Discrimination of SARS-CoV-2 omicron variant and its lineages by rapid detection of immune-escape mutations in spike protein RBD using asymmetric PCR-based melting curve analysis.

BACKGROUND: The SARS-CoV-2 Omicron strain has multiple immune-escape mutations in the spike protein receptor-binding domain (RBD). Rapid detection of these mutations to identify Omicron and its lineages is essential for guiding public health strategies and patient treatments. We developed a two-tube, four-color assay employing asymmetric polymerase chain reaction (PCR)-based melting curve analysis to detect Omicron mutations and discriminate the BA.1, BA.2, BA.4/5, and BA.2.75 lineages. METHODS: The presented technique involves combinatory analysis of the detection of six fluorescent probes targeting the immune-escape mutations L452R, N460K, E484A, F486V, Q493R, Q498R, and Y505H within one amplicon in the spike RBD and probes targeting the ORF1ab and N genes. After protocol optimization, the analytical performance of the technique was evaluated using plasmid templates. Sensitivity was assessed based on the limit of detection (LOD), and reliability was assessed by calculating the intra- and inter-run precision of melting temperatures (Tms). Specificity was assessed using pseudotyped lentivirus of common human respiratory pathogens and human genomic DNA. The assay was used to analyze 40 SARS-CoV-2-positive clinical samples (including 36 BA.2 and 4 BA.4/5 samples) and pseudotyped lentiviruses of wild-type and BA.1 viral RNA control materials, as well as 20 SARS-CoV-2-negative clinical samples, and its accuracy was evaluated by comparing the results with those of sequencing. RESULTS: All genotypes were sensitively identified using the developed method with a LOD of 39.1 copies per reaction. The intra- and inter-run coefficients of variation for the Tms were ≤ 0.69% and ≤ 0.84%, with standard deviations ≤ 0.38 °C and ≤ 0.41 °C, respectively. Validation of the assay using known SARS-CoV-2-positive samples demonstrated its ability to correctly identify the targeted mutations and preliminarily characterize the Omicron lineages. CONCLUSION: The developed assay can provide accurate, reliable, rapid, simple and low-cost detection of the immune-escape mutations located in the spike RBD to detect the Omicron variant and discriminate its lineages, and its use can be easily generalized in clinical laboratories with a fluorescent PCR platform.

Role of HLA class I and II alleles in susceptibility to ankylosing spondylitis in Chinese Han.

OBJECTIVE: The objective of the study was to clarify the associations of HLA class I and II alleles with ankylosing spondylitis (AS) among Chinese Han. METHODS: We performed HLA genotyping and Sanger sequencing for 68 HLA-B*27(-), 62 HLA-B*27(+) AS patients, and 70 controls. Case-control analyses and separate analyses of HLA-B*27(-) patients were performed. One-way ANOVA and Kruskal-Wallis multiple comparisons test were used to analyze the effects of HLA-A\B\C\DRB1\DQB1 alleles on clinical characteristics of HLA-B*27(-) and HLA-B*27(+) patients. RESULTS: In the HLA-B*27(+) group, positive associations were seen with A*11:02, B*27:04, B*27:05, C*02:02, C*12:02, and DRB1*04:01 and negative associations were seen with A*33:03, B*07:02, B*57:01, and C*07:02. The age at onset was greater in HLA-B*27(-) patients than in HLA-B*27(+) patients (30.03 ± 15.15 vs. 23.08 ± 7.79 years). In the HLA-B*27(-) group, those with A*01:01, B*13:01, B*13:02, C*01:02, C*04:01, DQB1*02:01, DQB1*06:01, and DRB1*03:01 had an earlier onset than those without these alleles, while patients carrying B*40:02, C*07:02, C*12:02, C*15:02, DQB1*05:02, and DQB1*05:03 had a delayed onset. In the HLA-B*27(-) group, A*32:01(+), C*08:01(+), and DRB1*04:05(-) women were likely to develop AS. In the HLA-B*27(+) group, DQB1*03:02(+) women may be more likely to develop AS. DRB1*12:02 and HLA-B*27 interacted with the distribution of AS-affected sites. In the HLA-B*27(+) group, DRB1*12:02(+) patients were likely to have peripheral joint involvement. CONCLUSION: HLA class I and II alleles other than HLA-B*27 contribute to AS predisposition and characteristics among Chinese Han patients.

Rapid detection of the irinotecan-related UGT1A1*28 polymorphism by asymmetric PCR melting curve analysis using one fluorescent probe.

BACKGROUND: Determination of UGT1A1 (TA)n polymorphism prior to irinotecan therapy is necessary to avoid severe adverse drug effects. Thus, accurate and reliable genotyping methods for (TA)n polymorphism are highly desired. Here, we present a new method for polymerase chain reaction (PCR) melting curve analysis using one fluorescent probe to discriminate the UGT1A1*1 [(TA)6 ] and *28 [(TA)7 ] genotypes. METHODS: After protocol optimization, this technique was applied for genotyping of 64 patients (including 23 with UGT1A1*1/*1, 22 with *1/*28, and 19 with *28/*28) recruited between 2016 and 2021 in China-Japan Friendship Hospital. The accuracy of the method was evaluated by comparing the results with those of direct sequencing and fragment analysis. The intra- and inter-run precision of the melting temperatures (Tm s) were calculated to assess the reliability, and the limit of detection was examined to assess the sensitivity. RESULTS: All genotypes were correctly identified with the new method, and its accuracy was higher than that of fragment analysis. The intra- and inter-run coefficients of variation for the Tm s were both ≤0.27%, with standard deviations ≤0.14°C. The limit of detection was 0.2 ng of input genomic DNA. CONCLUSION: The developed PCR melting curve analysis using one fluorescent probe can provide accurate, reliable, rapid, simple, and low-cost detection of UGT1A1 (TA)n polymorphism, and its use can be easily generalized in clinical laboratories with a fluorescent PCR platform.

Early-onset type 2 diabetes is associated with genetic variants of ß-cell function in the Chinese Han population.

AIMS: To investigate the genetic factors contributing to early-onset type 2 diabetes (EOD) in the Chinese Hans populations. MATERIALS AND METHODS: For 2734 newly diagnosed type 2 diabetes patients and 4041 normal glycemic controls, 25 single nucleotide polymorphisms from 24 genomic loci linked to diabetes were successfully genotyped. Three genetic risk scores (GRSs) were constructed, including the weighted type 2 diabetes-related GRS (wT-GRS), the weighted ß-cell function-related GRS (wB-GRS), and the weighted GRS constructed by risk alleles not related to ß-cell function (wNB-GRS). For patients with diabetes, EOD, middle-age-onset type 2 diabetes (MOD), and late-onset type 2 diabetes (LOD) were defined by onset ages ≤40, 40 to 60, and ≥60 years, respectively. RESULTS: From single marker analysis, different gene profiles were identified between EOD and LOD patients. EOD patients had greater wT-GRS and wB-GRS values than LOD patients. After adjustment for sex, elevated wT-GRS and wB-GRS values were significantly associated with an increased risk for EOD by 1.11- and 1.21-fold per allele (P = 1.69 × 10-7 ; 6.07 × 10-8 ). The wT-GRS and wNB-GRS were nominally related to an increased risk of LOD by 1.03-fold per allele (P = 1.03 × 10-2 , 1.78 × 10-2 ). In patients with diabetes, higher wT-GRS and wB-GRS were associated with younger onset age [wT-GRS: ß (SE) = -0.0033(0.0016), P = 3.74 × 10-2 ; wB-GRS: -0.0076(0.0028), 7.45 × 10-3 ] and decreased insulinogenic index [wT-GRS: -0.0384(0.0098), 9.39 × 10-5 ; wB-GRS: -0.0722(0.0176), 4.21 × 10-5 ]. CONCLUSION: Our findings indicate a strong genetic predisposition for EOD, which can be mainly attributed to genetic variants linked to ß-cell function, suggesting the ß-cell dysfunction plays a key role in the pathogenesis of EOD in Chinese Han individuals.

Interaction of MTHFR C677T polymorphism with smoking in susceptibility to diabetic nephropathy in Chinese men with type 2 diabetes.

We investigated the interaction of MTHFR C677T polymorphism (rs1801133) with smoking in susceptibility to diabetic nephropathy (DN) in Chinese men with type 2 diabetes mellitus (T2DM). We studied 655 Chinese men with T2DM, who were divided into two groups (321 with DN and 334 without DN). The genotype of MTHFR C677T polymorphism was detected by real-time polymerase chain reaction. MTHFR TT genotype carried a higher risk of DN compared with the CC genotype (OR = 2.05; P = 0.002). The T allele showed marked association with DN development in patients who smoked, using additive, recessive, and dominant models (OR = 1.60, 1.83, and 1.88, respectively; P = 0.006, 0.002, and 0.04, respectively), which was not observed in the nonsmoking group. Patients with TT and CT genotypes, who smoked had a higher risk of DN compared with the control group (non-smoking with CC genotype; OR = 3.73 and 2.28, respectively; P < 0.001 and P = 0.004, respectively), whereas the other groups were not observed. In conclusion, the T allele of rs1801133 may be a risk factor for DN in Chinese men with T2DM, and synergy appears to exist between the MTHFR rs1801133 and smoking in susceptibility to DN.

Liver X receptor ß increases aquaporin 2 protein level via a posttranscriptional mechanism in renal collecting ducts.

Liver X receptors (LXRs) including LXRα and LXRß are nuclear receptor transcription factors and play an important role in lipid and glucose metabolism. It has been previously reported that mice lacking LXRß but not LXRα develop a severe urine concentrating defect, likely via a central mechanism. Here we provide evidence that LXRß regulates water homeostasis through increasing aquaporin 2 (AQP2) protein levels in renal collecting ducts. LXRß-/- mice exhibited a reduced response to desmopressin (dDAVP) stimulation, suggesting that the diabetes insipidus phenotype is of both central and nephrogenic origin. AQP2 protein abundance in the renal inner medulla was significantly reduced in LXRß-/- mice but with little change in AQP2 mRNA levels. In vitro studies showed that AQP2 protein levels were elevated upon LXR agonist treatment in both primary cultured mouse inner medullary duct cells (mIMCD) and the mIMCD3 cell line with stably expressed AQP2. In addition, LXR agonists including TO901317 and GW3965 failed to induce AQP2 gene transcription but diminished its protein ubiquitination in primary cultured mIMCD cells, thereby inhibiting its degradation. Moreover, LXR activation-induced AQP2 protein expression was abolished by the protease inhibitor MG132 and the ubiquitination-deficient AQP2 (K270R). Taken together, the present study demonstrates that activation of LXRß increases AQP2 protein levels in the renal collecting ducts via a posttranscriptional mechanism. As such, LXRß represents a key regulator of body water homeostasis.

Mobile phone intervention and weight loss among overweight and obese adults: a meta-analysis of randomized controlled trials.

We conducted a meta-analysis of randomized controlled trials to examine the association of mobile phone intervention with net change in weight-related measures among overweight and obese adults. We searched electronic databases and conducted a bibliography review to identify articles published between the inception date of each database and March 27, 2014. Fourteen trials (including 1,337 participants in total) that met the eligibility criteria were included. Two investigators independently abstracted information on study characteristics and study outcomes. Net change estimates comparing the intervention group with the control group were pooled across trials using random-effects models. Compared with the control group, mobile phone intervention was associated with significant changes in body weight and body mass index (weight (kg)/height (m)(2)) of -1.44 kg (95% confidence interval (CI): -2.12, -0.76) and -0.24 units (95% CI: -0.40, -0.08), respectively. Subgroup analyses revealed that the associations were consistent across study-duration and intervention-type subgroups. For example, net body weight changes were -0.92 kg (95% CI: -1.58, -0.25) and -1.85 kg (95% CI: -2.99, -0.71) in trials of shorter (<6 months) and longer (≥6 months) duration, respectively. These findings provide evidence that mobile phone intervention may be a useful tool for promoting weight loss among overweight and obese adults.

Central diabetes insipidus associated with impaired renal aquaporin-1 expression in mice lacking liver X receptor ß.

The present study demonstrates a key role for the oxysterol receptor liver X receptor ß (LXRß) in the etiology of diabetes insipidus (DI). Given free access to water, LXRß(-/-) but not LXRα(-/-) mice exhibited polyuria (abnormal daily excretion of highly diluted urine) and polydipsia (increased water intake), both features of diabetes insipidus. LXRß(-/-) mice responded to 24-h dehydration with a decreased urine volume and increased urine osmolality. To determine whether the DI was of central or nephrogenic origin, we examined the responsiveness of the kidney to arginine vasopressin (AVP). An i.p. injection of AVP to LXRß(-/-) mice revealed a partial kidney response: There was no effect on urine volume, but there was a significant increase of urine osmolality, suggesting that DI may be caused by a defect in central production of AVP. In the brain of WT mice LXRß was expressed in the nuclei of magnocellular neurons in the supraoptic and paraventricular nuclei of the hypothalamus. In LXRß(-/-) mice the expression of AVP was markedly decreased in the magnocellular neurons as well as in urine collected over a 24-h period. The persistent high urine volume after AVP administration was traced to a reduction in aquaporin-1 expression in the kidney of LXRß(-/-) mice. The LXR agonist (GW3965) in WT mice elicited an increase in urine osmolality, suggesting that LXRß is a key receptor in controlling water balance with targets in both the brain and kidney, and it could be a therapeutic target in disorders of water balance.

Thyroid hormone-responsive SPOT 14 homolog promotes hepatic lipogenesis, and its expression is regulated by liver X receptor α through a sterol regulatory element-binding protein 1c-dependent mechanism in mice.

UNLABELLED: The protein, thyroid hormone-responsive SPOT 14 homolog (Thrsp), has been reported to be a lipogenic gene in cultured hepatocytes, implicating an important role of Thrsp in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Thrsp expression is known to be regulated by a variety of transcription factors, including thyroid hormone receptor, pregnane X receptor, and constitutive androstane receptor. Emerging in vitro evidence also points to a critical role of liver X receptor (LXR) in regulating Thrsp transcription in hepatocytes. In the present study, we showed that Thrsp was up-regulated in livers of db/db mice and high-fat-diet-fed mice, two models of murine NAFLD. Hepatic overexpression of Thrsp increased triglyceride accumulation with enhanced lipogenesis in livers of C57Bl/6 mice, whereas hepatic Thrsp gene silencing attenuated the fatty liver phenotype in db/db mice. LXR activator TO901317 induced Thrsp expression in livers of wild-type (WT) and LXR-ß gene-deficient mice, but not in LXR-α or LXR-α/ß double-knockout mice. TO901317 treatment significantly enhanced hepatic sterol regulatory element-binding protein 1c (SREBP-1c) expression and activity in WT mice, but failed to induce Thrsp expression in SREBP-1c gene-deficient mice. Sequence analysis revealed four LXR response-element-like elements and one sterol regulatory element (SRE)-binding site within a -2,468 ∼+1-base-pair region of the Thrsp promoter. TO901317 treatment and LXR-α overexpression failed to induce, whereas overexpression of SREBP-1c significantly increased Thrsp promoter activity. Moreover, deletion of the SRE site completely abolished SREBP-1c-induced Thrsp transcription. CONCLUSION: Thrsp is a lipogenic gene in the liver that is induced by the LXR agonist through an LXR-α-mediated, SREBP-1c-dependent mechanism. Therefore, Thrsp may represent a potential therapeutic target for the treatment of NAFLD.

Development of a Novel Multiplex PCR Method for the Rapid Detection of SARS-CoV-2, Influenza A Virus, and Influenza B Virus.

Objective: A sensitive and specific multiplex fluorescence rapid detection method was established for simultaneous detection of SARS-CoV-2, influenza A virus, and influenza B virus in a self-made device within 30 min, with a minimum detection limit of 200 copies/mL. Methods: Based on the genome sequences of SARS-CoV-2, influenza A virus (FluA), and influenza B virus (FluB) with reference to the Chinese Center for Disease Control and Prevention and related literature, specific primers were designed, and a multiplex fluorescent PCR system was established. The simultaneous and rapid detection of SARS-CoV-2, FluA, and FluB was achieved by optimizing the concentrations of Taq DNA polymerase as well as primers, probes, and Mg2+. The minimum detection limits of the nucleic acid rapid detection system for SARS-CoV-2, FluA, and FluB were evaluated. Results: By optimizing the amplification system, the N enzyme with the best amplification performance was selected, and the optimal concentration of Mg2+ in the multiamplification system was 3 mmol/L; the final concentrations of SARS-CoV-2 NP probe and primer were 0.15 µmol/L and 0.2 µmol/L, respectively; the final concentrations of SARS-CoV-2 ORF probe and primer were both 0.15 µmol/L; the final concentrations of FluA probe and primer were 0.2 µmol/L and 0.3 µmol/L, respectively; the final concentrations of FluB probe and primer were 0.15 µmol/L and 0.25 µmol/L, respectively. Conclusion: A multiplex real-time quantitative fluorescence RT-PCR system for three respiratory viruses of SARS-CoV-2, FluA, and FluB was established with a high amplification efficiency and sensitivity reaching 200 copies/mL for all samples. Combined with the automated microfluidic nucleic acid detection system, the system can achieve rapid detection in 30 minutes.

Tangshen formula improves diabetic nephropathy in STZ-induced diabetes rats fed with hyper-methionine by regulating the methylation status of kidney.

BACKGROUND: The objective of this study was to examine and analyze differential methylation profiles in order to investigate the influence of hyper-methioninemia (HM) on the development of diabetic nephropathy (DN). Male Wistar rats, aged eight weeks and weighing 250-300 g, were randomly assigned into four groups: a control group (Healthy, n = 8), streptozocin-induced rats (STZ group, n = 8), HM + STZ group (n = 8), and the Tangshen Formula (TSF) treatment group (TSF group, n = 8). Blood glucose levels and other metabolic indicators were monitored before treatment and at four-week intervals until 12 weeks. Total DNA was extracted from the aforementioned groups, and DNA methylation landscapes were analyzed via reduced representative bisulfite sequencing. RESULTS: Both the STZ group and HM + STZ group exhibited increased blood glucose levels and urinary albumin/creatinine ratios in comparison with the control group. Notably, the HM + STZ group exhibited a markedly elevated urinary albumin/creatinine ratio (411.90 ± 88.86 mg/g) compared to the STZ group (238.41 ± 62.52 mg/g). TSF-treated rats demonstrated substantial reductions in both blood glucose levels and urinary albumin/creatinine ratios in comparison with the HM + STZ group. In-depth analysis of DNA methylation profiles revealed 797 genes with potential therapeutic effects related to TSF, among which approximately 2.3% had been previously reported as homologous genes. CONCLUSION: While HM exacerbates DN through altered methylation patterns at specific CpG sites, TSF holds promise as a viable treatment for DN by restoring abnormal methylation levels. The identification of specific genes provides valuable insights into the underlying mechanisms of DN pathogenesis and offers potential therapeutic targets for further investigation.

Effects of PGE2 EP3/EP4 receptors on bladder dysfunction in mice with experimental autoimmune encephalomyelitis.

To investigate the expression of four subtypes of PGE2 E-prostanoid (EP) receptors (EP1-EP4) and the effects of EP3/EP4 on bladder dysfunction in a new neurogenic bladder model induced by experimental autoimmune encephalomyelitis (EAE), the mouse model of EAE was induced using a previously established method, and bladder function in mice with different defined levels of neurological impairment was then examined, including micturition frequencies and voiding weight. Bladders were then harvested for analysis of EP receptor expression by Western blot. Activities of agonists/antagonists of EP3 and EP4 receptors as well as PGE2 were also evaluated at different stages of EAE. The results showed that EAE mice developed profound bladder dysfunction characterized by significantly increased micturition and significantly decreased urine output per micturition. EAE-induced upregulation of EP3 and EP4 receptors in the bladder was accompanied by bladder dysfunction. However, EAE had no significant effect on EP1 and EP2 receptors. Moreover, PGE2 and agonists/antagonists of EP3 and EP4 receptors significantly affected bladder dysfunction in EAE mice. Thus, we believe that EAE mice are useful for investigations of the neurogenic bladder. In addition, EP3 and EP4 receptors play a role in EAE-induced bladder dysfunction, providing us with a new target for the treatment of neurogenic bladders.

Fast and Sensitive Detection of SARS-CoV-2 Nucleic Acid Using a Rapid Detection System Free of RNA Extraction.

Objectives: To establish and evaluate the analytical and clinical performance of the Flash20 SARS-CoV-2 nucleic acid rapid detection system free of RNA extraction. Methods: The limit of detection (LoD) was determined using a negative nasopharyngeal swab matrix spiked with different concentrations of SARS-CoV-2 virus; a total of 734,337 reference sequences of viral genomes from GenBank were used for the in-silico analysis to assess the inclusivity of the assay. The specificity of the system was evaluated by testing 27 medically relevant organisms. A total of 115 clinical specimens were collected and tested on the Flash20 SARS-CoV-2 detection system and with an FDA-approved comparator test to assess the clinical performance of the system. Results: The LoD of the Flash20 SARS-CoV-2 detection system is 250 copies/mL with a positive rate ≥90% (n = 20); alignments results showed that over 99% identity of the primer and probe of the Flash20 SARS-CoV-2 nucleic acid rapid detection system to the available SARS-CoV-2 sequences; the omicron samples tested 100% positive. None of the 27 organisms showed cross-reactivity with the Flash20 SARS-CoV-2 nucleic acid rapid detection system. Among all the 215 clinical samples, the Flash20 SARS-CoV-2 nucleic acid rapid detection system exhibits a high sensitivity of 99.24% (131/132) and 100% (83/83) specificity. Conclusion: The nucleic acid rapid detection system provides sensitive and accurate detection of SARS-CoV-2 free of RNA extraction. The high sensitivity and short time to results of approximately 35 minutes may impact earlier infection control and disease management.

A novel assay based on DNA melting temperature for multiplexed identification of SARS-CoV-2 and influenza A/B viruses.

Introduction: The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and influenza viruses can cause respiratory illnesses with similar clinical symptoms, making their differential diagnoses challenging. Additionally, in critically ill SARS-CoV-2-infected patients, co-infections with other respiratory pathogens can lead to severe cytokine storm and serious complications. Therefore, a method for simultaneous detection of SARS-CoV-2 and influenza A and B viruses will be clinically beneficial. Methods: We designed an assay to detect five gene targets simultaneously via asymmetric PCR-mediated melting curve analysis in a single tube. We used specific probes that hybridize to corresponding single-stranded amplicons at low temperature and dissociate at high temperature, creating different detection peaks representing the targets. The entire reaction was conducted in a closed tube, which minimizes the risk of contamination. The limit of detection, specificity, precision, and accuracy were determined. Results: The assay exhibited a limit of detection of <20 copies/µL for SARS-CoV-2 and influenza A and <30 copies/µL for influenza B, with high reliability as demonstrated by a coefficient of variation for melting temperature of <1.16% across three virus concentrations. The performance of our developed assay and the pre-determined assay showed excellent agreement for clinical samples, with kappa coefficients ranging from 0.98 (for influenza A) to 1.00 (for SARS-CoV-2 and influenza B). No false-positive, and no cross-reactivity was observed with six common non-influenza respiratory viruses. Conclusion: The newly developed assay offers a straightforward, cost-effective and nucleic acid contamination-free approach for simultaneous detection of the SARS-CoV-2, influenza A, and influenza B viruses. The method offers high analytical sensitivity, reliability, specificity, and accuracy. Its use will streamline testing for co-infections, increase testing throughput, and improve laboratory efficacy.

Targeted disruption of the prostaglandin E2 E-prostanoid 2 receptor exacerbates vascular neointimal formation in mice.

OBJECTIVE: Restenosis after angioplasty remains a major clinical problem. Prostaglandin E(2) (PGE(2)) plays an important role in vascular homeostasis. The PGE(2) receptor E-prostanoid 2 (EP2) is involved in the proliferation and migration of various cell types. We aimed to determine the role of EP2 in the pathogenesis of neointimal formation after vascular injury. METHODS AND RESULTS: Wire-mediated vascular injury was induced in the femoral arteries of male wild-type (EP2+/+) and EP2 gene-deficient (EP2-/-) mice. In EP2+/+ mice, EP2 mRNA expression was increased in injured vessels for at least 4 weeks after vascular injury. Neointimal hyperplasia was markedly accelerated in EP2-/- mice, which was associated with increased proliferation and migration of vascular smooth muscle cells (VSMCs) and increased cyclin D1 expression in the neointima layer. Platelet-derived growth factor-BB (PDGF-BB) treatment resulted in more significant cell proliferation and migration in VSMCs of EP2-/- mice than in those of EP2+/+ mice. Activation and overexpression of EP2 attenuated PDGF-BB-elicited cell proliferation and migration, induced G(1)→S-phase arrest and reduced PDGF-BB-stimulated extracellular signal-regulated kinase phosphorylation in EP2+/+ VSMCs. CONCLUSIONS: These findings reveal a novel role of the EP2 receptor in neointimal hyperplasia after arterial injury. The EP2 receptor may represent a potential therapeutic target for restenosis after angioplasty.

AMPK potentiates hypertonicity-induced apoptosis by suppressing NFκB/COX-2 in medullary interstitial cells.

Cells residing in the hypertonic, hypoxic renal medulla depend on dynamic adaptation mechanisms to respond to changes in energy supply and demand. The serine/threonine kinase 5'-AMP protein kinase (AMPK) is a sensor of cellular energy status, but whether it contributes to the survival of cells in the renal medulla is unknown. Here, hypertonic conditions induced a decrease in AMPK phosphorylation within 12 hours in renal medullary interstitial cells (RMIC), followed by a gradual return to baseline levels. Activation of AMPK markedly increased hypertonicity-induced apoptosis of RMICs and suppressed both hypertonicity-induced NFκB nuclear translocation and cyclooxygenase-2 (COX-2) activation; overexpression of COX-2 significantly attenuated these effects. AMPK activation also markedly reduced generation of reactive oxygen species and nuclear expression of tonicity-responsive enhancer-binding protein, which prevented upregulation of osmoprotective genes. In vivo, pharmacologic activation of AMPK led to massive apoptosis of RMICs and renal dysfunction in the setting of water deprivation in mice. Taken together, these results identify a critical role for AMPK in the maintenance of RMIC viability and suggest that AMPK modulates the NFκB-COX-2 survival pathway in the renal medulla. Furthermore, this study raises safety concerns for the development of AMPK activators as anti-diabetic drugs, especially for patients prone to dehydration.

Association of AGTR1 A1166C and CYP2C9∗3 Gene Polymorphisms with the Antihypertensive Effect of Valsartan.

Background: The differences in the antihypertensive treatment with angiotensin type II receptor blockers (ARBs) may be attributed to polymorphisms in genes involving drug-targeted receptor and drug metabolism. The present study aimed to investigate whether the antihypertensive effect of the ARB drug valsartan was associated with angiotensin II type 1 receptor (AGTR1) gene polymorphism (A1166 C) and cytochrome P450 enzyme 2C9 (CYP2C9) gene polymorphism (CYP2C9∗3). Methods: 281 patients with hypertension who received valsartan monotherapy in the past month were included in this retrospective study. Polymerase chain reaction-melting curve analysis was performed to genotype the AGTR1 and CYP2C9 gene polymorphisms. Based on the systolic blood pressure (SBP) and diastolic blood pressure (DBP) at the time of visit, the patients were divided into well-controlled group (n = 144, SBP/DBP <140/90 mmHg) and poorly controlled group (n = 137, SBP/DBP ≥140/90 mmHg). Results: Older age, decreased history of drinking, a higher proportion of mild-to-moderate hypertension, lower alanine aminotransferase levels, and higher high-density lipoprotein cholesterol levels were observed in the well-controlled group than the poorly controlled group. Higher frequencies of the C allele and AC + CC genotype of AGTR1 A1166C were detected in the well-controlled than the poorly controlled patients (P = 0.005 and P = 0.006). After adjustment for demographic and environmental factors, the CC + AC genotype of AGTR1 A1166C was markedly linked to better hypertension control with valsartan treatment compared to the AA genotype (odds ratio: 2.836, 95% confidence interval: 1.199-6.705, P = 0.018). No significant difference was observed in the allele or genotype distribution of CYP2C9∗3 polymorphism between well-controlled and poorly controlled patients. Conclusions: The current data suggested that the AGTR1 A1166 C polymorphism may be associated with the antihypertensive effect of valsartan, and carriers with AC and CC genotypes may have a better antihypertensive efficacy response to valsartan treatment.

A Novel Stool Methylation Test for the Non-Invasive Screening of Gastric and Colorectal Cancer.

Background: Because of poor compliance or low sensitivity, existing diagnostic approaches are unable to provide an efficient diagnosis of patients with gastric and colorectal cancer. Here, we developed the ColoCaller test, which simultaneously detects the methylation status of the SDC2, TFPI2, WIF1, and NDRG4 genes in stool DNA, to optimize the screening of gastric and colorectal cancer in high-risk populations. Methods: A total of 217 stool samples from patients with gastrointestinal cancer and from patients with negative endoscopy were prospectively collected, complete with preoperative and postoperative clinical data from patients. The methylation of these samples was detected using ColoCaller, which was designed by selecting CpGs with a two-step screening strategy, and was interpreted using a prediction model built using libSVM to evaluate its clinical value for gastric and colorectal cancer screening. Results: Compared to pathological diagnosis, the sensitivity and specificity of the ColoCaller test in 217 stool DNA samples were 95.56% and 91.86%, respectively, for colorectal cancer, and 67.5% and 97.81%, respectively, for gastric cancer. The detection limit was as low as 1% in 8 ng of DNA. Conclusion: In this study, we developed and established a new test, ColoCaller, which can be used as a screening tool or as an auxiliary diagnostic approach in high-risk populations with gastric and colorectal cancer to promote timely diagnosis and treatment.

Proteomics analysis reveals diabetic kidney as a ketogenic organ in type 2 diabetes.

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease. To date, the molecular mechanisms of DN remain largely unclear. The present study aimed to identify and characterize novel proteins involved in the development of DN by a proteomic approach. Proteomic analysis revealed that 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase 2 (HMGCS2), the key enzyme in ketogenesis, was increased fourfold in the kidneys of type 2 diabetic db/db mice. Consistently, the activity of HMGCS2 in kidneys and 24-h urinary excretion of the ketone body ß-hydroxybutyrate (ß-HB) were significantly increased in db/db mice. Immunohistochemistry, immunofluorescence, and real-time PCR studies further demonstrated that HMGCS2 was highly expressed in renal glomeruli of db/db mice, with weak expression in the kidneys of control mice. Because filtered ketone bodies are mainly reabsorbed in the proximal tubules, we used RPTC cells, a rat proximal tubule cell line, to examine the effect of the increased level of ketone bodies. Treating cultured RPTC cells with 1 mM ß-HB significantly induced transforming growth factor-ß1 expression, with a marked increase in collagen I expression. ß-HB treatment also resulted in a marked increase in vimentin protein expression and a significant reduction in E-cadherin protein levels, suggesting an enhanced epithelial-to-mesenchymal transition in RPTCs. Collectively, these findings demonstrate that diabetic kidneys exhibit excess ketogenic activity resulting from increased HMGCS2 expression. Enhanced ketone body production in the diabetic kidney may represent a novel mechanism involved in the pathogenesis of DN.

Peroxisome proliferator-activated receptor-α is renoprotective in doxorubicin-induced glomerular injury.

Doxorubicin (DOX) is an anthracycline antibiotic utilized in antitumor therapy; however, its clinical use is frequently impeded by renal toxic effects. As peroxisome proliferator-activated receptor-α (PPAR-α) has renoprotective effects in drug-related kidney injuries, we tested its ability to inhibit DOX-induced renal injury. Although both male PPAR-α knockout mice and their wild-type littermates (pure 129/SvJ background) had significant proteinuria 4 weeks after DOX treatment, those with deletion of PPAR-α had more severe proteinuria. This was associated with more serious podocyte foot process effacement compared with wild-type mice. In contrast, the PPAR-α agonist fenofibrate effectively reduced proteinuria and attenuated DOX-induced podocyte foot process effacement. Consistently, glomerular nephrin expression was significantly lower in the knockout compared with wild-type mice following DOX treatment. Fenofibrate therapy significantly blunted the reduction in glomerular nephrin levels in DOX-treated wild-type mice. In cultured podocytes, DOX induced apoptosis, increased cleaved caspase-3 levels, and decreased Bcl-2 expression, all attenuated by pretreatment with fenofibrate. Thus, PPAR-α deficiency exacerbates DOX-related renal injury, in part, due to increased podocyte apoptosis.