A Simple and Low-Cost CRISPR/Cas9 Knockout System Widely Applicable to Insects.

The CRISPR/Cas9 gene-editing system is a standard technique in functional genomics, with widespread applications. However, the establishment of a CRISPR/Cas9 system is challenging. Previous studies have presented numerous methodologies for establishing a CRISPR/Cas9 system, yet detailed descriptions are limited. Additionally, the difficulties in obtaining the necessary plasmids have hindered the replication of CRISPR/Cas9 techniques in other laboratories. In this study, we share a detailed and simple CRISPR/Cas9 knockout system with optimized steps. The results of gene knockout experiments in vitro and in vivo show that this system successfully knocked out the target gene. By sharing detailed information on plasmid sequences, reagent codes, and methods, this study can assist researchers in establishing gene knockout systems.

Identification and Functions of JHE 6 Specifically Expressed in Bombyx mori Silk Gland.

Juvenile hormone esterase (JHE) is the specific enzyme that degrades juvenile hormone (JH) and regulates the JH titer in insects. JH also regulates the development of the silk gland and the synthesis and secretion of silk proteins in Bombyx mori. Here, we identified nine possible JHE family members, Bmjhe1-9. Notably, Bmjhe6 is specifically expressed in the silk gland. Using semi-quantitative, quantitative real-time RT-PCR and Western blot, it was confirmed that Bmjhe6 was specifically expressed in the middle silk gland (MSG) with high levels in the anterior region of the MSG (A-MSG). The immunofluorescence localization analysis revealed that Bmjhe6 is produced within cells, secreted into the gland lumen, and co-transported with silk proteins into the anterior silk gland (ASG). In vitro hormone induction experiments demonstrated that Bmjhe6 responds to a JH analog, increasing its expression after 12-24 h, whereas 20-hydroxyecdysone inhibited it. In addition, Bmjhe6 knockdown using dsBmjhe6 injections accelerated larval development, resulting in increased larval body and silk gland weight. This induced disordered sericin genes (Ser2, Ser3) expression, and key genes in the JH synthesis pathway (BmKr-h1 and BmMet1) were significantly upregulated along with the transcription factors (SGF-1 and Sage). These results indicate that Bmjhe6 plays an important role in silk gland growth and silk protein synthesis by modulating JH signal.

Propofol-based total intravenous anesthesia is associated with better survival than desflurane anesthesia in limb-salvage surgery for osteosarcoma: A retrospective analysis.

Previous studies have demonstrated that anesthetic techniques can affect the outcomes of cancer surgery. We investigated the association between anesthetic techniques and patient outcomes after elective limb-salvage surgery for osteosarcoma (OS). This was a retrospective cohort study of patients who underwent elective limb-salvage surgery for OS between January 2007 and December 2018. Patients were grouped according to the administration of propofol-based total intravenous anesthesia (TIVA) or desflurane (DES) anesthesia. Kaplan-Meier analysis was performed, and survival curves were constructed from the date of surgery to death. Univariate and multivariate Cox regression models were applied to compare the hazard ratios (HRs) for death after propensity matching. Subgroup analyses were done for postoperative recurrence, metastasis, and tumor-node-metastasis (TNM) staging. A total of 30 patients (17 deaths, 56.7%) who received DES anesthesia and 26 (4 deaths, 15.4%) who received TIVA were eligible for analysis. After propensity matching, 22 patients were included in each group. In the matched analysis, patients who received TIVA had better survival with a HR of 0.30 (95% confidence interval [CI], 0.11-0.81; P = .018). Subgroup analyses also showed significantly better survival in the presence of postoperative metastasis (HR, 0.24; 95% CI, 0.06-0.87; P = .030) and with TNM stage II to III (HR, 0.26; 95% CI, 0.09-0.73; P = .011) in the matched TIVA group. In addition, patients administered with TIVA had lower risks of postoperative recurrence and metastasis than those administered with DES anesthesia in the matched analyses. Propofol-based TIVA was associated with better survival in patients who underwent elective limb-salvage surgery for OS than DES anesthesia. Prospective studies are needed to assess the effects of TIVA on oncological outcomes in patients with OS.

Comparison of 2 effect-site concentrations of remifentanil with midazolam during transrectal ultrasound-guided prostate biopsy under procedural analgesia and sedation: A randomized controlled study.

BACKGROUND: Until now, target-controlled infusion of remifentanil with midazolam for transrectal ultrasound-guided prostate biopsy has not been described. Here, we investigate 2 effect-site concentrations of remifentanil with intermittent bolus midazolam for transrectal ultrasound-guided prostate biopsy under procedural analgesia and sedation. METHODS: A prospective, randomized controlled trial including patients who received a transrectal ultrasound-guided prostate biopsy between February 2019 and January 2021 was conducted. Group 1 and Group 2 were respectively administered an initial effect-site concentration of remifentanil of 1.0 ng/mL and 2.0 ng/mL by a target-controlled infusion pump with Minto model. In both groups, maintenance of the effect-site concentration of remifentanil was adjusted upward and downward by 0.5 ng/mL to keep patient comfort with acceptable pain (remaining moveless), and mean arterial pressure and heart rate within baseline levels ±â€…30%, and using intermittent bolus midazolam to keep the Observer's Assessment of Alertness/Sedation scale between 2 and 4. The primary outcome was to determine which effect-site concentration of remifentanil provide adequate patient comfort with acceptable pain (remaining moveless) during the procedure. RESULTS: A total of 40 patients in Group 1 and 40 patients in Group 2 were eligible for analysis. Most parameters were insignificantly different between Group 1 and Group 2, except Group 1 having higher peripheral oxygen saturation while probe insertion compared with Group 2. Group 2 patients had less intraoperative movements affecting the procedure (2 vs 18; P < .001), and less total times of target-controlled infusion pump adjustment (0 [0-1] vs 1 [0-3], P < .001) compared with group 1. However, group 1 patients had less apnea with desaturation (peripheral oxygen saturation < 90%; 0 vs 9, P = .002) and less remifentanil consumption (94.9 ±â€…25.5 µg vs 106.2 ±â€…21.2 µg, P = .034) compared to Group 2. CONCLUSION: In transrectal ultrasound-guided prostate biopsy, target-controlled infusion with remifentanil Minto model target 2.0 ng/mL with 3 to 4 mg midazolam use provided sufficient analgesia and sedation, and appropriate hemodynamic and respiratory conditions.

HNRNPA2B1 as a trigger of RNA switch modulates the miRNA-mediated regulation of CDK6.

The functional inactivation of tumor suppressor microRNA (miRNA) is closely related to the tumorigenesis of cancer. There are instances where the miRNA and the corresponding target both exist in a cell, but the target gene silencing do not occur as expected. Herein, we found that both miR-506 and its target CDK6 are highly co-expressed in lung cancer cells. Sequence analyses suggested that a miR-506 binding site (1648-1654) and a cis-element (1785-1795) for binding by heterogeneous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1) are evolutionarily conserved and forms a stem structure in the 3' untranslated region (3'UTR) of CDK6. Furthermore, HNRNPA2B1 can bind to the stem structure to denature it and recruit the RNA helicase DExH-box helicase 9 (DHX9) to the 3'UTR, which ultimately facilitates miRNAs-mediated CDK6 silencing. These results indicate that the cis-element of the 3'UTR of CDK6, where HNRNPA2B1 binds, serves as an RNA switch to regulate miRNAs' function in cancer cells.

Propofol-Based Total Intravenous Anesthesia is Associated with Better Survival than Desflurane Anesthesia in Epithelial Ovarian Cancer Surgery: A Retrospective Cohort Study.

Background: Previous studies have shown that anesthetic techniques can affect outcomes of cancer surgery. We investigated the association between anesthetic techniques and patient outcomes after elective epithelial ovarian cancer surgery. Methods: This was a retrospective cohort study of patients who received elective open surgery for epithelial ovarian cancer between January 2009 and December 2014. Patients were grouped according to the administration of propofol or desflurane anesthesia. Kaplan-Meier analysis was performed, and survival curves were constructed from the date of surgery to death. Univariate and multivariate Cox regression models were used to compare hazard ratios for death after propensity matching. Subgroup analyses were performed for age, body mass index, preoperative carbohydrate antigen-125 level, International Federation of Gynecology and Obstetrics staging, and operation and anesthesia time. Results: In total, 165 patients (76 deaths, 46.1%) who received desflurane anesthesia and 119 (30 deaths, 25.2%) who received propofol anesthesia were eligible for analysis. After propensity matching, 104 patients were included in each group. In the matched analysis, patients who received propofol anesthesia had better survival with a hazard ratio of 0.52 (95% confidence interval, 0.33-0.81; p = 0.005). Subgroup analyses also showed significantly better survival with old age, high body mass index, elevated carbohydrate antigen-125 level, advanced International Federation of Gynecology and Obstetrics stage, and prolonged operation and anesthesia time in the matched propofol group. In addition, patients administered with propofol anesthesia had less postoperative recurrence and metastasis than those administered with desflurane anesthesia in the matched analysis. Conclusion: Propofol anesthesia was associated with better survival in patients who underwent elective epithelial ovarian cancer open surgery. Prospective studies are warranted to evaluate the effects of propofol anesthesia on oncological outcomes in patients with epithelial ovarian cancer.

Propofol-based total intravenous anesthesia is associated with better survival than desflurane anesthesia in glioblastoma surgery.

BACKGROUND: Previous research has shown that anesthetic techniques can influence patient outcomes following cancer surgery. However, the effects of anesthesia in patients undergoing glioblastoma surgery are still not known. We studied the relationship between the type of anesthesia and patient outcomes following elective glioblastoma surgery. METHODS: This was a retrospective cohort study of patients who underwent elective glioblastoma surgery between January 2008 and December 2018. Patients were grouped according to the anesthesia they received, desflurane or propofol. A Kaplan-Meier analysis was conducted, and survival curves were presented from the date of surgery to death. Univariable and multivariable Cox regression models were used to compare hazard ratios for death after propensity matching. RESULTS: A total of 50 patients (45 deaths, 90.0%) under desflurane anesthesia and 53 patients (38 deaths, 72.0%) under propofol anesthesia were included. Thirty-eight patients remained in each group after propensity matching. Propofol anesthesia was associated with improved survival (hazard ratio, 0.51; 95% confidence interval, 0.30-0.85; P = 0.011) in a matched analysis. Furthermore, patients under propofol anesthesia exhibited less postoperative recurrence than those under desflurane anesthesia (hazard ratio, 0.60; 95% confidence interval, 0.37-0.98; P = 0.040) in a matched analysis. CONCLUSIONS: In this limited sample size, we observed that propofol anesthesia was associated with improved survival and less postoperative recurrence in glioblastoma surgery than desflurane anesthesia. Further investigations are needed to examine the influence of propofol anesthesia on patient outcomes following glioblastoma surgery.

Comparison of 2 effect-site concentrations of remifentanil with midazolam during percutaneous transluminal balloon angioplasty under monitored anesthesia care: A randomized controlled study.

BACKGROUND: Until now, target-controlled infusion of remifentanil with midazolam in percutaneous transluminal balloon angioplasty for dysfunctional hemodialysis fistulas has not been described. Here, we investigate 2 effect-site concentrations of remifentanil with intermittent bolus midazolam for percutaneous transluminal balloon angioplasty under monitored anesthesia care. METHODS: A prospective, randomized controlled trial including patients who received a percutaneous transluminal balloon angioplasty between March 2019 and March 2021 was conducted. Group 1 and Group 2 were, respectively, administered an initial effect-site concentration of remifentanil of 1.0 and 2.0 ng/mL by a target-controlled infusion pump with Minto model. In both groups, maintenance of the effect-site concentration of remifentanil was adjusted upward and downward by 0.5 ng/mL with intermittent bolus midazolam to keep the Observer's Assessment of Alertness/Sedation scale between 2 and 4, mean arterial pressure and heart rate at baseline levels ±â€Š30%, and patient comfort (remaining moveless). The primary outcome was to determine the appropriate effect-site concentration of remifentanil for the procedure in terms of patient comfort (remaining moveless), hemodynamic conditions, and adverse events. Secondary endpoints included the total dosage of anesthetics and total times of target-controlled infusion pump adjustments. RESULTS: A total of 40 patients in Group 1 and 40 patients in Group 2 were eligible for analysis. Most parameters were insignificantly different between 2 groups, except Group 1 having higher peripheral oxygen saturation, while local anesthetic injection compared with Group 2. In addition, Group 1 patients had less apnea with desaturation (peripheral oxygen saturation < 90%; 0 vs 6, P = .034), less remifentanil consumption (189.65 ±â€Š69.7 vs 243.8 ±â€Š76.1 µg, P = .001), but more intraoperative movements affecting the procedure (14 vs 4; P = .016), total times of target-controlled infusion pump adjustment [2 (1-4) vs 1 (1-2), P < .001] compared with Group 2. CONCLUSION: In percutaneous transluminal balloon angioplasty for dysfunctional hemodialysis fistulas, target-controlled infusion with remifentanil Minto model target 2.0 ng/mL with 3 to 4 mg midazolam use provided appropriate hemodynamic conditions, sufficient sedation and analgesia, and acceptable apnea with desaturation.

The role of endotoxin in the pathogenesis of gastric mucosal damage in cirrhotic rats with portal hypertensive gastropathy.

OBJECTIVE: To study the role and the mechanism of endotoxin in the pathogenesis of gastric mucosa during portal vein hypertension gastrography (PHG) in the rats with cirrhosis. METHODS: Rat model for PHG was established by injection of tetrachloride. The animals were injected with endotoxin i.p. at 3 mg/kg and endotoxin antagonist BPI21 i.v. at 2.0 mg/kg. The plasma level of endotoxin as well as the gastric mucosal level of tumor necrosis factor alpha (TNF-α) was measured with azobenzene and ELISA respectively. Furthermore, the pathological changes of the gastric mucosa were studied with HE stainning. RESULTS: In rats with PHG, increased endotoxin and TNF-α as well as the gastric pathological lesion were observed. Injection of endotoxin remarkably increased plasma level of endotoxin as well as the gastric mucosal level of tumor necrosis TNF-α and induced more serious gastric lesion. Animals injected with endotoxin antagonist BPI21 showed improved gastric mucosal lesion, accompanied by the declining TNF-α level. CONCLUSIONS: Our results suggestes that endotoxin may play a pathogenetic role in PHG by inducing the expression of TNF-α.

[Clinical classify and therapy of unusual feeling symptom of pharynx].

OBJECTIVE: Through the observation, analysis, and treatment to lots of clinical patients with unusual feeling symptom of pharynx (UFSP), followed by the functional examination of autonomic nerve, putting forward the clinical classify and therapy of UFSP. METHODS: Through the clinical history inquired, routine examination, 24 hours pH value determined of esophagus, erect experiment, and coefficient of variation of the R-R(CVR-R) examination of electrocardiogram etc, divided the UFSP into two types: the type of pathogeny clear and the type of pathogeny unclear. The clear-type was further classified into five subtypes: the type of a mental scar, the type of morbid state of mind, the type of reflux esophagitis, the type of climacteric syndrome and menstrual disorder, and the type of functional disturbance of independence nerve. RESULTS: In the cases of 256, 106 were cured,76 were positive effective, 41 were effective, with total effective rate of 87.1%. Thirty three (12.9%) cases were not cured. Forty six (18.0%) of them with the type of a mental scar were whole cured. One hundred and thirty six (53.1%) of them with the type of morbid state of mind had a 93.4% (127) effective rate. Twenty one (8.2%) of them with the type of reflux esophagitis had 71.4% (15) effective rate. Among 35 (13.7%) cases of climacteric syndrome and menstrual disorder type, 29 cases were cured with a 82.9% effective rate. Twelve (4.7%) cases of the type of functional disturbance of independence nerve had 50% effective rate. Six (2.3%) cases of the pathogeny unclear type'were not cured. CONCLUSIONS: UFSP could be clinical classified and treated followed the detail inquiry of medical history and relative examination.