Integrated analysis of the relation to tumor immune microenvironment and predicted value of Stonin1 gene for immune checkpoint blockage and targeted treatment in kidney renal clear cell carcinoma.

BACKGROUND: Stonin1 (STON1) is an endocytic protein but its role in cancer remains unclear. Here, we investigated the immune role of STON1 in kidney renal clear cell carcinoma (KIRC). METHODS: We undertook bioinformatics analyses of the expression and clinical significance of STON1 in KIRC through a series of public databases, and the role of STON1 in the tumor microenvironment and the predictive value for immunotherapy and targeted treatment in KIRC were identified with R packages. STON1 expression was validated in clinical KIRC tissues as well as in KIRC and normal renal tubular epithelial cells. RESULTS: Through public databases, STON1 mRNA was found to be significantly downregulated in KIRC compared with normal controls, and decreased STON1 was related to grade, TNM stage, distant metastasis and status of KIRC patients. Compared with normal controls, STON1 was found to be downregulated in KIRC tissues and cell lines. Furthermore, OncoLnc, Kaplan-Meier, and GEPIA2 analyses also suggested that KIRC patients with high STON1 expression had better overall survival. The high STON1 group with enriched immune cells had a more favorable prognosis than the low STON1 group with decreased immune cells. Single sample Gene Set Enrichment Analysis and Gene Set Variation Analysis indicated that STON1 creates an immune non-inflamed phenotype in KIRC. Moreover, STON1 was positively associated with mismatch repair proteins and negatively correlated with tumor mutational burden. Furthermore, Single sample Gene Set Enrichment Analysis algorithm and Pearson analysis found that the low STON1 group was more sensitive to immune checkpoint blockage whereas the high STON1 group was relatively suitable for targeted treatment. CONCLUSIONS: Decreased STON1 expression in KIRC leads to clinical progression and poor survival. Mechanically, low STON1 expression is associated with an aberrant tumor immune microenvironment. Low STON1 is likely to be a favorable indicator for immunotherapy response but adverse indicator for targeted therapeutics in KIRC.

Simultaneous detection of four pesticides in agricultural products by a modified QuEChERS method and LC-MS/MS.

A modified QuEChERS pretreatment method and LC-MS/MS technique were performed to simultaneously determine four pesticide (Hexachlorophene, Dinex, Dinosam, Dinoterb) residues in agricultural products. Through the optimization of LC-MS/MS detection parameters in SIM mode, the optimal instrument conditions are obtained. The modified QuEChERS method was used to pretreat the samples. Solid phase extractants PSA, C18 and GCB were used for sample purification. The research results showed that the correlation coefficients of the four pesticides were all greater than 0.991, which had a good linear relationship. The limits of quantitation (LOQ) of the four pesticides were 0.05-0.56 µg/kg. The recoveries were 70.51-113.20% with relative standard deviations (RSDS) of 1.6-11.2%. The developed method can provide reliable data support for the subsequent simultaneous detection of these four pesticides.

Electron-Deficient Polycyclic Molecules via Ring Fusion for n-Type Organic Electrochemical Transistors.

The primary challenge for n-type small-molecule organic electrochemical transistors (OECTs) is to improve their electron mobilities and thus the key figure of merit µC*. Nevertheless, few reports in OECTs have specially proposed to address this issue. Herein, we report a 10-ring-fused polycyclic π-system consisting of the core of naphthalene bis-isatin dimer and the terminal moieties of rhodanine, which features intramolecular noncovalent interactions, high π-delocalization and strong electron-deficient characteristics. We find that this extended π-conjugated system using the ring fusion strategy displays improved electron mobilities up to 0.043 cm2 V-1 s-1 compared to our previously reported small molecule gNR, and thereby leads to a remarkable µC* of 10.3 F cm-1 V-1 s-1 in n-type OECTs, which is the highest value reported to date for small-molecule OECTs. This work highlights the importance of π-conjugation extension in polycyclic-fused molecules for enhancing the performance of n-type small-molecule OECTs.

Radiosynthesis of a novel antisense imaging probe targeting LncRNA HOTAIR in malignant glioma.

BACKGROUND: Long non-coding RNA (LncRNA) HOTAIR was amplified and overexpressed in many human carcinomas, which could serve as a useful target for cancer early detection and treatment. The 99mTc radiolabeled antisense oligonucleotides (ASON) could visualize the expression of HOTAIR and provide a diagnostic value for malignant tumors. The aim of this study was to evaluate whether liposome-coated antisense oligonucleotide probe 99mTc-HYNIC-ASON targeting HOTAIR can be used in in vivo imaging of HOTAIR in malignant glioma xenografts. METHODS: The ASON targeting LncRNA HOTAIR as well as mismatched ASON (ASONM) were designed and modified. The radiolabeling of 99mTc with two probes were via the conjugation of bifunctional chelator HYNIC. Then probes were purified by Sephadex G25 and tested for their radiolabeling efficiency and purity, as well as stability by ITLC (Instant thin-layer chromatography) and gel electrophoresis. Then the radiolabeled probes were transfected with lipofectamine 2000 for cellular uptake test and the next experimental use. Furthermore, biodistribution study and SPECT imaging were performed at different times after liposome-coated 99mTc-HYNIC-ASON/ASONM were intravenously injected in glioma tumor-bearing mice models. All data were analyzed by statistical software. RESULTS: The labeling efficiencies of 99mTc-HYNIC-ASON and 99mTc-HYNIC-ASONM measured by ITLC were (91 ± 1.5) % and (90 ± 0.6) %, respectively, and both radiochemical purities were more than 89%. Two probes showed good stability within 12 h. Gel electrophoresis confirmed that the oligomers were successfully radiolabeled no significant degradation were found. Biodistribution study demonstrated that liposome-coated antisense probes were excreted mainly through the kidney and bladder and has higher uptake in the tumor. Meanwhile, the tumor was clearly shown after injection of liposome coated 99mTc-HYNIC-ASON, and its T/M ratio was higher than that in the non-transfection group and mismatched group. No tumor was seen in mismatched and blocking group. CONCLUSION: The liposome encapsulated 99mTc-HYNIC-ASON probe can be used in the in vivo, real-time imaging of LncRNA HOTAIR expression in malignant glioma.

Occurrence, Molecular Characterization, and Antibiotic Resistance of Cronobacter sakazakii in Goat Milk-Based Infant Formula from Shaanxi Province, China.

This study aimed to investigate the prevalence of Cronobacter sakazakii in goat milk-based infant formula (GIF) collected from Shaanxi Province, China, and reveal the molecular characterization and antibiotic resistance profile of these isolates. A total of 750 GIF samples were collected from the retail markets in 5 cities in Shaanxi Province from February 2019 to February 2021. Molecular characterization was investigated using multilocus sequence typing and O-antigen serotyping. Antibiotic resistance of C. sakazakii isolates was assessed using antimicrobial susceptibility testing. Thirty-two strains of C. sakazakii were isolated from GIF samples with a prevalence rate of 4.27% and were divided into 16 sequence types (STs); among them, ST4 (6/32, 18.75%) and ST21 (5/32, 15.63%) were dominant. Five C. sakazakii serotypes (O2, O1, O7, O4, and O3) were detected, and C. sakazakii serotype O2 (15/32, 46.88%) was the main. Of the 21 antimicrobials, isolates showed higher resistance against cephalothin (87.5%), amoxicillin (25%), azithromycin (18.75%), oxytetracycline (18.75%), ampicillin (12.5%), and streptomycin (12.5%). In addition, three isolates were found to be resistant to three antimicrobials. These findings revealed the potential epidemiological risk and characterization of C. sakazakii in GIF from Shaanxi Province, China, and provided reference data for the effective prevention and control of C. sakazakii in powdered infant formula.

Antibacterial Effect of Chrysanthemum Buds' Crude Extract Against Salmonella Typhimurium and Potential Application in Cooked Chicken.

The objective of this study was to clarify the antibacterial activity and mechanism of Chrysanthemum buds' crude extract (CBCE) against Salmonella Typhimurium, and explore the potential application in cooked chicken. The zone of inhibition (ZI), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) were used to assess the in vitro antibacterial activity of CBCE against Salmonella Typhimurium. The antibacterial mechanism was elucidated by revealing the changes in intracellular adenosine 5'-triphosphate (ATP) concentration, membrane potential, content of biomacromolecule, and cell morphology. Furthermore, the effect of CBCE on the counts of Salmonella Typhimurium and color of cooked chicken during storage was studied. The results showed that the ZI, MIC, and MBC of CBCE against Salmonella Typhimurium were 12.9 ± 0.53-13.6 ± 0.14 mm, 40, and 80 mg/mL, respectively. In the process of inhibiting Salmonella Typhimurium by CBCE, the reduction of intracellular ATP concentration, cell membrane depolarization, leakage of protein and nucleic acid, and destruction of cell morphology were observed. Moreover, after treatments with CBCE, the growth of Salmonella Typhimurium in cooked chicken was significantly inhibited (p < 0.05) compared with the control group. No significant differences (p > 0.05) in lightness (L*), redness (a*), and yellowness (b*) values of cooked chicken were found between untreated and treated samples, as well as the color of cooked chicken treated with CBCE did not change significantly (p > 0.05) during the six days of storage. Overall, our findings suggested that CBCE exhibited the antibacterial effect against Salmonella Typhimurium, and had the potential to be used as a natural food preservative for the control of Salmonella Typhimurium in chicken products.

The Chemistry and Applications of Heteroisoindigo Units as Enabling Links for Semiconducting Materials.

ConspectusBecause of their low-temperature processing properties and inherent mechanical flexibility, semiconducting materials are promising candidates for enabling flexible displays, renewable energy, biological sensors, and healthcare. Progress has been made in materials performance by developing judicious materials design strategies. For example, improvements in electron transport have required new electron-deficient aromatics. Among them, isoindigo (IID) is an important functional group utilized in conjugated aromatics, where the structure combines two sets of five-membered electron-withdrawing lactam rings, exhibiting enhanced solubility, excellent chemical and thermal stabilities, broad absorption, and intriguing electron affinity.In the past decade, researchers have mainly focused on IID-based materials. However, the effect of heteroatom modification of the IID core has rarely been systemically investigated. In conventional conjugated polymers, single bonds connect the monomers, leading to energetic disorder and torsion defects, while ladder-type polymers are often intractable because of their fused nature. In this regard, the molecular design of new π scaffolds based on IID is central to the development of high-performance semiconductor polymers. Especially, a complete refresh of molecular design strategies and novel conjugated polymers with unique structures are needed to circumvent the disadvantages of the conventional ladder-type polymers.In this Account, we systematically summarize our recent progress in the design, synthesis, and structure-property relationships of IID- and particularly hetero-IID-based functional materials. More specifically, starting with molecular engineering of hetero-IIDs with variable electronic effects, conjugation lengths, and numbers of heterorings, we discuss the effect of the heteroring on the absorption spectra and energy levels. Additionally, we investigate a series of electron-withdrawing substitution of IIDs and hetero-IIDs and their molecular self-assembly behavior and the device performance. Furthermore, we discuss a series of IID-bis(EDOT) copolymers with hydrophilic ethylene glycol side chains for accumulation-mode organic thin-film electrochemical transistors, in which the relationships among the molecular structure, operational stability, film morphology, and device performance were revealed. Compared with IID polymers, the HOMO levels and optical band gaps of the thiophene and thienothiophene IID copolymers markedly decrease, and these polymers exhibit ambipolar charge transport. When we further expanded the IID core to a thieno[3,2-b][1]benzothiophene isoindigo (TBTI) core, such as in TBTIT, bulk-heterojunction solar cells employing this polymer class as the electron donor achieved good efficiency for additive- and annealing-free device conditions. When we introduced electron-deficient pyridine on the IID core, both the LUMO and HOMO energy levels of the copolymers markedly decreased, which significantly improved the electron mobility. In addition, we compare the correlation between the polymer structures of IID and hetero-IID copolymers with thiophene and benzothiophene as comonomers and their absorption spectra and energy levels. In particular, we evaluate the planarity and the dihedral angle between the repeat units, with systematic analysis by theoretical calculations to support our design concepts. We discuss polymer designs arising from simple aldol condensation, where the rigid backbone conformation has been locked by the double bonds. Our polymers display broad absorption from the visible to the NIR-II region, and more importantly, the high electron affinities of these polymers provide a platform to realize ambient-stable electron transport in solution-processed organic thin-film transistors. These exciting results are expected to open doors to new horizons of semiconducting materials in terms of other charming applications and the design and synthesis of superior materials.

Icariin-induced miR-875-5p attenuates epithelial-mesenchymal transition by targeting hedgehog signaling in liver fibrosis.

BACKGROUND: Hepatic fibrosis is the final endpoint for most chronic liver diseases and remains a significant public health problem worldwide. Icariin, a naturally occurring flavonol glucoside, has been reported to exhibit protective effects on liver injury and alleviate liver fibrosis. However, the underlying detail molecular mechanism is not fully revealed. METHODS: Mouse primary hepatic stellate cells (HSCs) and carbon tetrachloride (CCL4 )-induced liver fibrosis model in mice were used as in vitro and in vivo models in this study. The expression levels of miR-875-5p were detected by quantitative reverse transcription-PCR. The validation of the direct target of miR-875-5p was through dual-luciferase reporter assay and western blotting assay. The cell proliferation and cell mobility were determined using MTT assay and Transwell migration assay, respectively. RESULTS: We found that icariin inhibited epithelial-mesenchymal transition and collagen protein section of HSCs. Icariin exerted hepatoprotective effects on mice model of CCL4 -induced liver fibrosis. Our further results revealed that miR-875-5p was downregulated in human cirrhosis tissues and activated murine HSCs. Icariin induced miR-875-5p upregulation and subsequently decreased glioma-associated oncogene homolog 1 (GLI1) expression through direct binding to the three prime untranslated region of GLI1 mRNA. CONCLUSION: Our study highlighted the potential therapeutic application of icariin for liver fibrosis management.

An Intrinsically Disordered APLF Links Ku, DNA-PKcs, and XRCC4-DNA Ligase IV in an Extended Flexible Non-homologous End Joining Complex.

DNA double-strand break (DSB) repair by non-homologous end joining (NHEJ) in human cells is initiated by Ku heterodimer binding to a DSB, followed by recruitment of core NHEJ factors including DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4-like factor (XLF), and XRCC4 (X4)-DNA ligase IV (L4). Ku also interacts with accessory factors such as aprataxin and polynucleotide kinase/phosphatase-like factor (APLF). Yet, how these factors interact to tether, process, and ligate DSB ends while allowing regulation and chromatin interactions remains enigmatic. Here, small angle X-ray scattering (SAXS) and mutational analyses show APLF is largely an intrinsically disordered protein that binds Ku, Ku/DNA-PKcs (DNA-PK), and X4L4 within an extended flexible NHEJ core complex. X4L4 assembles with Ku heterodimers linked to DNA-PKcs via flexible Ku80 C-terminal regions (Ku80CTR) in a complex stabilized through APLF interactions with Ku, DNA-PK, and X4L4. Collective results unveil the solution architecture of the six-protein complex and suggest cooperative assembly of an extended flexible NHEJ core complex that supports APLF accessibility while possibly providing flexible attachment of the core complex to chromatin. The resulting dynamic tethering furthermore, provides geometric access of L4 catalytic domains to the DNA ends during ligation and of DNA-PKcs for targeted phosphorylation of other NHEJ proteins as well as trans-phosphorylation of DNA-PKcs on the opposing DSB without disrupting the core ligation complex. Overall the results shed light on evolutionary conservation of Ku, X4, and L4 activities, while explaining the observation that Ku80CTR and DNA-PKcs only occur in a subset of higher eukaryotes.

Nanospray HX-MS configuration for structural interrogation of large protein systems.

Hydrogen-deuterium exchange mass spectrometry (HX-MS) has made important contributions to the study of protein structure and function. Unfortunately, it is not known for low limits of detection, when compared with other forms of peptide-based or bottom-up protein MS methods. Systems perform poorly on sub-pmol quantities of protein states with greater than 300 kDa of unique sequences. The HX-MS analysis of complex protein states would be possible if proteomics-grade configurations could be used reliably, but temperature and temporal constraints have proven to be significant design challenges. Here, we describe an integrated HX-MS ion source operating on a vented-column geometry, which brings regulated column cooling right to the spray tip. The design offers chromatographic peak widths of 2-6 s (FWHM). It provides stable operation at 500 nL min-1, while retaining deuteration levels comparable to conventional geometries. We demonstrate at least a 50-fold improvement in protein consumption levels, and illustrate robustness by measuring peptide-averaged protection factors for 90% of DNA-PKcs, a 469 kDa protein, from 0.5 pmol injections.

Biological evaluation of two Keggin-type polyoxometalates containing glycine as mushroom tyrosinase inhibitors.

Two Keggin-type polyoxometalates (POMs) containing glycine, (HGly)3 PW12 O40 and (HGly)4 SiW12 O40 , were synthesized and evaluated as mushroom tyrosinase inhibitors. The spectrophotometric method results showed that both (HGly)3 PW12 O40 and (HGly)4 SiW12 O40 could strongly inhibit the diphenolase activity of the tyrosinase and that their inhibition mechanisms were reversible. Their half-inhibition concentration values were estimated to be 1.55 and 1.39 mmol/L, respectively. The inhibition kinetics analysis by Lineweaver-Burk plots indicated that (HGly)3 PW12 O40 was an uncompetitive inhibitor with KIS  = 0.046 mmol/L, whereas (HGly)4 SiW12 O40 was a noncompetitive inhibitor with KI  = KIS  = 2.17 mmol/L. This study may help to extend the application of POMs in the fields of medicine and food preservation.

Nepenthesin from monkey cups for hydrogen/deuterium exchange mass spectrometry.

Studies of protein dynamics, structure and interactions using hydrogen/deuterium exchange mass spectrometry (HDX-MS) have sharply increased over the past 5-10 years. The predominant technology requires fast digestion at pH 2-3 to retain deuterium label. Pepsin is used almost exclusively, but it provides relatively low efficiency under the constraints of the experiment, and a selectivity profile that renders poor coverage of intrinsically disordered regions. In this study we present nepenthesin-containing secretions of the pitcher plant Nepenthes, commonly called monkey cups, for use in HDX-MS. We show that nepenthesin is at least 1400-fold more efficient than pepsin under HDX-competent conditions, with a selectivity profile that mimics pepsin in part, but also includes efficient cleavage C-terminal to "forbidden" residues K, R, H, and P. High efficiency permits a solution-based analysis with no detectable autolysis, avoiding the complication of immobilized enzyme reactors. Relaxed selectivity promotes high coverage of disordered regions and the ability to "tune" the mass map for regions of interest. Nepenthesin-enriched secretions were applied to an analysis of protein complexes in the nonhomologous end-joining DNA repair pathway. The analysis of XRCC4 binding to the BRCT domains of Ligase IV points to secondary interactions between the disordered C-terminal tail of XRCC4 and remote regions of the BRCT domains, which could only be identified with a nepenthesin-based workflow. HDX data suggest that stalk-binding to XRCC4 primes a BRCT conformation in these remote regions to support tail interaction, an event which may be phosphoregulated. We conclude that nepenthesin is an effective alternative to pepsin for all HDX-MS applications, and especially for the analysis of structural transitions among intrinsically disordered proteins and their binding partners.

XRCC4's interaction with XLF is required for coding (but not signal) end joining.

XRCC4 and XLF are structurally related proteins important for DNA Ligase IV function. XRCC4 forms a tight complex with DNA Ligase IV while XLF interacts directly with XRCC4. Both XRCC4 and XLF form homodimers that can polymerize as heterotypic filaments independently of DNA Ligase IV. Emerging structural and in vitro biochemical data suggest that XRCC4 and XLF together generate a filamentous structure that promotes bridging between DNA molecules. Here, we show that ablating XRCC4's affinity for XLF results in DNA repair deficits including a surprising deficit in VDJ coding, but not signal end joining. These data are consistent with a model whereby XRCC4/XLF complexes hold DNA ends together--stringently required for coding end joining, but dispensable for signal end joining. Finally, DNA-PK phosphorylation of XRCC4/XLF complexes disrupt DNA bridging in vitro, suggesting a regulatory role for DNA-PK's phosphorylation of XRCC4/XLF complexes.

Overexpression of lactate dehydrogenase-A in human intrahepatic cholangiocarcinoma: its implication for treatment.

BACKGROUND: Previous studies have shown that lactate dehydrogenase-A (LDH-A) is strongly expressed in several malignancies, that LDH-A expression is associated with poor prognosis, and that LDH-A inhibition severely diminishes tumorigenicity. However, little is known about the implications of LDH-A expression in intrahepatic cholangiocarcinoma. The purpose of this study was to investigate the expression of LDH-A and to clarify its effect on intrahepatic cholangiocarcinoma. METHODS: We studied the expression of LDH-A in tissue samples from patients with intrahepatic cholangiocarcinoma (n = 54) using the ultrasensitive surfactant protein (S-P) immunohistochemical method. We then inhibited LDH-A using small hairpin RNA (shRNA) in the cholangiocarcinoma cell line HuCCT-1 in vitro to study the role it plays in promoting growth and escaping apoptosis. RESULTS: We report that LDH-A was overexpressed in 52 of 54 (96%) paraffin-embedded cancer tissue samples and 0 of 54 para-carcinoma tissue samples. Reduction of LDH-A by RNA interference (RNAi) inhibited cell growth and induced apoptosis in HuCCT-1 cells. This result correlated with the elevation of cytoplasmic reactive oxygen species (ROS) levels. CONCLUSIONS: LDH-A expression is closely correlated with histopathological variables of intrahepatic cholangiocarcinoma, indicating that LDH-A may serve as a new treatment target.

Correction of the Effect of Humic Acids on the Fluorescence Detection of Polycyclic Aromatic Hydrocarbons by Combination Spectroscopy.

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed in soil and water, but fluorescence spectroscopy for PAHs is often interfered with organic matter in the environment. The aim of this paper is to evaluate a correction method using combined spectral technology in an environment where humic acids and PAHs coexist. In the present work, humic acids and benzo[ghi]perylene were analyzed in various concentrations using fluorescence and near-infrared (NIR) spectroscopy from single and mixed samples. The NIR prediction model of humic acids in mixed samples was established based on synergy interval partial least squares, and the standard curve of fluorescence spectra for humic acids was established at 478 nm (characteristic wavelength of benzo[ghi]perylene). The fluorescence intensity of humic acids in the mixed sample was predicted from the content derived from the NIR spectra. The final correction was carried out by their exclusion from the fluorescence of the mixture at the same wavelength. The corrected fluorescence intensity was linearly correlated with the concentration of benzo[ghi]perylene with R2 = 0.8362, while R2 = 0.3538 before correction. These results give a new insight into the calibration modeling of the combined spectral method.

Dendrobium nobile active ingredient Dendrobin A against hepatocellular carcinoma via inhibiting nuclear factor kappa-B signaling.

OBJECTIVE: Dendrobin A, a typical active ingredient of the traditional Chinese medicine Dendrobium nobile, has potential clinical application in cancer treatment; however, its effect and mechanism in anti-hepatocellular carcinoma (HCC) remain unsolved. METHOD: The effects of Dendrobin A on the viability, migration, invasion, cycle, apoptosis, and epithelial-mesenchymal transition of HepG2 and SK-HEP-1 cells were verified by in vitro experiments. mRNA sequencing was performed to screen the differentially expressed genes (DEGs) of HCC cells before and after Dendrobin A treatment, following GO enrichment and KEGG signaling pathway analyses. Mechanistically, molecular docking was used to evaluate the binding of Dendrobin A with proteins p65 and p50, before further verifying the activation of nuclear factor kappa-B (NF-κB) signaling. Finally, the antiproliferative effect of Dendrobin A on HCC cells was explored through animal experiments. RESULTS: Dendrobin A arrested cell cycle, induced apoptosis, and inhibited proliferation, migration, invasion, and blocked epithelial-mesenchymal transition in HepG2 and SK-HEP-1 cells. mRNA sequencing identified 830 DEGs, involving various biological processes. KEGG analysis highlighted NF-κB signaling. Molecular docking revealed strong binding of Dendrobin A with p65 and p50 proteins, and western blotting confirmed reduced levels of p-p65 and p-p50 in HCC cells post Dendrobin A treatment. NF-κB agonist PMA reversed Dendrobin A-inhibited cell proliferation migration and invasion. In vivo experiments showed that Dendrobin A inhibited HCC cell growth. CONCLUSION: Our findings suggest that Dendrobin A exhibits anti-HCC properties by inhibiting the activation of the NF-κB pathway. These results provide a scientific basis for utilizing Dendrobium nobile in anti-HCC therapies.

Targeting protein for xenopus kinesin-like protein 2 (TPX2) regulates γ-histone 2AX (γ-H2AX) levels upon ionizing radiation.

The microtubule-associated protein targeting protein for Xenopus kinesin-like protein 2 (TPX2) plays a key role in spindle assembly and is required for mitosis in human cells. In interphase, TPX2 is actively imported into the nucleus to prevent its premature activity in microtubule organization. To date, no function has been assigned to nuclear TPX2. We now report that TPX2 plays a role in the cellular response to DNA double strand breaks induced by ionizing radiation. Loss of TPX2 leads to inordinately strong and transient accumulation of ionizing radiation-dependent Ser-139-phosphorylated Histone 2AX (γ-H2AX) at G(0) and G(1) phases of the cell cycle. This is accompanied by the formation of increased numbers of high intensity γ-H2AX ionizing radiation-induced foci. Conversely, cells overexpressing TPX2 have reduced levels of γ-H2AX after ionizing radiation. Consistent with a role for TPX2 in the DNA damage response, we found that the protein accumulates at DNA double strand breaks and associates with the mediator of DNA damage checkpoint 1 (MDC1) and the ataxia telangiectasia mutated (ATM) kinase, both key regulators of γ-H2AX amplification. Pharmacologic inhibition or depletion of ATM or MDC1, but not of DNA-dependent protein kinase (DNA-PK), antagonizes the γ-H2AX phenotype caused by TPX2 depletion. Importantly, the regulation of γ-H2AX signals by TPX2 is not associated with apoptosis or the mitotic functions of TPX2. In sum, our study identifies a novel and the first nuclear function for TPX2 in the cellular responses to DNA damage.

Phosphorylation of polynucleotide kinase/ phosphatase by DNA-dependent protein kinase and ataxia-telangiectasia mutated regulates its association with sites of DNA damage.

Human polynucleotide kinase/phosphatase (PNKP) is a dual specificity 5'-DNA kinase/3'-DNA phosphatase, with roles in base excision repair, DNA single-strand break repair and non-homologous end joining (NHEJ); yet precisely how PNKP functions in the repair of DNA double strand breaks (DSBs) remains unclear. We demonstrate that PNKP is phosphorylated by the DNA-dependent protein kinase (DNA-PK) and ataxia-telangiectasia mutated (ATM) in vitro. The major phosphorylation site for both kinases was serine 114, with serine 126 being a minor site. Ionizing radiation (IR)-induced phosphorylation of cellular PNKP on S114 was ATM dependent, whereas phosphorylation of PNKP on S126 required both ATM and DNA-PK. Inactivation of DNA-PK and/or ATM led to reduced PNKP at DNA damage sites in vivo. Cells expressing PNKP with alanine or aspartic acid at serines 114 and 126 were modestly radiosensitive and IR enhanced the association of PNKP with XRCC4 and DNA ligase IV; however, this interaction was not affected by mutation of PNKP phosphorylation sites. Purified PNKP protein with mutation of serines 114 and 126 had decreased DNA kinase and DNA phosphatase activities and reduced affinity for DNA in vitro. Together, our results reveal that IR-induced phosphorylation of PNKP by ATM and DNA-PK regulates PNKP function at DSBs.

Predictive value of neutrophil-to-apolipoprotein A1 ratio in all-cause and cardiovascular death in elderly non-valvular atrial fibrillation patients.

Neutrophil-to-apolipoprotein AI ratio's (NAR's) predictive value for the elderly non-valvular atrial fibrillation (NVAF) patients' death has not been fully recognized. We consecutively enrolled 1224 elderly patients with NVAF (≥75 years). With an average follow-up of 733.35 ± 271.39 days, 222 all-cause deaths were identified. Among these, 101 were caused by cardiovascular diseases. Cox regression showed that after correcting for potential confounders, patients in the Q4 group had an increased all-cause (hazard ratio [HR] = 1.90, 95% confidence interval [CI]: 1.20-2.99) and cardiovascular death (HR = 2.59, 95% CI: 1.30-5.15) risk compared to those in the lowest NAR quartile. Kaplan-Meier analysis indicated that all-cause and cardiovascular death were higher in the high NAR than those in the lowest NAR category (log rank, all, P < 0.001). A nonlinear association was observed between death and NAR. NAR may be a promising predictive biomarker for identifying elderly NVAF patients with poor clinical prognoses.

Integrating Network Pharmacology and Bioinformatics to Explore the Effects of Dangshen (Codonopsis pilosula) Against Hepatocellular Carcinoma: Validation Based on the Active Compound Luteolin.

Purpose: This study aimed to explore the pharmacological mechanism of Dangshen (Codonopsis pilosula) against hepatocellular carcinoma (HCC) based on network pharmacology and bioinformatics, and to verify the anticancer effect of luteolin, the active ingredient of Codonopsis pilosula, on HCC cells. Methods: The effective compounds and potential targets of Codonopsis pilosula were established using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database. The genes related to HCC were obtained through the GeneCards database. The interactive genes were imported into the Visualization and Integrated Discovery database for Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) signal enrichment, and the hub genes were screened out. The Cancer Genome Atlas database was used to construct a prognosis model, and the prognosis and clinicopathological correlation were analyzed. In in vitro experiments, we verified the effects of luteolin, an active compound of Codonopsis pilosula, on the proliferation, cell cycle, apoptosis and migration of HCC cells. Results: A total of 21 effective compounds of Codonopsis pilosula and 98 potential downstream target genes were screened through the TCMSP database, and 1406 HCC target genes were obtained through the GeneCards database. Finally, 53 interacting genes between the two databases were obtained, among which, the 10 key node genes were CASP3, TP53, MDM2, AKT1, ESR1, BCL2L1, MCL1, HSP90AA1, CASP9, and CCND1, involving 77 typical GO terms and 72 KEGG signals. The Kaplan-Meier survival curve of the model group showed that the overall survival of the low-risk group was significantly higher than that of the high-risk group. Luteolin significantly inhibited the proliferation and migration of HCC cells, induced apoptosis, and increased the G2/M phase ratio. Mechanistically, luteolin significantly inhibited the phosphorylation of MAPK-JNK and Akt (Thr308) and subsequently led to upregulation of ESR1. Pharmacological inhibition of ESR1 with fulvestrant enhanced cell viability and migration and attenuated apoptosis. Conclusion: Codonopsis pilosula has potential for clinical development due to its anti-HCC properties. Luteolin, the effective component of Codonopsis pilosula, plays anti-HCC role through AKT- or MAPK-JNK signaling mediated ESR1.