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BACKGROUND: Fed-batch mode is the standard culture technology for industrial bioprocesses. Nevertheless, most of the early-stage cell and process development is carried out in batch cultures, which can bias the initial selection of expression systems. Cell engineering can provide an alternative to fed-batch cultures for high-throughput screening and host selection. We have previously reported a library of Escherichia coli strains with single and multiple deletions of genes involved in glucose transport. Compared to their wild type (W3110), the mutant strains displayed lower glucose uptake, growth and aerobic acetate production rates. Therefore, when cultured in batch mode, such mutants may perform similar to W3110 cultured in fed-batch mode. To test that hypothesis, we evaluated the constitutive expression of the green fluorescence protein (GFP) in batch cultures in microbioreactors using a semi defined medium supplemented with 10 or 20 g/L glucose + 0.4 g yeast extract/g glucose. RESULTS: The mutant strains cultured in batch mode displayed a fast-growth phase (growth rate between 0.40 and 0.60 h-1) followed by a slow-growth phase (growth rate between 0.05 and 0.15 h-1), similar to typical fed-batch cultures. The phase of slow growth is most probably caused by depletion of key amino acids. Three mutants attained the highest GFP fluorescence. Particularly, a mutant named WHIC (ΔptsHIcrr, ΔmglABC), reached a GFP fluorescence up to 14-fold greater than that of W3110. Strain WHIC was cultured in 2 L bioreactors in batch mode with 100 g/L glucose + 50 g/L yeast extract. These cultures were compared with exponentially fed-batch cultures of W3110 maintaining the same slow-growth of WHIC (0.05 h-1) and using the same total amount of glucose and yeast extract than in WHIC cultures. The WHIC strain produced approx. 450 mg/L GFP, while W3110 only 220 mg/L. CONCLUSION: The combination of cell engineering and high throughput screening allowed the selection of a particular mutant that mimics fed-batch behavior in batch cultures. Moreover, the amount of GFP produced by the strain WHIC was substantially higher than that of W3110 under both, batch and fed-batch schemes. Therefore, our results represent a valuable technology for accelerated bioprocess development.

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N. Mirlean, L. Calliari, and K. Johannesson examined the REE paired La/Eu and Gd/Yb ratios in the region of Patos Lagoon estuary/Brazil and related coastal waters. They concluded that "the mud deposition events at the beach were most likely associated with dredging processes in the estuary especially where overflow is the source of fluid mud on the beach shoreface". In this response to that study, we reflect on several discrepancies in their methods, results and conclusions. In this work, we demonstrate that the approach adopted by the authors does not provide evidence that the selected REE ratios measured on muddy deposits on the beach were derived from dredged activities, particularly regarding overflow. On the contrary, based on their results, the REE ratios presented in the suspended sediments collected in the navigation channel are most likely associated to natural sediments.

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Deposits of fluid mud on wave-dominated beaches are rare. Where they happen frequently and periodically, they can be associated with anthropic processes that bring fluid mud to the shoreface and surf zone. To determine the reason for the frequent deposition of fluid mud at Cassino Beach (Southern Brazil) in the 2014-2020 period, the La/Eu and Gd/Yb ratios in marine, beach, and adjacent Patos estuary sediments were investigated. Mud from the beach showed similar ratios of REE to dredged sediments from the harbor basin and navigation channel within the estuary. Industrial and port activity changes REE ratio in sediments of the navigation channel, and they are distinct from naturally deposited marine and estuarine sediments. We conclude that the mud deposition events at the beach are most likely associated with dredging processes in the estuary especially where overflow is the source of fluid mud on the beach shoreface.

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Plasmid DNA (pDNA) vaccines require high supercoiled-pDNA doses (milligrams) to achieve an adequate immune response. Therefore, processes development to obtain high pDNA yields and productivity is crucial. pDNA production is affected by several factors including culture type, medium composition, and growth conditions. We evaluated the effect of kanamycin concentration and temperature on pDNA production, overflow metabolism (organic acids) and metabolic burden (neomycin phosphotransferase II) in batch and fed-batch cultures of Escherichia coli DH5α-pVAX1-NH36. Results indicated that high kanamycin concentration increases the volumetric productivity, volumetric and specific yields of pDNA when batch cultures were carried out at 42 °C, and overflow metabolism reduced but metabolic burden increased. Micrographs taken with a scanning electron microscope (SEM) were analyzed, showing important morphological changes. The high kanamycin concentration (300 mg/L) was evaluated in high cell density culture (50 gDCW/L), which was reached using a fed-batch culture with temperature increase by controlling heating and growth rates. The pDNA volumetric yield and productivity were 759 mg/L and 31.19 mg/L/h, respectively, two-fold greater than the control with a kanamycin concentration of 50 mg/L. A stress-based process simultaneously caused by temperature and high kanamycin concentration can be successfully applied to increase pDNA production.

RESUMO

The overflow of scientific literature stimulates poor reading habits which can aggravate science's reproducibility crisis. Thus, solving the reproducibility crisis demands not only methodological changes, but also changes in our relationship with the scientific literature, especially our reading habits. Importantly, this does not mean reading more, it means reading better.

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The purpose of this study was to evaluate the response of the tibialis anterior muscle (TAm) using surface electromyography in patients with Charcot-Marie-Tooth disease (CMT-IA), after ipsilateral proprioceptive neuromuscular patterns (PNF). Thirteen CMT-IA patients (both sexes) were treated twice a week, for 5 weeks, with bilateral PNF pattern, four times per treatment. During the execution of the patterns, we recorded the bilateral activation of the TAm in root mean square (RMS). We used the Student paired t-test for the first and last treatments, P-value set at <0.05. Clinical significance (CS) was obtained by subtracting the values of the first treatment from the last. Chopping pattern to the right side increased RMS for the right (t=-3.52, CS=52%), but not the left TAm (t= -3.35). Flexion-abduction with external rotation pattern to the right (t= -2.46, CS=55%) and left (t=-2.07, CS=53%) significantly increased RMS for TAm on both sides. Extension-adduction with internal rotation pattern to the right (t=-0.25) and left (t=-1.84) did not produce any changes in TAm. Ipsilateral PNF patterns selectively produce TAm overflow in peripheral polyneuropathy patients and can be successfully used as supportive therapy for foot drop.

RESUMO

La destruccion del músculo esquelético en la condición patológica conocida como rabdomiolisis resulta en la liberación al torrente sanguíneo de elevadas concentraciones de la proteína mioglobina de 17 kDa, la cual filtra libremente a través del glomérulo sobrepasando frecuentemente la capacidad de reabsorción del túbulo proximal. Por lo tanto, la identificación de mioglobina en orina es una herramienta esencial que complementa otros parámetros bioquímicos en el diagnóstico de la enfermedad. En el presente trabajo, mediante la combinación de electroforesis en geles de agarosa e inmunofijación empleando anticuerpos específicos, se provee evidencia directa de la presencia de mioglobina intacta en la orina de un paciente con insuficiencia renal aguda asociada a rabdomiolisis desencadenada por efecto secundario de una terapia reductora de lípidos. Los datos electroforéticos e inmunoquímicos fueron corroborados mediante secuencia N-terminal de aminoácidos, immunoblot y espectrometría de masa. La simple combinación de electroforesis e inmunofijación provee una estrategia flexible que puede extenderse a la identificación de diversas proteínas involucradas en proteinurias de sobrecarga.(AU)

The destruction of skeletal muscle in a condition known as rhabdomyolysis results in the release into the blood stream of large quantities of the 17 kDa myoglobin that freely filtrates through the glomeruli, often overwhelming the capacity of the proximal tubule for reabsorption. As a result, identification of myoglobin in the urine becomes an essential tool to fully complement other biochemical parameters in the diagnosis of the disease. Using a combination of protein electrophoresis in agarose gels and immunofixation with specific antibodies, direct evidence is provided for the presence of intact myoglobin in the urine of a patient with acute kidney injury due to rhabdomyolysis triggered by undesirable side effects of specific medications. The electrophoretic/immunochemical data was further corroborated by amino acid sequence, Western blot, and mass spectrometry analysis. The simple combination of electrophoresis and immunofixation protocols provides a flexible approach that can be extended to the identification of the various proteins known to be involved in overflow proteinuria.(AU)

A destruiþÒo do músculo esquelético na condiþÒo patológica conhecida como rabdomiólise resulta na liberaþÒo ao torrente sanguíneo de elevadas concentraþ§es da proteína mioglobina de 17 kDa a qual filtra livremente através do glomérulo ultrapassando frequentemente a capacidade de reabsorþÒo do túbulo proximal. Portanto, a identificaþÒo de mioglobina em urina é uma ferramenta essencial que complementa outros parÔmetros bioquímicos no diagnóstico da doenþa. No presente trabalho, mediante a combinaþÒo de eletroforese em géis de agarose e imunofixaþÒo empregando anticorpos específicos, é fornecida evidÛncia direta da presenþa de mioglobina intacta na urina de um paciente com insuficiÛncia renal aguda associada a rabdomiólise desencadeada por efeito secundário de uma terapia redutora de lipídeos. Os dados eletroforéticos e imunoquímicos foram confirmados mediante sequÛncia N-terminal de aminoácidos, immunoblot e espectrometria de massa. A simples combinaþÒo de eletroforese e imunofixaþÒo fornece uma estratégia flexível que pode se estender O identificaþÒo de diversas proteínas envolvidas em proteinúrias de sobrecarga.(AU)

RESUMO

Hombre de 71 años de edad con diabetes mellitus tipo II de 10 años de evolución, con tabaquismo intenso. Inició con derrame pleural e imagen radiológica de cavitación en lóbulo superior izquierdo.

Male of 71 years with 10 years evolution of diabetes mellitus II, heavy smoker. The case began with pleural overflow and with X ray cavitation in the left upper lobe.