A Two-color Single-molecule Sequencing Platform and Its Clinical Applications.

DNA sequencers have become increasingly important research and diagnostic tools over the past 20 years. In this study, we developed a single-molecule desktop sequencer, GenoCare 1600 (GenoCare), which utilizes amplification-free library preparation and two-color sequencing-by-synthesis chemistry, making it more user-friendly compared with previous single-molecule sequencing platforms for clinical use. Using the GenoCare platform, we sequenced an Escherichia coli standard sample and achieved a consensus accuracy exceeding 99.99%. We also evaluated the sequencing performance of this platform in microbial mixtures and coronavirus disease 2019 (COVID-19) samples from throat swabs. Our findings indicate that the GenoCare platform allows for microbial quantitation, sensitive identification of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, and accurate detection of virus mutations, as confirmed by Sanger sequencing, demonstrating its remarkable potential in clinical application.

Baoyuan decoction inhibits atherosclerosis progression through suppression peroxidized fatty acid and Src/MKK4/JNK pathway-mediated CD 36 expression.

BACKGROUND: Baoyuan decoction (BYD) has been widely utilized as a traditional prescription for the treatment of various conditions such as coronary heart disease, aplastic anemia, and chronic renal failure. However, its potential efficacy in improving atherosclerosis has not yet been investigated. PURPOSE: Our research aimed to assess the potential of BYD as an inhibitor of atherosclerosis and uncover the underlying mechanism by which it acts on foam cell formation. STUDY DESIGN AND METHODS: High-fat diet-induced ApoE-/- mice were employed to explore the effect of BYD on atherosclerosis. The differential metabolites in feces were identified and analyzed by LC-Qtrap-MS. In addition, we utilized pharmacological inhibition of BYD on foam cell formation induced by oxLDL in THP-1 cells to elucidate the underlying mechanisms specifically in macrophages. RESULTS: The atherosclerotic plaque burden in the aortic sinus of ApoE-/- mice was notably reduced with BYD treatment, despite no significant alterations in plasma lipids. Metabolomic analysis revealed that BYD suppressed the increased levels of peroxidized fatty acids, specifically 9/13-hydroxyoctadecadienoic acid (9/13-HODE), in the feces of mice. As a prominent peroxidized fatty acid found in oxLDL, we confirmed that 9/13-HODE induced the overexpression of CD36 in THP-1 macrophages by upregulating PPARγ. In subsequent experiments, the decreased levels of CD36 triggered by oxLDL were observed after BYD treatment. This decrease occurred through the regulation of the Src/MMK4/JNK pathway, resulting in the suppression of lipid deposition in THP-1 macrophages. CONCLUSIONS: These results illustrate that BYD exhibits potential anti-atherosclerotic effects by inhibiting CD36 expression to prevent foam cell formation.

G-Quadruplex-Programmed Versatile Nanorobot Combined with Chemotherapy and Gene Therapy for Synergistic Targeted Therapy.

Developing synergistic targeted therapeutics to improve treatment efficacy while reducing side effects has proven promising for anticancer therapies, but how to conveniently modulate multidrug cooperation remains a challenge. Here, a novel synergistic strategy using a G-quadruplex-programmed versatile nanorobot (G4VN) containing two subunits of DNAzyme (DzG4) and ligand-drug conjugates (LDCs) is proposed to precisely target tumors and then execute both gene silencing and chemotherapy. As the core module of this nanorobot, a well-designed G4 responding to a high level of K+ in tumor microenvironment smartly kills three birds with one stone, which makes two TfR aptamers proximate to improve their efficiency of targeting tumor cells, and in situ activates a split 10-23 DNAzyme to downregulate target mRNA expression, meanwhile promotes the cell uptake of a GSH-responsive LDCs to enhance drug efficacy. Such a design enables a potently synergistic anticancer therapy with low side effects in vivo, showing great promise for broad applications in precision disease treatment.

Target-induced DNA nanomachine operation for the detection of proteins.

Accurate and sensitive detection of disease-associated proteins in early stage of patients plays an important role in timely treatment and successfully extending patients' lives. To meet this demand, we herein rationally designed a flexible target-induced DNA nanomachine operation (TIDNMO) sensor for the detection of proteins. The TIDNMO system was composed of DNA nanoswitch and DNA walker. Triplex DNA nanoswitch was triggered by specific target, followed by the release of the walking strand, which initiated the DNA walker amplification as signal output. In addition, the Exo III could drive walking strand autonomously move on gold nanoparticle surface to realize 2 orders of magnitude signal amplification. What's more, this sensor could transform its suitable functional recognition element of DNA nanoswitch to recognize other specific molecule and realize different targets sensing based on identical walking tracks. Considering the facile reporter elements and efficient amplification performance, the present DNA nanomachine as a sensor could achieve a detection limit of 68 pM for anti-Dig antibody, 0.95 pM for mucin-1 respectively, along with a superb specificity. Furthermore, the method reported here opened a new chapter in disease-related protein sensing for the development of clinical early diagnosis.

Palmitoylation of PKCδ by ZDHHC5 in hypothalamic microglia presents as a therapeutic target for fatty liver disease.

The hypothalamus plays a fundamental role in controlling lipid metabolism through neuroendocrine signals. However, there are currently no available drug targets in the hypothalamus that can effectively improve human lipid metabolism. In this study, we found that the antimalarial drug artemether (ART) significantly improved lipid metabolism by specifically inhibiting microglial activation in the hypothalamus of high-fat diet-induced mice. Mechanically, ART protects the thyrotropin-releasing hormone (TRH) neurons surrounding microglial cells from inflammatory damage and promotes the release of TRH into the peripheral circulation. As a result, TRH stimulates the synthesis of thyroid hormone (TH), leading to a significant improvement in hepatic lipid disorders. Subsequently, we employed a biotin-labeled ART chemical probe to identify the direct cellular target in microglial cells as protein kinase Cδ (PKCδ). Importantly, ART directly targeted PKCδ to inhibit its palmitoylation modification by blocking the binding of zinc finger DHHC-type palmitoyltransferase 5 (ZDHHC5), which resulted in the inhibition of downstream neuroinflammation signaling. In vivo, hypothalamic microglia-specific PKCδ knockdown markedly impaired ART-dependent neuroendocrine regulation and lipid metabolism improvement in mice. Furthermore, single-cell transcriptomics analysis in human brain tissues revealed that the level of PKCδ in microglia positively correlated with individuals who had hyperlipemia, thereby highlighting a clinical translational value. Collectively, these data suggest that the palmitoylation of microglial PKCδ in the hypothalamus plays a role in modulating peripheral lipid metabolism through hypothalamus-liver communication, and provides a promising therapeutic target for fatty liver diseases.

Clinical Experience of Prenatal Chromosomal Microarray Analysis in 6159 Ultrasonically Abnormal Fetuses.

A single-center retrospective study of G-band karyotyping and chromosomal microarray analysis (CMA) for the invasive prenatal diagnosis of 6159 fetuses with ultrasound abnormalities was conducted. This study aimed to investigate the incidence rates of chromosomal abnormalities and pregnancy outcomes and postpartum clinical manifestations by long-term follow-up and to explore the correlation between different types of prenatal ultrasound abnormalities and pathogenic chromosomal abnormalities. The overall incidence of pathogenic chromosomal aberrations in fetuses with ultrasound abnormalities was 7.58% (467/6159), which comprised 41.7% (195/467) with chromosome number abnormalities, 57.6% (269/467) with pathogenic copy-number variations (pCNVs), and 0.64% (3/467) with uniparental disomy (UPD). In addition, 1.72% (106/6159) with likely pathogenic copy-number variations (lpCNVs) and 3.04% (187/6159) with variants of unknown significance (VOUS) were detected by CMA. Ultrasound abnormalities were categorized into structural anomalies and soft marker anomalies. The incidence rate of pathogenic and likely pathogenic chromosomal abnormalities was significantly higher among fetuses with structural anomalies than soft markers (11.13% vs 7.59%, p < 0.01). We retrospectively analyzed the prenatal genetic outcomes for a large cohort of fetuses with different types of ultrasound abnormalities. The present study showed that the chromosomal abnormality rate and clinical outcomes of fetuses with different types of ultrasound abnormalities varied greatly. Our data have important implications for prenatal genetic counseling for fetuses with different types of ultrasound abnormalities.

Confusion from prenatal diagnosis: Type C persistent fifth aortic arch or right-side ductus arteriosus.

We described a case of a double aortic arch (DAA) with a subaortic left brachiocephalic vein (LBCV) and right-side ductus arteriosus using high-definition (HD) flow render mode and spatiotemporal image correlation (STIC). We experienced uncertainty regarding this interesting case despite the diagnosis of right-sided ductus arteriosus. The ductus arteriosus originates from the right pulmonary artery (PA) and converges into the descending aorta (DAO), whereas the vessel originated from the PA and converged into the ascending aorta (AAO). Therefore, we assumed that the vessel connecting the PA to AAO may be a type-C persistent fifth aortic arch (PFAA).

Magnetic particle-based chemiluminescence immunoassay for serum human heart-type fatty acid binding protein measurement.

OBJECTIVES: Human heart-type fatty acid binding protein (HFABP) is a biomarker for diagnosis, risk assessment, and prognosis of acute myocardial infarction, and we aimed to establish an immunoassay for HFABP quantitation. METHODS: Human HFABP monoclonal antibodies (mAbs) were developed, evaluated by enzyme-linked immunosorbent assay, and a chemiluminescence enzyme immunoassay (CLEIA) generated. Analytical performance of the CLEIA was evaluated by measuring serum HFABP. RESULTS: The prokaryotically expressed rHFABP was purified and four anti-HFABP mAbs with superior detection performance were obtained after immunizing BALB/c mice. MAbs 2B8 and 6B3 were selected as respective capture and detection antibodies for HFABP measurement by CLEIA (detection range, 0.01-128 µg/L). Results using the CLEIA showed excellent correlation (r, 0.9622) and the correlation coefficient was 0.9809 (P < 0.05) by the Tukey test statistical analysis with those of latex-enhanced immunoturbidimetry in hospitals. CONCLUSION: Our mAbs and CLEIA for HFABP detection represent new diagnostic tools for measurement of human serum HFABP.

Benign Phyllodes Tumors: Comparison of Prognosis among Three Different Surgical Approaches.

Purpose: To evaluate the prognosis of patients with benign phyllodes tumors (PTs) treated by different surgical methods and to explore the influencing factors of local recurrence. Methods: We retrospectively analyzed 215 benign PTs from 193 patients who underwent surgery at Chinese PLA General Hospital between October 2008 and December 2020. We stratified our analysis according to surgical factors and explored the clinicopathological factors to influence local recurrence. Results: Among 193 patients, a total of 17 (8.8%, 17/193) recurred during follow-up. There were 89 patients in the US-VAE group, of whom 6 (6.7%) recurred; 8 of 57 patients (14%) in the local lumpectomy group recurred, while 3 of 47 patients (6.4%) in the extended lumpectomy group recurred (P=0.252). Multivariate logistic regression analysis showed that tumor diameter, mitosis, and history of breast myoma were independent risk factors for tumor recurrence (P=0.005, P=0.006, and P=0.004, respectively). The intraoperative blood loss, operation time, and scar length of the US-VAE group were shorter than those of the other two groups (P < 0.05). Conclusion: Negative surgical margins of benign PTs can obtain similar prognosis as negative surgical margins >10 mm. Therefore, we recommend that a follow-up observation policy be adopted for patients with unexpected benign PTs, rather than unnecessary open surgical resection. Patients' maximum tumor diameter, mitosis, and fibroadenoma history were independent predictors for recurrence of benign PTs.

Intraocular pressure effect of intravitreal conbercept injection for retinopathy of prematurity.

Purpose: Intravitreal injection of conbercept (IVC) is a novel anti-vascular endothelial growth factor (anti-VEGF) treatment for retinopathy of prematurity (ROP). This study aimed to assess the intraocular pressure (IOP) effect of IVC. Methods: All IVC surgeries were performed in the Department of Ophthalmology, Guangdong Women and Children Hospital, from January 2021 to May 2021. In this study, 30 eyes of 15 infants who received intravitreal injections of conbercept at a dose of 0.25 mg/0.025 mL were included. The IOP of all participants was measured prior to administering the injection and subsequently at 2 min, 1 h, 1 day, and 1 week thereafter. Results: We included 30 eyes (10 boys and 5 girls) with ROP. For the male group, the mean birth weight, mean gestational age at birth, and the mean time of postmenstrual age (PMA) at IVC treatment were 1,174.0 ± 446.0 g, 28.4 ± 3.0 weeks, and 37.1 ± 1.6 weeks, respectively; for the female group, they were 1,108 ± 285.5 g, 28.2 ± 2.5 weeks, and 36.8 ± 2.1 weeks, respectively. For the male group, the IOP at baseline, 2 min, 1 h, 1 day, and 1 week after IVC were 12.4 ± 1.5 mmHg, 49.0 ± 3.1 mmHg, 26.3 ± 2.5 mmHg, 13.4 ± 2.2 mmHg, and 11.6 ± 1.7 mmHg, respectively; for the female group, they were 10.7 ± 2.0 mmHg, 47.3 ± 3.2 mmHg, 26.4 ± 3.2 mmHg, 10.7 ± 1.8 mmHg, and 10.2 ± 1.8 mmHg, respectively. In both groups, the IOP immediately (2 min) after the operation was significantly higher than that at any other time point (p < 0.01). IOP values returned to the preoperative baseline level on the first day after surgery, with no significant difference compared with that before injection (p > 0.05). IOP continued to be maintained at the preoperative baseline level on the first week after surgery, with no significant difference compared with that before surgery (p > 0.05). Conclusion: Infants with ROP who received IVC experienced a sharp increase in the IOP immediately after injection, which decreased to below 30 mmHg after 1 h and maintain that level for 1 week or longer.

Preparation of monoclonal antibodies against Epstein-Barr virus glycoprotein 350.

Epstein-Barr virus (EBV) is the first identified human oncogenic herpesvirus infecting over 90% of the adults worldwide. However, the safe and effective prophylactic vaccine has not been licensed. The major glycoprotein 350 (gp350) on the EBV envelope is the main target for neutralizing antibodies, and gp350 (aa15-320) was used for the development of monoclonal antibodies in present study. The purified recombinant gp35015-320aa with an estimated molecular weight of 50 kDa was used to immunize six-week-old BALB/c mice, and the hybridoma cell lines that stably secreted monoclonal antibodies (mAbs) were obtained. The ability of developed mAbs for capturing and neutralizing EBV was evaluated, and mAb 4E1 presented better performance to block the infection of EBV in cell line Hone-1. The mAb 4E1 recognized the epitope. Its sequence of variable region genes (VH and VL) presented a unique identity which hadn't been reported. The developed mAbs might benefit the antiviral therapy and immunologic diagnosis for EBV infection.

[Chemical constituents of diterpenoids from Boswellia carterii].

This paper explored the chemical constituents of Boswellia carterii by column chromatography on silica gel, Sephadex LH-20, ODS column chromatography, and semi-preparative HPLC. The structures of the compounds were identified by physicochemical properties and spectroscopic data such as infrared radiation(IR), ultra violet(UV), mass spectrometry(MS), and nuclear magnetic resonance(NMR). Seven diterpenoids were isolated and purified from n-hexane of B. carterii. The isolates were identified as(1S,3E,7E,11R,12R)-11-hydroxy-1-isopropyl-4,8,12-trimethyl-15-oxabicyclo[10.2.1]pentadeca-3,7-dien-5-one(1),(1R,3S,4R,7E,11E)-4,8,12,15,15-pentamethyl-14-oxabicyclo[11.2.1]hexadeca-7,11-dien-4-ol(2), incensole(3),(-)-(R)-nephthenol(4), euphraticanoid F(5), dilospirane B(6), and dictyotin C(7). Among them, compounds 1 and 2 were new and their absolute configurations were determined by comparison of the calculated and experimental electronic circular dichroisms(ECDs). Compounds 6 and 7 were obtained from B. carterii for the first time.

Evolution of the germline mutation rate across vertebrates.

The germline mutation rate determines the pace of genome evolution and is an evolving parameter itself1. However, little is known about what determines its evolution, as most studies of mutation rates have focused on single species with different methodologies2. Here we quantify germline mutation rates across vertebrates by sequencing and comparing the high-coverage genomes of 151 parent-offspring trios from 68 species of mammals, fishes, birds and reptiles. We show that the per-generation mutation rate varies among species by a factor of 40, with mutation rates being higher for males than for females in mammals and birds, but not in reptiles and fishes. The generation time, age at maturity and species-level fecundity are the key life-history traits affecting this variation among species. Furthermore, species with higher long-term effective population sizes tend to have lower mutation rates per generation, providing support for the drift barrier hypothesis3. The exceptionally high yearly mutation rates of domesticated animals, which have been continually selected on fecundity traits including shorter generation times, further support the importance of generation time in the evolution of mutation rates. Overall, our comparative analysis of pedigree-based mutation rates provides ecological insights on the mutation rate evolution in vertebrates.

Isoquinoline alkaloids from Corydalis edulis Maxim. Exhibiting insulinotropic action.

Eleven undescribed isoquinoline analogues, namely edulisines A-K, along with sixteen known alkaloids, were isolated from the whole plants of Corydalis edulis. The structures of the isolated alkaloids were established on the basis of extensive spectroscopic data (1D and 2D NMR, UV, IR, and HRESIMS). Their absolute configurations were determined by single-crystal X-ray crystallographic analysis and ECD. Compounds (+)-1 and (-)-1 are a pair of undescribed isoquinoline alkaloids bearing a unique coupled pattern of coptisine and ferulic acid via Diels-Alder [4 + 2] cycloaddition, while compounds (+)-2 and (-)-2 feature benzo [1,2-d:3,4-d]bis [1,3]dioxole moiety. Compounds (+)-2, (-)-2, (-)-5, 10, 13, 15, 20, 22, and 23 significantly triggered the secretion of insulin in the HIT-T15 cells at a concentration of 40 µM.

Fas/FasL and Complement Activation are Associated with Chronic Active Epstein-Barr Virus Hepatitis.

Background and Aims: Chronic active Epstein-Barr virus hepatitis (CAEBVH) is a rare and highly lethal disease characterized by hepatitis and hepatomegaly. This study aimed to investigate the clinicopathological features and pathogenic mechanisms of CAEBVH. Methods: Ten patients with confirmed Epstein-Barr virus hepatitis infection were enrolled. The clinicopathological characteristics of these patients were summarized and analyzed. Flow cytometry was utilized to detect peripheral blood immune cell phenotypes and whole exome sequencing was used to explore pathogenic genetic mechanisms. Lastly, immunohistochemical staining was employed to verify pathogenic mechanisms. Results: Clinical features observed in all Epstein-Barr virus hepatitis patients included fever (7/10), splenomegaly (10/10), hepatomegaly (9/10), abnormal liver function (8/10), and CD8+ T cell lymphopenia (6/7). Hematoxylin and eosin staining revealed lymphocytic infiltration in the liver. Positive Epstein-Barr virus-encoded small RNA in-situ hybridization (EBER-ISH) of lymphocytes of liver tissues was noted. Whole exome sequencing indicated that cytotoxic T lymphocytes and the complement system were involved. The expression of CD8, Fas, FasL, and Caspase-8 expression as well as apoptotic markers was enhanced in the Epstein-Barr virus hepatitis group relative to the controls (p<0.05). Lastly, Complement 1q and complement 3d expression, were higher in CAEBVH patients relative to controls (p<0.05). Conclusions: CAEBVH patients developed fever, hepatosplenomegaly, and lymphadenopathy. Histopathological changes were a diffuse lymphocytic sinusoidal infiltrate with EBER-ISH positivity. Fas/FasL and complement activation were involved in CAEBVH patients.

Gastroprotective 2-(2-phenylethyl)chromone-sesquiterpene hybrids from the resinous wood of Aquilaria sinensis (Lour.) Gilg.

Six previously unprecedented 2-(2-phenylethyl)chromone-sesquiterpene hybrids, aquisinenins A-F (1 - 6), were isolated from the resinous wood of Aquilaria sinensis by a LC-MS-guided fractionation procedure. Their structures were determined by extensive spectroscopic analysis (1D and 2D NMR, UV, IR, and HRMS) and experimental and computed ECD data. Compounds 1 - 6 were rare dimeric 2-(2-phenylethyl)chromone-sesquiterpene derivatives featuring 5,6,7,8-tetrahydro-2-(2-phenylethyl)chromone hybridized with different sesquiterpene (eudesmane/guaiane type) moieties via ester bond. Furthermore, all the isolated compounds were evaluated for their protective effects on taurocholic acid (TCA)-induced GES-1 cell injury. The most effective aquisinenin F (6) was used to elucidate the involved mechanism on protection against TCA-induced gastric mucosal damage. Our results indicated that 6 protected against gastric mucosal cell insult by downregulation of the ER stress triggered by TCA.

Clinical Efficacy of Nutritional Intervention Combined with Muscle Exercise on Sarcopenia Patients with Femoral Fracture: A Pilot Randomized Controlled Trial.

Objective: To study the clinical efficacy of nutritional intervention combined with muscle exercise on sarcopenia patients with femoral fracture. Methods: From January 2019 to January 2021, a total of 100 sarcopenia patients with femoral fracture were included in this study and were divided into a control group (routine postoperative care) and a research group (nutritional intervention and muscle exercise), 50 cases in each group. Primary clinical outcomes included sarcopenia-related indicators and functional independence assessed by activities of daily living scale (ADL). Secondary clinical outcomes included time of fracture healing and hospital stay, pain score as assessed by visual analogue scale (VAS), and nursing satisfaction. Results: Before the intervention, there was no significant difference in the indicators of sarcopenia and the indicators of functional independence assessed by ADL between the two groups (P > 0.05). After 3 months of intervention, the BMI, grip strength, calf circumference, pace, and body muscle rate of the patients in the research group were significantly higher than those in the control group (P < 0.05), while body fat rates were significantly lower than those in the control group (P < 0.05), and the capability of eating, walking, bathing, and doing housework in research group were all significantly higher than those in control group (P < 0.05). In addition, the time of fracture healing and hospital stay in research group were all significantly lower than those in control group (P < 0.05), and the VAS scores of the control group at each time point after intervention were significantly higher than those of the research group (P < 0.05). The nursing satisfaction of the patients in the research group was significantly higher than that in the control group (94.00% vs. 76.00%, P < 0.05). Conclusion: Nutritional intervention combined with muscle exercise can help improve sarcopenia symptoms and promote fracture recovery in patients with sarcopenic femoral fractures.

Anti-inflammatory flavonoid derivatives from the heartwood of Dalbergia odorifera T. Chen.

Two pairs of flavonoid enantiomers (1a/1b and 2a/2b) together with three known analogues (3-5) were isolated from the heartwood of Dalbergia odorifera T. Chen. Their structures were elucidated by extensive spectroscopic analysis (1 D and 2 D NMR, UV, IR, and HRMS) and experimental and calculated ECD data. Compound 2 features an unusual 2-methyl-3(2H)-furanone moiety forming the C-ring of flavonoid, and its putative biosynthetic pathway is also proposed. Compounds 3‒5 exhibited significant inhibition of nitric oxide production in lipopolysaccharide-stimulated RAW264.7 cells with IC50 values of 14.7 ± 0.3 µM, 40.2 ± 1.1 µM, and 3.2 ± 0.1 µM, respectively.

Photo-sonodynamic therapy mediated with OLI_NPs to induce HPV16E7-specific immune response and inhibit cervical cancer in a Tc-1-grafted murine model.

Cervical carcinoma is the fourth most common gynecological cancer. Here we reported the synthesis of oxygen-carried and lipopolysaccharide (LPS)/ indocyanine green (ICG)-loaded nanoparticles (OLI_NPs) for photo-sonodynamic therapy (PSDT) mediated combination therapy to induce systemic antitumor immune responses. We effectively built a new nanoparticle system, a multifunctional nanoagent that integrated the ability of dual-model imaging and therapy for tumors. In this study, we confirmed that OLI_NPs can act as a multifunctional platform that enables not only to diagnose tumors conveniently but also to efficiently provide treatment of in situ tumors, permitting simultaneous dual-mode imaging and localization of the therapy in combination with PSDT-mediated drug release. Furthermore, our combined strategy could effectively depress the tumor development and extend mouse life by the combination of inducing immunogenic cell death (ICD) with encapsulated LPS. In conclusion, combining therapy of OLI_NPs plus PSDT can induce anti-tumor immune responses and tumor antigen-specific immunity in a common TC-1 graft tumor model. Therefore, this combination therapy is a viable technique for cervical cancer treatment.