Cancer Stem Cell and Aggressiveness Traits Are Promoted by Stable Endothelin-Converting Enzyme-1c in Glioblastoma Cells.

Glioblastoma (GBM) is the most common and aggressive type of brain tumor due to its elevated recurrence following treatments. This is mainly mediated by a subpopulation of cells with stemness traits termed glioblastoma stem-like cells (GSCs), which are extremely resistant to anti-neoplastic drugs. Thus, an advancement in the understanding of the molecular processes underlying GSC occurrence should contribute significantly towards progress in reducing aggressiveness. High levels of endothelin-converting enzyme-1 (ECE1), key for endothelin-1 (ET-1) peptide activation, have been linked to the malignant progression of GBM. There are four known isoforms of ECE1 that activate ET-1, which only differ in their cytoplasmic N-terminal sequences. Isoform ECE1c is phosphorylated at Ser-18 and Ser-20 by protein kinase CK2, which increases its stability and hence promotes aggressiveness traits in colon cancer cells. In order to study whether ECE1c exerts a malignant effect in GBM, we designed an ECE1c mutant by switching a putative ubiquitination lysine proximal to the phospho-serines Lys-6-to-Arg (i.e., K6R). This ECE1cK6R mutant was stably expressed in U87MG, T98G, and U251 GBM cells, and their behavior was compared to either mock or wild-type ECE1c-expressing clone cells. ECE1cK6R behaved as a highly stable protein in all cell lines, and its expression promoted self-renewal and the enrichment of a stem-like population characterized by enhanced neurospheroid formation, as well as increased expression of stem-like surface markers. These ECE1cK6R-derived GSC-like cells also displayed enhanced resistance to the GBM-related chemotherapy drugs temozolomide and gemcitabine and increased expression of the ABCG2 efflux pump. In addition, ECE1cK6R cells displayed enhanced metastasis-associated traits, such as the modulation of adhesion and the enhancement of cell migration and invasion. In conclusion, the acquisition of a GSC-like phenotype, together with heightened chemoresistance and invasiveness traits, allows us to suggest phospho-ECE1c as a novel marker for poor prognosis as well as a potential therapeutic target for GBM.

Acute meningoencephalitis associated with SARS-CoV-2 infection in Colombia.

We present the case of a patient in the third decade of life, with asthma as comorbidity, who presented to the emergency department due to odynophagia, dyspnea, and cough of 2 days of evolution, later developing acute ventilatory failure requiring orotracheal intubation. The high-resolution chest tomography study showed consolidation due to a pneumonic process towards the posterior segment of the right lower lobe with areas of ground-glass infiltrates with a peripheral distribution. During the clinical course, the patient presented multiple seizure episodes that met the criteria for status epilepticus with MRI compatible with changes due to leptomeningitis. Given symptoms and thorax imaging, tests for SARS-CoV-2 ensued, with both positive RT-PCR in bronchoalveolar lavage and cerebrospinal fluid for the virus also positive. RT-PCR multiplex panel of meningitis/encephalitis results negative for 14 common organisms. A diagnosis of acute meningoencephalitis associated with COVID-19 was considered, with an adequate response to corticosteroid management; to our knowledge, this is the first adult patient with CNS involvement and CSF positive test in Latin America.

A chymotrypsin from the Digestive Tract of California Spiny Lobster, Panulirus interruptus: Purification and Biochemical Characterization.

A chymotrypsin was purified from the gastric juice of California spiny lobster (Panulirus interrutpus), using preparative electrophoresis and affinity chromatography on agarose-p-aminobenzamidine. The molecular mass was estimated by polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions to be 28 kDa. Chymotrypsin activity was totally inhibited by phenylmethylsulfonyl fluoride (PMSF) and chymostatin. Lobster chymotrypsin had optimal pH 7.0-8.0 and temperature of 55 °C. The enzyme is highly stable under a wide range of pH (retaining up to 80 % of activity after 1 h of incubation at pH 3.0, 5.0, and 12.0), showing higher stability at pH 8.0, and was inactivated after 20 min at 55 °C. Lobster chymotrypsin was able to hydrolyze protein substrates at as low as pH 3.0. These results are consistent with the findings of enzyme stability. Activity was assessed after incubation of enzyme with different organic solvents (in the range of 10-50 %); when tested in the presence of acetone, ethanol, propanol, and butanol, lobster chymotrypsin residual activity was >80 %; whereas in the presence of dimethyl sulfoxide (DMSO) and toluene, lobster chymotrypsin residual activity was <80 %. Deduced amino acid sequence, corroborated by mass spectrometry, was determined.

Biochemical characterisation of chymotrypsin from the midgut gland of yellowleg shrimp, Penaeus californiensis.

Chymotrypsin from shrimp, Penaeus californiensis, was compared to Bos taurus chymotrypsin, and its structure-function relationship was studied. Catalytic efficiency toward synthetic substrate is lower, but it has a broad specificity and higher activity toward protein substrates, including collagen. It is active at pH 4-10 and fully active up to 50 °C for 2 h and at least nine days at room temperature. The activation peptide is twice as long as bovine chymotrypsinogen, has less disulfide bridges, and is a single polypeptide. Only one activation step is necessary from chymotrypsinogen to the mature enzyme. Postmortem implications in muscle softening and melanisation, resistance to temperature and pH and efficiency with proteinaceous substrates make chymotrypsin useful as a biotechnological tool in food processing. This makes shrimp processing wastes useful as a material for production of fine reagents.

Phenoloxidase activity of hemocyanin in whiteleg shrimp Penaeus vannamei: conversion, characterization of catalytic properties, and role in postmortem melanosis.

Latent phenoloxidase activity of hemocyanin (Hc) in whiteleg shrimp Penaeus vannamei was assayed to determine its potential involvement in postmortem melanosis. Conversion of pure 12-mer, but not 6-mer, hemocyanin to phenoloxidase by endogenous (serine proteinases) and exogenous (SDS) effectors demonstrated the need of complex aggregation for displaying enzyme activity. Because Hc was converted to Hc-phenoloxidase (HcPO) by hemocytes extracts, the mechanism of conversion seems to be the same for polyphenoloxidases. HcPO has similar biochemical and kinetic properties as real polyphenoloxidases and uses mono- and diphenols as substrates. The kinetics of hydroxygenation of monophenols has a lag phase, typical for tyrosinases, contrary to oxidation of diphenols. Regardless of the structure of the substrate, melanin is finally formed. Because of the abundance, distribution, and resistance of Hc to freezing-thawing, involvement of Hc in black spot formation postmortem is suggested. This has important implications for commercialization of shrimp and related seafood.

Clinical characteristics and outcomes of obstetric patients requiring ICU admission.

OBJECTIVES: To review a series of critically ill obstetric patients admitted to our ICU to assess the spectrum of disease, required interventions, and fetal/maternal mortality, and to identify conditions associated with maternal death. DESIGN: Retrospective cohort. SETTING: Medical-surgical ICU in a university-affiliated hospital. PATIENTS: Pregnant/postpartum admissions between January 1, 1998, and September 30, 2005. INTERVENTIONS: None. MEASUREMENTS AND RESULTS: We studied 161 patients (age, 28 +/- 9 years; mean gestational age, 29 +/- 9 weeks) [mean +/- SD], constituting 10% of 1,571 hospital admissions. APACHE (acute physiology and chronic health evaluation) II score was 14 +/- 8, with 24% predicted mortality; sequential organ failure assessment score was 5 +/- 3; and therapeutic intervention scoring system at 24 h was 25 +/- 9. Forty-one percent of patients required mechanical ventilation (MV). ARDS, shock, and organ dysfunction were present in 19%, 25%, and 48% of patients, respectively. Most patients (63%) were admitted postpartum, and 74% of admissions were of obstetric cause. Hypertensive disease (40%), major hemorrhage (16%), septic abortion (12%), and nonobstetric sepsis (10%) were the principal diagnoses. Maternal mortality was 11%, with multiple organ dysfunction syndrome (44%) and intracranial hemorrhage (39%) as main causes. There were no differences in death rate in patients admitted for obstetric and nonobstetric causes. Fetal mortality was 32%. Only 30% of patients received antenatal care, which was more frequent in survivors (33% vs 6% nonsurvivors, p = 0.014). CONCLUSIONS: Although ARDS, organ failures, shock, and use of MV were extremely frequent in this population, maternal mortality remains within an acceptable range. APACHE II overpredicted mortality in these patients. Septic abortion is still an important modifiable cause of mortality. Efforts should concentrate in increasing antenatal care, which was clearly underprovided in these patients.

Inactivación de lipoxigenasas e inhibidoeres de tripsina en un proceso de obtención de leche de soja por molienda directa y ultra alta temperatura (MD-UAT) / Lipoxygenases and trypsin inhibitors inactivation in a soymilk processing by direct milling and ultra high temperature (DM-UHT)

La elaboración de leche de soja mediante un nuevo proceso de Molienda Directa de sémola de soja y Ultra Alta Temperatura (MD-UAT), ha sido objeto de estudios de laboatorio en sus etapas de molienda y tratamientos térmicos, a los efectos de evaluar la extracción de sólidos y proteínas, como así también la inactivación de lipoxigenasas e inhibidores de tripsina (IT). Para la determinación de IT en extractos y leche de soja se modificó el método clásico de Kakade (17). Los mayores rendimientos de extracción en la molienda se obtienen a 70 grados Celsius durante 2 minutos. La inactivación de lipoxigenasas es notablemente más eficiente durante la molienda si se utiliza solución de carbonato de sodio 0,01 M en lugar de agua como medio dispersante (actividad residual de 14 por ciento en carbonato frente a 46 por ciento en agua), reduciéndose así la generación de sustancias indeseables. Se completa la destrucción de lipoxigenasas con un breve calentamiento de 30 segundos con vapor desde 70 grados Celsius hasta la temperatura de ebullición a 96 grados Celsius. Por otra parte, los inhibidores de tripsina no son adecuadamente inactivados en la molienda ni tampoco en el tratamiento con vapor a 96 degrees Celsius durante varios minutos. Posteriormente, en la etapa de UAT a 139 grados Celsius durante dos minutos se logra la inactivación de los IT hasta los valores considerados aceptables, posibilitando la destrucción simultánea de microorganismos. Estas condiciones se logran sin dificuldad en el proceso MD-UAT, cuya flexibilidad permite adecuar temperaturas y tiempos a requerimentos específicos.