The safety of plasma apheresis from donors recovering from COVID-19 infection in Japan.

PURPOSE: Since 2020, the novel coronavirus infection (COVID-19) has spread globally. A few studies have investigated the safety of COVID-19 convalescent plasma (CCP) apheresis from COVID-19. This study was the first retrospective observational study of CCP in Japan. METHODS: We recruit donors from April 2020 to November 2021 and plasmapheresis in our center (NCGM: national center for global health and medicine). We set the primary endpoint as the Donors Adverse Event (DAE) occurrence at the time of the CCP collection. Variable selection was used to explore the determinants of DAE. RESULTS: Mean and SD age was 50.5 (10.6) years old. Seventy-three (42.2 %) were female, and 87 (33.3 %) were multiple-times donors. Twelve (6.97 % by donors and 4.6 % in total collections) adverse events occurred. The DAEs were VVR (Vaso Vagal Reaction), paresthesia, hypotension, agitation, dizziness, malaise, and hearing impairment/paresthesia. Half of them were VVR during apheresis. DAE occurred only in first-time donors and more in severe illnesses such as using ventilation and ECMO. From the donor characteristics and variable selection, the risk factors are as follows: younger age, female, the severity of disease at the time of the disease, and lower SBP before initiation. Our DAE incidence did not differ from previous studies. DAEs were more likely to occur in CCP apheresis than in healthy donors. CONCLUSION: We confirm the safety of CCP apheresis in this study, although DAEs were more than healthy donors. More caution should be exercised in the plasma collection for future outbreaks of emerging infectious diseases.

How we secured a COVID-19 convalescent plasma procurement scheme in Japan.

BACKGROUND: In order to tackle the COVID-19 pandemic, a COVID-19 convalescent plasma (CCP) procurement program was initiated in Japan in April 2020. The program was a collaboration between a government-managed national hospital, an infectious disease research institute, and a blood banking organization. Each party assumed different responsibilities: recruitment, SARS-CoV-2 antibody profiling, and plasmapheresis; conduction of screening tests; and SARS-CoV-2 blood testing, respectively. METHODS: We adopted a two-point screening approach before the collected CCP was labeled as a CCP product for investigational use, for which we mainly tested anti-SARS-CoV-2 antibody eligibility and blood product eligibility. Anti-SARS-CoV-2 spike protein titer was measured using enzyme-linked immunosorbent assay, and the IC50 value was denoted as the neutralizing activity. Blood donor eligibility was extended beyond the normal blood donation guidelines to include a broader range of participants. After both eligibility criteria were confirmed, participants were asked to revisit the hospital for blood donation, which is a unique aspect of the Japanese CCP program, as most donations are taking place in normal blood donation venues in other countries. Some donors were re-scheduled for repeat plasma donations. As public interest in anti-SARS-CoV-2 antibodies increased, test results were given to the participants. RESULTS: As of September 17, 2020, our collection of CCP products was sufficient to treat more than 100 patients. As a result, projects for administration and distribution are also being conducted. CONCLUSIONS: We successfully implemented a CCP procurement scheme with the goal to expand to other parts of the country to improve treatment options for COVID-19.

Inhibition of ATP citrate lyase induces triglyceride accumulation with altered fatty acid composition in cancer cells.

De novo lipogenesis is activated in most cancers and several lipogenic enzymes have been implicated as therapeutic targets. Here, we demonstrate a novel function of the lipogenic enzyme, ATP citrate lyase (ACLY), in lipid metabolism in cancer cells. ACLY depletion by small interfering RNAs caused growth suppression and/or apoptosis in a subset of cancer cell lines. To investigate the effect of ACLY inhibition on lipid metabolism, metabolome and transcriptome analysis was performed. ACLY depletion blocks the fatty acid chain elongation from C16 to C18 in triglyceride (TG), but not in other lipid classes. Meanwhile, wild-type ACLY overexpression enhanced fatty acid elongation of TG, whereas an inactive mutant ACLY did not change it. ACLY depletion-mediated blockade of fatty acid elongation was coincident with downregulation of long-chain fatty acid elongase ELOVL6, which resides in endoplasmic reticulum (ER). Paradoxically, ACLY depletion-mediated growth suppression was associated with TG accumulation. ACLY depletion downregulated the expression of carnitine palmitoyltransferase 1A, which is a mitochondrial fatty acid transporter. Consistent with this finding, metabolome analysis revealed that ACLY positively regulates the carnitine system, which plays as an essential cofactor for fatty acid transport across mitochondrial membrane. AICAR, an activator of mitochondrial fatty acid oxidation (FAO), significantly reduced ACLY depletion-mediated TG accumulation. These data indicate that inhibition of ACLY might affect both fatty acid elongation in ER and FAO in mitochondria, thereby explaining the TG accumulation with altered fatty acid composition. This phenotype may be a hallmark of growth suppression mediated by ACLY inhibition.

Inhibition of ATP citrate lyase induces an anticancer effect via reactive oxygen species: AMPK as a predictive biomarker for therapeutic impact.

De novo lipogenesis is activated in most cancers. Inhibition of ATP citrate lyase (ACLY), the enzyme that catalyzes the first step of de novo lipogenesis, leads to growth suppression and apoptosis in a subset of human cancer cells. Herein, we found that ACLY depletion increases the level of intracellular reactive oxygen species (ROS), whereas addition of an antioxidant reduced ROS and attenuated the anticancer effect. ACLY depletion or exogenous hydrogen peroxide induces phosphorylation of AMP-activated protein kinase (p-AMPK), a crucial regulator of lipid metabolism, independently of energy status. Analysis of various cancer cell lines revealed that cancer cells with a higher susceptibility to ACLY depletion have lower levels of basal ROS and p-AMPK. Mitochondrial-deficient ρ(0) cells retained high levels of ROS and p-AMPK and were resistant to ACLY depletion, whereas the replenishment of normal mitochondrial DNA reduced the levels of ROS and p-AMPK and restored the sensitivity to ACLY depletion, indicating that low basal levels of mitochondrial ROS are critical for the anticancer effect of ACLY depletion. Finally, p-AMPK levels were significantly correlated to the levels of oxidative DNA damage in colon cancer tissues, suggesting that p-AMPK reflects cellular ROS levels in vitro and in vivo. Together, these data suggest that ACLY inhibition exerts an anticancer effect via increased ROS, and p-AMPK could be a predictive biomarker for its therapeutic outcome.

Metabolomic profiling rationalized pyruvate efficacy in cybrid cells harboring MELAS mitochondrial DNA mutations.

Pyruvate treatment was found to alleviate clinical symptoms of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome and is highly promising therapeutic. Using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), we measured time-changes of 161 intracellular and 85 medium metabolites to elucidate metabolic effects of pyruvate treatment on cybrid human 143B osteosarcoma cells harboring normal (2SA) and MELAS mutant (2SD) mitochondria. The results demonstrated dramatic and sustainable effects of pyruvate administration on the energy metabolism of 2SD cells, corroborating pyruvate as a metabolically rational treatment regimen for improving symptoms associated with MELAS and possibly other mitochondrial diseases.

Analysis of nucleotides by pressure-assisted capillary electrophoresis-mass spectrometry using silanol mask technique.

A method for the determination of nucleotides based on pressure-assisted capillary electrophoresis-electrospray ionization mass spectrometry (PACE-MS) is described. To prevent multi-phosphorylated species from adsorbing onto the fused-silica capillary, silanol groups were masked with phosphate ions by preconditioning the capillary with the background electrolyte containing phosphate. During preconditioning, nebulizer gas was turned off to avoid contamination of MS detector with phosphate ions. To detect nucleotides using the CE positive mode at a pH 7.5, it was necessary to apply air pressure to the inlet capillary during electrophoresis to supplement the electroosmotic flow (EOF) toward the cathode. Moreover, we exchanged the running electrolyte every analysis using the buffer replenishment system to obtain the required reproducibility. Under the optimized conditions, 14 phosphorylated species such as nucleotides, nicotinamide-adenine dinucleotides and coenzyme A (CoA) compounds were well determined in less than 20 min. The relative standard deviations (n=6) of the method were better than 0.9% for migration times and between 1.7% and 8.1% for peak areas. The detection limits for these species were between 0.5 and 1.7 micromol/L with pressure injection of 50 mbar for 30 s (30 nL) at a signal-to-noise ratio of 3. This approach is robust and quantitative compared to the previous method, and its utility is demonstrated by the analysis of intracellular nucleotides and CoA compounds extracted from Escherichia coli wild type, pfkA and pfkB knockout mutants. The methodology was used to suggest that pfkA is the main functional enzyme.

Direct measurement of isotopomer of intracellular metabolites using capillary electrophoresis time-of-flight mass spectrometry for efficient metabolic flux analysis.

We have developed a metabolic flux analysis method that is based on (13)C-labeling patterns of the intracellular metabolites directly measured by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). The flux distribution of the central carbon metabolism in Escherichia coli was determined by this new approach and the results were compared with findings obtained by conventional GC-MS analysis based on isotopomer of the proteinogenic amino acids. There were some differences in estimation results between new approach using CE-TOFMS and conventional approach using GC-MS. These were thought to be attributable to variations in measured mass distributions between amino acids and the corresponding precursors and to differences in the sensitivity of the exchange coefficients to mass distributions. However, our CE-TOFMS method facilitates high-throughput flux analysis without requiring complicated sample preparation such as hydrolysis of proteins and derivatization of amino acids.

Multiple high-throughput analyses monitor the response of E. coli to perturbations.

Analysis of cellular components at multiple levels of biological information can provide valuable functional insights. We performed multiple high-throughput measurements to study the response of Escherichia coli cells to genetic and environmental perturbations. Analysis of metabolic enzyme gene disruptants revealed unexpectedly small changes in messenger RNA and proteins for most disruptants. Overall, metabolite levels were also stable, reflecting the rerouting of fluxes in the metabolic network. In contrast, E. coli actively regulated enzyme levels to maintain a stable metabolic state in response to changes in growth rate. E. coli thus seems to use complementary strategies that result in a metabolic network robust against perturbations.