[(SIPr)Ag(CF2 H)]: A Shelf-Stable, Versatile Difluoromethylation Reagent.

Due to its unique physical and electrophilic properties, the difluoromethyl group (-CF2 H) has been playing an irreplaceable role in the field of pharmaceutical and agrochemical industry. Methods that could efficiently incorporate the difluoromethyl group into the target molecules are increasing in the recent years. Developing a stable and efficient difluoromethylating reagent is thus highly attractive. In this review, we describe the development of a nucleophilic difluoromethylation reagent [(SIPr)Ag(CF2 H)], including its elemental reaction, difluoromethylation reaction with different types of electrophiles, and its application in the synthesis of a nucleophilic and an electrophilic difluoromethylthiolating reagent.

[Ph4 P]+ [Cu(CF2 H)2 ]- : A Powerful Difluoromethylating Reagent Inspired by Mechanistic Investigation.

The quest of the active species in copper-mediated difluoromethylation of aryl halides led to the discovery of a powerful difluoromethylating reagent [Ph4 P]+ [Cu(CF2 H)2 ]- 2. Complex 2 was able to difluoromethylate a variety of electrophiles including electron-deficient and electron-rich aryl iodides, heteroaryl iodides, activated heteroaryl bromides, chloride and aryl bromides with a directing group, as well as other electrophiles such as alkenyl iodide, benzyl bromides, allyl bromides, alkyl iodide, allyl carbonates and acid chloride. In addition, in the presence of an oxidant, complex 2 reacted with various lithium nbutyl arylboronic acid pinacol esters, alkyne and heteroarene. Moreover, complex 2 could transmetalate the difluoromethyl reagent to other metals such as [Ph4 P]+ [CuCl2 ]- and [(DPPF)PdCl2 ].

Difluoromethylation of Alkyl Bromides and Iodides with TMSCF2H.

We describe, for the first time, two protocols for direct difluoromethylation of unactivated alkyl bromides and iodides. Reactions of alkyl iodides with TMSCF2H were mediated by a copper catalyst using CsF as the activator, while reactions of less reactive alkyl bromides required a combination of palladium and a stoichiometric amount of CuI as the catalysts. Preliminary mechanistic studies of the synergistic Pd/Cu-catalyzed difluoromethylation of alkyl bromides suggest that it proceeds likely via a Pd(I)/Pd(III) catalytic cycle.

Two Ligands Transfer from Ag to Pd: En Route to (SIPr)Pd(CF2H)(X) and Its Application in One-Pot C-H Borylation/Difluoromethylation.

A process for the concurrent transfer of both the NHC ligand and the difluoromethyl group from [(SIPr)Ag(CF2H)] to PdX2 (X = Cl, OAc, and OPiv) for the preparation of [(SIPr)Pd(CF2H)X] complexes is described. These complexes were air-stable and easily underwent transmetalation with aryl pinacol boronate/reductive elimination to generate ArCF2H in high yields. Based on this discovery, the first one-pot C-H borylation and difluoromethylation process for the preparation of difluoromethylated (hetero)arenes was developed.

Identification and Expression of Several Circular RNAs and Knockdown of hsa_circ_0005556 Exerts Oncogenic Functions by miR-767-5p in Gastric Cancer.

BACKGROUND Gastric cancer (GC) remains one of the most fatal digestive cancers in the world; nevertheless, its etiology remains vague. With the development of bioinformatics analysis, numerous circular RNAs (circRNAs) have been found to be dysregulated in GC. However, the functions of a large portion of dysregulated circRNAs in GC need further validation. In this study, we aimed to validate the biological functions of circ_0005556, which was previously identified to be dysregulated in GC. MATERIAL AND METHODS Levels of circRNAs and miRNAs in GC tissues and cells were estimated by qRT-PCR. The target miRNAs of circ_0005556 were predicted by bioinformatics methods. The interplay between circ_0005556 and miR-767-5p was validated by dual-luciferase reporter and circRNA immunoprecipitation assays. The effects of circ_0005556 and miR-767-5p on GC cell viability, apoptosis, migration, and invasion were assessed by MTT, flow cytometry, wound-healing and in vitro transwell experiments, respectively. RESULTS The upregulation of circ_0005556 was validated by qRT-PCR in GC tissues and cells, and a higher circ_0005556 level indicated a poorer prognosis. miR-767-5p was demonstrated to target circ_0005556 in GC cells, and a negative correlation was found between their expression levels in GC tissues. Knockdown of circ_0005556 promoted miR-767-5p expression in GC cells. Knockdown of circ_0005556 was revealed to repress GC cell viability, invasion, and migration and to promote GC cell apoptosis. Moreover, overexpression of miR-767-5p could significantly augment the repressive impacts of circ_0005556 knockdown on GC cell progression in vitro. CONCLUSIONS The in vitro knockdown of circ_0005556 remarkably repressed GC cell progression by increasing the expression of miR-767-5p.

The Core Knowledge and Skills of Nursing Competency Regarding Mealtime Assistance for Hemiplegic Patients in China.

PURPOSE: Hemiplegic patients often experience malnutrition and feeding risks due to disabilities and inadequate nursing support; nursing roles regarding mealtime assistance remain unclear in China. Therefore, this study aimed to clarify the core knowledge and skills of nursing competency regarding mealtime assistance for hemiplegic patients in China. METHODS: A cross-sectional survey of self-administered questionnaire to 640 nurses (response rate: 57.7%) from two tertiary teaching hospitals and two sanatoriums was conducted. Survey content included 25 items regarding the mealtime assistance competency plus 6 items on demographic characteristics. The factor structure of the 25 items was explored and verified by exploratory and confirmatory factor analysis. Its reliability was confirmed by Cronbach's alpha. RESULTS: Three factors including 22 items with a cumulative rate of 60.9% were identified: "Assistance knowledge and skills for acute period", "Knowledge about assistance and guidance for recovery period," and "Professional basic knowledge regarding hemiplegia." Its reliability was ensured with Cronbach's alpha ranging from .86 to .96. In addition, evidence for its construct validity was obtained, as structural equation modeling revealed a good fit to the data within the allowable range based on various fit indices. CONCLUSION: This study clarified the core knowledge and skills regarding mealtime assistance competency for hemiplegic patients, with "Assistance for acute period" most concerned, followed by "assistance and guidance for recovery period," and "Professional basic knowledge," and the latter two need more attention. Obtained results can provide useful evidence for competent nursing practice to improve the quality of mealtime assistance for hemiplegic patients in China.

Efficiency of unlocking or locking existing protected areas for identifying complementary areas for biodiversity conservation.

It is well known that existing protected areas (PAs) should function as focal areas for expanding PA systems. The optimal complementary conservation areas are often identified by implementing two approaches in systematic conservation planning, i.e., unlocking or locking existing PAs. However, evidence-based studies are lacking for clarifying the efficiencies of these two planning approaches. With Sichuan in southwest China - part of a global biodiversity hotspot - as one case, this study first assessed the ecological representativeness of existing nature reserves (NRs). Using 32 natural vegetation types as the conservation features, we then implemented a systematic conservation planning process by running Marxan software with NR-unlocked and NR-locked scenarios. A human disturbance index was also included as a penalty function in Marxan for achieving cost-effective planning. We finally investigated the efficiencies of the unlocking and locking planning approaches by comparing the outcomes of the NR-unlocked and NR-locked scenarios. We found that existing NRs were geographically biased towards the western mountainous regions with high elevations and low human disturbance levels. For achieving the same quantitative conservation targets, the total area of the NR-locked priority conservation areas was 18.6% larger than that of the NR-unlocked areas, whereas the area of NR-locked complementary areas to existing NRs was 15.3% smaller than that of NR-unlocked ones. Moreover, the NR-locked priority conservation areas had higher ecological representativeness than NR-unlocked areas. The results suggest that if a completely new PA system is to be established without considering existing PAs, the unlocking approach could more efficiently achieve the full conservation targets at lower costs of land area and with better connected habitats. When existing PAs must be used as focal areas for expansion, the locking approach is more cost-effective for filling conservation gaps by requiring smaller amounts of complementary areas. Our analysis provides evidence-based support for expanding the current PA systems in a cost-effective manner.

Application of enhanced recovery after surgery in patients undergoing radical cystectomy.

OBJECTIVE: This study was performed to evaluate the application of enhanced recovery after surgery (ERAS) in patients undergoing radical cystectomy (RC). METHODS: The clinical data of 192 patients who underwent RC were collected in this retrospective cohort study. Among them, 91 patients who underwent ERAS were allocated to the ERAS group, and the remaining 101 patients who underwent traditional postoperative care procedures were allocated to the non-ERAS group. Perioperative indexes in the two groups were compared. The ERAS components included rehabilitation exercise, carbohydrate fluid loading, cessation of nasogastric tubes, omission of oral bowel preparation, regional local anesthesia, body-warming procedures, reduced drainage use, and early postoperative drinking and eating. RESULTS: The times from RC to first water intake, first ambulation, first anal exhaust, first defecation, and pelvic drainage tube removal were significantly shorter and the hospitalization costs were significantly lower in the ERAS than non-ERAS group. The intraoperative blood loss volume, blood transfusion rate, readmission rate, and incidence of postoperative complications were also significantly lower in the ERAS than non-ERAS group. CONCLUSION: ERAS may effectively accelerate patient rehabilitation and reduce the length of stay, incidence of postoperative complications, readmission rates, and hospitalization costs for patients undergoing RC.

Clinical Significance of Peripheral Blood PCA3 Gene Expression in Early Diagnosis of Prostate Cancer.

Prostate cancer antigen 3 (PCA3) is one of the most promising genes currently investigated as a specific tumor biomarker for the diagnosis of prostate cancer. The purpose of this study was to investigate PCA3 gene expression in peripheral blood of prostate cancer (PCa) and benign prostate hyperplasia (BPH), and further to assess its clinical significance. We determined the copies of PCA3 mRNA in peripheral blood of PCa and BPH from 115 samples (PCa, n=78; BPH, n=37) using a quantitative reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) with TaqMan assay. The sensitivity and specificity of PCA3 for the diagnosis of prostate cancer was compared with that of prostate-specific antigen (PSA) by receiver operating characteristic (ROC) curve analysis. To evaluate the association between PCA mRNA and disease progression, we analyzed PCA3 levels in connection with Gleason score and TNM stage of PCa. The clinical data revealed that expression of PCA3 gene was significantly higher in PCa than in BPH. Moreover, PCA3 mRNA was significantly higher in PCa patients with a Gleason score ≥8 than in those with a Gleason score ≤7 (P<.01). The results showed that the area under the curve (AUC) was 0.790, 0.606, and 0.620 for the copy number of PCA3, PSA level, and significantly free PSA (fPSA) level, respectively. Increased PCA3 in peripheral blood is correlated with PCa, and the detection of PCA3 may significantly reduce adverse screening outcomes. PCA3 gene expression in peripheral blood had a promising clinical application in the early diagnosis of PCa.

Conditional Inducible Triple-Transgenic Mouse Model for Rapid Real-Time Detection of HCV NS3/4A Protease Activity.

Hepatitis C virus (HCV) frequently establishes persistent infections that can develop into severe liver disease. The HCV NS3/4A serine protease is not only essential for viral replication but also cleaves multiple cellular targets that block downstream interferon activation. Therefore, NS3/4A is an ideal target for the development of anti-HCV drugs and inhibitors. In the current study, we generated a novel NS3/4A/Lap/LC-1 triple-transgenic mouse model that can be used to evaluate and screen NS3/4A protease inhibitors. The NS3/4A protease could be conditionally inducibly expressed in the livers of the triple-transgenic mice using a dual Tet-On and Cre/loxP system. In this system, doxycycline (Dox) induction resulted in the secretion of Gaussia luciferase (Gluc) into the blood, and this secretion was dependent on NS3/4A protease-mediated cleavage at the 4B5A junction. Accordingly, NS3/4A protease activity could be quickly assessed in real time simply by monitoring Gluc activity in plasma. The results from such monitoring showed a 70-fold increase in Gluc activity levels in plasma samples collected from the triple-transgenic mice after Dox induction. Additionally, this enhanced plasma Gluc activity was well correlated with the induction of NS3/4A protease expression in the liver. Following oral administration of the commercial NS3/4A-specific inhibitors telaprevir and boceprevir, plasma Gluc activity was reduced by 50% and 65%, respectively. Overall, our novel transgenic mouse model offers a rapid real-time method to evaluate and screen potential NS3/4A protease inhibitors.

[Establishment and identification of human hepatocellular carcinoma line stably expressing hepatitis C virus core protein].

OBJECTIVE: To establish SMMC-7721 human hepatocellular carcinoma cell line stably expressing hepatitis C virus (HCV) core protein. METHODS: A lentiviral vector containing HCV core gene was constructed and transfected into HEK293T cells to package recombinant lentivirus (rLV-core) containing ZsGreen and HCV core genes. The SMMC-7721 cells were infected with the rLV-core. The expression of HCV core mRNA was examined by real-time fluorescent quantitative PCR and the HCV core protein was detected by immunofluorescence cytochemistry and Western blotting. The stably transfected cell line was screened. RESULTS: The lentiviral vector was confirmed by enzyme digestion and sequencing. The green fluorescence was seen under fluorescence microscope 48 hours after virus packaging. The SMMC-7721 cell line stably expressing HCV core protein was obtained after infected with the rLV-core. Real-time PCR showed the expression of HCV core mRNA, and both immunofluorescence cytochemistry and Western blotting verified the expression of HCV core protein. CONCLUSION: The SMMC-7721 human hepatocellular carcinoma cell line stably expressing HCV core protein has been established successfully.

Preliminary evaluation of a novel oral delivery system for rhPTH1-34: in vitro and in vivo.

rhPTH1-34 is clinically used for osteoporosis treatment. However, this peptide drug has no oral bioavailability because of proteolysis and low membrane permeability in gastrointestinal gut. This study explored the possibility of absorption enhancement for rhPTH1-34 through the oral delivery of the microemulsion. The microemulsion (85:15, oil/water) consisting of Labrasol, Crodamol GTCC, Solutol HS 15, d-α-tocopheryl acetate (6:2:1:1, w/w) and saline water was developed and characterized, including particle size, morphology, drug loading efficiency and permeability, stability and pharmacokinetics. The microemulsion showed high drug loading efficiency (83%) and permeability, and significantly higher resistance to proteolysis in vitro study. The relative oral bioavailability was 5.4% and 12.0% when delivered to gastric and ileum. Besides, osteoporosis rats were induced and treated with oral rhPTH1-34 microemulsion (0.05 mg/kg), injection (0.01 mg/kg) and vehicle, respectively, for 8 weeks. The proximal tibia bone mineral content and density in oral rats (0.188 ± 0.008 g, 0.283 ± 0.014 g/cm(2)) was significantly increased compared to the control rats (0.169 ± 0.006 g, 0.266 ± 0.011 g/cm(2)), reaching to the sham rats. And the proximal tibia microstructure of oral rats was improved greatly, approaching sham level too. These findings revealed that oral microemulsion may represent an effective oral delivery system for rhPTH1-34.