A Whole-Process Visible Strategy for the Preparation of Rhizomucor miehei Lipase with Escherichia coli Secretion Expression System and the Immobilization.

BACKGROUND: Rhizomucor miehei (RM) lipase is a regioselective lipase widely used in food, pharmaceutical and biofuel industries. However, the high cost and low purity of the commercial RM lipase limit its industrial applications. Therefore, it is necessary to develop cost-effective strategies for large-scale preparation of this lipase. The present study explored the high-level expression of RM lipase using superfolder green fluorescent protein (sfGFP)-mediated Escherichia coli secretion system. RESULTS: The sfGFP(-15) mutant was fused to the C-terminus of RM lipase to mediate its secretion expression. The yield of the fusion protein reached approximately 5.1 g/L with high-density fermentation in 5-L fermentors. Unlike conventional secretion expression methods, only a small portion of the target protein was secreted into the cell culture while majority of the fusion protein was still remained in the cytoplasm. However, in contrast to intracellular expression, the target protein in the cytoplasm could be transported efficiently to the supernatant through a simple washing step with equal volume of phosphate saline (PBS), without causing cell disruption. Hence, the approach facilitated the downstream purification step of the recombinant RM lipase. Moreover, contamination or decline of the engineered strain and degradation or deactivation of the target enzyme can be detected efficiently because they exhibited bright green fluorescence. Next, the target protein was immobilized with anion-exchange and macropore resins. Diethylaminoethyl sepharose (DEAE), a weak-basic anion-exchange resin, exhibited the highest bind capacity but inhibited the activity of RM lipase dramatically. On the contrary, RM lipase fixed with macropore resin D101 demonstrated the highest specific activity. Although immobilization with D101 didn't improve the activity of the enzyme, the thermostability of the immobilized enzyme elevated significantly. The immobilized RM lipase retained approximately 90% of its activity after 3-h incubation at 80 °C. Therefore, D101 was chosen as the supporting material of the target protein. CONCLUSION: The present study established a highly efficient strategy for large-scale preparation of RM lipase. This innovative technique not only provides high-purity RM lipase at a low cost but also has great potential as a platform for the preparation of lipases in the future.

Evaluation of the correlation between cerebral hemodynamics and blood pressure by comparative analysis of variation in cerebral blood flow in hypertensive versus normotensive individuals: A systematic review and meta-analysis.

Current understanding of the cerebral vascular response to variations in blood pressure (BP) among individuals with hypertension is limited. The aim of this meta-analysis was to determine the correlation between hypertension, risk of stroke, and cerebral blood flow (CBF). We reviewed studies published between 2000 and 2023 from PubMed, Google Scholar, and Science Direct that compared mean CBF in normotensive and hypertensive patients. A random effects model was used to construct the risk ratio (RR), 95% confidence interval (CI), forest plot, and inverse variance weighting. Additionally, a mixed-effects meta-regression was employed to examine the impact of study-specific patient variables. This meta-analysis included eight prospective cross-sectional studies published from 2002 to 2023. It revealed a significant average difference in the standard mean CBF of -0.45 (95% CI -0.60 to -0.30, I2 = 69%, P < 0.00001), distinguishing normotensive from hypertensive subjects. A RR of 0.90 (95% CI 0.63 to 1.30, I2 = 89%, P = 0.04) indicated a significant decrease in CBF among individuals with hypertension. We found a statistically significant relationship between changes in diastolic and systolic BPs and the mean CBF (R = -0.81, P = 0.001 and R = -0.90, P = 0.005, respectively). Our research demonstrates a strong relationship between elevated BP and reduced CBF, with hypertension reducing CBF compared to normotensive individuals, by increasing cerebrovascular resistance.

Endothelialization of PTFE-covered stents for aneurysms and arteriovenous fistulas created in canine carotid arteries.

To investigate the endothelialization of covered and bare stents deployed in the canine carotid arteries and subclavian arteries for treating experimental aneurysms and arteriovenous fistulas, twenty aneurysms were created in 10 dogs, and 20 fistulas in another 10 dogs. The Willis balloon-expandable covered stent and a self-expandable covered stent were used to treat these lesions, and a self-expandable bare stent was deployed in the subclavian artery for comparison. Followed up for up to 12 months, the gross observation, pathological staining, and scanning electronic microscopic data were analyzed. Two weeks after creation of animal model, thirty self-expandable covered stents and ten balloon-expandable covered stents were deployed. Fifteen bare stents were deployed within the left subclavian arteries. Twenty days after stenting, the aneurysm significantly shrank. At 6 months, the thrombi within the aneurysm cavity were organized. Three to 12 months later, most covered and bare stents were covered by a thin transparent or white layer of endothelial intima. Layers of intima or pseudomembrane were formed on the stent 20-40 days after stent deployment. Over three months, the pseudomembrane became organized, thinner, and merged into the vascular wall. Under scanning electronic microscopy, the surface of covered and bare stents had only deposition of collagen fibers and rare endothelial cells 20-40 days after stenting. From three to ten months, the endothelial cells on the internal surface of stent became mature, with spindle, stripe-like or quasi round morphology along the blood flow direction. Over time, the endothelial cells became mature. In conclusion, three months after deployment in canines' arteries, the self-expandable bare and covered stents have mostly been covered by endothelial cells which become maturer over time, whereas the balloon-expandable covered stents do not have complete coverage of endothelial cells at three months, especially for protruding stent struts and areas. Over time, the endothelialization will become mature.

Complications and long-term in-stent restenosis of endovascular treatment of severe symptomatic intracranial atherosclerotic stenosis and relevant risk factors.

To investigate the complications and in-stent restenosis of endovascular treatment of severe symptomatic intracranial atherosclerotic stenosis and relevant risk factors. Three hundred and fifty-four consecutive patients with intracranial atherosclerotic stenosis (70%-99%) were retrospectively enrolled. The clinical data, treatment outcomes, complications and in-stent restenosis at follow-up were analyzed. The endovascular treatment was composed of balloon dilatation only in 21 (5.93%) patients, and deployment of self-expandable stents in 232 (65.54%), balloon-expandable stents in 75 (21.19%), and both balloon- and self-expandable stents in 26 (7.34%), with a total of 359 stents being successfully deployed at the stenotic location. After treatment, the residual stenosis ranged 9.2%±1.5% (range 7%-19%), which was significantly (P < .05) smaller than that before treatment. Periprocedural complications occurred in 43 patients with a complication rate of 12.15% including arterial dissection in 4 (1.13%) patients, new cerebral infarction in 21 (5.93%), cerebral hemorrhage in 12 (3.3%), and subarachnoid hemorrhage in 6 (1.69%). Hyperlipidemia [odds ratio (OR) 10.35, 95% confidence interval (CI) 4.42-24.28, and P < .0001] and location at the middle cerebral artery (MCA) (OR 4.15, 95% CI 1.92-8.97, and P < .001) were significant (P < .05) risk factors for periprocedural complications, whereas hyperlipidemia (OR 11.28, 95% CI 4.65-30.60, and P < .0001), location at the MCA (or 5.26, 95% CI 2.03-15.08, and P < .001), and angulation (OR 1.02, 95% CI 1.00-1.04, and P = .02) were significant (P < .05) independent risk factors for periprocedural complications. Follow-up was performed in 287 (81.07%) patients at 6 to 36 (28 ± 6.7) months. In-stent restenosis was present in 36 (12.54%), and female sex (OR 2.53, and 95% CI 1.27-5.06) and periprocedural complications (OR 9.18, and 95% CI 3.52-23.96) were significant (P < .05) risk factors for in-stent restenosis, with periprocedural complication (OR 9.61, and 95% CI 3.48-27.23) as the only significant (P < .0001) independent risk factor for in-stent restenosis. A certain rate of periprocedural complications and in-stent stenosis may occur in endovascular treatment of severe intracranial stenosis, and the relevant risk factors may include hyperlipidemia, MCA location, angulation at the stenosis and female sex.

Circ VRK1/microRNA-17/PTEN axis modulates the angiogenesis of human brain microvascular endothelial cells to affect injury induced by oxygen-glucose deprivation/reperfusion.

BACKGROUND: Circular RNAs (circRNAs) can act as microRNA (miRNA) sponges, thus regulating gene expression. The role of circRNAs in the process of oxygen-glucose deprivation/reoxygenation (OGD/R) is unclear. Here, we explored the mechanism underlying Circ VRK1 in human brain microvascular endothelial cells (HBMVECs) injury induced by OGD/R. METHODS: The OGD/R cell model was established in HBMVECs. The microarray was applied to detect differentially expressed circRNAs, followed by subcellular fractionation assay. Colony formation assay, flow cytometry, ELISA, tube formation, Transwell and western blot assays were performed for loss-of-function assay. HE staining, TTC staining, immunohistochemistry and western blot were performed in an established mouse model. The relationships between Circ VRK1 and miR-17, and between miR-17 and PTEN were detected by bioinformatics and dual-luciferase assays. Rescue experiments were conducted in vitro and in vivo, and PI3K/AKT activity was detected by Western Blot. RESULTS: Circ VRK1, predominantly present in the cytoplasm of cells, was upregulated in the HBMVECs exposed to OGD/R. Circ VRK1 downregulation decreased proliferation, migration, tube formation, inflammatory factors and oxidative stress, while increased apoptosis in HBMVECs. Moreover, Circ VRK1 silencing reduced neurological damage, cerebral infarct size, CD34-positive cell counts and VEGF expression in mice. Circ VRK1 mediated PTEN expression and the PI3K/AKT pathway by targeting miR-17. Deletion of miR-17 inhibited the effects of Circ VRK1 siRNA, and silencing of PTEN suppressed the effects of miR-17 inhibitor. CONCLUSION: Circ VRK1 was upregulated during OGD/R. Circ VRK1 downregulation regulates PTEN expression by targeting miR-17, thereby promoting PI3K/AKT pathway activity to alleviate OGD/R injury.

Clinical effects of TP-based hyperthermic intraperitoneal chemotherapy (HIPEC) on CD133 and HE4 expression in advanced epithelial ovarian cancer.

Objectives: To investigate the clinical effects of TP-based hyperthermic intraperitoneal chemotherapy (HIPEC) on the levels of antigen cluster protein 133 (CD133) and human epididymal secretory protein 4 (HE4) in patients with advanced epithelial ovarian cancer (EOC). Methods: A total of 104 patients with advanced EOC hospitalized in Affiliated Hospital of Hebei Engineering University from April 2015 to December 2018 were assigned to two groups using a random number table. A control group (n =52) treated by the conventional postoperative TP regimen and an observation group (n =52) receiving HIPEC in addition to the conventional postoperative TP regimen. CD133 and HE4 expression in serum, overall response rate (ORR), long-term efficacy, and incidence of drug toxicity were measured for comparative analysis. Results: The serum levels of CD133 and HE4 expression in the observation group were lower than in the control group (P < 0.005, respectively); the observation group surpassed the control group in ORR, 2-year survival, and progression-free survival (PFS) (P < 0.005, respectively); however, the two groups had no statistically significant difference in the incidence of drug toxicity (P > 0.05). Conclusions: TP-based HIPEC can effectively inhibit CD133 and HE4 expression in advanced EOC, which thereby improves the clinical efficacy and encourages longer survival.

Effects of carboplatin combined with paclitaxel-based intraperitoneal hyperthermic perfusion chemotherapy on serum levels of HE4 and DJ-1 in patients with advanced recurrent ovarian cancer.

Objective: To analyze the effects of carboplatin combined with paclitaxel-based intraperitoneal hyperthermic perfusion chemotherapy (IPCH) on serum levels of human epididymis protein 4 (HE4) and mitogen-dependent oncogene-1 (DJ-1) in patients with advanced recurrent ovarian cancer (OC). Methods: From July 2019 to July 2020, patients with advanced recurrent OC (n=92) treated in Affiliated Hospital of Hebei Engineering University were selected as study subjects. According to the random number table method, patients were divided into control group and observation group. Patients in the control group were treated with carboplatin combined with paclitaxel-based intravenous chemotherapy, and patients in the observation group were treated with carboplatin combined with paclitaxel-based IPCH. The therapeutic effects, serum levels of HE4, DJ-1 and human kallikrein 10 (HK-10), peripheral blood immune indexes and adverse reactions of patients were compared between the two groups. Results: The response rate of the observation group was significantly higher than that of the control group (p<0.05); after treatment, the indexes of HE4, DJ-1 and HK-10 in the two groups were significantly decreased, while the indexes of CD3+CD4+, CD3+CD56+ and CD3+CD4+/CD3+CD8+ were significantly increased; moreover, significantly lower indexes of HE4, DJ-1 and HK-10 and significantly higher indexes of CD3+CD4+, CD3+CD56+ and CD3+CD4+/CD3+CD8+ were found in the observation group when compared with the control group (p<0.05). The severity of myelosuppression, nausea and vomiting in the observation group was significantly lower than that in the control group, and the total tumor metastasis rate in the observation group was significantly lower than that in the control group (p<0.05). Conclusions: Carboplatin combined with paclitaxel IPCH had obvious inhibitory effects on HE4, DJ-1 and other serum tumor markers in patients with advanced recurrent OC, with a more prominent clinical effect, and could further significantly reduce the risk of adverse reactions and metastasis.

Purification optimization for a recombinant single-chain variable fragment against type 1 insulin-like growth factor receptor (IGF-1R) by using design of experiment (DoE).

Design of experiment (DoE) is a statistics-based technique for experimental design that could overcome the shortcomings of traditional one-factor-at-a-time (OFAT) approach for protein purification optimization. In this study, a DoE approach was applied for optimizing purification of a recombinant single-chain variable fragment (scFv) against type 1 insulin-like growth factor receptor (IGF-1R) expressed in Escherichia coli. In first capture step using Capto L, a 2-level fractional factorial analysis and successively a central composite circumscribed (CCC) design were used to identify the optimal elution conditions. Two main effects, pH and trehalose, were identified, and high recovery (above 95%) and low aggregates ratio (below 10%) were achieved at the pH range from 2.9 to 3.0 with 32-35% (w/v) trehalose added. In the second step using cation exchange chromatography, an initial screening of media and elution pH and a following CCC design were performed, whereby the optimal selectivity of the scFv was obtained on Capto S at pH near 6.0, and the optimal conditions for fulfilling high DBC and purity were identified as pH range of 5.9-6.1 and loading conductivity range of 5-12.5 mS/cm. Upon a further gel filtration, the final purified scFv with a purity of 98% was obtained. Finally, the optimized conditions were verified by a 20-fold scale-up experiment. The purities and yields of intermediate and final products all fell within the regions predicted by DoE approach, suggesting the robustness of the optimized conditions. We proposed that the DoE approach described here is also applicable in production of other recombinant antibody constructs.

Microcatheter looping facilitates access to both the acutely angled parent artery and cerebral aneurysms for effective embolization.

Aneurysms with an acutely angled parent artery are difficult to access for coiling. This study aimed to investigate the safety and effectiveness of microcatheter looping for embolization of cerebral aneurysms with access difficulty. Ten patients (male:female=5:5) with cerebral aneurysms treated with the microcatheter looping technique were analyzed retrospectively. The parent artery formed an acute angle with the major artery in five aneurysms. The microcatheter was looped into a "α" loop for treatment in the anterior temporal artery aneurysm and a "U" loop in the remaining nine aneurysms. All ten aneurysms were successfully treated with the microcatheter looping technique. The microcatheter tip was successfully navigated into the aneurysm sac and remained stable throughout the embolization process. All aneurysms were occluded with total occlusion in five and near-total occlusion in five, and the parent artery remained patent in all cases. No complications occurred peri-procedurally. The Glasgow Outcome Scale was 5 in all patients before discharge. Follow-up angiography six to 12 months later revealed a good occlusion status of the aneurysms. The microcatheter looping technique is effective when the conventional embolization technique fails to treat cerebral aneurysms with difficult access especially when the parent artery forming an acute angle with the major artery exacerbates difficult access to the aneurysms.

The stent-assisted coil-jailing technique facilitates efficient embolization of tiny cerebral aneurysms.

OBJECTIVE: Tiny cerebral aneurysms are difficult to embolize because the aneurysm's sac is too small for a single small coil, and coils within the aneurysm may escape from the confinement of a stent. This study was performed to introduce the stent-assisted coil-jailing technique and to investigate its effect on the coil embolization of tiny intracranial aneurysms. MATERIALS AND METHODS: Sixteen patients with tiny intracranial aneurysms treated with the stent-assisted coil-jailing technique between January 2011 and December 2013 were retrospectively reviewed and followed-up. RESULTS: All aneurysms were successfully treated with the coil-jailing technique, and at the end of embolization, complete occlusion of the aneurysm was achieved in 9 cases (56.3%), incomplete occlusion in 6 (37.5%), and partial occlusion in 1 (6.3%). Intraprocedural complications included acute thrombosis in one case (6.3%) and re-rupture in another (6.3%). Both complications were managed appropriately with no sequela. Follow-up was performed in all patients for 3-24 months (mean, 7.7 months) after embolization. Complete occlusion was sustained in the 9 aneurysms with initial complete occlusion, progressive thrombosis to complete occlusion occurred in the 6 aneurysms with initial near-complete occlusion, and one aneurysm resulted in progressive thrombosis to complete occlusion after initial partial occlusion. No migration of stents or coils occurred at follow-up as compared with their positions immediately after embolization. At follow-up, all patients had recovered with no sequela. CONCLUSION: The stent-assisted coil-jailing technique can be an efficient approach for tiny intracranial aneurysms, even though no definite conclusion regarding its safety can be drawn from the current data.

Effects of (-)doxazosin on histomorphologic and cell apoptotic changes of the hyperplastic prostate in castrated rats.

BACKGROUND: (-)Doxazosin is one of the enantiomers of (+/-)doxazosin, and it was reported that (-)doxazosin possessed higher selectivity for lower urinary tract between the cardiovascular system and the urinary system in the animal experiments in comparison with that of (+/-)doxazosin and (+)doxazosin. Therefore, it is important to know whether (-)doxazosin has a therapeutic effect on the hyperplastic prostate. METHODS: (-)Doxazosin and (+/-)doxazosin were administered intragastrically to prostatic hyperplasia rats, induced by testosterone propionate for 4 weeks, and each experimental group contained 8 animals. The histomorphologic changes of the prostate were observed under light microscope, the quantitative analysis of the prostatic glandular cavity was performed using an image analysis system, and the cell apoptosis was detected by using flow cytometry. RESULTS: In comparison with model-control group, the volume index of the prostate in (-)doxazosin 3.0 mg/kg group became significantly smaller. The maximal diameter, perimeter, and area of the hyperplastic prostate glandular cavity, and the glandular epithelial cell height in (-)doxazosin (0.3, 1.0, and 3.0 mg/kg) groups and (+/-)doxazosin group were significantly reduced. (-)Doxazosin and (+/-)doxazosin did not significantly affect cell cycle distribution and cell proliferation index of the hyperplastic prostate. However, apoptotic rates of the prostatic cells in (-)doxazosin (0.3, 1.0, and 3.0 mg/kg) groups and (+/-)doxazosin group were significantly increased in comparison with those of model-control group. CONCLUSIONS: Both (-)doxazosin and (+/-)doxazosin inhibit the prostatic hyperplasia induced by testosterone propionate in castrated rats. The induction of prostate cell apoptosis might be one of the mechanisms underlying the therapeutic role of (-)doxazosin.