Lipopeptides from Bacillus velezensis induced apoptosis-like cell death in the pathogenic fungus Fusarium concentricum.

AIMS: Stem rot caused by Fusarium concentricum is a new disease of Paris polyphylla reported by our research group. The present study investigates the growth inhibitory and apoptotic effects of Bacillus velezensis FJAT-54560 lipopeptide against F. concentricum. METHODS AND RESULTS: HPLC preparation and LC-MS analysis results show that the crude lipopeptides secreted by Bacillus velezensis FJAT-54560 isolated from Jasminum sambac consist of C14-17 iturin A, C14 fengycin B, C16 fengycin A/A2, C18 fengycin A, C20 fengycin B2, C21 fengycin A2, C22-23 fengycin A, C12-16 surfactin A, and C15 surfactin A derivatives. The mass ratios (g/g) of iturin, fengycin, and surfactin in lipopeptides are 2.40, 67.51, and 30.08%, respectively. Through inhibition zone and inhibition rate experiments, we found that crude lipopeptides and purified fengycin exhibit strong antifungal activity against F. concentricum, including accumulation of reactive oxygen species, loss of mitochondrial membrane potential, DNA fragmentation, Ca2+ accumulation, chromatin condensation, and phosphatidylserine externalization. Transcriptomic analysis indicates that crude lipopeptide-induced apoptosis in F. concentricum cells may be mediated by apoptosis-inducing factors and apoptosis mediators and can serve as a metacaspase-independent model. CONCLUSION: Lipopeptides from Bacillus velezensis FJAT-54560 can control the pathogenic fungus F. concentricum by inducing apoptosis.

Medial patellofemoral ligament reconstruction using a digital tensiometer to determine graft tension: Surgical technique and mid-term follow-up.

PURPOSE: The optimal method for achieving proper graft tension during patellofemoral ligament reconstruction is a topic of debate. In the past, a digital tensiometer was used to simulate the knee structure, and a tension of approximately 2N was identified as suitable for restoring the patellofemoral track. However, it is unclear whether this tension level is sufficient during the actual surgery. The objective of this study was to verify the efficacy of graft tension using a digital tensiometer for medial patellofemoral ligament (MPFL) reconstruction and to conduct a mid-term follow-up. METHODS: The study enrolled 39 patients who had experienced recurrent patellar dislocation. Preoperative computed tomography scans and X-rays confirmed patellar instability, patellar tilt angle patellar congruence angle and the history of dislocation and patellar apprehension test. Knee function was evaluated using preoperative and postoperative Lysholm and Kujala scores. RESULTS: The study included 39 knees, comprising 22 females and 17 males, with an average age of 21.10 ± 7.26. The patients were followed up for at least 24 months through telephone or face-to-face questionnaires. All patients had a preoperative history of ≥2 patellar dislocations, none of which were surgically treated. During surgery, all patients underwent isolated MPFL reconstruction and lateral retinacula release. The mean Kujala and Lysholm scores were 91.28 ± 4.90 and 90.67 ± 5.15, respectively. The mean PTA and PCA were 11.5 ± 2.63 and 2.38 ± 3.58, respectively. The study found that a tension of approximately 27.39 ± 5.57N (14.3-33.5N) was required to restore the patellofemoral track in patients with recurrent patellar dislocation. No patients required reoperation during the follow-up period. Overall, 36 out of 39 patients (92.31%) reported no pain when completing daily activities at the last follow-up. CONCLUSION: In conclusion, a tension level of approximately 27.39 ± 5.57N is necessary to restore normal patellofemoral relationships during clinical practice, which indicates that using a tension of 2N is too low. The use of a tensiometer during patellofemoral ligament reconstruction is a more accurate and reliable surgical procedure for treating recurrent patellar dislocation.

Surgical site infection following elective orthopaedic surgeries in geriatric patients: Incidence and associated risk factors.

The purposes of this study were to investigate the incidence of surgical site infection (SSI) following geriatric elective orthopaedic surgeries and identify the associated risk factors This was a retrospective two-institution study. Between January 2014 and September 2017, patients aged 60 years or older undergoing elective orthopaedic surgeries were included for data collection and analysis. SSI was identified through the review of patients' medical records for the index surgery and through the readmission diagnosis of SSI. Patients' demographics, characteristics of disease, surgery-related variables, and laboratory examination indexes were inquired and documented. Univariate and multivariate logistic analyses were performed to determine independent risk factors for SSI. There were 4818 patients undergoing elective orthopaedic surgeries, and within postoperative 1 year, 74 patients were identified to develop SSIs; therefore, the overall incidence of SSI was 3.64%, with 0.4% for deep and 1.1% for superficial infection. Staphylococcus aureus (25/47, 53.2%) and coagulase-negative staphylococci (11/47, 23.4%) were the most common causative pathogens; half of S. aureus SSIs were caused by Methicillin-resistant Staphylococcus aureus (MRSA) (12/25, 48.0%). Five risk factors were identified to be independently associated with SSI, including diabetes mellitus (odds ratio [OR], 3.7; 95% confidence interval [95% CI], 1.7-5.6), morbid obesity (OR, 2.6; 95% CI, 1.3-3.9), tobacco smoking (OR, 4.2; 95% CI, 2.1-6.4), surgical duration>75th percentile (OR, 1.9; 95% CI, 1.0-2.9), and ALB < 35.0 g/L (OR, 2.3; 95% CI, 1.3-3.4). We recommend the optimisation of modifiable risk factors such as morbid obesity, tobacco smoking, and lower serum albumin level prior to surgeries to reduce the risk of SSI.

Intrathecal injection of lentivirus-mediated glial cell line-derived neurotrophic factor RNA interference relieves bone cancer-induced pain in rats.

Bone cancer pain is a common symptom in cancer patients with bone metastases and the underlying mechanisms are largely unknown. The aim of this study is to explore the endogenous analgesic mechanisms to develop new therapeutic strategies for bone-cancer induced pain (BCIP) as a result of metastases. MRMT-1 tumor cells were injected into bilateral tibia of rats and X-rays showed that the area suffered from bone destruction, accompanied by an increase in osteoclast numbers. In addition, rats with bone cancer showed apparent mechanical and thermal hyperalgesia at day 28 after intratibial MRMT-1 inoculation. However, intrathecal injection of morphine or lentivirus-mediated glial cell line-derived neurotrophic factor RNAi (Lvs-siGDNF) significantly attenuated mechanical and thermal hyperalgesia, as shown by increases in paw withdrawal thresholds and tail-flick latencies, respectively. Furthermore, Lvs-siGDNF interference not only substantially downregulated GDNF protein levels, but also reduced substance P immunoreactivity and downregulated the ratio of pERK/ERK, where its activation is crucial for pain signaling, in the spinal dorsal horn of this model of bone-cancer induced pain. In this study, Lvs-siGDNF gene therapy appeared to be a beneficial method for the treatment of bone cancer pain. As the effect of Lvs-siGDNF to relieve pain was similar to morphine, but it is not a narcotic, the use of GDNF RNA interference may be considered as a new therapeutic strategy for the treatment of bone cancer pain in the future.

[Systematic reviews of mini-invasive surgery versus standard approaches for total knee arthroplasty].

OBJECTIVE: To conduct a systematic review to compare the early efficacies of minimally invasive surgery (MIS) versus conventional approaches in TKA (total knee arthroplasty). METHODS: Randomized controlled trials (RCTs) and clinical controlled trials (CCTs) were retrieved from the databases of MEDLINE (1996.6 - 2010.12), EMBASE (1996.6 - 2010.12), PubMed (1996 - 2010.12) and Cochrane Library (Issue 2, 2012). Journal of Orthopedics (from establishment to December 2010) and Orthopedic Journal of China (from establishment to December 2010) were manually searched. Both RCTs and CCTs were included. The data were extracted by two reviewers with designed extraction form RevMan 4.2.8 software for data analysis. The criteria were as follows: (1) operative duration and reduced blood loss; (2) VAS (visual analog scale) score; (3) faster recovery of ROM (range of movement); (4) quadriceps muscle strength; (5) component positioning malalignment; (6) tibiofemoral angle; (7) rate of complications. RESULTS: A total of 18 RCTs were included. Compared with the standard TKA procedure, the MIS group had a longer operative duration (WMD (weighted mean difference) 14.16, 95%CI (confidence interval) (12.61, 15.71)); reduced blood loss (WMD 8.31, 95%CI (6.16, 10.46)); lower VAS score at Days 3-5 post-operation (WMD 4.99, 95%CI (4.19, 5.78)); better Mean Knee Society scores at Week 6 post-operation (WMD 4.99, 95%CI (4.19, 5.78)), improvement in ROM occurred more rapidly at Month 3 post-TKA (WMD 14.59, 95%CI (8.39, 20.80)). Although the differences were not statistically significant, tibiofemoral angle was more precise in the standard group and the rate of component malalignment occurred more frequently in the MIS group (WMD 0.20, 95%CI (-0.12, 0.52)) (RR 1.57, 95%CI (0.88, 2.83)). CONCLUSION: MIS leads to a faster recovery than conventional surgery with a shorter operative duration, a reduced blood loss, a lower VAS score and a faster recovery of ROM and quadriceps muscle strength. However, the rates of component malalignment and complications occur more frequently in the MIS group. Potential benefits in long-term survival rate and functional improvement require further investigations.

[Comparison of clinical results between high-flexion and standard cruciate-stabling prostheses in total knee arthroplasty].

OBJECTIVE: To compare the clinical results between high-flexion and standard cruciate-stabling prostheses in total knee arthroplasty (TKA) by using the 36-item short form health survey (SF-36). METHODS: Between August 2007 and January 2009, 98 patients (106 knees) underwent TKA with standard cruciate-stabling prostheses (standard group), and 46 patients (50 knees) underwent TKA with high-flexion prostheses (high-flexion group). In standard group, there were 30 males (32 knees) and 68 females (74 knees) with an age of (70.0 +/- 3.5) years, including 78 cases (82 knees) of osteoarthritis (OA) and 20 cases (24 knees) of rheumatoid arthritis (RA) with a disease duration of (14.5 +/- 3.3) years; the Hospital for Special Surgery Scoring System (HSS) and the range of motion (ROM) were 56.1 +/- 21.6 and (89.0 +/- 16.1) degrees, respectively. In high-flexion group, there were 8 males (10 knees) and 38 females (40 knees) with an age of (68.6 +/- 8.9) years, including 44 cases (47 knees) of OA and 2 cases (3 knees) of RA with a disease duration of (13.9 +/- 4.1) years; the HSS and ROM were 58.9 +/- 25.3 and (91.0 + 19.3) degrees, respectively. There was no significant difference in the general data (P > 0.05) between 2 groups, so the clinical data of 2 groups had comparability. RESULTS: In standard group, poor wound healing and persistent headache caused by cerebrospinal fluid leakage occurred in 1 case, respectively. In high-flexion group, transient common peroneal nerve palsy occurred in 1 case. There was significant difference (P < 0.05) in the hospitalization expense between standard group [yen(39,000 +/- 6000)] and high-flexion group [yen (52,000 +/- 8 000)]. The follow-up time was 12-26 months (18 months on average) in standard group (91 cases, 98 knees) and 11-19 months (13 months on average) in high-flexion group (44 cases, 47 knees). The SF-36 showed significant difference in role-physical score (P < 0.05), but no significant difference in other 7 indices scores (P > 0.05). At the final follow- up, the ROM was (129.1 +/- 19.2) degrees in high-flexion group and (123.6 +/- 16.7) degrees in standard group; showing significant difference (P < 0.05). The HSS was 91.2 +/- 17.6 in high-flexion group and 92.5 +/- 14.5 in standard group; showing no significant difference (P > 0.05). CONCLUSION: After TKA, the ROM in high-flexion group is superior to that in standard group, but there is no obvious advantages in terms of the HSS and SF-36 outcomes.

Liposomes incorporating sodium deoxycholate for hexamethylmelamine (HMM) oral delivery: development, characterization, and in vivo evaluation.

Liposomes incorporating sodium deoxycholate (NaDC) were prepared by the method of reverse phase evaporation and used for drug delivery by the oral route. Hexamethylmelamine (HMM), an anti-tumor agent, was chosen as a model drug and encapsulated into liposomes incorporating NaDC (NaDC-Lip). Several properties of NaDC-Lip containing HMM (HMM NaDC-Lip), such as particle size, entrapment efficiency, pinacyanol chloride (PIN) spectral characteristics with various molar ratio of NaDC/PC, as well as the vesicle stability measurements with calcein were evaluated. In vivo, the area under the plasma concentration-time curve obtained from the pharmacokinetics study of HMM NaDC-Lip was found to be approximately 9.76- and 1.21-fold higher than that of HMM solution and HMM Lip, respectively, indicating that NaDC-Lip can be used as a potential carrier for oral drug administration.

Lyophilization of water-in-oil emulsions to prepare phospholipid-based anhydrous reverse micelles for oral peptide delivery.

A novel procedure for the preparation of hydrophilic peptide-containing oily formulations involving the freeze-drying of water-in-oil emulsions (FWE) is described. A mixture of an aqueous phase containing insulin and oil phase containing phosphatidylcholine was emulsified to prepare water-in-oil emulsions, which were subsequently lyophilized. Upon addition of oil, the lyophilates formed a clear oily solution which was considered as an anhydrous reverse micelle (ARM) system since it contained no water but 20-nm insulin nanoparticles, as revealed by dynamic light scattering. The 20-nm insulin nanoparticles, existing in a crystal form, were also contained in the lyophilates, as proved by scanning electron microscopy, small angle X-ray scattering and differential scanning calorimetry analysis. The drug release from the oily formulation of SPC/insulin (10:1) was slow and less than 12% of the total insulin was released after 24h. A significant reduction in the plasma glucose level of fasting diabetic rats after oral administration of insulin-containing ARMs confirmed the bioactivity of the drug and the potential usefulness of phospholipid-based oily formulations. Both the ARMs and lyophilates were stable and could be stored at 4 degrees C for at least 6 months. Thus, this simple FWE procedure is suitable for the solubilization of hydrophilic peptides in oil to produce stable products for therapeutic applications.

Freeze drying of double emulsions to prepare topotecan-entrapping liposomes featuring controlled release.

Topotecan-entrapping liposomes were prepared by freeze drying double emulsions with hydrogenated soy phosphatidylcholine, N-(carbonyl-methoxypolyethyleneglycol2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and cholesterol. Different inner aqueous phases of different pH values containing topotecan together with different chemicals, such as citrate and sulfate, were used to modify the physicochemical state of the drug to prepare W1/O/W2 double emulsions that were then freeze dried to obtain dry products. Upon rehydration, the dry products, which were stable for at least 6 months, formed into unilamellar liposomes with a high encapsulation efficiency of up to 80% and a mean diameter below 200 nm. The in vitro release experiments demonstrated that different formulations displayed different drug release properties. Thus, stable submicron unilamellar topotecan-entrapping liposomes can be prepared by freeze drying double emulsions, and the drug release can be successfully controlled by altering the physicochemical state of the incorporated drug.

[Study on pharmacokinetics of PVP coated beta-elemene liposome in rats].

OBJECTIVE: To study the pharmacokinetics of PVP costed beta-elemente liposmes in rats. METHODS: Gas chromatography was established to determine the concentration of beta-elemene in plasma of rats after administered through i.g. RESULTS: The pharmacokinetics parameters was: T(1/2) = 95.07 +/- 20.46 min, AUC = 348.72 +/- 32.49 microg x min/ml, Cmax = 4.39 +/- 0.33 microg/ml, Tmax = 60 min. CONCLUSION: The bioavailability of PVP coated beta-elemene liposomes is 140.2 +/- 7.5% compared with conventional liposomes.

In-vitro cytotoxicity, in-vivo biodistribution and anti-tumour effect of PEGylated liposomal topotecan.

In attempt to increase the accumulation of topotecan in tumours and improve its anti-cancer activity, PEGylated liposome (H-PEG) containing topotecan was prepared. The in-vitro cytotoxicity, in-vivo biodistribution pattern and anti-tumour effect of H-PEG were studied systemically. Compared with free topotecan or conventional liposome (H-Lip), H-PEG improved the cytotoxic effect of topotecan against human ovarian carcinoma A2780 and human colon carcinoma HCT-8 cells. The IC50 value (concentration leading to 50% cell-killing) of H-PEG decreased 5 fold (P<0.01) and 9 fold (P<0.01) against A2780 and HCT-8 cells compared with H-Lip, respectively. The results of biodistribution studies in sarcoma S(180) tumour-bearing mice showed that liposomal encapsulation increased the concentration of total topotecan and the ratio of lactone form in plasma. H-PEG resulted in a 70-fold and 3.7-fold increase in AUC(0-->24 h) compared with free topotecan and H-Lip, respectively. Moreover, H-PEG increased the accumulation of topotecan in tumours and the relative tumour uptake ratio compared with free topotecan was 5.2, and higher than that of H-Lip. The anti-cancer effect studies in murine heptocarcinoma H(22) tumour-bearing mice showed that H-PEG improved the therapeutic efficiency of topotecan and decreased the toxicity of topotecan to a certain extent compared with H-Lip. These results indicated that PEG-modified liposome might be an efficient carrier of topotecan.

In vitro and in vivo studies of different liposomes containing topotecan.

Liposome as a carrier of topotecan (TPT), a promising anticancer drug, has been reported in attempt to improve the stability and antitumor activity of TPT. However, the biodistribution pattern of TPT liposome in vivo and PEG-modified liposome containing TPT have not been studied systemically. In this paper, the in vitro stability and in vivo biodistribution behavior of several liposomes containing TPT with different lipid compositions and PEG-modification were studied. Compared with the 'fluid' liposome (S-Lip) composed of soybean phosphatidylcholine (SPC), the 'solid' liposome (H-Lip) composed of hydrogenated soybean phosphatidylcholine HSPC decreased the leaking efficiency of TPT from liposome and enhanced the stability of liposome in fetal bovine serum (FBS) or human blood plasma (HBP). The results of biodistribution studies in S180 tumor-bearing mice showed that liposomal encapsulation increased the concentrations of total TPT and the ratio of lactone form in plasma. Compared with free TPT, S-Lip and H-Lip resulted in 5- and 19-fold increase in the area under the curve (AUC(0-->infinity)), respectively. PEG-modified H-Lip (H-PEG) showed 3.7-fold increase in AUC(0-->infinity) compared with H-Lip, but there was no significant increase in t(1/2) and AUC(0-->infinity) for PEG-modified S-Lip (S-PEG) compared with S-Lip. Moreover, the liposomal encapsulation changed the biodistribution behavior, and H-Lip and H-PEG dramatically increased the accumulation of TPT in tumor, and the relative tumor uptake ratios were 3.4 and 4.3 compared with free drug, respectively. There was also a marked increase in the distribution of TPT in lung when the drug was encapsulated into H-Lip and H-PEG. Moreover, H-PEG decreased the accumulation of TPT in bone marrow compared with unmodified H-Lip. All these results indicated that the membrane fluidity of liposome has an important effect on in vitro stability and in vivo biodistribution pattern of liposomes containing TPT, and PEG-modified 'solid' liposome may be an efficient carrier of TPT.

Oil-based formulations for oral delivery of insulin.

Several oil-based solution formulations of insulin were prepared, in which insulin was solubilized in the form of anhydrous reverse micelles. The preparation process involved micellar dissolution of insulin followed by freeze drying, then reconstitution of lyophilized product with an oil phase. These formulations were stable at room temperature for up to 12 months. No significant changes in the appearance were observed and no degradation products of insulin were detected during the course of the stability study. The efficacy of these formulations was evaluated in-vivo using diabetic Wistar rat as an animal model and then a specific formulation was chosen for further study in non-diabetic New Zealand rabbits. It was found that the efficacy of insulin oil solution was dose dependent and insulin oil solution had the same efficacy as insulin emulsion with the same formulation composition. If ethylene-diaminetetraacetic acid (EDTA) was pre-delivered 40 min before the delivery of insulin oil solution, the hypoglycaemic effect of insulin oil solution was greatly enhanced, with an AUC (% glucose reduced) value increase from 28.5 +/- 14.7 to 167.1 +/- 72.3. The improvement of oral absorption induced by predelivery of EDTA might be attributed to enzyme inhibition, reduced gut mobility and the opening of paracellular routes.