Simple and sensitive spectrophotometric method for estimating the nitrogen-fixing capacity of bacterial cultures.

Biological nitrogen fixation (BNF) is a process through which a group of microorganisms called diazotrophs convert unassimilable atmospheric nitrogen into ammonia. In aqueous media, ammonia yields ammonium ions that can be assimilated by microorganisms and plants. To reduce the application of nitrogen fertilizers and their environmental effects, an alternative approach toward sustainable agriculture is the induction of artificial associations between diazotrophs and plants. This has led to increased interest in the search for microorganisms capable of supplying nitrogen to crops. This article presents a simple, economical, and sensitive spectrophotometric method for estimating the BNF capacity of bacteria cultured in a liquid NFb medium, based on the variation of absorbance caused by the change in color of bromothymol blue in the culture medium. The structure and color of this indicator are modified by pH shifts, which depend on the concentration of fixed ammonium ions.•The nitrogen concentration (estimated from the ammonium in the culture medium) showed a positive correlation (R 2 = 0.984) with the absorbance measured at 610 nm. The regression equation obtained through the origin was y = 0.009682140x, where y is the absorbance and x is the nitrogen concentration in the culture medium.•The methods used at present to measure the efficiency of BNF require expensive equipment, which may not be affordable for many laboratories or companies working in this field.•This technique can be used for pure bacterial strains and microbial consortia from soil or commercial products.

A prime-boost combination of a three-protein cocktail and multiepitopic MVA as a vaccine against Babesia bigemina elicits neutralizing antibodies and a Th1 cellular immune response in mice.

In the intraerythrocytic protozoan parasites of the genus Babesia both innate and adaptive immune responses are necessary to confer protection against clinical disease. In particular, the adaptive immune response involves the production of neutralizing antibodies as well as the presentation of parasite antigens to CD4+ T lymphocytes by professional antigen-presenting cells. Therefore, the development of alternative vaccines that replace the use of live attenuated strains should include relevant epitopes targeting both B and T cell responses. The aim of this study was to design new Babesia bigemina immunogens and evaluate the humoral and cellular responses in mice. To achieve this, three B. bigemina recombinant antigens called Apical Membrane Antigen 1 (AMA-1), Rhoptry Associated Protein 1 (RAP-1) and the Thrombospondin Related Anonymous Protein 1 (TRAP-1) were obtained. Besides, two recombinant modified vaccinia virus Ankara vectors coding for chimeric constructs containing bioinformatically predicted B and T cell epitopes from the same three antigens were generated. These immunogens were evaluated in prime-boost heterologous schemes. Among the combinations tested, priming with a cocktail of the three proteins followed by a booster immunization with a mix of both viruses induced the highest activation of IFN-γ+ CD4+ and CD8+ antigen-specific T cell responses. Remarkably, all vaccine schemes containing antigen cocktails also induced antibodies that were capable of neutralizing merozoite invasion of bovine erythrocytes in vitro at a level comparable to an anti B. bigemina hyperimmune bovine serum. Our results offer a new perspective for vaccines against B. bigemina combining bioinformatics predictions and prime-boost immunization regimes for future control measures against bovine babesiosis.

TcVac1 vaccine delivery by intradermal electroporation enhances vaccine induced immune protection against Trypanosoma cruzi infection in mice.

The efforts for the development and testing of vaccines against Trypanosoma cruzi infection have increased during the past years. We have designed a TcVac series of vaccines composed of T. cruzi derived, GPI-anchored membrane antigens. The TcVac vaccines have been shown to elicit humoral and cellular mediated immune responses and provide significant (but not complete) control of experimental infection in mice and dogs. Herein, we aimed to test two immunization protocols for the delivery of DNA-prime/DNA-boost vaccine (TcVac1) composed of TcG2 and TcG4 antigens in a BALB/c mouse model. Mice were immunized with TcVac1 through intradermal/electroporation (IDE) or intramuscular (IM) routes, challenged with T. cruzi, and evaluated during acute phase of infection. The humoral immune response was evaluated through the assessment of anti-TcG2 and anti-TcG4 IgG subtypes by using an ELISA. Cellular immune response was assessed through a lymphocyte proliferation assay. Finally, clinical and morphopathological aspects were evaluated for all experimental animals. Our results demonstrated that when comparing TcVac1 IDE delivery vs IM delivery, the former induced significantly higher level of antigen-specific antibody response (IgG2a + IgG2b > IgG1) and lymphocyte proliferation, which expanded in response to challenge infection. Histological evaluation after challenge infection showed infiltration of inflammatory cells (macrophages and lymphocytes) in the heart and skeletal tissue of all infected mice. However, the largest increase in inflammatory infiltrate was observed in TcVac1_IDE/Tc mice when compared with TcVac1_IM/Tc or non-vaccinated/infected mice. The extent of tissue inflammatory infiltrate was directly associated with the control of tissue amastigote nests in vaccinated/infected (vs. non-vaccinated/infected) mice. Our results suggest that IDE delivery improves the protective efficacy of TcVac1 vaccine against T. cruzi infection in mice when compared with IM delivery of the vaccine.

RUNX3 gene polymorphisms and haplotypes in Mexican patients with colorectal cancer.

We analyzed a possible association between RUNX3 gene polymorphisms and haplotypes in Mexican patients with colorectal cancer (CRC). Genomic DNA samples were obtained from the peripheral blood of 176 Mexican patients with CRC at diagnosis and from 195 individuals that formed the control group. The polymorphisms were detected by polymerase chain reaction-restriction fragment length polymorphism. Association was estimated by odds ratio (OR). The haplotypes and linkage disequilibrium were established using the Arlequin v3.5 software. We found that the RUNX3 polymorphisms analyzed were in Hardy-Weinberg equilibrium. The RUNX3 rs2236852 AA genotype and A allele showed association with CRC (OR = 0.39, 95%CI = 0.21-0.73, P < 0.01; OR = 0.65, 95%CI = 0.49-0.87, P < 0.01, respectively), while the rs6672420, rs11249206, and rs760805 polymorphisms did not show significant association with CRC. The TA haplotype (SNPs rs760805 and rs2236852) showed an increased risk for CRC (OR = 2.52, 95%CI = 1.47-4.30, P < 0.001). In conclusion, we found that the AA genotype and A allele of rs2236852 polymorphism confer a decreased CRC risk, while the TA haplotype appears to increase the risk of CRC development in Mexican patients.

Effect of ZNF217 gene polymorphisms on colorectal cancer development in a Mexican population.

The ZNF217 gene, a potential oncogene amplified and overexpressed in several cancers including colorectal cancer (CRC), acts as a transcription factor that activates or represses target genes. The polymorphisms rs16998248 (T>A) and rs35720349 (C>T) in coronary artery disease have been associated with reduced expression of ZNF217. In this study, we analyzed the 2 polymorphisms in Mexican patients with CRC. Genotyping of rs16998248 and rs35720349 sites was performed by polymerase chain reaction-restriction fragment length polymorphism in 203 Mexican Mestizos, 101 CRC patients, and 102 healthy blood donors. Although no statistical differences regarding genotype and allele frequencies of ZNF217 polymorphisms were observed (P > 0.05), linkage disequilibrium was significant in CRC patients (r(2) = 0.39, P < 0.0001), as a result of reduced AC haplotype frequency. Thus, the AC haplotype may protect against CRC.

Association of MMP7-181A/G and MMP13-77A/G polymorphisms with colorectal cancer in a Mexican population.

Colorectal cancer (CRC) is characterized by enhanced expression and activity of several metalloproteinases (MMPs), including MMP13 and MMP7, which play an important role in tumor invasion and metastasis. The objective of this study was to analyze the association of functional MMP7-181A/G and MMP13-77A/G promoter polymorphisms with susceptibility to CRC in a Mexican population. Genomic DNA samples were obtained from peripheral blood of 102 CRC patients and 125 blood donors who were included as the control group. Identification of polymorphisms was based on polymerase chain reaction-restriction fragment length polymorphism methodology. The association was estimated by the odds ratio (OR) test. The results showed that MMP7-181A/G and MMP13-77A/G variants were associated with CRC. For MMP7-181A/G, the AA (P=0.02, OR=3.38, 95% confidence interval (CI)=1.16-9.84) and AG (P=0.01, OR=3.4, 95%CI=1.17-9.83) genotypes were associated with an increased risk of CRC. For MMP13-77A/G, the AA and AG genotypes were associated with CRC (AA genotype: P=0.04, OR=3.2, 95%CI=1.004-10.2; AG genotype: P=0.01, OR=4.08, 95%CI=1.3-13.07). In conclusion, AA and AG genotype carriers for both polymorphisms are at a higher risk of developing CRC in this Mexican population.

MLH1 and XRCC1 polymorphisms in Mexican patients with colorectal cancer.

DNA repair proteins maintain DNA integrity; polymorphisms in genes coding for these proteins can increase susceptibility to colorectal cancer (CRC) development. We analyzed a possible association of MLH1 -93G>A and 655A>G and XRCC1 Arg194Trp and Arg399Gln polymorphisms with CRC in Mexican patients. Genomic DNA samples were obtained from peripheral blood of 108 individuals with CRC (study group) at diagnosis and 120 blood donors (control group) from Western Mexico; both groups were mestizos. The polymorphisms were detected by PCR-RFLP. Association was estimated by calculating the odds ratio (OR). We found that the MLH1 and XRCC1 polymorphisms were in Hardy- Weinberg equilibrium. The MLH1 655A>G polymorphism in the 655G allele was associated with a 2-fold increase risk for CRC (OR = 2.04 and 95% confidence interval (95%CI) = 1.12-3.69; P < 0.01), while the MLH1 -93G>A polymorphism allele was associated with a protective effect (OR = 0.60, 95%CI = 0.40-0.89; P = 0.01 in the -93A allele and OR = 0.32, 95%CI = 0.13-0.79; P = 0.01 in the AA genotype). The XRCC1 Arg194Trp and Arg399Gln polymorphisms did not show any significant associations. In conclusion, we found that MLH1 -93G>A and 655A>G polymorphisms are associated with CRC in Mexican patients.

[Intubation using an illuminating stylet in a patient with a catecholamine-secreting paraganglioma]. / Intubación con estilete iluminado en una paciente con paraganglioma secretor de catecolaminas.

Laryngoscopy and tracheal intubation produce intense noxious stimuli and are associated with an adrenergic response that can be deleterious in patients with concomitant diseases. Illuminating stylets effectively aid blind intubation by lighting the trachea, and using such devices has been associated with lower incidences of sore throat, dysphonia, and adverse hemodynamic events in comparison with rigid laryngoscopy. We report the case of a female patient with a catecholamine-secreting vagal paraganglioma. She developed multiple hypertensive episodes that were difficult to control during surgical resection of the tumor. It was decided to perform orotracheal intubation with a Light Wand (Vital Signs, Inc., Totowa, NJ, USA) to avoid oropharyngeal stimulation and to attenuate the hypertension and arrhythmias associated with laryngoscopy in such patients. The procedure was carried out without difficulty on the first attempt, in spite of tracheal displacement. The maximum increases in blood pressure and heart rate were observed 1 minute after intubation and were less than 30% of basal levels. We believe that using this light wand while intubating this patient helped keep the expected hemodynamic response to a minimum during laryngoscopy.

Fístulas anales complejas: a propósito de 154 casos / Complex anal fistulae: regarding 154 cases

Introducción: Un buen referente de la complejidad de la fístula es el grado de compromiso del músculo esfinteriano. Son importantes, también, la presencia de trayectos secundarios, fístulas múltiples, multioperadas o con alteraciones previas de la continencia. Objetivo: Comunicar la clasificación empleada, el tratamiento y los resultados. Pacientes y métodos: Estudio observacional, prospectivo, de corte transverso de 154 casos de fístulas anales complejas tratadas en la 2º Cátedra de Clínica Quirúrgica del Hospital de Clínicas de la FCM, UNA y la Clínica Regional, durante 10 años. La clasificación empleada se basó en la relación entre la altura del trayecto con respecto al esfínter. Se consignaron antecedentes de operacion

Fístulas anales complejas: a propósito de 154 casos / Complex anal fistulae: regarding 154 cases

Introducción: Un buen referente de la complejidad de la fístula es el grado de compromiso del músculo esfinteriano. Son importantes, también, la presencia de trayectos secundarios, fístulas múltiples, multioperadas o con alteraciones previas de la continencia. Objetivo: Comunicar la clasificación empleada, el tratamiento y los resultados. Pacientes y métodos: Estudio observacional, prospectivo, de corte transverso de 154 casos de fístulas anales complejas tratadas en la 2§ Cátedra de Clínica Quirúrgica del Hospital de Clínicas de la FCM, UNA y la Clínica Regional, durante 10 años. La clasificación empleada se basó en la relación entre la altura del trayecto con respecto al esfínter. Se consignaron antecedentes de operaciones y alteraciones previas de la continencia, tipo de fistula, tratamiento y las complicaciones alejadas. El seguimiento promedio fue de 4,6 años. Resultados: Tenían antecedente de intervenciones previas por fístulas 57 pacientes, con promedio de 3,4 operaciones. Siete de estos pacientes refirieron disturbios de la continencia. Casi el 80 por ciento fueron fístulas altas. La mayoría fueron tratadas por fistulotomía en dos etapas. Se observaron 4 recidivas (2,6 por ciento) y hubo trastornos de la continencia en 21 pacientes (13,6 por ciento). Conclusiones: La mayoría de las fístulas fueron altas. El tratamiento en etapas tiene buenos resultados, con recurrencia poco frecuente y alteraciones menores de la continencia dentro de márgenes aceptables.

[Perioperative course of 17 cases of pheochromocytoma]. / El curso perioperatorio en 17 casos de feocromocitoma.

We analysed the perioperative course of 17 surgeries in 16 patients with pheochromocytoma between January 1981 and February 1995. The proportion of women was high (14/16 = 88%). All patients, except one, had hypertension (in three it was only sporadic). Twelve patients had complications or associated diseases. In 13 cases the pheochromocytoma was diagnosed before the operation: they were given preoperative pharmacological protection. The intraoperative monitoring included ECG, invasive blood pressure, CVP and when considered useful, a pulmonary catheter. During tumor handling, the increase in arterial pressure reached a higher level (p < 0.01) in the patients who did not receive preoperative adrenergic blockade versus those who did receive it (p < 0.05). Intraoperative nitroprussiate was needed in 13 cases (76%). Following tumour removal five patients required vasopressor support. One patient died two weeks after surgery due to septic shock. In the postoperative period eight patients had hypokalemia, a complication which has not been reported previously.

Distribución de colesterol, trigliceridos y lipoproteinas plasmaticas en dominicanos sanos: estudio retrospectivo de 1980 a 1990 en una población comprendida entre 20 y 79 años / Cholesterol tryglicerides and lipoproteines in the plama of healthy Dominicans : retrospective study form 1980 to 1990 in a population between 20 to 79 years old

Se estudiaron 551 pacientes entre 20 y 79 años de edad, los que fueron divididos por sexo y en grupos de 5 años. Hubo 309 hombres y 242 mujeres. Los grupos fueron también subdivididos en fumadores y no fumadores así como hijos y no hijos de padecederos de enfermedad coronaria. Se les tomó sangre para medir en el suero triglicéridos, colesterol total y fraccionado. El valor promedio de trigliceridos en la población masculina fue de 157 mg/dL mientras en la femenina fue de 123.9 mg/dL. El valor promedio de colestrol en la población masculina fue de 208mg/dL y en la femenina de 206 mg/dL. El valor promedio para las HDL en la población masculina fue de 40.7 mg/dL y en la femenina 44.7 mg/dL. Para las LDL el valor promedio en la población masculina fue de 133.5 mg/dL y para la femenina 134.9 mg/dL. La relación de riesgo fue mayor en la población masculina (5.6) en la relación con la femenina (5.0). De los 551 pacientes 53 resultaron ser hipertrigliceridémicos para un 9.3 por ciento con niveles superior a 250 mg/dL de trigliceridos. De estos 38 fueron masculinos y 15 femeninos. De los 551 pacientes, 141 o sea 23.8 por ciento tenían hipercolesterolemia con colestrol por encima de 240 mg/dL. De estos 71 correspondieron al sexo masculino y 60 al femenino. Un total de 161 pacientes (29.2 por ciento ) preentó hipoalfalipoproteinemia o sea un nivel de HDL de 35 mg o menos. De estos 112 pertenecían al sexo masculino y 49 al sexo femenino. Un total de 140 pacientes (25.4 por ciento ) presentaron hiperbetalipoproteinemia, esto es, niveles de LDL superiores a 160 mg/dL. De estos 74 correspondieron al sexo masculino y 66 al femenino. En la población fumadora masculina los niveles de HDL fueron mas bajos que en la no fumadora, 36.6 versus 42.4 mg/dL respectivamente. En la población famenina estos valores fueron para las fumadoras 40.4 y para las no fumadoras 45.8 mg/dL. No hubo relación con las variables y el factor hereditario de enfermedad coronaria

Dantroleno sodico en el tetanos. Informe de un caso. / Sodium dantrolene for tetanus. Report of a case

Se informa un caso de tetanos generalizado grave, tratado con dantroleno. Inicialmente el enfermo se manejo en la forma convencional con diazepan y cloropromazina, presentando depresion en el estado de conciencia y de la funcion respiratoria. El uso del dantroleno (100 mg/dia), produjo una excelente respuesta miorrelajante, con minima depresion respiratoria. Se corroboro la accion terapeutica al reaparecer el cuadro espastico 12 horas despues de suspender el dantroleno, y desaparecer al reiniciarlo