Amino acid feeding reduces ammonia production through rearrangement of metabolic fluxes in central carbon metabolism of CHO cells

Ammonia is a toxic byproduct of CHO cell metabolism, which inhibits cell growth, reduces cell viability, alters glycosylation, and decreases recombinant protein productivity. In an attempt to minimize the ammonium accumulation in cell culture media, different amino acids were added individually to the culture medium before the production phase to alleviate the negative effects of ammonium on cell culture performance. Among all the amino acids examined in this study, valine showed the most positive impact on CHO cell culture performance. When the cultured CHO cells were fed with 5 mM valine, EPO titer was increased by 25% compared to the control medium, and ammonium and lactate production were decreased by 23 and 26%, respectively, relative to the control culture. Moreover, the sialic acid content of the EPO protein in valine-fed culture was higher than in the control culture, most likely because of the lower ammonium concentration. Flux balance analysis (FBA) results demonstrated that the citric acid cycle was enriched by valine feeding. The analysis revealed that there might be a link between promoting tricarboxylic acid (TCA) cycle metabolism in valine-fed culture and reduction in lactate and ammonia accumulation. Furthermore, in valine-fed culture, FBA outcomes showed that alanine was excreted into the medium as the primary mechanism for reducing ammonium concentration. It was predicted that the elevated TCA cycle metabolism was concurrent with an increment in recombinant protein production. Taken together, our data demonstrate that valine addition could be an effective strategy for mitigating the negative impacts of ammonium and enhancing glycoprotein production in both quality and quantity.

Amino acid feeding reduces ammonia production through rearrangement of metabolic fluxes in central carbon metabolism of CHO cells

Ammonia is a toxic byproduct of CHO cell metabolism, which inhibits cell growth, reduces cell viability, alters glycosylation, and decreases recombinant protein productivity. In an attempt to minimize the ammonium accumulation in cell culture media, different amino acids were added individually to the culture medium before the production phase to alleviate the negative effects of ammonium on cell culture performance. Among all the amino acids examined in this study, valine showed the most positive impact on CHO cell culture performance. When the cultured CHO cells were fed with 5 mM valine, EPO titer was increased by 25% compared to the control medium, and ammonium and lactate production were decreased by 23 and 26%, respectively, relative to the control culture. Moreover, the sialic acid content of the EPO protein in valine-fed culture was higher than in the control culture, most likely because of the lower ammonium concentration. Flux balance analysis (FBA) results demonstrated that the citric acid cycle was enriched by valine feeding. The measurement of TCA cycle activity supported this finding. The analysis revealed that there might be a link between promoting tricarboxylic acid (TCA) cycle metabolism in valine-fed culture and reduction in lactate and ammonia accumulation. Furthermore, in valine-fed culture, FBA outcomes showed that alanine was excreted into the medium as the primary mechanism for reducing ammonium concentration. It was predicted that the elevated TCA cycle metabolism was concurrent with an increment in recombinant protein production. Taken together, our data demonstrate that valine addition could be an effective strategy for mitigating the negative impacts of ammonium and enhancing glycoprotein production in both quality and quantity.

Operational performance improvement through continuous improvement initiatives in micro-enterprises of Turkey

PurposeMicro-enterprises have a vital role in the development of a nation by creating employment, encouraging entrepreneurship, balancing social income and educating the workforce. Yet, micro-enterprises face significant operational challenges such as low productivity, high production costs and long changeover times. These challenges are often overlooked by researchers and practitioners but have a drastic impact on micro-enterprises' operational performance. With over 95% of the economy consisting of micro-enterprises, it is vital to improve operational performance and competitiveness of the micro-enterprises.Design/methodology/approachConsidering the data availability and practical challenges of gathering data from micro-enterprises, an action research methodology was selected as a suitable research method. Following the initial diagnostic visits, planned interventions were prepared and results were observed to gather data and draw conclusions.FindingsFindings suggest that commonly adapted performance improvement initiatives by large enterprises cannot be directly applied in micro-enterprises to enhance operational performance. Micro-enterprises lack the critical resources and company culture to easily adapt these initiatives. On the other hand, when these performance improvement initiatives are filtered according to specific needs of micro-enterprises, continuous improvement initiatives were often found to be effective in improving operational performance. Specifically, 5S, single-minute exchange of dies (SMED), suggestion schemes, layout improvements, management coaching, visual management, empowerment were found to be useful and suitable to address command and control culture, low productivity, unorganised workspace, undelegated authority, low skilled employees and unwillingness to take responsibility problems.Research limitations/implicationsFurther research is required to investigate the specific factors that affect the adaptation of continuous improvement initiatives such as culture and industry type.Originality/valueThis research extends and contributes the current literature on continuous improvement initiatives by revealing how performance improvement initiatives need to be filtered according to the specific needs of micro-enterprises, and how these initiatives can be used to address specific problems to improve operational efficiency. It provides a conceptual framework to guide the decision-making process on operational performance improvement in micro-enterprises.

Stimulation of Treg Cells to Inhibit Osteoclastogenesis in Gorham-Stout Disease

Gorham-Stout disease (GSD) is a very rare syndrome displaying excessive bone erosion and vascular lesion. Due to the rarity of the disease and to the limited studies, its etiopathogenesis is not entirely known. The involvement of immune system in the progressive osteolysis was recently suggested. Indeed, extensive reciprocal interactions between the immune and skeletal systems have been demonstrated. This study aimed to evaluate alterations of immune cells in GSD. An increase of CD8+ cells and reduction of CD4+ and CD4+CD25+CD127low cells was revealed in patients. Interestingly, patients’ regulatory T cells maintain the ability to respond to extracellular stimuli and to regulate osteoclastogenesis; GSD cells proliferate under aCD3/CD28 signal reaching similar levels to those observed in control culture and exert their immunomodulatory activity on effector T cells. GSD Treg cells preserved their inhibitory effects on the osteoclastogenesis. These results suggest that stimulation of Treg cells could open the way for the identification and testing of new therapeutic approaches for patients affected by GSD.

Image analysis for the automatic phenotyping of Orobanche cumana tubercles on sunflower roots

Background The parasitic plant Orobanche cumana is one of the most important threats to sunflower crops in Europe. Resistant sunflower varieties have been developed, but new O. cumana races have evolved and have overcome introgressed resistance genes, leading to the recurrent need for new resistance methods. Screening for resistance requires the phenotyping of thousands of sunflower plants to various O. cumana races. Most phenotyping experiments have been performed in fields at the later stage of the interaction, requiring time and space. A rapid phenotyping screening method under controlled conditions would need less space and would allow screening for resistance of many sunflower genotypes. Our study proposes a phenotyping tool for the sunflower/O. cumana interaction under controlled conditions through image analysis for broomrape tubercle analysis at early stages of the interaction. Results We optimized the phenotyping of sunflower/O. cumana interactions by using rhizotrons (transparent Plexiglas boxes) in a growth chamber to control culture conditions and Orobanche inoculum. We used a Raspberry Pi computer with a picamera for acquiring images of inoculated sunflower roots 3 weeks post inoculation. We set up a macro using ImageJ free software for the automatic counting of the number of tubercles. This phenotyping tool was named RhizOSun. We evaluated five sunflower genotypes inoculated with two O. cumana races and showed that automatic counting of the number of tubercles using RhizOSun was highly correlated with manual time-consuming counting and could be efficiently used for screening sunflower genotypes at the tubercle stage. Conclusion This method is rapid, accurate and low-cost. It allows rapid imaging of numerous rhizotrons over time, and it enables image tracking of all the data with time kinetics. This paves the way toward automatization of phenotyping in rhizotrons that could be used for other root phenotyping, such as symbiotic nodules on legumes.

Food Safety and Employee Health Implications of COVID-19

The COVID-19 pandemic has greatly impacted the US food supply and consumer behavior. Food production and processing are being disrupted as illnesses, proactive quarantines, and government-mandated movement restrictions cause labor shortages. In this environment, the food industry has been required to adopt new, additional practices to minimize the risk of COVID-19 cases and outbreaks among its workforce. Successfully overcoming these challenges requires a comprehensive approach that addresses COVID-19 transmission both within and outside the facility; possible interventions include strategies to (i) vaccinate employees, (ii) assure that employees practice social distancing, (iii) assure that employees wear face coverings, (iv) screen employees for COVID-19 (v) assure that employees practice frequent handwashing and avoid touching their faces, (vi) clean frequently touched surfaces, and (vii) assure proper ventilation. Compliance with these control strategies needs to be verified and an overall “COVID-19 control culture” needs to be established to facilitate an effective program. Despite some public misperceptions about SARS-CoV-2 presence on foods or food packaging representing a public health risk, it is important to note that both the virus’ biology and epidemiological data clearly support a negligible risk of COVID-19 transmission through food and food packing. However, COVID-19 pandemic related supply chain and workforce disruptions, as well as the shift in resources to protect food industry employees from COVID-19 may increase the actual food safety risks. The goal of this paper is to review the COVID-19 mitigation practices adopted by the food industry, and the potential impact of these practices and COVID-19 related disruptions on the industry’s food safety mission. A review of these impacts is necessary to ensure that the food industry is prepared to maintain a safe and nutritious food supply in the face of future global disruptions.

Manifestations and environmental implications of microbially-induced calcium carbonate precipitation (MICP) by the cyanobacterium Dolichospermum flosaquae

The aim of this work is to explore the ability and magnitude of the temperate cyanobacterium Dolichospermum flosaquae in microbially-induced calcium carbonate precipitation (MICP). Environmentally, MICP controls the availability of calcium, carbon and phosphorus in freshwater lakes and simultaneously controls carbon exchange with the atmosphere. Cultures of flosaquae were grown in BG11 medium containing 0, 1, 1.5, 2 and 4 mg Ca2+ L−1, as cardinal concentrations previously reported in freshwater lakes, in addition to a control culture (BG11 containing 13 mg Ca2+ L−1). Growth (cell number, chlorophyll a, and protein content) of D. flosaquae was generally reduced by elevating calcium concentrations of the different salts used (chloride, acetate, or citrate). D. flosaquae exhibited its ability to perform MICP as carbonate alkalinity was sharply increased up to its highest level (six times that of the control) at a citrate concentration of 4 mg Ca2+ L−1. Calcium carbonate was formed at a pre-precipitation stage as the minimum pH necessary for precipitation (8.7) has been scarcely approached under such conditions. In this work, MICP took place mostly empowered by photosynthesis and respiration. Residual calcium exhibited its lowest value at 4 mg Ca2+ citrate L−1, coinciding with the highest alkalinity level. Precipitated calcium was increased with chlorophyll a content, but not with increasing cell numbers.

Justice-System Monitoring Technologies and Victim Welfare

The evolution of criminal justice technologies is inextricably linked to the emergence of new modes of electronic and digital governance that have become essential components of a surveillance and crime control culture continually seeking out novel responses to actual and perceived threats. The slow emergence of these technologies in the second part of the 20th century was often theorized through a discourse of order and control that has subsequently evolved in the 21st century to emphasize the protective potential of technologies oriented toward the interests of victims. The potential of criminal justice technologies to improve public safety and address issues of repeat victimization has now been subjected to significant scrutiny from scholars across the globe. While it would be conceptually inaccurate to split offenders and victims into two discrete groups, there has been an increase in analytical focus upon the intersections between victims of crime and technology within the context of criminal justice processes that had traditionally been oriented toward offenders. A more sophisticated understanding of the psychological and behavioral potential of criminal justice technologies has emerged that has permanently adjusted the landscape of crime and disorder management and has had a transformative impact upon the relationship between victims, technology, and criminal justice. Yet, at the same time, the integration of digital technologies into the crime control and criminal justice infrastructure still is at an early stage in its evolution, with future trends and patterns uncertain.

Starch Production in Chlamydomonas reinhardtii through Supraoptimal Temperature in a Pilot-Scale Photobioreactor

An increase in temperature can have a profound effect on the cell cycle and cell division in green algae, whereas growth and the synthesis of energy storage compounds are less influenced. In Chlamydomonas reinhardtii, laboratory experiments have shown that exposure to a supraoptimal temperature (39 °C) causes a complete block of nuclear and cellular division accompanied by an increased accumulation of starch. In this work we explore the potential of supraoptimal temperature as a method to promote starch production in C. reinhardtii in a pilot-scale photobioreactor. The method was successfully applied and resulted in an almost 3-fold increase in the starch content of C. reinhardtii dry matter. Moreover, a maximum starch content at the supraoptimal temperature was reached within 1–2 days, compared with 5 days for the control culture at the optimal temperature (30 °C). Therefore, supraoptimal temperature treatment promotes rapid starch accumulation and suggests a viable alternative to other starch-inducing methods, such as nutrient depletion. Nevertheless, technical challenges, such as bioreactor design and light availability within the culture, still need to be dealt with.

Modification of heavy metals toxicity by Cyanobacteria Nostoc sp. N27P72 and Nostoc sp. FB71 in Culture Conditions

Purpose: Cyanobacteria are ecologically relevant prokaryotes that can be found in environments contaminated with heavy metals. As their photosynthetic machinery imposes high demands for metals, homeostasis of these micronutrients has been extensively considered in cyanobacteria. So far, most studies have focused on treatment of wastewaters using microalgae leads to remarkable reduction of an array of organic and inorganic nutrients, but what takes place in the extracellular environment when cells are exposed to external supplementation with heavy metals remains largely unknown. Methods: Here, extracellular polymeric substances (EPS) production in strains Nostoc sp. N27P72 and Nostoc sp. FB71 isolated from different habitats are reported and compared. Cultures of both strains, supplemented with either glucose, sucrose, lactose or maltose showed that production of EPS and cell dry weight was boosted by maltose supplementation. Result: Nostoc sp. N27P72 which was isolated from lime stones was higher, resulting in 9.1 ± 0.05 µg/ml and 1.01 ± 0.06 g/l in EPS and cell dry. The cell cultures tested for their ability to remove Cu(II), Cr(III) and Ni(II) in media culture containing the maltose and without maltose as control culture. Remarkably, we showed that although these elements can be toxic, supplementing the media culture can effectively sequester their toxic effects by increasing the production of EPSs, carbohydrates and total soluble proteins in comparison to control. The crude EPS showed metal adsorption capacity assuming the order Ni(II)> Cu(II)> Cr(III) from metal-binding experiments. Nickel was preferentially biosorbed with a maximal uptake of 188.8 ± 0.14 mg (g cell dry wt) -1 crude EPS. FT-IR spectroscopy revealed treatment with Ni made changes in the functional groups and glycoside linkages in both strains. Results of Gas Chromatography Mass Spectrometry (GC–MS) to determine the biochemical composition of Nostoc sp. N27P72 showed that strong Ni(II) removal capability is suspected to be associated with the high Cyclotrisiloxane and 1,2-Benzenedicarboxylic acid content. Conclusion: The results of these investigates specified that strains Nostoc sp. N27P72 is good candidates for the commercial production of EPS and might be utilized in bioremediation field as an alternative to synthetic and abiotic flocculants.