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Exclusively breastfed infants are at a high risk of vitamin D deficiency. Few studies have evaluated the effects of vitamin D supplementation. Hence, we conducted a prospective randomized controlled trial investigating the effects of oral vitamin D3 400 IU/d supplementation in exclusively breastfed newborns. Serum 25-hydroxy-vitamin D (25[OH]D) levels in pregnant women and their newborns were evaluated. Breastfed newborns were randomized to one of two regimens at age 10 days. One group received vitamin D3 supplementation at a dose of 400 IU/d (vD-400 group), whereas the placebo group received a liquid product without vitamin D3. Outcomes were assessed at 4 months of age. A total of 92 pregnant women and their infants were enrolled, and the data of 72 infants (37 in the vD-400 group and 35 in the placebo group) who completed the study at 4 months of age were assessed. The results showed severe vitamin D deficiency in 15.2% of mothers before delivery, while 54.3% had vitamin D deficiency. Moreover, 15.2% of newborns presented with severe vitamin D deficiency at birth, while 52.2% had vitamin D deficiency. Maternal vitamin D levels were significantly correlated with infant vitamin D levels at birth (r = 0.816, p 0.001). At 4 months of age, weight, head circumference, serum 25(OH)D, phosphorus, and intact parathyroid hormone levels significantly differed between the vD-400 and placebo groups. However, the body length and bone mineral density of the two groups did not differ significantly. Regardless of vitamin D supplementation, participants with severe vitamin D deficiency had significantly higher intact parathyroid hormone levels and lower bone mineral content. In conclusion, among exclusively breastfed infants, oral supplementation with vitamin D3 at a dose of 400 IU/d from age 10 days increased 25(OH)D concentrations at 4 months of age, but it did not affect bone mineralization. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Background Surgical interventions for congenital scoliosis are challenging for spine surgeons. The coordination of 3-dimensional (3D), real-time, image-guided navigation with an ultrasonic bone cutter allows surgeons to localize the affected area of the spine accurately and remove the lesion without damaging soft tissue structures. The goal of this technical paper is to report a previously undescribed method of hemivertebrectomy that combines 3D, real-time, image-guided navigation and an ultrasonic bone cutter. We highlight the feasibility and safety of this method in spinal surgery. Methods Three patients with congenital scoliosis were treated with this technique. We present three illustrative cases comprising hemivertebrectomies for congenital scoliosis. Intraoperative photos demonstrating the technique are also provided. Results All surgeries were completed without complications. The hemivertebrae were completely removed, and marked correction of congenital scoliosis was noted. Conclusions We believe that the combination of 3D, real-time image navigation and an ultrasonic bone cutter improves hemivertebrectomy by increasing accuracy and avoiding dura laceration, major organ damage, or potential vessel damage.

Background The risk of injury directly related to hospitalization for motor vehicle accidents (MVAs) in the obstructive sleep apnea (OSA) patients has not been thoroughly understood. Our study aimed to examine the association between the OSA and the hospitalization for an MVA injury. Methods This retrospective cohort study used Taiwan’s National Health Insurance Research Database (NHIRD) between 2000 and 2015. The OSA patients aged ≥20 years by age, sex, and index-year matched by non-OSA controls were enrolled (1:3). We used the Cox proportional regression model to evaluate the association between the OSA and the hospitalization for an MVA injury. Results The incidence rate of hospitalization for an MVA injury was higher in the OSA cohort (N = 3025) when compared with the non-OSA controls (N = 9075), as 575.3 and 372.0 per 100,000 person-years, respectively (p p p Conclusions This study provides evidence that patients with OSA are at a two-fold higher risk of developing hospitalization for an MVA injury, and the usage of modafinil and methylphenidate was associated with a lower risk of an overall hospitalization for the MVA injury.

Background. The pedicle screw is one of the main tools used in spinal deformity correction surgery. Robotic and navigated surgeries are usually used, and they provide superior accuracy in pedicle screw placement than free-hand and fluoroscopy-guided techniques. However, their high cost and space limitation are problematic. We provide a new solution using 3D printing technology to facilitate spinal deformity surgery. Methods. A workflow was developed to assist spinal deformity surgery using 3D printing technology. The trajectory and profile of pedicle screws were determined on the image system by the surgical team. The engineering team designed drill templates based on the bony surface anatomy and the trajectory of pedicle screws. Their effectiveness and safety were evaluated during a preoperative simulation surgery. The surgery consisted in making a pilot hole through the drill template on a computed tomography- (CT-) based, full-scale 3D spine model for every planned segment. Somatosensory evoke potential (SSEP) and motor evoke potential (MEP) were used for intraoperative neurophysiological monitoring. Postoperative CT was obtained 6 months after the correction surgery to confirm the screw accuracy. Results. From July 2015 to November 2016, we performed 10 spinal deformity surgeries with 3D printing technology assistance. In total, 173 pedicle screws were implanted using drill templates. No notable change in SSEP and MEP or neurologic deficit was noted. Based on postoperative CT scans, the acceptable rate was 97.1% (168/173). We recorded twelve pedicle screws with medial breach, six with lateral breach, and five with inferior breach. Medial breach (12/23) was the main type of penetration. Lateral breach occurred mostly in the concave side (5/6). Most penetrations occurred above the T8 level (69.6%, 16/23). Conclusion. 3D printing technology provides an effective alternative for spinal deformity surgery when expensive medical equipment, such as intraoperative navigation and robotic systems, is unavailable.

Background The aim of this study is to introduce a new method of percutaneous endoscopic decompression under 3D real-time image-guided navigation for spinal stenosis in degenerative kyphoscoliosis patients without instability or those who could not endure major surgery due to multiple comorbidities. Decompression along using endoscope for kyphoscoliosis patient is technical demanding and may result in unnecessary bone destruction leading to further instability. The O-arm/StealthStation system, a 3D real-time image-guided navigation system, is popular for its ability to provide automated registration with intraoperative, postpositioning computed tomography (CT) with no radiation exposure to operative staffs. With 3D real-time image-guided navigation, we could improve the accuracy of surgery and maintain the spinal stability as preoperative condition. Methods In this study, we presented four cases. All patients were over seventy years old female with variable degrees of kyphoscoliosis and multiple comorbidity who could not endure major spine fusion surgery. Percutaneous endoscopic unilateral laminotomy and bilateral decompression under 3D real-time image-guided navigation were successfully performed with following steps. First, the reference pin was introduced over iliac crest. Second, all navigational instruments were registered to the O-arm Stealth Station. Third. With the interlaminar approach, a 8.0 mm dilator followed by operative sleeve was introduced toward the ligamentum flavum. Then the endoscope attached with universal tracker was inserted, and the boundaries of lamina and facet joint were well-identified through the monitor. Finally, unitaleral laminotomy and resection of ligamentum flavum with joy-stick technique for bilateral decompression were performed under the navigation system. Patients’ demographics, image study parameters, and outcome measurements including pre- and post-operative serial visual analog scale (VAS), and Oswestry Disability Index (ODI) were well documented. The follow-up time was one year. Results All patients were successfully treated with this technique. Pre- and post-operative MRI showed average dural sac cross sectional area (DSCSA) improved from 81.62 (range 67.34–89.07) to 153.27 (range 127.96-189.73). All preoperative neurological symptoms including radicular leg pain and sciatica improved postoperatively. The mean ODI (%) were 85 (range 82.5–90) at initial visit, 35.875 (range 25–51) at 1 month post-operatively, 26.875 (range 22.5–35) at 6 months post-operatively and 22.5 (range 17.5–30) at 12 months post-operatively (p p

Objective To focus on the potential beneficial effects of the pleiotropic effects of dipeptidyl peptidase-4 inhibitors (DPP4is) on attenuating progression of diabetic kidney disease in reducing the long-term effect of the acute kidney injury (AKI) to chronic kidney disease (CKD) transition. Patients and Methods Data from the National Health Insurance Research Database from January 1, 1999, to July 31, 2011, were analyzed, and patients with diabetes weaning from dialysis-requiring AKI were identified. Cox proportional hazards models and inverse-weighted estimates of the probability of treatment were used to adjust for treatment selection bias. The outcomes were incident end-stage renal disease (ESRD) and mortality, major adverse cardiovascular events, and hospitalized heart failure. Results Of a total of 6165 patients with diabetes weaning from dialysis-requiring AKI identified, 5635 (91.4%) patients were DPP4i nonusers and 530 (8.6%) patients were DPP4i users. Compared with DPP4i nonusers, DPP4i users had a lower risk of ESRD (hazard ratio, 0.81; 95% CI, 0.70-0.94; P=.04) and all-cause mortality (hazard ratio, 0.28; 95% CI, 0.23-0.34; P

Yung-Fu Chen,1–3,* Hsuan-Hung Lin,4,* Chih-Sheng Lin,5 Battsetseg Turbat,1 Kuo-An Wang,4,6 Wei-Sheng Chung1,3,7 1Department of Healthcare Administration, 2Department of Dental Technology and Materials Science, Central Taiwan University of Science and Technology, Taichung, 3Department of Health Services Administration, China Medical University, Taichung, 4Department of Management Information Systems, Central Taiwan University of Science and Technology, Taichung, Taiwan, Republic of China; 5Department of Radiology, BenQ Medical Center, Nanjing, People’s Republic of China; 6Department of Industrial Education and Technology, National Changhua University of Education, Changhua, 7Department of Internal Medicine, Taichung Hospital, Ministry of Health and Welfare, Taichung, Taiwan, Republic of China *These authors contributed equally to this work Background: Bronchiectasis is characterized by permanent dilatation of the bronchial tree caused by recurrent airway infection and inflammation. The association of atherosclerosis and inflammation is well established. However, studies on the relationship between bronchiectasis and stroke are scant. Objective: We conducted a population-based cohort study to investigate the incidence and risk of ischemic stroke in patients with bronchiectasis. Methods: Data of 1,295 patients newly diagnosed as bronchiectasis between 2000 and 2008 were retrieved from the Taiwan National Health Insurance Research Database. A total of 6,475 controls without bronchiectasis at a ratio of 5:1 were randomly selected from the general population based on frequency-matched age and sex to the patients. All participants were followed up to the date of ischemic stroke development, censoring, or the end of 2010. The Cox proportional hazard model was used to identify the risk of ischemic stroke in patients with bronchiectasis compared with those without bronchiectasis. Results: The patients with bronchiectasis exhibited a higher incidence rate of ischemic stroke (9.18 vs 4.66 per 1,000 person-years) than the patients without bronchiectasis, with an adjusted hazard ratio of 1.74 (95% confidence interval =1.28–2.35). The patients with bronchiectasis and any comorbidities exhibited a 2.66-fold adjusted hazard ratio of ischemic stroke compared with those with neither bronchiectasis nor comorbidity (95% confidence interval =1.85–3.84). The patients with bronchiectasis carried a dose response of ischemic stroke according to the number of emergency visits and hospitalizations per year. Conclusion: This study indicated that bronchiectasis is an independent risk factor of ischemic stroke. Keywords: bronchiectasis, ischemic stroke, cohort study

Eradicating infection, protecting neurologic function, and maintaining structural alignment are the 3 objectives of treatment for infectious spondylodiscitis. For some patients, surgery may be necessary to achieve these goals; however, open surgeries are associated with high morbidity and mortality in elderly patients and those with multiple comorbidities. Endoscopic surgery provides a minimally invasive surgical option for obtaining a culture sample to aid identification of pathogens, while also providing a route for adequate decompression and drainage. The clinical results of this study were analyzed.To evaluate the efficacy and safety of spinal endoscopic surgery, the basic characteristics of patients analyzed and their inflammatory markers, pain levels, and local kyphotic angles were recorded before surgery and at regular intervals after surgery. The patients' cultured pathogens and previous antibiotic treatments were also recorded and analyzed.Retrospective observational study (institutional review board: CMUH 105-REC2-101).Inpatient surgery center.From October 2006 to March 2017, of 508 patients who received spinal endoscopic surgery, 60 with infectious spondylodiscitis were treated using this new strategy. All 60 patients underwent plain film radiography and enhanced magnetic resonance imaging of the affected region to obtain evidence of infectious spondylodiscitis. The role of a computed tomography-guided biopsy and some indications for open surgery were replaced with endoscopic surgery.All the patients reported rapid pain relief after endoscopic surgery and antibiotic treatment. No significant changes in sagittal alignment were observed in final follow-up radiography images. Causative pathogens were identified in 34 patients (culture rate: 77.27%) without previous antibiotic treatment. The patients' erythrocyte sedimentation rates and C-reactive protein levels had decreased significantly 3 months after endoscopic surgery. Two patients (3.3%) experienced infection relapse following initial endoscopic surgery; both of them were efficiently resolved through a second round of endoscopic surgery. No surgery-related complications were observed and no open spinal surgery was required during the follow-up period.This was a retrospective study; bias was unavoidable because of the single-center nature of the study design.Regarding the culture rate, recurrence rate, kyphotic change, and surgery-related complications, this new strategy for endoscopic surgery is safe and effective for treating infectious spondylodiscitis in the thoracic or lumbar spine and may be considered a new trend in treating diseases of this type.Spine, endoscopic, discectomy, spondylodiscitis, minimally invasive surgery.

Inflammation-mediated abnormalities in the renin-angiotensin system (RAS) and expression of matrix metalloproteinases (MMPs) are implicated in the pathogenesis of lung injury. Angiotensin converting enzyme II (ACE2), an angiotensin converting enzyme (ACE) homologue that displays antagonist effects on ACE/angiotensin II (Ang II) axis, could also play a protective role against lung diseases. However, the relationship between ACE2 and MMPs activation in lung injury is still largely unclear. The purpose of this study is to investigate whether MMPs activity could be affected by ACE2 and which ACE2 derived signaling pathways could be also involved via using a mouse model with lung injury induced by cigarette smoke (CS) exposure for 1 to 3 weeks. Wild-type (WT; C57BL/6) and ACE2 KO mice (ACE2(-/-)) were utilized to study CS-induced lung injury. Increases in the resting respiratory rate (RRR), pulmonary immunokines, leukocyte infiltration and bronchial hyperplasia were observed in the CS-exposed mice. Compared to WT mice, more serious physiopathological changes were found in ACE2(-/-) mice in the first week of CS exposure. CS exposure increased pulmonary ACE and ACE2 activities in WT mice, and significantly increased ACE in ACE2(-/-) mice. Furthermore, the activity of pulmonary MMPs was decreased in CS-exposed WT mice, whereas this activity was increased in ACE2(-/-) mice. CS exposure increased the pulmonary p-p38, p-JNK and p-ERK1/2 level in all mice. In ACE2(-/-) mice, a significant increase p-STAT3 signaling was detected; however, no effect was observed on the p-STAT3 level in WT mice. Our results support the hypothesis that ACE2 deficiency influences MMPs activation and STAT3 phosphorylation signaling to promote more pulmonary inflammation in the development of lung injury.

Angiotensin-converting enzyme (ACE) is the primary enzyme that converts angiotensin I (Ang I) to angiotensin II (Ang II) in the renin-angiotensin system (RAS). However, chymase hydrates Ang I to Ang II independently of ACE in some kidney diseases, and it may play an important role. The present study investigated whether chymase played a crucial role in aristolochic acid I (AAI)-induced nephropathy. C57BL/6 mice were treated with AAI via intraperitoneal injection for an accumulated AAI dosage of 45 mg/kg body weight (BW) (15 mg/kg BW per day for 3 days). The animals were sacrificed after acute kidney injury development, and blood, urine and kidneys were harvested for biochemical and molecular assays. Mice exhibited increased serum creatinine, BUN and urinary protein after the AAI challenge. Significant infiltrating inflammatory cells and tubular atrophy were observed in the kidneys, and high immunocytokine levels were detected. Renal RAS-related enzyme activities were measured, and a significantly increased chymase activity and slightly decreased ACE activity were observed in the AAI-treated mice. The renal Ang II level reflected the altered profile of RAS enzymes and was significantly increased in AAI-treated mice. Treatment of AAI-induced nephropathic mice with an ACE inhibitor (ACEI) or chymase inhibitor (CI; chymostatin) reduced renal Ang II levels. The combination of ACEI and CI (ACEI+CI) treatment significantly reversed the AAI-induced changes of Ang II levels and kidney inflammation and injuries. AAI treatment significantly increased renal p-MEK without increasing p-STAT3 and p-Smad3 levels, and p-MEK/p-ERK1/2 signalling pathway was significantly activated. CI and ACEI+CI treatments reduced this AAI-activated signaling pathway. AAI-induced nephropathy progression was significantly mitigated with CI and ACEI+CI treatment. This study elucidates the role of RAS in the pathogenesis of AAI-induced nephropathy.

More than 1 billion people suffer from chronic respiratory diseases worldwide, accounting for more than 4 million deaths annually. Inhaled corticosteroid is a popular medication for treating chronic respiratory diseases. Its side effects include decreased bone mineral density and osteoporosis. The aims of this study are to investigate the association of inhaled corticosteroids and fracture and to design a clinical support system for fracture prediction. The data of patients aged 20 years and older, who had visited healthcare centers and been prescribed with inhaled corticosteroids within 2002–2010, were retrieved from the National Health Insurance Research Database (NHIRD). After excluding patients diagnosed with hip fracture or vertebrate fractures before using inhaled corticosteroid, a total of 11645 patients receiving inhaled corticosteroid therapy were included for this study. Among them, 1134 (9.7%) were diagnosed with hip fracture or vertebrate fracture. The statistical results showed that demographic information, chronic respiratory diseases and comorbidities, and corticosteroid-related variables (cumulative dose, mean exposed daily dose, follow-up duration, and exposed duration) were significantly different between fracture and nonfracture patients. The clinical decision support systems (CDSSs) were designed with integrated genetic algorithm (GA) and support vector machine (SVM) by training and validating the models with balanced training sets obtained by random and cluster-based undersampling methods and testing with the imbalanced NHIRD dataset. Two different objective functions were adopted for obtaining optimal models with best predictive performance. The predictive performance of the CDSSs exhibits a sensitivity of 69.84–77.00% and an AUC of 0.7495–0.7590. It was concluded that long-term use of inhaled corticosteroids may induce osteoporosis and exhibit higher incidence of hip or vertebrate fractures. The accumulated dose of ICS and OCS therapies should be continuously monitored, especially for patients with older age and women after menopause, to prevent from exceeding the maximum dosage.

Background: The renin-angiotensin system (RAS) has significant influences on heart and renal disease progression. Angiotensin converting enzyme (ACE) and angiotensin converting enzyme II (ACE2) are major peptidases of RAS components and play counteracting functions through angiotensin II (Ang II)/ATIR and angiotensin-(1–7) (Ang-(1–7))/Mas axis, respectively. Methods: There were 360 uremic patients on regular hemodialysis (HD) treatment (inclusive of 119 HD patients with cardiovascular diseases (CVD) and 241 HD patients without CVD and 50 healthy subjects were enrolled in this study. Plasma ACE, ACE2, Ang II and Ang-(1–7) levels of the HD patients were determined. Results: We compared pre-HD levels of plasma ACE, ACE2, Ang II and Ang-(1–7) in the HD patients with and without CVD to those of the controls. The HD patients, particularly those with CVD, showed a significant increase in the levels of ACE and Ang II, whereas ACE2 and Ang-(1–7) levels were lower than those in the healthy controls. Therefore, imbalanced ACE/ACE2 was observed in the HD patients with CVD. In the course of a single HD session, the plasma ACE, ACE/ACE2 and Ang II levels in the HD patients with CVD were increased from pre-HD to post-HD. On the contrary, ACE2 levels were decreased after the HD session. These changes were not detected in the HD patients without CVD. Conclusions: Pathogenically imbalanced circulating ACE/ACE2 was detected in the HD patients, particularly those with CVD. HD session could increase ACE/Ang II/AT1R axis and decrease ACE2/Ang-(1–7)/Mas axis activity in the circulation of HD patients with CVD.

Single-incision laparoscopic surgery developed rapidly in recent years. We introduce an innovative technique: single-incision laparoscopic common bile duct exploration (SILCBDE) with conventional instruments. A retrospective comparison between SILCBDE and standard laparoscopic common bile duct exploration (LCBDE) was analyzed.Thirty-four patients who underwent LCBDE for choledocholithiasis in a period of 17 months were enrolled. Seventeen standard LCBDEs and 17 SILCBDEs were attempted. Simultaneous cholecystectomies were performed.The stone clearance rate was 94.1% (16 patients) in the standard LCBDE group and 100% in the SILCBDE group. There was no statistical difference in demographic distribution, clinical presentations, and operative results between the two groups, except the SILCBDE group had a higher rate of acute cholecystitis than the standard LCBDE group (76.5 vs. 35.3%; p 0.05). One procedure (5.9%) in the SILCBDE group was converted to a four-incision transcystic LCBDE. The complication rate was 11.8% (two patients) in the standard LCBDE group and 5.9% (one patient) in the SILCBDE group. The average follow-up period was 4.2 months.SILCBDE is as safe and efficacious as standard LCBDE in experienced hands. Choledochoscope manipulation and bile duct repair are the key skills. Long-term follow-up and further prospective randomized trials are anticipated.

Diosgenin, a traditional Yam extraction, has been used in hormone replacement for menopausal women. We aimed to investigate the influences of diosgenin administration upon the MMP-2 and -9 activity and expression and reproductive hormones of ovariectomized (OVX) rats, a model of menopausal status. Seven-week old female Wistar rats with bilateral OVX or sham operation (controls) were divided and administered different dosages of diosgenin (0, 10, 50, or 100 mg/kg/day) for 8 weeks. Serum was then sampled for progesterone (P4) and estradiol (E2) assay and uterine horns harvested. Myometrial MMP-2 and -9 activity and expression were surveyed and myometrial collagen expression was also assayed. The results show higher body weight in OVX rats across the 8 weeks post surgery and no significant differences were noted among OVX or Sham rats with diosgenin supplements. There were lower P4 and E2 concentrations in OVX rats compared to Sham rats, and higher P4 concentration of Sham rats post diosgenin supplement. MMP-2 and -9 mRNA expression and activity was lower in OVX rats, although higher MMP-2 and lower MMP-9 activity/mRNA expression was observed in OVX rats post diosgenin supplementation. Collagen mRNA expression was higher in OVX rats compared to Sham controls, and diosgenin administration decreased collagen mRNA expression in OVX rats. In conclusion, diosgenin is associated with gelatinase expression and collagen metabolism in OVX rats. Diosgenin administration can partially reverse the effects of OVX upon MMP functions and hormone status. Adequate diosgenin supplement might modulate myometrial gelatinase expression and collagen metabolism in menopausal subjects.

Condensation Both Gonal-F and Puregon, especially in their high-dosage administration, might inhibit the endometrial cell proliferation in the initial 48-hour culture. After 72-hour culture, Gonal-F persisted the inhibition of the endometrial growth, whereas Puregon reversed its effect to enhance endometrial growth. Objectives Endometrial proliferation or regeneration during menstrual cycle is regulated by sexual hormones. However, the effect of gonadotropins on the endometrial cell growth remains obscure. Herein, we aimed to investigate the effects of recombinant follicle-stimulating hormones (r-FSHs) (Gonal-F and Puregon) on the proliferation of human endometrial cells in vitro . Materials and Methods Human endometrial cells (RL95-2 cells) were obtained commercially and cultured in the serum-containing media in the presence of r-FSHs (Gonal-F and Puregon at concentrations of 0mIU/mL, 200mIU/mL, 400mIU/mL, and 600mIU/mL) up to 72 hours. According to the gonadotropin concentrations, all cultured endometrial cells were divided into four groups: (1) 0mIU/mL (control); (2) 200mIU/mL; (3) 400mIU/mL; and (4) 600mIU/mL. After 72-hour culture, endometrial cell proliferations were assessed overnight by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The influences of different r-FSH agents and dosages on endometrial cell proliferation in each group were evaluated and compared. Results In the four Gonal-F groups, the cell absorption (control and 200mIU/mL, 400mIU/mL, and 600mIU/mL Gonal-F) after 24/48/72-hour cultures were as follows: (1) 0.57/0.7/0.82; (2) 0.56/0.66/0.78; (3) 0.55/0.64/0.77; and (4) 0.51/0.61/0.78. After 48 hours, higher dosage of Gonal-F appeared to significantly inhibit the endometrial cell proliferation. After 72-hour culture, all three dosages of Gonal-F appeared to inhibit the endometrial cell proliferation similarly. In Puregon groups, the cell absorptions were as follows: (1) 0.62/0.53/0.62; (2) 0.61/0.5/0.66; (3) 0.61/0.49/0.66; and (4) 0.64/0.49/0.66. Puregon administration displayed initial inhibition and subsequent stimulation effects on the endometrial cells. Conclusions Both Gonal-F and Puregon, especially in their high-dosage administration, appeared to inhibit the endometrial cell proliferation in the initial 48-hour culture. After 72-hour culture, Gonal-F persisted the inhibition of the endometrium, whereas Puregon reversed its effect by enhancing the endometrial growth. The differences might be because of the different formulations or molecular structures existing between alpha and beta follitropins.

Summary Objective We aimed to investigate the effects of arsenic (As), benomyl (Ben), and carbendazim (Carb) on endometrial cells. Materials and Methods Human endometrial cells were obtained during diagnostic curettage. All cultured endometrial cells were divided into four groups: (1) 0 M (controls), (2) 10 −6 M, (3) 10 −5 M, (4) 10 −4 M for As, Ben and Carb. After 24 and 48 hours in culture, endometrial cell proliferations were assessed by diphenyltetrazolium bromide assay. The influences of different concentrations of As, Ben and Carb upon the endometrium were compared. Results During the first 24 hours, As, Ben and Carb appeared to have insignificant influences upon endometrial growth. After 48 hours in culture, all three agents significantly inhibited endometrial growth. In As groups, cell absorption after 48 hours culture were 100% (group 1), 82.1% (group 2), 43.6% (group 3) and 35.3% (group 4). In Ben groups, cell absorption was 100% (1), 75.9% (2), 66.4% (3) and 49. 6% (4). In the Carb groups, cell absorption was 100% (1), 70.4% (2), 73.0% (3) and 76.7% (4). Conclusion The agents As, Ben and Carb appear to have inhibitory effects upon endometrial cells after 48 hours in culture.

Hirudin, isolated from the leech Hirudo medicinalis, inhibits thrombin directly and several expression systems have been used to produce recombinant Hirudin (rHirudin) for pharmaceutical purposes. A DNA fragment containing the Hirudin coding sequence and goat beta-casein secretion signal was chemically synthesized in this study. The synthetic DNA then was further constructed into a goat beta-casein expression vector for mouse transgenesis. Four lines of transgenic mice were successfully developed and one line showed a meaningful anti-thrombin activity of 40,000 anti-thrombin units (ATU)/mL in their milk. In this animal line, Hirudin mRNA was found in samples of uterus and kidney with insignificant anti-thrombin activity (

Matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs) play important roles in the pathophysiology of renal diseases. Imbalanced MMPs/TIMPs are implicated in the vascular alterations of uremic patients on hemodialysis (HD). We have investigated the plasma levels of MMP-2, MMP-9, TIMP-1 and TIMP-2 in uremic patients and the effects of a course of HD on the changes in these factors. There were 382 uremic patients on regular HD treatment and 50 healthy controls enrolled in this study. The plasma MMP-2 and MMP-9 levels were detected by gelatin zymography, and TIMP-1 and TIMP-2 concentrations were determined by ELISA assay. Significantly higher plasma MMP-2 and MMP-9 and decreased TIMP-1 in the uremic patients were detected compared with those in the controls. Therefore, there were markedly higher MMP-2/TIMP-2 and MMP-9/TIMP-1 ratios in the uremic patients. In the course of a single HD session, the plasma MMP-2 level was significantly decreased from pre-HD to post-HD. TIMP-1 concentration was significantly increased from pre-HD to post-HD. Although the HD session did not have a significant effect on the levels of plasma MMP-9 and TIMP-2, both plasma MMP-2/TIMP-2 and MMP-9/TIMP-1 ratios were significantly decreased from pre-HD to post-HD levels. HD session could decrease MMP-2 and increase TIMP-1 level in the circulation of uremic patients. The physiological significance of reduced MMPs/TIMPs ratio due to a single HD session is required to further validate.

We have established a spontaneously immortalized porcine mammary epithelial cell line (SI-PMEC) from the mammary gland of a lactating sow and maintained it long-term in culture by continuous subculturing. SI-PMEC cells were maintained for more than 8 months (70 passages) in DMEM/F12 medium supplemented with 10% fetal calf serum, insulin, and hydrocortisone without obvious signs of senescence. When grown at low density on a plastic substratum, SI-PMEC cells formed islands, and when grown to confluency, the cells formed a monolayer and aggregated with the characteristic cobblestone morphology of epithelial cells. The subcultured SI-PMEC cells appeared to proliferate without changes in morphology or growth pattern, with an estimated population doubling time of 20-22 h. With increasing density, SI-PMEC cells organized into lumen-like structures with elongated cells at the margins. SI-PMEC cells from stocks frozen at Passage 30 were subcultured up to 20 times without changes in cell viability, proliferation rate, or morphology. Furthermore, SI-PMEC cells remained immunopositive to an antibody against cytokeratin AE3 and immunonegative to an antibody against a human fibroblast antigen. The SI-PMEC cells could form functional structures resembling ducts, lateral-buds, and alveoli in a Matrigel matrix-dependent manner in vitro. When grown on the Matrigel and stimulated by prolactin, the cells differentiated and formed mammary gland structures and strongly expressed transcripts encoding the milk proteins alpha-lactalbumin, beta-casein, and beta-lactoglobulin. Our results indicate that the SI-PMEC cell line can be subpassaged many times and still form functional differentiated secretory structures. To our knowledge, this is the first report of an immortalized mammary epithelial cell line from pig.

Leiomyoma and hyperprolactinaemia are both progesterone-dependent diseases. Hormone-related genes, such as the progesterone receptor (PGR), might be involved in their pathogenesis.Subjects were divided into three groups: (i) leiomyoma (n = 120); (ii) hyperprolactinaemia (n = 101); (iii) normal controls (n = 140). We investigated the Alu (306-bp DNA) insertion in intron G of the PGR gene in all individuals. PGR gene polymorphisms [T1 (wild-type); T2 (PROGINS, with Alu insertion)] were determined by PCR and electrophoresis. Genotype and allele frequencies of the PROGINS in each group were detected and compared.We observed no significant difference of the PGR*T1/T2 genotypes and allele frequencies between leiomyoma and other two groups. The proportions of T1 homozygote/heterozygote/T2 homozygote in each group were (i) 90/8.3/1.7%; (ii) 84.2/9.9/5.9%; (iii) 92.9/6.4/0.7%. In contrast, a higher percentage of T2-related genotype and allele were noted in hyperprolactinaemic women compared to other two groups. The proportions of T1/T2 alleles in each group were: (i) 94.2/5.8%; (ii) 89.1/10.9%; (iii) 96.1/3.9%.The PROGIN*T2-related genotype and allele are related to a higher susceptibility to hyperprolactinaemia. The PROGINS polymorphism is not associated with leiomyoma development.

BALB/c mice are widely used in genetic, tumour and immunological studies. However, the mice demonstrate a lower reproduction rate, low fertility and small litters, because of their highly genetic homozygoisty. Based on in vitro fertilization (IVF), a routine technique for biomedical studies, it is worth to evaluate the effects to BALB/c mice on IVF efficiency. In order to test the genetic factor affecting the IVF efficiency of BALB/c, four reciprocal IVF tests of BALB/cByJ and FVB/NCrl mice were performed. The results showed that the average fertility of IVF sponsored by FVB/NCrl spermatozoa was 69.6%, but only 12.1% was obtained from BALB/cByJ strain. Effect of glucose contained in the culture medium to the IVF efficiency of BALB/cByJ was also evaluated. The results showed that the fertility of BALB/cByJ spermatozoa incubated with 0, 2.7, 5.5, 11.1 and 22.2 mm of glucose in the TYH medium were 6.8, 9.9, 13.9, 32.7 and 22.2%, respectively. It is showed that IVF efficiency of BALB/cByJ spermatozoa could be improved depending on the concentration of glucose in the IVF medium. According to the results, it is beleived that lower IVF of BALB/cByJ mice might be due to the genetic defect in spermatozoa and increasing glucose in the IVF medium which significantly affect the IVF efficiency of BALB/cByl via activating the spermatozoa.

The aim of this study was to evaluate the correlation between embryonic early-cleavage status and the age of patients receiving either a GnRH agonist long protocol or a GnRH antagonist protocol. This retrospective study included 534 patients undergoing a fresh cycle of oocyte retrieval and day-3 embryo transfer. Of the 534 patients treated, 331 received a GnRH agonist long stimulation protocol (GnRH agonist group) for ovarian stimulation and 203 patients received a GnRH antagonist protocol (GnRH antagonist group). By logistic regression analysis, the rate of embryonic early-cleavage was significantly decreased with increasing age of women in the agonist (P

Background Single-incision laparoscopic cholecystectomy (SILC) has been shown to be safe for uncomplicated gallbladder diseases. Routinely applying SILC is debatable. Methods Two hundred SILCs were performed with single-incision multiple-port longitudinal-array and self-camera techniques. Results Eighty-eight (44%) procedures were scheduled for complicated diseases. The routine group had a higher comorbidity rate, a lower preoperative endoscopic retrograde cholangiopancreatography rate, a higher intraoperative cholangiography rate, a higher proportion of complicated gallbladder diseases, shorter operative time, more intraoperative blood loss, and lower postoperative pethidine dose than the selective group (the first 73 patients). The conversion and complication rates showed no statistical difference. It took fewer cases but longer time to pass the learning phase of SILC for complicated gallbladder diseases. The multivariate analysis showed that male sex and complicated gallbladder diseases were associated with a higher procedure conversion rate, and increased patient age was related to a higher complication rate. Conclusions Routine SILC for benign gallbladder diseases is feasible in the experienced phase. Practicing SILC for uncomplicated gallbladder diseases helps to achieve competence in this technique for complicated diseases.

Objective Anemia is associated with high mortality and poor prognosis after acute coronary syndrome (ACS). Increased red cell distribution width (RDW) is a strong independent predictor for adverse outcomes in ACS. The common underlying mechanism for anemia and increased RDW value is iron deficiency. It is not clear whether serum iron deficiency without anemia affects left ventricular (LV) performance after primary angioplasty for acute myocardial infarction (AMI). We investigated the prognostic value of serum iron concentration on LV ejection fraction (EF) at 6 months and its relationship to thrombolysis in myocardial infarction (TIMI) risk score in post MI patients. Methods We recruited 55 patients who were scheduled to undergo primary coronary balloon angioplasty after AMI and 54 age- and sex-matched volunteers. Serum iron concentration and interleukin-6 levels were measured before primary angioplasty. LVEF was measured by echocardiography at baseline and after 6 months. TIMI risk score was calculated for risk stratification. Results Serum iron concentration was significantly lower in those in whom LVEF had not improved ≥10% from baseline (52.7±24.1 versus 80.8±50.8 µg/dl, P = 0.016) regardless of hemoglobin level, and was significantly lower in the AMI group than in the control group (62.5±37.7 versus 103.0±38.1 µg/dl, P

Objectives Oxidized low-density lipoproteins (oxLDL) and oxidized low-density lipoprotein autoantibodies (OLAB) have been detected in human plasma and atherosclerotic lesions. OLAB appear to play a role in the clearance of oxLDL from circulation. Higher levels of OLAB appear to be associated with a reduced risk of a wide range of cardiovascular diseases. We investigated the prognostic value of plasma oxLDL and OLAB in patients undergoing primary coronary balloon angioplasty for acute ST-elevation myocardial infarction (STEMI). Methods Plasma oxLDL and OLAB concentrations were measured in 56 patients with acute STEMI before primary angioplasty, and then 3 days, 7 days and 1 month after the acute event. Follow-up angiography was repeated 6 months later to detect the presence of restensosis (defined as >50% luminal diameter stenosis). The thrombolysis in myocardial infarction (TIMI) risk score was calculated to determine the relationship between OLAB/oxLDL ratio and TIMI risk scores. Results Of the 56 patients, 18 (31%) had angiographic evidence of restenosis. Plasma OLAB concentrations were significantly lower in the restenosis group before angioplasty (181±114 vs. 335±257 U/L, p = 0.003), and at day 3 (155±92 vs. 277±185 U/L, p

Background Deep neck infections (DNIs) in HIV-infected patients often produce severe complications, even death. Data on the incidence rates and risks of DNI among HIV-infected patients are scarce, particularly with the widespread use of highly active antiretroviral therapy (HAART). We evaluated the incidence rates and risks for DNI among HIV-infected patients and observed the long-term trends. Methods A total of 9888 new HIV-infected patients diagnosed in 2001–2007 were included and matched with 49440 randomly selected subjects. The HIV-infected subjects were offered free access to HAART. All subjects were traced until December 2009. A Kaplan-Meier analysis generated the cumulative DNI incidence rate. The adjusted hazard ratio was computed using Cox proportional hazard regressions. Results From the HIV-infected and comparison cohorts, 222 individuals (57.01 cases per 10000 person-years) and 735 individuals (35.54 cases per 10000 person-years) developed DNI, respectively. The log rank test indicated that patients with HIV had a significantly higher 8-year incidence rate of DNI than the control group (P P Conclusion The risk of developing DNI is significantly elevated among HIV-infected patients, even with free access to HAART. Additional research is needed to examine the role of HAART in reducing the risk.

Atrial fibrillation (AF) is the most frequent cardiac arrhythmia seen in clinical practice. Several therapeutical approaches have been developed to terminate the AF and the effects are evaluated by the reduction of the wavelet number after the treatments. Most of the previous studies focus on modeling and analysis of the mechanism, and the characteristic of AF. But no one discusses about the prediction of the result after the drug treatment. This paper is the first study to predict whether the drug treatment for AF is active or not. In this paper, the linear autoregressive model with exogenous inputs (ARX) that models the system output-input relationship by solving linear regression equations with least-squares method was developed and applied to estimate the effects of pharmacological therapy on AF. Recordings (224-site bipolar recordings) of plaque electrode arrays placed on the right and left atria of pigs with sustained AF induced by rapid atrial pacing were used to train and test the ARX models. The cardiac mapping data from 12 pigs treated with intravenous administration of antiarrhythmia drug, propafenone (PPF), or dl-sotalol (STL) were evaluated. The recordings of cardiac activity before the drug treatment were input to the model and the model output reported the estimated wavelet number of atria after the drug treatment. The results show that the predicting accuracy rate corresponding to the PPF and STL treatments was 100% and 92%, respectively. It is expected that the developed ARX model can be further extended to assist the clinical staffs to choose the effective treatments for the AF patients in the future.

Endometrial proliferation or regeneration during menstrual cycle is regulated by sexual hormones. However, the effect of gonadotrophins on the endometrial cell growth remains obscure. Herein, we aimed to investigate the effects of r-FSH (Gonal-F, Puregon) and progesterone on the proliferation of human endometrial cells in-vitro. According as gonadotrophin concentrations, the follicular-phase endometrial cells were divided into six groups: (1) 0 (controls), (2) 1; (3) 10; (4) 100; (5) 1000; (6) 100,000 μIU/ml. The cell countings with microscopy and cell proliferation kit assay were used to assess the endometrial cell proliferations. In Gonal-F groups, the cell absorptions (%) after 24/48 h culture were: (1) 100/100; (2) 103.8/102.3; (3) 104.8/102.8; (4) 102.3/101.3; (5) 96.3/94.2; (6) 86.8/84.3. In Puregon groups, the cell absorptions were: (1) 100/100; (2) 102.8/101.9; (3) 103/102.3; (4) 103.9/103.5; (5) 102.9/102.4; (6) 103.7/103.2 (non-different). In progesterone groups, the cell absorptions were: (1) 100/100; (2) 99.1/101.9; (3) 83.5/80.4; (4) 80.7/82.4. Higher dosage of Gonal-F (100,000 μIU/ml) and progesterone (10, 100 μg/ml) appeared the significant inhibition upon endometrium. We conclude that lower dosages of Gonal-F, Puregon, and progesterone appear the non-significant influence upon endometrium. Higher dosage of Gonal-F (10,000 μIU/ml) and progesterone (10, 100 μg/ml), but not Puregon, might interfere with the endometrial proliferation during follicular phase.

Summary Leiomyomas (myoma or fibroid) are the most common gynecologic tumors that occur in women of reproductive age, but their molecular pathogenesis is still unknown. Since the growth of leiomyomas involve numerous vascular factors, an association between the leiomyoma and growth factors is suspected. Vascular endothelial growth factor (VEGF) is one of the most important angiogenic growth factors. VEGF regulates angiogenesis and mediates sex steroid-induced cell growth and differentiation. VEGF-mediated activities seem to contribute to the pathogenesis of leiomyoma. Genetic variations, including polymorphisms, in VEGF might also be associated with the complex pathogenesis of leiomyomas. Here, we performed a systematic review of the roles of VEGF and its polymorphisms in the pathogenesis of leiomyoma.

Objective To search for novel peptides and common binding motif that specifically bind to endometriosis. Design Prospective study. Setting Department of Biological Science and Technology in national university. Patient(s) Specimens were divided into [1] ectopic endometrium (n = 10); [2] eutopic endometrium (n = 10). Intervention(s) Peptides specifically binding to endometriosis are screened from a phage-displaying peptide library (Ph.D.-12) by using whole-cell screening technique after an adsorption elution amplification procedure. Main Outcome Measure(s) Combinatorial peptide libraries were used to identify small molecules that bind with high affinity to receptor molecules and mimic the interaction with natural ligands. Few pans of positive phage clones with significantly positive signals were identified by ELISA and analyzed by DNA sequencing. Result(s) During the biopanning processes, the recovered phage number (10 6 pfu/mL) in parts 1, 2, 3, 4, and 5 of the study were 9, 33, 82, 142, and 169. Nine phages consistently had residue Arg, whereas six clones had a consensus motif of Arg-X-Arg-X-X-X-X-Arg. The biotin-labeled peptide bound to endometriosis cells in a dose-dependent manner, yet the control peptide revealed lesser binding activity. Conclusion(s) The novel motif is associated with higher affinity of endometriosis, which might be useful in endometriosis targeting and as potential antiendometriosis therapies. We provide one potential approach for novel therapies toward endometriosis.

The purposes of this study were to investigate single strand conformation polymorphisms (SSCP) in the D-loop region of pig mitochondrial DNA (mtDNA) and to determinate their association with the reproductive traits of meishan pigs. A total of four types of band patterns, designed SSCP band pattern A, B, C and D, were identified. A type of SSCP band pattern was present in all European-American breeds, but not in East Asian breeds. This result showed the diversified sequence in the D-loop region between European-American and East Asian populations. Two types of band patterns, B and C, were found in Meishan pigs. The average body weight at day 21 of piglets from B type dams was significantly heavier than the body weight of C type (p < 0.05). We also tested whether the SSCP patterns would be suitable for paternity testing in a family group and found that bands of all the offspring were derived from their maternal parent. Therefore, we conclude that SSCP may be a marker for identification of maternal ancestors.

Background Atrial fibrillation (AF) is characterized by structural remodeling of the extracellular matrix (ECM) in cardiac atrium. Objective The purpose of this study was to gain further insight into atrial ECM remodeling at the molecular level and to test whether altered expression of ECM proteins was associated with the disease. Methods Sustained AF was induced in nine adult pigs after 3–4 weeks of continuous rapid atrial pacing at a rate of 600 bpm. Histologic studies and immunohistochemical stain were performed to identify the potential pathologic substrate underlying abnormalities in atrial tissues with sustained AF. Results In the pathologic findings, the fraction of myocardial ECM (ECM%) was measured, with a significantly greater ECM% found in the AF group compared with the sham operated group (n = 6; i.e., pigs with normal sinus rhythm [SR]). A set of 9,182 genes was screened with cDNA microarray analysis. In AF animals, expression of 121 genes increased and 24 genes decreased by ≥1.75-fold compared with SR animals. Significant up-regulation of fibronectin-1 (4.9-fold), fibrillin-1 (3.1-fold), and fibromodulin (1.9-fold) in the fibrillating atria was confirmed by quantitative real-time reverse transcriptase-polymerase chain reaction. Western blot analysis revealed significantly increased atrial fibronectin-1, fibrillin-1, and fibromodulin in the AF group compared with the SR group (1.5-, 2.7-, and 2.1-fold, respectively). Immunohistochemical staining of AF tissue displayed increased accumulation of fibronectin-1 and fibrillin-1 in the atrial interstitial space. Conclusion Increased expression of ECM proteins in fibrillating atria supports the hypothesis that ECM metabolism contributes to the development of AF.

Dysregulated p53 expression has been implicated as a major contributor to numerous tumorigenesis. Single nucleotide polymorphisms (SNPs) within the functional consequence of the novel p53 promoter region remains obscure. Herein, we aimed to establish the extent of genetic variability within the promoter region of p53 gene as well as their association with leiomyoma susceptibility. Women were divided into two groups, leiomyoma (n = 160) and nonleiomyoma controls (n = 200). Total DNA was isolated from the peripheral blood of subjects. The DNA fragment containing p53 promoter regions (+64 approximately -404 bp) were obtained by amplification of polymerase chain reaction. The variations of DNA fragments were detected by DNA sequencing or restriction fragment length polymorphism (RFLP). Sequence alignment was used to identify sequence variations in p53 promoter regions. Genotypes were analyzed by method of RFLP. Genotypes/allelic frequencies in the leiomyoma and control groups were compared. A total of 15 sequence variations within p53 promoter region were identified, including -408 T/C, -382 A/G, -359 A/G, -325 T/C, -250 A/G, -216 T/C, -205 G/A, -198 G/A, -177 T/C, -103 A/G, -81 G/A, -71 G/A, -51 T/A, -33 A/G, and -17 T/C. Among these variations, four SNPs (-250 A/G, -216 T/C, -103 A/G, and -33 A/G) were established. Allele frequencies of -250*G/-216*C/-103*G/-33*G in the leiomyoma group and control group 6.9/5.0/5.9/3.8% and 3.8/1.8/2.3/4.0%, respectively. Two of them (-216*C and -103*G) are associated with higher leiomyoma susceptibility. We concluded that some sequence variations were observed within the promoter region of p53 gene. The SNPs of -216*C and -103*G among the identical sequence variations are associated with leiomyoma development.

Endometriosis and leiomyoma are both common estrogen-related gynaecological diseases. We aimed to elucidate the association of estrogen receptor alpha (ERalpha)-351 A>G (XbaI) and -397 T>C (PvuII) gene polymorphisms with endometriosis and leiomyoma. Women were divided into three groups: (i) severe endometriosis (n = 112), (ii) leiomyoma (n = 106) and (iii) normal controls (n = 110). Genomic DNA was obtained from peripheral leukocytes. ERalpha-351 A/G XbaI and -397 T/C PvuII polymorphisms were assayed by the method of PCR and restriction fragment length polymorphism (RFLP). Genotypes and allelic frequencies in each group were compared. The genotype/allele frequencies of ERalpha-351 and -397 polymorphisms in endometriosis or leiomyoma groups were different from those of normal controls. ERalpha mutant-related genotypes/alleles (-351G and -397C) presented higher percentages in the endometriosis/leiomyoma population compared with normal controls. Proportions of ERalpha-351 AA/AG/GG genotypes and A/G alleles in each group were (i) 26.8/57.1/16.1 and 55.4/44.6%; (ii) 19.8/52.8/27.4 and 46.2/53.8% and (iii) 33.6/64.6/1.8 and 65.9/34.1%. Proportions of ERalpha-397 TT/TC/CC genotypes and T/C alleles in each group were (i) 24.1/60.7/15.2 and 54.5/45.5%; (ii) 23.6/70.8/5.6 and 59/41% and (iii) 54.5/40/5.5 and 74.5/25.5%. We concluded that ERalpha-351 XbaI*G- and -397 PvuII*C-related genotypes/alleles were correlated with higher susceptibilities of endometriosis or leiomyoma, which might be associated with related pathogeneses.

Atrial fibrillation (AF), the most common cardiac arrhythmia, is frequently accompanied by atrial interstitial fibrosis. Angiotensin II (Ang II) dependent signaling pathways have been implicated in interstitial fibrosis during the development of AF. However, Ang II could be further degraded by angiotensin converting enzyme II (ACE2). We examined expression of ACE2 in the fibrillating atria of pigs and its involvement in fibrotic pathogenesis during AF. Nine adult pigs underwent continuous rapid atrial pacing to induce sustained AF and six pigs were sham controls (i.e., sinus rhythm; SR). In the histological examinations, extensive accumulation of extracellular matrix in the interstitial space of the atria, as evidenced by Masson’s trichrome stain, were found in fibrillating atria. The relative amount of collagen type I in the atria with AF was significantly increased as compared with that in the SR. Local ACE activity in the fibrillating atria was also markedly higher than that in the SR subjects. ACE2 gene and protein expression in the AF subjects were significantly decreased compared with those in the SR subjects, whereas expression of mitogen-activated/ERK kinase 1/2 (MEK1/2), extracellular signal-regulated protein kinase 2 (ERK2), and activated ERK2 were significantly greater in the AF subjects. We propose that decreasing ACE2 expression during AF may affect the Ang II-dependent signaling pathway. In addition, our results suggest that atrial fibrosis in AF may be induced by antagonistic regulation between ACE and ACE2 expression.

Porcine mammary epithelial cells (PMECs) were isolated from lactating sow mammary glands and cultured on a matrix gel. Primary culture cells expressed significant amounts of the specific marker cytokeratin as determined by immunohistochemistry, and exhibited mammary-specific functions, such as transcription of alpha-lactalbumin, beta-casein and beta-lactoglobulin genes. They also formed mammospheres when the medium was supplemented with lactogenic hormones. The PMECs were used to study gene transfer and expression in vitro. A gene encoding enhanced green fluorescent protein (EGFP) was used as a reporter and two constructs were investigated, pEGFP-N1 (a vector constructed with a CMV promoter followed by the EGFP gene) and pGB562/GFP (a mammary gland-specific expression vector with regulatory sequences from the goat beta-casein gene linked to EGFP). The efficiency of DNA transfer into the cultured PMECs was about 20-30%. GFP expression in the pGB562/GFP-transfected PMECs was markedly stimulated by prolactin supplements in the medium. The established PMECs maintained optimal gene expression from 1 to 20 passages and appeared to provide an efficient and convenient system for assessing the expression of transgenes containing mammary gland-specific promoters.

Follicular growth and maturation is a complex process regulated by autocrine and paracrine factors. Follicular fluids may affect fertilization and early embryonic development (Ledee-Bataille et al., 2001). Follicular fluid may play an important role in the endocrine balance between the theca and granulosa cells (Ledee-Bataille et al., 2001). Cytokines have been shown to be present in human follicular fluid and have regulatory functions in follicular maturation and subsequent embryo development (Coskun et al., 1998). Leukemia inhibitory factor (LIF) is a pleiotropic cytokine of the interleukin-6 family and has different biological actions in various tissue systems. LIF regulates the growth and differentiation of embryonic stem cells, primordial germ cells, peripheral neurons, osteoblasts, adipocytes, and endothelial cells (Murray and Edgar, 2001). It also plays an important role in the physiology of ovulation, estrogen production, and early embryonic development (Arici et al., 1997). LIF is one of the key determinants of successful implantation (Arici et al., 1997). Follicular fluid LIF also enhances the rate of blastocyst formation (Dunglison et al., 1996). Follicular fluid LIF is a useful marker of oocyte quality (Arici et al., 1997). The rising level of follicular fluid LIF around the time of ovulation indicates that LIF may play a role in ovulatory events, early embryonic development, and

Objective To transfect the p53 and p21 gene into the leiomyoma cells isolated from patients and observe their influence on the cell proliferation, leukemia inhibitory factor production, and gene expression of collagen type I as well as tumor necrosis factor-α (TNF-α) of cultured cells. Design Prospective study. Setting An assisted reproductive technology (ART) and genetic unit of a medical center. Patient(s) Leiomyoma cells isolated from leiomyoma tissue of 12 patients were divided into three groups: [1] vehicle DNA, [2] p53 gene, and [3] p21 gene transfections. Intervention(s) The pcDNA3.1 was used as vector to carry p53 and p21 genes for transfer. After gene transfection, RNAs of the leiomyoma cells were extracted for further analyses of gene expression. Main outcome measure(s) Relative cell numbers were determined by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) assay. The leukemia inhibitory factor (LIF) concentration was determined with ELISA. Gene expressions of collagen type I and TNF-α were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Gene expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The cell proliferation, LIF production, as well as gene expressions of collagen type I and TNF-α in each group were compared. Results Relative cell numbers (%)/LIF production (in picograms per milliliter) in each group were: [1] 100/58, [2] 71/43, and [3] 106/65. The ratios of gene expression of collagen type I/TNF-α with GAPDH in each group were: [1] 1.64/0.335, [2] 1.25/0.434, and [3] 1.77/0.234. Conclusion(s) Transfection with p53 significantly inhibits proliferation of leiomyoma cells and decreases collagen type I gene expression and LIF production. The p21 transfection inhibits TNF-α gene expression.

AIM: To compare the clinical outcome of single-incision laparoscopic cholecystectomy (SILC) and three-incision laparoscopic cholecystectomy (3ILC) for acute cholecystitis. METHODS: From July 2009 to September 2012, 136 patients underwent SILC or 3ILC for acute cholecystitis at a tertiary referral hospital. One experienced surgeon performed every procedure using 5 or 10 mm 30-degree laparoscopes, straight instruments, and conventional ports. Five patients with perforated gallbladder and diffuse peritonitis and 23 patients with mild acute cholecystitis were excluded. The remaining 108 patients were divided into complicated and uncomplicated groups according to pathologic findings. Patient demography, clinical data, operative results and complications were recorded and analyzed. RESULTS: Fifty patients with gangrenous cholecystitis, gallbladder empyema, or hydrops were classified as the complicated group, and 58 patients with acute cholecystitis were classified as the uncomplicated group. Twenty-three (46.0%) of the patients in the complicated group (n = 50) and 39 (67.2%) of the patients in the uncomplicated group (n = 58) underwent SILC; all others underwent 3ILC. The postoperative length of hospital stay (PLOS) was significantly shorter in the SILC subgroups than the 3ILC subgroups (3.5 ± 1.1 d vs 4.6 ± 1.3 d, P < 0.01 in the complicated group; 2.9 ± 1.1 d vs 3.7 ± 1.4 d, P < 0.05 in the uncomplicated group). The maximum body temperature recorded at day 1 and at day 2 following the procedure was lower in the SILC subgroups, but the difference reached statistical significance only in the uncomplicated group (37.41 ± 0.56 °C vs 37.80 ± 0.72 °C, P < 0.05 on postoperative day 1; 37.10 ± 0.43 °C vs 37.57 ± 0.54 °C, P < 0.01 on postoperative day 2). The operative time, estimated blood loss, postoperative narcotic use, total length of hospital stay, conversion rates, and complication rates were similar in both SILC and 3ILC subgroups. The complicated group had longer operative time (122.2 ± 35.0 min vs 106.6 ± 43.6 min, P < 0.05), longer PLOS (4.1 ± 1.3 d vs 3.2 ± 1.2 d, P < 0.001), and higher conversion rates (36.0% vs 19.0%, P < 0.05) compared with the uncomplicated group. CONCLUSION: SILC is safe and efficacious for patients with acute cholecystitis. The main benefit is a faster recovery than that achieved with 3ILC.

The 5′-regulative sequence and intron 1 of the goat β-casein gene from −4044 to +2123 bp was cloned and fused with the reporter gene of green fluorescent protein (GFP) to create a plasmid termed pGB562/GFP. To detect GFP expression, pGB562/GFP was transfected in vitro via liposomes into the mammary epithelial cell line NMuMG. Cells could not express GFP unless the transfected NMuMG cells lined up to create functional alveoli. These functional cells were cultured with lactogenic hormones, including insulin, dexamethasone and prolactin, and were grown on a layer of the extracellular matrix Matrigel. Green fluorescent protein expression levels in NMuMG cells were 25-, 55- and 42-fold those in the control group at 24, 48, and 72 h after pGB562/GFP transfection respectively. In addition, pGB562/GFP was transfected ex vivo by electroporation into mammary gland fragments and cells were then cultured in vitro with a supplement of lactogenic hormones. Strong GFP expression localized in fragments of the mammary gland was observed 24 h after gene transfer. The novel strategy of ex vivo gene transfer into mammary tissue using GFP as a reporter gene to detect the function of a tissue-specific promoter is efficient and convenient. The data obtained herein reveal that the 5′-regulative sequence and intron 1 of the 6.2 kb goat β-casein gene can enhance the efficiency of transgene expression. Thus, the GB562 sequence may act as a good promoter and effectively elevate the production of exogenous protein in mammary glands.

Background To determine whether matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMP-1 and TIMP-2) in human follicular fluid, have any relationships with oocyte maturation in vivo and subsequent fertilization during in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) cycles. Methods The follicular fluids were obtained from 150 female patients undergoing IVF/ICSI cycles and a total of 1504 oocytes were retrieved for analysis. MMP-2 and MMP-9 activities were measured using zymography assay. TIMP-1 and TIMP-2 concentrations were quantitatively assessed using enzyme-linked immunosorbent assay (ELISA). Results Human follicular fluid MMP-2 level was significantly associated with the rate of maturity of oocytes (P