The study of DIEA three dimensional reconstruction technique based on CTA in preoperative perforator selection.

Preoperative selection of perforator is one of the key steps for successful surgery. The purpose of this study is to simulate the selection process of the perforator of the flap using the 3D models of the deep inferior epigastric artery (DIEA). A retrospective study was performed of women who underwent deep inferior epigastric perforator flap breast reconstruction from January 2011 to July 2021. Construct 3D models of the DIEA using computerized tomography angiography images, and then computational fluid dynamics simulations were performed. Correlation and regression analyses were used to analyze the geometric and hemodynamic parameters. Statistical analysis suggested that the outlet flow was positively correlated with the inlet area (r = 0.338, p = 0.000), outlet area (r = 0.840, p = 0.000), the average radius of the perforator (r = 0.592, p = 0.000), and negatively correlated with the length of perforator(r = -0.210, p = 0.024). The results of linear regression analysis showed that the outlet area (p = 0.000), the average radius (p = 0.000), and the length (p = 0.044) of the perforator were the influencing factors on outlet flow. In multiple perforators analysis, there was a significant difference in the total outlet flow among single perforator, double perforators, and triple perforators (p = 0.002). The successful implementation of this experiment provides a new approach for the selection of dominant perforators in the future.

Construction of a Quantum-Dot-Based FRET Nanosensor through Direct Encoding of Streptavidin-Binding RNA Aptamers for N6-Methyladenosine Demethylase Detection.

N6-Methyladenosine (m6A) demethylases can catalyze the removal of the methyl modification on m6A, and it is closely associated with the occurrence, proliferation, differentiation, and metastasis of malignancies. The m6A demethylases (e.g., fat mass and obesity-associated protein (FTO)) may act as a cancer biomarker and are crucial for anticancer drug screening and early clinical diagnosis. Herein, we demonstrate the construction of a quantum-dot-based Förster resonance energy-transfer (FRET) nanosensor through direct encoding of streptavidin-binding RNA aptamers (SA aptamers) for m6A demethylase detection. This nanosensor employs multiple Cy5-molecule-labeled SA aptamers as the building materials to construct the 605QD-RNA-Cy5 nanoassembly, and it exploits the hinder effect of m6A upon elongation and ligation reactions to distinguish m6A-containing RNA probes from demethylated RNA probes. When m6A demethylase is present, the m6A-containing RNA probes are demethylated to generate the demethylated RNA probes, initiating strand extension and ligation reactions to yield a complete transcription template for SA aptamers. Subsequently, a T7-assisted cascade transcription amplification reaction is activated to transcribe abundant SA aptamers with the incorporation of multiple Cy5 fluorophores. The Cy5-incorporated SA aptamers can self-assembly onto the streptavidin-coated 605QD surface to obtain the 605QD-SA aptamer-Cy5 nanoassemblies, resulting in the generation of distinct FRET signals. This nanosensor exhibits ultrahigh sensitivity and excellent specificity, and it can detect endogenous FTO at the single-cell level. Furthermore, this nanosensor can precisely measure enzyme kinetic parameters, screen m6A demethylase inhibitors, and differentiate the FTO expression between breast cancer patients and healthy individual tissues, offering a versatile platform for clinical diagnostic and drug discovery.

Corn-Based Fluorescent Light-Up Biosensors with Improved Signal-to-Background Ratio for Label-Free Detection of Long Noncoding RNAs.

Long noncoding RNAs (lncRNAs) play pivotal roles in multifarious physiological and pathological processes, and their aberrant expression may disturb the normal regulatory network of gene expression to induce diverse human diseases. Herein, we construct a fluorescent light-up biosensor with a low background for label-free detection of lncRNAs by coupling duplex-specific nuclease (DSN)-assisted target recycling amplification with transcription-driven synthesis of fluorogenic RNA aptamer-Corns. We design two linear probes, including a capture probe for initiating a cyclic cleavage reaction and a linear template for transcribing RNA aptamer-Corn. Target lncRNA is recognized by capture probes assembled on magnetic bead (MB) surfaces to trigger a DSN-assisted cyclic cleavage reaction, releasing abundant T7 promoter sequences. After magnetic separation, free T7 promoter hybridizes with a linear template to induce efficient transcription amplification with the assistance of T7 RNA polymerase, producing numerous fluorogenic RNA aptamer-Corns that can light up small-molecule fluorogens 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime (DFHO). Notably, the introduction of MBs facilitates both the separation of cleaved capture probes and the enrichment/isolation of target lncRNAs from the complex biological matrix. Benefiting from the high efficiency of DSN/T7 RNA polymerase-mediated cascade amplification and high signal-to-background ratio of the Corn-DFHO complex, this biosensor is capable of sensitively quantifying lncRNA with a detection limit of 31.98 aM. Moreover, it can precisely quantify lncRNA at the single-cell level and even in complex biological samples, and it can differentiate tumor cells from normal cells. Importantly, this Corn-based biosensor is readily extended to detect other lncRNAs by altering capture probe sequences, opening a new avenue for molecular diagnosis.

MnO2 nanosheet-mediated generalist probe: Cancer-targeted dual-microRNAs detection and enhanced CDT/PDT synergistic therapy.

While integrated nanoplatform for diagnosis and therapy has received much recent interest, its widespread application has been hampered by the complicated preparation process, high-cost and low-efficacy. Herein, we designed a MnO2 nanosheet-mediated generalist probe (MNSGP), for intracellular dual-microRNAs (miRNAs) imaging and enhanced synergistic therapy of chemodynamic therapy (CDT) and photodynamic therapy (PDT). Because MNSGP can specifically target nucleolin receptor overexpressed on the cancer cell surface, it can be internalized via a receptor-mediated endocytosis pathway. After entering the cells, MnO2 NS was degraded to Mn2+ by the excessive glutathione (GSH), releasing the DNA probes for cyclic amplification detection of miR-155 and miR-21 based on toehold-mediated strand displacement amplification (TSDA). Meanwhile, the produced O2 by MnO2 NS catalysis can promote the photosensitizer TMPyP4 to produce singlet oxygen (1O2) for PDT. The degraded Mn2+, as Fenton reagent, can convert endogenous H2O2 to cytotoxic hydroxyl radical (·OH) for CDT. In addition, the depletion of GSH impairs the antioxidant defense system (ADS), enhancing the CDT/PDT synergistic effect. The prepared generalist probe was fully characterized. Accuracy of dual-miRNAs detection and the high curative effect of enhanced CDT/PDT synergistic therapy were attested via in vitro and in vivo experiments. Unarguably, MNSGP broadens new horizons in the design of nucleic acid nanoplatform, cancer-targeted detection and theranostic application.

Deciphering the effective combinatorial components from Si-Miao-Yong-An decoction regarding the intervention on myocardial hypertrophy.

ETHNOPHARMACOLOGICAL RELEVANCE: Si-Miao-Yong-An decoction (SMYAD), a classical traditional Chinese medicine (TCM) formula, has been used to treat various cardiovascular diseases in clinics. AIM OF THE STUDY: The aim of this study is to investigate the effective combinatorial components from SMYAD and its mechanism regarding the intervention on myocardial hypertrophy. MATERIALS AND METHODS: SMYAD constituents absorbed in rat plasma and heart were identified using UHPLC Q-Exactive-Orbitrap MS/MS. The identified constituents in SMYAD were further analyzed using ADMET (absorption, distribution, metabolism, excretion and toxicity) prediction and molecular docking. The effective constituents were identified using isoproterenol (ISO)-induced H9c2 cardiomyocyte hypertrophy, and neochlorogenic acid (NCA), chlorogenic acid (CA), cryptochlorogenic acid (CCA), isochlorogenic acid C (ICAC), angoroside C (AGDC), isochlorogenic acid A (ICAA), sweroside (SRD), and harpagide (HPD) in SMYAD extract were quantified by HPLC for compatibility. Finally, anti-hypertrophic activities of candidate effective combinatorial components, which were prepared according to the determined molar concentration ratio of effective constituents using reference substance solution, were analyzed using immunofluorescence staining and Quantitative real-time PCR. The expression levels of PI3Kα, p-ERK, p-Akt, Akt, p-mTOR, mTOR and HIF-1α were measured using Western blot. RESULTS: 32 prototypes of SMYAD were identified from plasma and heart tissue of rat. Combining with ADMET prediction, 31 dominant constituents were focused. Based on HIF-1 pathway identified in preliminary result, 17 targets were focused, which were used to dock with 31 constituents. 27 constituents were therefore hit as the potential effective constituents of SMYAD in inhibiting myocardial hypertrophy. Bioactivity evaluation showed that NCA, CA, CCA, ICAC, AGDC, ICAA, SRD, and HPD significantly inhibited the increase of H9c2 cell surface area induced by ISO. Except for ICAA and AGDC, the remaining 6 effective constituents, showing a certain inhibitory effect on ISO-induced ANP mRNA overexpression at high and low concentrations, participated in compatibility based on the molar concentration ratio determined by HPLC. Effective combinatorial components composed of the 6 effective constituents (effective combinatorial components ABC) showed significant inhibitory effect on the increase of cell surface area, and the overexpression of ANP and ß-MHC mRNA in H9c2 cells induced by ISO. Moreover, effective combinatorial components ABC significantly inhibited the protein overexpressions of p-Akt, p-mTOR and HIF-1α. Based on the results, we put forward the strategy of "Focusing constituents" and "Focusing targets" for the effective constituents research of TCM formula. CONCLUSION: Effective combinatorial components ABC composed of NCA, CA, CCA, ICAC, SRD and HPD from SMYAD inhibited ISO-induced cardiomyocyte hypertrophy and down-regulated expression of ANP and ß-MHC mRNA through the inactivation of Akt/mTOR/HIF-1α pathway.

Long noncoding RNA HULC in acute ischemic stroke: Association with disease risk, severity, and recurrence-free survival and relation with IL-6, ICAM1, miR-9, and miR-195.

BACKGROUND: This study aimed to evaluate the clinical role of long noncoding RNA (lncRNA) HULC in acute ischemic stroke (AIS). METHODS: LncRNA HULC in plasma samples from 215 first episode AIS patients and 215 age/gender-matched non-AIS controls was detected by reverse transcriptional-quantitative polymerase chain reaction (RT-qPCR). Then, in AIS patients, interleukin-6 and intercellular adhesion molecule 1 (ICAM1), as well as microRNA (miR) target of lncRNA HUCL (miR-9 and miR-195), were detected by enzyme-linked immunosorbent assay and RT-qPCR, respectively. Disease severity was assessed by National Institution of Health stroke scale (NIHSS) score. AIS recurrence or death was recorded, and recurrence-free survival (RFS) was calculated. RESULTS: LncRNA HULC was increased in AIS patients compared to non-AIS controls (P < .001), and receiver operating characteristic curve showed that it was correlated with increased AIS risk (area under curve: 0.876, 95% confidence interval: 0.843-0.908). Meanwhile, lncRNA HULC was positively correlated with NIHSS score (P < .001, r = .456), interleukin-6 (P < .001, r = .275) and ICAM1 (P < .001, r = .383), whereas negatively correlated with miR-9 (P < .001, r = -.438) but not miR-195 (P = .205, r = -.087) in AIS patients. Additionally, miR-9 was negatively correlated with NIHSS score (P < .001, r = -.335), interleukin-6 (P = .001, r = -.231), and ICAM1 (P < .001, r = -.280), while miR-195 was only negatively associated with NIHSS score (P = .041, r = -.139) in AIS patients. Moreover, lncRNA HULC high expression predicted worse RFS (P = .013) in AIS patients. CONCLUSION: LncRNA HULC is correlated with higher AIS risk, increased disease severity and worse prognosis in AIS patients. Meanwhile, it associates with higher IL-6, elevated ICAM1, and lower miR-9 AIS patients.

[CRISPR/Cas9-based knockout of GPR43 gene in RAW264.7 cells inhibits their phagocytosis to Klebsiella pneumoniae].

Objective To construct cell line RAW264.7 with stable knockout of GPR43 gene using CRISPR/Cas9 system, and explore the role and mechanism of GPR43 gene in Klebsiella pneumoniae infection. Methods Three pairs of small-guide RNA (sgRNA) targeting the GPR43 gene were designed and inserted into plasmid pLenticrisprV2. The recombinant plasmid pLenticrisprV2 containing sgRNA was packaged using a lentivirus packaging system. RAW264.7 cells were transfected with viruses, and monoclonal cells were screened using puromycin. The genomic DNA was extracted from the amplified monoclonal cells. The GPR43 gene-related sequences were sequenced and compared with the wild-type GPR43 gene to confirm the cell line with successful knockout (GPR43-/- RAW264.7 cells). The expression of GPR43 protein was detected by Western blotting. After GPR43-/- RAW264.7 cells were transfected with Klebsiella pneumoniae, the changes in the expression of interleukin-1ß (IL-1ß), IL-6 and tumor necrosis factor-α (TNF-α) in the cells were detected using real-time quantitative PCR. Additionally, the phagocytic capacity of RAW264.7 cells after GPR43 knockout was observed. Results Western blotting confirmed that GPR43 protein was not expressed in the selected monoclonal cells, and DNA sequencing showed that 34 bases were missing at the insertion site of sgRNA, which proved that GPR43 gene was successfully knocked out. After GPR43-/- RAW264.7 cells were transfected with Klebsiella pneumoniae, the levels of IL-1ß, IL-6 and TNF-α expression in the cells were all lower than those in the control group, and the phagocytic capacity of GPR43-/- RAW264.7 cells to Klebsiella pneumoniae decreased. Conclusion CRISPR/Cas9-based knockout of GPR43 gene in RAW264.7 cells can inhibit their phagocytosis for Klebsiella pneumoniae and production of inflammatory cytokines.

Morusalones A-D, Diels-Alder Adducts with 6/7/6/6/6/6 Hexacyclic Ring Systems as Potential PTP1B Inhibitors from Cell Cultures of Morus alba.

Morusalones A-D (1-4), a new class of Diels-Alder adducts featuring unprecedented 6/7/6/6/6/6 hexacyclic core skeletons with a unique bridged cycloheptenone ring, were isolated from Morus alba cell cultures. The biosyntheses for 1-4 were proposed through an unusual Diels-Alder cycloaddition with quinostilbenes as dienophiles and prenyl 2-phenylbenzofuran as a diene to yield the typical methylhexene unit and a rare intramolecular nucleophilic addition to form the cycloheptenone ring. Compounds 1-4 exhibited protein tyrosine phosphatase 1B inhibitory activity.

Chlorogenic acid: A potent molecule that protects cardiomyocytes from TNF-α-induced injury via inhibiting NF-κB and JNK signals.

The traditional Chinese herb Lonicerae Japonicae Flos has shown significant clinical benefits in the treatment of heart failure, but the mechanism remains unclear. As the main active ingredient found in the plasma after oral administration of Lonicerae Japonicae Flos, chlorogenic acid (CGA) has been reported to possess anti-inflammatory, anti-oxidant and anti-apoptosis function. We firstly confirmed the cardioprotective effects of CGA in transverse aortic constriction (TAC)-induced heart failure mouse model, through mitigating the TNF-α-induced toxicity. We further used TNF-α-induced cardiac injury in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to elucidate the underlying mechanisms. CGA pre-treatment could reverse TNF-α-induced cellular injuries, including improved cell viability, increased mitochondrial membrane potential and inhibited cardiomyocytes apoptosis. We then examined the NF-κB/p65 and major mitogen-activated protein kinases (MAPKs) signalling pathways involved in TNF-α-induced apoptosis of hiPSC-CMs. Importantly, CGA can directly inhibit NF-κB signal by suppressing the phosphorylation of NF-κB/p65. As for the MAPKs, CGA suppressed the activity of only c-Jun N-terminal kinase (JNK), but enhanced extracellular signal-regulated kinase1/2 (ERK1/2) and had no effect on p38. In summary, our study revealed that CGA has profound cardioprotective effects through inhibiting the activation of NF-κB and JNK pathway, providing a novel therapeutic alternative for prevention and treatment of heart failure.

Iron encapsulated in 3D N-doped carbon nanotube/porous carbon hybrid from waste biomass for enhanced oxidative activity.

Novel iron encapsulated in nitrogen-doped carbon nanotubes (CNTs) supported on porous carbon (Fe@N-C) 3D structured materials for degrading organic pollutants were fabricated from a renewable, low-cost biomass, melamine, and iron salt as the precursors. SEM and TEM micrographs show that iron encapsulated bamboo shaped CNTs are vertically standing on carbon sheets, and thus, a 3D hybrid was formed. The catalytic activities of the prepared samples were thoroughly evaluated by activation of peroxymonosulfate for catalytic oxidation of Orange II solutions. The influences of some reaction conditions (pH, temperature, and concentrations of reactants, peroxymonosulfate, and dye) were extensively evaluated. It was revealed that the adsorption could enrich the pollutant which was then rapidly degraded by the catalytically generated radicals, accelerating the continuous adsorption of residual pollutant. Remarkable carbon structure, introduction of CNTs, and N/Fe doping result in promoted adsorption capability and catalytic performances. Due to the simple synthetic process and cheap carbon precursor, Fe@N-C 3D hybrid can be easily scaled up and promote the development of Fenton-like catalysts.

Regulation of L-type Ca2+ Channel Activity and Insulin Secretion by Huntingtin-associated Protein 1.

Huntingtin-associated protein 1 (Hap1) was originally identified as a protein that binds to the Huntington disease protein, huntingtin. Growing evidence has shown that Hap1 participates in intracellular trafficking via its association with various microtubule-dependent transporters and organelles. Recent studies also revealed that Hap1 is involved in exocytosis such as insulin release from pancreatic ß-cells. However, the mechanism underlying the action of Hap1 on insulin release remains to be investigated. We found that Hap1 knock-out mice had a lower plasma basal insulin level than control mice. Using cultured pancreatic ß-cell lines, INS-1 cells, we confirmed that decreasing Hap1 reduces the number of secreted vesicles and inhibits vesicle exocytosis. Electrophysiology and imaging of intracellular Ca2+ measurements demonstrated that Hap1 depletion significantly reduces the influx of Ca2+ mediated by L-type Ca2+ channels (Cav). This decrease is not due to reduced expression of Cav1.2 channel mRNA but results from the decreased distribution of Cav1.2 on the plasma membrane of INS-1 cells. Fluorescence recovery after photobleaching showed a defective movement of Cav1.2 in Hap1 silencing INS-1 cells. Our findings suggest that Hap1 is important for insulin secretion of pancreatic ß-cells via regulating the intracellular trafficking and plasma membrane localization of Cav1.2, providing new insight into the mechanisms that regulate insulin release from pancreatic ß-cells.

Anti-inflammatory dimeric furanocoumarins from the roots of Angelica dahurica.

Seven new dimeric furanocoumarins, dahuribiethrins A-G (1-7), were isolated from the roots of Angelica dahurica. Their structures were determined by chemical derivatization and extensive spectroscopic techniques, including (1)H NMR, (13)C NMR, HSQC, (1)H-(1)H COSY, HMBC, and NOESY experiments. Compounds 2, 3, 4, and 5 exhibited significant inhibition of nitric oxide production in the lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells with IC50 values in the range of 8.8-9.8 µM.