DNA Nanospheres Assisted Spatial Confinement Signal Amplification for MicroRNA Imaging in Live Cancer Cells.

DNA assemblies are commonly used in biosensing, particularly for the detection and imaging of microRNAs (miRNAs), which are biomarkers associated with tumor progression. However, the difficulty lies in the exploration of high-sensitivity analytical techniques for miRNA due to its limited presence in living cells. In this study, we introduced a DNA nanosphere (DS) enhanced catalytic hairpin assembly (CHA) system for the detection and imaging of intracellular miR-21. The single-stranded DNA with four palindromic portions and extending sequences at the terminal was annealed for assembling DS, which avoided the complex sequence design and high cost of long DNA strands. Benefiting from the multiple modification sites of DS, functional hairpins H1 (modified with Cy3 and BHQ2) and H2 were grafted onto the surface of DS for assembling DS-H1-H2 using a hybridization reaction. The DS-H1-H2 system utilized spatial confinement and the CHA reaction to amplify fluorescence signals of Cy3. This enabled highly sensitive and rapid detection of miR-21 in the range from 0.05 to 3.5 nM. The system achieved a limit of determination (LOD) of 2.0 pM, which was 56 times lower than that of the control CHA circuit with freedom hairpins. Additionally, the sensitivity was improved by 8 times. Moreover, DS-H1-H2 also showed an excellent imaging capability for endogenous miR-21 in tumor cells. This was due to enhanced cell internalization efficiency, accelerated reaction kinetics, and improved biostability. The imaging strategy was shown to effectively monitor the dynamic content of miR-21 in live cancer cells and differentiate various cells. In general, the simple nanostructure DS not only enhanced the detection and imaging capability of the conventional probe but also could be easily integrated with the reported DNA-free probe, indicating a wide range of potential applications.

Analysis of the phytochemical components of Prunella vulgaris using high-performance liquid chromatography quadrupole time-of-flight mass spectrometry combined with molecular networking and assessment of their antioxidant and anti-α-glucosidase activities.

Prunella vulgaris has long been used in traditional medicine and is consumed as a tea in China. Here, the total phenolic and flavonoid concentrations of plants from different geographical regions were measured. It was found that the total phenolic acid concentration ranged from 4.15 to 8.82 g of gallic acid equivalent per 100 g of dry weight (DW), and the total flavonoid concentration was 4.67-7.33 g of rutin equivalent per 100 g DW. Antioxidant activities were measured using 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, and the results ranged from 73.47% to 94.43% and 74.54% to 93.39%, respectively, whereas α-glucosidase inhibition was between 75.31% and 95.49%. Correlation analysis showed that the total flavonoids in P. vulgaris had superior antioxidant and anti-α-glucosidase activities compared to the total phenolic compounds. The active components of P. vulgaris were analyzed using high-performance liquid chromatography quadrupole time-of-flight mass spectrometry combined with both classical molecular networking and feature-based molecular networking on the Global Natural Products Social platform, identifying 32 compounds, namely 14 flavonoids, 12 phenolic compounds, and 6 other chemical components. These results could provide useful information on the use of P. vulgaris as a functional tea.

A General Signal Amplifier of Self-Assembled DNA Micelles for Sensitive Quantification of Biomarkers.

Owing to the excellent structural rigidity and programmable reaction sites, DNA nanostructures are more and more widely used, but they are limited by high cost, strict sequence requirements, and time-consuming preparation. Herein, a general signal amplifier based on a micelle-supported entropy-driven circuit (MEDC) was designed and prepared for sensitive quantification of biomarkers. By modifying a hydrophobic cholesterol molecule onto a hydrophilic DNA strand, the amphiphilic DNA strand was first prepared and then self-assembled into DNA micelles (DMs) driven by hydrophobic effects. The as-developed DM showed unique advantages of sequence-independence, easy preparation, and low cost. Subsequently, amplifier units DMF and DMTD were successfully fabricated by connecting fuel strands and three-strand duplexes (TDs) to DMs, respectively. Finally, the MEDC was triggered by microRNA-155 (miR-155), which herein acted as a model analyte, resulting in dynamic self-assembly of poly-DNA micelles (PDMs) and causing the recovery of cyanine 3 (Cy3) fluorescence as the DMTD dissociated. Benefiting from the "diffusion effect", the MEDC herein had a nearly 2.9-fold increase in sensitivity and a nearly 97-fold reduction in detection limit compared to conventional EDC. This amplifier exhibited excellent sensitivity of microRNAs, such as miR-155 detection in a dynamic range from 0.05 to 4 nM with a detection limit of 3.1 pM, and demonstrated outstanding selectivity with the distinguishing ability of a single-base mismatched sequence of microRNAs. Overall, the proposed strategy demonstrated that this sequence-independent DNA nanostructure improved the performance of traditional DNA probes and provided a versatile method for the development of DNA nanotechnology in biosensing.

Caffeine alleviates acute liver injury by inducing the expression of NEDD4L and deceasing GRP78 level via ubiquitination.

BACKGROUND: Acute liver injury is liver cell injury that occurs rapidly in a short period of time. Caffeine has been shown to maintain hepatoprotective effect with an unclear mechanism. Endoplasmic reticulum stress (ERS) has significant effects in acute liver injury. Induction of GRP78 is a hallmark of ERS. Whether or not caffeine's function is related to GRP78 remains to be explored. METHODS: Acute liver injury model was established by LPS-treated L02 cells and in vivo administration of LPS/D-Gal in mice. Caffeine was pre-treated in L02 cells or mice. Gene levels was determined by real-time PCR and western blot. Cell viability was tested by CCK-8 assay and cell apoptosis was tested by flow cytometry. The interaction of GRP78 and NEDD4L was determined by Pull-down and co-immunoprecipitation (Co-IP) assay. The ubiquitination by NEDD4L on GRP78 was validated by in vitro ubiquitination assay. RESULTS: Caffeine protected liver cells against acute injury induced cell apoptosis and ERS both in vitro and in vivo. Suppression of GRP78 could block the LPS-induced cell apoptosis and ERS. NEDD4L was found to interact with GRP78 and ubiquitinate its lysine of 324 site directly. Caffeine treatment induced the expression of NEDD4L, resulting in the ubiquitination and inhibition of GRP78. CONCLUSION: Caffeine mitigated the acute liver injury by stimulating NEDD4L expression, which inhibited GRP78 expression via ubiquitination at its K324 site. Low dose of caffeine could be a promising therapeutic treatment for acute liver injury.

Adjunctive vagus nerve stimulation for treatment-resistant depression: a preliminary study.

BACKGROUND: This study is the first to assess the safety and therapeutic efficacy of vagus nerve stimulation (VNS) as an adjunctive treatment for Chinese patients suffering from treatment-resistant depression (TRD). METHODS: A total of seven patients with TRD underwent surgical implantation of a VNS device were followed over a 9-month period. The 24-item Hamilton Rating Scale for Depression (HAMD-24) and the 14-item Hamilton Anxiety Scale (HAMA) were used to assess depressive and anxiety symptoms, respectively. Neurocognitive function was measured with the Wechsler Adult Intelligence Scale (WAIS) and the Wechsler Memory Scale (WMS). RESULTS: After 3 months of treatment with VNS, the antidepressant response and remission rates were 42.9% and 28.6%, respectively. After 9 months of treatment with VNS, the response and remission rates increased to 85.7% and 57.1%, respectively. Significant time main effects were identified for HAMD-24 scores, HAMA scores, the WMS memory quotient, and the full intelligence quotients measured with the WAIS (all ps < 0.05). The most frequent adverse effects of VNS treatment were voice alteration (100%) and cough frequency increase (71.4%). CONCLUSION: This preliminary study indicated that adjunctive VNS was effective and safe in treating Chinese patients who were suffering from TRD, and its efficacy increased with time.Key pointsThere is positive evidence to support the role of VNS as an adjunctive treatment in Chinese patients with TRD.The antidepressant efficacy of adjunctive VNS for Chinese patients with TRD increased with time.The most frequent adverse effects of VNS treatment were voice alteration and cough frequency increase.

Effects of high-fat diet on antioxidative status, apoptosis and inflammation in liver of tilapia (Oreochromis niloticus) via Nrf2, TLRs and JNK pathways.

Fatty liver injury (or disease) is a common disease in farmed fish, but its pathogenic mechanism is not fully understood. Therefore the present study aims to investigate high-fat diet (HFD)-induced liver injury and explore the underlying mechanism in fish. The tilapia were fed on control diet and HFD for 90 days, and then the blood and liver tissues were collected to determine biochemical parameter, gene expression and protein level. The results showed that HFD feeding signally increased the levels of plasma aminotransferases and pro-inflammatory factors after 60 days. In liver and plasma, HFD feeding significantly suppressed antioxidant ability, but enhanced lipid peroxidation formation, protein oxidation and DNA damage after 60 or 90 days. Further, the Nrf2 pathway and antioxidative function-related genes were adversely changed in liver of HFD-fed tilapia after 60 and/or 90 days. Meanwhile, HFD treatment induced apoptosis via initiating mitochondrial pathway in liver after 90 days. Furthermore, after 90 days of feeding, the expression of genes or proteins related to JNK pathway and TLRs-Myd88-NF-κB pathway was clearly upregulated in HFD treatment. Similarly, the mRNA levels of inflammatory factors including tumor necrosis factor (TNF-α), interleukin-1ß (IL-1ß), IL-6, IL-8 and IL-10 were also upregulated in liver of HFD-fed tilapia after 60 and/or 90 days. In conclusion, the current study suggested that HFD feeding impaired antioxidant defense system, induced apoptosis, enhanced inflammation and led to liver injury. The adverse influences of HFD in the liver might be due to the variation of Nrf2, JNK and TLRs-Myd88-NF-κB signaling pathways.

[Effects of Rg_1 on LPS-induced apoptosis and autophagy of lung epithelial cells].

This paper aimed to study the protective effect of ginsenoside Rg_1 on endotoxin(LPS)-induced apoptosis of lung epithelial cells and its mechanism of action. Mouse lung epithelial cells(MLE-12) were first treated with LPS. The autophagy changes and apoptosis and the relationship with concentration and time of LPS were observed. Then,the level of autophagy in MLE-12 was regulated at a specific concentration and action time of LPS,and the changes of apoptosis were observed. Secondly,ginsenoside Rg_1 and autophagy inhibitor 3-MA were added respectively at the same concentration and action time of LPS. The lung epithelial cells were grouped to observe the effect of ginsenoside Rg_1 on LPS-induced apoptosis of lung epithelial cells and its mechanism. In the animal experiment,the mice were grouped and tested by apoptosis protein,lung injury score and HE staining section to verify whether ginsenoside Rg_1 has a protective effect on LPS-induced lung injury. The results showed that apoptosis and autophagy increased as the rise of concentration after treatment with LPS for 12 h. The apoptosis increased gradually,and the autophagy increased first and then decreased over time at the LPS concentration of 25 g·L-1. The apoptosis of LPS group was higher than that of control group,and LPS+3-MA group increased further,while apoptosis decreased significantly in LPS+RAM(rapamycin,autophagy promoter) group. The autophagy increased in LPS group,decreased in LPS+3-MA group and increased in LPS+RAM group. The apoptosis of LPS group was higher than that of control group,and the apoptosis of LPS+Rg_1 group decreased. The apoptosis of LPS+Rg_1+3-MA group increased again. The autophagy of LPS group further increased after administration of ginsenoside Rg_1,but decreased after administration of 3-MA. In the in vivo experiments in mice,the apoptosis of LPS group increased significantly compared with the control group,while LPS + ginsenoside Rg_1 group decreased. Lung injury score and HE staining also conformed to the above trend. LPS can induce the apoptosis of lung epithelial cells in a time-dependent and concentration-dependent manner. The autophagy of lung epithelial cells increases with the rise of LPS concentration. At the specific concentration of LPS,autophagy increases first and then decreases after 12-16 hours. Proper increase of autophagy in lung epithelial cells within a certain period of time can reduce the apoptosis induced by LPS,while inhibition of autophagy can increase apoptosis. Ginsenoside Rg_1 has a protective effect on lung cancer epithelial cell apoptosis induced by autophagy.

Lovastatin protects neurite degeneration in LRRK2-G2019S parkinsonism through activating the Akt/Nrf pathway and inhibiting GSK3ß activity.

Parkinson's disease (PD) is a progressive neurodegenerative disorder that lacks a disease-modifying therapy. Leucine-rich repeat kinase 2 (LRRK2) was implicated as the most common genetic cause of PD. We previously established a LRRK2-G2019S Drosophila model that displayed the crucial phenotypes of LRRK2 parkinsonism. Here, we used a two-step approach to identify compounds from the FDA-approved licensed drug library that could suppress neurite degeneration in LRRK2-G2019S parkinsonism. Of 640 compounds, 29 rescued neurite degeneration phenotypes and 3 restored motor disability and dopaminergic neuron loss in aged LRRK2-G2019S flies. Of these three drugs, lovastatin had the highest lipophilicity, which facilitated crossing the blood-brain barrier. In LRRK2-G2019S knock-in mice and stably transfected human dopaminergic cells, lovastatin significantly rescued neurite degeneration in a dose-dependent manner, within a range of 0.05-0.1 µm The beneficial effect of lovastatin was exerted by activating anti-apoptotic Akt/Nrf signaling and decreasing caspase 3 levels. We also observed that lovastatin inhibited GSK3ß activity, a kinase downstream of Akt, by up-regulating GSK3ß (Ser9) phosphorylation. This inhibition subsequently decreased tau phosphorylation, which was linked to neuronal cytoskeleton instability. Conversely, pre-treatment with the Akt inhibitor, A6730, blocked the lovastatin-induced neuroprotective effect. The rescuing effects of lovastatin in dendritic arborization of LRRK2-G2019S neurons were abolished by co-expressing either a mutant allele of Akt (Akt104226) or a constitutively active form of GSK3ß (sggS9A). Our findings demonstrated that lovastatin restored LRRK2-G2019S neurite degeneration by augmenting Akt/NRF2 pathway and inhibiting downstream GSK3ß activity, which decreased phospho-tau levels. We suggested that lovastatin is a potential disease-modifying agent for LRRK2-G2019S parkinsonism.

Effects of Rhizoma Alismatis extract on biochemical indices and adipose gene expression in oleic acid-induced hepatocyte injury in Jian carp (Cyprinus carpio var. Jian).

Fatty liver is an increasingly serious disease of fish in aquaculture. However, the mechanisms responsible for the occurrence of fatty liver remain unclear, and no effective methods for the prevention and treatment of this disease have yet been found. In the present study, we aimed to develop an in vitro model of hepatocyte injury using oleic acid as hepatotoxicant and evaluate the protective effects of Rhizoma Alismatis extract (RAE) in Jian carp using this model. Primary hepatocytes from Jian carp were isolated and purified and cultured in vitro. The result indicated that 0.4 mmol L-1 oleic acid and 48 h could be the optimal conditions to induce hepatocyte injury model in cultured hepatocytes. Hepatocytes were exposed to oleic acid, followed by the addition of RAE at 0, 1, 5, 10, 20, or 50 µg mL-1. The hepatocytes and supernatant were then analyzed. RAE suppressed oleic acid-induced elevations in aspartate aminotransferase, alanine aminotransferase, triglycerides, total cholesterol, lactate dehydrogenase, alkaline phosphatase, cholinesterase, malondialdehyde, γ-glutamyl transferase, cytochrome P450 1A, cytochrome P450 2E1, liver-type fatty acid binding protein, free fatty acid, fatty acid synthetase, and tumor necrosis factor-α (P < 0.01 or P < 0.05); reduced protein levels of cytochrome P450 1A, nuclear factor (NF)-κB p65, and NF-κB c-Rel; and inhibited cytochrome P4503A, NF-κB c-Rel, nuclear factor erythroid-related factor 2, peroxisome proliferator-activated receptor-α, and cytochrome P4501A mRNA levels. In conclusion, RAE exhibited a protective effect against hepatocyte injury in Jian carp. Further in vivo studies are needed to provide more evidence for the use of RAE as a hepatoprotective agent for the treatment of hepatocyte injury.

LRRK2 overexpression alters glutamatergic presynaptic plasticity, striatal dopamine tone, postsynaptic signal transduction, motor activity and memory.

Mutations in leucine-rich repeat kinase 2 (Lrrk2) are the most common genetic cause of Parkinson's disease (PD), a neurodegenerative disorder affecting 1-2% of those >65 years old. The neurophysiology of LRRK2 remains largely elusive, although protein loss suggests a role in glutamatergic synapse transmission and overexpression studies show altered dopamine release in aged mice. We show that glutamate transmission is unaltered onto striatal projection neurons (SPNs) of adult LRRK2 knockout mice and that adult animals exhibit no detectable cognitive or motor deficits. Basal synaptic transmission is also unaltered in SPNs of LRRK2 overexpressing mice, but they do exhibit clear alterations to D2-receptor-mediated short-term synaptic plasticity, behavioral hypoactivity and impaired recognition memory. These phenomena are associated with decreased striatal dopamine tone and abnormal dopamine- and cAMP-regulated phosphoprotein 32 kDa signal integration. The data suggest that LRRK2 acts at the nexus of dopamine and glutamate signaling in the adult striatum, where it regulates dopamine levels, presynaptic glutamate release via D2-dependent synaptic plasticity and dopamine-receptor signal transduction.

Effectiveness of cognitive behavioral therapy in treating bipolar disorder: An updated meta-analysis with randomized controlled trials.

AIM: The aim of this updated meta-analysis was to further assess the effectiveness of cognitive behavioral therapy (CBT) in treating bipolar disorder (BD). METHODS: We carried out a literature search on PubMed, Embase, and the Cochrane Library up to October 2015. We calculated the pooled relative risk of relapse rate and standard mean difference (SMD) of mean change (data at a follow-up time-point - baseline) of the Beck Depression Inventory, Beck Hopelessness Scale, Hamilton Rating Scale for Depression, Young Mania Rating Scale (YMRS) and Mania Rating Scale scores with their 95% confidence interval (95%CI). Subgroup analyses based on follow-up time were performed. RESULTS: Nine randomized controlled trials with 520 bipolar I or II disorder patients were reanalyzed. Overall analysis showed that CBT did not significantly reduce the relapse rate of BD or improve the level of depression. However, significant efficacy of CBT in improving severity of mania was proved based on the YMRS (SMD = -0.54, 95%CI, -1.03 to -0.06, P = 0.03) but not based on MRS. Subgroup analyses showed that CBT had short-term efficacy in reducing relapse rate of BD (at 6 months' follow-up: relative risk = 0.49, 95%CI: 0.29-0.81, P = 0.006) and improving severity of mania based on YMRS score (post-treatment: SMD = -0.30, 95%CI, -0.59 to -0.01, P = 0.04). CONCLUSION: Short-term efficacy of CBT in reducing relapse rate of BD and improving the severity of mania was proved. But these effects could be weakened by time. In addition, there was no effect of CBT on level of depression in BD.

A Study of 2,3,7,8-Tetrachlorodibenzo-p-dioxin Induced Liver Injury in Jian Carp (Cyprinus carpio var. Jian) Using Precision-Cut Liver Slices.

The aim of this study was to establish a model for the study of liver injury induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in Jian carp using precision-cut liver slices (PCLS). PCLS were treated with TCDD at concentrations of 0, 0.05, 0.1, 0.3, and 0.6 µg/L for 6 h, followed by collection of the culture supernatant and PCLS for analysis. Several biochemical indices were analyzed, including glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), lactate dehydrogenase (LDH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA). Expression of mRNA was also estimated for cytochrome P4501A (CYP1A), aryl hydrocarbon receptor2 (AhR2), and aryl hydrocarbon receptor nuclear translocator2 (ARNT2). Results showed that some significant effects (p < 0.05) in MDA, GSH-Px and PCLS viability were observed at a TCDD concentration as low as 0.05 µg/L, and the observed effects increased with exposure concentration. Following exposure to TCDD for 6 h at a concentration of 0.3 µg/L, significant increases (p < 0.01) in the content of GPT, GOT, MDA, and LDH were observed, while SOD activity, GSH-Px activity, and PCLS viability were decreased (p < 0.01 or p < 0.05). Exposure to 0.3 µg/L TCDD also resulted in increased expression of mRNA for CYP1A, AhR2, and ARNT2. Overall, these results provide evidence of TCDD-induced liver injury and oxidative stress in Jian carp. These results also support the use of PCLS as an in vitro model for the evaluation of hepatotoxicity in Jian carp.

[Forensic Validation of the Goldeneye™ DNA ID 25A Kit].

OBJECTIVE: To test and estimate the forensic application of Goldeneye™ DNA ID 25A Kit. METHODS: The kit was validated by a series of tests for accuracy, sensitivity, consistency, peak height balance, stability, and mixed samples through measured blood samples and other samples in routine casework. RESULTS: The peak height balance of the different loci was ≥ 42%. The genotyping results of the positive control DNA was accurate. The complete STR genotyping result could be obtained from 0.125 ng positive control DNA. CONCLUSION: Goldeneye™ DNA ID 25A Kit is suitable for criminal cases and DNA database in forensic practice.

Growth inhibitory in vitro effects of glycyrrhizic acid in U251 glioblastoma cell line.

Despite dramatic advances in cancer therapy, the overall prognosis of glioblastoma (GBM) remains dismal. Nuclear factor kappa-B (NF-κB) has been previously demonstrated to be constitutively activated in glioblastoma, and it was suggested as a potential therapeutic target. Glycyrrhizic acid (GA) has been proved to have cytotoxic effects in many cancer cell lines. However, its role in glioblastoma has not yet been addressed. Therefore, this study aimed to investigate the effects of GA on human glioblastoma U251 cell line. The effects of GA on proliferation of U251 cells were measured by CCK-8 assay and plate colony-forming test. Cellular apoptosis was detected by Hoechst 33258 fluorescent staining and flow cytometry with annexin V-FITC/PI dual staining. The expression of nuclear p65 protein, the active subunit of NF-κB, was determined by Western blot and immunofluorescence. Our results demonstrated that the survival rate and colony formation of U251 cells significantly decreased in a time- and dose-dependent manner after GA addition, and the apoptotic ratio of GA-treated groups was significantly higher than that of control groups. Furthermore, the expression of NF-κB-p65 in the nucleus was remarkably reduced after GA treatment. In conclusion, our findings suggest that GA treatment can confer inhibitory effects on human glioblastoma U251 cell line including inhibiting proliferation and inducing apoptosis, which is possibly related to the NF-κB mediated pathway.

Estimation of Physiologic Ability and Surgical Stress scoring system for predicting complications following abdominal surgery: A meta-analysis spanning 2004 to 2022.

BACKGROUND: Postoperative complications remain a paramount concern for surgeons and healthcare practitioners. AIM: To present a comprehensive analysis of the Estimation of Physiologic Ability and Surgical Stress (E-PASS) scoring system's efficacy in predicting postoperative complications following abdominal surgery. METHODS: A systematic search of published studies was conducted, yielding 17 studies with pertinent data. Parameters such as preoperative risk score (PRS), surgical stress score (SSS), comprehensive risk score (CRS), postoperative complications, postoperative mortality, and other clinical data were collected for meta-analysis. Forest plots were employed for continuous and binary variables, with χ2 tests assessing heterogeneity (P value). RESULTS: Patients experiencing complications after abdominal surgery exhibited significantly higher E-PASS scores compared to those without complications [mean difference and 95% confidence interval (CI) of PRS: 0.10 (0.05-0.15); SSS: 0.04 (0.001-0.08); CRS: 0.19 (0.07-0.31)]. Following the exclusion of low-quality studies, results remained valid with no discernible heterogeneity. Subgroup analysis indicated that variations in sample size and age may contribute to heterogeneity in CRS analysis. Binary variable meta-analysis demonstrated a correlation between high CRS and increased postoperative complication rates [odds ratio (OR) (95%CI): 3.01 (1.83-4.95)], with a significant association observed between high CRS and postoperative mortality [OR (95%CI): 15.49 (3.75-64.01)]. CONCLUSION: In summary, postoperative complications in abdominal surgery, as assessed by the E-PASS scoring system, are consistently linked to elevated PRS, SSS, and CRS scores. High CRS scores emerge as risk factors for heightened morbidity and mortality. This study establishes the accuracy of the E-PASS scoring system in predicting postoperative morbidity and mortality in abdominal surgery, underscoring its potential for widespread adoption in effective risk assessment.

Whether Detection of Gene Mutations Could Identify Low- or High-Risk Papillary Thyroid Microcarcinoma? Data from 393 Cases Using the Next-Generation Sequencing.

Objective: The objective of this study is to explore the utilization of next-generation sequencing (NGS) technology in evaluating the likelihood of identifying individuals with papillary thyroid microcarcinoma (PTMC ≤10 mm) who are at high or low risk. Design: NGS was used to analyze 393 formalin-fixed, paraffin-embedded tissues of PTC tumors, all of which were smaller than 15 mm. Results: The study found that bilateralism, multifocality, intrathyroidal spread, and extrathyroidal extension were present in 84 (21.4%), 153 (38.9%), 16 (4.1%), and 54 (13.7%) cases, respectively. Metastasis of cervical lymph nodes was identified in 226 (57.5%) cases and 96 (24.4%) cases with CLNM >5. Out of the total number of cases studied, 8 cases (2.3%) showed signs of tumor recurrence, all of which were localized and regional. Genetic alterations were detected in 342 cases (87.0%), with 336 cases revealing single mutations and 6 cases manifesting compound mutations. 332 cases (84.5%) had BRAFV600E mutation, 2 cases had KRASQ61K mutation, 2 cases had NRASQ61R mutation, 8 cases had RET/PTC1 rearrangement, 3 cases had RET/PTC3 rearrangement, and 1 case had TERT promoter mutation. Additionally, six individuals harbored concurrent mutations in two genes. These mutations were of various types and combinations: BRAFV600E and NRASQ61R (n = 2), BRAFV600E and RET/PTC3 (n = 2), BRAFV600E and RET/PTC1 (n = 1), and BRAFV600E and TERT promoter (n = 1). The subsequent analysis did not uncover a significant distinction in the incidence of gene mutation or fusion between the cN0 and cN1 patient cohorts. The presence of BRAFV600E mutation and CLNM incidence rates were found to be positively correlated with larger tumor size in PTMC. Our data showed that gene mutations did not appear to have much to do with high-risk papillary thyroid microcarcinoma (PTMC). However, when we looked at tumor size, we found that if the tumor was at least 5 millimeters in size, there was a higher chance of it being at high risk for PTM (P < 0.001, odds ratio (OR) = 2.55, 95% confidence interval (CI): 1.57-4.14). Identification of BRAFV600E mutation was not demonstrated to be significantly correlated with advanced clinicopathological characteristics, although it was strongly associated with a bigger tumor diameter (OR = 4.92, 95% CI: 2.40-10.07, P < 0.001). Conclusion: In clinical practice, BRAFV600E mutation does not consistently serve as an effective biomarker to distinguish high-risk PTMC or predict tumor progression. The size of the tumor has a significant correlation with its aggressive characteristics. PTMC with a diameter of ≤5 mm should be distinguished and targeted as a unique subset for specialized treatment.

[Expression of pGSK-3α/ß Tyr279/216 and XIAP proteins in cholangiocarcinoma and their clinical significance].

OBJECTIVE: To investigate the expressions of the active form of glycogen synthase kinase-3(GSK-3)-pGSK-3α/ß (Tyr279/216) and its downstream moleculor X-linked inhibitor of apoptosis protein (XIAP) in cholangiocarcinoma and to analyze their correlation with clinicopathological and survival significance. METHODS: Immunohistoehemistry was used to detect the expressions of the active form of GSK-3- pGSK-3α/ß (Tyr279/216) and its downstream moleculor XIAP proteins in 50 cholangiocarcinoma tissues and 20 normal bile duct tissues. RESULTS: The positive rates of pGSK-3α/ß (Tyr279/216) and XIAP were 62.0% and 68.0% in cholangiocarcinoma, and 10.0% and 25.0% in normal bile duct tissues, respectively. The intensity of pGSK-3α/ß (Tyr279/216) and XIAP expressions in cholangiocarcinoma were significantly higher than that in the normal bile duct tissues (P < 0.001), and there was a significant correlation between pGSK-3α/ß (Tyr279/216) and XIAP expressions (r = 0.544, P < 0.001). The expression of pGSK-3α/ß(Tyr279/216) protein in cholangiocarcinoma was associated with TNM stage (P = 0.042), histological grade (P = 0.031), whereas the expression of XIAP protein in cholangiocarcinoma was correlated with CEA level (P = 0.006). Patients with positive expression of pGSK-3α/ß (Tyr279/216) and XIAP demonstrate a significantly worse prognosis than that of patients with negative expression of pGSK-3α/ß (Tyr279/216) and XIAP for overall survival (P = 0.002, P = 0.018). Multivariate survival analysis revealed that positive pGSK-3α/ß (Tyr279/216) expression provided significant independent prognostic value for overall survival (P = 0.002). CONCLUSIONS: The expressions of pGSK-3α/ß(Tyr279/216) and XIAP proteins were significantly associated with the development and progression of cholangiocarcinoma. pGSK-3α/ß(Tyr279/216) may be an important prognostic factor for survival of patients with cholangiocarcinoma.

[X-linked inhibitor of apoptosis protein (XIAP) and Survivin suppression on human pancreatic cancer cells Panc-1 proliferation and chemosensitivety].

OBJECTIVE: To investigate the effect on cell proliferation and chemosensitivity of human pancreatic cancer cells Panc-1 after X-linked inhibitor of apoptosis protein (XIAP) and Survivin are inhibited simultaneously, and to compare it with the separate gene suppression strategy by which expression of XIAP or Survivin is inhibited respectively. METHODS: Panc-1 (Panc-1-X, Panc-1-S and Panc-1-XS) in which expression of XIAP and/or Survivin was inhibited, was established by using XIAP-shRNA lentiviral and Survivin-shRNA lentiviral we had built. The expressions of XIAP and Survivin mRNA and protein were evaluated by Real-time PCR and Semi-quantitatively Western blot analysis; cell proliferation was investigated by cell counting and colony formation assay; cell apoptosis was investigated by Caspase-3/7 activity assay kit and flow cytometry; gemcitabine (Gem) chemosensitivity was investigated by MTT assay. RESULTS: The pancreatic cell line Panc-1 in which the expression of XIAP and/or Survivin was stablely inhibited was successfully established. The cell proliferation of Panc-1-XS cells decreased significantly. The colony formation rate of Panc-1-XS cells (10.12%± 1.33%), was significantly lower than that of Panc-1-XncSnc cells (96.61% ± 7.89%) and Panc-1 cells (100.28% ± 8.97%) respectively (P<0.05). After being treated by 0.5 mg/L Gem for 24 h, the Caspase-3/7 relative activity of Panc-1-XS cells (15.02 ± 0.57) was significantly higher than that of Panc-1 cells and Panc-1-XncSnc cells (8.87 ± 0.19 and 9.05 ± 0.23, respectively; P<0.05); and the rate of apoptosis of Panc-1-XS cells (24.09% ± 2.75%) was significantly higher than that of Panc-1-XncSnc cells and Panc-1 cells (12.09% ± 1.97% and 12.06% ± 1.22%, respectively; P<0.05). The IC50 value of Panc-1-XS cells [(0.47 ± 0.04) mg/L] was significantly lower than that of Panc-1-XncSnc cells [(2.18 ± 0.13) mg/L] and Panc-1 cells [(2.13 ± 0.18) mg/L, P<0.05]. Further testing also showed that, the IC50 value of Panc-1-XS cells [(0.47 ± 0.04) mg/L] was significantly lower than that of Panc-1-X cells [(0.76 ± 0.07) mg/L] and Panc-1-S cells [(0.87 ± 0.09) mg/L, P<0.05]. CONCLUSION: The cell proliferation of Panc-1 cells was significantly suppressed and the Gem chemosensitivity was significantly enhanced after expressions of XIAP and Survivin were inhibited simultaneously, and significantly better than the strategy in which expressions of XIAP and Survivin were inhibited separately.

Clinical significance of peritumoral lymphatic vessel density and lymphatic vessel invasion detected by D2-40 immunostaining in FIGO Ib1-IIa squamous cell cervical cancer.

The clinical significance of lymphangiogenesis in cervical cancer remains controversial. Our aim was to investigate the correlation between lymphangiogenesis, lymphatic vessel invasion (LVI) and tumor metastasis, invasion and prognosis in squamous cell cervical cancer. Paraffin sections of 90 patients with FIGO (Fédération Internationale de Gynécologie et d'Obstétrique) Ib1-IIa squamous cell cervical cancer were stained for immunohistochemistry with a D2-40 monoclonal antibody against the carcinoembryonic antigen M2A. The lymphatic vessel density (LVD) and LVI were measured, and their relationship with the clinicopathological data was analyzed. D2-40-positive lymphatic vessels were found in 75 of the 90 patients (83.3 %). All D2-40-positive vessels were located in peritumoral areas. The mean±SD of the peritumoral LVD was 10.08±4.16. The positive rate of LVI was 32.0 % (24/75). The recurrence rate of patients with LVD >10 (62.1 %, 18/29) was significantly higher than that of patients with LVD ≤10 (34.8 %, 16/46, P = 0.021). The 5-year recurrence-free survival rate of patients with LVD >10 (41.0 %) was significantly lower than that of patients with LVD ≤10 (67.0 %, P = 0.045). Univariate analysis showed that the peritumoral LVD (≤10 vs >10) was correlated with LVI (absent vs present, P = 0.016). The peritumoral LVD and LVI showed no correlation with age, FIGO stage, tumor size, tumor grade, depth of invasion, or pelvic lymph node metastasis (all: P > 0.05). Peritumoral lymphangiogenesis was correlated with the recurrence and recurrence-free survival in patients with squamous cell cervical cancer. Examination of peritumoral LVD in these patients might therefore help to estimate the risk of recurrence.

Polymorphisms in genes HSD17B1 and HSD17B2 and uterine leiomyoma risk in Chinese women.

PURPOSE: To evaluate the association of HSD17B1 and HSD17B2 gene polymorphisms with uterine leiomyoma in Chinese women. METHODS: 121 Chinese women with clinically diagnosed uterine leiomyoma and 217 healthy normal Chinese women were investigated to compare three single nucleotide polymorphisms (SNPs) (rs605059 and rs676387 of HSD17B1 gene and rs8191246 of HSD17B2 gene) by polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing method. RESULTS: All the SNPs were polymorphisms in Chinese women. Frequencies of rs605059 AA genotype and A allele were significantly increased in patients with uterine leiomyoma compared to healthy controls (GG vs. AA, OR 0.40, 95 % CI 0.20-0.82; G vs. A, OR 0.68, 95 % CI 0.50-0.94). CONCLUSION: The results suggest that the genotype of HSD17B1 rs605059 may play a role in the tumourgenesis of uterine leiomyoma.