A photoswitchable GPCR-based opsin for presynaptic inhibition

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Addressing amino acid-derived inhibitory metabolites and enhancing CHO cell culture performance through DOE-guided media modifications

Previously, we identified six inhibitory metabolites (IMs) accumulating in Chinese hamster ovary (CHO) cultures using AMBIC 1.0 community reference medium that negatively impacted culture performance. The goal of the current study was to modify the medium to control IM accumulation through design of experiments (DOE). Initial over-supplementation of precursor amino acids (AAs) by 100% to 200% in the culture medium revealed positive correlations between initial AA concentrations and IM levels. A screening design identified 5 AA targets, Lys, Ile, Trp, Leu, Arg, as key contributors to IMs. Response surface design analysis was used to reduce initial AA levels between 13% and 33%, and these were then evaluated in batch and fed-batch cultures. Lowering AAs in basal and feed medium and reducing feed rate from 10% to 5% reduced inhibitory metabolites HICA and NAP by up to 50%, MSA by 30%, and CMP by 15%. These reductions were accompanied by a 13% to 40% improvement in peak viable cell densities and 7% to 50% enhancement in IgG production in batch and fed-batch processes, respectively. This study demonstrates the value of tuning specific AA levels in reference basal and feed media using statistical design methodologies to lower problematic IMs.     

Chemical inhibitors of hexokinase-2 enzyme reduce lactate accumulation,alter glycosylation processing,and produce altered glycoforms in CHO cell cultures

Chinese hamster ovary (CHO) cells, predominant hosts for recombinant biotherapeutics production, generate lactate as a major glycolysis by-product. High lactate levels adversely impact cell growth and productivity. The goal of this study was to reduce lactate in CHO cell cultures by adding chemical inhibitors to hexokinase-2 (HK2), the enzyme catalyzing the conversion of glucose to glucose 6-phosphate, and examine their impact on lactate accumulation, cell growth, protein titers, and N-glycosylation. Five inhibitors of HK2 enzyme at different concentrations were evaluated, of which 2-deoxy- d -glucose (2DG) and 5-thio- d -glucose (5TG) successfully reduced lactate accumulation with only limited impacts on CHO cell growth. Individual 2DG and 5TG supplementation led to a 35%–45% decrease in peak lactate, while their combined supplementation resulted in a 60% decrease in peak lactate. Inhibitor supplementation led to at least 50% decrease in moles of lactate produced per mol of glucose consumed. Recombinant EPO-Fc titers peaked earlier relative to the end of culture duration in supplemented cultures leading to at least 11% and as high as 32% increase in final EPO-Fc titers. Asparagine, pyruvate, and serine consumption rates also increased in the exponential growth phase in 2DG and 5TG treated cultures, thus, rewiring central carbon metabolism due to low glycolytic fluxes. N-glycan analysis of EPO-Fc revealed an increase in high mannose glycans from 5% in control cultures to 25% and 37% in 2DG and 5TG-supplemented cultures, respectively. Inhibitor supplementation also led to a decrease in bi-, tri-, and tetra-antennary structures and up to 50% lower EPO-Fc sialylation. Interestingly, addition of 2DG led to the incorporation of 2-deoxy-hexose (2DH) on EPO-Fc N-glycans and addition of 5TG resulted in the first-ever observed N-glycan incorporation of 5-thio-hexose (5TH). Six percent to 23% of N-glycans included 5TH moieties, most likely 5-thio-mannose and/or 5-thio-galactose and/or possibly 5-thio-N-acetylglucosamine, and 14%–33% of N-glycans included 2DH moieties, most likely 2-deoxy-mannose and/or 2-deoxy-galactose, for cultures treated with different concentrations of 5TG and 2DG, respectively. Our study is the first to evaluate the impact of these glucose analogs on CHO cell growth, protein production, cell metabolism, N-glycosylation processing, and formation of alternative glycoforms.     

Mass Spectral Analysis of Synthetic Peptides: Implications in Proteomics

Sequence determination of peptides is a crucial step in mass spectrometry–based proteomics. Peptide sequences are determined either by database search or by de novo sequencing using tandem mass spectrometry. Determination of all the theoretical expected peptide fragments and eliminating false discoveries remains a challenge in proteomics. Developing standards for evaluating the performance of mass spectrometers and algorithms used for identification of proteins is important for proteomics studies. The current study is focused on these aspects by using synthetic peptides. A total of 599 peptides were designed from in silico tryptic digest with 1 or 2 missed cleavages from 199 human proteins, and synthetic peptides corresponding to these sequences were obtained. The peptides were mixed together, and analysis was carried out using liquid chromatography–electrospray ionization tandem mass spectrometry on a Q-Exactive HF mass spectrometer. The peptides and proteins were identified with SEQUEST program. The analysis was carried out using the proteomics workflows. A total of 573 peptides representing 196 proteins could be identified, and a spectral library was created for these peptides. Analysis parameters such as “no enzyme selection” gave the maximum number of detected peptides as compared with trypsin in the selection. False discoveries could be identified. This study highlights the limitations of peptide detection and the need for developing powerful algorithms along with tools to evaluate mass spectrometers and algorithms. It also shows the limitations of peptide detection even with high-end mass spectrometers. The mass spectral data are available in ProteomeXchange with accession no. PXD017992.     

Probiotics for preterm infants – time to end all controversies

Mortality, necrotising enterocolitis (NEC), late onset sepsis (LOS) and feeding intolerance are significant issues for very preterm (< 32 weeks) and extremely preterm (< 28 weeks) infants. The complications of ≥ Stage II NEC [e.g. Resection of the gangrenous gut, survival with intestinal failure, recurrent infections, prolonged hospital stay, and long-term neurodevelopmental impairment (NDI)] impose a significant health burden. LOS also carries significant burden including long-term NDI due to adverse effects of inflammation on the preterm brain during the critical phase of development. Frequent stopping of feeds due to feeding intolerance is a significant iatrogenic contributor to postnatal growth failure in extremely preterm infants. Over 25 systematic reviews and meta-analyses of RCTs (~12 000 participants) have reported that probiotics significantly reduce the risk of all-cause mortality, NEC ≥ Stage II, LOS and feeding intolerance in preterm infants. Systematic reviews and meta-analysis of non-RCTs have also shown that the benefits after adopting probiotics as a standard prophylaxis for preterm infants are similar to those reported in RCTs. No intervention comes close to probiotics when it comes to significant reduction in death, NEC, LOS and feeding intolerance at a cost of less than a dollar a day irrespective of the setting and baseline incidence of NEC. The common controversies that are preventing the rapid uptake of probiotics for preterm infants are addressed in this paper.     

A model for the regulation of expression of the potassium-transport operon,kdp, inEscherichia coli

The intracellular concentration of K+in Escherichia coli is known to be determined by osmolarity of the growth medium, and it is believed that the expression of the potassium-transport operon, kdp, is controlled by the turgor pressure differential between the cytoplasm and the extracellular environment. Several lines of evidence, however, argue against a strict turgor-regulation model for kdp expression. Instead, it is proposed here that kdp is controlled by one fraction of intracellular [K+], and that the size of this fraction is independent of the osmolarity of the culture medium.