Beyond Structural Bioinformatics for Genomics with Dynamics Characterization of an Expanded KRAS Mutational Landscape.

Current capabilities in genomic sequencing outpace functional interpretations. Our previous work showed that 3D protein structure calculations enhance mechanistic understanding of genetic variation in sequenced tumors and patients with rare diseases. The KRAS GTPase is among the critical genetic factors driving cancer and germline conditions. Because KRAS-altered tumors frequently harbor one of three classic hotspot mutations, nearly all studies have focused on these mutations, leaving significant functional ambiguity across the broader KRAS genomic landscape observed in cancer and non-cancer diseases. Herein, we extend structural bioinformatics with molecular simulations to study an expanded landscape of 86 KRAS mutations. We identify multiple coordinated changes strongly associated with experimentally established KRAS biophysical and biochemical properties. The patterns we observe span hotspot and non-hotspot alterations, which can all dysregulate Switch regions, producing mutation-restricted conformations with different effector binding propensities. We experimentally measured mutation thermostability and identified shared and distinct patterns with simulations. Our results indicate mutation-specific conformations which show potential for future research into how these alterations reverberate into different molecular and cellular functions. The data we present is not predictable using current genomic tools, demonstrating the added functional information derived from molecular simulations for interpreting human genetic variation.

Influence of Culture Media Formulated with Agroindustrial Wastes on the Antimicrobial Activity of Lactic Acid Bacteria.

The discarding of wastes into the environment is a significant problem for many communities. Still, food waste can be used for lactic acid bacteria (LAB) growth. Here, we evaluated three growth media equivalent to de Mann Rogosa Sharpe (MRS), using apple bagasse, yeast waste, fish flour, forage oats, and cheese whey. Cell-free supernatants of eight LAB strains were tested for antimicrobial activity against nine indicator microorganisms. The supernatants were also evaluated for protein content, reducing sugars, pH, and lactic acid concentration. Cell-free supernatants from fish flour broth (FFB) LAB growth were the most effective. The strain Leuconostoc mesenteroides PIM5 presented the best activity in all media. L. mesenteroides CAL14 completely inhibited L. monocytogenes and strongly inhibited Bacillus cereus (91.1%). The strain L. mesenteroides PIM5 consumed more proteins (77.42%) and reducing sugars (56.08%) in FFB than in MRS broth (51.78% and 30.58%, respectively). Culture media formulated with agroindustrial wastes positively improved the antimicrobial activity of selected LAB, probably due to the production of antimicrobial peptides or bacteriocins.

Production of Bioactive Peptides from Lactic Acid Bacteria: A Sustainable Approach for Healthier Foods.

Traditional fermented foods where lactic acid bacteria (LAB) are present have been associated with beneficial effects on human health, and some of those benefits are related to protein-derived products. Peptides produced by LAB have attracted the interest of food industries because of their diverse applications. These peptides include ribosomally produced (bacteriocins) and protein hydrolysates by-products (bioactive peptides), which can participate as natural preservatives and nutraceuticals, respectively. It is essential to understand the biochemical pathways and the effect of growth conditions for the production of bioactive peptides and bacteriocins by LAB, in order to suggest strategies for optimization. LAB is an important food-grade expression system that can be used in the simultaneous production of peptide-based products for the food, animal, cosmetic, and pharmaceutical industries. This review describes the multifunctional proteinaceous compounds generated by LAB metabolism and discusses a strategy to use a single-step production process, using an alternative protein-based media. This strategy will provide economic advantages in fermentation processes and will also provide an environmental alternative to industrial waste valorization. New technologies that can be used to improve production and bioactivity of LAB-derived peptides are also analyzed.

Improved method for qualitative screening of lipolytic bacterial strains.

Esterases and lipases are lipolytic enzymes that catalyse the hydrolysis of triacylglycerols, Determination of lipolysis on agar plates is a simple approach to determine lipase or esterase action, but visual evaluation of lipolysis is frequently difficult in practice. Therefore, the aim of this work was to improve the efficiency of lipolysis visualization in tributyrin agar (mTBA) by adding calcium and/or magnesium ions in the screening of lipolytic microbial strains. Lipolytic activity was evaluated in mTBA using the well diffusion technique, where a clear zone around the inoculated wells indicated lipid hydrolysis. Results suggest that the addition of 2.5 mM calcium and 5.0 mM magnesium was the best combination of ion addition to TBA. Lipolytic activity increased the clearing zone up to 38% more than without the addition of ions and the clear zone was clearly observed. The mTBA plate was used with culture collection microbial strains, as well as with a collection of soil microorganisms, to identify lipase producers. The addition of calcium and magnesium ions can provide an easier screening procedure for selection of lipolytic bacterial strains. •A modified tributyrin agar for screening of lipolytic bacteria was prepared by adding calcium and magnesium ions.•The modified TBA agar was tested with control bacterial strains, and, based on the results, 2.5 mM Ca and 5.0 mM Mg ions were added in the mTBA.•mTBA was validated with environmental bacterial strains for screening of lipolytic activity.

Microbiological Impact of the Use of Reclaimed Wastewater in Recreational Parks.

Reclaimed wastewater for irrigation is an opportunity for recovery of this natural resource. In this study, microbial risk from the use of treated wastewater for irrigation of recreational parks in the city of Chihuahua, evaluating the effect of distribution distance, season, and presence of storage tanks, was analyzed. Escherichia coli, Salmonella, and multidrug-resistant bacteria were recovered from samples of reclaimed water and soils at recreational parks in Chihuahua by the membrane filtration method, using selected agars for microbial growth. Samples were taken at three different seasons. No correlation in the presence of microbial indicators and multidrug-resistant bacteria (p > 0.05) was found between the distance from the wastewater treatment plant to the point of use. Presence of storage tanks in parks showed a significant effect (p < 0.05) with a higher level of E. coli. The highest count in wastewater occurred in summer. We isolated 392 multidrug-resistant bacteria from water and soil; cluster analysis showed that the microorganisms at each location were of different origins. Irrigation with reclaimed wastewater did not have a negative effect on the presence of microbial indicators of the quality of soils in the parks. However, the prevalence of multidrug-resistant bacteria still represents a potential risk factor for human health.

Expansion of CD25-Negative Forkhead Box P3-Positive T Cells during HIV and Mycobacterium tuberculosis Infection.

Tuberculosis (TB) and HIV alter the immune system, and coinfected (HIV-TB) individuals usually present deregulations of T-lymphocytic immune response. We previously observed an increased frequency of "unconventional" CD4+CD25-FoxP3+ Treg (uTreg) population during HIV-TB disease. Therefore, we aimed to explore the phenotype and function of uTreg and conventional CD4+CD25+FoxP3+ Treg subsets (cTreg) in this context. We evaluated the expression of CD39, programmed cell death protein 1 (PD1), glucocorticoid-induced tumor necrosis factor receptor (GITR), and the effector/memory distribution by flow cytometry in cTreg and uTreg. Also, IL-10, TGF-ß, IFN-γ production, and the suppressor capacity of uTregs were analyzed in cocultures with effector lymphocytes and compared with the effect of regulatory T cells (Tregs). We found diminished expression of CD39 and higher levels of PD1 on uTreg compared to cTreg in both HIV-TB and healthy donors (HD). In addition, uTreg and cTreg showed differences in maturation status in both HIV-TB and HD groups, due to the expansion of effector memory uTregs. Interestingly, both HIV-TB and HD showed a pronounced production of IFN-γ in uTreg population, though no significant differences were observed for IL-10 and TGF-ß production between uTreg and cTreg. Moreover, IFN-γ+ cells were restricted to the CD39- uTreg population. Finally, when the suppressor capacity was evaluated, both uTreg and cTreg inhibited polyclonal T cell-proliferation and IFN-γ production in a similar extent. These findings suggest that uTregs, which are expanded during HIV-TB coinfection, exert regulatory functions in a similar way to cTregs despite an altered surface expression of Treg characteristic markers and differences in cytokine production.

HIV-TB coinfection impairs CD8(+) T-cell differentiation and function while dehydroepiandrosterone improves cytotoxic antitubercular immune responses.

Tuberculosis (TB) is the leading cause of death among HIV-positive patients. The decreasing frequencies of terminal effector (TTE ) CD8(+) T cells may increase reactivation risk in persons latently infected with Mycobacterium tuberculosis (Mtb). We have previously shown that dehydroepiandrosterone (DHEA) increases the protective antitubercular immune responses in HIV-TB patients. Here, we aimed to study Mtb-specific cytotoxicity, IFN-γ secretion, memory status of CD8(+) T cells, and their modulation by DHEA during HIV-TB coinfection. CD8(+) T cells from HIV-TB patients showed a more differentiated phenotype with diminished naïve and higher effector memory and TTE T-cell frequencies compared to healthy donors both in total and Mtb-specific CD8(+) T cells. Notably, CD8(+) T cells from HIV-TB patients displayed higher Terminal Effector (TTE ) CD45RA(dim) proportions with lower CD45RA expression levels, suggesting a not fully differentiated phenotype. Also, PD-1 expression levels on CD8(+) T cells from HIV-TB patients increased although restricted to the CD27(+) population. Interestingly, DHEA plasma levels positively correlated with TTE in CD8(+) T cells and in vitro DHEA treatment enhanced Mtb-specific cytotoxic responses and terminal differentiation in CD8(+) T cells from HIV-TB patients. Our data suggest that HIV-TB coinfection promotes a deficient CD8(+) T-cell differentiation, whereas DHEA may contribute to improving antitubercular immunity by enhancing CD8(+) T-cell functions during HIV-TB coinfection.

Total phenolic content, in vitro antioxidant activity and chemical composition of plant extracts from semiarid Mexican region.

OBJECTIVE: To determine the extraction suitable conditions of total phenolic content (TPC) by heat-reflux system, antioxidant activities and HPLC characterization of the aqueous-ethanolic extracts of Jatropha dioica (J. dioica) (Dragon's blood), Flourensia cernua (F. cernua) (Tar bush), Eucalyptus camaldulensis (E. camaldulensis) (Eucalyptus) and Turnera diffusa (T. diffusa) (Damiana). METHODS: TPC was evaluated by the well-known colorimetric assay using Folin-Ciocalteu reagent. The antioxidant activities were assayed by three methods based on scavenging of DPPH, ABTS and by lipid oxidation inhibition. The chemical composition of the extracts obtained was subject to HPLC analysis. RESULTS: TPC in the plant extracts ranged from 2.3 to 14.12 mg gallic acid equivalents/g for J. dioica and E. camaldulensis, respectively. The plant extracts of F. cernua, E. camaldulensis and T. diffusa showed similar strong antioxidant activities on scavenging of DPPH and lipid oxidation inhibition. In contrast, J. dioica extracts had lowest potential antioxidant in three assays used. HPLC assay showed the presence of several phenolic compounds in the extracts used. CONCLUSIONS: The results obtained suggest that F. cernua, E. camaldulensis and T. diffusa are potential sources to obtain bioactive phenolic compounds with high antioxidant properties which can be used in the factories as antioxidant agents or for treatments in diseases.

Total phenolic content, in vitro antioxidant activity and chemical composition of plant extracts from semiarid Mexican region

OBJECTIVE@#To determine the extraction suitable conditions of total phenolic content (TPC) by heat-reflux system, antioxidant activities and HPLC characterization of the aqueous-ethanolic extracts of Jatropha dioica (J. dioica) (Dragon's blood), Flourensia cernua (F. cernua) (Tar bush), Eucalyptus camaldulensis (E. camaldulensis) (Eucalyptus) and Turnera diffusa (T. diffusa) (Damiana).@*METHODS@#TPC was evaluated by the well-known colorimetric assay using Folin-Ciocalteu reagent. The antioxidant activities were assayed by three methods based on scavenging of DPPH, ABTS and by lipid oxidation inhibition. The chemical composition of the extracts obtained was subject to HPLC analysis.@*RESULTS@#TPC in the plant extracts ranged from 2.3 to 14.12 mg gallic acid equivalents/g for J. dioica and E. camaldulensis, respectively. The plant extracts of F. cernua, E. camaldulensis and T. diffusa showed similar strong antioxidant activities on scavenging of DPPH and lipid oxidation inhibition. In contrast, J. dioica extracts had lowest potential antioxidant in three assays used. HPLC assay showed the presence of several phenolic compounds in the extracts used.@*CONCLUSIONS@#The results obtained suggest that F. cernua, E. camaldulensis and T. diffusa are potential sources to obtain bioactive phenolic compounds with high antioxidant properties which can be used in the factories as antioxidant agents or for treatments in diseases.

Total phenolic content, in vitro antioxidant activity and chemical composition of plant extracts from semiarid Mexican region

Objective: To determine the extraction suitable conditions of total phenolic content (TPC) by heat-reflux system, antioxidant activities and HPLC characterization of the aqueous-ethanolic extracts of Jatropha dioica (J. dioica) (Dragon's blood), Flourensia cernua (F. cernua) (Tar bush), Eucalyptus camaldulensis (E. camaldulensis) (Eucalyptus) and Turnera diffusa (T. diffusa) (Damiana). Methods: TPC was evaluated by the well-known colorimetric assay using Folin-Ciocalteu reagent. The antioxidant activities were assayed by three methods based on scavenging of DPPH, ABTS and by lipid oxidation inhibition. The chemical composition of the extracts obtained was subject to HPLC analysis. Results: TPC in the plant extracts ranged from 2.3 to 14.12 mg gallic acid equivalents/g for J. dioica and E. camaldulensis, respectively. The plant extracts of F. cernua, E. camaldulensis and T. diffusa showed similar strong antioxidant activities on scavenging of DPPH and lipid oxidation inhibition. In contrast, J. dioica extracts had lowest potential antioxidant in three assays used. HPLC assay showed the presence of several phenolic compounds in the extracts used. Conclusions: The results obtained suggest that F. cernua, E. camaldulensis and T. diffusa are potential sources to obtain bioactive phenolic compounds with high antioxidant properties which can be used in the factories as antioxidant agents or for treatments in diseases.

Experimental and quantum chemical studies of a novel synthetic prenylated chalcone.

BACKGROUND: Chalcones are ubiquitous natural compounds with a wide variety of reported biological activities, including antitumoral, antiviral and antimicrobial effects. Furthermore, chalcones are being studied for its potential use in organic electroluminescent devices; therefore the description of their spectroscopic properties is important to elucidate the structure of these molecules. One of the main techniques available for structure elucidation is the use of Nuclear Magnetic Resonance Spectroscopy (NMR). Accordingly, the prediction of the NMR spectra in this kind of molecules is necessary to gather information about the influence of substituents on their spectra. RESULTS: A novel substituted chalcone has been synthetized. In order to identify the functional groups present in the new synthesized compound and confirm its chemical structure, experimental and theoretical 1H-NMR and 13C-NMR spectra were analyzed. The theoretical molecular structure and NMR spectra were calculated at both the Hartree-Fock and Density Functional (meta: TPSS; hybrid: B3LYP and PBE1PBE; hybrid meta GGA: M05-2X and M06-2X) levels of theory in combination with a 6-311++G(d,p) basis set. The structural parameters showed that the best method for geometry optimization was DFT:M06-2X/6-311++G(d,p), whereas the calculated bond angles and bond distances match experimental values of similar chalcone derivatives. The NMR calculations were carried out using the Gauge-Independent Atomic Orbital (GIAO) formalism in a DFT:M06-2X/6-311++G(d,p) optimized geometry. CONCLUSION: Considering all HF and DFT methods with GIAO calculations, TPSS and PBE1PBE were the most accurate methods used for calculation of 1H-NMR and 13C-NMR chemical shifts, which was almost similar to the B3LYP functional, followed in order by HF, M05-2X and M06-2X methods. All calculations were done using the Gaussian 09 software package. Theoretical calculations can be used to predict and confirm the structure of substituted chalcones with good correlation with the experimental data.

Aryl hydrocarbon receptor activation by aminoflavone: new molecular target for renal cancer treatment.

Aminoflavone (AF; NSC 686288, AFP464, NSC710464) is a new anticancer drug that has recently entered phase II clinical trials. It has demonstrated antiproliferative effects in MCF-7 human breast cancer cells mediated by the aryl hydrocarbon receptor (AhR). AF also exhibits noteworthy evidence of antitumor activity in vitro and in vivo against neoplastic cells of renal origin. AF treatment of sensitive renal cells, in contrast to resistant cells, promotes the induction of CYP1A1, the covalent binding of AF-reactive intermediates and apoptosis. Based on this evidence, the aim of this study was to evaluate the role of AhR, the main transcriptional regulator of CYP1A1, in the antiproliferative effects of AF in human renal cancer cells. AF-cytoxicity in human renal cell lines and a renal cancer cell strain was assessed by MTS assay in the presence or absence of an Ahr inhibitor. Drug-induced AhR nuclear translocation was evaluated by western blotting of AhR in cytosolic and nuclear fractions and by measuring xenobiotic response element-driven luciferase activity. Apoptosis induced by the drug was evaluated by 4,6-diamidino-2-phenylindole and acridine orange/ethidium bromide staining and by measuring phosphorylated P53 (p-P53) and P21 levels, caspase 3 activation and poly(ADP-ribose) polymerase cleavage. AF inhibited cell growth in a dose-dependent manner in TK-10, Caki-1, SN12-C and A498 human renal cells but not in ACHN cells. The antiproliferative effect of AF was abrogated by pre-incubation of TK-10, Caki-1 and SN12-C cells with the AhR antagonist, α-naphthoflavone. AF treatment also induced apoptosis in TK-10, Caki-1 and SN12-C cells, which was not observed in ACHN cells. AF induced time-dependent AhR nuclear translocation and AhR transcriptional activity in sensitive renal cancer cell lines. A renal cell strain derived from a human papillary tumor also showed sensitivity to AF, as well as AhR pathway activation and drug-induced apoptosis. AhR translocation could be included as a marker of sensitivity to AF in sensitive renal tumor cells of different histological origin, in ongoing phase II clinical trials.

Effect of pH and salt gradient on the autolysis of Lactococcus lactis strains

The aim of this work was to assess in-vitro the effect of pH and salt concentration on the rate of autolysis in L. lactis strains. Regardless autolysis variation among L. lactis strains, statistical analysis showed evidence of increase of autolysis in L. lactis under low salt concentration and acidic conditions.

Effect of ph and salt gradient on the autolysis of Lactococcus lactis strains.

The aim of this work was to assess in-vitro the effect of pH and salt concentration on the rate of autolysis in L. lactis strains. Regardless autolysis variation among L. lactis strains, statistical analysis showed evidence of increase of autolysis in L. lactis under low salt concentration and acidic conditions.

Phenotypic and genotypic characteristics of Lactococcus lactis strains isolated from different ecosystems.

Lactococcus lactis is the lactic acid bacteria most used in the manufacture of dairy products because of its fast lactose fermentation and flavor production. In this study, L. lactis strains isolated from different sources and previously characterized and evaluated for their capability to produce flavor were compared with respect to their phenotypic and genotypic characteristics. The variability of phenotypes and genotypes in the L. lactis strains were influenced by the source of isolation. In general, strains that shared the same genotype presented different phenotypes, and only some genotypes were clearly related to the source of isolation. On the other hand, phenotypic characteristics such as coagulant activity and utilization of lactose, glucose, and arginine were closely related to the ecosystem of origin. Coagulant activity and lactose utilization, phenotypic characteristics that are of utmost importance for efficient flavor production by starters in the manufacture of dairy products, were better for strains isolated from raw milk dairy products than for those isolated from vegetables.

Incidencia de ojo seco post lasik / Incidence of dry eye after LASIK

Se define al ojo seco como los trastornos que comprometen la cantidad y/o la calidad de las lágrimas. Es la complicación más común asociada al LASIK. Más de un 50% de los pacientes a los que se le realizó un procedimiento LASIK presentan síntomas de sequedad ocular. Su fisiopatogenia se debe a la sección de los plexos nerviosos epiteliales, subepiteliales y estromales superficiales a nivel del margen y la interfase del flap. La epiteliopatia neurotrófica por LASIK es una forma de queratitis neurotrófica que se debe a esta denervación epitelial inducida iatrogénicamente. Entre los factores de riesgo para desarrollar ojo seco post LASIK encontramos el sexo femenino, la post menopausia, el ojo seco preexistente y los errores refractivos altos. Los signos y síntomas tienen una duración aproximada entre 6 a 9 meses. Antes de realizar un procedimiento LASIK, se deben identificar y tratar los casos de ojo seco. En cuanto al tratamiento farmacológico, actualmente se cuenta con lubricantes, geles, dispositivos de oclusión, suero autólogo y ciclosporina A tópica, la cual presenta cada vez más aceptación por parte de los cirujanos refractivos por su efecto antiinflamatorio y su mayor predictibilidad refractiva.

Incidencia de ojo seco post lasik / Incidence of dry eye after LASIK

Se define al ojo seco como los trastornos que comprometen la cantidad y/o la calidad de las lágrimas. Es la complicación más común asociada al LASIK. Más de un 50% de los pacientes a los que se le realizó un procedimiento LASIK presentan síntomas de sequedad ocular. Su fisiopatogenia se debe a la sección de los plexos nerviosos epiteliales, subepiteliales y estromales superficiales a nivel del margen y la interfase del flap. La epiteliopatia neurotrófica por LASIK es una forma de queratitis neurotrófica que se debe a esta denervación epitelial inducida iatrogénicamente. Entre los factores de riesgo para desarrollar ojo seco post LASIK encontramos el sexo femenino, la post menopausia, el ojo seco preexistente y los errores refractivos altos. Los signos y síntomas tienen una duración aproximada entre 6 a 9 meses. Antes de realizar un procedimiento LASIK, se deben identificar y tratar los casos de ojo seco. En cuanto al tratamiento farmacológico, actualmente se cuenta con lubricantes, geles, dispositivos de oclusión, suero autólogo y ciclosporina A tópica, la cual presenta cada vez más aceptación por parte de los cirujanos refractivos por su efecto antiinflamatorio y su mayor predictibilidad refractiva.(AU)