Docosahexaenoic Acid Reverses Epithelial-Mesenchymal Transition and Drug Resistance by Impairing the PI3K/AKT/ Nrf2/GPX4 Signalling Pathway in Docetaxel-Resistant PC3 Prostate Cancer Cells.

Drug resistance is a serious problem in cancer therapy. Growing evidence has shown that docosahexaenoic acid has anti-inflammatory and chemopreventive abilities. Studies have shown that autophagy inhibition and ferroptosis are promising therapeutic strategies for overcoming multidrug resistance. This study was aimed to examine whether docosahexaenoic acid (DHA) could reverse docetaxel resistance in prostate cancer cells. Cell survival was examined by MTT and colony formation. Protein expression was determined by Western blot. Reactive oxygen species (ROS) production was measured by flow cytometry. DHA displayed anti-cancer effects on proliferation, colony formation, migration, apoptosis, autophagy and epithelial mesenchymal transition. Glutathione-S-transferase π is an enzyme that plays an important role in drug resistance. DHA inhibited GSTπ protein expression and induced cytoprotective autophagy by regulating the PI3K/AKT signalling pathway in PC3R cells. DHA combined with PI3K inhibitor (LY294002) enhanced apoptosis by alleviating the expression of LC3B, (pro-) caspase- 3 and (uncleaved) PARP. DHA induced ferroptosis by attenuating the expression of glutathione peroxidase 4 (GPX4) and nuclear erythroid 2-related factor 2 (Nrf2). DHA-treated PC3R cells produced ROS. The ROS and cytotoxicity were reversed by treatment with ferrostatin-1. DHA combined with docetaxel inhibited EMT by regulating the expression of E-cadhein and N-cadherin. In summary, DHA reversed drug resistance and induced cytoprotective autophagy and ferroptosis by regulating the PI3K/AKT/Nrf2/GPX4 signalling pathway in PC3R cells. We propose that DHA could be developed as a chemosensitizer and that the PI3K/AKT /Nrf2/GPX4 signalling pathway might be a promising therapeutic target for overcoming cancer drug resistance.

[The relationship between serum HBV pgRNA and antigen status in patients with chronic hepatitis B after long-term nucleotide analogues treatment].

Objective: To study the relationship between serum HBV pgRNA and antigen status in patients with chronic hepatitis B treated with long-term nucleotide analogues, and to elucidate the reason and possible mechanism of high relapse rate in antiviral therapy of nucleotide analogues in chronic hepatitis B. Methods: 94 patients with chronic hepatitis B who had been treated with long-term antiviral therapy with nucleotide analogues (more than 2 years) were divided into 5 groups according to their HBeAg and HBsAg levels: e antigen positive group(group1), e antigen negative and HBsAg > 1 500 IU/L group(group2), e antigen negative and 100 IU/L< HBsAg < 1 500 IU/L group(group3), e antigen negative and HBsAg < 100 IU/L group(group4), e antigen negative and HBsAg negative group(group5). The level and detection rate of HBVpgRNA in different antigen states groups were analyzed and compared. In addition, in order to exclude the influence of other factors on the results of this study. The study was divided into groups according to age, gender and treatment time. Results: The detection rate of HBVpgRNA was 95.0% in patients with e antigen positive, while 43.2% in patients with e antigen seroconversion, which was significantly lower than that in patients with e antigen positive (P < 0.05). The detection rate of serum HBVpgRNA was 95.0% in e antigen positive group, 75.0% in group 2, 65.0% in e antigen negative with group 3, 15.0% in group 4 and 0% in group 5. Among them, group 1, group 2 and group 3 was significantly higher than that in group 4 and group 5. There was significant difference between the two groups (P < 0.05). However, there was no difference in the positive rate of serum HBV pgRNA among group 1, group 2 and group 3 (P > 0.05). Similarly, there was no difference in the positive rate of serum HBV pgRNA between group 4 and group 5 (P > 0.05). Moreover, the detection rate of serum HBV pgRNA was not correlated with age, gender and treatment time of nucleotide analogues (P > 0.05). Conclusion: There is a significant correlation between the serological antigen status and the presence of HBV pgRNA in chronic hepatitis B after long-term treatment of nucleotide analogues. The persistence of HBV pgRNA is closely related to the low seroconversion rate of e antigen and the high level of HBsAg. HBV pgRNA can be used as one of the biomarkers to judge the transcription activity and replication status of HBV cccDNA in liver.

Whole-genome resequencing reveals genetic structure and introgression in Pudong White pigs.

Pudong White (PDW) pigs, historically originating from Shanghai, are the only Chinese indigenous pigs characterised by their completely white coats, with the exception of Rongchang pigs. However, there is limited information concerning their overall genetic structure or relationship with other breeds, especially the East Chinese (ECN) and European pigs. To uncover the genetic structure, selection signatures, and potential exotic introgression in PDW pigs, we sampled 15 PDW pigs using whole-genome sequencing (~20×). We then conducted in-depth population genetic analyses in 320 pigs from 27 global pig groups, namely, European wild boars, Chinese wild boars, and outgroup. Neighbour-joining tree and principal component analysis confirmed that PDW pigs belonged to the ecotype of ECN pigs. Both f3, D-statistics, and structure analysis showed that PDW pigs shared apparent alleles with Large White (LW) pigs. Three statistics, rIBD, a haplotype heat map and copy number variation, further indicated that PDW pigs shared apparent alleles with LW pigs at the KIT Proto-Oncogene, Receptor Tyrosine Kinase (KIT) and PARG-MARCHF8 loci, suggesting that the lineage of European pigs in PDW originated from LW pigs. After further detecting the KIT mutations in different pig breeds, PDW was confirmed to have the same duplication region 1, duplication region 2, and the splicing mutation on intron 17 of KIT as LW pigs that determine the white coat colour phenotype in European white pigs. We hypothesised that LW pigs were imported to China ∼110-160 years ago according to the admixture time estimate and then crossed with ECN pigs, resulting in the introgression of the KIT alleles that produce the white coat colour phenotype in the PDW pig breed. To our knowledge, this study presents the first thorough description of the genetic structure of PDW pigs via whole-genome resequencing data; moreover, the results provide a basis for the national project for the conservation of this unique Chinese local population.

Determination of Azide Ions in Blood by Pentafluorobenzyl Derivation Followed by GC-MS.

ABSTRACT: Objective To establish a method for determination of the azide ions in blood by gas chromatography-mass spectrometry （GC-MS） following pentafluorobenzyl derivatization. Methods A blood sample of 0.2 mL was placed into a 10 mL glass test tube, and the internal standard sodium cyanide, derivatization reagent pentafluorobenzyl bromide and catalyst tetradecyl benzyl dimethyl ammonium chloride were added in turn. After vortex mixing, the mixture was heated with low-power microwave for 3 min. After centrifugation, the organic phase was taken for GC-MS analysis. Results The azide ions in blood had a good linear relationship in the mass concentration range of 0.5 to 20 µg/mL. The lowest detection limit was 0.25 µg/mL and the relative recovery was 91.36%-94.58%. The method was successfully applied to a case of death from sodium azide poisoning. The mass concentration of azide ions in the blood of the dead was 11.11 µg/mL. Conclusion The method developed in this paper has strong specificity and is easy to operate, which is suitable for the rapid detection of azide ions in blood.

Insolation triggered abrupt weakening of Atlantic circulation at the end of interglacials.

Abrupt cooling is observed at the end of interglacials in many paleoclimate records, but the mechanism responsible remains unclear. Using model simulations, we demonstrate that there exists a threshold in the level of astronomically induced insolation below which abrupt changes at the end of interglacials of the past 800,000 years occur. When decreasing insolation reaches the critical value, it triggers a strong, abrupt weakening of the Atlantic meridional overturning circulation and a cooler mean climate state accompanied by high-amplitude variations lasting for several thousand years. The mechanism involves sea ice feedbacks in the Nordic and Labrador Seas. The ubiquity of this threshold suggests its fundamental role in terminating the warm climate conditions at the end of interglacials.

Laser vibrational excitation of radicals to prevent crystallinity degradation caused by boron doping in diamond.

Pursuing high-level doping without deteriorating crystallinity is prohibitively difficult but scientifically crucial to unleashing the hidden power of materials. This study demonstrates an effective route for maintaining lattice integrity during the combustion chemical vapor deposition of highly conductive boron-doped diamonds (BDDs) through laser vibrational excitation of a growth-critical radical, boron dihydride (BH2). The improved diamond crystallinity is attributed to a laser-enabled, thermal nonequilibrium suppression of the relative abundance of boron hydrides (BH), whose excessive presence induces boron segregation and disturbs the crystallization. The BDDs show a boron concentration of 4.3 × 1021 cm-3, a film resistivity of 28.1 milliohm·cm, and hole mobility of 55.6 cm2 V-1 s-1, outperforming a commercial BDD. The highly conductive and crystalline BDDs exhibit enhanced efficiency in sensing glucose, confirming the advantages of laser excitation in producing high-performance BDD sensors. Regaining crystallinity with laser excitation in doping process could remove the long-standing bottlenecks in semiconductor industry.

LncRNA SNHG6 can regulate the proliferation and apoptosis of rat degenerate nucleus pulposus cells via regulating the expression of miR-101-3p.

OBJECTIVE: Intervertebral disc (IVD) degeneration (IDD) is a well-known consequence of low back pain, as characterized by aberrant cell proliferation and apoptosis of nucleus pulposus (NP) cells. In the present study, we aimed to investigate the effect of lncRNA small nucleolar RNA host gene 6 (SNHG6) on deregulated functions of degenerative NP cells. MATERIALS AND METHODS: After the establishment of rat IDD models, the mRNA and protein levels of collagen-I (Col-I) and collagen II (Col-II), and mRNA level of SNHG6 were detected by using reverse transcription quantitative Real Time-PCR (RT-qPCR) and Western blot. We further investigated the role and molecular mechanisms of SNHG6 by overexpressing or silencing it in degenerative NP cells. Cell proliferation was measured by MTT assay and EdU staning, and apoptosis was measured by flow cytometry. The target of SNHG6 was identified by starBase and Dual-Luciferase reporter assay. RESULTS: Upregulation of SNHG6 was found in IDD NP cells than in normal cells, associated with higher level of Col-I and lower level of Col-II. Overexpression of SNHG6 inhibited cell proliferation and enhanced apoptosis, accompanied by increased expression of Bax, caspase-3, and p21, as well as decreased expression of Bcl-2, which was in reverse to the treatment of SNHG6 silencing. Moreover, miR-101-3p was indicated as a target of SNHG6, and inhibition of miR-101-3p reversed the effects on proliferation and apoptosis induced by SNHG6. CONCLUSIONS: SNHG6 suppressed cell proliferation and induced apoptosis by increasing expression of Bax, caspase-3, p21 and decreasing Bcl-2 through targeting miR-101-3p, which suggested that SNHG6 could be a potential target in the treatment of IDD.

Expression of SIRT5 protein in gastric cancer cells.

The purpose of this paper was to investigate the dynamic trend of SIRT5 (Sirtuins 5) protein expression in gastric cancer cells, for which a hypoxic gastric cancer cell line was established. Afterward, the parental gastric cancer cell group and hypo group were designed, and the levels of related proteins and mRNAs were detected by using Western blot along with real-time quantitative fluorescence PCR (RT-PCR). The results showed that the expression of SIRT5 in hypoxic gastric cancer cells increased significantly, which could increase the phosphorylation level of c-MET (hepatocyte growth factor receptor) and promote the migration of gastric cancer cells. When the expression of SIRT5 protein was observed under hypoxic conditions, SIRT5 silencing significantly reduced the migration ability of MGC803/hypo cells. It could be predicted that SIRT5-mediated protein desuccinylation played an important role in promoting the migration of hypoxic gastric cancer cells. Therefore, the migration rate of gastric cancer cells could be affected by controlling the expression of SIRT5 protein, which provides a novel idea for the treatment of gastric cancer in the future.

MiR-204 promotes fracture healing via enhancing cell viability of osteoblasts.

OBJECTIVE: To investigate the effects and related mechanisms of miR-204 on fracture healing. MATERIALS AND METHODS: Mouse osteoblastic cell line MC3T3-E1 was used in our experiment. Three groups were established to investigate the potential function between miR-204 and osteoblastic cells: miR-NC group (negative control), miR-204 mimics group (MC3T3-E1 cells transfected with miR-204 mimics) and miR-204 mimics + inhibitor group (MC3T3-E1 cells transfected with miR-204 mimics and inhibitor). After incubation, cell viability, activity of caspase-3, and migration ability of MC3T3-E1 cells, were measured. Further, the expression levels of Runt-related transcription factor 2 (RUNX2) and Osterix (OSX) were detected and analyzed. RESULTS: Compared with miR-NC group, the cell viability and migration ability of MC3T3-E1 cells were enhanced while the activity of caspase-3 was respectively mitigated. Besides, the expression level of RUNX2 and OSX was increased by treatment of miR-204 mimics. However, these variations of the indicators were reversed by the intervention using miR-204 inhibitor. CONCLUSIONS: We revealed the promotion effect of miR-204 on fracture healing, indicating that miR-204 could be a potential therapeutic target for the treatment of a fracture.

Optimization of Growth Temperature of ß-Ga2O3 Thin Films for Solar-Blind Photodetectors.

Monoclinic gallium oxide thin films were deposited on c-plane sapphire substrates at various substrate temperatures ranging from 450 °C to 700 °C by radio frequency magnetron sputtering technology. X-ray diffraction results showed that the deposited ß-Ga2O3 films were oriented at ( 2 ¯ 01) direction. As the substrate temperature increased, the intensity of ß-Ga2O3 peaks increased and bandgap decreased accordingly. Metal/semiconductor/metal structured solar-blind photodetectors based on ß-Ga2O3 thin films growing at various substrate temperatures had been fabricated. The growth temperatures of thin films had no obvious influence on dark current and response to 365 nm light illuminations. The photoelectric properties such as responsivity and response speed of the thin films to 254 nm light illuminations were growth temperature dependent. At an applied bias of 50 V, the photodetectors prepared with 450 °C grown film had the highest responsivity of 2.18 A/W, and the photodetectors prepared with 700 °C grown film had the shortest rising time of 0.95 s under 254 nm light illuminations.

Electrical and Optical Properties of In2O3 Thin Films Deposited on Sapphire Substrate.

In2O3 thin films were prepared on c-plane sapphire substrates using laser molecular beam epitaxy technique. The X-ray diffraction (XRD) patterns revealed that the In2O3 thin films were highly oriented along the (111) direction. The intensity of (222) diffraction peaks mainly depend on growth temperature, and the crystallite sizes mainly depend on oxygen pressure. The carrier concentrations exhibit a decrease with increasing growth temperature and oxygen pressure, meantime, the resistivity increase. The red shift of In2O3 thin films respect to that of bulk In2O3 can be explained by defect energy levels formation, the blue shift of In2O3 thin films depends on carrier concentration, can be explained by Burstein-Moss band-filling effect.

Factors associated with a failed closed reduction for supracondylar fractures in children.

PURPOSE OF THE STUDY: The aim of this retrospective study is to analyze the risk factors causing the failure of closed reduction of children supracondylar fracture. PATIENTS AND METHODS: The children with supracondylar humerus fractures who were treated in our hospital from February 2008 to February 2013, were recorded as well as their age, sex, BMI, injured side, mechanism of injury, associated injuries, fracture type, delay from injury to surgery. Mean comparisons or Chi(2) test were used for univariate analysis of the above factors, and then multivariate logistic regression analysis was used to analyse the possible risk factors, in order to elicit the risk factors associated with a failed closed reduction for supracondylar fractures in children. RESULTS: Univariate analysis showed that BMI, fracture type, duration from injury to surgery, and mechanism of injury had statistically significant association with the failure of closed reduction for children supracondylar fracture (*P=0.021, 0.044, 0.000 and 0.037 respectively). Multivariate logistic regression analysis demonstrated that fracture type (P=0.027, OR=1.177), time from injury to surgery (P=0.022, OR=2.003), and mechanism of injury (P=0.044, OR=4.182) were independent risk factors of a failed closed reduction for paediatric supracondylar fractures. DISCUSSIONS: Gartland type III supracondylar fractures, the peak period of soft tissue swelling and high-energy injury are significant risk factors to warrant open reduction. Treating surgeons should preoperatively carefully evaluate these risk factors and be prepared to treat these injuries accordingly. LEVEL OF EVIDENCE: Level IV retrospective study.

[A study on dental and cranioficial structure of normal shanghai adults occlusion-I the craniofacial structure analysis with posterioanterior cephalometric roentgenography]

OBJECTIVE: To evaluate the characteristics of normal transverse craniofacial structure in Shanghai area and to establish the database of normal Shanghai adults occlusion for posterioanterior cephalometric roentgenography.METHODS:Posterioanterior-cephalometric of the 92 adults subjects with normalocclusion were measured and analysed by Jiffy orthodontic evaluation 5.0 software package.RESULTS:The database of normal Shanghai adults occlusion for posterioanterior cephalometric roentgenography was established. Transverse measurements were intercorrelated, no statistically significant gender differences were demonstrated in craniofacial width, while the molar overjet of male was larger than that of female.CONCLUSION:The normal transverse cranioficial structure in Shanghai area has its own characteristics. The established database would provide a foundation for the diagnosis and treatment of malocclusion.

[Quantitative determination of mass ratio of additives in special super-high pressure hydraulic oil].

Two additives, phenol, 2,6-bis(1,1-dimethylethyl)-4-methyl- and triphenyl phosphate with mass ratio of less than one thousandth in special super-high pressure hydraulic oil were determined by GC-MS. Tributyl phosphate ester was used as internal standard. The correction factor of each additive was determined before analysis. Each sample was analysed for 5 times to get good precision. It is satisfactory to use SIM as the detecting mode, and the CVs of correction factors and mass ratio were about 5%. The problems of how to select the monitoring ion in SIM mode and the sample size required are discussed in this paper. This method is satisfactory in analyzing low mass ratio constituents in a mixture.

Determination of free digoxin in sera of 8 patients with chronic cardiac insufficiency.

AIM: To establish a method for the determination of free digoxin in serum for clinical use and to study the relationship between the free and total digoxin concentrations in chronic cardiac insufficiency patients receiving digoxin with different renal function. METHODS: The ultrafiltration with fluorescence polarization immunoassay was used to determine the concentration of free digoxin. RESULTS: The concentrations of digoxin standards in serum were 0.96, 1.92, and 3.84 nmol.L-1. The relative standard deviation was < 7% for intra-day and < 6% for inter-day determinations. The average recovery was 99.95 +/- 2.18%. The ratio of free/total digoxin in chronic cardiac insufficiency patients with renal dysfunction was lower than that in patients with normal kidneys (63.5 +/- 4.7% vs 75.1 +/- 3.9%, P < 0.01). CONCLUSION: The present method is simple and reliable. In these patients there is an over-measurement for total digoxin concentration, suggesting the presence of elevated endogenous digoxin-like immunoreactive substances.

Luminescent oxygen channeling immunoassay: measurement of particle binding kinetics by chemiluminescence.

A method for monitoring formation of latex particle pairs by chemiluminescence is described. Molecular oxygen is excited by a photosensitizer and an antenna dye that are dissolved in one of the particles. 1 delta gO2 diffuses to the second particle and initiates a high quantum yield chemiluminescent reaction of an olefin that is dissolved in it. The efficiency of 1 delta gO2 transfer between particles is approximately 3.5%. The technique permits real-time measurement of particle binding kinetics. Second-order rate constants increase with the number of receptor binding sites on the particles and approach diffusion control. By using antibody-coated particles, a homogeneous immunoassay capable of detecting approximately 4 amol of thyroid-stimulating hormone in 12 min was demonstrated. Single molecules of analyte produce particle heterodimers that are detected even when no larger aggregates are formed.

Experimental and clinical studies on one-stage arterialization of the venous channels for revascularization of severely ischemic limbs.

A new approach to perfuse arterial blood through venous channels for revascularization of severely ischemic limbs is reported. The procedure studied and used consists of creating an arteriovenous fistula between the normal arterial trunk proximal to the occlusion and the deep venous trunk of the diseased limb, constricting the venous trunk proximal to the anastomosis to one third of its lumen diameter, ligating the communicating and small tributary veins distal to the constriction in the operative field. The results of the experimental and clinical studies have shown that the treated ischemic limb was quickly revascularized without undesirable influence on cardiac function. This new approach has been used in the treatment of severe ischemia involving total 212 limbs in 156 patients, and the results appeared more satisfactory than those treated with staged arteriovenous reversal.

Crystal structure of selenosubtilisin at 2.0-A resolution.

The three-dimensional structure of selenosubtilisin, an artificial selenoenzyme, has been solved at 2.0-A resolution by the method of molecular replacement. Selenosubtilisin is a chemical derivative of the bacterial serine protease subtilisin in which the catalytically essential serine residue has been replaced with a selenocysteine. Its unique hydrolytic and redox properties reflect the intrinsic chemical reactivity of the selenium prosthetic group. Structural analysis of the modified protein reveals that the selenium moiety is selectively incorporated into the side chain of residue 221 and confirms the seleninic acid oxidation state expected from treatment of the enzyme with hydrogen peroxide prior to crystallization. Although the seleninic acid replaces the essential nucleophile in the enzyme's catalytic triad and introduces a negative charge into the active site, the interaction between His64 and Asp32 is not altered by the modification. Hydrogen bonds from the oxygen atoms of the seleninic acid to His64 and to Asn155 in the oxyanion hole confine the prosthetic group to a single well-defined conformation within the active site. These interactions thus provide a structural basis for understanding the seleninic acid's unusually low pKa, the enzyme's relatively sluggish rate of reaction with thiols, and its much more efficient peroxidase activity. Aside from the active site region, the structure of the protein is essentially the same as that previously reported for native subtilisin Carlsberg, indicating the viability of chemical modification strategies for incorporating site-specific changes into the protein backbone. Comparison of the three-dimensional structures of selenosubtilisin and glutathione peroxidase, an important naturally occurring selenoenzyme, provides the means to evaluate how the function of the selenium prosthetic group varies with molecular context.

Kinetic studies on the peroxidase activity of selenosubtilisin.

Selenosubtilisin, a semisynthetic selenoenzyme produced by chemical modification of the serine protease subtilisin, acts as a mimic of glutathione peroxidase, catalyzing the reduction of tert-butyl hydroperoxide by 3-carboxy-4-nitrobenzenethiol. To clarify the mechanism of action of this catalyst, detailed kinetic studies have been carried out. Thiol-mediated reduction converts the seleninic acid form of selenosubtilisin (ESeO2H) into a selenenyl sulfide (ESeSAr). Investigations into the reduction of ESeO2H by the aromatic thiol revealed saturation kinetics and were consistent with a significant lowering of the pKa of the seleninic acid in the enzyme active site. While the reduction of ESeO2H was slow compared with a simple model system, the reduced selenoenzyme (ESeSAr) exhibited a much greater peroxidase activity than model compounds. The enzymic selenocysteine residue was shown to be crucial for this activity, and ping-pong kinetics were observed. A catalytic cycle involving interconversion of the ESeSAr, ESeH, and ESeOH forms of the enzyme has been proposed that is consistent with all the available data. The pH-rate profile for the peroxidase activity indicates the involvement of the active site histidine (His64) in the rate-determining step, which these investigations suggest is attack of ArS- on ESeSAr. The results presented here correlated well with crystallographic and spectroscopic data and provide more detailed information about crucial interactions within the active site of selenosubtilisin.