Detoxification of pulping black liquor with Pleurotus ostreatus or recombinant Pichia pastoris followed by CuO/TiO2/visible photocatalysis.

Cellulose-pulping requires chemicals such as Cl2, ClO2, H2O2, and O2. The black liquor (BL) generated exhibits a high chemical oxygen demand (COD), five-day biochemical oxygen demand (BOD5), and chlorophenol content, along with an augmented colour and increased pH. BL is often discharged into water bodies, where it has a negative impact on the environment. Towards that end, laccases are of great interest for bioremediation, since they can degrade aromatic and non-aromatic compounds while reducing O2 to water instead of H2O2. As such, we evaluated Pleurotus ostreatus and Pichia pastoris (which produces rPOXA 1B laccase) in the treatment of synthetic BL (SBL) in an "in vitro" modified Kraft process followed by CuO/TiO2/visible light photocatalysis. Treating SBL with P. ostreatus viable biomass (VB) followed by CuO/TiO2/visible light photocatalysis resulted in 80.3% COD removal and 70.6% decolourisation. Toxic compounds such as 2-methylphenol, 4-methylphenol, and 2-methoxyphenol were eliminated. Post-treated SBL exhibited low phytotoxicity, as evidenced by a Lactuca sativa L seed germination index (GI) > 50%. Likewise, SBL treatment with P. pastoris followed by VB/CuO/TiO2/visible light photocatalysis resulted in 63.7% COD removal and 46% decolourisation. Moreover, this treatment resulted in the elimination of most unwanted compounds, with the exception of 4-chlorophenol. The Lactuca sativa L seed GI of the post-treated SBL was 40%, indicating moderate phytotoxicity.

Malachite Green and Crystal Violet Decolorization by Ganoderma lucidum and Pleurotus ostreatus Supernatant and by rGlLCC1 and rPOXA 1B Concentrates: Molecular Docking Analysis.

Laccases catalyze the oxidation of various aromatic organic compounds concomitantly with molecular oxygen reduction to water. Triphenylmethane dyes are synthetic compounds widely used in diverse industries. Their removal from effluents is difficult, due to their high degree of structural complexity; hence, their high concentration in effluents cause a negative impact on the environment. In the present work, molecular docking was used to evaluate interactions between rGlLCC1 or rPOXA 1B enzymes with Crystal Violet (CV) or Malachite Green (MG) dyes. In addition, removal tests of the two dyes were performed. Van der Waals interactions were obtained for only the CV dye for both GlLCC1 and POXA 1B enzymes. Nevertheless, in the GlLCC1 model, two π-π interactions were observed. For the MG dye only, Van der Waals interactions were obtained. Moreover, amino acid composition interacting in each model with each dye was similar. It is important to highlight that by molecular docking, none of the estimated ligand configurations generated hydrogen bonds. Thus, explaining the difficulty to degrade CV and MG. Regarding CV, maximum decolorization percentage was 23.6 ± 1.0% using Ganoderma lucidum supernatant and 5.0 ± 0.5% with Pleurotus ostreatus supernatant. When using recombinant laccase enzyme concentrates, decolorization percentages were 9.9 ± 0.1 and 7.5 ± 1.0% for rGlLCC1 and rPOXA 1B, respectively. On the other hand, for the MG dye, maximum decolorization percentages were 52.1 ± 5.1 and 2.3 ± 0.2% using G. lucidum and P. ostreatus concentrates, respectively. Whereas with recombinant laccase enzymatic concentrates, values of 9.4 ± 0.8% were obtained, with rGlLCC1, and 2.1 ± 0.1% when using rPOXA 1B. These findings represent an important step in bioremediation processes improvement and efficiency of industry-generated products, using environmentally friendly alternatives.

Correction to: Malachite Green and Crystal Violet Decolorization by Ganoderma lucidum and Pleurotus ostreatus Supernatant and by rGlLCC1 and rPOXA 1B Concentrates: Molecular Docking Analysis.

The original version of this article unfortunately contained a mistake. The replacement image of Fig. 4 provided by the first corresponding author, Aura M. Pedroza-Rodríguez, is incorrect and that the originally submitted Fig. 4 should have been retained. The original article has been corrected.

Plackett-Burman Design for rGILCC1 Laccase Activity Enhancement in Pichia pastoris: Concentrated Enzyme Kinetic Characterization.

Laccases are multicopper oxidases that catalyze aromatic and nonaromatic compounds with concomitant reduction of molecular oxygen to water. They are of great interest due to their potential biotechnological applications. In this work we statistically improved culture media for recombinant GILCC1 (rGILCC1) laccase production at low scale from Ganoderma lucidum containing the construct pGAPZαA-GlucPost-Stop in Pichia pastoris. Temperature, pH stability, and kinetic parameter characterizations were determined by monitoring concentrate enzyme oxidation at different ABTS substrate concentrations. Plackett-Burman Design allowed improving enzyme activity from previous work 36.08-fold, with a laccase activity of 4.69 ± 0.39 UL-1 at 168 h of culture in a 500 mL shake-flask. Concentrated rGILCC1 remained stable between 10 and 50°C and retained a residual enzymatic activity greater than 70% at 60°C and 50% at 70°C. In regard to pH stability, concentrated enzyme was more stable at pH 4.0 ± 0.2 with a residual activity greater than 90%. The lowest residual activity greater than 55% was obtained at pH 10.0 ± 0.2. Furthermore, calculated apparent enzyme kinetic parameters were a Vmax of 6.87 × 10-5 mM s-1, with an apparent Km of 5.36 × 10-2 mM. Collectively, these important stability findings open possibilities for applications involving a wide pH and temperature ranges.

"In Silico" Characterization of 3-Phytase A and 3-Phytase B from Aspergillus niger.

Phytases are used for feeding monogastric animals, because they hydrolyze phytic acid generating inorganic phosphate. Aspergillus niger 3-phytase A (PDB: 3K4Q) and 3-phytase B (PDB: 1QFX) were characterized using bioinformatic tools. Results showed that both enzymes have highly conserved catalytic pockets, supporting their classification as histidine acid phosphatases. 2D structures consist of 43% alpha-helix, 12% beta-sheet, and 45% others and 38% alpha-helix, 12% beta-sheet, and 50% others, respectively, and pI 4.94 and 4.60, aliphatic index 72.25 and 70.26 and average hydrophobicity of -0,304 and -0.330, respectively, suggesting aqueous media interaction. Glycosylation and glycation sites allowed detecting zones that can affect folding and biological activity, suggesting fragmentation. Docking showed that H59 and H63 act as nucleophiles and that D339 and D319 are proton donor residues. MW of 3K4Q (48.84 kDa) and 1QFX (50.78 kDa) is similar; 1QFX forms homodimers which will originate homotetramers with several catalytic center accessible to the ligand. 3K4Q is less stable (instability index 45.41) than 1QFX (instability index 33.66), but the estimated lifespan for 3K4Q is superior. Van der Waals interactions generate hydrogen bonds between the active center and O2 or H of the phytic acid phosphate groups, providing greater stability to these temporal molecular interactions.

Partial removal and detoxification of Malachite Green and Crystal Violet from laboratory artificially contaminated water by Pleurotus ostreatus / Eliminación parcial y desintoxicación de Verde Malaquita y Cristal Violeta del laboratorio con agua contaminada artificialmente por Pleurotus ostreatus / Extração e desintoxicação de Verde Malaquita e Cristal Violeta de água contaminada artificialmente em laboratório por Pleurotus ostreatus

Abstract The triphenylmethane Malachite Green (MG) and Crystal Violet (CV) dyes are cationic dyes and mix with domestic wastewater when dumped; increasing, among others, the chemical and biological oxygen demand and can cause acute toxicity at different trophic levels. Promoting the removal (decolorization) of MG and CV, and laccase activity (54.8 ± 8.9 and 30.6 ± 2.9 UL-1 respectively) by using P. ostreatus viable biomass needed parameters such as pH (4.5 and 6.0), temperature (25 to 30 °C), stirring speed (120 rpm), percentage of inoculum (2% v/v), and dye concentration (20 and 10 mg L-1). In adsorption studies, it was showed that an acidic pH favors the adsorption of both dyes and the model of pseudo-second order describes best the phenomenon of adsorption. Finally, the germination index (GI), using Lactuca sativa seeds for the initial dyes solutions, was < 50%; demonstrating its high phytotoxic effect. When dye solutions were treated with viable biomass, the GI increased, leaving open the possibility to perform future research to determine if the aqueous solutions, post-treated with P. ostreatus, could be used in treatments that generate less toxic water which could be used in processes that do not require potable water.

Resumen Los colorantes trifenilmetánicos Verde Malaquita (MG) y Crystal Violeta (CV) son catiónicos y al ser vertidos se mezclan con aguas residuales domésticas, incrementando, entre otros, la demanda química y biológica de oxígeno; pudiendo causar toxicidad aguda en diferentes niveles tróficos. En este estudio se encontró que los parámetros pH (4,5 y 6,0), temperatura (25 y 30 °C), velocidad de agitación (120 r.p.m.), porcentaje de inóculo (2 % v/v) y concentración de colorante (20 y 10 mgL-1), presentaron un efecto significativo (p < 0.05) para favorecer la remoción (decoloración) de MG y CV, así como la actividad lacasa (54,76 ± 8,91 y 30,59 ± 2,89 UL-1 respectivamente) al utilizar biomasa viable de P. ostreatus. En los estudios de adsorción se evidenció que pH ácidos favorecen la adsorción de ambos colorantes y que el modelo de Pseudo-segundo orden describe mejor el fenómeno de quimisorción. Finalmente los índices de germinación (IG) empleando semillas de Lactuca sativa, para los colorantes iniciales fueron < 50 %; demostrando su efecto fitotóxico elevado. Cuando las soluciones de colorantes fueron tratadas con biomasa viable, el IG aumentó, dejando abierta la puerta para la realización de investigaciones futuras con la intensión de determinar si las soluciones acuosas, postratadas con P ostreatus, pueden ser utilizadas en tratamientos que generen aguas menos tóxicas y que estas puedan ser empleadas en otros procesos que no requieran agua potable.

Resumen Os corantes de tipo trifenilmetano Verde Malaquita (VM) e Cristal Violeta (CV) são corantes catiônicos e se misturam com águas residuais domésticas quando descartadas; aumentando, entre outros, as demandas químicas e biológicas de oxigênio, podendo causar toxicidade aguda em diferentes níveis tróficos. Promoveu-se a remoção (descoloração) de VM e CV, e atividade da lacase (54.8 ± 8.9 e 30.6 ± 2.9 UL-1 respectivamente) utilizando como parâmetros necessários para a biomassa viável de P. ostreatus como pH (4,5 e 6,0), temperatura (25 a 30 °C), velocidade de agitação (120 RPM), porcentagem de inócuo (2 % v/v), e concentração de corante (20 e 10 mg L-1). Em estudos de absorção, se demonstrou que um pH mais ácido favorece a absorção de ambos corantes e o modelo de pseudo-segunda ordem descreve melhor o fenômeno da absorção. Finalmente, o índice de germinação (IG), utilizando sementes de Lactuca sativa para as soluções iniciais dos corantes, foi < 50 %; demonstrando assim seu alto efeito fitotóxico. Quando as soluções de corante foram tratadas com a biomassa viável, o IG aumentou, deixando em aberto a possibilidade de realizar futuras investigações para determinar se as soluções aquosas, tratadas com P. ostreatus, poderiam ser utilizadas em tratamentos que gerem águas menos tóxicas, que poderia ser utilizada em processos que não requerem água potável.

Computational analysis and low-scale constitutive expression of laccases synthetic genes GlLCC1 from Ganoderma lucidum and POXA 1B from Pleurotus ostreatus in Pichia pastoris.

Lacasses are multicopper oxidases that can catalyze aromatic and non-aromatic compounds concomitantly with reduction of molecular oxygen to water. Fungal laccases have generated a growing interest due to their biotechnological potential applications, such as lignocellulosic material delignification, biopulping and biobleaching, wastewater treatment, and transformation of toxic organic pollutants. In this work we selected fungal genes encoding for laccase enzymes GlLCC1 in Ganoderma lucidum and POXA 1B in Pleurotus ostreatus. These genes were optimized for codon use, GC content, and regions generating secondary structures. Laccase proposed computational models, and their interaction with ABTS [2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)] substrate was evaluated by molecular docking. Synthetic genes were cloned under the control of Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase (GAP) constitutive promoter. P. pastoris X-33 was transformed with pGAPZαA-LaccGluc-Stop and pGAPZαA-LaccPost-Stop constructs. Optimization reduced GC content by 47 and 49% for LaccGluc-Stop and LaccPost-Stop genes, respectively. A codon adaptation index of 0.84 was obtained for both genes. 3D structure analysis using SuperPose revealed LaccGluc-Stop is similar to the laccase crystallographic structure 1GYC of Trametes versicolor. Interaction analysis of the 3D models validated through ABTS, demonstrated higher substrate affinity for LaccPost-Stop, in agreement with our experimental results with enzymatic activities of 451.08 ± 6.46 UL-1 compared to activities of 0.13 ± 0.028 UL-1 for LaccGluc-Stop. This study demonstrated that G. lucidum GlLCC1 and P. ostreatus POXA 1B gene optimization resulted in constitutive gene expression under GAP promoter and α-factor leader in P. pastoris. These are important findings in light of recombinant enzyme expression system utility for environmentally friendly designed expression systems, because of the wide range of substrates that laccases can transform. This contributes to a great gamut of products in diverse settings: industry, clinical and chemical use, and environmental applications.

Low-scale expression and purification of an active putative iduronate 2-sulfate sulfatase-Like enzyme from Escherichia coli K12.

The sulfatase family involves a group of enzymes with a large degree of similarity. Until now, sixteen human sulfatases have been identified, most of them found in lysosomes. Human deficiency of sulfatases generates various genetic disorders characterized by abnormal accumulation of sulfated intermediate compounds. Mucopolysaccharidosis type II is characterized by the deficiency of iduronate 2-sulfate sulfatase (IDS), causing the lysosomal accumulation of heparan and dermatan sulfates. Currently, there are several cases of genetic diseases treated with enzyme replacement therapy, which have generated a great interest in the development of systems for recombinant protein expression. In this work we expressed the human recombinant IDS-Like enzyme (hrIDS-Like) in Escherichia coli DH5α. The enzyme concentration revealed by ELISA varied from 78.13 to 94.35 ng/ml and the specific activity varied from 34.20 to 25.97 nmol/h/mg. Western blotting done after affinity chromatography purification showed a single band of approximately 40 kDa, which was recognized by an IgY polyclonal antibody that was developed against the specific peptide of the native protein. Our 100 ml-shake-flask assays allowed us to improve the enzyme activity seven fold, compared to the E. coli JM109/pUC13-hrIDS-Like system. Additionally, the results obtained in the present study were equal to those obtained with the Pichia pastoris GS1115/pPIC-9-hrIDS-Like system (3 L bioreactor scale). The system used in this work (E. coli DH5α/pGEX-3X-hrIDS-Like) emerges as a strategy for improving protein expression and purification, aimed at recombinant protein chemical characterization, future laboratory assays for enzyme replacement therapy, and as new evidence of active putative sulfatase production in E. coli.

Análisis de la actualización del Plan Obligatorio de Salud del Sistema de Seguridad Social en Salud colombiano, 2009-2012 / An analysis of the updating of the Colombian social security system's mandatory health plan, 2009-2012

Objetivo Analizar, desde la perspectiva del derecho a la salud, la actualización del Plan Obligatorio de Salud (POS) del Sistema General de Seguridad Social en Salud (SGSSS) colombiano, comparando los contenidos del POS vigente hasta el 31 de diciembre de 2009 (POS-2009) con los contenidos del POS vigente a partir del 1 de enero del año 2012. Metodología Estudio descriptivo que indagó las modificaciones cuantitativas y las características de esas modificaciones del POS-2012 en comparación con el POS-2009. Resultados Hubo variación importante en el número y en las características de los servicios y de los medicamentos entre los POS comparados. En total se incluyeron 1 724 nuevos procedimientos y 128 nuevos medicamentos en el POS-2012, al tiempo que se excluyeron 366 procedimientos y 79 medicamentos. Algunas de esas inclusiones se corresponden con la frecuencia de demandas mediante acciones de tutela en el período 2009-2011. Discusión y conclusiones Hay avances significativos en los contenidos del POS-2012 que responden a algunas de las más frecuentes demandas de la población.(AU)

Objective Analyzing the updating the Colombian social security system's mandatory health plan (MHP),from a perspective of the right to health, by comparing and contrasting MHP content valid until 31st December 2009 (POS-2009) with MHP content from 1st January 2012 onwards (POS-2012). Methodology This was a descriptive study aimed at ascertaining the quantitative changes made and comparing the characteristics of modifications made in POS-2012 to POS-2009. Results Variation was observed between the two versions of the MHP regarding the number and characteristics of services and drugs;1,724 new procedures and 128 new medicines were included in the 2012 MHP, while 366 procedures and 79 medicines present in the 2009 MHP were excluded from MHP 2012. Some inclusions were related to frequent writs being issued from 2009-2011demanding access to such procedures and drugs. Discussion and Conclusions Significant progress was found regarding the content of the 2012 MHP compared to 2009 provision, thereby addressing some of the population's most frequent writs and plaints.(AU)

[An analysis of the updating of the Colombian social security system's mandatory health plan, 2009-2012]. / Análisis de la actualización del Plan Obligatorio de Salud del Sistema de Seguridad Social en Salud colombiano, 2009-2012.

OBJECTIVE: Analyzing the updating the Colombian social security system's mandatory health plan (MHP),from a perspective of the right to health, by comparing and contrasting MHP content valid until 31st December 2009 (POS-2009) with MHP content from 1st January 2012 onwards (POS-2012). METHODOLOGY: This was a descriptive study aimed at ascertaining the quantitative changes made and comparing the characteristics of modifications made in POS-2012 to POS-2009. RESULTS: Variation was observed between the two versions of the MHP regarding the number and characteristics of services and drugs;1,724 new procedures and 128 new medicines were included in the 2012 MHP, while 366 procedures and 79 medicines present in the 2009 MHP were excluded from MHP 2012. Some inclusions were related to frequent writs being issued from 2009-2011 demanding access to such procedures and drugs. DISCUSSION AND CONCLUSIONS: Significant progress was found regarding the content of the 2012 MHP compared to 2009 provision, thereby addressing some of the population's most frequent writs and plaints.

Eficacia del Tratamiento Homeopático en la Regeneración Clínica de la Heridas Post- Exodoncias Complejas en Pacientes del Centro de Salud Francisco Bolognesi de Cayma. Arequipa ­ 2006 / Efficacy of Homeopathic Treatment in the Clinical Regeneration of Post-Complex Excess Wounds in Patients of the Francisco Bolognesi Health Center of Cayma. Arequipa - 2006

La presente investigación que pongo a consideración, es para demostrar que la Homeopatía es de gran ayuda en el tratamiento de heridas post exodoncias complejas, por la acción podemos usarlo como analgésicos, Antiinflamatorios e inclusive como antibióticos, sin causar ningún efecto secundario a los pacientes; éste trabajo fue realizado en las personas que acudían al consultorio odontológico del Centro de Salud Francisco Bolognesi de Cayma de la ciudad de Arequipa. El tratamiento fue aplicado a 130 pacientes de ambos sexos entre 20 y 40 años de edad, los cuales tenían impactados sus terceros molares inferiores de ambos lados de la arcada y ellos mismos formaron parte del grupo experimental y del grupo control; para lo cual se plantearon los siguientes objetivos: Identificar los componentes del tratamiento homeopático, precisar las características de la regeneración clínica en las heridas post- exodoncias complejas con tratamiento homeopático, así como las características con el tratamiento convencional. Posteriormente pasados 30 días de haber realizado las exodoncias respectivas, del lado derecho se procede a dar tratamiento homeopático tres días antes de la intervención y luego realizábamos las exodoncias del lado izquierdo y se continúa con la medicación homeopática; realizando los controles de la misma manera que cuando administraba el tratamiento convencional. Analizados los resultados, la investigación reveló que con tratamiento homeopático se controla el dolor, la inflamación y la infección igual o incluso mejor que con el tratamiento alopático; y el retiro de puntos fue en menor tiempo que con el tratamiento convencional, sin poner en riesgo la salud de los pacientes, siendo factible la investigación porque se contó con la colaboración amplia y desinteresada del personal del Centro de Salud y de los pacientes. Como conclusión se confirmó la hipótesis que el tratamiento Homeopático es significativamente eficaz en la regeneración clínica de las heridas post- exodoncias complejas; por lo tanto es recomendable difundir la aplicación del tratamiento homeopático porque se puede garantizar una mejor regeneración de las heridas post - exodoncias complejas sin causar efectos secundarios en nuestros pacientes.

Espina bífida

Se presenta el caso de una paciente de 14 años, quien desde el nacimiento presentó una masa a nivel de la región lumbar, la misma que progresivamente aumentó de tamaño. Desde los nueve años manifiesta dificultad para la deambulación e incapacidad de control esfínteres, cuadro que se exacerba en los últimos dos años. Al examen físico evidencia disbasia, parestesias y mialgias en miembros inferiores con predominio en el izquierdo, disfunción de esfínteres y dermatitis de Jaquet; a la palpación se detecta una masa renitente de 5 cm por 6 cm por 8 cm, hipotrofia de pie izquierdo. Se solicitaron estudios de imagen; en la radiografía anteroposterior se observa un defecto en los cuerpos vertebrales de S2 y S3...