Clinical utility of cerebrospinal fluid-derived circular RNAs in lung adenocarcinoma patients with brain metastases.

BACKGROUND: Free circular RNAs(circRNAs) escaping from primary lesion of cancer to brain are strictly regulated by blood-brain barrier and therefore cerebrospinal fluid (CSF) circRNAs have potential advantage in exploring biomarkers and mechanism of brain metastasis in lung cancer. METHODS: We collected paired cerebrospinal fluid, plasma and tumor tissues from 21 lung adenocarcinoma (ADC) patients with brain metastases (BM) and performed RNA sequencing. RESULTS: Compared to tumor tissue and plasma, circRNAs in CSF were characterized by lower number of spieces but higher abundance. Notably, CSF-circRNAs displayed high heterogeneity among different BM lung ADC patients. A total of 60 CSF-circRNAs was identified and associated with shorten overall survival. The circRNA-miRNA-mRNA network analysis revealed that the 60 CSF-circRNAs involved in cancer-associated pathways, and five of them showed strong association with WNT signaling pathway. Validation by RT-PCR of CSF and in vitro experiments of the five candidate circRNAs support their potential roles in cell proliferation and invasion. CONCLUSIONS: In summary, our results depicted the heterogenous CSF-circRNAs profiles among BM lung ADC and implied that CSF-circRNAs may be promising prognosis-related biomarkers.

Skip participates in formation and lipid metabolism of beige adipose and might mediate the effects of SIRT1 activator BTM-0512 on beige remodeling.

Dissipating energy by activating thermogenic adipose to combating obesity attracts many interests. Ski-interacting protein (Skip) has been known to play an important role in cell proliferation and differentiation, but whether it participates in energy metabolism is not known. Our previous study revealed that BTM-0512 could induce beige adipose formation, accompanying with up-regulation of Skip, but the role of Skip in metabolism was unknown. In this study, we mainly investigated whether Skip was involved in beige remodeling of subcutaneous white preadipocytes as well as in lipid metabolism of differentiated beige adipocytes. The results showed that in high fat diet-induced obesity mice, the protein levels of Skip in subcutaneous and visceral white adipose as well as in brown adipose were all down-regulated, especially in subcutaneous white adipose. Then we cultured subcutaneous adipose derived-stem cells (ADSCs) and found knock-down of Skip (siSkip) inhibited the expressions of thermogenic adipose specific genes including PRDM16 and UCP1 in both undifferentiated ADSCs and differentiated beige adipocytes, which could abolish the effects of BTM-0512 on beige remodeling. We further observed that siSkip affected multiple rate-limiting enzymes in lipid metabolism. The expressions of ACC, GPAT-1, HSL and ATGL were down-regulated, while CPT1α expression was up-regulated by siSkip. The expression of AMPK was also decreased by siSkip. In conclusion, our study demonstrated that Skip might play an important role in the beige remodeling of white adipocytes as well as lipid metabolism of beige adipose.

Alpha-glucosidase inhibitor 1-Deoxynojirimycin promotes beige remodeling of 3T3-L1 preadipocytes via activating AMPK.

Obesity is a serious health challenge in the world, and searching effective drugs to cure obesity is of great importance. 1-Deoxynojirimycin (DNJ) is extracted from mulberry leaves and acts as an α-glucosidase inhibitor to lower blood glucose. Recent studies demonstrated that it also has anti-obesity effect, but the mechanisms remain unknown. In our present study, we mainly examined the effects of DNJ on beige remodeling of 3T3-L1 preadipocytes. We observed that DNJ didn't affect the mRNA levels of fatty acid binding protein 4 (aP2), peroxisome proliferator-activated receptor γ (PPARγ), preadipocyte factor-1 (Pref-1) as well as the mitochondrial uncoupling protein 1 (UCP1), PR domain containing protein 16 (PRDM16), transmembrane protein 26 (TMEM26) in undifferentiated preadipocytes. But after inducing 3T3-L1 preadipocytes to differentiation with white or beige adipogenic medium, DNJ significantly reduced aP2, PPARγ and Pref-1 expressions, while up-regulated the expressions of UCP1, PRDM16 and TMEM26, accompanying with decreased lipid deposition. The ratio of p-AMPK/AMPK was up-regulated by DNJ (10 µM) treatment for 10 days, and the effects of DNJ on p-AMPK/AMPK, UCP1 and PRDM16 could be blocked by AMPK inhibitor Compound C. These results demonstrated that hypoglycemic agent DNJ could suppress the adipogenesis during the differentiation of white preadipocytes, and promote the switch of white preadipocytes to beige adipocytes via activating AMPK, which provided new mechanisms for explaining the benefits of DNJ on obesity-related disorders.

Effect of scrotal heating on sperm quality, seminal biochemical substances, and reproductive hormones in human fertile men.

At present, male contraceptive methods are only vasectomy and condoms, so it is necessary to research on male contraceptive techniques. The aim of this study is to observe the effects of scrotal heating (SH) on semen parameters, seminal l-carnitine (LC), epidermal growth factor (EGF), macrophage migration inhibitory factor (MIF), reproductive hormones and sperm chromosome numbers of adult healthy men, and to provide the experimental data for male contraception. The scrotums of 30 healthy male volunteers were exposed to the condition of 40 to 43°C SH belt warming 40 minutes each day for successive 2 days per week. The course of SH was continuous for 3 months. Computer-assisted semen analysis and hypo-osmotic swelling test, sperm DNA integrity, l-carnitine, MIF and EGF, and sperm fluorescence in situ hybridization were performed before, during, and after SH. The serum level of follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) were measured by chemiluminescent immunoassay. The mean parameters of sperm concentration, vitality, and normal morphological sperm were significantly decreased in groups with sperms being collected during 1, 2, and 3 months of SH when compared with those in groups of pre-SH (P < 0.01). Statistically significant differences of sperm DNA fragmentation, normal sperm membrane functionality, levels of LC and MIF in semen, and LH, FSH, and T in serum were observed between the groups of before SH and after SH 3 months and the groups of during SH 1, 2, and 3 months (P < 0.001). The total rate of chromosome number for 13, 18, 21, X, and Y in the 3 months of SH was 13.7-fold greater (13.72%/1.69%) than before SH (P < 0.001). The constant SH can impact the semen quality, sperm DNA integrity, sperm chromosome, LC and MIF, and LH, FSH, and T in serum. Transient SH may be a new method for male contraception.

[Yam polysaccharide improves sperm viability and protects sperm DNA integrity in vitro].

OBJECTIVE: To clarify the roles of yam polysaccharide (YPS) in improving sperm viability and protecting sperm DNA integrity in vitro and provide a new approach to the treatment of oligoasthenozoospermia. METHODS: We collected samples by masturbation from 36 normal fertile males aged 27－39 years. Each sample was divided into six groups: blank control or treated with normal saline, vitamin C solution, and YPS solution at low (0.25 mg/ml), medium (1.0 mg/ml) or high concentration (5.0 mg/ml). Using eosin-Y staining, sperm hypotonic swelling (HOS) and sperm chromatin diffusion (SCD) test, we observed the effects of different concentrations of YPS on sperm viability, membrane integrity and nuclear DNA. RESULTS: After 24 and 48 hours of treatment, sperm viability was markedly reduced in the vitamin C (ï¼»28.5 ± 3.1ï¼½ and ï¼»6.5 ± 1.2ï¼½%), low-YPS (ï¼»31.3 ± 3.5ï¼½ and ï¼»6.5 ± 2.2ï¼½%), medium-YPS (ï¼»37.1 ± 3.5ï¼½ and ï¼»9.5 ± 2.8ï¼½%) and high-YPS groups (ï¼»38.3 ± 3.3ï¼½ and ï¼»9.0 ± 3.2ï¼½%) as compared with the blank control (ï¼»17.3 ± 2.1ï¼½ and ï¼»3.2 ± 1.3ï¼½%) (P <0.01) and normal saline groups (ï¼»13.4 ± 4.1ï¼½ and ï¼»3.1 ± 2.0ï¼½%) (P <0.01), and it was significantly higher in the medium- and high-YPS than in the vitamin C group (P <0.05 and P <0.01). The rate of sperm DNA fragmentation was remarkably decreased at 48 hours in the vitamin C (ï¼»30.5 ± 3.1ï¼½%), low-YPS (ï¼»29.4 ± 2.6ï¼½%), medium-YPS (ï¼»28.5 ± 2.3ï¼½%) and high-YPS groups (ï¼»27.9 ± 1.9ï¼½%) in comparison with the blank control (ï¼»41.7 ± 2.2ï¼½%) (P <0.01) and normal saline groups (ï¼»42.1 ± 3.3ï¼½%), markedly lower in the medium- and high-YPS than in the blank control, normal saline and vitamin C groups (P <0.05 or P <0.01), but with no statistically significant difference between the low-YPS and vitamin C groups (P >0.05). CONCLUSIONS: Yam polysaccharide can improve sperm viability and protect sperm DNA integrity in vitro.

Optimization of Ultrasound-Assisted Extraction of Natural Antioxidants from the Osmanthus fragrans Flower.

An ultrasound-assisted extraction (UAE) method was developed to extract natural antioxidants from the Osmanthus fragrans flower. The effect of UAE on antioxidant activity of the extract from the Osmanthus fragrans flower was studied using a Trolox equivalent antioxidant capacity (TEAC) assay. Optimization conditions were firstly determined using a single-factor experiment, and response surface methodology was then used to evaluate interaction of several experimental parameters. Analysis of the coefficient of determination showed that second-order polynomial models produced a highly satisfactory fitting of the experimental data with regard to TEAC values (R² = 0.9829, p < 0.0001). The optimal conditions were 39.1% ethanol, and extraction for 35.2 min at 59.4 °C. Under these conditions, the maximum TEAC value was 584.9 ± 6.0 µmol Trolox/g DW, which was higher than those obtained by the conventional extracting method (486.4 ± 12.6 µmol Trolox/g DW) and the Soxhlet extraction method (339.1 ± 16.2 µmol Trolox/g DW). The crude extract obtained could be used either as a food additive or in pharmaceuticals for the prevention and treatment of diseases caused by oxidative stress.

Optimization of Ultrasound-Assisted Extraction of Natural Antioxidants from the Flower of Jatropha integerrima by Response Surface Methodology.

An ultrasound-assisted extraction (UAE) method was developed for the efficient extraction of natural antioxidants from the flowers of Jatropha integerrima. Four independent variables, including ethanol concentration, solvent/material ratio, ultrasound irradiation time and temperature were studied by single factor experiments. Then, the central composite rotatable design and response surface methodology were employed to investigate the effect of three key parameters (ethanol concentration, solvent/material ratio, and ultrasound irradiation time) on the antioxidant activities of the flower extracts. The optimal extraction conditions were an ethanol concentration of 59.6%, solvent/material ratio of 50:1, ultrasound irradiation time of 7 min, and ultrasound irradiation temperature of 40 °C. Under these conditions, the optimized experimental value was 1103.38 ± 16.11 µmol Trolox/g dry weight (DW), which was in accordance with the predicted value (1105.49 µmol Trolox/g DW). Furthermore, the antioxidant activities of flower extracts obtained by UAE were compared with those produced by the traditional maceration and Soxhlet extraction methods, and UAE resulted in higher antioxidant activities after a shorter time at a lower temperature. The results obtained are helpful for the full utilization of Jatropha integerrima, and also indicate that ultrasound-assisted extraction is an efficient method for the extraction of natural antioxidants from plant materials.

Antioxidant Phytochemicals for the Prevention and Treatment of Chronic Diseases.

Overproduction of oxidants (reactive oxygen species and reactive nitrogen species) in the human body is responsible for the pathogenesis of some diseases. The scavenging of these oxidants is thought to be an effective measure to depress the level of oxidative stress of organisms. It has been reported that intake of vegetables and fruits is inversely associated with the risk of many chronic diseases, and antioxidant phytochemicals in vegetables and fruits are considered to be responsible for these health benefits. Antioxidant phytochemicals can be found in many foods and medicinal plants, and play an important role in the prevention and treatment of chronic diseases caused by oxidative stress. They often possess strong antioxidant and free radical scavenging abilities, as well as anti-inflammatory action, which are also the basis of other bioactivities and health benefits, such as anticancer, anti-aging, and protective action for cardiovascular diseases, diabetes mellitus, obesity and neurodegenerative diseases. This review summarizes recent progress on the health benefits of antioxidant phytochemicals, and discusses their potential mechanisms in the prevention and treatment of chronic diseases.

Circulating Tumor DNA-Guided De-Escalation Targeted Therapy for Advanced Non-Small Cell Lung Cancer: A Nonrandomized Controlled Trial.

Importance: Uninterrupted targeted therapy until disease progression or intolerable toxic effects is currently the routine therapy for advanced non-small cell lung cancer (NSCLC) involving driver gene variations. However, drug resistance is inevitable. Objective: To assess the clinical feasibility of adaptive de-escalation tyrosine kinase inhibitor (TKI) treatment guided by circulating tumor DNA (ctDNA) for achieving complete remission after local consolidative therapy (LCT) in patients with advanced NSCLC. Design, Setting, and Participants: This prospective nonrandomized controlled trial was conducted at a single center from June 3, 2020, to July 19, 2022, and included 60 patients with advanced NSCLC with driver variations without radiologically detectable disease after TKI and LCT. The median (range) follow-up time was 19.2 (3.8-29.7) months. Data analysis was conducted from December 15, 2022, to May 10, 2023. Intervention: Cessation of TKI treatment and follow-up every 3 months. Treatment was restarted in patients with progressive disease (defined by the Response Evaluation Criteria in Solid Tumors 1.1 criteria), detectable ctDNA, or elevated carcinoembryonic antigen (CEA) levels, whichever manifested first, and treatment ceased if all indicators were negative during follow-up surveillance. Main Outcomes and Measures: Progression-free survival (PFS). Secondary end points were objective response rate, time to next treatment, and overall survival. Results: Among the total study sample of 60 participants (median [range] age, 55 [21-75] years; 33 [55%] were female), the median PFS was 18.4 (95% CI, 12.6-24.2) months and the median (range) total treatment break duration was 9.1 (1.5-28.1) months. Fourteen patients (group A) remained in TKI cessation with a median (range) treatment break duration of 20.3 (6.8-28.1) months; 31 patients (group B) received retreatment owing to detectable ctDNA and/or CEA and had a median PFS of 20.2 (95% CI, 12.9-27.4) months with a median (range) total treatment break duration of 8.8 (1.5-20.6) months; and 15 patients (group C) who underwent retreatment with TKIs due to progressive disease had a median PFS of 5.5 (95% CI, 1.5-7.2) months. For all participants, the TKI retreatment response rate was 96%, the median time to next treatment was 29.3 (95% CI, 25.3-35.2) months, and the data for overall survival were immature. Conclusions and Relevance: The findings of this nonrandomized controlled trial suggest that this adaptive de-escalation TKI strategy for patients with NSCLC is feasible in those with no lesions after LCT and a negative ctDNA test result. This might provide a de-escalation treatment strategy guided by ctDNA for the subset of patients with advanced NSCLC. Trial Registration: ClinicalTrials.gov Identifier: NCT03046316.

Copper-catalyzed asymmetric C(sp2)-H arylation for the synthesis of P- and axially chiral phosphorus compounds.

Transition metal-catalyzed C-H bond functionalization is an important method in organic synthesis, but the development of methods that are lower cost and have a less environmental impact is desirable. Here, a Cu-catalyzed asymmetric C(sp2)-H arylation is reported. With diaryliodonium salts as arylating reagents, a range of ortho-arylated P-chiral phosphonic diamides were obtained in moderate to excellent yields with high enantioselectivities (up to 92% ee). Meanwhile, enantioselective C-3 arylation of diarylphosphine oxide indoles was also realized under similar conditions to construct axial chirality.

First-line pyrotinib in advanced HER2-mutant non-small-cell lung cancer: a patient-centric phase 2 trial.

To explore targeted treatment options in patients with non-small-cell lung cancer (NSCLC) with rare genetic mutations in the context of a patient-centric clinical trial, we initiated, in parallel, a phase 2 adaptive umbrella trial consisting of a criteria-fulfilled (CF) cohort and a compassionate use (CU) cohort under expanded eligibility criteria, and a prospective real-world study (RWS). Here, we present efficacy and safety data from 48 patients with treatment-naive, advanced HER2-mutant NSCLC treated with the pan-HER receptor tyrosine kinase inhibitor pyrotinib (CF and CU cohorts) or physician's therapy of choice (RWS cohort). In the phase 2 trial CF cohort (n = 28), the primary endpoint was reached with an objective response rate of 35.7% after pyrotinib treatment. Secondary endpoints included disease control rate (89.3%), median progression-free survival (PFS) (7.3 months), median overall survival (OS) (14.3 months) and toxicity, which was acceptable, with grade 3 or 4 treatment-related adverse events occurring in three patients (10.7%). The phase 2 trial CU cohort (n = 12) showed an objective response rate of 16.7%, disease control rate of 83.4%, median PFS of 4.7 months and median OS of 14.2 months after pyrotinib treatment. The RWS cohort (n = 8) had no responses to physician's therapy of choice, while median PFS and OS were 3.0 and 12.2 months, respectively. Phase 2 umbrella trial, clinicaltrials.gov identifier: NCT03574402 . RWS, clinicaltrials.gov identifier: NCT03605602 .

Directed evolution and structural prediction of cellobiohydrolase II from the thermophilic fungus Chaetomium thermophilum.

Cellulases can be engineered with enhanced properties for broad use in scientific and industrial applications. In this study, the wild-type cbh2 gene of the thermophilic fungus Chaetomium thermophilum encoding cellobiohydrolase II (CBHII) was mutagenized through in vitro directed evolution. The resulting Pichia pastoris yeast library was screened, and two transformants were selected for enhanced CBHII activities that were not attributed to increased gene copy numbers. The optimum fermentation times of the two mutant transformants were shortened to 4-5 days after methanol induction compared to 6 days for the wild-type. The optimum reaction temperature (60 °C) and pH level (5 or 6) of the mutant CBHII proteins, designated CBHIIX16 and CBHIIX305, were higher than those of wild-type CBHII (50 °C and pH 4). Kept at 80 °C for 1 h, CBHIIX16 and CBHIIX305 retained >50% of their activities, while the wild-type CBHII lost all activity. Sequence analysis of CBHIIX16 and CBHIIX305 revealed that they contained five and six mutated amino acids, respectively. Structural modeling confirmed the presence of carbohydrate binding type-1 and catalytic domains, where the hydrogen bond numbers between the 227th and 203rd amino acids were increased, which perhaps contributed to the elevated enzyme stability. Therefore, the two CBHII mutants selected for increased enzymatic activities also demonstrated elevated optimum reaction temperature and pH levels and enhanced thermal stability. These properties may be beneficial in practical applications for CBHII.

Expression of a novel thermostable Cu, Zn-superoxide dismutase from Chaetomium thermophilum in Pichia pastoris and its antioxidant properties.

A new superoxide dismutase (SOD) gene from the thermophilic fungus Chaetomium thermophilum (Ctsod) was cloned and expressed in Pichia pastoris and its gene product was characterized. The specific activity of the purified CtSOD was 2,170 U/mg protein. The enzyme was inactivated by KCN and H(2)O(2) but not by NaN(3), confirming that it belonged to the type of Cu, ZnSOD. The amino acid residues involved in coordinating copper and zinc were conserved. The recombinant CtSOD exhibited optimum activity at pH 6.5 and 60°C. The enzyme retained 65% of the maximum activity at 70°C for 60 min and the half-life was 22 and 7 min at 80 and 90°C, respectively. The recombinant yeast exhibited higher stress resistance than the control yeast cells to salt and superoxide-generating agents, such as paraquat and menadione.

MET amplification identified by next-generation sequencing and its clinical relevance for MET inhibitors.

BACKGROUND: MET amplification plays an important role in the development of non-small-cell lung cancer (NSCLC) either de novo or in resistance to epidermal growth factor receptor tyrosine-kinase inhibitor (EGFR-TKI) settings. Fluorescence in situ hybridization (FISH) is the standard method for MET amplification. With more and more discoveries of oncogenic driver genes, next-generation sequencing (NGS) plays a significant role in precision oncology. Meanwhile, the role of NGS in MET amplification remains uncertain. METHODS: Forty patients diagnosed with advanced NSCLC were included. FISH and NGS were conducted prior to MET inhibitors treatment. MET amplification by FISH was defined as a MET/CEP7 ratio of > 2.0 and/or copy number (CN) > 5. MET amplification by NGS was defined as gene copy number (GCN) ≥ 5. RESULTS: The concordance rate among FISH and NGS was 62.5% (25/40). MET amplification identified by FISH showed the optimal predictive value. The partial response (PR) rate was 68.0% (17/25 with MET amplification) vs. 6.7% (1/15 without MET amplification); the median progression-free survival (PFS) was 5.4 months versus 1.0 months (P < 0.001). MET amplification identified by NGS failed to distinguish significant clinical outcomes. The PR rate was 60.0% (6/10, with MET GCN ≥ 5) vs. 40.0% (12/30, with MET GCN < 5); the median PFS was 4.8 months vs. 2.2 months (P = 0.357). The PR rate was 68.8% (11/16) and the median PFS was 4.8 months in patients with focal amplification by NGS. CONCLUSIONS: MET amplification identified by FISH remains the optimal biomarker to identify suitable candidates for MET-TKI therapy. In comparison, amplification identified by NGS seems not as robust to be effective predictive biomarker. Further exploration is needed regarding the focal amplification by NGS in predicting the efficacy.

[Characterization and Size Distribution of Carbonaceous Aerosols at Mountain Dinghu].

To understand the characterization and sources of carbonaceous aerosols at Mountain Dinghu, organic carbon (OC) and elemental carbon (EC) in size-resolved aerosol samples were measured at a regional background site in South China using a DRI Model 2001A analyzer. The average mass concentrations of organic carbon (OC) are (5.6±2.0) µg ·m-3 in PM1.1, (7.3±2.4) µg ·m-3 in PM2.1, and (12.8±4.0) µg ·m-3 in PM9.0; the average mass concentrations of elemental carbon (EC) are (2.3±1.4) µg ·m-3in PM1.1, (2.7±1.6) µg ·m-3 in PM2.1, and (3.4±1.7) µg ·m-3 in PM9.0. OC concentrations in PM1.1 and PM2.1 account for 43.8% and 57.0% of OC in PM9.0, and EC concentrations in PM1.1 and PM2.1 account for 67.6% and 79.4%, respectively. OC and EC are enriched with fine particles. In PM1.1 and PM2.1, the highest concentrations of OC and EC are measured in autumn, and the lowest concentration of OC is measured in winter and EC in summer. In PM9.0, the highest OC concentration is measured in summer. Carbonaceous aerosols are mainly composed of OC2, EC1, OC3, and OC4. In summer, the concentration of OC3 is higher than that of EC1, suggesting that biogenic sources are dominant during summer. The concentration of EC1 in winter is the highest, indicating that the impacts of motor vehicle emissions are prominent in the local area during winter. OC and EC both show bimodal distributions in four seasons, with peaks in the size ranges of 0.43-0.65 µm for fine particles and 3.3-5.8 µm for coarse particles. In PM1.1 and PM2.1, the sources of OC are mainly primary emissions. In PM2.1, the highest concentration of SOC is measured in spring at (3.0±1.4) µg ·m-3 and the lowest in winter at (1.3±1.4) µg ·m-3, indicating that the secondary aerosol formation is significant in spring. At the Mountain Dinghu background site, OC is mainly from coal combustion and motor vehicle emissions for fine particles and from biogenic sources for coarse particles. EC is mainly from coal combustion, motor vehicle emissions, and dust.

Purification and characterization of two thermostable proteases from the thermophilic fungus Chaetomium thermophilum.

Thermostable protease is very effective to improve the industrial processes in many fields. Two thermostable extracellular proteases from the culture supernatant of the thermophilic fungus Chaetomium thermophilum were purified to homogeneity by fractional ammonium sulfate precipitation, ion-exchange chromatography on DEAE-Sepharose, and PhenylSepharose hydrophobic interaction chromatography. By SDS-PAGE, the molecular mass of the two purified enzymes was estimated to be 33 kDa and 63 kDa, respectively. The two proteases were found to be inhibited by PMSF, but not by iodoacetamide and EDTA. The 33 kDa protease (PRO33) exhibited maximal activity at pH 10.0 and the 63 kDa protease (PRO63) at pH 5.0. The optimum temperature for the two proteases was 65 degrees C. The PRO33 had a K(m) value of 6.6 mM and a V(max) value of 10.31 micromol/l/min, and PRO63 17.6 mM and 9.08 micromol/l/min, with casein as substrate. They were thermostable at 60 degrees C. The protease activity of PRO33 and PRO63 remained at 67.2% and 17.31%, respectively, after incubation at 70 degrees C for 1 h. The thermal stability of the two enzymes was significantly enhanced by Ca2+. The residual activity of PRO33 and PRO63 at 70 degrees C after 60 min was approximately 88.59% and 39.2%, respectively, when kept in the buffer containing Ca2+. These properties make them applicable for many biotechnological purposes.

Soluble c-Met Levels Correlated With Tissue c-Met Protein Expression in Patients With Advanced Non-Small-Cell Lung Cancer.

BACKGROUND: Immunohistochemistry (IHC) and fluorescent in situ hybridization are reliable methods for identifying c-Met protein expression or c-Met gene amplification. However, each technique requires a high-quality tissue sample, which might not be available. The aim of the present study was to investigate the correlation between the soluble c-Met level and tissue c-Met protein expression and the relationship between these markers and patient prognosis. MATERIALS AND METHODS: In 198 patients with advanced non-small-cell lung cancer, tumor tissue c-Met expression was determined using IHC according to the H score criteria. Positivity was defined as ≥ 50% of cells with strong staining (IHC 3+). The concentration of c-Met protein in paired plasma samples was measured using a human soluble c-Met quantitative enzyme-linked immunosorbent assay kit, and the predictive value was determined using receiver operating characteristic curve analysis. RESULTS: Of the 198 patients, 140 (70.7%) had tissue c-Met- findings and 58 (29.3%) tissue c-Met+ findings. Receiver operating characteristic curve analysis showed 67.2% specificity and 65.0% sensitivity for predicting tissue c-Met positivity at a plasma c-Met cutoff of 766 ng/mL. The correlation between the soluble c-Met level and tissue c-Met protein expression was significant (Pearson's r = 0.309; P < .001). Patients with high soluble c-Met levels (> 766 ng/mL) had poorer overall survival than patients with low soluble c-Met levels (9.5 vs. 22.2 months; P < .001). Multivariate analyses demonstrated the same result (hazard ratio, 2.15; 95% confidence interval, 1.334-3.446; P = .002). CONCLUSION: A significant correlation was found between the plasma soluble c-Met levels and tissue c-Met protein expression in patients with advanced non-small-cell lung cancer. A high level of soluble c-Met was associated with a poor prognosis.

The Unique Characteristics of MET Exon 14 Mutation in Chinese Patients with NSCLC.

INTRODUCTION: Predictive biomarkers of mesenchymal-to-epithelial transition factor (MET)-targeted therapy remain elusive. Since the discovery of the MNNG HOS Transforming gene (MET) exon 14 mutation, it has been found to have the best potential to become one precise biomarker for MET-targeted therapy. Here, we present the unique characteristics of MET exon 14 mutations in Chinese patients with NSCLC. METHODS: A total of 1296 patients with NSCLC were screened for MET exon 14 mutations. Next-generation sequencing was performed on the DNA of 968 patients and Sanger sequencing was conducted on complementary DNA of the other 328 patients. Immunohistochemical analysis and fluorescence in situ hybridization were also performed on all specimens. RESULTS: Twelve patients had MET exon 14 mutations. These accounted for only 0.9% of adenocarcinoma. Thus, the mutations were present at less than half the frequency of their occurrence in Western patients (0.9% versus 3% in Chinese and white patients, respectively, χ(2) = 15.1, p < 0.001). Samples from six patients with MET exon 14 mutations were analyzed using immunohistochemical analysis and fluorescence in situ hybridization. We found no significant relationships among the mutation, MET amplification, and MET overexpression. In two patients who received crizotinib, only one patient (who exhibited MET amplification) experienced a partial response; the progression-free survival was 9 months. However, it remains unclear whether the sensitivity of this patient to crizotinib was conferred by the MET exon 14 mutation per se or by MET amplification. In the other patient with concomitant MET exon 14 skipping and KRAS G12D mutation, the disease progressed in only 1 month. CONCLUSIONS: MET exon 14 mutation per se may not be sufficiently robust for use in defining a subset of NSCLCs. Further research on MET exon 14 mutations, MET amplification, and MET overexpression is required. Maybe a panel of biomarkers will be necessary in the future.

The coexistence of MET over-expression and an EGFR T790M mutation is related to acquired resistance to EGFR tyrosine kinase inhibitors in advanced non-small cell lung cancer.

MET overexpression and the EGFR T790M mutation are both associated with acquired resistance (AR) to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in advanced non-small cell lung cancer (NSCLC). We characterized the frequency, underlying molecular mechanisms, and subsequent treatment for AR in MET overexpressing NSCLC patients with or without the T790M mutation. The study participants were 207 patients with advanced NSCLC and AR to EGFR-TKIs. The percentages of MET-, T790M- and MET/T790M-positive patients were 20.3% (42/207), 34.8% (72/207) and 6.8% (14/207), respectively. The disease control rate was 100% (5/5) for five patients with MET overexpression who received EGFR-TKIs plus a MET inhibitor. Among the MET/T790M-positive patients, seven received EGFR-TKIs plus a MET inhibitor and four received a T790M inhibitor, but no response was observed. The median post-progression survival (PPS) was 14.1, 24.5, and 10.7 months for MET-overexpressing, T790M-positive and MET/T790M-positive patients, respectively (P=0.044). c-Met, p-Met, ERBB3, and p-ERBB3 were highly expressed in MET-positive and MET/T790M-positive patients, but were poorly expressed in T790M-positive patients. EGFR, p-EGFR, AKT, p-AKT, MAPK, and p-MAPK were highly expressed in all three groups. These results suggest that MET/T790M-positive patients are at higher risk of AR to EGFR-TKIs, and have a worse PPS than patients with only MET overexpression or the T790M mutation alone. Clinical trials are needed to determine the best treatment for patients with both MET overexpression and the EGFR T790M mutation.

Resources and biological activities of natural polyphenols.

The oxidative stress imposed by reactive oxygen species (ROS) plays an important role in many chronic and degenerative diseases. As an important category of phytochemicals, phenolic compounds universally exist in plants, and have been considered to have high antioxidant ability and free radical scavenging capacity, with the mechanism of inhibiting the enzymes responsible for ROS production and reducing highly oxidized ROS. Therefore, phenolic compounds have attracted increasing attention as potential agents for preventing and treating many oxidative stress-related diseases, such as cardiovascular diseases, cancer, ageing, diabetes mellitus and neurodegenerative diseases. This review summarizes current knowledge of natural polyphenols, including resource, bioactivities, bioavailability and potential toxicity.