The pre-induction temperature affects recombinant HuGM-CSF aggregation in thermoinducible Escherichia coli.

The overproduction of recombinant proteins in Escherichia coli leads to insoluble aggregates of proteins called inclusion bodies (IBs). IBs are considered dynamic entities that harbor high percentages of the recombinant protein, which can be found in different conformational states. The production conditions influence the properties of IBs and recombinant protein recovery and solubilization. The E. coli growth in thermoinduced systems is generally carried out at 30 °C and then recombinant protein production at 42 °C. Since the heat shock response in E. coli is triggered above 34 °C, the synthesis of heat shock proteins can modify the yields of the recombinant protein and the structural quality of IBs. The objective of this work was to evaluate the effect of different pre-induction temperatures (30 and 34 °C) on the growth of E. coli W3110 producing the human granulocyte-macrophage colony-stimulating factor (rHuGM-CSF) and on the IBs structure in a λpL/pR-cI857 thermoinducible system. The recombinant E. coli cultures growing at 34 °C showed a ~ 69% increase in the specific growth rate compared to cultures grown at 30 °C. The amount of rHuGM-CSF in IBs was significantly higher in cultures grown at 34 °C. Main folding chaperones (DnaK and GroEL) were associated with IBs and their co-chaperones (DnaJ and GroES) with the soluble protein fraction. Finally, IBs from cultures that grew at 34 °C had a lower content of amyloid-like structure and were more sensitive to proteolytic degradation than IBs obtained from cultures at 30 °C. Our study presents evidence that increasing the pre-induction temperature in a thermoinduced system allows obtaining higher recombinant protein and reducing amyloid contents of the IBs. KEY POINTS: • Pre-induction temperature determines inclusion bodies architecture • In pre-induction (above 34 °C), the heat shock response increases recombinant protein production • Inclusion bodies at higher pre-induction temperature show a lower amyloid content.

Threat cues and attentional bias in blood-injection-injury phobia and snake phobia / Señales de amenaza y sesgo atencional en la fobia a la sangre, inyecciones y heridas y en la fobia a las serpientes

The aim of this research was to study the effect of a cue signalling the upcoming of a phobic picture on the electrocortical activity provoked by the disorder-relevant stimulus in in blood-injection-injury (BII) phobia and snake phobia. A sample of 13 BII phobia participants, 12 snake phobia individuals and 14 non-phobic controls underwent an S1-S2 task, where S1 was a word that described the content of a subsequent picture (blood-related, snake and neutral) that appeared 2 seconds later (S2). We obtained the P200 and P300 ERP amplitudes provoked by the pictures. Our results reveal that P200 did not differentiate between picture contents in BII phobia while, in contrast, snake and blood-related pictures provoked the largest responses in snake phobia participants. Both blood-related and snake pictures provoked greater P300 amplitudes than neutral pictures in all the groups. Threat cues reduced the electrocortical reaction of the BII phobia participants, possibly by the elicitation of anticipatory or regulatory responses. These results are indicative of a low automatic, exogenous attention towards the feared stimuli in BII phobia, as revealed by P200, probably related to a lack of attentional bias to the phobic object.(AU)

El objetivo de esta investigación fue estudiar el efecto de una señal que indica la aparición de una imagen fóbica sobre la actividad electrocortical provocada por el estímulo fóbico en personas con fobia a la sangre, inyecciones y heridas (SIH) y en fóbicos a las serpientes. Una muestra de 13 participantes con fobia SIH, 12 con fobia a las serpientes y 14 controles no fóbicos se sometieron a una tarea S1-S2, donde S1 era una palabra que describía el contenido de una imagen posterior (relacionada con sangre, serpientes o neutra) que aparecía 2 segundos después (S2). Obtuvimos las amplitudes de los potenciales evocados P200 y P300 provocados por las imágenes. Nuestros resultados revelaronn que P200 no diferenciaba entre el contenido de las imágenes en la fobia SIH mientras que, por el contrario, las imágenes relacionadas con serpientes y con sangre provocaron las mayores respuestas en los participantes con fobia a las serpientes. Tanto las imágenes relacionadas con sangre como las relacionadas con serpientes provocaron amplitudes de P300 mayores que las imágenes neutras en todos los grupos. Las señales de amenaza redujeron la reacción electrocortical de los fóbicos SIH, posiblemente porque desencadenaron respuestas anticipatorias o reguladoras. Estos resultados indican una baja atención exógena hacia los estímulos temidos en la fobia SIH, como lo revela P200, probablemente relacionada con una pérdida del sesgo atencional hacia su objeto fóbico.(AU)

Thermoinducible expression system for producing recombinant proteins in Escherichia coli: advances and insights.

Recombinant protein (RP) production from Escherichia coli has been extensively studied to find strategies for increasing product yields. The thermoinducible expression system is commonly employed at the industrial level to produce various RPs, which avoids the addition of chemical inducers, thus minimizing contamination risks. Multiple aspects of the molecular origin and biotechnological uses of its regulatory elements (pL/pR promoters and cI857 thermolabile repressor) derived from bacteriophage λ provide knowledge to improve the bioprocesses using this system. Here, we discuss the main aspects of the potential use of the λpL/pR-cI857 thermoinducible system for RP production in E. coli, focusing on the approaches of investigations that have contributed to the advancement of this expression system. Metabolic and physiological changes that occur in the host cells caused by heat stress and RP overproduction are also described. Therefore, the current scenario and the future applications of systems that use heat to induce RP production are discussed to understand the relationship between the activation of the bacterial heat shock response, RP accumulation and its possible aggregation to form inclusion bodies.

Integrative overview of antibodies against SARS-CoV-2 and their possible applications in COVID-19 prophylaxis and treatment.

SARS-CoV-2 is a novel ß-coronavirus that caused the COVID-19 pandemic disease, which spread rapidly, infecting more than 134 million people, and killing almost 2.9 million thus far. Based on the urgent need for therapeutic and prophylactic strategies, the identification and characterization of antibodies has been accelerated, since they have been fundamental in treating other viral diseases. Here, we summarized in an integrative manner the present understanding of the immune response and physiopathology caused by SARS-CoV-2, including the activation of the humoral immune response in SARS-CoV-2 infection and therefore, the synthesis of antibodies. Furthermore, we also discussed about the antibodies that can be generated in COVID-19 convalescent sera and their associated clinical studies, including a detailed characterization of a variety of human antibodies and identification of antibodies from other sources, which have powerful neutralizing capacities. Accordingly, the development of effective treatments to mitigate COVID-19 is expected. Finally, we reviewed the challenges faced in producing potential therapeutic antibodies and nanobodies by cell factories at an industrial level while ensuring their quality, efficacy, and safety.

Recombinant production of ESAT-6 antigen in thermoinducible Escherichia coli: the role of culture scale and temperature on metabolic response, expression of chaperones, and architecture of inclusion bodies.

The heat-inducible expression system has been widely used to produce recombinant proteins in Escherichia coli. However, the rise in temperature affects cell growth, activates the bacterial Heat-Shock Response (HSR), and promotes the formation of insoluble protein aggregates known as inclusion bodies (IBs). In this work, we evaluate the effect of the culture scale (shake flasks and bioreactors) and induction temperature (39 and 42 °C) on the kinetic behavior of thermoinducible recombinant E. coli ATCC 53606 producing rESAT-6 (6-kDa early-secretory antigenic target from Mycobacterium tuberculosis), compared with cultures grown at 30 °C (without induction). Also, the expression of the major E. coli chaperones (DnaK and GroEL) was analyzed. We found that almost twice maximum biomass and rESAT-6 production were obtained in bioreactors (~ 3.29 g/L of biomass and ~ 0.27 g/L of rESAT-6) than in shake flasks (~ 1.41 g/L of biomass and ~ 0.14 g/L of rESAT-6) when induction was carried out at 42 °C, but similar amounts of rESAT-6 were obtained from cultures induced at 39 °C (~ 0.14 g/L). In all thermo-induced conditions, rESAT-6 was trapped in IBs. Furthermore, DnaK was preferably expressed in the soluble fraction, while GroEL was present in IBs. Importantly, IBs formed at 39 °C, in both shake flasks and bioreactors, were more susceptible to degradation by proteinase-K, indicating a lower amyloid content compared to IBs formed at 42 °C. Our work presents evidence that the culture scale and the induction temperature modify the E. coli metabolic response, expression of chaperones, and structure of the IBs during rESAT-6 protein production in a thermoinducible system.

Autonomic reactivity in blood-injection-injury and snake phobia.

OBJECTIVE: This research aimed to study the salivary flow and other autonomic reactions -heart rate (HR) and skin conductance response (SCR)- in blood-injection-injury (BII) phobia and snake phobia participants, under the assumption that exposure to blood-related pictures in BII phobia will provoke an increase in parasympathetic activity that, in turn, will lead to a greater saliva production than other affective contents. METHODS: We selected 18 BII phobia and 14 snake phobia participants along with 22 non-phobia individuals. All participants were exposed to 3 blocks of pictures (12 pictures per block) depicting either mutilations, snakes or neutral, household objects. Saliva samples were taken in the 2-min interval before and after each block. RESULTS: In comparison to other contents, blood-related pictures provoked an increase in salivary flow in BII phobia participants, as well as an increase in the number of SCRs. In the snake phobia group, snake pictures provoked HR acceleration, but the SCRs they elicited did not differ from the SCRs provoked by the blood-related pictures. CONCLUSION: BII phobia individuals react to their phobic object with a series of physiological changes resulting from a sympathetic-parasympathetic co-activation. This is in contrast with other specific phobias (e.g., small animal phobias) that usually show a sympathetically mediated, defensive reactivity when exposed to their disorder-relevant stimuli. These data support the use of therapeutic interventions in BII phobia that may differ in some respect from those used in other specific phobias.

Production of a recombinant phospholipase A2 in Escherichia coli using resonant acoustic mixing that improves oxygen transfer in shake flasks.

BACKGROUND: Shake flasks are widely used during the development of bioprocesses for recombinant proteins. Cultures of recombinant Escherichia coli with orbital mixing (OM) have an oxygen limitation negatively affecting biomass growth and recombinant-protein production. With the aim to improve mixing and aeration in shake flask cultures, we analyzed cultures subjected to OM and the novel resonant acoustic mixing (RAM) by applying acoustic energy to E. coli BL21-Gold (DE3): a producer of recombinant phospholipase A2 (rPLA2) from Micrurus laticollaris snake venom. RESULTS: Comparing OM with RAM (200 rpm vs. 7.5g) at the same initial volumetric oxygen transfer coefficient (kLa ≈ 80 h-1) ~69% less biomass was obtained with OM compared with RAM. We analyzed two more conditions increasing agitation until maximal speed (12.5 and 20g), and ~1.6- and ~1.4-fold greater biomass was obtained as compared with cultures at 7.5g. Moreover, the specific growth rate was statistically similar in all cultures carried out in RAM, but ~1.5-fold higher than that in cultures carried out under OM. Almost half of the glucose was consumed in OM, whereas between 80 and 100% of the glucose was consumed in RAM cultures, doubling biomass per glucose yields. Differential organic acid production was observed, but acetate production was prevented at the maximal RAM (20g). The amount of rPLA2 in both, OM and RAM cultures, represented 38 ± 5% of the insoluble protein. A smaller proportion of α-helices and ß-sheet of purified inclusion bodies (IBs) were appreciated by ATR-FTIR from cultures carried out under OM, than those from RAM. At maximal agitation by RAM, internal E. coli localization patterns of protein aggregation changed, as well as, IBs proteolytic degradation, in conjunction with the formation of small external vesicles, although these changes did not significantly affect the cell survival response. CONCLUSIONS: In moderate-cell-density recombinant E. coli BL21-Gold (DE3) cultures, the agitation increases in RAM (up to the maximum) was not enough to avoid the classical oxygen limitation that happens in OM shake flasks. However, RAM presents a decrease of oxygen limitation, resulting in a favorable effect on biomass growth and volumetric rPLA2 production. While under OM a higher recombinant protein yield was obtained.

Prescripción de nuevos anticoagulantes orales en un área de salud / Prescription of new oral anticoagulants in a health area

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Preattentive processing of feared stimuli in blood-injection-injury fearful subjects.

This research aimed to study the defence responses of blood-injection-injury (BII) fearful subjects elicited by the preattentive processing of their feared objects and by an abrupt acoustic stimulus. We selected 21 BII fearful subjects and 25 non-fearful controls from an initial sample of 128 women, according to their scores on the Fear Survey Scale (damage subscale) and the Mutilation Questionnaire. Subjects were exposed to a burst of white noise to promote a defence response, and to 48 pictures, depicting mutilations, as well as other affective contents, displayed through a backward masking procedure. Heart rate (HR), skin conductance response (SCR) and corrugator supercilii activity were continuously recorded throughout the task. Both groups showed similar SCRs, EMG activity and cardiac defence responses to the acoustic stimulus, though fearful subjects showed greater initial HR deceleration than controls. While BII fearful subjects displayed the usual defence response when exposed to a non-feared threatening stimulus, the preattentive processing of the pictures did not reveal autonomic differences between fearful subjects and controls. Mutilation pictures, however, evoked the greatest EMG activity, but only in the fearful group. These data further extend previous research on conscious perception of blood-related stimuli in BII fearful subjects, by showing a failure to recruit autonomic defence responses when blood-related pictures appear outside of conscious awareness.

Staphylococcus aureus resistentes a meticilina (SARM) positivos para PVL aislados en individuos sanos de Montería-Córdoba / PVL positive methicillin-resistant Staphylococcus aureus (MRSAs) strains isolated from healthy individuals in Montería, Córdoba / Staphylococcus aureus resistente à meticilina (SARM) positivos para PVL isolados em indivíduos saudáveis de "Montería, Córdoba"

Objetivo. Estudiar cepas asociadas a la comunidad de SARM y productoras de PVL en individuos sanos de la ciudad de Montería. Materiales y métodos. Estudio descriptivo, prospectivo de corte transversal, a partir de un total de 253 muestras obtenidas de hisopados faríngeos en tres comunidades: 91 internos de la cárcel de Montería (19-58 años), 100 estudiantes adultos jóvenes de la Universidad de Córdoba (17-30 años) y 62 niños en edad escolar (4-9 años) del Colegio de la Universidad de Córdoba. Los individuos participantes no estuvieron hospitalizados en los últimos meses, ni recibieron tratamiento antimicrobiano y no presentaron signos y/o síntomas clínicos. Los aislamientos fueron identificados por pruebas convencionales microbiológicas y se determinó la susceptibilidad antimicrobiana para los diferentes antibióticos a través de MicroScan™ combo 1A (SIEMENS). Para la detección de los genes nuc, mecA y PVL se utilizaron los protocolos de Brakstad et al (5), Oliveira et al (6) y Gerard et al (7) respectivamente. Resultados. De las 253 muestras analizadas, 62 (24,5%) resultaron positivas para S. aureus, de éstas 4 (6,45%) fueron resistentes a meticilina, de las cuales 2 (25%) resultaron PVL positivas; 58 (93,54%) fueron sensibles y 6 (75%) positivas para PVL. Conclusiones. Los resultados obtenidos demuestran la colonización por SARM-AC en individuos sanos, la colonización fue mayor en la población infantil y adultos jóvenes.

Objective. To study SARM and PVL producing strains in healthy individuals of Montería city. Materials and methods. This was a descriptive, prospective and transversal study. A total of 253 samples from pharyngeal swabs in three communities were studied: 91 prisoners of the jail of Montería (19-58 years old), 100 young adult students of the University of Cordoba (17-30 years old) and 62 children in school age (4-9 years old) of the primary school of the University of Cordoba. Individuals analyzed had not been hospitalized, had not received any antimicrobial treatment and did not show any clinical signs or symptoms in the last months. The isolates were identified by conventional microbiological tests and the antimicrobial susceptibility was determined for different antibiotics with MicroScan™ kit 1A (SIEMENS). For the detection of the nuc, mecA and PVL genes, we followed the protocols of Brakstad et al., Oliveira et al. and Gerard et al. respectively. Results. Of the 253 analyzed samples, 62 (24.5%) were positive for S. aureus, out of those 4 (6.45%) were resistant to methicillin and 2 (25%) of them were PVL positive; 58 (93.54%) were sensitive to methicillin and 6 (75%) positive for PVL. Conclusions. Results obtained in this study show the colonization by SARM-AC of healthy individuals. Colonization was higher in young adults and children in school-age.

Objetivo. Estudar cepas associada à comunidade de SARM e produtoras de PVL em indivíduos saudáveis da cidade de Monteria. Materiais e métodos. Estudo descriptivo, prospectivo de corte transversal, de um total de 253 amostras obtidas em esfregaços faríngeos em três comunidades: 91 reclusos em Monteria (19-58 anos), 100 alunos adultos jovens da "Universidad de Córdoba" (17 - 30 anos) e 62 crianças em idade escolar (4-9 anos) do "Colégio de la Universidad de Córdoba". Os indivíduos envolvidos não foram internados nos últimos meses, ou receberam tratamento antimicrobiano e não tinham sinais e / ou sintomas clínicos. Os isolados foram identificados por testes microbiológicos convencionais e se determinou a susceptibilidade antimicrobiana aos diferentes antibióticos através de MicroScan™ combo 1A (SIEMENS). Para a detecção de genes nuc, mecA e LPV foram utilizados protocolos Brakstad et al (5), Oliveira et al (6) e Gerard et al (7), respectivamente. Resultados. De 253 amostras testadas, 62 (24,5%) foram positivas para S. aureus, destas 4(6,45%) foram resistentes à meticilina, dois das quais (25%) foram LPV positivas, 58 (93,54%) foram sensíveis e seis (75%) foram positivas para PVL. Conclusões. Os resultados demonstram a colonização de SARM-AC em indivíduos saudáveis, a colonização foi maior em crianças e adultos jovens.