To establish a prokaryotic expression and purification protocol for nuclease P1 (NP1), we first obtained a synthetic NP1 by splicing 22 oligonucleotides with overlapping PCR. We constructed and transformed a secretory expression vector pMAL-p4X-NP1 into Escherichia coli host strains T7 Express and Origami B (DE3) separately. Then, the recombinant NP1 was purified by amylose affinity chromatography, and its activity, thermo-stability and metal-ion dependence were investigated systematically. The results indicated that the expressed fusion proteins MBP-NP1 (Maltose binding protein-NP1) existed mainly in soluble form both in host strains T7 Express and Origami B (DE3), but the specific activity of recombinant protein from Origami B(DE3) strain was higher than T7 Express strain (75.48 U/mg : 51.50 U/mg). When the MBP-tag was cleaved by protease Factor Xa, the specific activity both increased up to 258.1 U/mg and 139.2 U/mg. The thermal inactivation experiments demonstrated that the recombinant NP1 was quite stable, and it retained more than 90% of original activity after incubated for 30 min at 80 degrees C. Zn2+ (2.0 mmol/L) could increase enzyme activity (to 119.1%), on the contrary, the enzyme activity was reduced by 2.0 mmol/L Cu2+ (to 63.12%). This research realized the functional expression of NP1 in the prokaryotic system for the first time, and provided an alternative pathway for NP1 preparation.
Cloning, Molecular ; Enzyme Stability ; Escherichia coli ; genetics ; metabolism ; Fungal Proteins ; biosynthesis ; genetics ; metabolism ; Genes, Synthetic ; Genetic Vectors ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism ; Single-Strand Specific DNA and RNA Endonucleases ; biosynthesis ; genetics ; metabolism

Objective:To investigate the influencing factors of headache degree in migraine patients with patent foramen ovale (PFO).Methods:The clinical data of 124 migraine patients with PFO from January 2013 to June 2019 in Dalian Central Hospital Affiliated to Dalian Medical University were retrospectively analyzed. The right-to-left shunt of PFO was assessed by contrast-enhanced transcranial Doppler (c-TCD); the diameter of PFO, length of PFO tunnel and the presence of atrial septal aneurysm (ASA) were detected by transesophageal echocardiography (TEE); the degree of migraine was assessed by headache impact test-6 (HIT-6) scale. The relations between right-to-left shunt volume of PFO, diameter of PFO and degree of migraine were analyzed by Goodman-Kruskal γ test; the relations between the length of PFO tunnel, ASA and degree of migraine were analyzed by Spearman rank correlation analysis.Results:Among the 124 migraine patients with PFO, the c-TCD test result showed that small shunt volume was in 20 cases, medium shunt volume in 31 cases, and large shunt volume in 73 cases. The TEE test result showed that small foramen ovale in 76 cases, medium foramen ovale in 47 cases, and large foramen ovale in 1 case; long tunnel in 57 cases, and short tunnel in 67 cases; without ASA in 91 cases, and with ASA in 33 cases. The HIT-6 score result showed that some influence in 9 cases, significantly influence in 22 cases, and serious influence in 93 cases. The Goodman-Kruskal γ test result showed that right-to-left shunt volume of PFO and diameter of PFO were positive correlation with degree of migraine ( γ = 0.66, P<0.01; γ = 0.38, P<0.05). The Spearman rank correlation analysis result showed that ASA was positive correlation with degree of migraine ( r = 0.18, P<0.05), while the length of PFO tunnel was negative correlation with degree of migraine ( r = -0.23, P<0.05). Conclusions:The right to left shunt amount of PFO, diameter of PFO and the presence of ASA are positive correlation with degree of migraine, while the length of PFO tunnel is negative correlation with degree of migraine.

ObjectiveTo observe the clinical efficacy of modified Shenqi Pill （肾气丸） plus Tongdu Tiaoshen Acupuncture （通督调神针刺） in the treatment of neurogenic bladder after spinal cord injury of kidney-yang deficiency syndrome. MethodsForty-six patients were randomly divided into 23 cases each in the control group and the treatment group. Both groups were given conventional treatment， i.e. oral methylcobalamin tablets （0.5 mg each time， 3 times a day） and paraplegic conventional acupuncture （once a day， 6 consecutive days a week）. The control group was given simple bladder function rehabilitation training on the basis of the conventional treatment； and the treatment group was given modified Shenqi Pill orally （1 dose a day， 150 ml each time， taken warmly in morning and evening） and Tongdu Tiaoshen Acupuncture （once a day， 6 consecutive days per week） in addition to what were given to the control group. The treatment course lasted for 4 weeks. The 24 h urination frequency， 24 h urine leakage frequency， 24 h single urine volume， bladder residual urine volume， international lower urinary tract symptom （LUTS） score， traditional Chinese medicine （TCM） syndrome score were compared between the two groups， and clinical effectiveness and TCM syndrome effectiveness were compared between the two groups after treatment. ResultsTwenty patients in each group were finally analyzed in this study. The number of 24 h urination， the number of 24 h urine leakage， bladder residual urine volume， LUTS score， and the TCM syndrome scores decreased after treatment in both groups， and the 24 h single urine volume increased （P＜0.01）； and much more improvement was found of each index in the treatment group than in the control group （P＜0.05 or P＜0.01）. The total clinical effectiveness and TCM syndrome effectiveness in the treatment group was 85.00% （17/20） respectively， which were statistically significantly higher than 45.00% （the total clinical effectiveness， 9/20） and 60.00% （TCM syndrome effectiveness， 12/20） in the control group （P＜0.01）. ConclusionModified Shenqi Pill plus Tongdu Tiaoshen Acupuncture can signi-ficantly improve the clinical symptoms of neurogenic bladder patients after spinal cord injury of kidney-yang deficiency syndrome， having better effectiveness than simple bladder function rehabilitation training， and its mechanism may be related to the improvement of the injured nerve function innervating the bladder.

Objective:To explore the gap between the clinical nursing status and practice guidelines of elderly functional constipation, propose corresponding action strategies, and provide reference for the clinical transformation of the guidelines.Methods:From July to October 2019, totally 330 clinical nurses and nursing managers of elderly patients with functional constipation admitted into the general hospitals, hospitals of traditional Chinese medicine, and hospitals of traditional Chinese medicine and western medicine in Beijing were selected by convenient sampling and investigated with self-made questionnaires. The standard and algorithm of recommendations were formulated, and Excel software was used to calculate the compliance rate, partial compliance rate, and non-compliant rate of recommendations. A total of 330 questionnaires were distributed in this study, and 330 questionnaires were recovered, with a recovery rate of 100.0% (330/330) . After excluding invalid questionnaires such as items with missing values greater than 10% and all items having the same answers, there were 317 valid questionnaires, with an effective rate of 96.1% (317/330) .Results:Among the recommendations of the 12 guidelines, the 3 items with the highest compliance rate were health education related to toileting patterns, health education related to activities and sports, satisfaction and effect evaluation, while the 4 items with the highest non-compliance rates were building interdisciplinary teams, comprehensive education and program promotion, health assessment, and physical examination of medication history. There was a statistically significant difference in the clinical practice status between nurses who had and had not received relevant training ( P<0.01) . Conclusions:Most nurses can provide comprehensive care for elderly patients with functional constipation, but there is a big gap between the practice and the guidelines. Measures such as strengthening nurse training and talent training and standardizing the nursing process of constipation can help promote the clinical translation of evidence.

AIM: To understand the publication status, research trends, and cutting-edge and hot topics in this field by conducting a bibliometrics analysis of relevant literatures on the pathogenesis of primary open angle glaucoma(POAG)in the past 30 a.METHODS:A total of 986 relevant literatures on the pathogenesis of POAG published on the core databases of China National Knowledge Infrastructure(CNKI)and Web of Science(WOS)from 1 September 1993 to 1 September 2023 were retrieved. CiteSpace(6.2.R.4)and VOSviewer(1.6.18)software were used to conduct knowledge graph analysis on the retrieved literature, including publication volume, author, research institution, country/region, and keywords.RESULTS:The United States(243 articles)has the highest number of publications, followed by China(121 articles). The foreign institution with the highest number of publications is Harvard University(37 articles), while domestic institutions such as Zhongshan Ophthalmic Center, Sun Yat-sen University, ophthalmology department of Xuanwu Hospital of Capital Medical University, and Peking University First Hospital tied for the highest number of publications. Louis R. Pasquale(21 articles)is the most prolific English author. Wang Ningli is the most active Chinese researcher in this field. Keywords include trabecular meshwork, intraocular pressure, aqueous humor, glucocorticoid, hemorheology, etc.CONCLUSION: The research on the pathogenesis of POAG is in a period of vigorous development. The United States has the largest number of publications in this field, and Harvard University is a leading institution in this field. The research focus in the field of POAG has shifted from the structural aspect to the genetic level, and gene research and traditional Chinese medicine treatment have broad application prospects in this field.

Inflammatory caspase-11 senses and is activated by intracellular lipopolysaccharide (LPS) leading to pyroptosis that has critical role in defensing against bacterial infection, whereas its excess activation under pathogenic circumstances may cause various inflammatory diseases. However, there are few known drugs that can control caspase-11 activation. We report here that scutellarin, a flavonoid from Erigeron breviscapus, acted as an inhibitor for caspase-11 activation in macrophages. Scutellarin dose-dependently inhibited intracellular LPS-induced release of caspase-11p26 (indicative of caspase-11 activation) and generation of N-terminal fragment of gasdermin D (GSDMD-NT), leading to reduced pyroptosis. It also suppressed the activation of non-canonical nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome as evidenced by reduced apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and decreased interleukin-1 beta (IL-1β) and caspase-1p10 secretion, whereas the NLRP3-specific inhibitor MCC950 only inhibited IL-1β and caspase-1p10 release and ASC speck formation but not pyroptosis. Scutellarin also suppressed LPS-induced caspase-11 activation and pyroptosis in RAW 264.7 cells lacking ASC expression. Moreover, scutellarin treatment increased Ser/Thr phosphorylation of caspase-11 at protein kinase A (PKA)-specific sites, and its inhibitory action on caspase-11 activation was largely abrogated by PKA inhibitor H89 or by adenylyl cyclase inhibitor MDL12330A. Collectively, our data indicate that scutellarin inhibited caspase-11 activation and pyroptosis in macrophages at least partly via regulating the PKA signaling pathway.