patients with both null genotypes (P= 0.035). CONCLUSIONS • The data obtained in the present study suggest a higher risk of bladder cancer in individuals with the GSTM1 null genotype. • This risk is twofold higher when GSTM1 and GSTT1 null genotypes are both present and is also higher in smokers. • A greater predisposition for more aggressive tumours appears to exist, particularly when both null genotypes are combined. • Longer-term longitudinal studies are needed to confirm these results.

Objective To determine the prognostic value of p53 gene mutations and P53 overexpression for predicting the incidence of recurrence, progression and long-term survival of patients with transitional cell carcinoma (TCC) of the bladder. Methods Prospective cohort study with 94 consecutive patients diagnosed and treated for TCC. DNA was obtained from tumor tissue to perform PCR-SSCP of p53 exons 5–9, with automatic sequencing of any mutated samples. Immunohistochemistry using anti-human P53 monoclonal antibody was also performed. Survival was analyzed and the survival curves compared (Mantel-Haenszel). Lastly, a Cox proportional hazards model was constructed. Results Mutations were found in 46.8% of samples, with 61.8% in infiltrating tumors. Exon 8 was involved in 42.3%. P53 overexpression (cutoff ≥20%) was found in 52.1%. Mean follow-up was 44.1 months; 43.6% had died by the end of this period. Mean survival was lower in patients with exon 8 mutations (38.4 months), compared with patients without this exon mutated ( P = 0.016). There were no differences in patient survival based on positive or negative immunohistochemistry (cutoff ≥20%), although survival was lower in patients with a percentage higher than 50% of antibody-stained cells ( P = 0.02). In the Cox analysis, tumor stage, pM stage, and interaction between stage ≥pT2 and mutated p53 gene were independent risk factors, with a 6.13-fold risk of death in these patients ( P = 0.019). The number of tumors, nuclear grade, pTa stage, and the interaction between GI degree and nonmutated p53 gene remained in the Cox model for superficial tumors, such that these patients had a lower risk of recurrence or progression ( P = 0.008). Conclusions Alterations in the p53 gene may be indicative of poorer prognosis and greater recurrence in patients with urothelial bladder tumor, in particular, the presence of mutations in exon 8 and a greater percentage of stained cells in the immunohistochemistry. Nevertheless, the classic prognostic factors (primarily, pTNM stage) should still be considered the most useful factors for follow-up of these patients.

Fundamento y objetivo: La metilacion de los islotes CpG (secuencias del genoma ricas en guanina y citosina) presentes en las regiones promotoras constituye la forma de inactivacion epigenetica mas comun y es una alteracion en el mecanismo de regulacion de los genes que no requiere cambios en la secuencia del ADN. Se ha descrito hipermetilacion del promotor del gen hMLH1 en algunas lineas celulares procedentes de carcinoma de celulas renales (CCR). Nuestro objetivo ha sido determinar la existencia de hipermetilacion en el promotor del gen hMLH1 en muestras de ADN tumoral de pacientes con CCR esporadico. Material y metodo: Se recogieron consecutivamente 65 muestras de tejido tumoral procedente de pacientes diagnosticados de CCR. Tras la obtencion y purificacion del ADN se procedio a su digestion con las enzimas de restriccion HpaII y MspI, seguido de amplificacion mediante reaccion en cadena de la polimerasa de 3 regiones del promotor del gen hMLH1, electroforesis en gel de agarosa y analisis densitometrico de las bandas amplificadas. Resultados: La edad media de los pacientes fue de 63,7 anos. El tipo celular mas frecuente fue el carcinoma de celulas claras (67,7%). Un 73,9% de los tumores se diagnosticaron en estadios inferiores a pT2, un 9,3% tenian afectacion ganglionar y un 20%, metastasis a distancia. No se detecto hipermetilacion somatica en la region promotora del gen hMLH1 en ninguno de los pacientes estudiados. Conclusiones: La hipermetilacion del promotor del gen hMLH1 no parece intervenir en la patogenia del CCR esporadico, y es necesario analizar la existencia de otro tipo de mutaciones, inestabilidad de microsatelites y/o perdida de heterocigosis para determinar el posible papel de este gen en la patogenia del CCR esporadico.

This study aimed to evaluate the usefulness of carbonic anhydrase IX (CA-IX) expression in clear cell renal cell carcinoma (CCRCC) using two different techniques to detect protein expression.An experimental, cross-sectional, analytical study was conducted to analyse proteins in renal tumour and healthy tissue specimens from 38 consecutive patients who underwent nephrectomy for renal cancer. CA-IX protein expression was measured by immunohistochemistry and Western blot analysis and quantified. Statistical analysis was performed with the positive and negative specific agreements and kappa coefficient. The sensitivity and specificity of both techniques were assessed. Statistical tests were conducted to analyse the association between CA-IX expression quantitation and normal prognosis factors (TNM stage and Fuhrman nuclear grade), only in CCRCC.The mean patient age was 65 years, 78.9% of patients were men and 57.9% of tumours were CCRCC. CA-IX protein expression was positive in 63.2% of tumours by immunohistochemistry and in 60.5% by Western blot. Both techniques detected CA-IX expression only in CCRCC and unclassifiable tumours. High concordance indices were observed for CCRCC diagnosis. Western blot and immunohistochemistry had a sensitivity of 95.5% and 100%, respectively; the specificity was 100% in both techniques. CA-IX expression quantitation did not correlate with tumour stage or Fuhrman nuclear grade.Immunochemistry and Western blot techniques can be used to detect abnormal CA-IX protein expression in CCRCC and to support morphology-based diagnostic techniques.

OBJECTIVES To analyse the implications of DNA mismatch repair genes hMLH1 and hMSH2 in sporadic renal cell carcinoma (RCC). MATERIALS AND METHODS Specimens of tumour and healthy renal tissue were collected from 89 patients treated for sporadic RCC. Another 95 blood samples taken from individuals with no history of cancer were also analysed. After DNA extraction and PCR amplification, microsatellite instability (MSI) was determined using the Bethesda microsatellite panel, two exonic microsatellites of the TGFbRII and BAX genes, and the microsatellite D3S1611. The promoter methylation status of hMLH1 was investigated using the HpaII and MspI restriction enzymes. In addition, a sequencing analysis of complete coding region of hMLH1 and hMSH2 genes was performed. RESULTS MSI and promoter hypermethylation of hMLH1 were not detected. Interestingly, loss of heterozygosity (LOH) was common among patients with RCC, particularly in microsatellite D3S1611 (34.9%). Mutations were identified in eight patients: K618A and V716M in gene hMLH1; and I145V, G322D, and the novel mutation P349A, in gene hMSH2. The mutations also appeared in healthy renal tissue and therefore, were considered as germline DNA sequence variations. There were G322D and K618A changes in >1% of the healthy control subjects, suggesting that they are DNA polymorphisms. CONCLUSIONS Our data show that loss of function of both hMLH1 and hMSH2 is not involved in sporadic RCC, either by promoter methylation or mutation in their exons. However, LOH indicated that chromosomal instability affecting large fragments of DNA was the main genetic alteration we detected associated with RCC.

Epigenetic inactivation is a gene function abnormality that produces no changes in the DNA sequence, with the most frequent epigenetic alteration being hypermethylation of CpG islands in the promoter regions of the genes. Based on recent indications of a potential relationship between mismatch repair genes and renal cell carcinoma (RCC), we were interested in investigating the existence of promoter hypermethylation of the hMLH1 gene in tumor DNA samples from patients with sporadic RCC.Sixty-five tumor tissue specimens were collected consecutively. The DNA was first obtained and purified, then digested with the restriction enzymes Hpa II and Msp I, followed by polimerase chain reaction amplification of 3 promoter regions of the hMLH1 gene, agarose gel electrophoresis, and densitometric analysis of the images of the amplified bands.Mean patient age was 63.7 years. The most frequent cell type was clear cell carcinoma (67.7%). 73.9% of tumors were diagnosed in stages below pT2, 9.3% had gland involvement and 20%, distant metastasis. No somatic hypermethylation was detected in the promoter region of the hMLH1 gene in any of the patients studied.Our data indicate that promoter hypermethylation of the hMLH1 gene is not implicated in the pathogenesis of sporadic RCC, and therefore the existence of another type of mutation, microsatellite instability and/or loss of heterozygosity should be examined to determine the possible role of this gene in sporadic RCC.

Under a Creative Commons license.-- et al., Extracellular signal-regulated kinase 5 (ERK5), also known as big mitogen-activated protein kinase (MAPK) 1, is implicated in a wide range of biologic processes, which include proliferation or vascularization. Here, we show that ERK5 is degraded through the ubiquitin-proteasome system, in a process mediated by the tumor suppressor von Hippel-Lindau (VHL) gene, through a prolyl hydroxylation-dependent mechanism. Our conclusions derive from transient transfection assays in Cos7 cells, as well as the study of endogenous ERK5 in different experimental systems such as MCF7, HMEC, or Caki-2 cell lines. In fact, the specific knockdown of ERK5 in pVHL-negative cell lines promotes a decrease in proliferation and migration, supporting the role of this MAPK in cellular transformation. Furthermore, in a short series of fresh samples from human clear cell renal cell carcinoma, high levels of ERK5 correlate with more aggressive and metastatic stages of the disease. Therefore, our results provide new biochemical data suggesting that ERK5 is a novel target of the tumor suppressor VHL, opening a new field of research on the role of ERK5 in renal carcinomas., This work was supported by grants from Fundación Leticia Castillejo Castillo and Ministerio de Economía y Competitividad (SAF2009-07329 and SAF2012-30862) and grant JCCM PPII10-0141-040 to R.S.P., grant FIS PI080432 and a grant from Fundación Para la Investigación en Urología to A.S.S.-S., grant FISCAM PI2007-38 to J.M.G.-B., and grant FIS PS09/00868 to A.E.-O. R.S.P. and A.P. Research Institutes and the work carried out in their laboratories receive support from the European Community through the Regional Development Funding Program (FEDER).

UDP-N-acetylglucosamine (UDP-GlcNAc) is an important sugar nucleotide used as a precursor of cell wall components in bacteria, and as a substrate in the synthesis of oligosaccharides in eukaryotes. In bacteria UDP-GlcNAc is synthesized from the glycolytic intermediate D-fructose-6-phosphate (fructose-6P) by four successive reactions catalyzed by three enzymes: glucosamine-6-phosphate synthase (GlmS), phosphoglucosamine mutase (GlmM) and the bi-functional enzyme glucosamine-1-phosphate acetyltransferase/ N-acetylglucosamine-1-phosphate uridyltransferase (GlmU). We have previously reported a metabolic engineering strategy in Lactobacillus casei directed to increase the intracellular levels of UDP-GlcNAc by homologous overexpression of the genes glmS, glmM and glmU. One of the most remarkable features regarding the production of UDP-GlcNAc in L. casei was to find multiple regulation points on its biosynthetic pathway: (1) regulation by the NagB enzyme, (2) glmS RNA specific degradation through the possible participation of a glmS riboswitch mechanism, (3) regulation of the GlmU activity probably by end product inhibition and (4) transcription of glmU.