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Objetivo: Conocer sobre la auditoría y su influencia en el control de las actividades realizadas por las empresas. Método: El enfoque del estudio fue cuantitativo, el tipo de investigación descriptiva, correlacional y exploratoria; el diseño transeccional no experimental. Se realizó una investigación de campo, con visitas a las empresas, empleando encuestas y conociendo su realidad; a su vez se recurrió a la investigación histórica, con la cual se obtuvo información auténtica de las empresas y de la utilización de auditorías a nivel empresarial. Resultados: Las auditorías influyen en el control de las actividades que realizan las empresas, ya que pueden ayudar a las entidades a mejorar su funcionalidad y alcanzar significativos ahorros en los costos. Conclusiones: Se logró determinar la situación actual de las empresas en estudio, conociendo sus fortalezas y debilidades, lo cual permitió conocer su operatividad, con el propósito de verificar la veracidad de las auditorías y justificar el uso de sus recursos de manera eficiente constatando la importancia de llevar a cabo este tipo de control minucioso y exhaustivo cada determinado tiempo en las organizaciones.

The effect of gradients of elevated glucose and low dissolved oxygen in the addition zone of fed-batch E. coli thermoinduced recombinant high cell density cultures can be evaluated through two-compartment scale-down models. Here, glucose was fed in the inlet of a plug flow bioreactor (PFB) connected to a stirred tank bioreactor (STB). E. coli cells diminished growth from 48.2 ± 2.2 g/L in the stage of RP production if compared to control (STB) with STB-PFB experiments, when residence time inside the PFB was 25 s (34.1 ± 3.5 g/L) and 40 s (25.6 ± 5.1 g/L), respectively. The recombinant granulocyte-macrophage colony-stimulating factor (rHuGM-CSF) production decreased from 34 ± 7% of RP in inclusion bodies (IB) in control cultures to 21 ± 8%, and 7 ± 4% during the thermoinduction production phase when increasing residence time inside the PFB to 25 s and 40 s, respectively. This, along with the accumulation of acetic and formic acid (up to 4 g/L), indicates metabolic redirection of central carbon routes through metabolic flow and mixed acid fermentation. Special care must be taken when producing a recombinant protein in heat-induced E. coli , because the yield and productivity of the protein decreases as the size of the bioreactors increases, especially if they are carried at high cell density.

This paper introduces a new variable structure controller designed for depth control of an autonomous underwater sensor platform equipped with a variable buoyancy module. To that end, the prototype linear model is presented, and a finite element-based method is used to estimate one of its parameters, the hull deformation due to pressure. To manage potential internal disturbances like hull deformation or external disturbances like weight changes, a disturbance observer is developed. An analysis of the observer steady-state estimation error in relation to input disturbances and system parameter uncertainties is developed. The locations of the observer poles according to its parameters are also identified. The variable structure controller is developed, keeping energy savings in mind. The proposed controller engages when system dynamics are unfavorable, causing the vehicle to deviate from the desired reference, and disengages when dynamics are favorable, guiding the vehicle toward the target reference. A detailed analysis determines the necessary switching control actions to ensure the system reaches the desired reference. Finally, simulations are run to compare the proposed controller's performance with that of PID-based controllers recently developed in the literature, assessing dynamic response and energy consumption under various operating conditions. Both the VBM- and propeller-actuated vehicles were evaluated. The results demonstrate that the proposed controller achieves an average energy consumption reduction of 22% compared to the next most efficient PID-based controller for the VBM-actuated vehicle, though with some impact on control performance.

Underwater long-endurance platforms are crucial for continuous oceanic observation, allowing for sustained data collection from a multitude of sensors deployed across diverse underwater environments. They extend mission durations, reduce maintenance needs, and significantly improve the efficiency and cost-effectiveness of oceanographic research endeavors. This paper investigates the closed-loop depth control of actuation systems employed in underwater vehicles, focusing on the energy consumption of two different mechanisms: variable buoyancy and propeller actuated devices. Using a prototype previously developed by the authors, this paper presents a detailed model of the vehicle using both actuation solutions. The proposed model, although being a linear-based one, accounts for several nonlinearities that are present such as saturations, sensor quantization, and the actuator brake model. Also, it allows a simple estimation of the energy consumption of both actuation solutions. Based on the developed models, this study then explores the intricate interplay between energy consumption and control accuracy. To this end, several PID-based controllers are developed and tested in simulation. These controllers are used to evaluate the dynamic response and power requirements of variable buoyancy systems and propeller actuated devices under various operational conditions. Our findings contribute to the optimization of closed-loop depth control strategies, offering insights into the trade-offs between energy efficiency and system effectiveness in diverse underwater applications.

Bacterial extracellular vesicles (BEVs) are non-replicative nanostructures released by Gram-negative and Gram-positive bacteria as a survival mechanism and inter- and intraspecific communication mechanism. Due to BEVs physical, biochemical, and biofunctional characteristics, there is interest in producing and using them in developing new therapeutics, vaccines, or delivery systems. However, BEV release is typically low, limiting their application. Here, we provide a biotechnological perspective to enhance BEV production, highlighting current strategies. The strategies include the production of hypervesiculating strains through gene modification, bacteria culture under stress conditions, and artificial vesicles production. We discussed the effect of these production strategies on BEVs types, morphology, composition, and activity. Furthermore, we summarized general aspects of BEV biogenesis, functional capabilities, and applications, framing their current importance and the need to produce them in abundance. This review will expand the knowledge about the range of strategies associated with BEV bioprocesses to increase their productivity and extend their application possibilities., (© 2024. The Author(s).)

Microsporum canis is a widely distributed dermatophyte, which is among the main etiological agents of dermatophytosis in humans and domestic animals. This fungus invades, colonizes and nourishes itself on the keratinized tissues of the host through various virulence factors. This review will bring together the known information about the mechanisms, enzymes and their associated genes relevant to the pathogenesis processes of the fungus and will provide an overview of those virulence factors that should be better studied to establish effective methods of prevention and control of the disease. Public databases using the MeSH terms " Microsporum canis ", "virulence factors" and each individual virulence factor were reviewed to enlist a series of articles, from where only original works in English and Spanish that included relevant information on the subject were selected. Out of the 147 articles obtained in the review, 46 were selected that reported virulence factors for M. canis in a period between 1988 and 2023. The rest of the articles were discarded because they did not contain information on the topic (67), some were written in different languages (3), and others were repeated in two or more databases (24) or were not original articles (7). The main virulence factors in M. canis are keratinases, fungilisins and subtilisins. However, less commonly reported are biofilms or dipeptidylpeptidases, among others, which have been little researched because they vary in expression or activity between strains and are not considered essential for the infection and survival of the fungus. Although it is known that they are truly involved in resistance, infection and metabolism, we recognize that their study could strengthen the knowledge of the pathogenesis of M. canis with the aim of achieving effective treatments, as well as the prevention and control of infection.

Background: Developing effective vaccines against SARS-CoV-2 that consider manufacturing limitations, equitable access, and acceptance is necessary for developing platforms to produce antigens that can be efficiently presented for generating neutralizing antibodies and as a model for new vaccines., Results: This work presents the development of an applicable technology through the oral administration of the SARS-CoV-2 RBD antigen fused with a peptide to improve its antigenic presentation. We focused on the development and production of the recombinant receptor binding domain (RBD) produced in E. coli modified with the addition of amino acids extension designed to improve antigen presentation. The production was carried out in shake flask and bioreactor cultures, obtaining around 200 mg/L of the antigen. The peptide-fused RBD and peptide-free RBD proteins were characterized and compared using SDS-PAGE gel, high-performance chromatography, and circular dichroism. The peptide-fused RBD was formulated in an oil-in-water emulsion for oral mice immunization. The peptide-fused RBD, compared to RBD, induced robust IgG production in mice, capable of recognizing the recombinant RBD in Enzyme-linked immunosorbent assays. In addition, the peptide-fused RBD generated neutralizing antibodies in the sera of the dosed mice. The formulation showed no reactive episodes and no changes in temperature or vomiting., Conclusions: Our study demonstrated the effectiveness of the designed peptide added to the RBD to improve antigen immunostimulation by oral administration., (© 2024. The Author(s).)

In the socioeconomic sphere, the concept of informality has been used to address issues pertaining to economic dynamics, institutions, work, poverty, settlements, the use of space, development, and sustainability, among others. This thematic range has given way to multiple discourses, definitions and approaches that mostly focus on a single phenomenon and conform to traditional disciplinary lines, making it difficult to fully understand informality and adequately inform policymaking. In this article, we carried out a multilevel co-word analysis with the purpose of unveiling the intellectual structure of socioeconomic informality. Co-occurring document keywords were used, initially, to delimit the scope of the socioeconomic dimension of informality (macro level) and, later, to identify its main concepts, themes (meso level) and sub-themes (micro level). Our results show that there is a corpus of research on socioeconomic informality that is sufficiently differentiable from other types of informality. This corpus, at the same time, can be divided into six major themes and 31 sub-themes related, more prominently, to the informal economy, informal settlements and informal institutions. Looking forward, the analysis suggests, an increasing focus on context and on the experience of multiple 'informalities' has the potential, on the one hand, to reveal links that help unify this historically fragmented corpus and, on the other hand, to give informality a meaning and identity that go beyond the traditional formal-informal dualism., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Gómez-Cruz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Micro-, small-, and medium-sized enterprises (MSMEs) account for the vast majority of firms in most economies, particularly in developing nations, and are key contributors to job creation and global economic development. However, the most significant impediment to MSME development in low- and middle-income countries is a lack of access to both investment and working capital financing. Due to a lack of essential track record, appropriate collateral, and credit history, MSMEs are frequently denied business loans by traditional lending institutions. In addition, SMEs' inability to access funding is hindered by institutional, structural, and non-financial factors. To address this, both the public and private sectors employ indirect and direct finance interventions to help MSMEs in developing and emerging economies enhance and increase their financing needs. Given the importance of MSMEs in the economy, a comprehensive overview of and systematic synthesizing of the evidence of the effects of financial access interventions for MSMEs, capturing a wide variety of outcome variables, is useful., Objectives: The objective of this evidence and gap map (EGM) is to describe the existing evidence on the effects of various interventions dedicated to supporting and improving MSMEs' access to credit, as well as the corresponding firm performance and/or welfare outcomes., Methods: An EGM is a systematic evidence product that displays the existing evidence relevant to a specific research question. An EGM's end product is a research article or report, but it can also be shared via an interactive map drawn as a matrix of included studies and their corresponding interventions and outcomes. Interventions in low- and middle-income countries that target specific population subgroups are included on the map. The EGM considers five types of interventions: (i) strategy, legislation and regulatory; (ii) systems and institutions; (iii) facilitate access; (iv) lending instruments or financial products; and (v) demand-side interventions. The map, on the other hand, covers outcome domains for policy environment, financial inclusion, firm performance, and welfare. Impact evaluations or systematic reviews of relevant interventions for a previously defined target population are included in the EGM. Studies using experimental or non-experimental designs, as well as systematic reviews, are eligible. The EGM excludes before-and-after study designs with no suitable comparison group. Furthermore, the map excludes literature reviews, key informant interviews, focus group discussions, and descriptive analyses. Search strings were used to conduct electronic searches in databases. To ensure that the research team had identified a significant portion of relevant research works, the search strategy was supplemented with gray literature searches and systematic review citation tracking. We have compiled studies that are either completed or in progress. For practical reasons, studies are limited to papers written in English and are not restricted by publication date., Selection Criteria: We included studies that examined interventions to enhance MSMEs' access to finance in low- and middle-income countries targeting MSMEs including households, smallholder farmers and single person enterprise as well as financial institutions/agencies and their staff. The EGM considers five types of interventions that aim to: (i) deliver strategy, legislation, and regulatory aspects; (ii) systems and institutions that enable financing; (iii) facilitate access to finance; (iv) deliver different lending instruments or financial products, including traditional forms of microcredit; and (v) demand-side interventions such as programs on financial literacy. The map includes outcome domains surrounding policy environment, financial inclusion, firm performance, and welfare. Eligible studies must be experimental, non-experimental, or systematic reviews. In addition, the study designs must have a suitable comparison group before and after the implementation of interventions., Results: The EGM includes 413 studies. The majority of the studies (379 studies) analyzed microenterprises, such as households and smallholder farmers; 7 studies analyzed community groups; while 109 studies analyzed small and medium enterprises. There were 147 studies on interventions that targeted multiple firm sizes. Lending instruments/financial products are the most common intervention across all firm types. When it comes to the types of firms that receive the said financial intervention, the data is overwhelmingly in favor of microenterprises (278 studies), followed by systems and organizations (138 studies) that support better access to such financial products and services. Welfare outcomes have the most evidence out of all of the outcomes of interest, followed by firm performance and financial inclusion. Among all firm types, welfare outcomes are primarily targeted at microenterprises. With 59 studies, we can say that small businesses have a significantly large number of enterprise performance outcomes. of the 413 studies, 243 used non-experimental or quasi-experimental designs (mainly propensity score matching and instrumental variable approaches), 136 used experimental methods, and 34 were systematic reviews. 175 studies (43%) provided evidence from Sub-Saharan Africa, 142 studies (35%) from South Asia, 86 studies (21%) from East Asia and the Pacific, 66 studies (16%) from Latin America and the Caribbean, 28 studies (7%), Europe and Central Asia, and 21 studies (5%) from the Middle East and North Africa. Most of the included evidence covers low-income (26%) and lower-middle income countries (66%), and to a lesser extent upper-middle-income countries (26%)., Conclusion: This map depicts the existing evidence and gaps on the effects of interventions to enhance MSMEs' access to financial services in low and middle-income countries. Interventions directed at microenterprises with welfare outcomes have a significant number of research outcomes in the literature. SME evaluations have looked at firm performance, with less focus to employment and the welfare effects on owners and employees, including poverty reduction. Microcredit/loans have been the focus of a large number of research papers (238 studies), indicating the field's growing popularity. However, emerging financial interventions such as facilitating access to digital financial services are relatively under-studied. Several studies also investigate rural or population in remote areas with 192 studies, 126 studies on poor and disadvantaged, and 114 papers on women. Most of the research is conducted in Sub-Saharan Africa (175 studies) and South Asia (142 studies) so further research in other regions could be conducted to allow a more holistic understanding of the effects of financial inclusion interventions. Credit lines, supply chain finance, and trade financing, which are some of the ADB's financial tools have limited evidence. Future studies should look into strategy, law, and regulation interventions, as well as interventions targeted at SMEs, and examine policy and regulatory environment outcomes as well as welfare outcomes. Interventions on the demand side and their impact on the policy and regulatory environment, as well as facilitating access are relatively understudied., (© 2023 The Authors. Campbell Systematic Reviews published by John Wiley & Sons Ltd on behalf of The Campbell Collaboration.)

Terpenes and terpenoids are a group of isoprene-derived molecules that constitute the largest group of natural products and secondary metabolites produced by living things, with more than 25,000 compounds reported. These compounds are synthesized by enzymes called terpene synthases, which include several families of cyclases and enzymes. These are responsible for adding functional groups to cyclized structures. Fungal terpenoids are of great interest for their pharmacological properties; therefore, understanding the mechanisms that regulate their synthesis (regulation of the mevalonate pathway, regulation of gene expression, and availability of cofactors) is essential to direct their production. For this reason, this review addresses the detailed study of the biosynthesis of fungal terpenoids and their regulation by various physiological and environmental factors., (© 2023. The Author(s).)

Inflammation and oxidative stress are critical events involved in neurodegeneration. In animal models, it has been shown that chronic consumption of a hypercaloric diet, which leads to inflammatory processes, affects the hippocampus, a brain region fundamental for learning and memory processes. In addition, advanced age and menopause are risk factors for neurodegeneration. Hormone replacement therapy (HRT) ameliorates menopause symptoms. Tibolone (TB), a synthetic hormone, exerts estrogenic, progestogenic and androgenic effects on different tissues. We aimed to determine the effect of short-term TB administration on oxidative stress and inflammation markers in the hippocampus of ovariectomized rats fed a high-fat-and-fructose diet (HFFD). Adult female rats were ovariectomized (OVX) and fed standard diet or HFFD-consisting of 10% lard supplemented chow and 20% high-fructose syrup in the drinking water-and administered vehicle or TB (1 mg/kg for seven days). Finally, we administered hormone receptor antagonists (MPP, RU486 or FLU) to each of the OVX + HFFD + TB groups. Bodyweight, triglycerides and cholesterol, oxidative stress and inflammation markers, and the activity and expression of antioxidant enzymes were quantified in the hippocampus of each experimental group. We observed that short-term TB administration significantly reduced body weight, AGEs, MDA levels, increased SOD and GPx activity, improved GSH/GSSG ratio, and reduced IL-6 and TNF-α. Our findings suggest that short-term administration of TB decreases oxidative stress and reduces inflammation caused by HFFD and early estrogenic decline. These effects occurred via estrogen receptor alpha.

The temperature-inducible λpL/pR-cI857 expression system has been widely used to produce recombinant proteins (RPs), especially when it is necessary to avoid the addition of exogenous materials to induce the expression of recombinant genes, preventing contamination of bioprocesses. The temperature increase favors the formation of inclusion bodies (IBs). The temperature upshift could change the metabolism, productivities, cell viability, IBs architecture, and the host cell proteins inside IBs, affecting downstream to obtain the final product. In this contribution, we focus on the relationship between the bioprocesses using temperature increase as inducer, the heat shock response associated with temperature up-shift, the RP accumulation, and the formation of IBs. Here, we describe how to produce IBs and how culture conditions can modulate the composition and architecture of IBs by modifying the induction temperature in RP production., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Low temperature and sodium butyrate (NaBu) are two of the most used productivity-enhancing strategies in CHO cell cultures during biopharmaceutical manufacturing. While these two approaches alter the balance in the reciprocal relationship between cell growth and productivity, we do not fully understand their mechanisms of action beyond a gross cell growth inhibition. Here, we used continuous culture to evaluate the differential effect of low temperature and NaBu supplementation on CHO cell performance and gene expression profile. We found that an increase in cell-productivity under growth-inhibiting conditions was associated with the arrest of cells in the G1/G0 phase. A transcriptome analysis revealed that the molecular mechanisms by which low temperature and NaBu arrested cell cycle in G1/G0 differed from each other through the deregulation of different cell cycle checkpoints and regulators. The individual transcriptome changes in pattern observed in response to low temperature and NaBu were retained when these two strategies were combined, leading to an additive effect in arresting the cell cycle in G1/G0 phase. The findings presented here offer novel molecular insights about the cell cycle regulation during the CHO cell bioprocessing and its implications for increased recombinant protein production. This data provides a background for engineering productivity-enhanced CHO cell lines for continuous manufacturing., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Avello et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

There are known disparities in the burden of illness and access/quality of care for African, Latino/a, Asian, and Native American (ALANA) patients diagnosed with depressive disorders, which may occur because of health inequities. Racial stress and trauma (RST), or the significant fear and distress that can be imparted from exposure to racism, is one such inequity linked to the development of depression. The current review summarizes past research examining the association between racism, RST, and depression, as well as avenues in which RST becomes biologically embedded in ALANA individuals. We describe multimodal research that supports vigilance as a potential mediator of the association between RST and depression and consider the nuanced role that vigilance plays during experiences with racism. Finally, we describe methodological advances in the assessment of vigilance evoked by RST and the clinical implications that may be generated by future improvements. In each of these areas, we present examples of how ongoing and future research can be leveraged to provide support for psychosocial programs that facilitate autonomous community healing and resilience, increase calls for public policy changes, and support clinical interventions that lessen the burden of racism on ALANA communities., Competing Interests: Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2022.)

The selection of highly recombinant protein (RP)-productive Chinese hamster ovary (CHO) cell lines is widely carried out in shake flasks. It is assumed that increases in the operating parameters in shake flasks lead to impairments in cell growth and RP production. These effects in cells metabolism are widely associated with high mass transfers and hydrodynamic stress. This study examined the impact of commonly used operational parameters on growth and specific productivity (q P ) of two CHO cell lines differentially secreting a humanized anti-hIL8 monoclonal antibody (mAb) and cultured in 250 ml flasks. The evaluated parameters are filling volume (10, 15, and 20%), shaking frequency (60 and 120 revolutions per minute -rpm-), and orbital diameter (25.4 and 19 mm). The analysis of the oxygen transfer was done in terms of the measured volumetric mass transfer coefficient (k L a) and of the hydrodynamics in terms of power input per unit volume of liquid (P/V), the turbulent eddy length scale measured by the Kolmogorov's microscale of turbulence, the energy dissipation rate, the average shear stress, and the shear rate. Though almost all measured kinetic and stoichiometric parameters remained unchanged, mAb titer included, significant differences were found in maximum cell concentration, 10-45% higher in conditions with lower values of k L a and P/V. Changes in glucose metabolism contributing to q P were only shown in the higher producer cell line. Non-lethal responses to elevated oxygen transfer and shear stress might be present and must be considered when evaluating CHO cell cultures in shake flasks., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

With the uncontrolled growth of multidrug-resistant bacteria, there is an urgent need to search for new therapeutic targets, to develop drugs with novel modes of bactericidal action. FoF1-ATP synthase plays a crucial role in bacterial bioenergetic processes, and it has emerged as an attractive antimicrobial target, validated by the pharmaceutical approval of an inhibitor to treat multidrug-resistant tuberculosis. In this work, we aimed to design, through two types of in silico strategies, new allosteric inhibitors of the ATP synthase, by targeting the catalytic β subunit, a centerpiece in communication between rotor subunits and catalytic sites, to drive the rotary mechanism. As a model system, we used the F1 sector of Escherichia coli, a bacterium included in the priority list of multidrug-resistant pathogens. Drug-like molecules and an IF1-derived peptide, designed through molecular dynamics simulations and sequence mining approaches, respectively, exhibited in vitro micromolar inhibitor potency against F1. An analysis of bacterial and Mammalia sequences of the key structural helix-turn-turn motif of the C-terminal domain of the β subunit revealed highly and moderately conserved positions that could be exploited for the development of new species-specific allosteric inhibitors. To our knowledge, these inhibitors are the first binders computationally designed against the catalytic subunit of FOF1-ATP synthase.

Background: Providing optimal care for type 2 diabetes (DM2) patients remains a challenge for all healthcare systems. Patients often encounter various barriers in adhering to self-management programs due to lack of knowledge and understanding of self-care activities, lack of individualized and coordinated care, inconvenient and costly education sessions, and poor patient-provider communication. Mobile technologies such as cell phones/smartphones, handheld tablets, and other wireless devices offer new and exciting opportunities for addressing some of these challenges. The purpose of this study is to compare a diabetes management strategy using an information board and a mobile application versus standard care in patients with uncontrolled DM2., Method: The SANENT (Sistema de Análisis de Enfermedades No Transmisibles) trial is a primary care-based, prospective, two-arm, randomized controlled, open-label, blinded-endpoint study. We aim to recruit 1440 DM2 patients during a period of 6 months until the requested number of participants has been achieved. The total length of the intervention will be 1 year. Both men and women treated for DM2 with an HbA1c > 8.5% and ≥ 20 years of age are eligible to participate in the study. The primary outcome of the study is improved diabetes control measured by changes in HbA1c in the study participants. HbA1c will be measured at baseline, 3-month, 6-month, 9-month, and 12-month follow-up visits in all participants. The main analysis will be based on the intention-to-treat principle. The primary endpoint of the study will be the change in HbA1C within the groups and the differences between the groups. This will be assessed by a repeated measurement approach based on mixed models which contain both fixed effects and random effects., Discussion: The overall goal of this project is to contribute to the evidence for the use of mobile technology to improve the treatment and regulation of poorly controlled DM2 patients living in Mexico. Our proposed project will show how mobile health technology tools can be used in the treatment of patients with uncontrolled DM2 in primary health care in a Latin American population, and particularly how they could help diabetes patients take better care of themselves., Trial Registration: ClinicalTrials.gov , US National Institutes of Health NCT04974333 . Prospectively registered on July 13, 2021. Protocol version number 1, dated August 15th, 2021., (© 2022. The Author(s).)

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., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Arbuscular mycorrhizal fungi (AMF) are obligate biotrophs, and the difficulty of growing them in asymbiotic or monoxenic (AMF + root) conditions limits research and their large-scale production as biofertilizer. We hypothesized that a combination of flavanols and strigolactones can mimic complex root signaling during the presymbiotic stages of AMF. We evaluated the germination, mycelial growth, branching, and auxiliary cell clusters formation by Gigaspora margarita during the presymbiotic stage in the presence (or absence) of transformed Cichorium intybus roots in basal culture medium enriched with glucose, a flavonol (quercetin or biochanin A) and a strigolactone analogue (1-Methyl-2-oxindole or indole propionic acid). With quercetin (5 µM), methyl oxindole (2.5 nM), and glucose (8.2 g/L) in the absence of roots, the presymbiotic mycelium of G. margarita grew without cytoplasmic retraction and produced auxiliary cells over 71 days similar to presymbiotic mycelium in the presence of roots but without glucose, strigolactones, and flavonols. Our results indicate that glucose and a specific combination of certain concentrations of a flavonol and a strigolactone might be used in asymbiotic or monoxenic liquid or semisolid cultures to stimulate AMF inoculant bioprocesses., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose of Review: The objective of this review was to summarize the current scientific evidence of mobile health technology in the primary prevention of type 2 diabetes in patients with prediabetes derived from randomized clinical trials., Recent Findings: Few randomized clinical trials are available using mobile health technologies in the prevention of type 2 diabetes. There is heterogeneity in regard to the main study outcomes, duration of interventions, and study findings. Inconsistent findings have been reported whether mobile health technologies are effective in reducing HbA1C levels or the incidence of type 2 diabetes in patients with prediabetes. However, results are promising that mobile health interventions may decrease body weight. Future study may consistently measure changes in glycemic indicators as well as develop elements that better address behavior changes., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Bacillus spp. are well known plant growth promoting bacteria (PGPB) and biological control agents (BCA) due to their capacity to synthesize a wide variety of phytostimulant and antimicrobial compounds. B. velezensis 83 is a strain marketed in Mexico as a foliar biofungicide (Fungifree AB™) which has been used for biological control of five different genera of phytopathogenic fungi ( Colletotrichum, Erysiphe, Botrytis, Sphaerotheca, Leveillula ) in crops of agricultural importance such as mango, avocado, papaya, citrus, tomato, strawberry, blueberry, blackberry and cucurbits, among others. In this work, the potential of plant growth promotion of B. velezensis 83 was evaluated on different phenological stages of tomato plants as well as the biocontrol efficacy of B. velezensis 83 formulations (cells and/or metabolites) against B. cinerea infection on leaves and postharvest fruits. Greenhouse grown tomato plants inoculated with a high concentration (1 × 10 8 CFU/plant) of B. velezensis 83 yielded 254 tons/Ha•year of which the 64% was first quality tomato (≥100 g/fruit), while the control plants produced less than 184 tons/Ha•year with only 55% of first quality tomato. Additionally, in vitro assays carried out with leaves and fruits, shown that the B. velezensis 83 cells formulation had an efficacy of control of B. cinerea infection of ∼31% on leaves and ∼89% on fruits, while the metabolites formulation had an efficacy of control of less than 10%. Therefore, it was concluded that spores (not the metabolites) are the main antagonism factor of Fungifree AB™. The high effectivity of B. cinerea control on fruits by B. velezensis 83, opens the possibility for a postharvest use of this biofungicide., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors. Published by Elsevier B.V.)

Different cellular processes that contribute to protein production in Chinese hamster ovary (CHO) cells have been previously investigated by proteomics. However, although the classical secretory pathway (CSP) has been well documented as a bottleneck during recombinant protein (RP) production, it has not been well represented in previous proteomic studies. Hence, the significance of this pathway for production of RP was assessed by identifying its own proteins that were associated to changes in RP production, through subcellular fractionation coupled to shot-gun proteomics. Two CHO cell lines producing a monoclonal antibody with different specific productivities were used as cellular models, from which 4952 protein groups were identified, which represent a coverage of 59% of the Chinese hamster proteome. Data are available via ProteomeXchange with identifier PXD021014. By using SAM and ROTS algorithms, 493 proteins were classified as differentially expressed, of which about 80% was proposed as novel targets and one-third were assigned to the CSP. Endoplasmic reticulum (ER) stress, unfolded protein response, calcium homeostasis, vesicle traffic, glycosylation, autophagy, proteasomal activity, protein synthesis and translocation into ER lumen, and secretion of extracellular matrix components were some of the affected processes that occurred in the secretory pathway. Processes from other cellular compartments, such as DNA replication, transcription, cytoskeleton organization, signaling, and metabolism, were also modified. This study gives new insights into the molecular traits of higher producer cells and provides novel targets for development of new sub-lines with improved phenotypes for RP production., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

SARS-CoV-2 is a novel β-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.

A careful selection of culture mediums and feeds has become necessary to maximize yields of recombinant proteins during bioprocesses of mammalian cells. Supplements contain a variety of concentrate nutrients, and their beneficial effects vary according to recombinant cell lines. In this study, the effects of PowerFeed A on growth kinetics, productivity and cellular metabolism were evaluated for two Chinese hamster ovary cell lines producing a monoclonal antibody in a batch culture. Supplemented cultures increased integral viable cell density of CRL-12444 and CRL-12445 cells by 2.4 and 1.6 times through extension of culture time at which viability was above 90% in 72 and 36 h, respectively, and increment of maximal cell concentration in 3.25 × 10 6 cells/ml (69%) for CRL-12445 cells. Product titer augmented 1.9 and 2.5 times for CRL-12444 and CRL-12445 cells, respectively, without changes in growth rate and specific productivity. Feed supplementation also stimulated full consumption of glucose and free glutamine and reduced 10 times lactate accumulation, while ammonium, sodium and potassium remained at similar concentrations at the end of the culture. About 44% of calcium, mainly provided by feed, was consumed by both cell lines. Maximization of cellular growth, viability and protein titer through feeding encourages extending its use to other cell lines and exploring novel combinations with other basal mediums or feeds. A thorough investigation of its impact on protein quality and the molecular mechanisms behind these effects will allow designing effective feeds and strategies to rationally optimize protein production in the biomanufacturing industry.

Estrogen replacement therapy decreases some risk factors of the metabolic syndrome but increases the risk of some types of cancer. Tibolone (TIB) has shown similar neuroprotective effects as estrogens. This study aimed to evaluate the effects of TIB on metabolic parameters and the expression of sex hormone receptors in the CNS in ovariectomised rats fed with a hypercaloric diet. Sprague-Dawley female rats were ovariectomised and fed for 30 days with a standard diet (SD) or high-fat high-fructose diet (HFFD) and treated with TIB (1 mg/kg) or vehicle. At the end of the treatments, HFFD increased body weight, glucose tolerance, triglycerides and cholesterol levels, while TIB treatment decreased these parameters. Subsequently, the hippocampus, the hypothalamus and the frontal cortex were dissected. RT-PCR was performed for progesterone receptor (PR), androgen receptor (AR), estrogen receptors alpha and beta (ERα, ERβ), insulin receptor (IR) and insulin-like growth factor 1 (IGF-1). HFFD altered the expression of sex hormone receptors in specific brain structures involved in the regulation of homeostasis and cognition, which highlights the importance of a healthy diet. In turn, TIB modulated the expression of these receptors, particularly in the hypothalamus., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Chinese hamster ovary cells have been the workhorse for the production of recombinant proteins in mammalian cells. Since biochemical, cellular and omics studies are usually affected by the lack of suitable fractionation procedures to isolate compartments from these cells, differential and isopycnic centrifugation based techniques were characterized and developed specially for them. Enriched fractions in intact nuclei, mitochondria, peroxisomes, cis-Golgi, trans-Golgi and endoplasmic reticulum (ER) were obtained in differential centrifugation steps and subsequently separated in discontinuous sucrose gradients. Nuclei, mitochondria, cis-Golgi, peroxisomes and smooth ER fractions were obtained as defined bands in 30-60% gradients. Despite the low percentage represented by the microsomes of the total cell homogenate (1.7%), their separation in a novel sucrose gradient (10-60%) showed enough resolution and efficiency to quantitatively separate their components into enriched fractions in trans-Golgi, cis-Golgi and ER. The identity of these organelles belonging to the classical secretion pathway that came from 10-60% gradients was confirmed by proteomics. Data are available via ProteomeXchange with identifier PXD019778. Components from ER and plasma membrane were the most frequent contaminants in almost all obtained fractions. The improved sucrose gradient for microsomal samples proved being successful in obtaining enriched fractions of low abundance organelles, such as Golgi apparatus and ER components, for biochemical and molecular studies, and suitable for proteomic research, which makes it a useful tool for future studies of this and other mammalian cell lines., Competing Interests: GE Healthcare Life Sciences provided support in cell culture media and supplies, GeneTex provided the generous gift of antibodies, and Dr. Paola Toledo Ibelles from Inolab Especialistas en Servicio provided support in cell culture media. There are no patents, products in development or marketed products to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Modern imaging plays a central role in the care of obese patients, and there is an integral focus on its use and accessibility in individuals who have alterations of various in various organs. The objective in this study was to perform an echographic analysis of musculoskeletal system disorders, endothelial dysfunction and the left ventricle (LV) in obese rats., Methods: Sprague Dawley rats (250 ± 5 g) were obtained and divided into two groups: the control (C) group was fed with a standard diet, and the obese (Ob) group was fed hyper caloric diet with a high fructose-fat content for 4 months. Body weight, cholesterol, triglycerides, glucose, inflammatory cytokines and adhesion molecules (ICAM-1, VCAM-1) were measured. Additionally, two-dimensional echocardiography, abdominal ultrasound and musculoskeletal system studies were performed in the lower extremities., Results: The body weight in the Ob group was increased compared to that in the control group, (p < 0.001); in addition, increased glucose, cholesterol and triglyceride concentrations (p < 0.05) as well as increased levels of the adhesion molecules ICAM-1 and, VCAM-1 (p < 0.01) were found in the Ob group vs the C group. On ultrasound, 75% of the Ob group presented fatty liver and distal joint abnormalities., Conclusion: Obese rats exhibit endothelial dysfunction and musculoskeletal changes, also, fatty liver and articular cysts in the posterior region of the distal lower- extremity joints.

The metabolic syndrome includes changes in blood glucose levels, arterial hypertension, triglycerides, dyslipidemia and central obesity. Countless reports have described the correlation between the metabolic syndrome and cognitive impairment. However, only a few reports have assessed cognitive impairment associated with the metabolic syndrome in animals of both sexes. For this purpose, Sprague-Dawley male and female rats were fed either with a hypercaloric diet as model of the metabolic syndrome or with a standard chow diet as controls. Subsequently, spatial learning and memory (Morris water maze) as well as short- and long-term memory (passive avoidance task) were evaluated. Body weight, blood pressure, triglycerides, and total cholesterol significantly increased (F(1, 36) = 94.89, p < .001) in rats fed with hypercaloric diet compared to control rats. Furthermore, cognitive impairment was observed in spatial learning and spatial memory on male rats but not on female rats fed with hypercaloric diet. In addition, a long-term memory impairment was observed in both groups fed with hypercaloric diet in comparison to their respective control group (F(1, 32) = 10.61, p = .0027). Immunohistochemistry results showed no changes in the number of positive cells for NeuN, GFAP and Ox-42. In males fed with a hypercaloric diet, a decrease in testosterone levels was observed, whereas estradiol levels decreased in females when compared with their respective control group (p < .0001). In this MetS animal model, metabolic and cognitive differences were observed in males and females, which demonstrates that sex hormones play a significant role in metabolic regulation and neuroprotection related to the CA1 region of the hippocampus., (© 2020 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

In order to increase survival rates of greenhouse seedlings destined for restoration and conservation programs, successful mycorrhization of the seedlings is necessary. To reforest forest ecosystems, host trees must be inoculated with ectomycorrhizal fungi and, in order to guarantee a sufficient supply of ectomycorrhizal inoculum, it is necessary to develop technologies for the mass production of ectomycorrhizal fungi mycelia. We selected the ectomycorrhizal fungus Laccaria trichodermophora, due to its ecological traits and feasible mycelia production in asymbiotic conditions. Here, we report the field sampling of genetic resources, as well as the highly productive nutritional media and cultivation parameters in solid cultures. Furthermore, in order to achieve high mycelial production, we used strain screening and evaluated pH, carbon source concentration, and culture conditions of submerged cultures in normal and baffled shake flasks. The higher productivity culture conditions in shake flasks were selected for evaluation in a pneumatic bioreactor, using modified BAF media with a 10 g/L glucose, pH 5.5, 25 °C, and a volumetric oxygen transfer coefficient (K L a) of 36 h -1 . Under those conditions less biomass (12-37 %) was produced in the pneumatic bioreactor compared with the baffled shake flasks. This approach shows that L. trichodermophora can generate a large biomass concentration and constitute the biotechnological foundation of its mycelia mass production., Competing Interests: Declaration of Competing Interest On behalf of all the authors, the corresponding author states that there is no conflict of interest., (Copyright © 2020 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

The global rise in urbanization and industrial activity has led to the production and incorporation of foreign contaminant molecules into ecosystems, distorting them and impacting human and animal health. Physical, chemical, and biological strategies have been adopted to eliminate these contaminants from water bodies under anthropogenic stress. Biotechnological processes involving microorganisms and enzymes have been used for this purpose; specifically, laccases, which are broad spectrum biocatalysts, have been used to degrade several compounds, such as those that can be found in the effluents from industries and hospitals. Laccases have shown high potential in the biotransformation of diverse pollutants using crude enzyme extracts or free enzymes. However, their application in bioremediation and water treatment at a large scale is limited by the complex composition and high salt concentration and pH values of contaminated media that affect protein stability, recovery and recycling. These issues are also associated with operational problems and the necessity of large-scale production of laccase. Hence, more knowledge on the molecular characteristics of water bodies is required to identify and develop new laccases that can be used under complex conditions and to develop novel strategies and processes to achieve their efficient application in treating contaminated water. Recently, stability, efficiency, separation and reuse issues have been overcome by the immobilization of enzymes and development of novel biocatalytic materials. This review provides recent information on laccases from different sources, their structures and biochemical properties, mechanisms of action, and application in the bioremediation and biotransformation of contaminant molecules in water. Moreover, we discuss a series of improvements that have been attempted for better organic solvent tolerance, thermo-tolerance, and operational stability of laccases, as per process requirements.

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.

In addition to oxidative stress due to the increase of free radicals, estrogen deficiency is associated with changes in enzymatic activity, glutathione redox ratio (GSH/GSSG), and the content of oxidative markers such as malondialdehyde. Tibolone, a synthetic steroid, has been used as an elective treatment for the relief of menopausal symptoms. However, the acute effects of hormonal therapy with tibolone on metabolic parameters and oxidative stress markers associated with the first stages of estrogen deficiency are still unknown. The study aimed to evaluate if the acute administration of tibolone reduces oxidative stress in ovariectomized rats fed high-fat-and-fructose diet. Rats were fed a standard diet or a diet consisting of 10% lard-supplemented chow and 20% high-fructose syrup in the drinking water plus tibolone or vehicle for seven days. Weight, cholesterol, triglycerides, and glucose levels, as well as antioxidant enzymes and oxidative stress markers were quantified in the serum of each experimental group. It was observed that seven days of diet and tibolone treatment in the ovariectomized group reduced weight, triglycerides, cholesterol, glucose levels and advanced glycation end products but did not change GSH/GSSG ratio nor the enzymatic activity of superoxide dismutase. Also, both glutathione peroxidase and glutathione reductase activity decreased, as well as malondialdehyde levels. These results suggest that the acute treatment with tibolone prevented the changes in the metabolic parameters analyzed as well as the increase in the levels of malondialdehyde and AGEs induced by ovariectomy and high-fat diet., Competing Interests: The authors declare that they have no conflict of interest., (© Georg Thieme Verlag KG Stuttgart · New York.)

Zoanthids of the genus Palythoa are distributed worldwide in shallow waters around coral reefs. Like all cnidarians, they possess nematocysts that contain a large diversity of toxins that paralyze their prey. This work was aimed at isolating and functionally characterizing a cnidarian neurotoxic phospholipase named A2-PLTX-Pcb1a for the first time. This phospholipase was isolated from the venomous extract of the zoanthid Palythoa caribaeorum . This enzyme, which is Ca 2+ -dependent, is a 149 amino acid residue protein. The analysis of the A2-PLTX-Pcb1a sequence showed neurotoxic domain similitude with other neurotoxic sPLA₂´s, but a different catalytic histidine domain. This is remarkable, since A2-PLTX-Pcb1a displays properties like those of other known PLA₂ enzymes.

Background: Pichia pastoris (syn. Komagataella phaffii) is one of the most highly utilized eukaryotic expression systems for the production of heterologous glycoproteins, being able to perform both N- and O-mannosylation. In this study, we present the expression in P. pastoris of an O-mannosylated recombinant version of the 38 kDa glycolipoprotein PstS-1 from Mycobacterium tuberculosis (Mtb), that is similar in primary structure to the native secreted protein., Results: The recombinant PstS-1 (rPstS-1) was produced without the native lipidation signal. Glycoprotein expression was under the control of the methanol-inducible promoter pAOX1, with secretion being directed by the α-mating factor secretion signal. Production of rPstS-1 was carried out in baffled shake flasks (BSFs) and controlled bioreactors. A production up to ~ 46 mg/L of the recombinant protein was achieved in both the BSFs and the bioreactors. The recombinant protein was recovered from the supernatant and purified in three steps, achieving a preparation with 98% electrophoretic purity. The primary and secondary structures of the recombinant protein were characterized, as well as its O-mannosylation pattern. Furthermore, a cross-reactivity analysis using serum antibodies from patients with active tuberculosis demonstrated recognition of the recombinant glycoprotein, indirectly indicating the similarity between the recombinant PstS-1 and the native protein from Mtb., Conclusions: rPstS-1 (98.9% sequence identity, O-mannosylated, and without tags) was produced and secreted by P. pastoris, demonstrating that this yeast is a useful cell factory that could also be used to produce other glycosylated Mtb antigens. The rPstS-1 could be used as a tool for studying the role of this molecule during Mtb infection, and to develop and improve vaccines or kits based on the recombinant protein for serodiagnosis.

Understanding the structure, functionalities and biology of functional amyloids is an issue of emerging interest. Inclusion bodies, namely protein clusters formed in recombinant bacteria during protein production processes, have emerged as unanticipated, highly tunable models for the scrutiny of the physiology and architecture of functional amyloids. Based on an amyloidal skeleton combined with varying amounts of native or native-like protein forms, bacterial inclusion bodies exhibit an unusual arrangement that confers mechanical stability, biological activity and conditional protein release, being thus exploitable as versatile biomaterials. The applicability of inclusion bodies in biotechnology as enriched sources of protein and reusable catalysts, and in biomedicine as biocompatible topographies, nanopills or mimetics of endocrine secretory granules has been largely validated. Beyond these uses, the dissection of how recombinant bacteria manage the aggregation of functional protein species into structures of highly variable complexity offers insights about unsuspected connections between protein quality (conformational status compatible with functionality) and cell physiology.

Background: In Streptomyces, understanding the switch from primary to secondary metabolism is important for maximizing the production of secondary metabolites such as antibiotics, as well as for optimizing recombinant glycoprotein production. Differences in Streptomyces lividans bacterial aggregation as well as recombinant glycoprotein production and O-mannosylation have been reported due to modifications in the shake flask design. We hypothetized that such differences are related to the metabolic switch that occurs under oxygen-limiting conditions in the cultures., Results: Shake flask design was found to affect undecylprodigiosin (RED, a marker of secondary metabolism) production; the RED yield was 12 and 385 times greater in conventional normal Erlenmeyer flasks (NF) than in baffled flasks (BF) and coiled flasks (CF), respectively. In addition, oxygen transfer rates (OTR) and carbon dioxide transfer rates were almost 15 times greater in cultures in CF and BF as compared with those in NF. Based on these data, we obtained respiration quotients (RQ) consistent with aerobic metabolism for CF and BF, but an RQ suggestive of anaerobic metabolism for NF., Conclusion: Although the metabolic switch is usually related to limitations in phosphate and nitrogen in Streptomyces sp., our results reveal that it can also be activated by low OTR, dramatically affecting recombinant glycoprotein production and O-mannosylation and increasing RED synthesis in the process.

Objective: To identify risk factors for chronic kidney disease progression in Brazilian children and to evaluate the interactions between factors., Methods: This was a multicenter prospective cohort in São Paulo, involving 209 children with CKD stages 3-4. The study outcome included: (a) death, (b) start of kidney replacement therapy, (c) eGFR decrease >50% during the followup. Thirteen risk factors were tested using univariate regression models, followed by multivariable Cox regression models. The terms of interaction between the variables showing significant association with the outcome were then introduced to the model., Results: After a median follow-up of 2.5 years (IQR=1.4-3.0), the outcome occurred in 44 cases (21%): 22 started dialysis, 12 had >50% eGFR decrease, seven underwent transplantation, and three died. Advanced CKD stage at onset (HR=2.16, CI=1.14-4.09), nephrotic proteinuria (HR=2.89, CI=1.49-5.62), age (HR=1.10, CI=1.01-1.17), systolic blood pressure Z score (HR=1.36, CI=1.08-1.70), and anemia (HR=2.60, CI=1.41-4.77) were associated with the outcome. An interaction between anemia and nephrotic proteinuria at V1 (HR=0.25, CI=0.06-1.00) was detected., Conclusions: As the first CKD cohort in the southern hemisphere, this study supports the main factors reported in developed countries with regards to CKD progression, affirming the potential role of treatments to slow CKD evolution. The detected interaction suggests that anemia may be more deleterious for CKD progression in patients without proteinuria and should be further studied., (Copyright © 2017 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.)

In the biopharmaceutical sector, Chinese hamster ovary (CHO) cells have become the host of choice to produce recombinant proteins (r-proteins) due to their capacity for correct protein folding, assembly, and posttranslational modification. However, the production of therapeutic r-proteins in CHO cells is expensive and presents insufficient production yields for certain proteins. Effective culture strategies to increase productivity (qp) include a high glucose concentration in the medium and mild hypothermia (28-34 °C), but these changes lead to a reduced specific growth rate. To study the individual and combined impacts of glucose concentration, specific growth rate and mild hypothermia on culture performance and cell metabolism, we analyzed chemostat cultures of recombinant human tissue plasminogen activator (rh-tPA)-producing CHO cell lines fed with three glucose concentrations in feeding media (20, 30 and 40 mM), at two dilution rates (0.01 and 0.018 1/h) and two temperatures (33 and 37 °C). The results indicated significant changes in cell growth, cell cycle distribution, metabolism, and rh-tPA productivity in response to the varying environmental culture conditions. High glucose feed led to constrained cell growth, increased specific rh-tPA productivity and a higher number of cells in the G2/M phase. Low specific growth rate and temperature (33 °C) reduced glucose consumption and lactate production rates. Our findings indicated that a reduced specific growth rate coupled with high feed glucose significantly improves r-protein productivity in CHO cells. We also observed that low temperature significantly reduced qp, but not cell growth when dilution rate was manipulated, regardless of the glucose concentration or dilution rate. In contrast, we determined that feed glucose concentration and consumption rate were the dominant aspects of the growth and productivity in CHO cells by using multivariate analysis., Competing Interests: The authors have declared that no competing interests exist.

Aggregation of recombinant proteins into inclusion bodies (IBs) is the major drawback of heterologous expression in Escherichia coli. Here, we evaluated the effects of a pH shift after expression induction on recombinant phospholipase A2 production and its aggregation in IBs in E. coli Origami™, as compared to cultures with pH maintained at 7.5 or uncontrolled pH. Cultures shifted from 7.5 to pH 6.5 or 8.5 produced ∼15-25% less biomass as compared with those kept at 7.5 or without pH control. The cultures shifted to pH 8.5 showed a ∼50% higher yield of acetate per biomass, and the rPLA2 yield was improved 2.4-fold. Purified IBs formed at pH 8.5 containing ∼50% of rPLA2, were more susceptible to proteinase-K cleavage and bound less thioflavin-T, indicating lower amyloid content, with the concomitant enrichment of α-helical and random-coil secondary structures, as demonstrated by FTIR. Moreover, only one IB per cell was formed at pH 8.5; instead, more than two were observed under the other culture pH conditions. Nevertheless, under uncontrolled pH conditions, ∼300nm larger IBs were observed. Our work presents evidence of the usefulness of recombinant protein expression cultivated at pH 8.5 allowing the reduction of amyloid content in IBs., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Progress has been made in resource-limited countries in treating acute lymphoblastic leukemia, but advances in solid malignancies have been slower. Multidisciplinary care coordination is challenging, assessing adherence to guidelines through quality improvement initiatives is essential. We characterized deviations from guidelines in the delivery of radiation in a middle-income country program as a pilot for evaluating adequacy of local control and as surrogate for integration of multidisciplinary care. One-third of patients for whom it was indicated did not receive radiation. Of the patients who received radiation, 95% had a deviation. This study underscores the importance of quality assessment in resource-limited settings., (© 2017 Wiley Periodicals, Inc.)

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 (k L a ≈ 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 β-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.

Recombinant proteins are widely used as biopharmaceuticals, but their production by mammalian cell culture is expensive. Hence, improvement of bioprocess productivity is greatly needed. A temperature downshift (TDS) from 37°C to 28-34°C is an effective strategy to expand the productive life period of cells and increase their productivity (qp). Here, TDS in Chinese hamster ovary (CHO) cell cultures, initially grown at 37°C and switched to 30°C during the exponential growth phase, resulted in a 1.6-fold increase in the qp of recombinant human tissue plasminogen activator (rh-tPA). The transcriptomic response using next-generation sequencing (NGS) was assessed to characterize the cellular behavior associated with TDS. A total of 416 (q > 0.8) and 3,472 (q > 0.9) differentially expressed transcripts, with more than a 1.6-fold change at 24 and 48 h post TDS, respectively, were observed in cultures with TDS compared to those at constant 37°C. In agreement with the extended cell survival resulting from TDS, transcripts related to cell growth arrest that controlled cell proliferation without the activation of the DNA damage response, were differentially expressed. Most upregulated genes were related to energy metabolism in mitochondria, mitochondrial biogenesis, central metabolism, and avoidance of apoptotic cell death. The gene coding for rh-tPA was not differentially expressed, but fluctuations were detected in the transcripts encoding proteins involved in the secretory machinery, particularly in glycosylation. Through NGS the dynamic processes caused by TDS were assessed in this biological system.

Azospirillum brasilense has significance as a growth promoter in plants of commercial interest. Two industrial native strains (Start and Calf), used as a part of an inoculant formulation in Mexico during the last 15 years, were incubated in laboratory-scale pneumatic bioreactors at different aeration rates. In both strains, the positive effect of decreased aeration was observed. At the lowest (0.1 vvm, air volume/liquid volume×minute), the highest biomass were obtained for Calf (7.8 × 10(10)CFU/ml), and Start (2.9 × 10(9)CFU/ml). These were higher in one magnitude order compared to cultures carried out at 0.5 vvm, and two compared to those at 1.0 vvm. At lower aeration, both stereoisomeric forms of malic acid were consumed, but at higher aeration, just L-malate was consumed. A reduction in aeration allows an increase of the shelf life and the microorganism saved higher concentrations of polyhydroxybutyrate. The selected fermentation conditions are closely related to those prevalent in large-scale bioreactors and offer the possibility of achieving high biomass titles with high shelf life at a reduced costs, due to the complete use of a carbon source at low aeration of a low cost raw material as DL-malic acid mixture in comparison with the L-malic acid stereoisomer., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Inclusion bodies (IBs) are aggregated proteins that form clusters when protein is overexpressed in heterologous expression systems. IBs have been considered as non-usable proteins, but recently they are being used as functional materials, catalytic particles, drug delivery agents, immunogenic structures, and as a raw material in recombinant therapeutic protein purification. However, few studies have been made to understand how culture conditions affect the protein aggregation and the physicochemical characteristics that lead them to cluster. The objective of our research was to understand how pH affects the physicochemical properties of IBs formed by the recombinant sphingomyelinase-D of tick expressed in E. coli BL21-Gold (DE3) by evaluating two pH culture strategies., Results: Uncontrolled pH culture conditions favored recombinant sphingomyelinase-D aggregation and IB formation. The IBs of sphingomyelinase-D produced under controlled pH at 7.5 and after 24 h were smaller (<500 nm) than those produced under uncontrolled pH conditions (>500 nm). Furthermore, the composition, conformation and β-structure formation of the aggregates were different. Under controlled pH conditions in comparison to uncontrolled conditions, the produced IBs presented higher resistance to denaturants and proteinase-K degradation, presented β-structure, but apparently as time passes the IBs become compacted and less sensitive to amyloid dye binding., Conclusions: The manipulation of the pH has an impact on IB formation and their physicochemical characteristics. Particularly, uncontrolled pH conditions favored the protein aggregation and sphingomyelinase-D IB formation. The evidence may lead to find methodologies for bioprocesses to obtain biomaterials with particular characteristics, extending the application possibilities of the inclusion bodies.