Sarcopenia is an independent risk factor for all-cause mortality rate in patients with diabetic foot ulcers.

Objective: This study aimed to determine whether sarcopenia affects the all-cause mortality rate of patients with diabetic foot ulcers (DFUs). Research design and methods: The clinic-based observational study included 217 patients treated at the Department of Endocrinology, the First Affiliated Hospital of Chongqing Medical University during a 4-year period. All subjects underwent dual-energy X-ray absorptiometry to determine their body composition during hospitalization. Diagnosis of sarcopenia was based on the Baumgartner diagnostic criteria. Patients were followed up regularly by phone calls until April 1, 2019, and their survival status was recorded.Univariate and multivariate Cox risk ratio regression models were used to analyze factors influencing the all-cause mortality rate of patients with DFUs. Results: Of the 217 patients, 158 people survived (82.7%), 33 died (17.3%), and 26 were lost to follow-up. The median follow-up time was 23 (Range 11-34) months. The majority of patients were male (68.6%), with a mean age of 67.29 ± 11.14 years. The 5-year survival rate was 68.3% and 45.9% for all study patients (n = 217) and sarcopenia patients (n = 81), respectively. Multivariate Cox risk regression model showed that age (HR 1.042[95%CI:1.006, 1.078], P = 0.021), sarcopenia (HR 5.051[95%CI:1.968, 12.961], P = 0.001), and serum creatinine (HR 1.007[95%CI: 1.003, 1.010], P < 0.001) were independent risk factors for all-cause mortality rate of patients with DFUs. Kaplan-Meier survival curve indicated that the survival rate of patients with sarcopenia was significantly lower than non-sarcopenia patients (P < 0.001). Conclusions: Sarcopenia is an independent risk factor for all-cause mortality of patients with DFUs and hence an important prognostic factor for patients with DFUs. Active prevention and improvement of sarcopenia can potentially improve the survival outcomes of this patient population.

Prognostic Value of Coronary Angiography-Derived Fractional Flow Reserve Immediately After Stenting.

Objectives: The aim of this study was to investigate the potential prognostic value of post-percutaneous coronary intervention (PCI) angiography-derived fractional flow reserve (FFR) and its gradient across the stent. Background: Post-PCI FFR and its gradient across the stent have been proved to be associated with clinical outcomes. However, little is known about the prognostic value of post-PCI coronary angiography-derived FFR and its gradient across the stent. Methods: Patients diagnosed with coronary heart disease and participated in drug-eluting stent (DES) clinical trials for stent implantation in a single center were included for this retrospective analysis. A novel coronary angiography-derived FFR (caFFR) and its gradient across the stent were calculated offline using two projections from coronary angiography performed after PCI. Clinical follow-up was completed at 9 months after the index procedure and the primary outcome was target vessel failure (TVF), defined as a composite of target vessel-related myocardial infarction (MI), target vessel-related revascularization (TVR), and cardiac death. Coronary angiography was also performed at the 9 months follow-up time to get data of late lumen loss (LLL) and percent diameter stenosis (%DS). Results: A total of 159 vessels in 136 patients were analyzed. The mean value of post-PCI caFFR was 0.90 ± 0.06. The median value of trans-stent caFFR gradient (ΔcaFFRstent) was 0.04 (interquartile range 0.02-0.08). ΔcaFFRstent>0 was demonstrated in 147 vessels (92.45%). The TVF rate was significantly higher in patients with post-PCI caFFR < 0.90 (4 [8.16%] vs. 1 [1.15%], P = 0.037), which was mainly achieved by the difference between the TVR rate. In the subgroup with lesions located in the left anterior descending coronary artery (LAD), post-PCI caFFR was an independent predictor of LLL (ß = -1.07, 95% CI: -1.74 to -0.39, P = 0.002) and %DS at follow-up (ß = -30.24, 95% CI: -56.44 to -4.04, P = 0.025), ΔcaFFRstent was an independent predictor of LLL (ß=0.98, 95% CI:0.13-1.83, P = 0.026). Conclusion: Suboptimal post-PCI caFFR and trans-stent caFFR gradient were common among vessels immediately after stenting. Lower post-PCI caFFR was associated with a higher rate of 9-month TVF. After LAD PCI, both post-PCI caFFR and its gradient across stent were independent predictors of the neointimal proliferation of the target vessel evaluated by LLL and %DS at follow-up.

Direct Oral Anticoagulants Compared with Vitamin K Antagonists for Left Ventricular Thrombus: A Systematic Review and Meta-analysis.

BACKGROUND: Direct oral anticoagulants (DOACs) are the guideline-recommended therapy for some hypercoagulable diseases but are used off-label for left ventricular thrombus (LVT) owing to a paucity of evidence. We performed a meta-analysis to assess the safety and efficacy of DOACs compared with vitamin K antagonists (VKAs) for LVT treatment. METHODS: We comprehensively searched PubMed, EMBASE, Cochrane Library, and Web of Science databases for studies that compared DOACs with VKAs for LVT treatment. Outcome indicators included stroke or systemic embolism (SSE), thrombus resolution, bleeding, and death. The Newcastle-Ottawa scale was used to evaluate the quality of included studies. Data were analyzed using Review Manager 5.3, and the meta-analysis is registered at PROSPERO (CRD 42020211376). RESULTS: We included 12 observational studies (n = 2262 patients). SSE was similar for DOACs and VKAs groups (odds ratio (OR) = 1.01, 95% confidence interval (CI) 0.66-1.54, P = 0.95). For thrombus resolution, DOACs were not significantly different to VKAs (OR = 1.15, 95% CI 0.54-2.45, P = 0.71). DOACs and VKAs had a similar bleeding risk (OR = 0.78, 95% CI 0.45-1.35, P = 0.37). DOACs and VKAs groups had a comparable mortality (OR = 0.91, 95% CI 0.50-1.65, P = 0.76). Subgroup analysis showed that post-acute myocardial infarction (AMI) patients using DOACs had a lower risk of SSE (OR = 0.24, 95% CI 0.07-0.87, P = 0.03) and bleeding (OR = 0.38, 95% CI 0.18-0.81, P = 0.01). CONCLUSION: DOACs and VKAs showed no difference in the safety and efficacy of patients with LVT. DOACs might be superior to VKAs for LVT treatment in post-AMI patients.

The Destruction of the Anaerobic Environment Caused by Rumen Fistula Surgery Leads to Differences in the Rumen Microbial Diversity and Function of Sheep.

This experiment was to study the impact of rumen fistula surgery on the rumen microbios in sheep. Six male adult Hu sheep (48.8 ± 0.23 kg, 0.5 years) were fed at 0700 and 1,800 with ad libitum access to water. The rumen fistula was installed in the same batch from 0600 to 0900. Monitoring the dry mater intake and the output of dry mater faces 1, 2, 4, 6, 8, 10, 12, 14 days after fistulated surgery. The collection of rumen fluid was arranged at 1d during rumen surgery (DRS1), 3d after rumen surgery (ARS3), and 14d after rumen surgery (ARS14) for volatile fatty acid (VFA) and DNA extraction for sequencing and bioinformatics analysis. There was no difference in DMI, the pH apparent digestibility of dry matter, organic matter, neutral detergent fiber, acid detergent fiber both before and 14 days after surgery. Increases were observed in the acetate and total VFA at ARS3. There was no difference in digestion of dry material, organic material, neutral detergent fiber, and acid detergent fiber before and after surgery. The relative abundance of Bacteroides decreased from 61.96% at DRS1 to 28.85% at ARS3. In comparison with the DRS1 and ARS3, the relative abundance of Firmicutes in the ARS14 increased to 44.58% (P < 0.01). Proteobacteria increased from 11.33% at DRS1 to 51.66% at ARS3 and then decreased to 11.39% at ARS14. Prevotella decreased form 61.06% at DRS1 to 28.04% in the ARS3. Succinivibrio increased from 8.32% at DRS1 to 48.58% at ARS3, but decreased to 10.43% in the ARS14. Compared with DRS1 and ARS3, the ARS14 was higher in the Simpson and Shannon index. As for the BugBase function prediction, rumen fistula surgery increased the microorganism abundance of aerobic and facultative anaerobic phenotype, and anaerobic phenotype was decreased in the ARS3. There was higher microorganism abundance of aerobic phenotype in the ARS14 than before fistula installation. In conclusion, the rumen fistula surgery destroys the anaerobic environment of rumen, leading to differences in rumen microbial diversity and function, but the apparent digestibility and total VFA were not affected.

Expanded flap to repair facial scar left by radiotherapy of hemangioma.

This study explored the feasibility and clinical efficacy of expanded flap to repair facial scar left by radiotherapy of hemangioma. From March 2000 to April 2011, 13 cases of facial cicatrices left by radiotherapy of hemangioma have been treated with implantation surgery of facial skin dilator under local anesthesia. After water flood expansion for 1-2 months, resection of facial scar was performed, and wound repairing with expansion flap transfer was done. Thirteen patients were followed up from 5 months to 3 years. All patients tolerated flap transfer well; no contracture occurred during the facial expansion flap transfer. The incision scar was not obvious, and its color and texture were identical to surrounding skin. In conclusion, the use of expanded flap transfer to repair the facial scar left by radiotherapy of hemangioma is advantageous due to its simplicity, flexibility, and large area of repairing. This method does not affect the subsequent facial appearance.

Quantification of puerarin in plasma by on-line solid-phase extraction column switching liquid chromatography-tandem mass spectrometry and its applications to a pharmacokinetic study.

A highly precise, automatic and rapid method for quantification of puerarin in canine and human plasma using an on-line solid-phase extraction (SPE) column switching procedure combined with liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS) was developed. The eluent of SPE column consisted of acetonitrile/methanol/0.1% formic acid (25/25/50) at a flow rate of 0.2mLmin(-1). Puerarin was analyzed by a linear ion trap mass spectrometer, LTQ-MS, operating in the negative ion and selective reaction monitoring (SRM) acquisition mode. Method validation results demonstrated that the linear calibration curve covered a wide range of 0.39-400.00ngmL(-1), the correlation coefficients (r(2)) were above 0.999. The lower limit of detection (LLOD) with the signal-to-noise (S/N) ratio higher than 12 was 0.39ngmL(-1). The intra- and inter-batch precisions were less than 7.61% and 6.42%, respectively. The accuracy was well within the accept limit. The on-line SPE column switching HPLC-MS system was applied to pharmacokinetic (PK) study of puerarin after a single orally dose in beagles. And the optimum conditions were successfully utilized to quantify puerarin in human plasma, which indicated the feasibility and the reliability of this method for application in preclinical and clinical PK studies of isoflavone drugs.

[Producing recombinant adenovirus encoding green fluorescent protein (Ad-GFP) by suspension cultured HEK-293 N3S cells].

Adenovirus vectors are one of the most promising gene transfer systems. They are of great value for gene therapy because these vectors achieve temporal high-level transgene expression and high gene transfer efficiency. To meet increasing needs of adenovirus vectors for gene therapy programs, parallel development of efficient, scalable and reproducible production processes is required. Perfusion cultivation of 293 cells is one of the most commonly used methods to produce adenovirus vectors and it is suitable for industrialized production specially. Experimental studies had been carried out to produce recombinant adenovirus containing the green fluorescent protein gene (Ad-GFP) by perfusion cultivation of HEK-293 N3S cells in a 5L stirring bioreactors. Perfusion rate was 1-2 volume/day. To infect the 293 N3S cells with Ad-GFP at the density of (2-4) x 10(6) cells/ ml. The time of collecting cells was 48 hours post infection. After three rounds of freeze/thaw and centrifugation, the crude viral lysates were stored at--80 degrees C until use. Then to get the Ad-GFP products by 2 x CsCl-gradient purification. The purity of the products was determined by the A260/A280 ratio and a high performance liquid chromatography (HPLC) assay. The infective titer was determined by a TCID50 assay. The culture term was 10-12 days. The infectious titer, the number of virus particle and the ratio of infectious titer to virus particle for the product were 1.0 x 10(11) IU/mL, 1.68 x 10(12) VP/mL and 6.0% IU/VP respectively. The A260/A280 ratio was 1.33, and the purity determined by HPLC was 99.2%. The cell specific productivity was around 1000 IU/cell. By perfusion cultivation of 293 N3S cells in a 5L stirring bioreactors, we established the production process for Ad-GFP, which paves a way to produce other recombinant adenovirus for gene therapy.

Pharmacokinetics of His-tag recombinant human endostatin in Rhesus monkeys.

AIM: To study the pharmacokinetics and accumulation of an Escherichia coli expressed His-tag fused recombinant human endostatin (rh-endostatin) in Rhesus monkeys. METHODS: Rh-endostatin was iv or sc injected in Rhesus monkeys, and the rh-endostatin concentration in serum samples was determined by an enzyme immunoassay (EIA) method. The serum drug concentration-time data were analyzed by compartmental analysis using the practical pharmacokinetic program 3p97. RESULTS: Following iv administration at a dose rate of 1.5, 4.5, and 13.5 mg/kg in rhesus monkeys, the concentration-time curves of rh-endostatin were best fitted to a three-compartment open model. AUC(0-infinity) linearly increased with dose, while Cls exhibited no significant difference among different dose groups. The terminal half-lives (lambda3) were 8+/-8, 3.1+/-1.4, and 20+/-14 h, respectively. After sc administration at a dose rate of 1.5 mg/kg, the concentration-time curve was best fitted to a two-compartment open model, with a terminal half-life (T(1/2beta)) of 8+/-3 h. Bioavailability following sc injection was approximately 70%+/-3%. After consecutive iv injection of rh-endostatin at a dose rate of 1.5 mg.kg(-1).d(-1) for 7 d, the AUC(0-24 h) substantially increased from 22+/-13 mg.h.L(-1) (d 1) to 50+/-29 mg.h.L(-1) (d 7), with an accumulation factor of 2.3+/-0.6 (P < 0.05). CONCLUSION: The pharmacokinetic behavior of rh-endostatin in Rhesus monkeys complies with linear kinetics within the examined dose range. It tends to be accumulated in bodies after repeated administration at a dose level of 1.5 mg.kg(-1).d(-1) for more than 7 consecutive days.

Antisense candidates against protein kinase C-alpha designed based on phylogenesis and simulant structure of mRNA.

AIM: To optimize the antisense drug design by the combined method of phylogenetic analysis and secondary structure prediction and to get ideal candidates. METHODS: The phylogenetic analysis and the secondary structure simulation were performed by computer. Oligodeoxynucleotides (ODN) were designed against the full-conserved blocks with low local reaction free energy of protein kinase C (PKC)-alpha mRNA. The in vitro effects of ODN were evaluated by human A549 lung carcinoma cells and mouse B16-BL6 melanoma cells, the expression of target mRNA was detected by in situ hybridization and RT-PCR. The in vivo effects of ODN were also evaluated by models of A549 xenografts in nude mice and B16 melanoma in mice. RESULTS: Three ODN had significantly lower IC50 values than that of ISIS3521, the positive control, on A549 cells in vitro. Five ODN inhibited the growth of B16-BL6 cells with IC50 <100 nmol/L, while IC50 of ISIS3521 was >200 nmol/L. In situ hybridization and RT-PCR showed that the best candidate AP1261 inhibited the expression of PKC-alpha at mRNA level in a dose-dependent manner. AP1261 inhibited the growth of A549 and B16 tumors in vivo at 0.005-0.5 mg.kg(-1).d(-1). The inhibitory rate of AP1261 on A549 tumors was greater than that of ISIS3521 at the same dose. ISIS3521 did not affect the growth of B16 tumors. CONCLUSION: AP1261 may be of value as an antitumor agent or adjuvant and the combined method of phylogenetic analysis and secondary structure prediction is a potential helpful tool for antisense drug design.

Pharmacokinetics and partial thromboplastin time after intravenous recombinant hirudin variant-2 in rhesus monkeys.

AIM: To study the pharmacokinetics (PK) and changes of kaolin partial thromboplastin time (KPTT) following single or multiple (7 d) dosing of a novel recombinant hirudin variant-2 (rHV-2) via the route of iv bolus injection (50 % of the total dose) plus infusion (the remained 50 % of the dose) in rhesus monkeys. METHODS: A crossover design was applied to research the PK and KPTT profiles of rHV-2 after single (with total dose at 1, 3, and 6 mg/kg, respectively) and multiple dosing (3 mg/kg). An enzyme-linked immunosorbent assay (ELISA) method was utilized to determine the level of rHV-2 in plasma. RESULTS: The concentration profiles of rHV-2 during or after administration were dependent both on the loading dose and the infusion rate. Mean Cmax after bolus in three single dose groups were 2.90, 9.78, and 15.68 mg/L, respectively. Infusions at rate of 8.35, 25, and 50 g/kg/h in 1 h resulted in steady-state levels of 0.73-0.86, 1.94-2.04, and 5.41-5.59 mg/L, respectively. The plasma rHV-2 levels during or after administration among doses were significantly different at most of the time points. Area under concentration-time curve (AUC) increased linearly with dose but systemic clearances were similar among different groups. KPTT was significantly prolonged (compared with baseline) at all dose levels, and trended to increase with dose. CONCLUSION: Both the loading dose and the infusion rate are very important for controlling the rHV-2 level, and the data may be helpful for optimizing dosage-regimen in clinical trials.

Sequencing Analysis of Oligodeoxynucleotide by Matrix-assisted Laser Desorption Ionization Time-of-flight Mass Spectrometry.

A mixed matrix alpha-cyano 4-hydroxycinnamic acid (alpha-Cyano)/3-hydroxypicolinic acid (3HPA) was found to be a good matrix for the analysis of oligodeoxynucleotides by matrix-assisted laser desorption ionization time-of-flight mass spectrometry ( MALDI-TOF-MS). Using the mixed matrix, a similar sensitivity was obtained for the different oligodeoxynucleotides: d(T)(10), d(A)(10) and d(C)(10). This methodology can be used in combination with the partial digestion of oligodeoxynucleotides by 5'- and 3'-exonucleases as a powerful tool for sequencing analysis of oligonucleotides.