Antibody-ligand interactions on a high-capacity staphylococcal protein A resin.

Staphylococcal protein-A affinity chromatography has been optimized for antibody purification, achieving a current capacity of up to 90 mg/ml in packed bed. The morphology of the particles, the number of antibodies bound per ligand and the spatial arrangement of the ligands were assessed by in-situ Small-angle X-ray scattering (SAXS) and scanning electron microscopy (SEM) combined with measurement of adsorption isotherms. We employed SAXS measurements to probe the nanoscale structure of the chromatographic resin. From scanning electron microcopy, the morphology and area of the beads were obtained. The adsorption isotherm revealed a bi-Langmuirian behavior where the association constant varied with the critical bulk concentration, indicating multilayer adsorption. Determining the antibody-ligand stoichiometry was crucial for understanding the adsorption mechanism, which was estimated to be 4 at lower concentrations and 4.5 at higher concentrations, suggestive of reversible protein-protein interactions. The same results were reached from the in-situ small angle X-ray scattering measurements. A stoichiometry of 6 cannot be achieved since the two protein A monomers are anchored to the stationary phase and thus sterically hindered. Normalization through ellipsoids facilitated SAXS analysis, enabling the determination of distances between ligands and antibody-ligand complexes. Density fluctuations were examined by subtracting the elliptical fit, providing insights into ligand density distribution. The dense ligand packing of TOYOPEARL® AF-rProtein A HC was confirmed, making further increases in ligand density impractical. Additionally, SAXS analysis revealed structural rearrangements of the antibody-ligand complex with increasing antibody surface load, suggesting reversible association of antibodies.

Erythrocytes' surface properties and stiffness predict survival and functional decline in ALS patients.

Erythrocytes play a fundamental role in oxygen delivery to tissues and binding to inflammatory mediators. Evidences suggest that dysregulated erythrocyte function could contribute to the pathophysiology of several neurodegenerative diseases. We aimed to evaluate changes in morphological, biomechanical, and biophysical properties of erythrocytes from amyotrophic lateral sclerosis (ALS) patients, as new areas of study in this disease. Blood samples were collected from ALS patients, comparing with healthy volunteers. Erythrocytes were assessed using atomic force microscopy (AFM) and zeta potential analysis. The patients' motor and respiratory functions were evaluated using the revised ALS Functional Rating Scale (ALSFRS-R) and percentage of forced vital capacity (%FVC). Patient survival was also assessed. Erythrocyte surface roughness was significantly smoother in ALS patients, and this parameter was a predictor of faster decline in ALSFRS-R scores. ALS patients exhibited higher erythrocyte stiffness, as indicated by reduced AFM tip penetration depth, which predicted a faster ALSFRS-R score and respiratory subscore decay. A lower negative charge on the erythrocyte membrane was predictor of a faster ALSFRS-R and FVC decline. Additionally, a larger erythrocyte surface area was an independent predictor of lower survival. These changes in morphological and biophysical membrane properties of ALS patients' erythrocytes, lead to increased cell stiffness and morphological variations. We speculate that these changes might precipitate motoneurons dysfunction and accelerate disease progression. Further studies should explore the molecular alterations related to these observations. Our findings may contribute to dissect the complex interplay between respiratory function, tissue hypoxia, progression rate, and survival in ALS.

Invasive Pulmonary Aspergillosis after COVID-19 Pneumonia.

COVID-19 emerged in China in late 2019 and quickly spread worldwide. The severe immunomodulation and depletion of lymphocytes caused by the virus and its therapy led to an increase in the incidence of superinfections. COVID-19-associated pulmonary aspergillosis (CAPA) is a new entity with increasing incidence and high associated mortality. We present the case of a 68-year-old patient admitted to our ward after recovering from severe COVID-19 pneumonia. Due to worsening of her clinical condition, chest computed tomography was performed and a lung abscess was documented with the identification of Aspergillus niger. Despite therapy with voriconazole, the patient's condition deteriorated, culminating in her death. LEARNING POINTS: COVID-19-associated pulmonary aspergillosis (CAPA) is a new entity with an increasing incidence.It is a serious and life-threatening complication in patients with severe COVID-19 even in the absence of the classic risk factors for invasive pulmonary aspergillosis.Clinical suspicion is crucial since a timely diagnosis and treatment have a major impact on prognosis.

Antibody adsorption in protein-A affinity chromatography - in situ measurement of nanoscale structure by small-angle X-ray scattering.

Protein-A chromatography is the most widely used chromatography step in downstream processing of antibodies. A deeper understanding of the influence of the surface topology on a molecular/nanoscale level on adsorption is essential for further improvement. It is not clear if the binding is homogenous throughout the entire bead network. We followed the protein absorption process and observed the formation of a protein layer on fibers of chromatography resin in a time-resolved manner in nanoscale. To characterize the changes in the antibody-protein-A ligand complex, small angle X-ray scattering was employed using a miniaturized X-ray-transparent chromatography column packed with a MabSelect SuRe resin. Antibody-free MabSelect SuRe resin fiber had an average radius of 12 nm and the protein layer thickness resulting from antibody adsorption was 5.5 and 10.4 nm for fiber and junctions, respectively under applied native conditions. We hypothesize that an average of 1.2 antibodies were adsorbed per protein-A ligand tetramer bound to the outermost units. In contrast to previous studies, it was therefore possible for the first time to directly correlate the nanostructure changes inside the column, which is otherwise a black box, with the adsorption and elution process.

From inulin to fructose syrups using sol-gel immobilized inulinase.

The present work aims to provide the basic characterization of sol-gel immobilized inulinase, a biocatalyst configuration yet unexploited, using as model system the hydrolysis of inulin to fructose. Porous xerogel particles with dimensions in slight excess of 10 µm were obtained, yielding an immobilization efficiency of roughly 80%. The temperature- and pH-activity profiles displayed a broader bell-shaped pattern as a result of immobilization. In the latter case, a shift of the optimal pH of 0.5 pH units was observed towards a less acidic environment. The kinetic parameters estimated from the typical Michaelis-Menten kinetics suggest that immobilization in sol-gel did not tamper with the native enzyme conformation, but on the other hand, entrapment brought along mass transfer limitations. The sol-gel biocatalyst displayed a promising operational stability, since it was used in more than 20 consecutive 24-hour batch runs without noticeable decay in product yield. The performance of sol-gel biocatalyst particles doped with magnetite roughly matched the performance of simple sol-gel particles in a single batch run. However, the operational stability of the former proved poorer, since activity decay was evident after four consecutive 24-hour batch runs.

[The persistence of the left vena cava superior and the absence of the right vena cava superior: the implications in the implantation of a definitive pacemaker]. / Persistência da veia cava superior esquerda e ausência da veia cava superior direita: implicações na implantação de pacemaker definitivo.

Persistent left superior vena cava and associated absence of the right superior vena cava is exceedingly rare. The authors present a case, diagnosed when a DDD pacemaker system was being implanted, and point out its rarity, possibility of diagnosis by non invasive methods and technical implications in transvenous electrode implantation.

[Echocardiographic evaluation of the aortic and pulmonary valve areas in the newborn infant. Normal patterns]. / Avaliação ecocardiográfica das áreas valvulares aórtica e pulmonar do recém-nascido. Padrões de normalidade.

OBJECTIVES: To determine normal values of the aortic and pulmonary functional valve areas in healthy newborn children. MATERIAL AND METHODS: We prospectively studied 32 newborns (17 boys) who were included in the following criteria: healthy parents, normal pregnancy, eutocic delivery, Apgar index 10 at 5 minutes weight between 2.500 and 4.000 kg, normal physical and echocardiographical examinations. The echocardiographical examination was executed during the first 48 hours of life. We used the continuity equation to calculate the aortic and pulmonary functional valve areas. As a reference we used the anatomic (pi r2) aortic and pulmonary valve areas, calculated in the bidimensional images from the distance measured between the two insertion points of the sigmoid valves. RESULTS: We had echocardiographic images and Doppler registrations of excellent quality, in all the newborn children. The functional pulmonary valve area ranged between 0.30 and 0.50 cm2 (mean +/- SD = 0.41 +/- 0.06) and the anatomical one ranged between 0.29 and 0.49 cm2 (mean +/- SD = 0.49 +/- 0.05), without statistical significance and with a correlation index 0.92. The functional aortic valve area ranged between 0.20 and 0.40 cm2 (mean +/- SD = 0.31 +/- 0.05) and the anatomical ranged between 0.21 and 0.36 cm2 (mean +/- SD = 0.29 +/- 0.05), without statistical significance and with a correlation index 0.91. CONCLUSION: There is a good correlation between the functional aortic and pulmonary valve areas, calculated from the continuity equation, and the anatomical ones. These values will be useful in characterizing the critical stenosis of the newborn child with decreased ventricular function, where the transvalvular gradient is inaccurate in the quantification of the obstruction.

[Atrial myxoma. Apropos a clinical case]. / Mixoma auricular. A propósito de um caso clínico.

The authors report a case of a 61-years-old woman with hypertension and palpitations, who was diagnosed as having a left auricular myxoma. This diagnosis was established by echocardiography and confirmed afterwards by an histological survey. On the basis of this case the authors reviewed the subject and discuss the role of echocardiography in the diagnosis and follow-up of this kind of patients.

[Cardiac changes in a case of mitochondrial encephalomyopathy]. / Alterações cardíacas num caso de encefalomiopatia mitocondrial.

In the present article the authors describe a case of a twenty-one year-old patient with a mitochondrial encephalomyopathy presenting cardiac changes. Mitochondrial encephalomyopathy a multisystemic disease is described in various publications having cardiac involvement although there are sparse references about cardiomyopathy in this same disease. In the present case, the patient had a syndrome of both Mitochondrial Encephalopathy, Lactic Acidosis and Stroke like episodes (MELAS) and Myoclonic Epilepsy and Ragged Red Fibers (MERRF). There were no significant changes in the cardiac conductivity studied by ECG but an echocardiographic pattern of hypertrophic cardiomyopathy was found.