Risk factors of COVID-19 mortality: a systematic review of current literature and lessons from recent retracted articles.

OBJECTIVE: Recently, two influential articles that reported the association of (hydroxy)chloroquine or angiotensin converting enzyme (ACE) inhibitors and coronavirus disease 2019 (COVID-19) mortality were retracted due to significant methodological issues. Therefore, we aimed to analyze the same clinical issues through an improved research method and to find out the differences from the retracted papers. We systematically reviewed pre-existing literature, and compared the results with those of the retracted papers to gain a novel insight. MATERIALS AND METHODS: We extracted common risk factors identified in two retracted papers, and conducted relevant publication search until June 26, 2020 in PubMed. Then, we analyzed the risk factors for COVID-19 mortality and compared them to those of the retracted papers. RESULTS: Our systematic review demonstrated that most demographic and clinical risk factors for COVID-19 mortality were similar to those of the retracted papers. However, while the retracted paper indicated that both (hydroxy)chloroquine monotherapy and combination therapy with macrolide were associated with higher risk of mortality, our study showed that only combination therapy of hydroxychloroquine and macrolide was associated with higher risk of mortality (odds ratio 2.33; 95% confidence interval 1.63-3.34). In addition, our study demonstrated that use of ACE inhibitors or angiotensin receptor blockers (ARBs) was associated with reduced risk of mortality (0.77; 0.65-0.91). CONCLUSIONS: When analyzing the same clinical issues with the two retracted papers through a systematic review of randomized controlled trials and relevant cohort studies, we found out that (hydroxy)chloroquine monotherapy was not associated with higher risk of mortality, and that the use of ACE inhibitors or ARBs was associated with reduced risk of mortality in COVID-19 patients.

The triglyceride-lowering effect of supplementation with dual probiotic strains, Lactobacillus curvatus HY7601 and Lactobacillus plantarum KY1032: Reduction of fasting plasma lysophosphatidylcholines in nondiabetic and hypertriglyceridemic subjects.

BACKGROUND AND AIMS: This study evaluated the triglyceride (TG)-lowering effects of consuming dual probiotic strains of Lactobacillus curvatus (L. curvatus) HY7601 and Lactobacillus plantarum (L. plantarum) KY1032 on the fasting plasma metabolome. METHODS AND RESULTS: A randomized, double-blind, placebo-controlled study was conducted on 92 participants with hypertriglyceridemia but without diabetes. Over a 12-week testing period, the probiotic group consumed 2 g of powder containing 5 × 10(9) colony-forming units (cfu) of L. curvatus HY7601 and 5 × 10(9) cfu of L. plantarum KY1032 each day, whereas the placebo group consumed the same product without probiotics. Fasting plasma metabolomes were profiled using UPLC-LTQ-Orbitrap MS. After 12 weeks of treatment, the probiotic group displayed a 20% reduction (p = 0.001) in serum TGs and 25% increases (p=0.001) in apolipoprotein A-V (apoA-V). At the 12-week follow-up assessment, the following 11 plasma metabolites were significantly reduced in the probiotic group than the placebo group: palmitoleamide, palmitic amide, oleamide, and lysophosphatidyl choline (lysoPC) containing C14:0, C16:1, C16:0, C17:0, C18:3, C18:2, C18:1, and C20:3. In the probiotic group, changes (▵) in TG were negatively correlated with ▵ apoA-V, which was positively correlated with ▵ FFA. In addition, ▵ FFA was strongly and positively correlated with ▵ lysoPCs in the probiotic group but not the placebo group. CONCLUSIONS: The triglyceride-lowering effects of probiotic supplementation, partly through elevated apoA-V, in borderline to moderate hypertriglyceridemic subjects showed reductions in plasma metabolites, fatty acid primary amides and lysoPCs (NCT02215694; http://www.clinicaltrials.gov). Clinical trials: NCT02215694; http://www.clinicaltrials.gov.

Effects of dietary levels of glycine, threonine and protein on threonine efficiency and threonine dehydrogenase activity in hepatic mitochondria of chicks.

This study was carried out to evaluate the relationship between threonine (Thr) efficiency and Thr dehydrogenase (TDG) activity as an indicator of Thr oxidation on chicks fed with levels of diets (CP [17.5% and 21.5%] and Thr [3.8 and 4.7 g/100 g CP]; glycine [Gly][0.64% and 0.98%] and true digestible Thr [dThr] [0.45% and 0.60%]). Calculation of the Thr efficiency was based on N-balance data and an exponential N-utilization model, and TDG activity was determined as accumulation of aminoacetone and Gly during incubation of hepatic mitochondria. This study found that in the liver of chicks who received a diet containing up to 0.79% Thr (4.7 g Thr/100 g of CP) in the 17.5% CP diet, no significant (p>0.05) effect on TDG activity was observed. However, significantly (p = 0.014) increased TDG activity was observed with a diet containing 21.5% CP (4.7 g Thr/100 g of CP) and the efficiency of Thr utilization showed a significant (p = 0.001) decrease, indicating the end of the Thr limiting range. No significant (p>0.05) effect on the total TDG activity and accumulation of Gly was observed with addition of Gly to a diet containing 0.45% dThr. In addition, addition of Gly to a diet containing 0.60% dThr also did not result in a change in accumulation of Gly. Due to an increase in accumulation of aminoacetone, an elevated effect on total TDG activity was also observed. No significant (p>0.05) reduction in the efficiency of Thr utilization was observed after addition of Gly at the level of 0.45% dThr. However, significantly (p<0.001) reduced efficiency of Thr utilization was observed after addition of Gly at the level of 0.60% dThr. Collectively, we found that TDG was stimulated not only by addition of Thr and protein to the diet, but also by addition of Gly, and efficiency of Thr utilization was favorably affected by addition of Gly at the level near to the optimal Thr concentration. In addition, no metabolic requirement of Gly through the TDG pathway was observed with almost the same accumulation of Gly and a slight increase in TDG activity by addition of Gly. Thus, our findings suggest that determination of TDG activity and parameter of efficiency of Thr utilization may be useful for evaluation of dietary Thr level.

The anti-diabetic activity of Bifidobacterium lactis HY8101 in vitro and in vivo.

AIMS: The aim of this study was to evaluate the effects of Bifidobacterium lactis HY8101 on insulin resistance induced using tumour necrosis factor-α (TNF-α) in rat L6 skeletal muscle cells and on the KK-A(Y) mouse noninsulin-dependent diabetes mellitus (NIDDM) model. METHODS AND RESULTS: The treatment using HY8101 improved the insulin-stimulated glucose uptake and translocation of GLUT4 via the insulin signalling pathways AKT and IRS-1(Tyr) in TNF-α-treated L6 cells. HY8101 increased the mRNA levels of GLUT4 and several insulin sensitivity-related genes (PPAR-γ) in TNF-α-treated L6 cells. In KK-A(Y) mice, HY8101 decreased fasting insulin and blood glucose and significantly improved insulin tolerance. HY8101 improved diabetes-induced plasma total cholesterol and triglyceride (TG) levels and increased the muscle glycogen content. We observed concurrent transcriptional changes in the skeletal muscle tissue and the liver. In the skeletal muscle tissue, the glycogen synthesis-related gene pp-1 and GLUT4 were up-regulated in mice receiving HY8101 treatment. In the liver, the hepatic gluconeogenesis-regulated genes (PCK1 and G6PC) were down-regulated in mice receiving HY8101 treatment. CONCLUSIONS: Bifidobacterium lactis HY8101 can be used to moderate glucose metabolism, lipid metabolism and insulin sensitivity in mice and in cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Bifidobacterium lactis HY8101 might have potential as a probiotic candidate for alleviating metabolic syndromes such as diabetes.

Application of PTFE membrane for ammonia removal in a membrane contactor.

The feasibility of a membrane contactor system for ammonia removal was studied. The mass transfer coefficient was used to quantitatively compare the effect of various operation conditions on ammonia removal efficiency. Effective removal of ammonia was possible with a Polytetrafluoroethylene (PTFE) membrane contactor system at all tested conditions. Among the various operation parameters, contact time and solution pH showed significant effect on the ammonia removal mechanism. The overall ammonia removal rate was not affected by influent suspended solution concentration unlike other pressure driven membrane filtration processes. Also the osmotic distillation phenomena which deteriorate the mass transfer efficiency can be minimized by preheating of influent wastewater. A membrane contactor system can be a possible alternative to treat high strength nitrogen wastewater by optimizing operation conditions such as stripping solution flow rate, influent wastewater temperature, and influent pH.

Fate of nitrogen species in nitrate reduction by nanoscale zero valent iron and characterization of the reaction kinetics.

This study investigates the fate of nitrogen species during nitrate reduction by nanoscale zero valent iron (NZVI) as well as the related kinetics. The NZVI used for the experiments was prepared by chemical reduction without a stabilizing agent. The pseudo first order kinetic constant of nitrate reduction at 30 degrees C with an NZVI/nitrate ratio of 1.25:1, which were the reference conditions of this study, was 4.08 h(-1) (R(2)=0.955). A nitrogen mass balance was established by quantitative analysis of aqueous-phase and gas-phase nitrogen species. The results confirm that the nitrate was converted to ammonium ion, that ammonia stripping subsequently occurred under a strong alkaline condition, and that the total amount of aqueous nitrogen was consequently reduced. The nitrate reduction rate also increased with a lower pH and a higher temperature when microscale ZVI was used. However, in contrast to the reaction by microscale ZVI, the nitrate reduction rate by NZVI was higher for an unbuffered condition, possibly due to the abundance of surface atoms and the smaller size.

Modeling of extracellular polymeric substances and soluble microbial products production in a submerged membrane bioreactor at various SRTs.

Extracelluar polymeric substances (EPSs) and soluble microbial products (SMPs) produced by microbial cultures involved in membrane biofouling have been widely investigated. A mathematical model of EPS and SMP formation and degradation was established based on the activated sludge model no. 1 (ASM1) and was applied to the membrane bioreactor sludge with different sludge retention times (SRTs). The unified theory that the distinct products from the EPS and SMP overlapped each other was integrated into the ASM1. Two components, five processes and eight parameters were newly added to set up the model. To increase the accuracy of model simulation, microbial kinetic parameters were determined by respirometry method and applied to the model instead of microbial kinetic constant offered in ASM1. From the respirometry result, both of heterotroph and autotroph showed different yield value, growth rate and decay rate from activated sludge. There was no significant effect of SRT on SMP production and the experimental results showed good agreement with the predicted values by the model simulation. With the developed unified EPS and SMP model, EPS and SMP production could be simulated so well that it can be applied for the membrane biofouling control.

Effect of internal recycle rate on the high-strength nitrogen wastewater treatment in the combined UBF/MBR system.

An anaerobic/aerobic system combining an anaerobic upflow-sludge bed filter (UBF) and an aerobic membrane bioreactor (MBR) was operated to enhance organic and nitrogen removal efficiency. The internal recycle rate, which is one of the most important operation factors that affects overall removal efficiency, was varied from 100% to 300% of the influent flow. Under these conditions, the overall removal efficiencies of organic and nitrogen and characteristics of membrane fouling in the combined system treating the synthetic wastewater including high concentration of organics and nutrients were studied. As a result, nitrogen removal efficiency was increased to 67% when the internal recycle rate was 300% of influent flow rate. As the internal recycle ratio increased from 100% to 200%, protein content decreased by 17% and carbohydrate content increased by 12%. However, there was no remarkable difference in total extracellular polymeric substances (EPS) content. At the high recycle rate of 300%, the surface charge of sludge was decreased while hydrophobicity (specific ultraviolet absorbance, SUVA) was increased. The differences in SUVA and surface charge were 11% and 1%, respectively. It is concluded that SUVA and EPS composition were important parameters affecting membrane fouling in the combined system.

DNA recognition, strand selectivity, and cleavage mode during integrase family site-specific recombination.

We have probed the association of Flp recombinase with its DNA target using protein footprinting assays. The results are consistent with the domain organization of the Flp protein and with the general features of the protein-DNA interactions revealed by the crystal structures of the recombination intermediates formed by Cre, the Flp-related recombinase. The similarity in the organization of the Flp and Cre target sites and in their recognition by the respective recombinases implies that the overall DNA-protein geometry during strand cleavage in the two systems must also be similar. Within the functional recombinase dimer, it is the interaction between two recombinase monomers bound on either side of the strand exchange region (or spacer) that provides the allosteric activation of a single active site. Whereas Cre utilizes the cleavage nucleophile (the active site tyrosine) in cis, Flp utilizes it in trans (one monomer donating the tyrosine to its partner). By using synthetic Cre and Flp DNA substrates that are geometrically restricted in similar ways, we have mapped the positioning of the active and inactive tyrosine residues during cis and trans cleavage events. We find that, for a fixed substrate geometry, Flp and Cre cleave the labile phosphodiester bond at the same spacer end, not at opposite ends. Our results provide a model that accommodates local heterogeneities in peptide orientations in the two systems while preserving the global functional architecture of the reaction complex.

The 2 micrometer plasmid stability system: analyses of the interactions among plasmid- and host-encoded components.

The stable inheritance of the 2 micrometer plasmid in a growing population of Saccharomyces cerevisiae is dependent on two plasmid-encoded proteins (Rep1p and Rep2p), together with the cis-acting locus REP3 (STB). In this study we demonstrate that short carboxy-terminal deletions of Rep1p and Rep2p severely diminish their normal capacity to localize to the yeast nucleus. The nuclear targeting, as well as their functional role in plasmid partitioning, can be restored by the addition of a nuclear localization sequence to the amino or the carboxy terminus of the shortened Rep proteins. Analyses of deletion derivatives of the Rep proteins by using the in vivo dihybrid genetic test in yeast, as well as by glutathione S-transferase fusion trapping assays in vitro demonstrate that the amino-terminal portion of Rep1p (ca. 150 amino acids long) is responsible for its interactions with Rep2p. In a monohybrid in vivo assay, we have identified Rep1p, Rep2p, and a host-encoded protein, Shf1p, as being capable of interacting with the STB locus. The Shf1 protein expressed in Escherichia coli can bind with high specificity to the STB sequence in vitro. In a yeast strain deleted for the SHF1 locus, a 2 micrometer circle-derived plasmid shows relatively poor stability.

Flp ribonuclease activities. Mechanistic similarities and contrasts to site-specific DNA recombination.

The ribonuclease active site harbored by the Flp site-specific recombinase can act on two neighboring phosphodiester bonds to yield mechanistically distinct chain breakage reactions. One of the RNase reactions apparently proceeds via a covalent enzyme intermediate and targets the phosphodiester position involved in DNA recombination (Flp RNase I activity). The second activity (Flp RNase II) targets the phosphodiester immediately to the 3' side but appears not to involve an enzyme-linked intermediate. Flp RNase I is absolutely dependent upon Tyr-343 of Flp and is competitive with respect to the normal strand joining reaction. It can utilize the 2'-hydroxyl group from any one of the four ribonucleotides with comparable efficiencies in the cleavage reaction. On the other hand, the RNase II reaction mediated by Flp(Y343F) is specific for U and cannot utilize the 2'-hydroxyl group from ribo-A, -G, or -C under standard reaction conditions. The RNase II activity is also sensitive to the 3'-neighboring base. Although dT is functional, the activity is stimulated by U or U-2'-OMe. The Flp RNase II reaction effectively competes with the normal strand cleavage reaction mediated by Tyr-343, even though their phosphodiester targets are not the same.

The 2microm-plasmid-encoded Rep1 and Rep2 proteins interact with each other and colocalize to the Saccharomyces cerevisiae nucleus.

The efficient partitioning of the 2microm plasmid of Saccharomyces cerevisiae at cell division requires two plasmid-encoded proteins (Rep1p and Rep2p) and a cis-acting locus, REP3 (STB). By using protein hybrids containing fusions of the Rep proteins to green fluorescent protein (GFP), we show here that fluorescence from GFP-Rep1p or GFP-Rep2p is almost exclusively localized in the nucleus in a cir+ strain. Nuclear localization of GFP-Rep1p and GFP-Rep2p, though discernible, is less efficient in a cir(0) host. GFP-Rep2p or GFP-Rep1p is able to promote the stability of a 2microm circle-derived plasmid harboring REP1 or REP2, respectively, in a cir(0) background. Under these conditions, fluorescence from GFP-Rep2p or GFP-Rep1p is concentrated within the nucleus, as is the case in cir+ cells. This characteristic nuclear accumulation is not dependent on the expression of the FLP or RAF1 gene of the 2microm circle. Nuclear colocalization of Rep1p and Rep2p is consistent with the hypothesis that the two proteins directly or indirectly interact to form a functional bipartite or high-order protein complex. Immunoprecipitation experiments as well as baiting assays using GST-Rep hybrid proteins suggest a direct interaction between Rep1p and Rep2p which, in principle, may be modulated by other yeast proteins. Furthermore, these assays provide evidence for Rep1p-Rep1p and Rep2p-Rep2p associations as well. The sum of these interactions may be important in controlling the effective cellular concentration of the Rep1p-Rep2p complex.

Renal cell carcinoma development in the rat independent of alterations at the VHL gene locus.

Germline alterations of the human von Hippel-Lindau (VHL) tumor suppressor gene predispose to renal cell carcinoma and a constellation of other tumor types found in VHL disease. This gene is also mutated or deleted in a high proportion of sporadic nonpapillary renal cell carcinomas. In the Eker rat model, spontaneous renal cell carcinoma develops with a high frequency. We therefore investigated the role of this tumor suppressor gene in the development of these hereditary rat tumors. By using reverse transcriptase (RT)-polymerase chain reaction (PCR) analysis, the sequence of the rat VHL gene was determined over the portion of the gene homologous to regions where most mutations in the human VHL gene occur. The sequence homology was 90% and the amino-acid identity 99% between the rat and human genes. A developmental and tumor-specific pattern of expression for the VHL gene was found; a ubiquitous 3.2-kb transcript was expressed in all rat tissues examined (neonatal kidney, lung, liver, brain, heart, kidney, spleen, testis, and stomach), and an additional 4.5-kb transcript was expressed in neonatal kidney and cell lines derived from Eker rat renal cell carcinomas (ERC cell lines). To determine whether mutations in the VHL gene were involved in tumor development in the Eker model, RT-PCR, single-strand conformation polymorphism (SSCP) analysis, and direct sequencing were used to search for alterations in this gene in the ERC cell lines. Alterations in the VHL gene were not detected by SSCP, and these data were confirmed by direct sequencing. Transformed rat kidney epithelial cell lines derived from Fisher rats also expressed the VHL gene but like the ERC cell lines did not contain mutations in the VHL gene. These data indicate that in the rat, transformation of kidney epithelial cells and the development of solid, nonpapillary renal cell carcinoma can occur via pathways that are independent of alterations at the VHL gene locus.