Establishment of a markerless multiple-gene deletion method based on Cre/loxP mutant system for Bacillus pumilus.

In this study, we established a Cre/loxP mutant recombination system (Cre/lox71-66 system) for markerless gene deletion to facilitate our follow-up rational genetic engineering to the strain Bacillus pumilus W3. This modified method uses two mutant loxP sites, which after recombination creates a double-mutant loxP site that is poorly recognized by Cre recombinase, facilitating multiple gene deletions in a single genetic background. Two selected genes, cotA and sigF, were continuously knocked out and verified at different levels using this method. This method is simple and efficient and can be easily implemented for multiple gene deletions in B. pumilus.

Comparison of 2 Anesthetic Methods for Transforaminal Endoscopic Lumbar Discectomy: A Prospective Randomized Controlled Study.

STUDY DESIGN: Randomized controlled trial. OBJECTIVES: To compare the effect and safety of 2 anesthetic methods using in the operation of Transforaminal Endoscopic Lumbar Discectomy. METHODS: From the January of 2020 to the December of 2021, 230 consecutive patients that underwent TELD were applied with two methods of anesthesia. All the patients were divided into two groups. The Monitored Anesthesia Care (MAC) group used the local anesthesia (LA) with MAC that based on the combination of dexmedetomidine and butorphanol tartrate. The LA group used the local anesthesia only. Then the Visual Analogic Scale (VAS) through the operating period was compared between the two groups at the time points of before operation (T0), inserting of the puncture needle (T1), establishing of the working cannula (T2), excision of the fibrous rings (T3) and immediately postoperatively (T4). Also, the satisfaction degree of the patients for the course of the operations and the occurrence of the complications were compared between the two groups. RESULTS: There were no differences of the VAS around the operating area at the time point of T0. Then the MAC group expressed lower scores at all other points of T1, T2, T3 and T4. Then the satisfaction degree of the MAC group was superior than the LA group. No difference was observed for the occurrence of the complications. CONCLUSIONS: MAC based on the combination of dexmedetomidine and butorphanol tartrate is an ideal method of anesthesia for TELD with enough effect and safety.

Analysis of the efficacy of microwave ablation in the treatment of early hepatic alveolar echinococcosis: A propensity score matching based study.

BACKGROUND: To explore the efficacy of microwave ablation (MWA) in the treatment of hepatic alveolar echinococcosis (HAE) with a diameter of ≤5 cm. METHOD: From June 2014 to January 2020, patients diagnosed with HAE were retrospectively analyzed. After balancing the confounding factors by propensity score matching (PSM) , the patients were divided into MWA group (n = 20) and radical operation group (n = 20) by 1:1 matching. The safety and effectiveness of MWA were assessed by comparing the differences between the two groups in terms of postoperative general laboratory indices, grading of postoperative complications, length of postoperative hospitalization, the outcome of treatment, and disease recurrence. RESULT: After PSM, all confounders were not statistically different (P>0.05) . Compared with the radical surgery group, patients in the MWA group had lower postoperative ALT and WBC elevations (P<0.001) , shorter postoperative hospital stay (P<0.001) ) , lower hospital costs (P<0.001) . The effective rate of the two groups was 100%. There was no statistical difference in the incidence of postoperative complications and recurrence rate (P>0.05). CONCLUSION: MWA is a safe and effective means of treating HAE ≤ 5 cm in diameter.

One-pot synthesis and enzyme-responsiveness of amphiphilic doxorubicin prodrug nanomicelles for cancer therapeutics.

In this study, we report a one-pot synthesis and enzyme-responsiveness of polyethylene glycol (PEG) and glutamic acid (Glu)-based amphiphilic doxorubicin (DOX) prodrug nanomicelles for cancer therapeutics. The nanomicelles were accomplished by esterification and amidation reactions. The nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR) data confirmed the structure of nanomicelles. The DOX-loaded nanomicelles showed a DLS-measured average size of 107 nm and excellent stability in phosphate-buffered saline (PBS) for 7 days. The drug loading and cumulative release rates were measured by ultraviolet-visible (UV-vis) spectrophotometry at 481 nm. The cumulative release rate could reach 100% in an enzyme-rich environment. Further, the therapeutic efficiency of nanomicelles to cancer cells was determined by cell viability and cellular uptake and distribution using HeLa cells. The cell viability study showed that the DOX-loaded nanomicelles could effectively inhibit the HeLa cell proliferation. The cellular uptake study confirmed that the nanomicelles could be effectively ingested by HeLa cells and distributed into cell nuclei. Based on the collective experimental data, this study demonstrated that the synthesized nanomicellar prodrug of DOX is a potential candidate for cancer therapeutics.

[Electroacupuncture of both single- and multi-acupoints promotes recovery of gastrointestinal function in laparoscopic cholecystectomy patients].

OBJECTIVE: To compare the therapeutic effect of electroacupuncture (EA) of single-acupoint Zusanli (ST36) and multi-acupoints Zusanli (ST36), Shangjuxu (ST37) and Neiguan (PC6) in promoting the recovery of gastrointestinal movement in laparoscopic cholecystectomy (LC) patients undergoing general anesthesia. METHODS: A total of 70 LC patients (American Society of Anesthesiologists [ASA] grade I and II) were recruited and randomly divided into control (n＝23), single ST36 (n＝23) and ST36＋ST37＋PC6 (n＝24) groups. The patients in the control group only received routine basic treatments (postoperative fasting and water deprivation, intravenous drip of biotics, water-electrolyte and acid-base balancing, oxygen uptake, etc). EA (10 Hz, 5 mA, 30 min every time) was applied to the abovementioned single-acupoint or multi-acupoints groups before, and 4, 22, 34 and 46 h after the operation. The time-points of postoperative borborygmus recovery, first anal exhaust and defecation, post-operative abdominal distension (mild, moderate and severe), nausea and vomiting (grade Ⅰ， Ⅱ， Ⅲ and Ⅳ) at 6, 24 and 48 h after surgery were recorded and analyzed. RESULTS: Compared to the control group, the time of borborygmus recovery, first anal exhaust and defecation were markedly earlier in both single ST36 and ST36＋ST37＋PC6 groups (P<0.01, P<0.05, P<0.001). The number of patients who had mild plus moderate abdominal distention, and nausea (grade Ⅱ＋Ⅲ) at 24 h after ope-ration was significantly lower in both single ST36 and ST36＋ST37＋PC6 groups than in the control group (P<0.05). No significant differences were found between the two EA groups in the time of borborygmus recovery, first anal exhaust and defecation, and in the number of patients with mild plus moderate abdominal distention and those with nausea (P>0.05). CONCLUSION: EA of both single ST36 and ST36＋ST37＋PC6 can promote gastrointestinal function recovery in LC patients, without remarkable difference between them.

Functional expression enhancement of Bacillus pumilus CotA-laccase mutant WLF through site-directed mutagenesis.

Bacterial laccases are potential enzymes for biotechnological applications, such as detoxification of industrial effluents, decolorization of textile, and dimerization of phenolic acids, due to their remarkable advantages, including broad substrate spectrum, high thermostability, wide pH scope, and tolerance to alkaline environments. L386W/G417L/G57F (abbreviated as WLF), a good mutant of CotA-laccase from Bacillus pumilus W3, has been constructed and reported by our laboratory with highly improved catalytic efficiency. However, the low-functional expression level of mutant WLF in Escherichia coli was a shortcoming. Three mutants, namely, K317N/WLF, D501G/WLF, and K317N/D501G/WLF, were constructed through site-directed mutagenesis to improve the functional expression of WLF in this study. The soluble and active expression of D501G/WLF and K317N/D501G/WLF in E. coli enhanced 4.48-fold and 3.63-fold level, respectively. The K317N/WLF failed to increase the soluble expression level, but slightly improved the stability of CotA-laccase. Results showed that not only the position 501 is significant for functional expression of B. pumilus W3 CotA, but also these mutants still remained its high thermostability, resistance of alkaline with salt, and conspicuous decolorizing efficiency. This work is the first to improve the soluble expression of B. pumilus CotA-laccase in E. coli by site-directed mutagenesis. The D501G/WLF and K317N/D501G/WLF will be suitable candidates for biotechnological applications.

Changes in chemical interactions and protein conformation during heat-induced wheat gluten gel formation.

In order to elucidate the heat-induced wheat gluten gel formation mechanism, changes in chemical interactions and protein conformation were investigated during gelation. The contribution of ionic and hydrogen bonds were found to decrease from 0.746 and 4.133g/L to 0.397 and 2.733g/L, respectively, as the temperature increased from 25 to 90°C. Moreover, the free SH content remarkably decreased from 37.91 to 19.79µmol/g during gelation. Ultraviolet absorption spectra and intrinsic fluorescence spectra suggested that wheat gluten unfolded during the heating process. In addition, wheat gluten gels treated at 80 and 90°C exhibited a "steric hindrance" effect, which can be attributed to the formation of aggregates. Fourier transform infrared spectra suggested that the random coil content increased at low temperatures (40 and 50°C), whereas the content of intermolecular ß-sheets due to protein aggregation increased from 38.10% to 44.28% when the gelation temperature was 90°C.

Effect of Modified Wheat Gluten on Boiling Resistance Capacity of Pork Meatballs.

The effect of the modified wheat gluten (MWG) extender, prepared by alcalase-based hydrolysis and transglutaminase cross-linking, on meatballs was analyzed in this study. Here, we studied the effect of MWG addition on the boiling resistance capacity of pork meatballs (MB-MWG) at high temperature (100 °C) and increasing cooking time; meatballs with added soy protein isolates (MB-SPI) and raw wheat gluten (MB-WG) were used as references. The cooking loss, water-holding capacity (WHC), and textural properties of meatballs were investigated. The results revealed that MB-MWG showed lower cooking loss, which decreased by 49.16% compared to meatballs without added extenders when treated for 30 min. The WHC of MB-MWG significantly increased from 80.68% to 95.42%. The hardness, springiness, and chewiness (textural properties) of MB-MWG were also significantly increased by 97.05%, 6.68%, and 121.96%, respectively. The addition of MWG increased the cross-linking in meatballs during the cooking process, as indicated by the higher G'. SDS-PAGE indicated an obvious decrease in myosin heavy chain in MB-MWG cooked for 30 min at 100 °C, which was attributed to the interaction of myofibrillar proteins in pork meat with MWG. The nuclear magnetic resonance T2 relaxation time patterns indicated that MWG addition caused an increase in the bound water content, and decrease in the free water content, of meatballs. An analysis of the microstructures revealed that the MB-MWG formed the most regular and compact network. Therefore, MWG could be used as an ingredient to facilitate the processing of meat products.