A mutation-sensitive, multiplexed and amplification-free detection of nucleic acids by stretching single-molecule tandem hairpin probes.

The detection of nucleic acid sequences in parallel with the discrimination of single nucleotide variations (SNVs) is critical for research and clinical applications. A few limitations make the detection technically challenging, such as too small variation in probe-hybridization energy caused by SNVs, the non-specific amplification of false nucleic acid fragments and the few options of dyes limited by spectral overlaps. To circumvent these limitations, we developed a single-molecule nucleic acid detection assay without amplification or fluorescence termed THREF (hybridization-induced tandem DNA hairpin refolding failure) based on multiplexed magnetic tweezers. THREF can detect DNA and RNA sequences at femtomolar concentrations within 30 min, monitor multiple probes in parallel, quantify the expression level of miR-122 in patient tissues, discriminate SNVs including the hard-to-detect G-U or T-G wobble mutations and reuse the probes to save the cost. In our demonstrative detections using mock clinic samples, we profiled the let-7 family microRNAs in serum and genotyped SARS-CoV-2 strains in saliva. Overall, the THREF assay can discriminate SNVs with the advantages of high sensitivity, ultra-specificity, multiplexing, reusability, sample hands-free and robustness.

Discovery of FO-4-15, a novel 1,2,4-oxadiazole derivative, ameliorates cognitive impairments in 3×Tg mice by activating the mGluR1/CaMKIIα pathway.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder characterized by cognitive impairments. Despite the limited efficacy of current treatments for AD, the 1,2,4-oxadiazole structure has garnered significant attention in medicinal chemistry due to its potential impact on mGluR1 and its association with AD therapy. In this study, a series of novel 1,2,4-oxadiazole derivatives were designed, synthesized, and evaluated for the neuroprotective effects in human neuroblastoma (SH-SY5Y) cells. Among all the derivatives tested, FO-4-15 (5f) existed the lowest cytotoxicity and the highest protective effect against H2O2. Based on these in vitro results, FO-4-15 was administered to 3×Tg mice and significantly improved the cognitive impairments of the AD mice. Pathological analysis showed that FO-4-15 significantly reduced Aß accumulation, Tau hyper-phosphorylation, and synaptic impairments in the 3×Tg mice. Dysfunction of the CaMKIIα/Fos signaling pathway in 3×Tg mice was found to be restored by FO-4-15 and the necessity of the CaMKIIα/Fos for FO-4-15 was subsequently confirmed by the use of a CaMKIIα inhibitor in vitro. Beyond that, mGluR1 was identified to be a potential target of FO-4-15, and the interaction of FO-4-15 and mGluR1 was displayed by Ca2+ flow increase, molecular docking, and interaction energy analysis. The target of FO-4-15 was further confirmed in vitro by JNJ16259685, a nonselective inhibitor of mGluR1. These findings suggest that FO-4-15 may hold promise as a potential treatment for Alzheimer's disease.

Design, synthesis and biological activity of oxyevodiamine-based histone deacetylase 6 inhibitors.

Histone deacetylase 6 (HDAC6) was a potential target for Alzheimer's disease (AD). In this study, a series of novel oxyevodiamine-based HDAC6 inhibitors with a variety of linker moieties were designed, synthesized and evaluated. Compound 12 with a benzyl linker was identified as a high potent and selective HDAC6 inhibitor. It inhibited HDAC6 with an IC50 value of 6.2 nM and was more than 200 fold selectivity over HDAC1. It also had lower cytotoxicity and higher anti-H2O2 activity in vitro comparing with other derivatives. Compound 12 might be a good lead as novel HDAC6 inhibitor for the treatment of AD.

Discovery of palmatine derivatives as potent neuroprotective agents.

Alzheimer's disease is a neurodegenerative disorder characterized by the presence of neurodegenerative lesions and cognitive impairment. In this study, a series of novel palmatine derivatives were designed and synthesized through the introduction of a heteroatom using carbodiimide-mediated condensation. The synthesized compounds were then screened for toxicity and potency, leading to the identification of compound 2q, which exhibited low toxicity and high potency. Our findings demonstrated that compound 2q displayed significant neuroprotective activity in vitro, emerging as a promising candidate for Alzheimer's disease treatment.

Fingolimod Inhibits Exopolysaccharide Production and Regulates Relevant Genes to Eliminate the Biofilm of K. pneumoniae.

Klebsiella pneumoniae (K. pneumoniae) exhibits the ability to form biofilms as a means of adapting to its adverse surroundings. K. pneumoniae in this biofilm state demonstrates remarkable resistance, evades immune system attacks, and poses challenges for complete eradication, thereby complicating clinical anti-infection efforts. Moreover, the precise mechanisms governing biofilm formation and disruption remain elusive. Recent studies have discovered that fingolimod (FLD) exhibits biofilm properties against Gram-positive bacteria. Therefore, the antibiofilm properties of FLD were evaluated against multidrug-resistant (MDR) K. pneumoniae in this study. The antibiofilm activity of FLD against K. pneumoniae was assessed utilizing the Alamar Blue assay along with confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), and crystal violet (CV) staining. The results showed that FLD effectively reduced biofilm formation, exopolysaccharide (EPS), motility, and bacterial abundance within K. pneumoniae biofilms without impeding its growth and metabolic activity. Furthermore, the inhibitory impact of FLD on the production of autoinducer-2 (AI-2) signaling molecules was identified, thereby demonstrating its notable anti-quorum sensing (QS) properties. The results of qRT-PCR analysis demonstrated that FLD significantly decreased the expression of genes associated with the efflux pump gene (AcrB, kexD, ketM, kdeA, and kpnE), outer membrane (OM) porin proteins (OmpK35, OmpK36), the quorum-sensing (QS) system (luxS), lipopolysaccharide (LPS) production (wzm), and EPS production (pgaA). Simultaneously, FLD exhibited evident antibacterial synergism, leading to an increased survival rate of G. mellonella infected with MDR K. pneumoniae. These findings suggested that FLD has substantial antibiofilm properties and synergistic antibacterial potential for colistin in treating K. pneumoniae infections.

Protective Effect and Mechanism of Aspirin Eugenol Ester on Lipopolysaccharide-Induced Intestinal Barrier Injury.

Intestinal inflammation is a complex and recurrent inflammatory disease. Pharmacological and pharmacodynamic experiments showed that aspirin eugenol ester (AEE) has good anti-inflammatory, antipyretic, and analgesic effects. However, the role of AEE in regulating intestinal inflammation has not been explored. This study aimed to investigate whether AEE could have a protective effect on LPS-induced intestinal inflammation and thus help to alleviate the damage to the intestinal barrier. This was assessed with an inflammation model in Caco-2 cells and in rats induced with LPS. The expression of inflammatory mediators, intestinal epithelial barrier-related proteins, and redox-related signals was analyzed using an enzyme-linked immunosorbent assay (ELISA), Western blotting, immunofluorescence staining, and RT-qPCR. Intestinal damage was assessed by histopathological examination. Changes in rat gut microbiota and their functions were detected by the gut microbial metagenome. AEE significantly reduced LPS-induced pro-inflammatory cytokine levels (p < 0.05) and oxidative stress levels in Caco-2 cells and rats. Compared with the LPS group, AEE could increase the relative expression of Occludin, Claudin-1, and zonula occludens-1 (ZO-1) and decrease the relative expression of kappa-B (NF-κB) and matrix metalloproteinase-9. AEE could significantly improve weight loss, diarrhea, reduced intestinal muscle thickness, and intestinal villi damage in rats. Metagenome results showed that AEE could regulate the homeostasis of the gut flora and alter the relative abundance of Firmicutes and Bacteroidetes. Flora enrichment analysis indicated that the regulation of gut flora with AEE may be related to the regulation of glucose metabolism and energy metabolism. AEE could have positive effects on intestinal inflammation-related diseases.

The Transport and Uptake of Resveratrol Mediated via Glucose Transporter 1 and Its Antioxidant Effect in Caco-2 Cells.

Resveratrol has anti-inflammatory, anti-cancer, and anti-aging pharmacological activities. There is currently a gap in academic research regarding the uptake, transport, and reduction of H2O2-induced oxidative damage of resveratrol in the Caco-2 cell model. This study investigated the role of resveratrol in the uptake, transport, and alleviation of H2O2-induced oxidative damage in Caco-2 cells. In the Caco-2 cell transport model, it was observed that the uptake and transport of resveratrol (10, 20, 40, and 80 µM) were time dependent and concentration dependent. Different temperatures (37 °C vs. 4 °C) could significantly affect the uptake and transportation of resveratrol. The apical to basolateral transport of resveratrol was markedly reduced by STF-31, a GLUT1 inhibitor, and siRNA intervention. Furthermore, resveratrol pretreatment (80 µM) improves the viability of Caco-2 cells induced by H2O2. In a cellular metabolite analysis combined with ultra-high performance liquid chromatography-tandem mass spectrometry, 21 metabolites were identified as differentials. These differential metabolites belong to the urea cycle, arginine and proline metabolism, glycine and serine metabolism, ammonia recycling, aspartate metabolism, glutathione metabolism, and other metabolic pathways. The transport, uptake, and metabolism of resveratrol suggest that oral resveratrol could prevent intestinal diseases caused by oxidative stress.

Multivalent Cations Reverse the Twist-Stretch Coupling of RNA.

When stretched, both DNA and RNA duplexes change their twist angles through twist-stretch coupling. The coupling is negative for DNA but positive for RNA, which is not yet completely understood. Here, our magnetic tweezers experiments show that the coupling of RNA reverses from positive to negative by multivalent cations. Combining with the previously reported tension-induced negative-to-positive coupling reversal of DNA, we propose a unified mechanism of the couplings of both RNA and DNA based on molecular dynamics simulations. Two deformation pathways are competing when stretched: shrinking the radius causes positive couplings but widening the major groove causes negative couplings. For RNA whose major groove is clamped by multivalent cations and canonical DNA, their radii shrink when stretched, thus exhibiting positive couplings. For elongated DNA whose radius already shrinks to the minimum and canonical RNA, their major grooves are widened when stretched, thus exhibiting negative couplings.

Development and Validation of a Nomogram for Predicting Postoperative Pulmonary Infection in Patients Undergoing Lung Surgery.

OBJECTIVES: To develop and validate a nomogram for predicting postoperative pulmonary infection (PPI) in patients undergoing lung surgery. DESIGN: Single-center retrospective cohort analysis. SETTING: A university-affiliated cancer hospital PARTICIPANTS: A total of 1,501 adult patients who underwent lung surgery from January 2018 to December 2020. INTERVENTIONS: Observation for PPI within 7 days after lung surgery. MEASUREMENTS AND MAIN RESULTS: A complete set of demographics, preoperative variables, and postoperative follow-up data was recorded. The primary outcome was PPI; a total of 125 (8.3%) out of 1,501 patients developed PPI. The variables with p < 0.1 in univariate logistic regression were included in the multivariate regression, and multivariate logistic regression analysis showed that surgical procedure, surgical duration, the inspired fraction of oxygen in one-lung ventilation, and postoperative pain were independent risk factors for PPI. A nomogram based on these factors was constructed in the development cohort (area under the curve: 0.794, 95% CI 0.744-0.845) and validated in the validation cohort (area under the curve: 0.849, 95% CI 0.786-0.912). The calibration slope was 1 in the development and validation cohorts. Decision curve analysis indicated that when the threshold probability was within a range of 0.02-to-0.58 and 0.02-to-0.42 for the development and validation cohorts, respectively, the nomogram model could provide a clinical net benefit. CONCLUSIONS: The authors developed and validated a nomogram for predicting PPI in patients undergoing lung surgery. The prediction model can predict the development of PPI and identify high-risk groups.

Genetic substructure analysis of three isolated populations in southwest China.

Southwest China is home to numerous ethnic minorities, as well as many geographically and genetically isolated groups. However, the genetic substructure of these ethnic groups, especially the paternal genetic structure between groups, has not been comprehensively analyzed. In this study, we used Y chromosome capture and Illumina sequencing technologies to investigate the paternal genetic structure of three isolated groups of male unrelated individuals, including Baima in Pingwu, Sichuan Province, Muya in Shimian, Sichuan Province, and Kongge in Jinghong, Yunnan Province. We calculated the frequencies of related haplogroups by the fixed-point compound amplification method and direct counting method, and used the Past3.0 software to perform principal component analysis to draw a population clustering tree. we observed that Kongge had 3 Y chromosome haplogroups, Baima had 4 Y chromosome haplogroups, and Muya had 5 Y chromosome haplogroups. The results showed that Kongge was most closely related to the Wa, and the Y chromosome types of the Baima and Muya were mainly concentrated in the D haplogroup and its lower reaches. It has the closest relationship with the Tibetans in Qamdo and Nyingchi. The study on the genetic structure of different ethnic groups has enriched the genetic relationship of isolated populations and provided a new perspective for understanding Chinese ethnic groups.

Design, synthesis and biological evaluation of dipeptides as novel non-covalent 20S proteasome inhibitors.

Based on the interaction modes of the natural 20S proteasome inhibitors TMC-95A, we have previously discovered a dipeptide 1. To explore the SAR around compound 1, we designed and synthesized a series of dipeptides (8-38) with a fragment-based strategy. Among them, nine compounds showed significant inhibitory activities against the chymotrypsin-like activity of human 20S proteasome with IC50 values at the submicromolar level, which were comparable or even superior to the parent compound 1. Meanwhile, they displayed no significant inhibition against trypsin-like and caspase-like activities of 20S proteasome. The results suggested the feasibility to design dipeptides as novel and potent 20S proteasome inhibitors.[Formula: see text].

Cytosine Methylation Enhances DNA Condensation Revealed by Equilibrium Measurements Using Magnetic Tweezers.

CpG methylation of DNA is common in mammalian cells. In sperm, the DNA has the highest level of CpG methylation and is condensed into toroidal structures. How CpG methylation affects DNA structures and interactions is important to understand its biological roles but is largely unknown. Using an RNA-DNA-RNA structure, we observed the equilibrium hopping dynamics between the condensed and extended states of DNA in the presence of polyamines or polylysine peptide as a reduced model of histone tails. Combing with the measured DNA elasticities, we report that CpG methylation of each cytosine nucleotide substantially increases DNA-DNA attraction by up to 0.2 kBT. For the DNA with 57% GC content, the relative increase caused by CpG methylation is up to 32% for the spermine-induced DNA-DNA attraction and up to 9% for the polylysine-induced DNA-DNA attraction. These findings help us to evaluate the energetic contributions of CpG methylation in sperm development and chromatin regulation.

Opposite Effects of High-Valent Cations on the Elasticities of DNA and RNA Duplexes Revealed by Magnetic Tweezers.

We report that trivalent cobalt hexammine cations decrease the persistence length, stretching modulus, helical density, and size of plectonemes formed under torque of DNA but increase those of RNA. Divalent magnesium cations, however, decrease the persistence lengths, contour lengths, and sizes of plectonemes while increasing the helical densities of both DNA and RNA. The experimental results are explained by different binding modes of the cations on DNA and RNA in our all-atom molecular dynamics simulations. The significant variations of the helical densities and structures of DNA and RNA duplexes induced by high-valent cations may affect interactions of the duplexes with proteins.

Synthesis and antiviral activity of lycorine derivatives.

There are no effective antiviral drugs to treat hand, foot, and mouth disease. In this study, a series of lycorine derivatives were synthesized and evaluated against enterovirus 71 and coxsackievirus A16 in vitro. Derivatives 7c-m with the phenoxyacyl group at the C-1 position showed higher efficacy and lower toxicity than lycorine. In addition, derivative 7e enhanced the survival rate to 40% in the mouse model of the lethal EV71 infection.

S-DNA and RecA/RAD51-Mediated Strand Exchange in Vitro.

S-DNA (stretched DNA) is an elongated base-paired DNA conformation under high tension. Because the RecA/Rad51 family DNA recombinases form helical filaments on DNA and mediate the formation of the DNA triplex (D-loop), in which the DNA is stretched, and because the extension of these nucleoprotein filaments is similar to the extension of S-DNA, S-DNA has long been hypothesized as a possible state of DNA that participants in RecA/Rad51-mediated DNA strand exchange in homologous recombination. Such a hypothesis, however, is still lacking direct experimental studies. In this work, we have studied the polymerization and strand exchange on S-DNA mediated by Escherichia coli RecA, human Rad51, and Saccharomyces cerevisiae Rad51 by single-molecule magnetic tweezers. We report that RecA/Rad51 polymerizes faster on S-DNA than on B-DNA with the same buffer conditions. Furthermore, the RecA/Rad51-mediated DNA triplex forms faster from S-DNA than from B-DNA together with the homologous single-stranded DNA. These results provide evidence that S-DNA can interact with RecA and Rad51 and shed light on the possible functions of S-DNA.

ELP2 negatively regulates osteoblastic differentiation impaired by tumor necrosis factor α in MC3T3-E1 cells through STAT3 activation.

Tumor necrosis factor-α (TNF-α) is a pluripotent signaling molecule. The biological effect of TNF-α includes slowing down osteogenic differentiation, which can lead to bone dysplasia in long-term inflammatory microenvironments. Signal transducer and activator of transcription 3 (STAT3)-interacting protein 1 (StIP1, also known as elongator complex protein 2, ELP2) play a role in inhibiting TNF-α-induced osteoblast differentiation. In the present study, we investigated whether and how ELP2 activation mediates the effects of TNF-α on osteoblastic differentiation. Using in vitro cell cultures of preosteoblastic MC3T3-E1 cells, we found that TNF-α inhibited osteoblastic differentiation accompanied by an increase in ELP2 expression and STAT3 activation. Forced ELP2 expression inhibited osteogenic differentiation of MC3T3-E1 cells, with a decrease in the expression of osteoblast marker genes, alkaline phosphatase activity, and matrix mineralization capacity. In contrast, ELP2 silencing ameliorated osteogenic differentiation in MC3T3-E1 cells, even after TNF-α stimulation. The TNF-α-induced inhibitory effect on osteoblastic differentiation was therefore mediated by ELP2, which was associated with Janus kinase 2 (JAK2)/STAT3 activation. These results suggest that ELP2 is upregulated at the differentiation of MC3T3-E1 cells into osteoblasts and inhibits osteogenic differentiation in response to TNF-α through STAT3 activation.

An LC-MS/MS method for the quantification of diclofenac sodium in dairy cow plasma and its application in pharmacokinetics studies.

An LC-MS/MS method with internal standard tolfenamic acid for determining diclofenac sodium (DCF) in dairy cow plasma was developed and validated. Samples were processed with protein precipitation by cold formic acid-acetonitrile. Determination of DCF was performed using LC-ESI+ -MS/MS with the matrix-matched calibration curve. The results showed that the method was sensitive (LOD 2 ng mL-1 , LOQ 5 ng mL-1 ), accurate (97.60 ± 5.64%), precise (<10%) and linear in the range of 5-10,000 ng mL-1 . A single intravenous (i.v.) or intramuscular (i.m.) administration of 5% diclofenac sodium injection at a dose of 2.2 mg kg-1 was performed in six healthy dairy cows according to a two-period crossover design. The main pharmacokinetic (PK) parameters after a single i.v. administration were as follows: t1/2ß , 4.52 ± 1.71 h; AUC, 77.79 ± 16.76 h µg mL-1 ; mean residence time, 5.16 ± 1.11 h. The main PK parameters after a single i.m. administration were as follows: Tmax , 2.38 ± 1.19 h; Cmax , 7.46 ± 1.85 µg mL-1 ; t1/2ß , 9.46 ± 2.86 h; AUC 67.57 ± 13.07 h µg mL-1 . The absolute bioavailability was 87.37 ± 5.96%. The results showed that the diclofenac sodium injection had PK characteristics of rapid absorption and slow elimination, and high peak concentration and bioavailability in dairy cows, and that the recommended clinical dosage of diclofenac sodium injection is 2.2 mg kg-1 .

Cellular Metabolomics Reveal the Mechanism Underlying the Anti-Atherosclerotic Effects of Aspirin Eugenol Ester on Vascular Endothelial Dysfunction.

Aspirin eugenol ester (AEE) possesses anti-thrombotic, anti-atherosclerotic and anti-oxidative effects. The study aims to clarify the mechanism underlying the anti-atherosclerotic effects of AEE on vascular endothelial dysfunction. Both the high-fat diet (HFD)-induced atherosclerotic rat model and the H2O2-induced human umbilical vein endothelial cells (HUVECs) model were used to investigate the effects of AEE on vascular endothelial dysfunction. UPLC/QTOF-MS coupled with a multivariate data analysis method were used to profile the variations in the metabolites of HUVECs in response to different treatments. Pretreatment of HUVECs with AEE significantly ameliorated H2O2-induced apoptosis, the overexpression of E-selectin and VCAM-1, and the adhesion of THP-1 cells. Putative endogenous biomarkers associated with the inhibition of endothelial dysfunction were identified in HUVECs pretreated with AEE in the absence or presence of H2O2, and these biomarkers were involved in important metabolic pathways, including amino acid metabolism, carbohydrate metabolism, and glutathione metabolism. Moreover, in vivo, AEE also significantly reduced vascular endothelial dysfunction and decreased the overexpression of VCAM-1 and E-selectin. Based on our findings, the mechanism underlying the anti-atherosclerotic effects of AEE might be related to a reduction in vascular endothelial dysfunction mediated by ameliorating alterations in metabolism, inhibiting oxidative stress, and decreasing the expression of adhesion molecules.

Whole sequence analysis indicates a recent southern origin of Mongolian Y-chromosome C2c1a1a1-M407.

The Y-chromosome haplogroup C2c1a1a1-M407 is a predominant paternal lineage in Mongolic-speaking populations, especially in Buryats and Kalmyks. However, the origin and internal phylogeny of C2c1a1a1-M407 have not been investigated in detail. In this study, we analyzed twenty-three Y-chromosome sequences of haplogroup C2c1a1a1-M407 and its most closely related clades. We generated a high-resolution phylogenetic tree of haplogroup C2c1a1a1-M407 and its upstream clade C2c1a1-CTS2657, including 32 subclades and 144 non-private Y-chromosome polymorphisms. We discover that all available C2c1a1a1-M407 samples from Mongolic-speaking populations belong to its newly defined downstream clade C2c1a1a1b-F8465, whereas all samples of C2c1a1-CTS2657(xF8465) come from northern Han Chinese, Korean, and Japanese. Furthermore, we observe that C2c1a1a1b-F8465 and its subclade C2c1a1a1b1-F8536 expanded at approximately 0.86 and 0.44 thousand years ago, respectively. Therefore, we conclude that C2c1a1a1-M407 in Mongolic-speaking populations has originated from northeastern Asia. C2c1a1a1b1-F8536, the newly defined subclade of C2c1a1a1-M407, probably represents the genetic relationships between ancient Oyrats, modern Kalmyks, Mongolians, and Buryats.

Genome-wide identification and characterization of ALTERNATIVE OXIDASE genes and their response under abiotic stresses in Camellia sinensis (L.) O. Kuntze.

MAIN CONCLUSION: Four typical ALTERNATIVE OXIDASE genes have been identified in tea plants, and their sequence features and gene expression profiles have provided useful information for further studies on function and regulation. Alternative oxidase (AOX) is a terminal oxidase located in the respiratory electron transport chain. AOX catalyzes the oxidation of quinol and the reduction of oxygen into water. In this study, a genome-wide search and subsequent DNA cloning were performed to identify and characterize AOX genes in tea plant (Camellia sinensis (L.) O. Kuntze cv. Longjing43). Our results showed that tea plant possesses four AOX genes, i.e., CsAOX1a, CsAOX1d, CsAOX2a and CsAOX2b. Gene structure and protein sequence analyses revealed that all CsAOXs share a four-exon/three-intron structure with highly conserved regions and amino acid residues, which are necessary for AOX secondary structures, catalytic activities and post-translational regulations. All CsAOX were shown to localize in mitochondria using the green fluorescent protein (GFP)-targeting assay. Both CsAOX1a and CsAOX1d were induced by cold, salt and drought stresses, and with different expression patterns in young and mature leaves. Reactive oxygen species (ROS) accumulated strongly after 72 and 96 h cold treatments in both young and mature leaves, while the polyphenol and total catechin decreased significantly only in mature leaves. In comparison to AtAOX1a in Arabidopsis thaliana, CsAOX1a lost almost all of the stress-responsive cis-acting regulatory elements in its promoter region (1500 bp upstream), but possesses a flavonoid biosynthesis-related MBSII cis-acting regulatory element. These results suggest a link between CsAOX1a function and the metabolism of some secondary metabolites in tea plant. Our studies provide a basis for the further elucidation of the biological function and regulation of the AOX pathway in tea plants.