PAC1 is the neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) preferring receptor, which belongs to class B G protein-coupled receptors (GPCR) family. PAC1 mediates the most effects of PACAP as neurotransmitter, neuroregulator and neuroprotectant, while its high expression has close relationship with some physiological and pathological processes such as nerve-injury and tumor. To further understand the function of PAC1, a cell line that expressed inducible PAC1 was constructed to achieve Doxycycline (Dox) dependent expression of PAC1 in CHO (Chinese hamster ovary) cell using the improved Tet (tetracycline)-on Advanced System. First, the PAC1-EYFP fusion gene composed of PAC1 gene and gene encoding EYFP (enhanced yellow fluorescent protein) was sub-cloned to the tetracycline response element pTRE-Tight vector to construct the recombinant vector pEYFP-PAC1-EYFP by double enzyme digestion. Second, the tetracycline regulation components pTet-On advanced vector and the response element pTRE-PAC1-EYFP vector were both introduced into CHO cells successively and the positive clones were screened with G418 and hygromycin respectively. Third, the controlled expression of PAC1-EYFP in CHO was induced by tetracycline analogues Dox in different concentrations and the different levels of receptor PAC1-EYFP were detected. The results of fluorescence analysis and western blotting show that the cell strain with Dox dependent expression of PAC1-EYFP named PAC1-Tet-CHO was obtained. Moreover, in PAC1-Tet-CHO cells the expression of PAC1-EYFP was induced by Dox in a dose-dependent manner. The inducible expression of PAC1 still was stable after sub-culturing for more than 10 passages. It was also found by MTT assay that the higher expression level of PAC1 endowed the cells with higher proliferative viabilities. The construction of controlled expression system of PAC1 will lay a foundation for the further research on PAC1 profiles.
Animals ; Blotting, Western ; CHO Cells ; Cloning, Molecular ; Cricetinae ; Cricetulus ; Genetic Vectors ; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I ; biosynthesis

Objective:To compare the therapeutic effect of fluid resuscitation strategy guided by pulse-indicated continuous cardiac output (PiCCO) monitoring and early goal-directed therapy (EGDT) on renal function of acute kidney injury (AKI) patients caused by septic shock.Methods:Septic shock patients with AKI admitted to the intensive care unit (ICU) of Tianjin Fifth Central Hospital and Teda International Cardiovascular Hospital from March 2017 to February 2020 were enrolled. All patients were given fluid resuscitation. Patients were divided into PiCCO-guided fluid resuscitation group [PiCCO group, intrathoracic blood volume index (ITBVI) was maintained between 850-1 000 mL/m 2] and EGDT-guided fluid resuscitation group [EGDT group, central venous pressure (CVP) was maintained between 8-12 mmHg (1 mmHg = 0.133 kPa) or CVP ≤ 15 mmHg when patients received mechanical ventilation (MV)] according to both the patient's condition and the informed consent of the patient's family. The changes of heart rate (HR), mean arterial pressure (MAP), CVP, blood lactic acid (Lac), fluid balance, urine volume and serum creatinine (SCr) at 6, 24, and 48 hours after fluid resuscitation in the two groups were observed, and the renal replacement therapy (RRT), duration of MV, length of ICU stay and 28-day mortality between the two group were compared. Results:① A total of 94 patients were enrolled, including 51 in the EGDT group and 43 in the PiCCO group. There was no significant difference in gender, age, acute physiology and chronic health evaluation Ⅱ (APACHE Ⅱ) score, sequential organ failure assessment (SOFA) score, procalcitonin (PCT), HR, MAP, CVP, Lac or SCr at ICU admission between the two groups. ② The parameters of hemodynamics, fluid balance, urine volume and SCr were improved with the time of resuscitation in the two groups, and there was no significant difference in HR, MAP or Lac between the two groups. Compared with the EGDT group, the CVP decreased significantly at 24 hours and 48 hours after fluid resuscitation in the PiCCO group (mmHg: 9.1±0.9 vs. 12.0±1.3 at 24 hours, 8.0±1.0 vs. 10.2±1.3 at 48 hours), the fluid balance significantly decreased (mL: 2 929.8±936.3 vs. 3 898.4±923.5 at 24 hours, 3 143.5±1 325.4 vs. 4 843.8±1 326.7 at 48 hours), and the condition of urine volume and SCr were better in the PiCCO group [urine volume (mL·kg -1·h -1): 1.02±0.21 vs. 0.79±0.14 at 24 hours, 1.28±0.18 vs. 0.94±0.22 at 48 hours; SCr (μmol/L): 145.7±37.6 vs. 164.3±46.4 at 24 hours, 128.4±33.6 vs. 143.5±37.7 at 48 hours), with significant differences (all P < 0.05). ③ Compared with the EGDT group, the rate of RRT in the PiCCO group was lower [11.6% (5/43) vs. 17.6% (9/51)], the duration of MV and the length of ICU stay were shorter [duration of MV (days): 4.64±1.31 vs. 6.50±2.19, length of ICU stay (days): 10.35±3.50 vs. 14.50±5.78), with significant differences (all P < 0.05). There was no significant difference in the 28-day mortality between the PiCCO group and EGDT group [14.0% (6/43) vs. 15.7% (8/51), P > 0.05]. Conclusion:Fluid resuscitation strategy guided by PiCCO in septic shock patients with AKI can reduce the amount of fluid load, improve renal function, shorten the MV duration and length of ICU stay, and shows clinical significance.

Objective:To compare the clinical effects of external-internal four paper splints fixation at a fixed point with the routine paper splint fixation in the treatment of the unstable distal radius fracture.Methods:A total of 66 patients with type C1 or C2 distal radial fractures, from November 2016 to May 2019, were analyzed retrospectively. According to different external fixation methods, patients were divided into 2 groups with 33 patients in each group. Modified group treated with external-internal four paper splint fixation at a fixed point and normal group treated with routine paper splint fixation. The 4th and 6th weeks after the operation, the reduction of the distal radius fracture was evaluated based on anteroposterior and lateral wrist radiographs. After 3 months follow-up, the radial height, radial inclination and palm dip angle were measured and the healing time were recorded. The Gartland-Werley functional scale was used to evaluate the wrist function from four aspects of residual malformation, subjective evaluation, objective evaluation and complications, and to evaluate the clinical efficacy.Results:After 3 months follow-up, all patients got clinical healing. The loss of palm dip angle [(2.9 ± 2.4)° vs. (4.6 ± 3.2)°, t=-2.524], radial inclination [(3.4 ± 2.2)° vs. (5.0 ± 3.0)°, t=-2.506] and radial height [(2.4 ± 1.9) mm vs. (3.6 ± 2.1) mm, t=-2.302] in modified group were significantly less than those in normal group ( P<0.05). There was a lower Gartland-Werley score in modified group compared with normal group [(1.52 ± 2.75) vs. (5.76 ± 8.68); t=-2.677, P<0.05]. The excellent and good rate was 97.0% (32/33) in the modified group and 78.8% (26/33) in the nomal group. There was significant difference between two groups ( Z=-2.315, P=0.021). Conclusions:The external-internal four paper splint fixation at a fixed point can control the redisplacement of unstable distal radius fractures effectively, and maintain the reduction. The effect of modified paper splint fixation at a fixed point is better than routine paper splint fixation.

Objective To investigate the imaging characteristics of nontraumatic spontaneous intramural hematoma of small bowel secondary to warfarin therapy.Methods The retrospective and descriptive study was conducted.The clinical data of 12 patients with nontraumatic spontaneous intramural hematoma of small bowel secondary to warfarin therapy who were admitted to the First People's Hospital of Wenling (11 patients) and Taizhou Hospital of Zhejiang Province (1 patient) between January 2010 and December 2016 were collected.Patients received plain and enhanced scans of computed tomography (CT).Patients stopped warfarin therapy,received intramuscular injection of vitamin K1 and fresh-frozen plasma transfusion.Observation indicators:(1) primary signs of CT:① small bowel wall thickening;② increased density of small bowel wall;(2) secondary signs of CT:intestine lumen stenosis,intestinal pneumatosis,perienteric changes and other concomitant signs;(3) treatment situations.Measurement data with normal distribution were represented as (x)±s.Measurement data with skewed distribution were described as M (range).Results Twelve patients received total abdomen plain scans of CT,and 7 received total abdomen enhanced scans of CT.(1) Primary signs of CT:① Small bowel wall thickening.CT of 12 patients showed solitary,homogeneously symmetric,consecutive and diffuse thickening of small bowel wall,including ileal involvement in 7 patients and jejunum involvement in 5 patients.Ascending part of duodenum of 3 patients involved the 2nd and 3rd groups small bowel.The average length of involved small bowel was 30.7 cm (range,11.0-58.0 cm).There was no multifocal thickening or colonic involvement.Of 12 patients,10 and 2 showed moderate thickening (thickness was 1.0-2.0 cm) and severe thickening (thickness ＞ 2.0 cm),and the thickest small bowel wall was 2.5 cm.② Increased density of small bowel wall.Twelve patients showed varying degrees of increased density of small bowel wall,with an average value of 49.7 HU (range,36.0-63.4 HU).Of 12 patients,homogeneously increased whole layer density of small bowel wall were detected in 4 patients,higher submucous layer density of small bowel compared with serosal layer density in 4 patients,and all of the above characteristics in 4 patients.Enhanced scans of CT in 7 patients showed enhancement in small bowel mucosal layer and serosal layer,no obvious enhancement in the submucous layer of small bowel wall,clear layers,and a target sign when small bowel lumen was perpendicular to scan slice,including 5 patients with obvious enhancement in small bowel mucosal layer,spring-like change,and clear imaging in portal vein phase.(2) Secondary signs of CT:① Intestine lumen stenosis:12 patients had varying degrees of intestine lumen stenosis.Five patients were complicated with small bowel obstruction,showing a gas-liquid level.② Intestinal pneumatosis:2 of 12 patients showed patchy and mottled gas shadow.③ Perienteric changes and other concomitant signs:12 patients showed obviously increased fat density in surrounding area of involved small bowel and corresponding mesenteric area,and indistinct outside small bowel,including 8 with lots of lath-like high density shadow in surrounding area of small bowel and mesenteric area.The varying degrees of hemoperitoneum were seen in 12 patients.(3) Treatment situations:of 12 patients,9 underwent correct treatments,8 of them were improved,and 1 of them with persistently severe abdominal pain underwent urgent explorative laparotomy,showing hemorrhage and necrosis,and then underwent surgery;3 didn't undergo correct treatment,without improved or severe symptoms.Conclusion Patients undergoing anticoagulant therapy show acute abdominal pain with abnormal coagulation function,small bowel wall thickening and increased density by CT scans,hemoperitoneum complicated with intestine lumen stenosis,small bowel obstruction and hematocele in surrounding area of small bowel and mesenteric area,these are highly indicative of nontraumatic spontaneous intramural hematoma of small bowel secondary to warfarin therapy.

For wild natural medicine, unanticipated biodiversity as species or varieties with similar morphological characteristics and sympatric distribution may co-exist in a single batch of medical materials, which affects the efficacy and safety of clinical medication. DNA barcoding as an effective species identification tool is limited by its low sample throughput nature. In this study, combining DNA mini-barcode, DNA metabarcoding and species delimitation method, a novel biological sources consistency evaluation strategy was proposed, and high level of interspecific and intraspecific variations were observed and validated among 5376 Amynthas samples from 19 sampling points regarded as "Guang Dilong" and 25 batches of proprietary Chinese medicines. Besides Amynthas aspergillum as the authentic source, 8 other Molecular Operational Taxonomic Units (MOTUs) were elucidated. Significantly, even the subgroups within A. aspergillum revealed here differ significantly on chemical compositions and biological activity. Fortunately, this biodiversity could be controlled when the collection was limited to designated areas, as proved by 2796 "decoction pieces" samples. This batch biological identification method should be introduced as a novel concept regarding natural medicine quality control, and to offer guidelines for in-situ conservation and breeding bases construction of wild natural medicine.