Addition of ß-glucans in diets for tropical gar (Atractosteus tropicus) larvae: effects on growth, digestive enzymes and gene expression of intestinal epithelial integrity and immune system.

The effect of ß-glucans 1,3/1,6 from Saccharomyces cerevisiae yeast at different inclusion percentages (0.0, 0.2, 0.4, 0.6, and 0.8%) in the diet for tropical gar (Atractosteus tropicus) larvae was evaluated on growth, digestive enzyme activity and, relative expression of the immune system genes. The bioassay started on the third day after hatching (DAH) and lasted 21 days, using a total of 1500 larvae of 0.055 ± 0.008 g and, a total length of 2.46 ± 0.26 cm. Larviculture was carried out in a recirculation system with 15 tanks of 70 L using a density of 100 organisms per experimental unit. No significant differences in larval growth were observed by the inclusion of ß-glucans (p > 0.05). Digestive enzymes showed changes in lipase and trypsin activities, presenting higher values in fish fed 0.6% and 0.8% ß-glucans diets compared to the other treatments (p < 0.05). Leucine-aminopeptidase, chymotrypsin, acid phosphatase, and alkaline phosphatase activity showed higher activities in larvae fed with a 0.4% ß-glucan diet compared to the control group. The relative expression of intestinal membrane integrity (mucin 2) muc-2, (occludins) occ, (nucleotide-binding oligomerization domain) nod-2, and immune system lys (lysosome) genes showed over-expression in larvae fed the 0.4% ß-glucan diet to the rest of the treatments (p < 0.05). The inclusion of ß-glucans at 0.4-0.6% in diets for A. tropicus larvae could improve larviculture, as effects on the increase in the activity of several digestive enzymes and the expression of genes of the immune system.

Digestive proteases and in vitro protein digestibility of feed ingredients for the Central American river turtle, Dermatemys mawii.

Functional characteristics of digestive proteases and in vitro digestibility of several protein sources were studied in hatchlings of Central American river turtles, Dermatemys mawii. Acidic and alkaline proteases from the stomach and intestines were used, and optimums for acidic proteases were registered at 55°C and pH 2, while alkaline proteases were found at 55°C and pH 9. Ten protein ingredients, both vegetable and animal sources, were explored by the pH-STAT method, only for alkaline proteases. The degree of hydrolysis was at its highest for squid meal and lowest for blood meal, while the total free amino acids level was at its highest for squid meal, and lowest for wheat gluten meal. Our results indicate that D. mawii has a broad capacity to digest both animal and vegetable sources, and suggests some ingredients more suitable to design artificial diets for this species.

Epidermal growth factor receptor and epididymis invasion as prognostic biomarkers in clinical stage I testicular germ cell tumours.

BACKGROUND: Inguinal orchiectomy is curative in 70-80% of clinical stage I testicular germ cell tumours (CS I TGCT). The identification of patients who are at low risk of relapse is critical to avoid unnecessary treatment. The aim of this study is to explore EGFR, hMLH-1/hMSH-2 and microsatellite instability (MSI) as potential prognostic factors of recurrence in CS I TGCT. METHODS: Fifty-six CS I TGCT patients who underwent inguinal orchiectomy were included in this study. We analysed the relationship between clinicopathological and molecular factors with survival. Analysis of hMLH1, hMSH2 and EGFR expression was carried out by immunohistochemistry. Methylation status of the hMLH1 promoter was determined by pyrosequencing analysis in selected cases. EGFR exons 19, 20, 21 were analysed by PCR labeled-fragments and MSI status was determined using standard Multiplex MSI assays. RESULTS: Classical pathological factors such as lymphovascular invasion, high percentage of embryonal carcinoma, rete testis invasion or tumour size ≥4 cm showed a significant relationship with a higher risk of relapse. Additionally, it was found that an epididymis invasion proved to be a significant independent poor prognostic factor of recurrence (p = 0.001). hMLH1 or hMSH2 expression showed no significant association with risk of relapse and no MSI was found. EGFR expression was observed in 30.4% of samples and its expression was associated with higher risk of relapse (HR 3.5; 95% CI 1.3-9.8; p = 0.016). None of the cases presented EGFR kinase domain mutations. CONCLUSIONS: Epididymis invasion and EGFR expression, but not hMLH-1/hMSH-2 or MSI, could be potentially useful as new prognostic factors of recurrence for CS I TGCT.

Changes on digestive enzymes during initial ontogeny in the three-spot cichlid Cichlasoma trimaculatum.

A study was performed in order to understand the development of digestive enzymes during initial ontogeny of Cichlasoma trimaculatum, for which the activity of acidic and alkaline proteases, lipases, amylases and phosphatases was determined by means of biochemical and electrophoretic analysis. Our results showed that the activity of alkaline proteases, trypsin and chymotrypsin is present from day 6 after hatching (dah) during exogenous feeding with Artemia nauplii. The activities of carboxypeptidase A and leucine aminopeptidase are present from the first days, increasing at 6 dah and reaching their maximum activity at 9 dah while acid protease activity started at 9 dah. Furthermore, the lipase activity is detected on 6 dah and keeps increasing and decreasing on 17 dah. Amylase activity is detected on 3 dah, presenting fluctuations until 45 dah, where it reaches its maximum activity. Acid and alkaline phosphatases are detected from 3 dah and reach a maximum activity between 13 and 19 dah. The SDS-PAGE electrophoresis revealed six types of bands in the alkaline proteases, with molecular weight between 113.4 and 20.4 kDa. First three bands appear on 6 dah, but it is until 11 dah when all isoforms appear. Based on these results, it is considered that this species completes its digestive enzymatic machinery from day 9 after hatching, therefore is recommended to perform the transition from live feed to inert feed at 15 dah.

Neonatal exposure to estradiol valerate increases dopamine content in nigrostriatal pathway during adulthood in the rat.

Research in programming has focused in the study of stimuli that affect sensitive periods of development such as prenatal and neonatal stage. We previously showed that exposure to estradiol valerate to female rats during the first 12 h of life increased catecholamine content in ventromedial-arcuatus hypothalamus of the adult rat. However, changes in others dopaminergic circuits have not been studied. The purpose of this work was to determine the neurotransmitters changes induced by neonatal estradiol valerate (0.1 mg/50 µl s. c. per rat) administration on nigrostriatal pathway of adult female rats. Sesame oil (50 µl s. c. per rat) was administered in a control parallel group. EV-1 adult rats presented effective markers of long-term estrogenization as decreased serum levels of progesterone and a reduction in the size of estrogen-sensitive organs. In the brain, neonatal estradiol valerate administration led to a significant increase in dopamine content in striatum, substantia nigra and ventral tegmental area. With respect to the contents of dopamine metabolites, only 3-methoxytyramine content increased in substantia nigra and ventral tegmental area. In addition, the content of noradrenaline increased only in striatum. Interestingly, estrogenized rats lacked locomotor activity induced by acute dose of amphetamine (1 mg/kg i. p.). Altogether, these results show that neonatal exposure to estradiol valerate permanently modified the content of monoamine neurotransmitters in nigrostriatal pathway and amphetamine-induced locomotor activity of adult female rats. This might imply that estrogenized rats could have changes in the expression of key proteins in dopaminergic regulation, as tyrosine hydroxylase and dopamine transporter.

Partial characterization of digestive proteases in tropical gar Atractosteus tropicus juveniles.

Tropical gar (Atractosteus tropicus) is an economically and socially important freshwater species from Southeastern Mexico, with a high aquaculture potential. With this in mind, the purpose of this study was to characterize the digestive proteases of tropical gar juveniles through biochemical and electrophoretic analyses. Twenty specimens with an average weight of 73.6 ± 12.7 g were used to obtain stomach and intestinal tissue from which multienzymatic extracts were prepared. The general activities of the acid and alkaline proteases were evaluated, as well as the specific activities of trypsin, chymotrypsin, leucine aminopeptidase and carboxypeptidase A. The effect of the pH and temperature on the proteases was also analyzed, together with the composition of the multienzymatic extracts using protease inhibitors and electrophoretic tests. Results showed that A. tropicus have a functional stomach in which protein hydrolysis starts with pepsin and which contains endo- and exopeptidases (trypsin, chymotrypsin, leucine aminopeptidase and carboxypeptidase A) and proteases that are resistant to high temperatures (45 and 55 °C for alkaline and acid proteases, respectively) and pH values. Using zymogram technique, we found two acid protease isoforms (0.35 and 0.71 rf) and five alkaline protease isoforms (83.7, 43.7, 27.5, 24.0 and 19.4 kDa), which decrease or disappear with the different inhibitors. Thus, this species is considered to be a carnivore capable of adapting to its environment by consuming different types of proteins from preys and also could adapt rapidly to consume a compound diet with different animal protein sources.

Partial characterisation of digestive proteases of the Mayan cichlid Cichlasoma urophthalmus.

The characterisation of digestive proteases in native freshwater fish such as the Mayan cichlid Cichlasoma urophthalmus provides scientific elements that may be used to design balanced feed that matches with the digestive capacity of the fish. The purpose of this study was to characterise the digestive proteases, including the effect of the pH and the temperature on enzyme activity and stability, as well as the effect of inhibitors using multienzymatic extracts of the stomach and intestine of C. urophthalmus juveniles. Results showed that the optimum activities of the acid and alkaline proteases occurred at pH values of 3 and 9, respectively, whereas their optimum temperatures were 55 and 65 °C, respectively. The acid proteases were most stable at pH values of 2­3 and at temperatures of 35­45 °C, whereas the alkaline proteases were most stable at pH values of 6­9 and at 25­55 °C. The inhibition assays recorded a residual activity of 4% with pepstatin A for the acid proteases. The inhibition of the alkaline proteases was greater than 80% with TPCK, TLCK, EDTA and ovalbumin, and of 60 and 43.8% with PMSF and SBT1, respectively. The results obtained in this study make it possible to state that C. urophthalmus has a sufficiently complete digestive enzyme machinery to degrade food items characteristic of an omnivorous fish species, although specimens showed a tendency to carnivory.

Structural characterization, stability, and cytocompatibility study of chitosan BaTiO3@ZnO:Er heterostructures.

New imaging agents are required in cancer diagnosis to enhance the diagnostic accuracy, classification, and therapeutic management of tumors. Nanomaterials have emerged as a promising alternative to developing new nanostructures with imaging applications. In this study, a heterostructure based on barium titanate (BT), zinc oxide (ZnO), and erbium (Er) was prepared and coated with Chitosan (CS) to investigate their stability and compatibility with biological systems. The structure, particle morphology, luminescence properties, stability, and cytotoxicity of different nanoparticles (NPs) were assessed. The results demonstrated the formation of a [BT@ZnO:Er]-CS heterostructure, which is consistent with the relative intensities and positions of peaks in the X-ray diffraction (XRD) with an average crystallite size of ~76 nm. The electrokinetic measurement results indicate that the coated NPs are the most stable and have an average size close to 200 nm when the pH is between 3 and 5. Finally, we presented a cytotoxicity study of naked and CS-coated NPs. The results indicate that naked NPs exhibit varying cellular toxicity, as indicated by decreased cell viability, morphological changes, and an increase in an apoptotic marker. The CS-coated NPs prevented the cytotoxic effect of the naked NPs, demonstrating the significance of CS as a stabilizing agent.

Kanamycin treatment in the pre-symptomatic stage of a Drosophila PD model prevents the onset of non-motor alterations.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor alterations, which is preceded by a prodromal stage where non-motor symptoms are observed. Over recent years, it has become evident that this disorder involves other organs that communicate with the brain like the gut. Importantly, the microbial community that lives in the gut plays a key role in this communication, the so-called microbiota-gut-brain axis. Alterations in this axis have been associated to several disorders including PD. Here we proposed that the gut microbiota is different in the presymptomatic stage of a Drosophila model for PD, the Pink1B9 mutant fly, as compared to that observed in control animals. Our results show this is the case: there is basal dysbiosis in mutant animals evidenced by substantial difference in the composition of midgut microbiota in 8-9 days old Pink1B9 mutant flies as compared with control animals. Further, we fed young adult control and mutant flies kanamycin and analyzed motor and non-motor behavioral parameters in these animals. Data show that kanamycin treatment induces the recovery of some of the non-motor parameters altered in the pre-motor stage of the PD fly model, while there is no substantial change in locomotor parameters recorded at this stage. On the other hand, our results show that feeding young animals the antibiotic, results in a long-lasting improvement of locomotion in control flies. Our data support that manipulations of gut microbiota in young animals could have beneficial effects on PD progression and age-dependent motor impairments. This article is part of the Special Issue on "Microbiome & the Brain: Mechanisms & Maladies".

Identification of predictive circulating biomarkers of bevacizumab-containing regimen efficacy in pre-treated metastatic colorectal cancer patients.

BACKGROUND: To identify whether circulating levels of angiogenesis-related factors may be predictive of bevacizumab efficacy in pre-treated metastatic colorectal cancer (mCRC) patients. METHODS: Pre-treatment serum levels of 24 cytokines were measured using a multiplex bead assay (MBA) in 32 pre-treated mCRC patients treated with irinotecan plus bevacizumab-based salvage therapy. Macrophage-derived chemokine (MDC), interleukins (ILs) 8 and 6 levels were also validated by enzyme-linked immunosorbent assay (ELISA) at different time points during therapy. RESULTS: Higher epidermal growth factor (EGF) and MDC baseline levels (2.2- and 1.4-fold, respectively) and lower IL-10, IL-6 and IL-8 levels (0.2-, 0.6-, and 0.6-fold, respectively, P<0.05) were observed in patients responding to therapy. Baseline levels of these five serum factors compose a risk signature that may define the subset of patients most likely to benefit from bevacizumab-based therapy in terms of response rate and survival times. A positive correlation was found between MBA and ELISA results (P<0.01). Treatment exposure increased MDC and had opposite effects on IL-8 levels, which were decreased (P<0.05). CONCLUSION: This study suggests that a set of inflammatory and angiogenesis-related serum markers may be associated with the efficacy of bevacizumab-containing regimen.

Influence of TiO2 on prebiotic thermal synthesis of the Gly-Gln polymer.

The role of the titanium dioxide (rutile and anatase) with and without room light on the thermal synthesis of the glycine-L-glutamine (Gly-Gln) polymer is described. The efficiency in percentage of polymerization with room light was increased in 6% in the presence of rutile and in 23% in the presence of anatase. The thermal synthesis in the molten state was carried out in the absence and presence of both oxides. In all cases, the vibrational spectra showed characteristic group frequencies corresponding to a polypeptide structure. No spectral differences were observed by room light effect on the polymer on rutile. However, the polymer obtained in the presence of anatase and room light shows spectral changes associated with the formation of shorter new abundant and conformationally different species compared with the original polymer. The SEM-EDX characterization of the solid phase involved in the thermal synthesis showed that the morphology of the polypeptide is different in the presence of rutile compared to anatase. The SDS-PAGE and GPC results suggest that smaller chains are formed in the presence of both oxides and the distribution of the size and weight of each polymer molecule is completely different when the condensation is performed in the presence of anatase or rutile. Nuclear magnetic resonance analyses confirmed the incorporation of both Gly and Gln residues in the polymers, with a prevalence of Gly. Both possible sequences N-GlyGln-C and N-GlnGly-C were also detected.

Role of primary miRNA polymorphic variants in metastatic colon cancer patients treated with 5-fluorouracil and irinotecan.

MicroRNAs are non-coding RNAs that can block mRNA translation and influence mRNA stability. Recent evidence indicates that miRNA variations can affect drug resistance, efficacy, and metabolism, opening new avenues of pharmacogenomics research. We investigated associations between polymorphisms in both miRNA-containing genomic regions (primary and precursor miRNA) and in genes related to miRNA biogenesis with clinical outcome in metastatic colorectal cancer (mCRC) patients treated with 5-fluorouracil and irinotecan (CPT-11). Eighteen single-nucleotide polymorphisms (SNPs) were analyzed in 61 patients. A significant association with tumor response and time to progression (TTP) was found for SNP rs7372209 in pri-miR26a-1 (P=0.041 and P=0.017, respectively). The genotypes CC and CT were favorable when compared with the TT variant genotype. In addition, SNP rs1834306, located in the pri-miR-100 gene, significantly correlated with a longer TTP (P=0.04). In the miRNA-biogenesis pathway, a trend was identified between SNP rs11077 in the exportin-5 gene and disease control rate (P=0.076). This study is the first to suggest a relationship between treatment outcome and SNPs in the miRNA-biogenesis machinery, in both primary and precursor miRNAs. Our results suggest that miRNA polymorphic variants might be useful predictors of clinical outcome in mCRC patients treated with 5-fluorouracil and CPT-11 combination.

Dose-finding study and pharmacogenomic analysis of fixed-rate infusion of gemcitabine, irinotecan and bevacizumab in pretreated metastatic colorectal cancer patients.

BACKGROUND: To determine the dose-limiting toxicity (DLT), maximum tolerated dose, recommended dose (RD) and preliminary evidence of activity of escalating doses of irinotecan (CPT-11) fixed-dose-rate infusional gemcitabine (FDR-GMB) and bevacizumab in pretreated metastatic colorectal cancer (mCRC) patients. Pharmacogenomic analysis was performed to investigate the association between VEGF single-nucleotide polymorphisms and clinical outcome. PATIENTS AND METHODS: A total of 89 mCRC patients were recruited in a two-step study design; 28 were included in the dose-finding study and 59 in the pharmacogenomic analysis. The FDR-GMB of 1000 mg m⁻², bevacizumab 5 mg kg⁻¹ and CPT-11 doses ranging from 100 to 160 mg m⁻² were explored. The VEGF protein serum levels were quantified by EIA. Allelic discrimination was performed to genotype polymorphisms in the VEGF gene. RESULTS: CPT-11 RD was 150 mg m⁻². Diarrhoea and neutropenia were the DLT. After a median follow-up of 42 months, the median time to progression (TTP) and overall survival were 5.2 and 19.9 months, respectively. VEGF levels were significantly correlated with VEGF-2578AA and VEGF-460CC genotypes, and a trend was observed with VEGF+405GG genotype. The presence of any of these genotypes correlated with a longer median TTP (8.8 vs 4.5 months, P=0.04). CONCLUSION: The triplet combination tested in this study is effective and well tolerated. A possible predictive role for VEGF gene polymorphisms and baseline VEGF circulating levels is suggested.

Oxaliplatin, irinotecan and capecitabine as first-line therapy in metastatic colorectal cancer (mCRC): a dose-finding study and pharmacogenomic analysis.

BACKGROUND: A dose-finding study was performed to evaluate the dose-limiting toxicity (DLT), maximum-tolerated dose (MTD) and the recommended dose (RD) of escalating the doses of capecitabine and fixed doses of irinotecan and oxaliplatin on a biweekly schedule for metastatic colorectal cancer patients (mCRC). A pharmacogenomic analysis was performed to investigate the association between SNPs and treatment outcome. METHODS: Eighty-seven chemotherapy-naïve mCRC patients were recruited through a two-step study design; 27 were included in the dose-finding study and 60 in the pharmacogenomic analysis. Oxaliplatin (85 mg m(-2)) and CPT-11 (150 mg m(-2)), both on day 1, and capecitabine doses ranging from 850 to 1500 mg m(-2) bid on days 1-7 were explored. Peripheral blood samples were used to genotype 13 SNPs in 10 genes related to drug metabolism or efficacy. Univariate and multivariate Cox analysis was performed to examine associations between SNPs, ORR and PFS. RESULTS: The capecitabine RD was 1000 mg m(-2) bid. Diarrhoea and neutropenia were the DLTs. After a median follow-up of 52.5 months, the median PFS and OS were 12 (95% CI; 10.6-13.4) and 27 months (95% CI; 17.2-36.8), respectively.The GSTP1-G genotype, the Köhne low-risk category and use of a consolidation approach strongly correlated with decreased risk of progression. Patients with all favourable variables showed a median PFS of 42 months vs 3.4 months in the group with all adverse factors. A superior clinical response was obtained in patients with one GSTP1-G allele as compared with GSTP1-AA carriers (P=0.004). CONCLUSION: First-line therapy with oxaliplatin, irinotecan and capecitabine is efficient and well-tolerated. The GSTP1 polymorphism A>G status was significantly associated with ORR and PFS in mCRC treated with this triplet therapy.

Receptors involved in dexketoprofen analgesia in murine visceral pain.

Various animal models, especially rodents, are used to study pain, due to the difficulty of studying it in humans. Many drugs that produce analgesia have been studied and there is evidence among which NSAIDs deserve to be highlighted. Dexketoprofen (DEX) provides a broad antinociceptive profile in different types of pain; therefore, this study was designed to evaluate the profile of antinociceptive potency in mice. Analgesic activity was evaluated using the acetic acid abdominal constriction test (writhing test), a chemical model of visceral pain. Dose-response curves for i.p. DEX administration (1, 3, 10, 30 and 100 mg/kg), using at least six mice in each of at least five doses, was obtained before and 30 min after pre-treatment with different pharmacological agents. Pretreatment of the mice with opioid receptor antagonists was not effective; however, the serotonin receptor antagonist and nitric oxide synthase inhibitor produce a significant increase in DEX-induced antinociception. The data from the present study shows that DEX produces antinociception in the chemical twisting test of mice, which is explained with difficulty by the simple inhibition of COX. This effect appears to be mediated by other mechanisms in which the contribution of the NO and 5-HT pathways has an important effect on DEXinduced antinociception.

MicroRNAs as a new potential therapeutic opportunity in gastrointestinal cancer.

MicroRNAs (miRNAs) comprise an abundant class of small non-coding RNAs that act as important post-transcriptional regulators of gene expression. Accumulating evidence shows that aberrantly expressed miRNAs play important roles in human cancers and underscores them as potential targets for therapeutic intervention. Basic research has provided strong evidence about the role of miRNA as oncogenes and as tumor suppressor genes in cancer. In accord with this finding, miRNA-based cancer therapy is a very interesting field of investigation that offers the appeal of targeting multiple gene networks controlled by a single, aberrantly expressed miRNA. A new door in cancer research is opening and it may lead to the modulation of miRNAs.

G protein ßγ subunits play a critical role in the actions of amphetamine.

Abnormal levels of dopamine (DA) are thought to contribute to several neurological and psychiatric disorders including drug addiction. Extracellular DA levels are regulated primarily via reuptake by the DA transporter (DAT). Amphetamine, a potent psychostimulant, increases extracellular DA by inducing efflux through DAT. Recently, we discovered that G protein ßγ subunits (Gßγ) interact with DAT, and that in vitro activation of Gßγ promotes DAT-mediated efflux. Here, we investigated the role of Gßγ in the actions of amphetamine in DA neurons in culture, ex vivo nucleus accumbens (NAc), and freely moving rats. Activation of Gßγ with the peptide myr-Ser-Ile-Arg-Lys-Ala-Leu-Asn-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp (mSIRK) in the NAc potentiated amphetamine-induced hyperlocomotion, but not cocaine-induced hyperlocomotion, and systemic or intra-accumbal administration of the Gßγ inhibitor gallein attenuated amphetamine-induced, but not cocaine-induced hyperlocomotion. Infusion into the NAc of a TAT-fused peptide that targets the Gßγ-binding site on DAT (TAT-DATct1) also attenuated amphetamine-induced but not cocaine-induced hyperlocomotion. In DA neurons in culture, inhibition of Gßγ with gallein or blockade of the Gßγ-DAT interaction with the TAT-DATct1 peptide decreased amphetamine-induced DA efflux. Furthermore, activation of Gßγ with mSIRK potentiated and inhibition of Gßγ with gallein reduced amphetamine-induced increases of extracellular DA in the NAc in vitro and in freely moving rats. Finally, systemic or intra-accumbal inhibition of Gßγ with gallein blocked the development of amphetamine-induced, but not cocaine-induced place preference. Collectively, these results suggest that interaction between Gßγ and DAT plays a critical role in the actions of amphetamine and presents a novel target for modulating the actions of amphetamine in vivo.