Variation in presepsin and thrombomodulin levels for predicting COVID-19 mortality.

Coronavirus disease (COVID-19) has caused extensive mortality globally; therefore, biomarkers predicting the severity and prognosis of COVID-19 are essential. This study aimed to evaluate the application of presepsin (P-SEP) and thrombomodulin (TM), which are biomarkers of sepsis and endothelial dysfunction, respectively, in the prognosis of COVID-19. Serum P-SEP and TM levels from COVID-19 patients (n = 183) were measured. Disease severity was classified as mild, moderate I, moderate II, or severe based on hemoglobin oxygen saturation and the history of intensive care unit transfer or use of ventilation at admission. Patients in the severe group were further divided into survivors and non-survivors. P-SEP and TM levels were significantly higher in the severe group than those in the mild group, even after adjusting for creatinine values. In addition, TM levels were significantly higher in non-survivors than in survivors. Changes in the P-SEP levels at two time points with an interval of 4.1 ± 2.2 days were significantly different between the survivors and non-survivors. In conclusion, TM and continuous P-SEP measurements may be useful for predicting mortality in patients with COVID-19. Moreover, our data indicate that P-SEP and TM values after creatinine adjustment could be independent predictive markers, apart from renal function.

Viral load of SARS-CoV-2 Omicron BA.5 is lower than that of BA.2 despite the higher infectivity of BA.5.

BACKGROUND: Sublineage BA.5 of the SARS-CoV-2 Omicron variant rapidly spread and replaced BA.2 in July 2022 in Tokyo. A high viral load can be a possible cause of high transmissibility. METHODS AND RESULTS: The copy numbers of SARS-CoV-2 in nasopharyngeal swab samples obtained from all patients visiting the hospital where this research was conducted were measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Viral genotypes were determined using PCR-based melting curve analysis. Next, whole-genome sequencing was performed using approximately one fifth of the samples to verify the viral genotypes determined using PCR. Then, the copy numbers of the BA.1, BA.2, and BA.5 cases were compared. Contrary to expectations, the copy numbers of the BA.5 cases (median 4.7 × 104 copies/µL, n = 291) were significantly (p = .001) lower than those of BA.2 cases (median 1.1 × 105 copies/µL, n = 184). There was no significant difference (p = .44) between the BA.5 and BA.1 cases (median, 3.3 × 104 copies/µL; n = 215). CONCLUSION: The results presented here suggest that the increased infectivity of BA.5 is not caused by higher viral loads, but presumably by other factors such as increased affinity to human cell receptors or immune escape due to its L452R mutation.

Viral load of SARS-CoV-2 Omicron is not high despite its high infectivity.

Patients infected with the Omicron variant of severe acute respiratory syndrome coronavirus 2 has increased worldwide since the beginning of 2022 and the variant has spread more rapidly than the Delta variant, which spread in the summer of 2021. It is important to clarify the cause of the strong transmissibility of the Omicron variant to control its spread. In 694 patients with coronavirus disease 2019, the copy numbers of virus in nasopharyngeal swab-soaked samples and the viral genotypes were examined using quantitative polymerase chain reaction (PCR) and PCR-based melting curve analysis, respectively. Whole-genome sequencing was also performed to verify the viral genotyping data. There was no significant difference (p = 0.052) in the copy numbers between the Delta variant cases (median 1.5 × 105 copies/µl, n = 174) and Omicron variant cases (median 1.2 × 105 copies/µl, n = 328). During this study, Omicron BA.1 cases (median 1.1 ×105 copies/µl, n = 275) began to be replaced by BA.2 cases (median 2.3 × 105 copies/µl, n = 53), and there was no significant difference between the two groups (p = 0.33). Our results suggest that increased infectivity of the Omicron variant and its derivative BA.2 is not caused by higher viral loads but by other factors, such as increased affinity to cell receptors or immune escape.

Effect of myeloperoxidase oxidation and N-homocysteinylation of high-density lipoprotein on endothelial repair function.

Endothelial cell (EC) migration is essential for healing vascular injuries. Previous studies suggest that high-density lipoprotein (HDL) and apolipoprotein A-I (apoA-I), the major protein constituent of HDL, have endothelial healing functions. In cardiovascular disease, HDL is modified by myeloperoxidase (MPO) and N-homocysteine, resulting in apoA-I/apoA-II heterodimer and N-homocysteinylated (N-Hcy) apoA-I formation. This study investigated whether these modifications attenuate HDL-mediated endothelial healing. Wound healing assays were performed to analyze the effect of MPO-oxidized HDL and N-Hcy HDL in vitro. HDL obtained from patients with varying troponin I levels were also examined. MPO-oxidized HDL reduces EC migration compared to normal HDL in vitro, and N-Hcy HDL showed a decreasing trend toward EC migration. EC migration after treatment with HDL from patients was decreased compared to HDL isolated from healthy controls. Increased apoA-I/apoA-II heterodimer and N-Hcy apoA-I levels were also detected in HDL from patients. Wound healing cell migration was significantly negatively correlated with the ratio of apoA-I/apoA-II heterodimer to total apoA-II and N-Hcy apoA-I to total apoA-I. MPO-oxidized HDL containing apoA-I/apoA-II heterodimers had a weaker endothelial healing function than did normal HDL. These results indicate that MPO-oxidized HDL and N-Hcy HDL play a key role in the pathogenesis of cardiovascular disease.

Effect of the recording condition on the quality of a single-lead electrocardiogram.

Although many wearable single-lead electrocardiogram (ECG) monitoring devices have been developed, information regarding their ECG quality is limited. This study aimed to evaluate the quality of single-lead ECG in healthy subjects under various conditions (body positions and motions) and in patients with arrhythmias, to estimate requirements for automatic analysis, and to identify a way to improve ECG quality by changing the type and placement of electrodes. A single-lead ECG transmitter was placed on the sternum with a pair of electrodes, and ECG was simultaneously recorded with a conventional Holter ECG in 12 healthy subjects under various conditions and 35 patients with arrhythmias. Subjects with arrhythmias were divided into sinus rhythm (SR) and atrial fibrillation (AF) groups. ECG quality was assessed by calculating the sensitivity and positive predictive value (PPV) of the visual detection of QRS complexes (vQRS), automatic detection of QRS complexes (aQRS), and visual detection of P waves (vP). Accuracy was defined as a 100% sensitivity and PPV. We also measured the amplitude of the baseline, P wave, and QRS complex, and calculated the signal-to-noise ratio (SNR). We then focused on aQRS and estimated thresholds to obtain an accurate aQRS in more than 95% of the data. Finally, we sought to improve ECG quality by changing electrode placement using offset-type electrodes in 10 healthy subjects. The single-lead ECG provided 100% accuracy for vQRS, 87% for aQRS, and 74% for vP in healthy subjects under various conditions. Failure for accurate detection occurred in several motions in which the baseline amplitude was increased or in subjects with low QRS or P amplitude, resulting in low SNR. The single-lead ECG provided 97% accuracy for vQRS, 80% for aQRS in patients with arrhythmias, and 95% accuracy for vP in the SR group. The AF group showed higher baseline amplitude than the SR group (0.08 mV vs. 0.02 mV, P < 0.01) but no significant difference in accuracy for aQRS (79% vs. 81%, P = 1.00). The thresholds to obtain an accurate aQRS were a QRS amplitude > 0.42 mV and a baseline amplitude < 0.20 mV. The QRS amplitude was significantly influenced by electrode placement and body position (P < 0.01 for both, two-way analysis of variance), and the maximum reduction by changing body position was estimated as 30% compared to the sitting posture. The QRS amplitude significantly increased when the inter-electrode distance was extended vertically (1.51 mV for vertical extension vs. 0.93 mV for control, P < 0.01). The single-lead ECG provided at least 97% accuracy for vQRS, 80% for aQRS, and 74% for vP. To obtain stable aQRS in any body positions, a QRS amplitude > 0.60 mV and a baseline amplitude < 0.20 mV were required in the sitting posture considering the reduction induced by changing body position. Vertical extension of the inter-electrode distance increased the QRS amplitude.

Viral loads and profile of the patients infected with SARS-CoV-2 Delta, Alpha, or R.1 variants in Tokyo.

The rapid spread of the Delta variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) became a serious concern worldwide in summer 2021. We examined the copy number and variant types of all SARS-CoV-2-positive patients who visited our hospital from February to August 2021 using polymerase chain reaction (PCR) tests. Whole genome sequencing was performed for some samples. The R.1 variant (B.1.1.316) was responsible for most infections in March, replacing the previous variant (B.1.1.214); the Alpha (B.1.1.7) variant caused most infections in April and May; and the Delta variant (B.1.617.2) was the most prevalent in July and August. There was no significant difference in the copy numbers among the previous variant cases (n = 29, median 3.0 × 104 copies/µl), R.1 variant cases (n = 28, 2.1 × 105 copies/µl), Alpha variant cases (n = 125, 4.1 × 105 copies/µl), and Delta variant cases (n = 106, 2.4 × 105 copies/µl). Patients with Delta variant infection were significantly younger than those infected with R.1 and the previous variants, possibly because many elderly individuals in Tokyo were vaccinated between May and August. There was no significant difference in mortality among the four groups. Our results suggest that the increased infectivity of Delta variant may be caused by factors other than the higher viral loads. Clarifying these factors is important to control the spread of Delta variant infection.

SARS-CoV-2 R.1 lineage variants that prevailed in Tokyo in March 2021.

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, such as B.1.1.7 and B.1.351, has become a crucial issue worldwide. Therefore, we began testing all patients with COVID-19 for the N501Y and E484K mutations by using polymerase chain reaction (PCR)-based methods. Nasopharyngeal swab samples from 108 patients who visited our hospital between February and April 2021 were analyzed. The samples were analyzed using reverse transcription-PCR with melting curve analysis to detect the N501Y and E484K mutations. A part of the samples was also subjected to whole-genome sequencing (WGS). Clinical parameters such as mortality and admission to the intensive care unit were analyzed to examine the association between increased disease severity and the E484K mutation. The ratio of cases showing the 501N + 484K mutation rapidly increased from 8% in February to 46% in March. WGS revealed that the viruses with 501N + 484K mutation are R.1 lineage variants. Evidence of increased disease severity related to the R.1 variants was not found. We found that the R.1 lineage variants rapidly prevailed in Tokyo in March 2021, which suggests the increased transmissibility of R.1 variants, while they showed no increased severity.

Apolipoprotein C-II and C-III preferably transfer to both high-density lipoprotein (HDL)2 and the larger HDL3 from very low-density lipoprotein (VLDL).

Triglyceride hydrolysis by lipoprotein lipase (LPL), regulated by apolipoproteins C-II (apoC-II) and C-III (apoC-III), is essential for maintaining normal lipid homeostasis. During triglyceride lipolysis, the apoCs are known to be transferred from very low-density lipoprotein (VLDL) to high-density lipoprotein (HDL), but the detailed mechanisms of this transfer remain unclear. In this study, we investigated the extent of the apoC transfers and their distribution in HDL subfractions, HDL2 and HDL3. Each HDL subfraction was incubated with VLDL or biotin-labeled VLDL, and apolipoproteins and lipids in the re-isolated HDL were quantified using western blotting and high-performance liquid chromatography (HPLC). In consequence, incubation with VLDL showed the increase of net amount of apoC-II and apoC-III in the HDL. HPLC analysis revealed that the biotin-labeled apolipoproteins, including apoCs and apolipoprotein E, were preferably transferred to the larger HDL3. No effect of cholesteryl ester transfer protein inhibitor on the apoC transfers was observed. Quantification of apoCs levels in HDL2 and HDL3 from healthy subjects (n = 8) showed large individual differences between apoC-II and apoC-III levels. These results suggest that both apoC-II and apoC-III transfer disproportionately from VLDL to HDL2 and the larger HDL3, and these transfers might be involved in individual triglyceride metabolism.

Culturing Chinese hamster ovary cells on cyclo olefin polymer triggers epithelial-mesenchymal transition and spheroid formation, which increases the foreign gene expression driven by the Moloney murine leukemia virus long terminal repeat promoter.

Chinese hamster ovary (CHO) cells are frequently used for recombinant protein production (RPP) as a host. While the RPP has been proven successful, there is still a compelling need for further improvement. Cyclo olefin polymer (COP) is a plastic material widely utilized due to its properties including its low protein absorption. We applied this as a raw material for RPP cell culture to see if the COP is suitable. A recombinant CHO cell line expressing the human erythropoietin (hEPO) gene under the control of the Moloney murine leukemia virus-long terminal repeat (MMLV-LTR) was established. When the cells were cultured in a dish made from COP, the cells attached to the bottom, and then started to float and form spheroids. RNASeq data analysis suggested the epithelial-mesenchymal transition (EMT) was triggered with receptor tyrosine kinase activation shortly after cultivation. It coincided with the hEPO transcription increase. After the cell floating, though EMT marker gene expression subsided, a hEPO expression increase sustained. When fibronectin was applied to COP dish surface, the cell floating was suppressed and hEPO expression decreased. We then treated cells with MßCD, a drug that destroys the lipid raft, eliminating molecules in the raft. This facilitated cell floating and spheroid formation coincided with hEPO expression enhancement. These results suggest interactions between a cell and COP surface might trigger the EMT and the subsequent event, both of which activated the MMLV-LTR promoter. Thus, employing COP for culturing cells, a potent RPP system could be established with its advantage for efficient protein purification.

Marked Changes in Serum Amyloid A Distribution and High-Density Lipoprotein Structure during Acute Inflammation.

High-density lipoprotein- (HDL-) cholesterol measurements are generally used in the diagnosis of cardiovascular diseases. However, HDL is a complicated heterogeneous lipoprotein, and furthermore, it can be converted into dysfunctional forms during pathological conditions including inflammation. Therefore, qualitative analysis of pathophysiologically diversified HDL forms is important. A recent study demonstrated that serum amyloid A (SAA) can remodel HDL and induce atherosclerosis not only over long periods of time, such as during chronic inflammation, but also over shorter periods. However, few studies have investigated rapid HDL remodeling. In this study, we analyzed HDL samples from patients undergoing orthopedic surgery inducing acute inflammation. We enrolled 13 otherwise healthy patients who underwent orthopedic surgery. Plasma samples were obtained on preoperative day and postoperative days (POD) 1-7. SAA, apolipoprotein A-I (apoA-I), and apolipoprotein A-II (apoA-II) levels in the isolated HDL were determined. HDL particle size, surface charge, and SAA and apoA-I distributions were also analyzed. In every patient, plasma SAA levels peaked on POD3. Consistently, the HDL apoA-I : apoA-II ratio markedly decreased at this timepoint. Native-polyacrylamide gel electrophoresis and high-performance liquid chromatography revealed the loss of small HDL particles during acute inflammation. Furthermore, HDL had a decreased negative surface charge on POD3 compared to the other timepoints. All changes observed were SAA-dependent. SAA-dependent rapid changes in HDL size and surface charge were observed after orthopedic surgery. These changes might affect the atheroprotective functions of HDL, and its analysis can be available for the qualitative HDL assessment.

Cholesterol Efflux Capacity of Apolipoprotein A-I Varies with the Extent of Differentiation and Foam Cell Formation of THP-1 Cells.

Apolipoprotein A-I (apoA-I), the main protein component of high-density lipoprotein (HDL), has many protective functions against atherosclerosis, one of them being cholesterol efflux capacity. Although cholesterol efflux capacity measurement is suggested to be a key biomarker for evaluating the risk of development of atherosclerosis, the assay has not been optimized till date. This study aims at investigating the effect of different states of cells on the cholesterol efflux capacity. We also studied the effect of apoA-I modification by homocysteine, a risk factor for atherosclerosis, on cholesterol efflux capacity in different states of cells. The cholesterol efflux capacity of apoA-I was greatly influenced by the extent of differentiation of THP-1 cells and attenuated by excessive foam cell formation. N-Homocysteinylated apoA-I indicated a lower cholesterol efflux capacity than normal apoA-I in the optimized condition, whereas no significant difference was observed in the cholesterol efflux capacity between apoA-I in the excessive cell differentiation or foam cell formation states. These results suggest that cholesterol efflux capacity of apoA-I varies depending on the state of cells. Therefore, the cholesterol efflux assay should be performed using protocols optimized according to the objective of the experiment.

High-Density Lipoprotein Binds to Mycobacterium avium and Affects the Infection of THP-1 Macrophages.

High-density lipoprotein (HDL) is involved in innate immunity toward various infectious diseases. Concerning bacteria, HDL is known to bind to lipopolysaccharide (LPS) and to neutralize its physiological activity. On the other hand, cholesterol is known to play an important role in mycobacterial entry into host cells and in survival in the intracellular environment. However, the pathogenicity of Mycobacterium avium (M. avium) infection, which tends to increase worldwide, remains poorly studied. Here we report that HDL indicated a stronger interaction with M. avium than that with other Gram-negative bacteria containing abundant LPS. A binding of apolipoprotein (apo) A-I, the main protein component of HDL, with a specific lipid of M. avium might participate in this interaction. HDL did not have a direct bactericidal activity toward M. avium but attenuated the engulfment of M. avium by THP-1 macrophages. HDL also did not affect bacterial killing after ingestion of live M. avium by THP-1 macrophage. Furthermore, HDL strongly promoted the formation of lipid droplets in M. avium-infected THP-1 macrophages. These observations provide new insights into the relationship between M. avium infection and host lipoproteins, especially HDL. Thus, HDL may help M. avium to escape from host innate immunity.

Effects of serum amyloid A on the structure and antioxidant ability of high-density lipoprotein.

Serum amyloid A (SAA) levels increase during acute and chronic inflammation and are mainly associated with high-density lipoprotein (HDL). In the present study, we investigated the effect of SAA on the composition, surface charge, particle size and antioxidant ability of HDL using recombinant human SAA (rhSAA) and HDL samples from patients with inflammation. We confirmed that rhSAA bound to HDL3 and released apolipoprotein A-I (apoA-I) from HDL without an apparent change in particle size. Forty-one patients were stratified into three groups based on serum SAA concentrations: Low (SAA ≤ 8 µg/ml), Middle (8 < SAA ≤ 100 µg/ml) and High (SAA > 100 µg/ml). The ratios of apoA-I to total protein mass, relative cholesterol content and negative charge of HDL samples obtained from patients with high SAA levels were lower than that for samples from patients with low SAA levels. Various particle sizes of HDL were observed in three groups regardless of serum SAA levels. Antioxidant ability of rhSAA, evaluated as the effect on the formation of conjugated diene in low-density lipoprotein (LDL) induced by oxidation using copper sulfate, was higher than that of apoA-I. Consistent with this result, reconstituted SAA-containing HDL (SAA-HDL) indicated higher antioxidant ability compared with normal HDL. Furthermore, HDL samples obtained from High SAA group patients also showed the highest antioxidant ability among the three groups. Consequently, SAA affects the composition and surface charge of HDL by displacement of apoA-I and enhances its antioxidant ability.

Identification of valine- or leucine-containing glycopeptidolipids from Mycobacterium avium-intracellulare complex.

Mycobacterium avium-intracellulare complex is a species of acid-fast microorganisms that cause opportunistic infections in immuno-compromised hosts. The cell wall of this microbe is rich in glycopeptidolipids (GPLs), which are composed of a fatty acyl moiety, several sugar moieties and a tripeptide-amino alcohol, D-Phe-D-alloThr-D-Ala-L-Alaninol. GPLs have molecular diversity in the hydrocarbon chain variety of the acyl moiety, and methyl and acetyl modifications of the sugar moiety, but there has been no report of any variety in the tripeptide-amino alcohol component. In this study, we showed two atypical GPL ions of 34 or 48 Da less than the dominant ions of GPLs by mass spectrometry. These ions could not be explained as resulting from conventional molecular diversity. To investigate the reasons why these ions appeared, we made a preparation of the lipopeptide component from intact GPLs and structurally analyzed the molecules. The results suggested that these atypical ions differed from the typical ions in amino acid composition. We further determined its composition by amino acid analysis, and the results showed that the tripeptide portion of the two atypical ions is composed of the Val-alloThr-Ala or the Leu-alloThr-Ala amino acid sequence. In this study, we present novel variations in the tripeptide portion of GPL molecules.

Safety evaluation of a heavy oil-degrading bacterium, Rhodococcus erythropolis C2.

The safety of an oil-degrading bacterium, C2 strain, was evaluated for utilization in an open system for bioremediation of oil-contaminated environments. The C2 strain was identified as Rhodococcus erythropolis by performing an alignment analysis of the whole 16S rRNA sequence. R. erythropolis was classified as a nonpathogenic (category 1) bacterium. Biological and biochemical properties of the C2 strain also confirmed its nonpathogenicity. The pathogenicity and basic ecotoxicity were studied in laboratory animals and in a variety of test species, respectively. General and inhalation toxicities were not detected; additionally, there was no evidence of skin irritation, mutagenic potential, eye irritation, skin sensitization, ecotoxicity or notable pathogenicity. The comparison of these results with human exposure levels and previously published data indicates that the C2 strain appears to be safe for utilization in bioremediation of polluted environments, requires no special occupational health precautions during the application process, and has a low environmental impact. This study suggests that the C2 strain could be suitable for bioremediation of oil-contaminated environments.

Negative regulation of Wnt signalling by HMG2L1, a novel NLK-binding protein.

BACKGROUND: Wnt signalling plays a critical role in many developmental processes and tumorigenesis. Wnt/beta-catenin signalling induces the stabilization of cytosolic beta-catenin, which interacts with TCF/LEF-1 transcription factors, thereby inducing expression of Wnt-target genes. Recent evidence suggests that a specific MAP kinase pathway involving the MAP kinase kinase kinase TAK1 and the MAP kinase NLK counteract Wnt signalling. RESULTS: To identify NLK-interacting proteins, we performed yeast two-hybrid screening. We isolated the gene HMG2L1 and showed that injection of Xenopus HMG2L1 (xHMG2L1) mRNA into Xenopus embryos inhibited Wnt/beta-catenin-induced axis duplication and expression of Wnt/beta-catenin target genes. Moreover, xHMG2L1 inhibited beta-catenin-stimulated transcriptional activity in mammalian cells. CONCLUSIONS: Our findings indicate that xHMG2L1 may negatively regulate Wnt/beta-catenin signalling, and that xHMG2L1 may play a role in early Xenopus development together with NLK.