DESARROLLO DE UNA METODOLOGÍA POR CROMATOGRAFÍA DE EXCLUSIÓN MOLECULAR PARA LA DETERMINACIÓN DE POLIRRIBOSIL-RIBITOL-FOSFATO (PRP) LIBRE Y CONJUGADO A TOXOIDE TETÁNICO (PRP-TTD) EN VACUNAS ANTI HAEMOPHILUS INFLUENZAE TIPO B / Development of a size exclusion chromatography method for a determination of free polyribosyl-ribitol-phosphate (PRP) and conjugated to tetanus toxoid (PRP-TTD) in Haemophilus influenzae type B vaccines

Haemophilus influenzae tipo b es un patógeno oportunista responsable de infecciones invasivas y graves como bacteriemia y meningitis bacteriana aguda en poblaciones susceptibles, principalmente en niños menores de 5 años de edad. Su antígeno capsular, el polirribosil-ribitol-fosfato, es un polisacárido específico del serotipo b y ha sido utilizado para la producción de vacunas, inicialmente en forma aislada, con escaso éxito debido a la pobre estimulación inmune en la población objetivo, dando una protección poco sostenida en el tiempo. Surgieron así las vacunas conjugadas, usadas actualmente, donde el polisacárido capsular se une a una proteína carrier para generar una respuesta inmune timo-dependiente, con memoria inmunológica, y apta para el uso en niños a partir de los 2 meses de edad. El polirribosil-ribitol-fosfato se caracteriza por ser una secuencia polimérica del azúcar fosfatado de largo variable, formando una mezcla con diferente abundancia de pesos moleculares. El largo de las cadenas polisacarídicas es un parámetro de calidad del proceso productivo de estas vacunas, y se controla mediante técnicas de cromatografía de exclusión molecular. El presente trabajo consistió en desarrollar una técnica analítica para la determinación de polirribosil-ribitol-fosfato en su forma libre, y conjugado a toxoide tetánico, presentes en vacunas anti Haemophilus influenzae tipo b, utilizando cromatografía de exclusión molecular acoplada a detectores por índice de refracción y por luz ultravioleta a longitudes de onda de 215 y 280 nm. El desarrollo se planteó sobre antecedentes bibliográficos que describen una metodología similar como control de la instancia de conjugación en el proceso productivo del ingrediente farmacéutico activo de vacunas anti Haemophilus influenzae tipo b. En el procedimiento se utilizaron estándares internacionales de los analitos libres y estándares de pesos moleculares, y se evaluó la capacidad del sistema para discriminar los componentes de la vacuna y detectar diferencias que podrían indicar un compromiso en la calidad de la misma.

Haemophilus influenzae type b is an opportunistic pathogen responsible for invasive and severe infections such as bacteremia and acute bacterial meningitis in susceptible populations, mainly children under 5 years of age. Its capsular antigen, polyribosylribitol-phosphate, is a specific polysaccharide of serotype b and has been used to produce vaccines, initially in isolation, with little success due to poor immune stimulation in the target population, and providing little sustained protection over time. Thus, arose the conjugated vaccines currently used, where the capsular polysaccharide binds to a carrier protein to generate a thymusdependent immune response with immunological memory and suitable for use in children from 2 months of age. Polyribosyl-ribitolphosphate is characterized by being a polymeric sequence of the phosphated sugar of variable length, forming a mixture with Emilia Ojeda Zachara*, Gisela Severino*, Ana Carolina Abba*, Patricia Aprea. Departamento del Laboratorio Nacional para el Estudio y Control de Biológicos, Dirección de Evaluación y Control de Biológicos y Radiofármacos, Instituto Nacional de Medicamentos. Administración Nacional de Medicamentos, Alimentos y Tecnología Médica, Buenos Aires. Argentina. Correspondencia: Emilia Ojeda Zachara (emilia.ojeda@anmat.gob.ar) *Estos autores contribuyeron equitativamente al desarrollo de este trabajo. Recibido: 26 de julio de 2021. Aprobado: 1 de diciembre de 2021. Revista Científica ANMAT. Año 5 (vol 2), 2021 different abundances of various molecular weights. The length of the polysaccharide chains is a quality parameter of the production process of these vaccines, and is controlled by size-exclusion chromatography techniques. The presented work consisted in developing an analytical technique for the determination of polyribosyl-ribitol-phosphate in its free form and conjugated to tetanus toxoid present in vaccines against Haemophilus influenzae type b, using size-exclusion chromatography coupled to refractive index and UV detectors at wavelengths of 215 and 280 nm. The development was based on bibliographic backgrounds that use a similar methodology as a control of the conjugation step in the production process of the active pharmaceutical ingredient of anti-Haemophilus influenzae type b vaccines. In the procedure, international standards for free analytes and molecular weight standards were used, and the ability of the system to discriminate the vaccine components and to detect differences that could indicate a compromise in the quality of this vaccine was evaluated

Cognitive impairment in neuromuscular diseases: A systematic review.

Neuromuscular diseases are multifactorial pathologies characterized by extensive muscle fiber damage that leads to the activation of satellite cells and to the exhaustion of their pool, with consequent impairment of neurobiological aspects, such as cognition and motor control. To review the knowledge and obtain a broad view of the cognitive impairment on Neuromuscular Diseases. Cognitive impairment in neuromuscular disease was explored; a literature search up to October 2017 was conducted, including experimental studies, case reports and reviews written in English. Keywords included Cognitive Impairment, Neuromuscular Diseases, Motor Neuron Diseases, Dystrophinopathies and Mitochondrial Disorders. Several cognitive evaluation scales, neuroimaging scans, genetic analysis and laboratory applications in neuromuscular diseases, especially when it comes to the Motor Neuron Diseases, Dystrophinopathies and Mitochondrial Disorders. In addition, organisms model using rats in the genetic analysis and laboratory applications to verify the cognitive and neuromuscular impacts. Several studies indicate that congenital molecular alterations in neuromuscular diseases promote cognitive dysfunctions. Understanding these mechanisms may in the future guide the proper management of the patient, evaluation, establishment of prognosis, choice of treatment and development of innovative interventions such as gene therapy.

Morphology of mucosa-associated lymphoid tissue in odontocetes.

This study describes the mucosa-associated lymphoid tissue (MALT) in odontocetes from the Brazilian coast and freshwater systems. Seven species were evaluated and tissue samples were analyzed by light, scanning and transmission electron microscopy, and immunohistochemistry. Laryngeal tonsil was a palpable oval mass located in the larynx, composed of a lymphoepithelial complex. Dense collections of lymphocytes were found in the skin of male fetus and calf. Clusters of lymphoid tissue were found in the uterine cervix of a reproductively active juvenile female and along the pulmonary artery of an adult female. Lymphoid tissues associated with the gastrointestinal tract were characterized by diffusely arranged or organized lymphocytes. The anal tonsil was composed of an aggregate of lymphoid tissue occurring exclusively in the anal canal, being composed of squamous epithelium branches. MALT was present in different tissues and organic systems of cetaceans, providing constant protection against mucosal pathogens present in their environment.

Culture-independent characterization of novel psychrophilic magnetotactic cocci from Antarctic marine sediments.

Magnetotactic bacteria (MTB) are a heterogeneous group of ubiquitous aquatic microorganisms capable of biomineralizing nano-sized, membrane-bound, magnetic iron-rich mineral particles called magnetosomes. MTB are found in chemically-stratified aquatic sediments and/or water columns with a wide range of salinities, moderate to high temperatures, and pH varying from neutral to strongly alkaline. MTB from very cold environments have not been investigated to any great degree and here we characterize MTB from the low temperature Antarctic maritime region. Sediment samples were collected at nine sampling sites within Admiralty Bay, King George Island (62°23'S 58°27'W) from 2009 to 2013. Samples from five sites contained MTB and those from two of these sites contained large number of magnetotactic cocci that were studied using electron microscopy and molecular techniques. The magnetotactic cocci contained magnetosomes either arranged as two or four chains or as a disorganized cluster. The crystalline habit and composition of all magnetosomes analyzed with high-resolution transmission electron microscopy and energy dispersive X-ray microanalysis were consistent with elongated prismatic crystals of magnetite (Fe3 O4 ). The retrieved 16S rRNA gene sequences from magnetically-enriched magnetotactic cocci clustered into three distinct groups affiliated with the Alphaproteobacteria class of the Proteobacteria. Novel sequences of each phylogenetic cluster were confirmed using fluorescent in situ hybridization. Metagenomic data analysis of magnetically-enriched magnetotactic cocci revealed the presence of mam genes and MTB-specific hypothetical protein coding genes. Sequence homology and phylogenetic analysis indicated that predicted proteins are related to those of cultivated alphaproteobacterial MTB. The consistent and continuous low temperature of the sediment where the magnetotactic cocci are present (always below 1°C) suggests that these MTB from maritime Antarctica are psychrophiles. Moreover, similar morphotypes and 16S gene sequences were retrieved from samples collected from different sites from maritime Antarctica for several years suggesting that these new strains of MTB are indigenous members of Antarctic microbiota.

Influence of copigment derived from Tasmannia pepper leaf on Davidson's plum anthocyanins.

UNLABELLED: Davidson's plum (Davidsonia pruriens, F. Muell.), a native to Australian rainforests, large, crimson-red fruit, which superficially resembles plum, has been commercially cultivated in Australia since 1990s. The current production volume exceeds market demands therefore this study was designed to evaluate the suitability of Davidson's plum extract as a source of anthocyanin-based food colorant. The stability of the Davidson's plum extract towards heat treatment at 95 °C was higher than that of commercial mulberry colorant, but inferior to colorants derived from red cabbage and purple sweetpotato. An addition of a variety of phenolic acids significantly increased color intensity indicating the formation of copigmentation complexes. Commercial chlorogenic acid as well as extract from a native Australian herb rich in chlorogenic acid, Tasmannia pepper leaf (Tasmannia lanceolata, R. Br.), were both tested in model soft drink solutions subjected to light irradiation and heat treatment. In both cases, the addition of the copigment resulted in a lasting increase in color intensity. In conclusion, Davidson's plum extract can successfully be utilized as a source of natural food color. Extract from Tasmania pepper leaf can be used as a co-pigment for Davidson's plum anthocyanins. PRACTICAL APPLICATION: The color properties of an anthocyanin colorant derived from the native Australian fruit Davidson's plum are comparable to those of mulberry, which is currently applied as a food colorant in Australian food products. Utilization of Davidson's plum fruit as a source of natural color will allow the industry to increase the range of natural pigments and will create new opportunities for the emerging native food industry.

Comparison of apolipoprotein-B/apolipoprotein-AI in subjects with versus without the metabolic syndrome.

Recent studies have suggested that the apolipoprotein-B (apo-B)/apolipoprotein-AI (apo-AI) ratio predicts cardiovascular risk better than any of the cholesterol indexes. The aim of the present study was to assess if the apo-B/apo-AI ratio is related to the metabolic syndrome and its components. Data were analyzed from 2,964 subjects (mean age 48 years; 1,516 men, 1,448 women) from the National Health and Nutrition Examination Survey III with apolipoprotein data who were evaluated for the metabolic syndrome and its components. The metabolic syndrome was defined according to the criteria of the National Cholesterol Education Program Adult Treatment Panel III and the International Diabetes Federation. The mean values of the apo-B/apo-AI ratio in subjects with and without the metabolic syndrome were compared. Overall, the median distribution of the apo-B/apo-AI ratio was significantly greater (p <0.0001) in subjects with the Adult Treatment Panel III metabolic syndrome (0.90) than without (0.69). The apo-B/apo-AI ratio was associated significantly with each of the metabolic syndrome components, in descending order of magnitude: low high-density lipoprotein cholesterol (odds ratio [OR] 5.7), high triglycerides (OR 4.7), high waist circumference (OR 2.6), high fasting glucose (OR 1.9), and high blood pressure (OR 1.5). The apo-B/apo-AI ratio was also different between subjects with and without the metabolic syndrome. Mean values of apo-B/apo-AI increased significantly as the numbers of metabolic syndrome components increased in men (p <0.0001) and women (p <0.0001). After excluding high-density lipoprotein cholesterol and triglycerides as criteria for the metabolic syndrome, the association between means persisted (analysis of variance p <0.0001) in men and women. Apo-B/apo-AI was significantly associated with the presence of the metabolic syndrome (OR 5.1, p <0.0001). In conclusion, the apo-B/apo-AI ratio is strongly associated with the presence of individual metabolic syndrome components, with the metabolic syndrome itself, and with insulin resistance. An elevated apo-B/apo-AI ratio may constitute an important feature of the metabolic syndrome and may provide an additional mechanism to explain the increased cardiovascular risk in subjects with this syndrome.

Repercussoes respiratorias da aplicaçao da tecnica de isostretching em individuos sadios / Resporatory repercussions of isostrecching technique application in healthy individuals

O objetivo deste estudo foi avaliar o impacto da aplicaçao do isostretching, um exercicio postural global, sobre o comportamento resparatorio toraco-abdominal numa populaçao sadia. A tecnica foi aplicada em seis jovens sedentarias, com idade media de 22,33+ ou -2,42 anos, previamente submetidas a uma avaliaçao respiratoria pontuada e a analise angular fotogrametrica da inspiraçao e expiraçao maxima na postura ortostatica e caracterizaçao do padrao muscular ventilatorio utilizado. O isostretching foi aplicado em sessoes de 45 minutos, durante 10 dias, perfazendo duas semanas. Apos a plicaçao da tecnica, a avaliaçao foi repetida e os resultados comparados. Houve um aumento de 0,9 por cento e 0,73 por cento respectivamente, para a pontuaçao registrada para exame fisico e avaliaçao funcional. A analise angular dos valores encontrados nos registros pos-aplicaçao da tecnica mostrou uma maior contribuiçao do comportamento obtido no registros pre-intervençao. Os resultados sao compativeis com a melhora da atuaçao diafragmatica durante a mibilizaçao de medios a altos volumes respiratorios, e sugerem que o isostretching pode promover impacto efetivo e mensuravel sobre a funçao respiratoria de sue praticantes, mesmo em regimes de aplicaçao diferenciados daqueles estabelecidos pelos seus precursores.