Harnessing metabolic plasticity in CHO cells for enhanced perfusion cultivation.

Chinese Hamster Ovary (CHO) cells have rapidly become a cornerstone in biopharmaceutical production. Recently, a reinvigoration of perfusion culture mode in CHO cell cultivation has been observed. However, most cell lines currently in use have been engineered and adapted for fed-batch culture methods, and may not perform optimally under perfusion conditions. To improve the cell's resilience and viability during perfusion culture, we cultured a triple knockout CHO cell line, deficient in three apoptosis related genes BAX, BAK, and BOK in a perfusion system. After 20 days of culture, the cells exhibited a halt in cell proliferation. Interestingly, following this phase of growth arrest, the cells entered a second growth phase. During this phase, the cell numbers nearly doubled, but cell specific productivity decreased. We performed a proteomics investigation, elucidating a distinct correlation between growth arrest and cell cycle arrest and showing an upregulation of the central carbon metabolism and oxidative phosphorylation. The upregulation was partially reverted during the second growth phase, likely caused by intragenerational adaptations to stresses encountered. A phase-dependent response to oxidative stress was noted, indicating glutathione has only a secondary role during cell cycle arrest. Our data provides evidence of metabolic regulation under high cell density culturing conditions and demonstrates that cell growth arrest can be overcome. The acquired insights have the potential to not only enhance our understanding of cellular metabolism but also contribute to the development of superior cell lines for perfusion cultivation.

A Socioecological-Genetic Framework of Culture and Personality: Their Roots, Trends, and Interplay.

Culture and personality are two central topics in psychology. Individuals are culturally influenced influencers of culture, yet the research linking culture and personality has been limited and fragmentary. We integrate the literatures on culture and personality with recent advances in socioecology and genetics to formulate the Socioecological-Genetic Framework of Culture and Personality. Our framework not only delineates the mutual constitution of culture and personality but also sheds light on (a) the roots of culture and personality, (b) how socioecological changes partly explain temporal trends in culture and personality, and (c) how genes and culture/socioecology interact to influence personality (i.e., nature × nurture interactions). By spotlighting the roles of socioecology and genetics, our integrative framework advances the understanding of culture and personality.

Hepatitis C virus NS5A and core protein induce fibrosis-related genes regulation on Huh7 cells through activation of LX2 cells.

INTRODUCTION AND OBJECTIVES: Liver fibrosis remains a complication derived from a chronic Hepatitis C Virus (HCV) infection even when it is resolved, and no liver antifibrotic drug has been approved. Molecular mechanisms on hepatocytes and activation of hepatic stellate cells (HSCs) play a central role in liver fibrogenesis. To elucidate molecular mechanisms, it is important to analyze pathway regulation during HSC activation and HCV infection. MATERIALS AND METHODS: We evaluate the fibrosis-associated molecular mechanisms during a co-culture of human HSCs (LX2), with human hepatocytes (Huh7) that express HCV NS5A or Core protein. We evaluated LX2 activation induced by HCV NS5A or Core expression in Huh7 cells during co-culture. We determined a fibrosis-associated gene expression profile in Huh7 that expresses NS5A or Core proteins during the co-culture with LX2. RESULTS: We observed that NS5A induced 8.3-, 6.7- and 4-fold changes and that Core induced 6.5-, 1.8-, and 6.2-fold changes in the collagen1, TGFß1, and timp1 gene expression, respectively, in LX2 co-cultured with transfected Huh7. In addition, NS5A induced the expression of 30 genes while Core induced 41 genes and reduced the expression of 30 genes related to fibrosis in Huh7 cells during the co-culture with LX2, compared to control. The molecular pathways enriched from the gene expression profile were involved in TGFB signaling and the organization of extracellular matrix. CONCLUSIONS: We demonstrated that HCV NS5A and Core protein expression regulate LX2 activation. NS5A-induced LX2 activation, in turn, regulates diverse fibrosis-related gene expression at different levels in Huh7, which can be further analyzed as potential antifibrotic targets during HCV infection.

Uncovering a novel mechanism: Butyrate induces estrogen receptor alpha activation independent of estrogen stimulation in MCF-7 breast cancer cells.

Butyrate is a promising candidate for an antitumoral drug, as it promotes cancer cell apoptosis and reduces hormone receptor activity, while promoting differentiation and proliferation in normal cells. However, the effects of low-dose butyrate on breast cancer cell cultures are unclear. We explored the impact of sub-therapeutic doses of butyrate on estrogen receptor alpha (ERα) transcriptional activity in MCF-7 cells, using RT-qPCR, Western blot, wound-healing assays, and chromatin immunoprecipitation. Our results showed that sub-therapeutic doses of sodium butyrate (0.1 - 0.2 mM) increased the transcription of ESR1, TFF1, and CSTD genes, but did not affect ERα protein levels. Moreover, we observed an increase in cell migration in wound-healing assays. ChIP assays revealed that treatment with 0.1 mM of sodium butyrate resulted in estrogen-independent recruitment of ERα at the pS2 promoter and loss of NCoR. Appropriate therapeutic dosage of butyrate is essential to avoid potential adverse effects on patients' health, especially in the case of estrogen receptor-positive breast tumors. Sub-therapeutic doses of butyrate may induce undesirable cell processes, such as migration due to low-dose butyrate-mediated ERα activation. These findings shed light on the complex effects of butyrate in breast cancer and provide insights for research in the development of antitumoral drugs.

The topology of genome-scale metabolic reconstructions unravels independent modules and high network flexibility.

The topology of metabolic networks is recognisably modular with modules weakly connected apart from sharing a pool of currency metabolites. Here, we defined modules as sets of reversible reactions isolated from the rest of metabolism by irreversible reactions except for the exchange of currency metabolites. Our approach identifies topologically independent modules under specific conditions associated with different metabolic functions. As case studies, the E.coli iJO1366 and Human Recon 2.2 genome-scale metabolic models were split in 103 and 321 modules respectively, displaying significant correlation patterns in expression data. Finally, we addressed a fundamental question about the metabolic flexibility conferred by reversible reactions: "Of all Directed Topologies (DTs) defined by fixing directions to all reversible reactions, how many are capable of carrying flux through all reactions?". Enumeration of the DTs for iJO1366 model was performed using an efficient depth-first search algorithm, rejecting infeasible DTs based on mass-imbalanced and loopy flux patterns. We found the direction of 79% of reversible reactions must be defined before all directions in the network can be fixed, granting a high degree of flexibility.

Workers' individual and dyadic coping with the COVID-19 health emergency: A cross cultural study.

The aim of this study was to examine workers' psychological distress during the COVID-19 pandemic as a function of their individual coping, dyadic coping, and work-family conflict. We also tested the moderating role of gender and culture in these associations. To achieve this aim, we run HLM analyses on data from 1521 workers cohabiting with a partner, coming from six countries (Italy, Spain, Malta, Cyprus, Greece, and Russia) characterized by various degrees of country-level individualism/collectivism. Across all six countries, findings highlighted that work-family conflict as well as the individual coping strategy social support seeking were associated with higher psychological distress for workers, while the individual coping strategy positive attitude and common dyadic coping were found to be protective against workers' psychological distress. This latter association, moreover, was stronger in more individualistic countries.

Managing the unexpected: Bicultural identity integration during the COVID-19 emergency.

Unexpected and sudden emergency situations such as COVID-19 may render ethnic minorities particularly vulnerable to experiencing negative outcomes. Yet, we put forward that Bicultural Identity Integration (BII) - the degree to which bicultural individuals perceive their cultural identities as compatible and overlapping - may represent a resource in times of emergencies, since it may positively influence, through enhancement of psychological well-being, how bicultural individuals respond in terms of distress and coping strategies. Based on this assumption, the present study aimed at examining the relationship between BII and responses to COVID-19. N = 370 bicultural individuals (mean age = 26.83, SD = 8.74) from different cultural backgrounds were recruited online and completed measures of BII, psychological well-being, COVID-19 distress and coping strategies (positive attitudes, avoidance, social support seeking) during the second wave of the COVID-19 pandemic in Italy. We tested a model in which BII was the predictor, psychological well-being was the mediator and reactions to the COVID-19 emergency (distress, use of coping) were the outcomes. This model was tested against two alternative models. The proposed model showed a better fit to the data compared to the alternative models. In this model, psychological well-being mediated the relationship between BII (harmony) and coping strategies, except social support seeking. These findings highlight the important role played by BII in emergency situations, as it may indirectly, through enhancement of psychological well-being, contribute to enhance biculturals' adaptive reactions in terms of distress as well as affect coping strategies during highly stressful events.

Perfusion culture of Chinese Hamster Ovary cells for bioprocessing applications.

Much of the biopharmaceutical industry's success over the past 30 years has relied on products derived from Chinese Hamster Ovary (CHO) cell lines. During this time, improvements in mammalian cell cultures have come from cell line development and process optimization suited for large-scale fed-batch processes. Originally developed for high cell densities and sensitive products, perfusion processes have a long history. Driven by high volumetric titers and a small footprint, perfusion-based bioprocess research has regained an interest from academia and industry. The recent pandemic has further highlighted the need for such intensified biomanufacturing options. In this review, we outline the technical history of research in this field as it applies to biologics production in CHO cells. We demonstrate a number of emerging trends in the literature and corroborate these with underlying drivers in the commercial space. From these trends, we speculate that the future of perfusion bioprocesses is bright and that the fields of media optimization, continuous processing, and cell line engineering hold the greatest potential. Aligning in its continuous setup with the demands for Industry 4.0, perfusion biomanufacturing is likely to be a hot topic in the years to come.

Modeling apoptosis resistance in CHO cells with CRISPR-mediated knockouts of Bak1, Bax, and Bok.

Chinese hamster ovary (CHO) cells are the primary platform for the production of biopharmaceuticals. To increase yields, many CHO cell lines have been genetically engineered to resist cell death. However, the kinetics that governs cell fate in bioreactors are confounded by many variables associated with batch processes. Here, we used CRISPR-Cas9 to create combinatorial knockouts of the three known BCL-2 family effector proteins: Bak1, Bax, and Bok. To assess the response to apoptotic stimuli, cell lines were cultured in the presence of four cytotoxic compounds with different mechanisms of action. A population-based model was developed to describe the behavior of the resulting viable cell dynamics as a function of genotype and treatment. Our results validated the synergistic antiapoptotic nature of Bak1 and Bax, while the deletion of Bok had no significant impact. Importantly, the uniform application of apoptotic stresses permitted direct observation and quantification of a delay in the onset of cell death through Bayesian inference of meaningful model parameters. In addition to the classical death rate, a delay function was found to be essential in the accurate modeling of the cell death response. These findings represent an important bridge between cell line engineering strategies and biological modeling in a bioprocess context.

Bicultural identity in childhood: Preliminary validation of the Bicultural Identity Integration Scale for Children (BIIS-C).

OBJECTIVE: In the present study, we adapted and validated the Bicultural Identity Integration Scale for Children (BIIS-C). METHOD: 259 bicultural children (119 males, 140 females; Mage = 11.07, SD = 1.24) were provided with a questionnaire. Based on adult versions of the scale, we tested the factorial structure of a set of 11 nonreversed items tapping into harmony (vs. conflict; six items) and blendedness (vs. compartmentalization; five items) dimensions. RESULTS: A two-factor model was compared with a one-factor model. In line with research on adults, results showed that the two-factor model (with nine items) fitted the data better than the one-factor model. The two dimensions yielded reliable scores and were correlated in the expected direction with personality variables, acculturation attitudes, and perceived discrimination. CONCLUSIONS: The BIIS-C provides valid and reliable scores for research on biculturalism in childhood. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Ethnocultural diversity of immigrants' personal social networks, bicultural identity integration and global identification.

There is some evidence that ethnocultural diversity encourages superordinate levels of categorisation, such as feeling identified with people globally. A remaining question is what type of engagement with diversity facilitates this link and why. We use immigrants' personal social network data and examine the link between global identification and ethnocultural diversity among closer relationships (i.e. strong network contacts, such as friendships) and more distant ones (i.e. weak contacts, including neighbours and acquaintances). Furthermore, following exposure to diversity, individuals may internalise more than one culture and differ how they integrate their multiple cultural socialisation into the self (i.e. vary in their degree of bicultural identity integration). We thus test whether relational ethnocultural diversity is linked to a stronger global identification through either cultural blendedness (i.e. combining two cultures) or harmony (i.e. perceiving two cultures as compatible). Relying on a culturally diverse community sample of 216 immigrants residing in Barcelona (53% female, Mage  = 31 years, SD = 10.4), we found that ethnocultural diversity among strong (but not weak) contacts was associated with stronger global identification and that this association is mediated by cultural harmony (but not blendedness). These results attest to the link between having ethnoculturally diverse close social relationships and superordinate identification.

Hepatitis E virus infection and waste pickers: A case-control seroprevalence study.

Whether waste pickers are a risk group for hepatitis E virus (HEV) infection is largely unknown. This study aimed to determine the association between HEV exposure and the occupation of waste pickers and the work characteristics of waste pickers. An age-and gender-matched case-control seroprevalence study of 86 waste pickers and 86 control subjects of the general population was performed. We determined anti-HEV IgG antibodies in sera of cases and controls using a commercially available enzyme-linked immunoassay. The McNemar's test was used to assess the association between HEV seropositivity and the occupation of waste picker. The association between HEV seropositivity and work characteristics of waste pickers was assessed by bivariate and logistic regression analyses. Anti-HEV IgG antibodies were detected in 14 (16.3%) of the 86 waste pickers and in 8 (9.3%) of the 86 control subjects (McNemar's pair test: odds ratio (OR) = 13.0; 95% confidence interval (CI): 0.73-230.77; p = .02). Bivariate analysis showed that HEV exposure was associated with an ill status (p = .01) and reflexes impairment (p = .009). Logistic regression analysis showed that HEV seropositivity was associated with increasing age (OR = 6.52; 95% CI: 1.95-21.78; p = .002) and raising pigs (OR = 12.01; 95% CI: 1.48-97.26; p = .02). This is the first age- and gender-matched case-control study on the association between HEV infection and the occupation of waste picker. Waste pickers represent a risk group for HEV infection. Factors associated with HEV seropositivity found in this study may help in the design of optimal planning to avoid HEV infection.

'Omics driven discoveries of gene targets for apoptosis attenuation in CHO cells.

Chinese hamster ovary (CHO) cells are widely used in biopharmaceutical production. Improvements to cell lines and bioprocesses are constantly being explored. One of the major limitations of CHO cell culture is that the cells undergo apoptosis, leading to rapid cell death, which impedes reaching high recombinant protein titres. While several genetic engineering strategies have been successfully employed to reduce apoptosis, there is still room to further enhance CHO cell lines performance. 'Omics analysis is a powerful tool to better understand different phenotypes and for the identification of gene targets for engineering. Here, we present a comprehensive review of previous CHO 'omics studies that revealed changes in the expression of apoptosis-related genes. We highlight targets for genetic engineering that have reduced, or have the potential to reduce, apoptosis or to increase cell proliferation in CHO cells, with the final aim of increasing productivity.

Valorization of pelagic sargassum biomass into sustainable applications: Current trends and challenges.

Since long ago, pelagic Sargassum mats have been known to be abundant in the Sargasso Sea, where they provide habitat to diverse organisms. However, over the last few years, massive amounts of pelagic Sargassum have reached the coast of several countries in the Caribbean and West Africa, causing economic and environmental problems. Aiming for lessening the impacts of the blooms, governments and private companies remove the seaweeds from the shore, but this process results expensive. The valorization of this abundant biomass can render Sargassum tides into an economic opportunity and concurrently solve their associated environmental problems. Despite the diverse fields where algae have found applications and the relevance of this recurrent situation, Sargassum biomass remains without large scale applications. Therefore, this review aims to present the potential uses of these algae, identifying the limitations that must be assessed to effectively valorize this bioresource. Due to the constraints identified for each of the presented applications, it is concluded that a biorefinery approach should be developed to effectively valorize this abundant biomass. However, there is an urgent need for investigations focusing on holopelagic Sargassum to be able to truly valorize this seaweed.

Spectral Characteristics Related to Chemical Substructures and Structures Indicative of Organic Precursors from Fulvic Acids in Sediments by NMR and HPLC-ESI-MS.

The aim of this work was to determine Fulvic Acids (FAs) in sediments to better know their composition at the molecular level and to propose substructures and structures of organic precursors. The sediment samples were obtained from a priority area for the conservation of ecosystems and biodiversity in Mexico. FAs were extracted and purified using modifications to the International Humic Substances Society method. The characterization was carried out by 1D and 2D nuclear magnetic resonance (NMR) and high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) in positive (ESI+) and negative (ESI-) modes. Twelve substructures were proposed by the COSY and HSQC experiments, correlating with compounds likely belonging to lignin derivatives obtained from soils as previously reported. The analysis of spectra obtained by HPLC-ESI-MS indicated likely presence of compounds chemically similar to that of the substructures elucidated by NMR. FAs studied are mainly constituted by carboxylic acids, hydroxyl, esters, vinyls, aliphatics, substituted aromatic rings, and amines, presenting structures related to organic precursors, such as lignin derivatives and polysaccharides.

Alteration of Fermentative Metabolism Enhances Mucor circinelloides Virulence.

The fungus Mucor circinelloides undergoes yeast-mold dimorphism, a developmental process associated with its capability as a human opportunistic pathogen. Dimorphism is strongly influenced by carbon metabolism, and hence the type of metabolism likely affects fungus virulence. We investigated the role of ethanol metabolism in M. circinelloides virulence. A mutant in the adh1 gene (M5 strain) exhibited higher virulence than the wild-type (R7B) and the complemented (M5/pEUKA-adh1+) strains, which were nonvirulent when tested in a mouse infection model. Cell-free culture supernatant (SS) from the M5 mutant showed increased toxic effect on nematodes compared to that from R7B and M5/pEUKA-adh1+ strains. The concentration of acetaldehyde excreted by strain M5 in the SS was higher than that from R7B, which correlated with the acute toxic effect on nematodes. Remarkably, strain M5 showed higher resistance to H2O2, resistance to phagocytosis, and invasiveness in mouse tissues and induced an enhanced systemic inflammatory response compared with R7B. The mice infected with strain M5 under disulfiram treatment exhibited only half the life expectancy of those infected with M5 alone, suggesting that acetaldehyde produced by M. circinelloides contributes to the toxic effect in mice. These results demonstrate that the failure in fermentative metabolism, in the step of the production of ethanol in M. circinelloides, contributes to its virulence, inducing a more severe tissue burden and inflammatory response in mice as a consequence of acetaldehyde overproduction.

Attenuating apoptosis in Chinese hamster ovary cells for improved biopharmaceutical production.

Chinese hamster ovary (CHO) cells are the predominant host cell line for the production of biopharmaceuticals, a growing industry currently worth more than $188 billion USD in global sales. CHO cells undergo programmed cell death (apoptosis) following different stresses encountered in cell culture, such as substrate limitation, accumulation of toxic by-products, and mechanical shear, hindering production. Genetic engineering strategies to reduce apoptosis in CHO cells have been investigated with mixed results. In this review, a contemporary understanding of the real complexity of apoptotic mechanisms and signaling pathways is described; followed by an overview of antiapoptotic cell line engineering strategies tested so far in CHO cells.

A parallel-group, multicenter randomized, double-blinded, placebo-controlled, phase 2/3, clinical trial to test the efficacy of pyridostigmine bromide at low doses to reduce mortality or invasive mechanical ventilation in adults with severe SARS-CoV-2 infection: the Pyridostigmine In Severe COvid-19 (PISCO) trial protocol.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the causative agent of coronavirus disease 2019 (COVID-19), may lead to severe systemic inflammatory response, pulmonary damage, and even acute respiratory distress syndrome (ARDS). This in turn may result in respiratory failure and in death. Experimentally, acetylcholine (ACh) modulates the acute inflammatory response, a neuro-immune mechanism known as the inflammatory reflex. Recent clinical evidence suggest that electrical and chemical stimulation of the inflammatory reflex may reduce the burden of inflammation in chronic inflammatory diseases. Pyridostigmine (PDG), an ACh-esterase inhibitor (i-ACh-e), increases the half-life of endogenous ACh, therefore mimicking the inflammatory reflex. This clinical trial is aimed at evaluating if add-on of PDG leads to a decrease of invasive mechanical ventilation and death among patients with severe COVID-19. METHODS: A parallel-group, multicenter, randomized, double-blinded, placebo-controlled, phase 2/3 clinical trial to test the efficacy of pyridostigmine bromide 60 mg/day P.O. to reduce the need for invasive mechanical ventilation and mortality in hospitalized patients with severe COVID-19. DISCUSSION: This study will provide preliminary evidence of whether or not -by decreasing systemic inflammation- add-on PDG can improve clinical outcomes in patients with severe COVID-19. TRIAL REGISTRATION: ClinicalTrials.gov NCT04343963 (registered on April 14, 2020).

Preparation and mechanistic studies of 2-substituted Bisthiazolidines by imine exchange.

Bisthiazolidines (BTZ) are bicyclic compounds considered penicillin analogs that inhibit the full range of Metallo-ß-Lactamases (MBLs) and potentiate ß-lactam activity against resistant bacteria. Herein we present a new methodology to prepare 2-substituted bisthiazolidines by aldehyde exchange. Thirteen new bisthiazolidines were prepared using this methodology, with yields ranged from 31 to 75%. The reaction is based on in situ imines formation, which are able to exchange side chains. The reaction intermediates were studied based on NMR experiments and a key imine 1b-II could be detected in the reaction mixture. Furthermore, a DFT computational analysis was performed to gain insights into the reaction mechanism, allowing us to unveil the different pathways and their activation barriers within the synthetic route. The results suggest that the most favorable route involve the formation of the thiazolidine 1b-III by i) a N-assisted N-C bond cleavage, and ii) a thiol-mediated 5 endo-trig cyclization followed by a C-N bond cleavage. In contrast with previously reported evidence, the imine metathesis was discarded as a plausible pathway. Finally, the reaction of 1b-III with aldehyde 2a leads to bicycle 4a via the iminium ion 1b-V.

Low prevalence of anti-hepatitis E virus IgG antibodies in Tepehuanos in Mexico.

INTRODUCTION AND OBJECTIVE: The epidemiology of infection with hepatitis E virus (HEV) in Tepehuanos (a Mexican ethnic group living in rural areas) is largely unknown. This study aimed to determine the seroprevalence of and risk factors associated with HEV infection in Tepehuanos in Durango, Mexico, and to compare this seroprevalence with that in non-Tepehuanos. MATERIALS AND METHODS: Through a case-control seroprevalence study, we studied 146 Tepehuanos and 146 age- and gender-matched control subjects of the general population from rural settings. The frequency of anti-HEV IgG antibodies was determined using an enzyme-linked immunoassay. Bivariate and multivariate analyses were used to assess the association between seropositivity and socio-demographic, clinical and behavioral characteristics of the Tepehuanos. RESULTS: IgG antibodies against HEV were found in 5 (3.4%; 95% CI: 1.1-7.8) of 146 Tepehuanos and in 46 (31.5%; 95% CI: 24.1-39.7) of 146 control subjects (OR=0.01; 95% CI: 0.0007-0.20; P<0.000001). Bivariate analysis showed that HEV seropositivity was associated with age, consumption of meat from goat, sheep, boar, turkey and pigeon, and concrete flooring at home. However, these variables were no longer significant when analyzed by logistic regression. CONCLUSIONS: This is first study on the epidemiology of HEV exposure in Tepehuanos. We demonstrated serological evidence of HEV infection in this ethnic group. The seroprevalence of HEV exposure in Tepehuanos is low as compared with that found in non-Tepehuano people living in rural Durango. Further studies to determine the risk factors associated with HEV exposure in Tepehuanos are needed.