Serum cystatin C and CRP are early predictive biomarkers for emergence of hypoxia in COVID-19.

BACKGROUND: In Japan, during the coronavirus disease 2019 (COVID-19) pandemic, patients with non-hypoxia are recommended to recuperate at home or in pre-hospital facilities. However, it was observed that unexpected hypoxia may occur and become severe subsequently in patients whose symptoms were initially expected to improve naturally. The aim of this study is to validate biomarkers that can predict at an early stage the emergence of hypoxia in COVID-19 patients without hypoxia. METHODS: We retrospectively enrolled 193 patients with COVID-19, excluding patients with hypoxia and severe disease from the onset. Participants were classified into two groups according to the emergence of hypoxia during the clinical course, and the laboratory data were compared to identify biomarkers that could predict early the emergence of hypoxia. RESULTS: The areas under the curve for serum cystatin C (CysC) and C-reactive protein (CRP) levels for the emergence of hypoxia during the clinical course were higher than those for other biomarkers (CysC, 0.84 and CRP, 0.83). Multivariate analysis showed that high serum CysC and CRP levels were associated with the emergence of hypoxia during the clinical course. CONCLUSIONS: Elevated serum CysC and CRP levels were associated with the emergence of hypoxia during the clinical course in COVID-19 patients without hypoxia. These findings may help determine the need for hospitalization in initially non-hypoxic COVID-19 patients.

Genetic epidemiology using whole genome sequencing and haplotype networks revealed the linkage of SARS-CoV-2 infection in nosocomial outbreak.

BACKGROUND: A characteristic feature of SARS-CoV-2 is its ability to transmit from pre- or asymptomatic patients, complicating the tracing of infection pathways and causing outbreaks. Despite several reports that whole genome sequencing (WGS) and haplotype networks are useful for epidemiologic analysis, little is known about their use in nosocomial infections. AIM: We aimed to demonstrate the advantages of genetic epidemiology in identifying the link in nosocomial infection by comparing single nucleotide variations (SNVs) of isolates from patients associated with an outbreak in Showa University Hospital. METHODS: We used specimens from 32 patients in whom COVID-19 had been diagnosed using clinical reverse transcription-polymerase chain reaction tests. RNA of SARS-CoV-2 from specimens was reverse-transcribed and analysed using WGS. SNVs were extracted and used for lineage determination, phylogenetic tree analysis, and median-joining analysis. FINDINGS: The lineage of SARS-CoV-2 that was associated with outbreak in Showa University Hospital was B.1.1.214, which was consistent with that found in the Kanto metropolitan area during the same period. Consistent with canonical epidemiological observations, haplotype network analysis was successful for the classification of patients. Additionally, phylogenetic tree analysis revealed three independent introductions of the virus into the hospital during the outbreak. Further, median-joining analysis indicated that four patients were directly infected by any of the others in the same cluster. CONCLUSION: Genetic epidemiology with WGS and haplotype networks is useful for tracing transmission and optimizing prevention strategies in nosocomial outbreaks.

Eribulin upregulates miR-195 expression and downregulates Wnt3a expression in non-basal-like type of triple-negative breast cancer cell MDA-MB-231.

Triple-negative breast cancer (TNBC), which does not show hormone sensitivity, is a poor prognosis disease without an established targeted treatment, so that establishing a therapeutic target for each subtype is desired. In addition, microRNA (miRNA), a non-cording RNA 19-25 nucleotide-longs in length, is known to be involved in regulating gene expression. We examined miRNA expression after exposure to eribulin, MDA-MB-231 cells, non-basal-like type of TNBC cell lines, and HCC1143 cells, basal-like type of TNBC cell lines. The activity of caspase-3 significantly increased compared to the control in MDA-MB-231, whereas no significant difference was observed in HCC1143. The expression level of 20-miRNAs significantly increased compared to the control in MDA-MB-231 after exposure to eribulin. The expression level of 6-miRNAs also significantly increased compared to the control in HCC1143. In these 2 cell types, miR-125b-1 and miR-195 were commonly expressed. While the expression level of miR-125b-1 decreased in both cells, the expression level of miR-195 increased in MDA-MB-231 and decreased in HCC1143. The expression level of miR-195 targeting Wnt3a significantly decreased compared to the control in MDA-MB-231, whereas it significantly increased in HCC1143. These results showed that exposure to eribulin highly increased the expression of miR-195 while it decreased the expression of Wnt3a in non-basal-like type of TNBC. Some miRNAs are known to regulate other signaling pathways involved in human pathogenesis by regulating the Wnt signaling pathway, and miRNA can act as a tumor-suppressing gene; therefore, miR-195 may serve as a therapeutic target in non-basal-like type of TNBC.

Reduced PTEN expression and overexpression of miR-17-5p, -19a-3p, -19b-3p, -21-5p, -130b-3p, -221-3p and -222-3p by glioblastoma stem-like cells following irradiation.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor gene that induces cell apoptosis by inhibiting the PI3K/Akt signaling pathway. Glioblastoma (GBM) is a brain tumor that is resistant to irradiation and chemotherapy and, thus, is difficult to cure. GBM stem-like cells (GSCs) have been implicated as a cause of this resistance. microRNA (miRNA/miR) inhibits the expression of proteins. The objective of the present study was to identify miRNAs that target PTEN, which induces apoptosis, in irradiation-resistant GSCs. When the expression of miRNAs was examined in GSCs irradiated at 60 Gy using the human GBM A172 cell line, the expression of PTEN-targeting miR-17-5p, -19a-3p, -19b-3p, -21-5p, -130b-3p, -221-3p and -222-3p was significantly higher in irradiated GSCs than in non-irradiated cells, and the PTEN expression levels, as revealed by immunostaining, were lower in the irradiated GSCs than in the non-irradiated cells. These results suggested that the expression of PTEN was suppressed through the overexpression of PTEN-targeting miRNAs in GSCs following irradiation.

Diabetes therapies in hemodialysis patients: Dipeptidase-4 inhibitors.

Although several previous studies have been published on the effects of dipeptidase-4 (DPP-4) inhibitors in diabetic hemodialysis (HD) patients, the findings have yet to be reviewed comprehensively. Eyesight failure caused by diabetic retinopathy and aging-related dementia make multiple daily insulin injections difficult for HD patients. Therefore, we reviewed the effects of DPP-4 inhibitors with a focus on oral antidiabetic drugs as a new treatment strategy in HD patients with diabetes. The following 7 DPP-4 inhibitors are available worldwide: sitagliptin, vildagliptin, alogliptin, linagliptin, teneligliptin, anagliptin, and saxagliptin. All of these are administered once daily with dose adjustments in HD patients. Four types of oral antidiabetic drugs can be administered for combination oral therapy with DPP-4 inhibitors, including sulfonylureas, meglitinide, thiazolidinediones, and alpha-glucosidase inhibitor. Nine studies examined the antidiabetic effects in HD patients. Treatments decreased hemoglobin A1c and glycated albumin levels by 0.3% to 1.3% and 1.7% to 4.9%, respectively. The efficacy of DPP-4 inhibitor treatment is high among HD patients, and no patients exhibited significant severe adverse effects such as hypoglycemia and liver dysfunction. DPP-4 inhibitors are key drugs in new treatment strategies for HD patients with diabetes and with limited choices for diabetes treatment.

Decreased miR-206 expression in BRCA1 wild-type triple-negative breast cancer cells after concomitant treatment with gemcitabine and a Poly(ADP-ribose) polymerase-1 inhibitor.

No targeted-therapy has been established for triple-negative breast cancer accompanied by mutations in breast cancer susceptibility gene1 (BRCA1) mutation. In the present study, using BRCA1 wild-type cells (MDA-MB-231) and BRCA1-mutated cells (MDA-MB-436), we investigated miRNA expression and apoptosis on day 1 after addition of gemcitabine-alone and in combination with poly ADP-ribose polymerase-1 (PARP1) inhibitor. After drug treatment, there were significantly fewer apoptotic BRCA1 wild-type cells than BRCA1-mutated cells. Expression of miRNA-26a, -29b, -100, and -148a increased in BRCA1 wild-type cells exposed to gemcitabine-alone and in combination with the PARP1 inhibitor. The addition of PARP1 inhibitor reduced miR-206 expression in BRCA1 wild-type cells but increased it in BRCA1-mutated cells. It was suggested that miR-206 serves as a target molecule of PARP1 inhibitor combination therapy for BRCA1 wild-type triple-negative breast cancer cells.

Gene analysis and dynamics of tumor stem cells in human glioblastoma cells after radiation.

Glioblastoma is the most malignant central nervous system tumor. Patients with glioblastoma are treated with a combination of surgery, radiotherapy and chemotherapy; however, this effect is not satisfactory with regard to the prognosis. It is reported that the tumor stem cells affect recurrence, and radio- and chemotherapy resistance of the tumor, and that these cells play an important role in tumorigenesis and tumor progression. Using human glioblastoma cell lines (T98G and A172), irradiated (0, 30, 60 Gy) glioblastoma cells were prepared under the same conditions as clinical therapy. We analyzed cell proliferation rate, side population analysis by fluorescence-activated cell sorting and isolation of CD133⁺ cells, and performed genetic analysis (human stem cells) on these cells. We also investigated the difference in gene expression in the cells after radiation. The stem cell-related genes were highly expressed in the CD133⁺ cells compared with the CD133⁻ cells, suggesting that the cancer stem cells may be located in these CD133⁺ cells. In the T98G cell line, the cell proliferation rate of 30-Gy irradiated cells was higher than those of non-irradiated cells and 60-Gy irradiated cells. Stem cell-related genes were highly expressed in 30-Gy irradiated CD133⁺ T98G cells. In conclusion, we suggest that CD133⁺ cells may strongly affect tumor proliferation and the resistance against radiation therapy.

Analysis of p53 and miRNA expression after irradiation of glioblastoma cell lines.

Glioblastoma is a malignant brain tumor that is difficult to completely cure by surgical treatment alone. However, resistance to anticancer drugs and radiation may be acquired during treatment. For instance, miRNAs involved in regulating the expression of genes inducing apoptosis and other specific genes have been proposed for use, in order to induce the apoptosis of radioresistant cancer cells. A172 glioblastoma cells, expressing wild-type p53 were irradiated to a total dose of up to 60 Gy allowing us to analyze the activities of apoptosis-related proteins. The miR-34a expression levels in cells after irradiation at 30 and 60 Gy were 0.17- and 18.7-times the BCL2 and caspase-9 expression levels, respectively. The high miR-34a expression level in the cells after irradiation at 60 Gy reduced the p53 expression level. This study suggests that apoptosis might be promoted by regulating the action of miRNAs, even in cells that have acquired radioresistance.

Individual difference in the pharmacokinetics of a drug, pravastatin, in healthy subjects.

The purpose of this investigation was to determine whether there were individual pharmacokinetic differences of a drug, pravastatin. Furthermore, the percentage of subjects who showed pharmacokinetic differences was determined. A single oral dose of pravastatin 10 mg was administered to 84 Japanese healthy male subjects. Serum concentrations of pravastatin were measured for 8 hours postdose. Area under the concentration-time curve (AUC) and peak concentration (Cmax) were determined as primary evaluation parameters. An outlier was defined as follows: Outlier 1 < Q1 - (Q3 - Q1) x 1.5 or Q3+(Q3-Q1) x 1.5 < Outlier 1, Outlier 2 < Q1 - (Q3 - Q1) x 3 or Q3+ (Q3-Q1) x 3 < Outlier 2. Subjects who were outliers were regarded as having an individual difference in pharmacokinetic behavior. In AUC and Cmax, 4 of 84 subjects (4.8%) were higher outliers. Of these 4 subjects, 2 were high outliers in both AUC and Cmax. No subjects were low outliers. It was concluded that a significant individual difference in the pharmacokinetics of pravastatin was observed in 4.8% of the subjects (4/84).

Increase in plasma fibrinogen and plasminogen activator inhibitor level in diabetic KK and KK-Ay mice.

Coagulation activity in KK mice and KK-Ay mice produced by transferring the yellow obese gene (Ay) into KK mice, was studied to examine whether both mice are useful as a model of diabetic atherosclerosis. Plasma levels of hemoglobin A1c (HbA1c), insulin, fibrinogen, plasminogen activator inhibitor (PAI) and thrombomodulin were significantly high in KK and KK-Ay mice compared with age-matched non-diabetic mice (ddY mice). The changes in the plasma levels of fibrinogen at each time-point correlated with the increases in HbA1c levels. Pathological observation by Oil red O staining of aorta tissue from 4-month-old KK and KK-Ay mice revealed the early stages of atherosclerosis such as lipid deposition. These age-related increases in the plasma level of fibrinogen and PAI suggested that KK-Ay mice may contribute to help elucidate the early stages of diabetic atherosclerosis.

Diabetic nephropathy in KK and KK-Ay mice.

KK mice and KK-Ay mice were examined for age related changes in blood and urinary biophysiological parameters. Blood hemoglobin A1c levels were significantly higher in KK-Ay and KK mice as compared to non-diabetic ddY mice. In both diabetic mice, especially KK-Ay mice, plasma insulin levels markedly increased at 2 to 4 months of age, and the urinary glucose and microalbumin levels and albumin-to-creatinine ratios increased dependent on age. Plasma thrombomodulin levels significantly increased at 2 to 4 months of age in both KK and KK-Ay mice. Mild enlargement of mesangial matrix and segmental proliferative glomerular nephritis were revealed in KK and KK-Ay mice, respectively, at 4 months of age. KK-Ay mice with insulin resistance and high urine mAlb level might be useful as models for the early stage of diabetic nephropathy.