Protective effect of oleic acid against very long-chain fatty acid-induced apoptosis in peroxisome-deficient CHO cells.

Very long-chain fatty acids (VLCFAs) are degraded exclusively in peroxisomes, as evidenced by the accumulation of VLCFAs in patients with certain peroxisomal disorders. Although accumulation of VLCFAs is considered to be associated with health issues, including neuronal degeneration, the mechanisms underlying VLCFAs-induced tissue degeneration remain unclear. Here, we report the toxic effect of VLCFA and protective effect of C18: 1 FA in peroxisome-deficient CHO cells. We examined the cytotoxicity of saturated and monounsaturated VLCFAs with chain-length at C20-C26, and found that longer and saturated VLCFA showed potent cytotoxicity at lower accumulation levels. Furthermore, the extent of VLCFA-induced toxicity was found to be associated with a decrease in cellular C18:1 FA levels. Notably, supplementation with C18:1 FA effectively rescued the cells from VLCFA-induced apoptosis without reducing the cellular VLCFAs levels, implying that peroxisome-deficient cells can survive in the presence of accumulated VLCFA, as long as the cells keep sufficient levels of cellular C18:1 FA. These results suggest a therapeutic potential of C18:1 FA in peroxisome disease and may provide new insights into the pharmacological effect of Lorenzo's oil, a 4:1 mixture of C18:1 and C22:1 FA.

Proliferation of mammalian cells with Chlorococcum littorale algal compounds without serum support.

In recent years, serum-free medium for mammalian cell cultivation has attracted a lot of attention, considering the high cost of production and environmental load involved in developing the conventional animal sera. The use of alternative growth-promoting products in mammalian cell cultivation such as extracts from microalgae has proven to be quite beneficial and environmental-friendly. This research aims to cultivate mammalian cells with growth-promoting factors derived from Chlorococcum littorale. We have established a simple extraction using the ultrasonication method and applied the extract in place of serum on mammalian C2C12 cell lines, 3T3 cell lines, and CHO cell lines to compare and analyze the effectiveness of the extract. Cell passage was conducted in a suspended culture condition with the addition of the extract. The results indicate that the extract from microalgae shows a high proliferation rate in all cell lines without fetal bovine serum. Moreover, it is eco-friendly and has huge potential to replace the traditional cell culture system. It could be applied in the fields of regenerative medicine, gene/cell therapies, as well as cultured meat production.

Design, optimization, production and activity testing of recombinant immunotoxins expressed in plants and plant cells for the treatment of monocytic leukemia.

Antibody-drug conjugates (ADCs) can improve therapeutic indices compared to plain monoclonal antibodies (mAbs). However, ADC synthesis is complex because the components are produced separately in CHO cells (mAb) and often by chemical synthesis (drug). They are individually purified, coupled, and then the ADC is purified, increasing production costs compared to regular mAbs. In contrast, it is easier to produce recombinant fusion proteins consisting of an antibody derivative, linker and proteinaceous toxin, i.e. a recombinant immunotoxin (RIT). Plants are capable of the post-translational modifications needed for functional antibodies and can also express active protein toxins such as the recombinant mistletoe lectin viscumin, which is not possible in prokaryotes and mammalian cells respectively. Here, we used Nicotiana benthamiana and N. tabacum plants as well as tobacco BY-2 cell-based plant cell packs (PCPs) to produce effective RITs targeting CD64 as required for the treatment of myelomonocytic leukemia. We compared RITs with different subcellular targeting signals, linkers, and proteinaceous toxins. The accumulation of selected candidates was improved to ~ 40 mg kg-1 wet biomass using a design of experiments approach, and corresponding proteins were isolated with a purity of ~ 80% using an optimized affinity chromatography method with an overall yield of ~ 84%. One anti-CD64 targeted viscumin-based drug candidate was characterized in terms of storage stability and cytotoxicity test in vitro using human myelomonocytic leukemia cell lines. We identified bottlenecks in the plant-based expression platform that require further improvement and assessed critical process parameters that should be considered during process development for plant-made RITs.

Toxin type and domain sequence affect accumulation of recombinant immunotoxins.Transient expression in plant cell packs and intact plants correlates well.IC₅₀ values of toxicity correlate with the cell surface receptor concentration.

Automated Raman feed-back control of multiple supplemental feeds to enable an intensified high inoculation density fed-batch platform process.

Biologics manufacturing is increasingly moving toward intensified processes that require novel control strategies in order to achieve higher titers in shorter periods of time compared to traditional fed-batch cultures. In order to implement these strategies for intensified processes, continuous process monitoring is often required. To this end, inline Raman spectroscopy was used to develop partial least squares models to monitor changes in residual concentrations of glucose, phenylalanine and methionine during the culture of five different glutamine synthetase piggyBac® Chinese hamster ovary clones cultured using an intensified high inoculation density fed-batch platform process. Continuous monitoring of residual metabolite concentrations facilitated automated feed-rate adjustment of three supplemental feeds to maintain glucose, phenylalanine, and methionine at desired setpoints, while maintaining other nutrient concentrations at acceptable levels across all clones cultured on the high inoculation density platform process. Furthermore, all clones cultured on this process achieved high viable cell concentrations over the course of culture, indicating no detrimental impacts from the proposed feeding strategy. Finally, the automated control strategy sustained cultures inoculated at high cell densities to achieve product concentrations between 5 and 8.3 g/L over the course of 12 days of culture.

Cell-Free Synthesis and Electrophysiological Analysis of Multipass Voltage-Gated Ion Channels Tethered in Microsomal Membranes.

Cell-free protein synthesis (CFPS) has emerged as a powerful tool for the rapid synthesis and analysis of various structurally and functionally distinct proteins. These include 'difficult-to-express' membrane proteins such as large multipass ion channel receptors. Owing to their membrane localization, eukaryotic CFPS supplemented with endoplasmic reticulum (ER)-derived microsomal vesicles has proven to be an efficient system for the synthesis of functional membrane proteins. Here we demonstrate the applicability of the eukaryotic cell-free systems based on lysates from the mammalian Chinese Hamster Ovary (CHO) and insect Spodoptera frugiperda (Sf21) cells. We demonstrate the efficiency of the systems in the de novo cell-free synthesis of the human cardiac ion channels: ether-a-go-go potassium channel (hERG) KV11.1 and the voltage-gated sodium channel hNaV1.5.

Trace metal optimization in CHO cell culture through statistical design of experiments.

A majority of the biotherapeutics industry today relies on the manufacturing of monoclonal antibodies from Chinese hamster ovary (CHO) cells, yet challenges remain with maintaining consistent product quality from high-producing cell lines. Previous studies report the impact of individual trace metal supplemental on CHO cells, and thus, the combinatorial effects of these metals could be leveraged to improve bioprocesses further. A three-level factorial experimental design was performed in fed-batch shake flasks to evaluate the impact of time wise addition of individual or combined trace metals (zinc and copper) on CHO cell culture performance. Correlations among each factor (experimental parameters) and response variables (changes in cell culture performance) were examined based on their significance and goodness of fit to a partial least square's regression model. The model indicated that zinc concentration and time of addition counter-influence peak viable cell density and antibody production. Meanwhile, early copper supplementation influenced late-stage ROS activity in a dose-dependent manner likely by alleviating cellular oxidative stress. Regression coefficients indicated that combined metal addition had less significant impact on titer and specific productivity compared to zinc addition alone, although titer increased the most under combined metal addition. Glycan analysis showed that combined metal addition reduced galactosylation to a greater extent than single metals when supplemented during the early growth phase. A validation experiment was performed to confirm the validity of the regression model by testing an optimized setpoint of metal supplement time and concentration to improve protein productivity.

Tuning the immune response: sulfated archaeal glycolipid archaeosomes as an effective vaccine adjuvant for induction of humoral and cell-mediated immunity towards the SARS-CoV-2 Omicron variant of concern.

Liposomes composed of sulfated lactosyl archaeol (SLA) have been shown to be a safe and effective vaccine adjuvant with a multitude of antigens in preclinical studies. In particular, SLA-adjuvanted SARS-CoV-2 subunit vaccines based on trimeric spike protein antigens were shown to be immunogenic and efficacious in mice and hamsters. With the continued emergence of SARS-CoV-2 variants, we sought to evaluate next-generation vaccine formulations with an updated antigenic identity. This was of particular interest for the widespread Omicron variant, given the abundance of mutations and structural changes observed within its spike protein compared to other variants. An updated version of our resistin-trimerized SmT1 corresponding to the B.1.1.529 variant was successfully generated in our Chinese Hamster Ovary (CHO) cell-based antigen production platform and characterized, revealing some differences in protein profile and ACE2 binding affinity as compared to reference strain-based SmT1. We next evaluated this Omicron-based spike antigen for its immunogenicity and ability to generate robust antigen-specific immune responses when paired with SLA liposomes or AddaS03 (a mimetic of the AS03 oil-in-water emulsion adjuvant system found in commercialized SARS-CoV-2 protein vaccines). Immunization of mice with vaccine formulations containing this updated antigen with either adjuvant stimulated neutralizing antibody responses favouring Omicron over the reference strain. Cell-mediated responses, which play an important role in the neutralization of intracellular infections, were induced to a much higher degree with the SLA adjuvant relative to the AddaS03-adjuvanted formulations. As such, updated vaccines that are better capable of targeting towards SARS-CoV-2 variants can be generated through an optimized combination of antigen and adjuvant components.

Wuzi Yanzong Pill protects neural tube defects by activating PI3K/Akt signaling pathway.

Neural tube defects (NTDs) are severe congenital malformations that can lead to lifelong disability. Wuzi Yanzong Pill (WYP) is an herbal formula of traditional Chinese medicine (TCM) that has been shown to have a protective effect against NTDs in a rodent model induced by all-trans retinoic acid (atRA), but the mechanism remains unclear. In this study, the neuroprotective effect and mechanism of WYP on NTDs were investigated in vivo using an atRA-induced mouse model and in vitro using cell injury model induced by atRA in Chinese hamster ovary (CHO) cells and Chinese hamster dihydrofolate reductase-deficient (CHO/dhFr) cells. Our findings suggest that WYP has an excellent preventive effect on atRA-induced NTDs in mouse embryos, which may be related to the activation of the PI3K/Akt signaling pathway, improved embryonic antioxidant capacity, and anti-apoptotic effects, and this effect is not dependent on folic acid (FA). Our results demonstrated that WYP significantly reduced the incidence of NTDs induced by atRA; increased the activity of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and content of glutathione (GSH); decreased the apoptosis of neural tube cells; up-regulated the expression of phosphatidylinositol 3 kinase (PI3K), phospho protein kinase B (p-Akt), nuclear factor erythroid-2 related factor (Nrf2), and b-cell lymphoma-2 (Bcl-2); and down-regulated the expression of bcl-2-associated X protein (Bax). Our in vitro studies suggested that the preventive effect of WYP on atRA-treated NTDs was independent of FA, which might be attributed to the herbal ingredients of WYP. The results suggest that WYP had an excellent prevention effect on atRA-induced NTDs mouse embryos, which may be independent of FA but related to the activation of the PI3K/Akt signaling pathway and improvement of embryonic antioxidant capacity and anti-apoptosis.

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.

Next generation CANCAN focusing for remote stimulation by nanosecond electric pulses.

Focusing electric pulse effects away from electrodes is a challenge because the electric field weakens with distance. Previously we introduced a remote focusing method based on bipolar cancellation, a phenomenon of low efficiency of bipolar nanosecond electric pulses (nsEP). Superpositioning two bipolar nsEP into a unipolar pulse canceled bipolar cancellation ("CANCAN" effect), enhancing bioeffects at a distance despite the electric field weakening. Here, we introduce the next generation (NG) CANCAN focusing with unipolar nsEP packets designed to produce bipolar waveforms near electrodes (suppressing electroporation) but not at the remote target. NG-CANCAN was tested in CHO cell monolayers using a quadrupole electrode array and labeling electroporated cells with YO-PRO-1 dye. We routinely achieved 1.5-2 times stronger electroporation in the center of the quadrupole than near electrodes, despite a 3-4-fold field attenuation. With the array lifted 1-2 mm above the monolayer (imitating a 3D treatment), the remote effect was enhanced up to 6-fold. We analyzed the role of nsEP number, amplitude, rotation, and inter-pulse delay, and showed how remote focusing is enhanced when re-created bipolar waveforms exhibit stronger cancellation. Advantages of NG-CANCAN include the exceptional versatility of designing pulse packets and easy remote focusing using an off-the-shelf 4-channel nsEP generator.

Cottonseed hydrolysate supplementation alters metabolic and proteomics responses in Chinese hamster ovary cell cultures.

Hydrolysates are used as media supplements although their role is not well characterized. In this study, cottonseed hydrolysates, which contained peptides and galactose as supplemental substrates, were added to Chinese hamster ovary (CHO) batch cultures, enhancing cell growth, immunoglobulin (IgG) titers, and productivities. Extracellular metabolomics coupled with tandem mass tag (TMT) proteomics revealed metabolic and proteomic changes in cottonseed-supplemented cultures. Shifts in production and consumption dynamics of glucose, glutamine, lactate, pyruvate, serine, glycine, glutamate, and aspartate suggest changes in tricarboxylic acid (TCA) and glycolysis metabolism following hydrolysate inputs. Quantitative proteomics revealed 5521 proteins and numerous changes in relative abundance of proteins related to growth, metabolism, oxidative stress, protein productivity, and apoptosis/cell death at day 5 and day 6. Differential abundance of amino acid transporter proteins and catabolism enzymes such as branched-chain-amino-acid aminotransferase (BCAT)1 and fumarylacetoacetase (FAH) can alter availability and utilization of several amino acids. Also, pathways involved in growth including the polyamine biosynthesis through higher ornithine decarboxylase (ODC1) abundance and hippo signaling were upregulated and downregulated, respectively. Central metabolism rewiring was indicated by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) downregulation, which corresponded with re-uptake of secreted lactate in the cottonseed-supplemented cultures. Overall, cottonseed hydrolysate supplementation modified culture performance by altering cellular activities critical to growth and protein productivity including metabolism, transport, mitosis, transcription, translation, protein processing, and apoptosis. HIGHLIGHTS: Cottonseed hydrolysate, as a medium additive, enhances Chinese hamster ovary (CHO) cell culture performance. Metabolite profiling and tandem mass tag (TMT) proteomics characterize its impact on CHO cells. Rewired nutrient utilization is observed via glycolysis, amino acid, and polyamine metabolism. Hippo signaling pathway impacts cell growth in the presence of cottonseed hydrolysate.

Reconstruction of reverse transsulfuration pathway enables cysteine biosynthesis and enhances resilience to oxidative stress in Chinese Hamster Ovary cells.

Cysteine is a critically important amino acid necessary for mammalian cell culture, playing key roles in nutrient supply, disulfide bond formation, and as a precursor to antioxidant molecules controlling cellular redox. Unfortunately, its low stability and solubility in solution make it especially problematic as an essential medium component that must be added to Chinese hamster ovary and other mammalian cell cultures. Therefore, CHO cells have been engineered to include the capacity of endogenously synthesizing cysteine by overexpressing multiple enzymes, including cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CTH) and glycine N-methyltransferase (GNMT) to reconstruct the reverse transsulfuration pathway and overcome a key metabolic bottleneck. Some limited cysteine biosynthesis was obtained by overexpressing CBS and CTH for converting homocysteine to cysteine but robust metabolic synthesis from methionine was only possibly after incorporating GNMT which likely represents a key bottleneck step in the cysteine biosynthesis pathway. CHO cells with the reconstructed pathway exhibit the strong capability to proliferate in cysteine-limited and cysteine-free batch and fed-batch cultures at levels comparable to wildtype cells with ample cysteine supplementation, providing a selectable marker for CHO cell engineering. GNMT overexpression led to the accumulation of sarcosine byproduct, but its accumulation did not affect cell growth. Furthermore, pathway reconstruction enhanced CHO cells' reduced and glutathione levels in cysteine-limited conditions compared to unmodified cells, and greatly enhanced survivability and maintenance of redox homeostasis under oxidative stress induced by addition of menadione in cysteine-deficient conditions. Such engineered CHO cell lines can potentially reduce or even eliminate the need to include cysteine in culture medium, which not only reduces the cost of mammalian media but also promises to transform media design by solving the challenges posed by low stability and solubility of cysteine and cystine in future mammalian biomanufacturing processes.

NAD+ supplementation improves mAb productivity in CHO cells via a glucose metabolic shift.

Aerobic glycolysis and its by-product lactate accumulation are usually associated with adverse culture phenotypes such as poor cell viability and productivity. Due to the lack of knowledge on underlying mechanisms and accompanying biological processes, the regulation of aerobic glycolysis has been an ongoing challenge in culture process development for therapeutic protein productivity. Nicotinamide adenine dinucleotide (NAD+ ), a coenzyme and co-substrate in energy metabolism, promotes the conversion of inefficient glycolysis into an efficient oxidative phosphorylation (OXPHOS) pathway. However, the effect of NAD+ on Chinese hamster ovary (CHO) cells for biopharmaceutical production has not been reported yet. In this work, we aimed to elucidate the influence of NAD+ on cell culture performance by examining metabolic shifts and mAb productivity. The supplementation of NAD+ increased the intracellular concentration of NAD+ and promoted SIRT3 expression. Antibody titer and the specific productivity in the growth phase were improved by up to 1.82- and 1.88-fold, respectively, with marginal restrictions on cell growth. NAD+ significantly reduced the accumulation of reactive oxygen species (ROS) and the lactate yield from glucose, determined by lactate accumulation versus glucose consumption (YLAC/GLC ). In contrast, OXPHOS capacity and amino acid consumption rate increased substantially. Collectively, these results suggest that NAD+ contributes to improving therapeutic protein productivity in bioprocessing via inducing an energy metabolic shift.

Modulation of nutrient precursors for controlling metabolic inhibitors by genome-scale flux balance analysis.

Therapeutic protein productivity and glycosylation pattern highly rely on cell metabolism. Cell culture medium composition and feeding strategy are critical to regulate cell metabolism. In this study, the relationship between toxic metabolic inhibitors and their nutrient precursors was explored to identify the critical medium components toward cell growth and generation of metabolic by-products. Generic CHO metabolic model was tailored and integrated with CHO fed-batch metabolomic data to obtain a cell line- and process-specific model. Flux balance analysis study was conducted on toxic metabolites cytidine monophosphate, guanosine monophosphate and n-acetylputrescine-all of which were previously reported to generate from endogenous cell metabolism-by mapping them to a compartmentalized carbon utilization network. Using this approach, the study projected high level of inhibitory metabolites accumulation when comparing three industrially relevant fed-batch feeding conditions one against another, from which the results were validated via a dose-dependent amino acids spiking study. In the end, a medium optimization design was employed to lower the amount of supplemented nutrients, of which improvements in critical process performance were realized at 40% increase in peak viable cell density (VCD), 15% increase in integral VCD, and 37% increase in growth rate. Tight control of toxic by-products was also achieved, as the study measured decreased inhibitory metabolites accumulation across all conditions. Overall, the study successfully presented a digital twin approach to investigate the intertwined relationship between supplemented medium constituents and downstream toxic metabolites generated through host cell metabolism, further elucidating different control strategies capable of improving cellular phenotypes and regulating toxic inhibitors.

Nimbin analog N2 alleviates high testosterone induced oxidative stress in CHO cells and alters the expression of Tox3 and Dennd1a signal transduction pathway involved in the PCOS zebrafish.

Polycystic ovarian syndrome (PCOS) is a hormonal disorder that causes enlargement of ovaries and follicular maturation arrest, which lacks efficient treatment. N2, a semi-natural triterpenoid from the neem family, was already reported to have antioxidant and antiinflammatory properties in our previous report. This study investigated the anti-androgenic property of N2 on testosterone-induced oxidative stress in Chinese Hamster Ovarian cells (CHO) and PCOS zebrafish model. The testosterone exposure disrupted the antioxidant enzymes and ROS level and enhanced the apoptosis in both CHO cells and PCOS zebrafish. However, N2 significantly protected the CHO cells from ROS and apoptosis. N2 improved the Gonado somatic index (GSI) and upregulated the expression of the SOD enzyme in zebrafish ovaries. Moreover, the testosterone-induced follicular maturation arrest was normalized by N2 treatment in histopathology studies. In addition, the gene expression studies of Tox3 and Denndla in zebrafish demonstrated that N2 could impair PCOS condition. Furthermore, to confirm the N2 activity, the in-silico studies were performed against PCOS susceptible genes Tox3 and Dennd1a using molecular docking and molecular dynamic simulations. The results suggested that N2 alleviated the oxidative stress and apoptosis in-vitro and in-vivo and altered the expression of PCOS key genes.

Cellular transport of uranium and its cytotoxicity effects on CHO-k1 cells.

Uranium is a radioactive heavy metal and a significant public health concern; however, its associated underlying toxicological mechanisms remain largely unknown. In this work, the uptake and efflux processes of uranium in CHO-k1 cells were studied and the cytotoxicity effects were explored. It was found that both the uptake and efflux processes took place rapidly and half of the internalized uranium was expelled within 8 h. The uranium exposure caused a decrease of cell viability and adhesion ability in a dose-dependent manner and blocked the cell cycle at the G1 stage. In addition, gene expression analysis revealed relative changes in the transcription of metabolism related genes. Further studies revealed that the cytotoxicity of uranium could be alleviated by exposing cells to a lower temperature or by the addition of amantadine-HCl, an endocytosis inhibitor. Interestingly, after uranium exposure, needle-like precipitates were observed in both intracellular and extracellular regions. These findings collectively suggest that the cellular transport of uranium is a rapid process that disturbs cell metabolism and induces cytotoxicity, and this impact could be reduced by slowing down endocytic processes.

In Vitro and In Vivo Inhibitory Activities of Selected Traditional Medicinal Plants against Toxin-Induced Cyto- and Entero- Toxicities in Cholera.

Careya arborea, Punica granatum, Psidium guajava, Holarrhena antidysenterica, Aegle marmelos, and Piper longum are commonly used traditional medicines against diarrhoeal diseases in India. This study investigated the inhibitory activity of these plants against cytotoxicity and enterotoxicity induced by toxins secreted by Vibrio cholerae. Cholera toxin (CT) and non-membrane damaging cytotoxin (NMDCY) in cell free culture filtrate (CFCF) of V. cholerae were quantified using GM1 ELISA and cell-based assays, respectively. Hydro-alcoholic extracts of these plants and lyophilized juice of P. granatum were tested against CT-induced elevation of cAMP levels in CHO cell line, binding of CT to ganglioside GM1 receptor and NMDCY-induced cytotoxicity. Significant reduction of cAMP levels in CFCF treated CHO cell line was observed for all extracts except P. longum. C. arborea, P. granatum, H. antidysenterica and A. marmelos showed >50% binding inhibition of CT to GM1 receptor. C. arborea, P. granatum, and P. guajava effectively decreased cytotoxicity and morphological alterations caused by NMDCY in CHO cell line. Further, the efficacy of these three plants against CFCF-induced enterotoxicity was seen in adult mice ligated-ileal loop model as evidenced by decrease in volume of fluid accumulation, cAMP levels in ligated-ileal tissues, and histopathological changes in intestinal mucosa. Therefore, these plants can be further validated for their clinical use against cholera.

Enhancement of recombinant human IL-24 (rhIL-24) protein production from site-specific integrated engineered CHO cells by sodium butyrate treatment.

Interleukin-24 (IL-24) has specific inhibitory effects on the proliferation of various tumor cells with almost no toxicity to normal cells. The antitumor activity of recombinant human IL-24 protein produced in mammalian cells is much higher than that of bacteria, but its expression level is extremely low. Sodium butyrate (NaBu) was utilized as a media additive to increase protein expression in Chinese hamster ovary cells. The site-specific integrated engineered cells FCHO/IL-24 were treated with NaBu under different culture conditions (10% and 0.5% serum adherent culture, 0.5% serum suspension culture). First, 3 days of 1 mmol/L NaBu treatment significantly increased rhIL-24 expression level in FCHO/IL-24 cells by 119.94 ± 1.5% (**p < 0.01), 57.49 ± 2.4% (**p < 0.01), and 20.17 ± 3.03% (*p < 0.05) under the above culture conditions. Second, NaBu has a time- and dose-dependent inhibitory effect on FCHO/IL-24 proliferation and induces G0/G1 phase arrest. Under 10% and 0.5% serum adherent culture, G0/G1 phase cells were increased by 11.3 ± 0.5% (**p < 0.01) and 15.0 ± 2.6% (**p < 0.01), respectively. No induction of apoptosis was observed under a high dosage of NaBu treatment. These results suggest that NaBu increases rhIL-24 secretion via inhibiting cell cycle progression, thereby trapping cells in the highly productive G0/G1 phase. Finally, with increasing NaBu dose, glucose concentration increased (**p < 0.01) while lactic acid and ammonia concentrations reduced significantly (**p < 0.01) in 10% and 0.5% serum adherent culture supernatant. RNA-seq showed that NaBu treatment affected multiple tumor and immune-related pathways. In conclusion, NaBu treatment dramatically promoted rhIL-24 production in engineered FCHO/IL-24 cells by altering downstream pathways and inducing G0/G1 cell arrest with little effect on apoptosis.

Sub-MHz bursts of nanosecond pulses excite neurons at paradoxically low electric field thresholds without membrane damage.

Neuromodulation applications of nanosecond electric pulses (nsEP) are hindered by their low potency to elicit action potentials in neurons. Excitation by a single nsEP requires a strong electric field which injures neurons by electroporation. We bypassed the high electric field requirement by replacing single nsEP stimuli with high-frequency brief nsEP bursts. In hippocampal neurons, excitation thresholds progressively decreased at nsEP frequencies above 20-200 kHz, with up to 20-30-fold reduction at sub-MHz and MHz rates. For a fixed burst duration, thresholds were determined by the duty cycle, irrespective of the specific nsEP duration, rate, or number of pulses per burst. For 100-µs bursts of 100-, 400-, or 800-ns pulses, the threshold decreased as a power function when the duty cycle exceeded 3-5 %. nsEP bursts were compared with single "long" pulses whose duration and amplitude matched the duration and the time-average amplitude of the burst. Such pulses deliver the same electric charge as bursts, within the same time interval. High-frequency nsEP bursts excited neurons at the time-average electric field 2-3 times below the threshold for a single long pulse. For example, the excitation threshold of 139 ± 14 V/cm for a single 100-µs pulse decreased to 57 ± 8 V/cm for a 100-µs burst of 100-ns, 0.25-MHz pulses (p < 0.001). Applying nsEP in bursts reduced or prevented the loss of excitability in multiple stimulation attempts. Stimulation by high-frequency nsEP bursts is a powerful novel approach to excite neurons at paradoxically low electric charge while also avoiding the electroporative membrane damage.

TNB-738, a biparatopic antibody, boosts intracellular NAD+ by inhibiting CD38 ecto-enzyme activity.

Cluster of differentiation 38 (CD38) is an ecto-enzyme expressed primarily on immune cells that metabolize nicotinamide adenine dinucleotide (NAD+) to adenosine diphosphate ribose or cyclic ADP-ribose and nicotinamide. Other substrates of CD38 include nicotinamide adenine dinucleotide phosphate and nicotinamide mononucleotide, a critical NAD+ precursor in the salvage pathway. NAD+ is an important coenzyme involved in several metabolic pathways and is a required cofactor for the function of sirtuins (SIRTs) and poly (adenosine diphosphate-ribose) polymerases. Declines in NAD+ levels are associated with metabolic and inflammatory diseases, aging, and neurodegenerative disorders. To inhibit CD38 enzyme activity and boost NAD+ levels, we developed TNB-738, an anti-CD38 biparatopic antibody that pairs two non-competing heavy chain-only antibodies in a bispecific format. By simultaneously binding two distinct epitopes on CD38, TNB-738 potently inhibited its enzymatic activity, which in turn boosted intracellular NAD+ levels and SIRT activities. Due to its silenced IgG4 Fc, TNB-738 did not deplete CD38-expressing cells, in contrast to the clinically available anti-CD38 antibodies, daratumumab, and isatuximab. TNB-738 offers numerous advantages compared to other NAD-boosting therapeutics, including small molecules, and supplements, due to its long half-life, specificity, safety profile, and activity. Overall, TNB-738 represents a novel treatment with broad therapeutic potential for metabolic and inflammatory diseases associated with NAD+ deficiencies.Abbreviations: 7-AAD: 7-aminoactinomycin D; ADCC: antibody dependent cell-mediated cytotoxicity; ADCP: antibody dependent cell-mediated phagocytosis; ADPR: adenosine diphosphate ribose; APC: allophycocyanin; cADPR: cyclic ADP-ribose; cDNA: complementary DNA; BSA: bovine serum albumin; CD38: cluster of differentiation 38; CDC: complement dependent cytotoxicity; CFA: Freund's complete adjuvant; CHO: Chinese hamster ovary; CCP4: collaborative computational project, number 4; COOT: crystallographic object-oriented toolkit; DAPI: 4',6-diamidino-2-phenylindole; DNA: deoxyribonucleic acid; DSC: differential scanning calorimetry; 3D: three dimensional; εNAD+: nicotinamide 1,N6-ethenoadenine dinucleotide; ECD: extracellular domain; EGF: epidermal growth factor; FACS: fluorescence activated cell sorting; FcγR: Fc gamma receptors; FITC: fluorescein isothiocyanate; HEK: human embryonic kidney; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IgG: immunoglobulin; IFA: incomplete Freund's adjuvant; IFNγ: Interferon gamma; KB: kinetic buffer; kDa: kilodalton; KEGG: kyoto encyclopedia of genes and genomes; LDH: lactate dehydrogenase; M: molar; mM: millimolar; MFI: mean fluorescent intensity; NA: nicotinic acid; NAD: nicotinamide adenine dinucleotide; NADP: nicotinamide adenine dinucleotide phosphate; NAM: nicotinamide; NGS: next-generation sequencing; NHS/EDC: N-Hydroxysuccinimide/ ethyl (dimethylamino propyl) carbodiimide; Ni-NTA: nickel-nitrilotriacetic acid; nL: nanoliter; NK: natural killer; NMN: nicotinamide mononucleotide; OD: optical density; PARP: poly (adenosine diphosphate-ribose) polymerase; PBS: phosphate-buffered saline; PBMC: peripheral blood mononuclear cell; PDB: protein data bank; PE: phycoerythrin; PISA: protein interfaces, surfaces, and assemblies: PK: pharmacokinetics; mol: picomolar; RNA: ribonucleic acid; RLU: relative luminescence units; rpm: rotations per minute; RU: resonance unit; SEC: size exclusion chromatography; SEM: standard error of the mean; SIRT: sirtuins; SPR: surface plasmon resonance; µg: microgram; µM: micromolar; µL: microliter.