Emodin alleviates CRS4-induced mitochondrial damage via activation of the PGC1α signaling.

Cardiorenal syndrome type 4 (CRS4), a progressive deterioration of cardiac function secondary to chronic kidney disease (CKD), is a leading cause of death in patients with CKD. In this study, we aimed to investigate the cardioprotective effect of emodin on CRS4. C57BL/6 mice with 5/6 nephrectomy and HL-1 cells stimulated with 5% CKD mouse serum were used for in vivo and in vitro experiments. To assess the cardioprotective potential of emodin, we employed a comprehensive array of methodologies, including echocardiography, tissue staining, immunofluorescence staining, biochemical detection, flow cytometry, real-time quantitative PCR, and western blot analysis. Our results showed that emodin exerted protective effects on the function and structure of the residual kidney. Emodin also reduced pathologic changes in the cardiac morphology and function of these mice. These effects may have been related to emodin-mediated suppression of reactive oxygen species production, reduction of mitochondrial oxidative damage, and increase of oxidative metabolism via restoration of PGC1α expression and that of its target genes. In contrast, inhibition of PGC1α expression significantly reversed emodin-mediated cardioprotection in vivo. In conclusion, emodin protects the heart from 5/6 nephrectomy-induced mitochondrial damage via activation of the PGC1α signaling. The findings obtained in our study can be used to develop effective therapeutic strategies for patients with CRS4.

Development and validation of a prediction model for all-cause mortality in maintenance dialysis patients: a multicenter retrospective cohort study.

BACKGROUND: The mortality risk varies considerably among individual dialysis patients. This study aimed to develop a user-friendly predictive model for predicting all-cause mortality among dialysis patients. METHODS: Retrospective data regarding dialysis patients were obtained from two hospitals. Patients in training cohort (N = 1421) were recruited from the Fifth Affiliated Hospital of Sun Yat-sen University, and patients in external validation cohort (N = 429) were recruited from the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine. The follow-up endpoint event was all-cause death. Variables were selected by LASSO-Cox regression, and the model was constructed by Cox regression, which was presented in the form of nomogram and web-based tool. The discrimination and accuracy of the prediction model were assessed using C-indexes and calibration curves, while the clinical value was assessed by decision curve analysis (DCA). RESULTS: The best predictors of 1-, 3-, and 5-year all-cause mortality contained nine independent factors, including age, body mass index (BMI), diabetes mellitus (DM), cardiovascular disease (CVD), cancer, urine volume, hemoglobin (HGB), albumin (ALB), and pleural effusion (PE). The 1-, 3-, and 5-year C-indexes in the training set (0.840, 0.866, and 0.846, respectively) and validation set (0.746, 0.783, and 0.741, respectively) were consistent with comparable performance. According to the calibration curve, the nomogram predicted survival accurately matched the actual survival rate. The DCA showed the nomogram got more clinical net benefit in both the training and validation sets. CONCLUSIONS: The effective and convenient nomogram may help clinicians quantify the risk of mortality in maintenance dialysis patients.

Astragaloside IV attenuates myocardial dysfunction in diabetic cardiomyopathy rats through downregulation of CD36-mediated ferroptosis.

Diabetic cardiomyopathy (DCM), one of the major complications of type 2 diabetes, is a leading cause of heart failure and death in advanced diabetes. Although there is an association between DCM and ferroptosis in cardiomyocytes, the internal mechanism of ferroptosis leading to DCM development remains unknown. CD36 is a key molecule in lipid metabolism that mediates ferroptosis. Astragaloside IV (AS-IV) confers various pharmacological effects such as antioxidant, anti-inflammatory, and immunomodulatory. In this study, we demonstrated that AS-IV was able to recover the dysfunction of DCM. In vivo experiments showed that AS-IV ameliorated myocardial injury and improved contractile function, attenuated lipid deposition, and decreased the expression level of CD36 and ferroptosis-related factors in DCM rats. In vitro experiments showed that AS-IV decreased CD36 expression and inhibited lipid accumulation and ferroptosis in PA-induced cardiomyocytes. The results demonstrated that AS-IV decreased cardiomyocyte injury and myocardial dysfunction by inhibiting ferroptosis mediated by CD36 in DCM rats. Therefore, AS-IV regulated the lipid metabolism of cardiomyocytes and inhibited cellular ferroptosis, which may have potential clinical value in DCM treatment.

Detection of Cancer Marker Flap Endonuclease 1 Using One-Pot Transcription-Powered Clustered Regularly Interspaced Short Palindromic Repeat/Cas12a Signal Expansion.

As a critical functional protein in DNA replication and genome stability, flap endonuclease 1 (FEN1) has been considered a promising biomarker and druggable target for multiple cancers. We report here a transcription-powered clustered regularly interspaced short palindromic repeat (CRISPR)/Cas12a signal expansion platform for rapid and sensitive detection of FEN1. In this method, the probe cleavage by FEN1 generated a free 5' flap single-stranded DNA which could hybridize with the single-stranded T7 promoter-bearing template and trigger the extension. Then, the CRISPR guide RNA (crRNA) transcribed from the extended template activated the collateral DNase activity of Cas12a, releasing the fluorophore from the quenched DNA signal probe to report the FEN1 detection result. The high specificity for FEN1 was validated by comparing with other repair-relevant proteins. The limit of detection (LOD) could be as low as 0.03 mU, which is sensitive enough to detect the FEN1 activity in biological samples. In addition, the inhibition assay of FEN1 was also successfully achieved with this platform, proving its potential in inhibitor screening. In summary, this study provides a novel biosensor for FEN1 activity analysis and provides new insights into the development of CRISPR-based biosensors for non-nucleic acid targets.

Frame-shifted proteins of a given gene retain the same function.

Frameshift mutations are generally considered to be lethal because it could result in radical changes of the protein sequence behind. However, the protein of frameshift mutants of a type I toxin (ibsc) was found to be still toxic to bacteria, retaining the similar function as wild-type protein to arrest the cellular growth by impairing the membrane's integrity. Additionally, we have verified that this observation is not an individual event as the same phenomenon had been found in other toxins subsequently. After analyzing the coding sequence of these genes, we proposed a hypothesis to search this kind of hidden gene, through which a dihydrofolate reductase-encoding gene (dfrB3) was found out. Like the wild-type reductase, both +1 and -1 frame-shifted proteins of dfrB3 gene were also proved to catalyze the reduction of dihydrofolate to tetrahydrofolate by using NADPH.

Long non-coding RNA PWRN2 regulates cytotoxicity in an in vitro model of age-related macular degeneration.

BACKGROUND: Age-related macular degeneration (AMD) may lead to irreversibly vision loss among aging populations. In this work, in an in vitro AMD cell model, we examined the expression and function of long non-coding RNA, Prader-Willi Region Non-Protein Coding RNA 2 (PWRN2) in injured human retinal pigment epithelial cells. METHOD: ARPE-19 cell line was maintained in vitro and treated with multi-module stressful conditions, including hydrogen peroxide (H2O2) tert-butylhydroperoxide (t-BuOOH) and ultraviolet B (UVB). Multi-module-stressor-induced cell death was monitored by a viability assay, and PWRN2 expression by qRT-PCR. PWRN2 was either downregulated or upregulated in ARPE-19 cells. The effects of PWRN2 downregulation or upregulation on t-BuOOH-induced cell death, cellular apoptosis and mitochondrial injuries were then quantitatively evaluated. RESULTS: Multi-module stressful conditions induced cell death and PWRN2 upregulation in ARPE-19 cells in vitro. We created ARPE-19 subpopulations with either downregulated or upregulated PWRN2 expressions. Quantitative assays demonstrated that, PWRN2 downregulation effectively alleviated t-BuOOH-induced cell death, apoptosis and various-type of mitochondrial injuries. On the other hand, PWRN2 upregulation worsened t-BuOOH-induced cellular damages in ARPE-19 cells. CONCLUSION: We demonstrated that downregulating PWRN2 protected multi-module-stressor-induced cell death, apoptosis and mitochondrial injuries in human retinal pigment epithelial cells, suggesting PWRN2 may be an active factor in human AMD.

Intracellular selection of trans-cleaving hammerhead ribozymes.

Hammerhead ribozyme is the smallest and best characterized catalytic RNA-cleaving ribozyme. It has been reported as potential therapeutic tools to manipulate the expression of target genes. However, most of naturally occurring hammerhead ribozymes process self-cleavage rather than cleave substrate RNA in trans, and its high intracellular activity relies on the tertiary interaction of Loop II and steam I bulge, resulting in decreased performance as applied in gene silencing. We described a direct intracellular selection method to evolve hammerhead variants based on trans-cleavage mode via using a toxin gene as the reporter. And a dual fluorescence proteins system has also been established to quantitatively evaluate the efficiency of selected ribozymes in the cell. Based on this selection strategy, we obtained three mutants with enhanced intracellular cleaving activity compared to wide type hammerhead ribozyme. The best one, TX-2 was revealed to possess better and consistent gene knockdown ability at different positions on diverse targeted mRNA either in prokaryotic or eukaryotic cells than wild-type hammerhead ribozyme. These observations imply the efficiency of the intracellular selection method of the trans-acting ribozyme and the potentials of improved ribozyme variants for research and therapeutic purposes.

Ion-induced PCR strategy for mercury detection.

Mercury contamination is one of the most serious environmental problems. It can cause serious effects on the human health, such as case damage in the brain, nervous system, immune system, and kidney failure. Therefore, development of an accurate, sensitive, and simple operational detection method for mercury is very necessary. Herein, we report a new strategy for mercury ion detection based on commonly used PCR technique. High selectivity and sensitivity were achieved by the formation of the thymine-Hg-thymine (T-Hg-T) unnatural base pair at the 3'-end of PCR primers. The detection results of PCR amplification in presence of mercury ion could be reported either by using agarose gel analysis or through real-time fluorometric dye tracing for different detection purposes. To our knowledge, this study represents the first application of PCR based technique to the detection of metal ions.

Recombinase assisted loop-mediated isothermal DNA amplification.

Polymerase chain reaction (PCR) and isothermal amplification methods such as LAMP and RPA are widely used for genetic detection. However, there are some shortcomings of these methods such as dependence on thermocycler instruments for PCR, complexity of primer design, the possibility for nonspecific amplification in LAMP and complexity of components in RPA. We develop a novel isothermal DNA detection system named Recombinase Assisted Loop-mediated Amplification (RALA). Recombinase from Thermus thermophilus (TthRecA) was used to open target double-stranded DNA to initiate loop-mediated amplification under isothermal conditions, which simplified the primer design and circumvented pre-denaturation. A FRET sensor named ProofMan and a proofreading enzyme Pfu were introduced to produce fluorescence signals by cleaving the sensor from the 3' end. Consequently, sequence-specific detection based on the RALA system was achieved, and even a single nucleotide polymorphism (SNP) could be identified. By introducing additional loop primers, the fast RALA version can amplify 102 DNA targets in 30 minutes. In addition to high sensitivity and specificity, the flexibility of choosing different reporting sensors makes this method versatile in either quantitative or qualitative DNA detection.

Long noncoding RNA IGF2AS regulates high-glucose induced apoptosis in human retinal pigment epithelial cells.

High-glucose-induced retinal tissue impairment is the major pathological phenotype of diabetic retinopathy. In an in vitro diabetic apoptosis cell model, we evaluated the function of long noncoding RNA, insulin growth factor 2 antisense (IGF2-AS) in high-glucose-injured human retinal pigment epithelial cells. A human retinal pigment epithelial cell line, ARPE-19 was incubated with high-glucose in vitro to induce apoptosis. SiRNA-mediated IGF2-AS downregulation was conducted in ARPE-19 cells to evaluate its effect on high-glucose induced apoptosis, assessed by a TUNEL assay. qRT-PCR and western blot assays were applied to examine the functional effect of IGF2-AS on IGF2/AKT/Casp-9 expressions in glucose-injured ARPE-19 cells. ART was further knocked down, specifically in IGF2-AS-downregualted ARPE-19 cells, to investigate its functional involvement in IGF2-AS-inhibition-mediated apoptotic protection in glucose-injured ARPE-19 cells. High-glucose induced apoptosis in ARPE-19 cells, and upregulated IGF-2AS in a dose-dependent manner. SiRNA-mediated IGF2-AS downregulation ameliorated apoptosis, upregulated IGF2/AKT and decreased Casp-9, in high-glucose-treated ARPE-19 cells. AKT knockdown was shown to dramatically reverse the preventive effect of IGF2-AS-downregulation on high-glucose-induced apoptosis in ARPE-19 cells. Moreover, it was demonstrated that AKT knockdown directly upregulated Casp-9 in IGF2-AS-downregulated and high-glucose-treated ARPE-19 cells. We demonstrated that inhibiting IGF2-AS, possibly also through activation of AKT signaling pathway, has a protective function in high-glucose-induced apoptosis in human retinal pigment epithelial cells in diabetic retinopathy.

Selective tumor cell death induced by irradiated riboflavin through recognizing DNA G-T mismatch.

Riboflavin (vitamin B2) has been thought to be a promising antitumoral agent in photodynamic therapy, though the further application of the method was limited by the unclear molecular mechanism. Our work reveals that riboflavin was able to recognize G-T mismatch specifically and induce single-strand breaks in duplex DNA targets efficiently under irradiation. In the presence of riboflavin, the photo-irradiation could induce the death of tumor cells that are defective in mismatch repair system selectively, highlighting the G-T mismatch as potential drug target for tumor cells. Moreover, riboflavin is a promising leading compound for further drug design due to its inherent specific recognition of the G-T mismatch.

Colorimetric PCR-Based microRNA Detection Method Based on Small Organic Dye and Single Enzyme.

microRNAs (miRNAs) have been a class of promising disease diagnostic biomarkers and therapeutic targets for their important biological functions. However, because of the high homology, interference from precursors (pri-miRNA, pre-miRNA), as well as limitations in the current assay technologies, it poses high demand and challenge for a specific, efficient, and economic miRNA assay method. Here, we propose a new miRNA detection method based on a label-free probe and a small organic dye with sequence dependence, realizing the sequence-specific and colorimetric detection of target miRNA. What is pleasantly surprising, only one enzyme is enough to propel the whole miRNA assay process, greatly simplifying the reaction component and detection process. Together with PCR amplification for the high enough sensitivity and three checks for specificity control, a detection limit of 5 fM was obtained and even one mutation could be discriminated visually. Overall, the new method makes much progress in convenience and economy of PCR-based miRNA assay method so that miRNA assay is going to be more friendly and affordable.

Effectiveness of antiplatelet drugs for the prevention of diabetic nephropathy: A meta-analysis of randomized controlled trials
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AIMS: To evaluate the effects of antiplatelet drugs on proteinuria, renal function, and blood pressure (BP) in patients with diabetic nephropathy (DN). MATERIALS AND METHODS: A meta-analysis of randomized controlled trials (RCTs) was performed. Relevant RCTs were identified by a systematic search of the PubMed, Cochrane Library, and Embase databases. A random-effects model was applied to pool the results. RESULTS: Antiplatelet drugs significantly reduced urinary albumin excretion (weighted mean difference (WMD) = -69.25 mg/24, p = 0.005) and the urinary albumin/creatinine ratio (standardized mean difference (SMD) = -0.33, p = 0.009). However, renal function as reflected by the estimated glomerular filtration rate (WMD = -1.15 mL/min/1.73m2, p = 0.46) and BP (systolic BP: WMD = 1.53 mmHg, p = 0.35; diastolic BP: WMD = 0.40 mmHg, p = 0.70) were not significantly affected by antiplatelet drugs within 12 months of use (p > 0.05). CONCLUSION: Antiplatelet drugs may benefit patients with DN by reducing proteinuria. The long-term effects of antiplatelet drugs on the progression of DN warrant further evaluation.
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Selection of Intracellularly Functional RNA Mimics of Green Fluorescent Protein Using Fluorescence-Activated Cell Sorting.

Fluorescence-activated cell sorting (FACS) was exploited to isolate Escherichia coli cells that were highly fluorescent due to the expression of RNA aptamers that induce fluorescence of 3,5-difluoro-4-hydroxybenzylidene imidazolinone. Two different aptamers, named ZT-26 and ZT-324, were identified by this method and compared to the fluorescence-signaling properties of Spinach, a previously reported RNA aptamer. Aptamer ZT-26 exhibits significantly enhanced fluorescence over Spinach only in vitro. However, aptamer ZT-324 is 36% brighter than Spinach when expressed in E. coli. The FACS-based selection strategy presented here is attractive for deriving fluorescent RNA aptamers that function in cells as it directly selects for cells with a high level of fluorescence due to the expression of the RNA aptamer.

Metal dimer nanojunction-magnetic material composites for magnetic field sensing.

Noble metal nanocrystals are used as high sensitivity optoelectronic sensors, such as surface-enhanced Raman scattering, SERS. The sensing performance of metal nanocrystals can be further improved by forming dimer nanojunctions with strong "plasmonic coupling". Since the strength of "plasmonic coupling" is highly sensitive to the sub-nanoscale spacing between plasmonic nanocrystals in nanojunctions, nanojunctions can be used to detect external stimuli that can change the spacing of nanocrystals in the nanojunction and thus change the sensitivity of the Raman scattering spectrum. Here, we utilize this principle to detect the direction and strength of an external magnetic field (MF) using dimer nanojunctions surrounded by magnetic materials as a sensing platform. The results reveal that the changes in nanocrystal spacing in the nanojunction are caused by the rearrangement of the magnetic material under an external MF, which strongly depends on the interaction between the magnetic material and the ligands on the nanocrystal surface and the steric repulsion generated by the ligand configuration on the nanocrystal surface. Compared with the Raman spectrum without an external MF, the enhancement factors of the Raman scattering spectrum under an external MF can reach up to ∼900%, which makes dimer nanojunctions with magnetic materials suitable for "magnetic field" sensing applications.

Relationship between immune cells and diabetic nephropathy: a Mendelian randomization study.

OBJECTIVE: Although previous studies have suggested potential correlations between immunocytes and diabetic nephropathy (DN), the causal correlations between them remain unclarified. In this study, we employed Mendelian randomization (MR) to analyze the potential causative correlations between immune 731 cells and DN. METHODS: We used the Genome-Wide Association Studies (GWAS) database to aggregate signatures of immune cells and DN from European and East Asian populations. Single-nucleotide polymorphisms (SNPs) were used as instrumental variables. MR analysis was conducted using Mendelian randomization-Egger (MR-Egger) regression and the random-effects inverse-variance weighted (IVW) method. RESULTS: A total of 3,571 SNPs were included as instrumental variables. The MR-Egger regression model indicated no genetic pleiotropy (P = 0.6284). The results of the IVW method indicated a statistically significant causal relationship between immune cell HLA-DR on CD14-CD16- (P = 0.029), CD45RA-CD28-CD8 + T cell% T cells (P = 0.0278), CD11c on myeloid dendritic cells (P = 0.0352), HLA-DR on CD14+ monocytes (P < 0.001), CD27 on CD24 + CD27 + B cells (P = 0.0334), CD27 on IgD + CD24 + B cells (P = 0.0137), CD4 on CD39 + CD4 + T cells (P = 0.0347), CD28 on CD39 + CD4 + T cells (P = 0.0414), CD39 on CD39 + CD4 + T cells (P = 0.0426), and DN. Additionally, there was no heterogeneity in SNPs related HLA-DR on CD14-CD16-cells and DN(I2 = 32%, Cochran's Q = 2.9476, P = 0.2291). Moreover, leave-one-out analysis showed a causal correlation between HLA-DR on CD14-CD16- cells and DN. CONCLUSION: Higher expression of immune cell HLA-DR on CD14-CD16- cells may indicate a lower risk of DN.

Shengqing Jiangzhuo capsule ameliorates diabetic nephropathy by improving Keap1/Nrf2 signaling pathway.

BACKGROUND: Diabetic nephropathy (DN) is a major contributor to end-stage renal failure, and lacking effective treatment options. Shengqing Jiangzhuo capsule (SQJZJN), a traditional Chinese medicine prescription with known efficacy in chronic kidney disease, has not been thoroughly investigated for its potential in DN protection. METHODS: Eight-week-old male C57BLKS/J db/db, C57BLKS/J db/m mice, and human glomerular mesangial cell (HMC) cells cultured with high glucose were used as experimental models in this study. RESULTS: The in vivo investigation showed that SQJZJN can significantly ameliorate renal pathological damage, reduce serum creatinine, and lower urinary microalbumin levels in db/db mice. In vitro, SQJZJN treatment mitigated advanced glycation end products (AGEs) and reactive oxygen species (ROS), leading to a reduction in renal cell apoptosis. Mechanistically, SQJZJN activated the Keap1/Nrf2/ARE pathway by promoting nuclear factor erythroid-derived 2-related factor 2 (Nrf2), γ-glutamylcysteine synthetase heavy subunit (γ-GCS), and Heme oxygenase-1 (HO-1) expressions, while decreasing Kelch-like ECH-associated protein 1 (KEAP1) expressions. CONCLUSION: These findings suggest that SQJZJN exerts a protective effect on DN, potentially through the activation of the Keap1/Nrf2/ARE pathway.

(+)-Catechin ameliorates diabetic nephropathy injury by inhibiting endoplasmic reticulum stress-related NLRP3-mediated inflammation.

Endoplasmic reticulum (ER) stress and chronic sterile inflammation are associated with the pathogenesis of diabetic nephropathy (DN). Catechins are natural polyphenolic compounds found in green tea that possess some health benefits. However, whether (+)-catechin can reduce tubular injury in DN by regulating ER stress and NLRP3-associated inflammation remains uncertain. This study examined the effects of (+)-catechin on streptozotocin (STZ)-induced diabetic mice and on palmitic acid (PA)-treated HK-2 cells. In vivo, a DN mouse model was generated by injecting STZ. The biochemical indicators of serum and urine, as well as renal histopathology and ultrastructure were analysed. To predict the mechanisms associated with (+)-catechin, network pharmacology and molecular docking were used. Finally, quantitative real-time PCR (qPCR), western blot analysis and immunofluorescence analysis were performed to measure the mRNA and protein expressions of specific targets in the renal tissue of DN mice and PA-treated HK-2 cells to validate the predicted results. (+)-Catechin significantly ameliorated renal function and pathological changes associated with tubular injury by inhibiting ER stress by downregulating of GRP78, PEAK, CHOP, ATF6 and XBP1. In addition, (+)-catechin inhibited renal inflammation by suppressing NLRP3 associated inflammation, which was characterized by the downregulation of NLRP3, ASC, AIM2, Caspase1, IL-1ß and IL-18 in DN mice and PA-treated HK-2 cells. Collectively, these findings suggested that (+)-catechin exerted a renoprotective effect against DN by inhibiting ER stress and NLRP3-related inflammation to ameliorate tubular injury, suggesting the therapeutic potential of (+)-catechin.

Astragaloside IV attenuates renal tubule injury in DKD rats via suppression of CD36-mediated NLRP3 inflammasome activation.

Background: In recent years, diabetic kidney disease (DKD) has emerged as a prominent factor contributing to end-stage renal disease. Tubulointerstitial inflammation and lipid accumulation have been identified as key factors in the development of DKD. Earlier research indicated that Astragaloside IV (AS-IV) reduces inflammation and oxidative stress, controls lipid accumulation, and provides protection to the kidneys. Nevertheless, the mechanisms responsible for its protective effects against DKD have not yet been completely elucidated. Purpose: The primary objective of this research was to examine the protective properties of AS-IV against DKD and investigate the underlying mechanism, which involves CD36, reactive oxygen species (ROS), NLR family pyrin domain containing 3 (NLRP3), and interleukin-1ß (IL-1ß). Methods: The DKD rat model was created by administering streptozotocin along with a high-fat diet. Subsequently, the DKD rats and palmitic acid (PA)-induced HK-2 cells were treated with AS-IV. Atorvastatin was used as the positive control. To assess the therapeutic effects of AS-IV on DKD, various tests including blood sugar levels, the lipid profile, renal function, and histopathological examinations were conducted. The levels of CD36, ROS, NLRP3, Caspase-1, and IL-1ß were detected using western blot analysis, PCR, and flow cytometry. Furthermore, adenovirus-mediated CD36 overexpression was applied to explore the underlying mechanisms through in vitro experiments. Results: In vivo experiments demonstrated that AS-IV significantly reduced hyperglycemia, dyslipidemia, urinary albumin excretion, and serum creatinine levels in DKD rats. Additionally, it improved renal structural abnormalities and suppressed the expression of CD36, NLRP3, IL-1ß, TNF-α, and MCP-1. In vitro experiments showed that AS-IV decreased CD36 expression, lipid accumulation, and lipid ROS production while inhibiting NLRP3 activation and IL-1ß secretion in PA-induced HK-2 cells. Conclusion: AS-IV alleviated renal tubule interstitial inflammation and tubule epithelial cell apoptosis in DKD rats by inhibiting CD36-mediated lipid accumulation and NLRP3 inflammasome activation.

Ultrafast DNA detection based on turn-back loop primer-accelerated LAMP (TLAMP).

Rapid DNA detection is a long-pursuing goal in molecular detection, especially in combating infectious diseases. Loop-mediated isothermal amplification (LAMP) is a robust and prevailing DNA detection method in pathogen detection, which has been drawing broad interest in improving its performance. Herein, we reported a new strategy and developed a new LAMP variant named TLAMP with a superior amplification rate. In this strategy, the turn-back loop primers (TLPs) were devised by ingeniously extending the 5' end of the original loop primer, which conferred the new role of being the inner primer for TLPs while retaining its original function as the loop primer. In theory, based on the bifunctional TLPs, a total of eight basic dumbbell-like structures and four cyclic amplification pathways were produced to significantly enhance the amplification efficiency of TLAMP. With the enhancing effect of TLPs, TLAMP exhibited a significantly reduced amplification-to-result time compared to the conventional six-primer LAMP (typically 1 h), enabling rapid DNA detection within 20 min. Furthermore, TLAMP proved to be about 10 min faster than the fast LAMP variants reported so far, while still presenting comparable sensitivity and higher repeatability. Finally, TLAMP successfully achieved an ultrafast diagnosis of Monkeypox virus (MPXV), capable of detecting as few as 10 copies (0.67copies/µL) of pseudovirus within 20 min using real-time fluorescence assay or within 30 min using a colorimetric assay, suggesting that the proposed TLAMP offers a sensitive, specific, reliable, and, most importantly, ultrafast DNA detection method when facing the challenges posed by infectious diseases.