Optimization of reaction condition of recombinase polymerase amplification to detect SARS-CoV-2 DNA and RNA using a statistical method.

Recombinase polymerase amplification (RPA) is an isothermal reaction that amplifies a target DNA sequence with a recombinase, a single-stranded DNA-binding protein (SSB), and a strand-displacing DNA polymerase. In this study, we optimized the reaction conditions of RPA to detect SARS-CoV-2 DNA and RNA using a statistical method to enhance the sensitivity. In vitro synthesized SARS-CoV-2 DNA and RNA were used as targets. After evaluating the concentration of each component, the uvsY, gp32, and ATP concentrations appeared to be rate-determining factors. In particular, the balance between the binding and dissociation of uvsX and DNA primer was precisely adjusted. Under the optimized condition, 60 copies of the target DNA were specifically detected. Detection of 60 copies of RNA was also achieved. Our results prove the fabrication flexibility of RPA reagents, leading to an expansion of the use of RPA in various fields.

Cross-platform comparison of SYBR Green real-time PCR with TaqMan PCR, microarrays and other gene expression measurement technologies evaluated in the MicroArray Quality Control (MAQC) study.

BACKGROUND: The MicroArray Quality Control (MAQC) project evaluated the inter- and intra-platform reproducibility of seven microarray platforms and three quantitative gene expression assays in profiling the expression of two commercially available Reference RNA samples (Nat Biotechnol 24:1115-22, 2006). The tested microarrays were the platforms from Affymetrix, Agilent Technologies, Applied Biosystems, GE Healthcare, Illumina, Eppendorf and the National Cancer Institute, and quantitative gene expression assays included TaqMan Gene Expression PCR Assay, Standardized (Sta) RT-PCRtrade mark and QuantiGene. The data showed great consistency in gene expression measurements across different microarray platforms, different technologies and test sites. However, SYBR Green real-time PCR, another common technique utilized by half of all real-time PCR users for gene expression measurement, was not addressed in the MAQC study. In the present study, we compared the performance of SYBR Green PCR with TaqMan PCR, microarrays and other quantitative technologies using the same two Reference RNA samples as the MAQC project. We assessed SYBR Green real-time PCR using commercially available RT2 Profilertrade mark PCR Arrays from SuperArray, containing primer pairs that have been experimentally validated to ensure gene-specificity and high amplification efficiency. RESULTS: The SYBR Green PCR Arrays exhibit good reproducibility among different users, PCR instruments and test sites. In addition, the SYBR Green PCR Arrays have the highest concordance with TaqMan PCR, and a high level of concordance with other quantitative methods and microarrays that were evaluated in this study in terms of fold-change correlation and overlap of lists of differentially expressed genes. CONCLUSION: These data demonstrate that SYBR Green real-time PCR delivers highly comparable results in gene expression measurement with TaqMan PCR and other high-density microarrays.

Effects of insulin replacements, inhibitors of angiotensin, and PKCbeta's actions to normalize cardiac gene expression and fuel metabolism in diabetic rats.

High-density oligonucleotide arrays were used to compare gene expression of rat hearts from control, untreated diabetic, and diabetic groups treated with islet cell transplantation (ICT), protein kinase C (PKC)beta inhibitor ruboxistaurin, or ACE inhibitor captopril. Among the 376 genes that were differentially expressed between untreated diabetic and control hearts included key metabolic enzymes that account for the decreased glucose and increased free fatty acid utilization in the diabetic heart. ICT or insulin replacements reversed these gene changes with normalization of hyperglycemia, dyslipidemia, and cardiac PKC activation in diabetic rats. Surprisingly, both ruboxistaurin and ACE inhibitors improved the metabolic gene profile (confirmed by real-time RT-PCR and protein analysis) and ameliorated PKC activity in diabetic hearts without altering circulating metabolites. Functional assessments using Langendorff preparations and (13)C nuclear magnetic resonance spectroscopy showed a 36% decrease in glucose utilization and an impairment in diastolic function in diabetic rat hearts, which were normalized by all three treatments. In cardiomyocytes, PKC inhibition attenuated fatty acid-induced increases in the metabolic genes PDK4 and UCP3 and also prevented fatty acid-mediated inhibition of basal and insulin-stimulated glucose oxidation. Thus, PKCbeta or ACE inhibitors may ameliorate cardiac metabolism and function in diabetes partly by normalization of fuel metabolic gene expression directly in the myocardium.

Effects of endothelin receptor blockade on hypervasoreactivity in streptozotocin-diabetic rats: vessel-specific involvement of thromboxane A2.

Increased vasoconstrictor response to norepinephrine (NE) and endothelin (ET)-1 in arteries from diabetic animals is ameliorated by chronic endothelin receptor blockade with bosentan and was absent in endothelium-denuded arteries, suggesting the involvement of ET-1 and an endothelium-derived contracting factor such as thromboxane A2 (TxA2). To examine this possibility, we determined the effects of acute blockade of ET receptors or inhibition of TxA2 synthesis on the vascular function of superior mesenteric arteries (SMA) and renal arteries (RA) isolated from nondiabetic and 11-week streptozotocin (STZ) diabetic rats chronically treated with either bosentan or vehicle. Both in vitro incubation with bosentan and a selective ETA receptor blocker, BQ123, eradicated the increase in NE contractile responses in diabetic SMA. Additionally, in vitro incubation with the thromboxane synthase inhibitor, dazmegrel, abrogated the exaggerated NE and ET-1 contractile responses in diabetic SMA. Conversely, in RA, no significant acute effect of bosentan, BQ123, nor dazmegrel on vascular responses to NE was observed. Dazmegrel incubation attenuated the maximum contractile responses to ET-1 in diabetic RA; however, these responses in diabetic RA remained significantly greater than those of other groups. Diabetic RA but not SMA exhibited an enhanced contractile response to the TxA2 analogue U46619, which was corrected by chronic bosentan treatment. Immunohistochemical analyses in diabetic SMA revealed an increase in ETA receptor level that was normalized by chronic bosentan treatment. These data indicate that an interaction between ET-1 and TxA2 may be involved in mediating the exaggerated vasoconstrictor responses in diabetic arteries. Furthermore, the underlying mechanisms appear to be vessel specific.

Reduction of diabetes-induced oxidative stress, fibrotic cytokine expression, and renal dysfunction in protein kinase Cbeta-null mice.

Diabetes induces the activation of several protein kinase C (PKC) isoforms in the renal glomeruli. We used PKC-beta(-/-) mice to examine the action of PKC-beta isoforms in diabetes-induced oxidative stress and renal injury at 8 and 24 weeks of disease. Diabetes increased PKC activity in renal cortex of wild-type mice and was significantly reduced (<50% of wild-type) in diabetic PKC-beta(-/-) mice. In wild-type mice, diabetes increased the translocation of PKC-alpha and -beta1 to the membrane, whereas only PKC-alpha was elevated in PKC-beta(-/-) mice. Increases in urinary isoprostane and 8-hydroxydeoxyguanosine, parameters of oxidative stress, in diabetic PKC-beta(-/-) mice were significantly reduced compared with diabetic wild-type mice. Diabetes increased NADPH oxidase activity and the expressions of p47(phox), Nox2, and Nox4 mRNA levels in the renal cortex and were unchanged in diabetic PKC-beta(-/-) mice. Increased expression of endothelin-1 (ET-1), vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-beta, connective tissue growth factor (CTGF), and collagens IV and VI found in diabetic wild-type mice was attenuated in diabetic PKC-beta(-/-) mice. Diabetic PKC-beta(-/-) mice were protected from renal hypertrophy, glomerular enlargement, and hyperfiltration observed in diabetic wild-type mice and had less proteinuria. Lack of PKC-beta can protect against diabetes-induced renal dysfunction, fibrosis, and increased expressions of Nox2 and -4, ET-1, VEGF, TGF-beta, CTGF, and oxidant production.

Chronic estrogen treatment modifies insulin-induced insulin resistance and hypertension in ovariectomized rats.

BACKGROUND: Gender differences have been found in the development of hypertension. The role of estrogen in the association between hyperinsulinemia/insulin resistance and hypertension was investigated in an insulin-induced, insulin-resistant, and hypertensive model. METHODS: Ovariectomized or sham operated female Wistar rats were chronically treated with insulin and/or estrogen via subcutaneous implants (insulin, 2 U/day; 17beta-estradiol 0.5 mg/pellet, 60-day release). Systolic blood pressure was monitored at weeks 0, 3, and 6. At week 7, an oral glucose tolerance test was performed. RESULTS: Ovariectomy resulted in the development of insulin resistance and blood pressure elevation in chronically insulin-treated female rats. Chronic estrogen treatment prevented the elevation in blood pressure and the development of insulin resistance. CONCLUSION: The results indicate that chronic estrogen treatment modifies the insulin-induced hypertension by increasing insulin sensitivity in ovariectomized rats.

Differential regulation of angiotensin II-induced expression of connective tissue growth factor by protein kinase C isoforms in the myocardium.

Protein kinase C (PKC) and angiotensin II (AngII) can regulate cardiac function in pathological conditions such as in diabetes or ischemic heart disease. We have reported that expression of connective tissue growth factor (CTGF) is increased in the myocardium of diabetic mice. Now we showed that the increase in CTGF expression in cardiac tissues of streptozotocin-induced diabetic rats was reversed by captopril and islet cell transplantation. Infusion of AngII in rats increased CTGF mRNA expression by 15-fold, which was completely inhibited by co-infusion with AT1 receptor antagonist, candesartan. Similarly, incubation of cultured cardiomyocytes with AngII increased CTGF mRNA expression by 2-fold, which was blocked by candesartan and a general PKC inhibitor, GF109203X. The role of PKC isoform-dependent action was further studied using adenoviral vector-mediated gene transfer of dominant negative (dn) PKC or wild type PKC isoforms. Expression of dnPKCalpha, -epsilon, and -zeta isoforms suppressed AngII-induced CTGF expression in cardiomyocytes. In contrast, expression of dominant negative PKCdelta significantly increased AngII-induced CTGF expression, whereas expression of wild type PKCdelta inhibited this induction. This inhibitory effect was further confirmed in the myocardium of transgenic mice with cardiomyocyte-specific overexpression of PKCdelta (deltaTg mice). Thus, AngII can regulate CTGF expression in cardiomyocytes through a PKC activation-mediated pathway in an isoform-selective manner both in physiological and diabetic states and may contribute to the development of cardiac fibrosis in diabetic cardiomyopathy.

Androgens are necessary for the development of fructose-induced hypertension.

Hyperinsulinemia and insulin resistance are closely associated with hypertension in humans and in animal models. Gender differences have been found in the development of hypertension in fructose-fed rats. The objectives of the present study were, first, to clarify whether androgens are required in the development of hyperinsulinemia, insulin resistance, and hypertension in fructose-fed rats, and second, to determine if cyclooxygenase-1 and cyclooxygenase-2 are also increased in the arteries of these rats. Male rats were gonadectomized or sham-operated and fed a 60% fructose diet beginning at age 7 weeks. Blood pressure was measured by a tail-cuff method, and an oral glucose tolerance test was performed to assess insulin sensitivity after 8 weeks of fructose feeding. Cyclooxygenase-1 and cyclooxygenase-2 mRNA expression was also assessed in the thoracic aortae and mesenteric arteries. Gonadectomy prevented hypertension from developing in the fructose-fed rats, but hyperinsulinemia and insulin resistance developed. There was an increase in cyclooxygenase-2 expression in the thoracic aortae and mesenteric arteries of the fructose-fed sham-operated rats while the expression of cyclooxygenase-1 remained unchanged. Gonadectomy prevented the mRNA overexpression of vascular cyclooxygenase-2 in the fructose-fed rats. These results suggest that the presence of androgens is necessary for the development of fructose-induced hypertension. Androgens apparently act as a link between hyperinsulinemia/insulin resistance and hypertension in fructose-hypertensive rats. Furthermore, an increase in the expression of cyclooxygenase-2 is implicated in the development of hypertension. The mechanisms involved require further study.

Exaggerated coronary reactivity to endothelin-1 in diabetes: reversal with bosentan.

We previously demonstrated that chronic endothelin receptor blockade (with bosentan) improved functional cardiac performance in streptozotocin-diabetic rats, suggesting a novel role of endothelin-1 (ET-1) in modulating diabetic heart dysfunction. To gain insight into the mechanism(s) underlying this effect, we examined the coronary vascular responses to ET-1 in hearts from diabetic and control rats treated with or without bosentan. Rats were divided into control, control-treated, diabetic, and diabetic-treated groups. The control-treated and diabetic-treated groups received bosentan (100 mg x kg(-1) x d(-1)) for 8 weeks. Following treatment, hearts were isolated and perfused, and coronary reactivity to ET-1 was assessed by measuring the changes in coronary perfusion pressure in response to ET-1 (50 and 100 pM). Additionally, maximal coronary blood flow (assessed with 10(-5) M adenosine) was measured in isolated perfused hearts. The key observation is that coronary reactivity to ET-1 was significantly higher in the diabetic than the control rats. This effect was normalized in diabetic rats chronically receiving bosentan. Maximal coronary vasodilation did not differ between the four groups. In conclusion, the reactivity of ET-1 is altered in the isolated perfused coronary vascular bed from diabetic rats, and chronic ET receptor blockade restores this reactivity to control values. These observations provide a possible mechanism for the improvement in diabetic heart function observed after chronic bosentan treatment.