Two-dimensional liquid chromatography measurement of meropenem concentration in synovial fluid of patients with periprosthetic joint infection.

Periprosthetic joint infection (PJI) is a catastrophic complication following joint replacement surgery. One potential treatment approach for PJI could be the combination of one-stage revision and intra-articular infusion of antibiotics. Meropenem is one of the commonly used intra-articular antibiotics in our institution. Determining the concentration of meropenem in the joint cavity could be crucial for optimizing its local application, effectively eradicating biofilm infection, and improving PJI treatment outcomes. In this study, we developed a simple, precise, and accurate method of two-dimensional liquid chromatography (2D-LC) for determining the concentration of meropenem in human synovial fluid. The method was then validated based on the guidelines of the Food and Drug Administration and the Chinese Pharmacopoeia. Meropenem showed good linearity in the range of 0.31-25.01 µg/mL (r ≥ .999). Selectivity, intra-day and inter-day precision and accuracy, extraction recovery, and stability validation results were all within the acceptance range. This method has been successfully applied to the determination of synovial fluid samples from PJI patients, providing a useful detection method for meropenem therapeutic drug monitoring (TDM) in PJI patients.

First report of Cercospora cf. citrulina causing leaf spot of Ipomoea pes-caprae in China.

Ipomoea pes-caprae plays an important role in protecting the tropical and subtropical coastal beach of the world. In 2018, a leaf spot was observed on I. pes-caprae in Xisha islands of China, 13.2-25.8% of leaves were infected. The initial symptoms were small (1-3 mm diameter), single, circular, dark gray spots with a light-yellow center on the leaves. The lesions enlarged and were scattered or confluent, distinct and circular, subcircular or irregular, occasionally vein-limited, pale to dark gray-brown, with a narrow dark brown border surrounded by a diffuse yellow margin. Microscopic observations of the spots revealed that caespituli were dark brown and amphigenous, but abundant on the underside of the leaves. Mycelia were internal. Conidiophores were fasciculate, occasionally solitary, pale olivaceous-brown throughout, 0- to 3-septate, 27.9-115.8 (63.4±22.5) µm × 3.2-5.3 (4.3±0.87) µm (n=100). Conidial scars were conspicuously thickened. Conidia were solitary, hyaline, filiform, acicular to obclavate, straight to slightly curved, subacute to obtuse at the apex, truncate at the base, multi-septate, 21.0-125.5 (60.2±20.1) µm × 2.0-5.0 (3.8±0.83) µm (n=100). Single-conidium isolates were obtained from representative colonies grown on potato dextrose agar (PDA) incubated at 25℃ in the dark. The colonies grew slowly and were dense, white to gray and flat with aerial mycelium. Mycelia were initially white, and then became gray. Conidia were borne on the conidiophores directly. The pure isolate HTW-1 was selected for molecular identification and pathogenicity test, which were deposited in Microbiological Culture Collection Center of Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences. The internal transcribed spacer (ITS) region of rDNA, translation elongation factor 1-alpha (tef1) and histone H3 (his3) genes were amplified with ITS1/ITS4, EF-1 / EF-2, and CYLH3F / CYLH3R primers, respectively (Groenewald et al. 2013). The obtained sequences of HTW-1 were all deposited in GenBank with accession numbers MT410467 for ITS, MT418903 for tef1 and MT418904 for his3. The ITS, tef1 and his3 genes all showed 100% similarity for ITS (JX143582), tef1 (JX143340) and his3 (JX142602) with C. cf. citrulina (MUCC 588; MAFF 239409) from I. pes-caprae in Japan. Based on the morphological characteristics and molecular identification, the pathogen was identified as Cercospora cf. citrulina (Groenewald et al. 2013). The pathogenicity test was conducted by spraying conidial suspension (1×104 conidia/mL) on wounded and unwounded leaves for seedling of I. pes-caprae in greenhouse and in sterile vitro condition. The conidial suspension was prepared using conidia from 30-day-old culture grown on PDA at 25℃ in the dark. Leaf surfaces of seedling in greenhouse were wounded by lightly rubbing with a steel sponge and detached leaf surfaces were wounded by sterile needles. the treatments were sprayed with conidial suspensions on wounded and unwounded leaf surfaces. The control was sprayed with sterile water. After eight days, the typical symptoms of spots which were small, single, circular and dark gray appeared on the inoculated wounded leaves, while the inoculated unwounded leaves and the control leaves were symptomless. The pathogen was only re-isolated from the inoculated wounded leaves. The pathogen may be infected by wound. A total of 20 Cercospora and related species was found on Ipomoea spp. (García et al. 1996). Cercospora cf. citrulina has been reported on I. pes-caprae in Japan, although it was unclear if it was a pathogen or saprophyte (Groenewald et al. 2013). To our knowledge, this is the first report of C. cf. citrulina causing leaf spot of I. pes-caprae in China. This disease could threat the cultivation of I. pes-caprae in China.

First report of Colletotrichum siamense causing anthracnose on Erythrina crista-galli in China.

Erythrina crista-galli L. (Fabaceae) is a popular ornamental plant in tropical and subtropical regions of South Asia. In October 2019, anthracnose-like lesions were observed on the leaves of E. crista-galli planted in Haikou, China. 5-30% of leaves were infected. At first, the circular spots of 1-2 mm in diameter were reddish-brown on the leaves, and then enlarged to circular, subcircular or irregular spots with reddish-brown center and surrounded by a diffuse yellow margin. Neighboring spots sometimes coalesced. Under continuously wet or humid conditions, the lesions expanded quickly, and became gray, subcircular or irregular spots covered by grayish-white mycelium and orange-pink conidial masses. Diseased leaves eventually fell off the trees. To identify the pathogen, diseased leaves were sampled from four gardens. Leaf tissues (5×5 mm) were cut from the margins of typical symptomatic lesions, surface-sterilized in 1% sodium hypochlorite for 1 min, plated on potato dextrose agar (PDA) medium, and incubated at 28.0±0.5℃ in the dark. Similar fungal colonies were obtained from all plated tissues after 3 days. The single-conidium colonies of all isolates were white to pale gray and cottony with visible orange conidial masses. Conidia were one-celled, aseptate, hyaline, straight, cylindrical to fusiform with obtuse ends, and ranged from 14.2-18.6 µm (16.4 µm)× 3.8-5.4 µm (4.7 µm) (n=100). After germination, conidia formed single, brown, oval or slightly irregular appressoria ranging from 8.0 to 11.8 µm (9.6 µm), and from 4.8 to 6.0 µm (5.4 µm). Sexual stage was absent. These characteristics of conidia and appressoria were matched with C. siamense belonging to the C. gloeosporioides complex (Prihastuti et al. 2009; Yang et al. 2009; Weir et al. 20012; Hu et al. 2015). To accurately identify the species, DNA was extracted from four purified isolates (JG-1, JG-3-1, SWS-1-3, SWS-2-1) （Fu et al. 2019）. The internal transcribed spacer of rDNA region (ITS), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), actin (ACT) and chitin synthase (CHS) genes were amplified and sequenced. The nucleotide sequences were all deposited in GenBank (ITS: MT229427-MT229430, GAPDH: MT250821-MT250824, CAL: MT258893-MT258896, ACT: MT258897-MT258900 and CHS: MT258901-MT258904). Multi-locus phylogenetic analyses (ITS, GAPDH, CAL, ACT and CHS) (Weir et al. 2012) showed that the four isolates were clustered with C. siamense, which was in accordance with BLAST results. Pathogenicity tests of the four isolates were repeated three times on detached leaves (Ji et al. 2019). The conidial suspension (1×106 conidia/mL) was prepared using the conidia from 10-day-old cultures grown on PDA. Two 20-µL drops of conidial suspension were inoculated on non-wounded young healthy leaves, and each isolate was inoculated on 10 leaves. Two 20-µL drops of sterile water were inoculated on non-wounded young healthy leaves as control. The samples were maintained in containers at a relative humidity of 90± 5 per cent inside and 28℃ with a 12-h photoperiod. Gray, subcircular spots similar to the field disease symptoms were observed on the all inoculated leaves after 7 days, whereas no visible symptoms appeared on the non-inoculated leaves. The pathogen was re-isolated from inoculated leaves thus fulfilling Koch's postulates. C. gloeosporioides has been previously reported as a pathogen causing leaf spot on Erythrina (E. indica var. picta, E. variegata var. orientalis) in Guam in 1983 and Brazil in 2012. (Russo et al. 1983; Oliveira et al. 2012). To our knowledge, this is the first report of C. siamense causing leaf spot of E. crista-galli in China.

Differential metabolite analysis of the pharmacodynamic differences between different ratios of Dahuang ()-Taoren () herb pair.

OBJECTIVE: To explore the material basis of the difference of efficacy of Dahuang (Radix Et Rhizoma Rhei Palmati)-Taoren (Semen Persicae) (DT) drugs with different proportions. METHODS: Samples of different ratios of Dahuang (Radix et Rhizoma Rhei Palnati, DH) to Taoren (Semen Persicae, TR) (Group A 1:1, B 2:3, C 3:2) were analyzed based on gas chromatography time-of-flight mass spectrometry untargeted metabolomics technique. RESULTS: A total of 240 primary metabolites were detected. Forty-one differential metabolites involved nine differential metabolic pathways, of which four were closely related to the efficacy of DT in the treatment of heat and blood stasis syndrome. These pathways included the biosynthesis of amino acid (phenylalanine tyrosine and tryptophan), flavonoids, unsaturated fatty acids, and the glycolysis/glycogenesis pathway. CONCLUSION: There are significant differences in the efficacy of different ratios of DT drugs, and their optimal ratio for the treatment of heat and blood stasis syndrome should be 1:1.

Effect of calcitonin gene-related peptide on osteoblast differentiation in an osteoblast and endothelial cell co-culture system.

We have investigated the in vitro effects and regulatory mechanism of CGRP (calcitonin gene-related peptide) on the differentiation of OB (osteoblasts) in co-culture with HUVEC (human umbilical vein endothelial cells). Primary human MOB (mandibular OB) and OB-like cells (MG-63) were either cultured directly or indirectly co-cultured with HUVEC at a 1:1 ratio. Expression of OC (osteocalcin) was measured by ELISA, and expression of ALP (alkaline phosphatase) and collagen mRNA was measured by quantitative fluorescent PCR. For mineralization nodus, OB were stained with Alizarin Red-S. When co-cultured with HUVEC, expression of OC and ALP mRNA were increased in MG-63 (P<0.01), and the expression of OC, ALP and collagen mRNA were increased in MOB (P<0.01 or 0.05). When treated with CGRP, OC and ALP mRNA and mineralization nodus numbers were increased in the MG-63 co-culture system (P<0.01 or 0.05); OC, ALP and collagen mRNA, and mineralization nodus numbers were increased in the MOB co-culture system (P<0.01 or 0.05). The effect of CGRP regulation on the differentiation of OB is not only direct but also indirect, via its effect on HUVEC and stimulation of OB.

One-stage total knee arthroplasty for patients with osteoarthritis of the knee and extra-articular deformity.

PURPOSE: The purpose of this paper is to review our experience and study the feasibility and clinical results of one-stage total knee arthroplasty (TKA) for patients with osteoarthritis of the knee with extra-articular deformity. METHODS: Nine patients with osteoarthritis of the knee associated with extra-articular deformity underwent one-stage TKA from June 2006 to April 2010. There were two men and seven women, with an average age of 51 years (range 34-69 years); four of them had tibial deformities and five had femoral deformities. Eight of the cases resulted from malunion after fracture healing and one from femoral recurvatum. Six of the cases had uniplanar and three had biplanar deformities. The average angles of the femoral deformities were 13.3° in the coronal plane (8-22) and 11.3° in the sagittal plane (6-15); one femur had 10° external rotational deformity. Tibial deformity of 16° in the coronal plane (11-22) was noted, and one had sagittal plane deformity of 21°. RESULTS: All patients were followed for an average of 29 months. The average Hospital for Special Surgery (HSS) knee score improved from 18.7 points pre-operatively to 89.8 points at the time of last follow-up; the range of knee motion improved from 46.7° preoperatively to 100.6° postoperatively. The average angle of mechanical axis deviation was restored from 11.8° preoperatively to 1° postoperatively. One of the patients had unsatisfactory clinical results due to delayed union at the osteotomy site. No complications such as infection, deep vein thrombosis, ligament instability, low level or subluxed/dislocated patella or component loosening were observed. One-stage TKA with intra-articular correction of the extra-articular deformity was performed in seven patients, included proper planning, appropriate bone cuts to restore alignment and the necessary soft tissue releases to balance the knee in flexion and extension. Two patients underwent simultaneous extra-articular correctional osteotomy and TKA because the deformity was so large. Five knees that had good collateral ligamentous stability and balance received a posterior stabilised prosthesis; four knees that had ligamentous instability received a constrained condylar knee (CCK) prosthesis. CONCLUSIONS: One-stage TKA is a technically difficult but effective treatment for patients with osteoarthritis of the knee and extra-articular deformity. If feasible we recommend TKA with intra-articular bone resection and soft tissue balancing.

Prolonged duration of epidural labour analgesia decreases the success rate of epidural anaesthesia for caesarean section.

OBJECTIVE: To summarise the process of conversion of epidural labour analgesia to anaesthesia for caesarean delivery and explore the relationship between duration of labour analgesia and conversion. METHODS: Parturients who underwent conversion from epidural labour analgesia to anaesthesia for caesarean delivery between May 2019 and April 2020 at the Chengdu Women's and Children's Central Hospital, Sichuan Maternal and Child Health Hospital, and Jinjiang District Maternal and Child Health Hospital were selected. If the position of the epidural catheter was correct and the effect was good, patients were converted to epidural surgical anaesthesia. If epidural labour analgesia was ineffective, spinal anaesthesia (SA) was administered immediately. For category-1 emergency caesarean sections, general anaesthesia (GA) was administered. RESULTS: A total of 1084 parturients underwent conversion. Of these, 19 (1.9%) received GA due to the initiation of category-1 emergency caesarean section. 704 (64.9%) were converted to epidural surgical anaesthesia, 2 (0.2%) had failed conversions and were administered GA before delivery, and 357 (32.9%) were converted to SA. Logistic regression analysis showed that prolonged duration of epidural labour analgesia ([Crude odds ratio (OR)=1.065; 95% confidence interval (CI), 1.037-1.094; p < .01]; [Adjusted OR = 1.060; 95% CI, 1.031-1.091; p < .01]) was an independent risk factor for conversion failure. A receiver operating characteristic curve constructed using duration of epidural labour analgesia showed that parturients with a duration of epidural labour analgesia ≥8 h, more frequently required a change of anaesthesia technique during conversion, and the relative risk of conversion failure was 1.54 (95% CI, 1.23-1.93; p < .01). CONCLUSION: Prolonged duration of epidural labour analgesia increases the possibility of having an invalid epidural catheter, resulting in an increased risk of conversion failure from epidural labour analgesia to epidural surgical anaesthesia. Further, this risk is higher when the time exceeds 8 h. KEY MESSAGESProlonged duration of epidural labour analgesia > 8 h is associated with conversion failure.If it is impossible to judge whether the conversion is successful immediately, spinal anaesthesia should be administered to minimise complications.

Effect of calcitonin gene-related peptide on nitric oxide production in osteoblasts: an experimental study.

The aim of this study was to investigate the in vitro effects and regulatory mechanism of CGRP (calcitonin gene-related peptide) on NO (nitric oxide) production in osteoblasts. MOB (primary human mandibular osteoblasts) and osteoblast-like cells (MG-63) were either cultured with CGRP or co-incubated with inhibitors targeting eNOS (endothelial nitric oxide synthase), iNOS (inducible nitric oxide synthase), nNOS (neuronal nitric oxide synthase) and [Ca2+]i (intracellular Ca2+). The NO concentration in cell culture supernatants was measured during the first 24 h using the Griess test; cellular NO was marked with the fluorescent marker DAF-FM, DA (3-amino, 4-aminomethyl-2',7'-difluorescein; diacetate) and measured by fluorescence microscopy from 1 to 4 h after treatment. eNOS and iNOS mRNA expression levels were measured by quantitative RT-PCR during the first 24 h after treatment. CGRP-induced NO production in the supernatants was high between 1 to 12 h, while cellular NO was highest between 1 to 2 h after treatment and returned to basal levels by 3 h. Both in MG-63 cells and MOBs, the most effective CGRP concentration was 10 nM with a peak time of 1 h. CGRP-induced NO production decreased when eNOS activity was inhibited or when voltage-dependent L-type Ca2+ channels were blocked at 4 h. CGRP was not able to induce changes in iNOS or eNOS mRNA levels and had no effect on the cytokine-induced increase of iNOS expression. Our results suggest that CGRP transiently induces NO production in osteoblasts by elevating intracellular Ca2+ to stimulate the activity of eNOS in vitro.

Significant elevation of survivin and livin expression in human colorectal cancer: inverse correlation between expression and overall survival.

BACKGROUND: Survivin and livin are novel members of the inhibitor of apoptosis protein family, which have rarely been studied in human colorectal cancer (CRC). This study aims to examine their expression and association with clinicopathological factors and prognosis in CRC, and evaluate the possibility of their use as biomarkers for CRC. PATIENTS AND METHODS: We investigated the expression of survivin and livin in 61 CRC samples using immunohistochemical staining (Envision) and correlated it with the survival of these patients using log-rank test and correlation analysis. RESULTS: Among the 61 cases, 60.7 and 54.1% were positive for expression of survivin (p < 0.05) and livin (p < 0.05). No expression of survivin and livin was detected in normal colorectal mucosa. An inverse correlation (r = -0.9916) between the increased survivin and livin levels and overall survival was observed in univariate survival analysis. The expression of both proteins was not correlated with age, gender, degrees of differentiation, and TNM stage (p < 0.05) in malignancies. CONCLUSIONS: High expression of both survivin and livin may influence the prognosis of CRC. This finding opens new perspectives for CRC prognosis because survivin and livin can both be used as biomarkers or potential therapeutic targets.

Evaluation and Recommendation of the 8th Edition of American Joint Committee on Cancer (AJCC) Staging System for Intrahepatic Cholangiocarcinoma (ICC) in 820 Patients from the Surveillance, Epidemiology, and End Results (SEER) Database.

BACKGROUND: The AJCC made four changes to T category in the 8th AJCC stage for ICC, but this is a topic of debate. METHODS: Data from 820 patients with ICC were extracted from the SEER database. Survival analysis of the 8th AJCC stage was examined. RESULTS: To verify the four T staging changes by survival analysis: prognosis of patients with tumor size > 5 cm was poorer than that with tumor size ≤ 5 cm (P < 0.05); in N0M0 cohort, there was no significant difference in survival between solitary tumor with vascular invasion and multiple tumors (P = 0.092), tumor perforating the visceral peritoneum with and without involving local extrahepatic structures by direct invasion (P = 0.470), and tumor with and without periductal invasion (PI) (P = 0.220). The prognosis of patients with ≥ 4 positive lymph nodes was relatively poor compared with 1-3 positive lymph nodes (P = 0.037) and similar to patients with stage IV (8th AJCC, P = 0.585). CONCLUSION: This study found that there was no significant difference in survival between tumor perforating the visceral peritoneum with and without involving local extrahepatic structures by direct invasion, whereas other T staging changes were effective. The inclusion of the number of positive lymph nodes in the 8th AJCC stage may improve prognostic discrimination in ICC patients.

Assessing the effects of acetochlor on soil fungal communities by DGGE and clone library analysis.

We assessed the potential eco-toxicological risks of the herbicide acetochlor on fungal communities in the microcosm of black soil using 28S rRNA gene-PCR-DGGE and clone library analysis. The acetochlor was applied to black soil at four concentrations (0-control, 50, 150, and 250 mg/kg). The DGGE fingerprint patterns indicated that acetochlor stimulated fungal communities at day 7 after application, after which there was a suppression effect. The fungal communities in acetochlor-treated soil gradually became more like that of the control during the 60-day experimental period. Diversity indices in the 50 and 150 mg/kg acetochlor treatments changed more rapidly than in the 250 mg/kg acetochlor treatment. The cluster analysis indicated a significant change in fungal community structure after application of acetochlor. The impacts were markedly greater in the 150 and 250 mg/kg acetochlor treatments compared with the 50 mg/kg acetochlor treatment. Sequencing of clones showed that acetochlor application resulted in an increase in pathogenic and non-cultivatable fungal populations, which could increase the risk of plant disease outbreaks.

Settling behaviour of pellet flocs in pelleting flocculation process: analysis through operational conditions.

Pellet flocs' settling velocity is an important parameter in the pelleting flocculation blanket (PFB) process, hence, it is necessary to investigate flocs' settling behaviour to achieve the optimum operation parameters of the process. To investigate the settling behaviour of pellets under different operational conditions, a dynamic experiment was carried out to concentrate ferric flocs sludge by pelleting flocculation blanket (PFB) process with the scale of 0.5-1.2 m3/h. Under different operating conditions such as raw water concentration, polyacrylamide (PAM) dosage, up-flow rate, and agitation speed, pellet particles were sampled from different locations of the blanket in various operating stages to analyze pellet size, setting velocity, and porosity. Experimental results indicated that, when the PAM dosage increased from 0.59 mg/L to 1.18 mg/L, pellets size would flocculated from 2.25 mm to 3.52 mm with the settling velocity accelerated from 3.28 mm/s to 7.37 mm/s, while under the same up-flow rate, agitation intensity and PAM dosage, accompany with the raw water concentration increased from 216 mg/L to 840 mg/L, pellets settling velocity would improved from 6.03 mm/s to 13.6 mm/s. Under the experimental condition, along with the up-flow rate increased from 13.3 m/h to 40 m/h, pellets settling velocity would decreased from 4.39 mm/s to 3.42 mm/s due to its lower density.

Complete ureteral necrosis after injury sustained during lumbar disc surgery: A case report.

INTRODUCTION: Reports pertaining to ureteral injury sustained during lumbar disc surgery are rare; most ureteral injuries in this setting involve laceration or transection. PATIENT CONCERNS: We report a rare case of a 55-year-old man who presented with complete left ureteral necrosis 20 days after sustaining ureteral transection during lumbar disc surgery. DIAGNOSIS: The patient presented with seroperitoneum caused by left ureteral injury; post-operative histopathological examination of surgical specimen after discectomy had revealed ureter-like tissue. Exploratory laparoscopic surgery revealed necrosis of a long segment of ureter, which was not amenable to treatment with conventional methods. INTERVENTION: We used a spiral bladder muscle flap with vascular pedicles to repair the ureteral defect. OUTCOMES: Post-operative period was uneventful and the patient showed good recovery. CONCLUSION: Spiral bladder muscle flap with vascular pedicles may be used to repair extensive ureteric injury.

[Cloning, expression and immunity analysis of transketolase of Echinococcus granulosus].

OBJECTIVE: To obtain the prokaryotic expression of transketolase genes and analyze its value as a diagnostic antigen for echinococcosis. METHODS: TK gene was amplified by PCR and cloned into prokaryotic vector pMD19-EgTK, and then subcloned into the expression vector pET-28a. The target gene TK prokaryotic expression plasmid pET-28a was constructed and transferred into BL21. The purified protein was identified by SDS-PAGE and Western blotting. The blood samples of patients with cystic echinococcosis (CE group), alveolar echinococcosis (AE group) and healthy people (healthy group) were collected and detected by ELISA with the recombinant EgTK protein as a diagnostic antigen. RESULTS: The recombinant plasmid pET-28a (+)-EgTK was constructed successfully, and there was a band around 70 kDa by using Western blotting. ELISA showed that the difference among the 3 groups of sera reaction A450 was significantly different (F = 44.47, P < 0. 01), and the A450 values ofthe CE group (1.46±0.41) and AE group (1.28±0.29) were higher than that of the healthy group (0.66±0.23), but there was no significant difference between the former two. CONCLUSIONS: The recombinant EgTK protein is better to distinguish the echinococcosis group and healthy group, but it can't do a differential diagnosis between CE and AE cases.

Prospective, randomized, blinded, and placebo-controlled study of Cerebrolysin dose-response effects on long-term functional outcomes in a rat model of mild traumatic brain injury.

Using a prospective, randomized, blinded, placebo-controlled protocol, the authors demonstrated that Cerebrolysin at doses of 0.8-7.5 ml/kg, administered 4 hours after injury and then once daily for a total of 10 consecutive days, improves long-term functional outcomes in a rat model of mild closed head injury; a 2.5-ml/kg dose was identified as optimal. These findings suggest that Cerebrolysin has the potential to treat mild traumatic brain injury, the incidence of which is high without effective treatments.

Angiogenesis and improved cerebral blood flow in the ischemic boundary area detected by MRI after administration of sildenafil to rats with embolic stroke.

To dynamically investigate the long-term response of an ischemic lesion in rat brain to the administration of sildenafil, male Wistar rats subjected to embolic stroke were treated with sildenafil (n=11) or saline (n=10) at a dose of 10 mg/kg administered subcutaneously 24-h after stroke and daily for an additional 6 days. Magnetic resonance images were acquired and functional performance was measured in all animals at 1 day, 2 days and weekly for 6 weeks post-stroke. All rats were sacrificed 6 weeks after stroke and endothelial barrier antigen immunostaining was employed for morphological analysis and quantification of cerebral vessels. Map-ISODATA was computed from T(1), T(2) and T(1sat) maps. ISODATA derived tissue signatures characterize the degree of ischemic injury. Based on the map-ISODATA calculated at 6 weeks, the ischemic lesion for each animal was divided into two specific regions, the ischemic boundary and ischemic core. The temporal profiles of cerebral blood flow (CBF) and tissue signature were retrospectively tracked in these two regions and were compared with histological evaluation and functional outcome. After 1 week of sildenafil treatment, the ischemic lesion exhibited two significantly different regions, with higher CBF level and correspondingly, lower tissue signature value in the boundary region than in the core region. Sildenafil treatment did not significantly reduce the lesion size, but did enhance angiogenesis. Functional performance was significantly increased after sildenafil treatment compared with the control group. Administration of sildenafil to rats with embolic stroke enhances angiogenesis and selectively increases the CBF level in the ischemic boundary, and improves neurological functional recovery compared to saline-treated rats.

[Effect of calcitonin gene-related peptide on MC3T3-E1 osteoblast apoptosis and autophagy induced by serum starvation].

OBJECTIVE: To study the effect of calcitonin gene-related peptide (CGRP) on apoptosis and autophagy of mouse MC3T3-E1 osteoblast and their interaction and to further clarify protective mechanism of CGRP on osteoblasts. METHODS: MC3T3-E1 osteoblasts of mouse were cultured in vitro. Western blot and flow cytometry were used to detect expressions of microtubule-associated protein 1 light chain 3 (LC3) and P62 protein of MC3T3-E1 osteoblasts cultured with serum culture and serum-free (serum starvation) culture. Western blot was also used to detect expressions of LC3 and P62 protein of MC3T3-E1 osteoblast cultured at different concentrations (10⁻¹°, 10⁻9, 10⁻8, and 10⁻7 mol·L⁻¹) or without added CGRP. MC3T3-E1 osteoblasts were treated with 10⁻8 mol·L⁻¹ CGRP at different times (2, 6, 12, 24, 48, and 72 h), protein expression levels of LC3 were assessed by Western blot and flow cytometry, and changes in autophagosome in cells were detected by monodansylcadaverin staining. Autophagy inhibitor 3-methyladenine (3-MA) was used to pretreat MC3T3-E1 osteoblasts. Cells were then treated with or without CGRP for 24 h. Flow cytometry was used to detect apoptosis level. RESULTS: Under serum starvation conditions, LC3Ⅱ expression and apoptosis of osteoblasts increased compared with that of serum culture. Under 3-MA pretreatment and serum starvation conditions, LC3Ⅱ expression of osteoblasts increased compared with that of serum culture (P<0.01). Compared with serum culture, serum starvation culture with or without CGRP significantly increased expression level of LC3 and reduced expression level of P62. LC3Ⅱ/Ⅰ of osteoblasts was the highest under serum starvation and 10⁻8 mol·L⁻¹ CGRP conditions. Serum starvation and 10⁻8 mol·L⁻¹ CGRP culture inhibited apoptosis of osteoblasts and promoted synthesis of autophagosome. Apoptosis of osteoblasts increased after 3-MA pretreatment, and CGRP reversed inhibitory effects of 3-MA CGRP on apoptosis. CONCLUSIONS: CGRP can increase autophagy of MC3T3-E1 osteoblasts under serum starvation conditions. CGRP may also inhibit apoptosis of MC3T3-E1 osteoblasts by promoting autophagy.
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Whole Exome Sequencing Identified a Novel IGFBP6 Variant in a Disc Degeneration Pedigree.

OBJECTIVE: To identify the likely causal mutation that results in disc degeneration in a pedigree with a high incidence of disc degeneration. MATERIALS AND METHODS: A large pedigree with a high incidence of disc degeneration was recruited for this study. Exome sequencing was completed on four family members with disc degeneration to screen for potential causal gene variants. Detected variants were filtered against the 1000 Genomes Project, the Short Genetic Variations database (dbSNP), and the Beijing Genomics Institute (BGI) in-house database. After removing synonymous variants, Sanger sequencing was used to verify the lack of the candidate single nucleotide polymorphism (SNP) in five healthy subjects of the study family. RESULTS: We identified a novel SNP variant, Chr12:g.53494591T>C. c.T430C (p.S144P) in the insulin-like growth factor binding protein-6 (IGFBP6) gene. This variant was shared by all four affected family members, but not by five unaffected members in the same pedigree. Furthermore, this variant was not detected in 200 unrelated healthy people. CONCLUSIONS: The c.T430C (p.S144P) variant of IGFBP6 was identified as the likely causal variant associated with increased risk of familial disc degeneration in the studied pedigree.

Two new compounds from Trollius chinensis Bunge.

Two new compounds, 2″-O-feruloylisoswertiajaponin (1) and (2E)-2-methyl-1-O-vaniloyl-4-ß-D-glucopyranoside-2-butene (2), along with one indole alkaloid and five known flavonoids, were isolated from the flowers of Trollius chinensis Bunge. Their structures were elucidated on the basis of spectroscopic evidence (UV, IR, HR-ESI-MS, NMR).

Detection of BBB disruption and hemorrhage by Gd-DTPA enhanced MRI after embolic stroke in rat.

Thrombolytic therapy with rtPA increases the risk of hemorrhagic transformation (HT) after cerebral ischemia. We employed contrast enhancement MRI with Gd-DTPA to detect HT in a rat model of embolic stroke treated with rtPA and a glycoprotein IIb/IIIa receptor antagonist, 7E3 F(ab')2, at 4 h after embolic stroke. Male Wistar rats were subjected to embolic stroke and treated with the combination of rtPA and 7E3 F(ab')2 (n=12) or with saline (n=10) at 4 h after onset of stroke. MRI studies were performed immediately and at 24 h after embolization using a 7-T system. Histological measurements were obtained at 48 h. With Gd-DTPA, T1WI images and permeability related MRI parameters (the blood-to-brain transfer constant, Ki, and the distribution volume of mobile protons, Vp) of 15 out of 18 animals showed hyperintensity regions in gross or microscopic HT areas at 24 h, confirmed histologically at 48 h post stroke. Contrast enhancement MRI detected six of seven (86%) animals with gross HT and nine of eleven (82%) animals with microscopic HT at 24 h after ischemia. Two of eighteen animals with HT, had MRI indices of hemorrhage at 3 h post stroke. However, compared to HT data measured histologically at 48 h in embolic stroke rats, the enhanced areas by Gd-DTPA at 24 h were larger, and the patterns (time, intensity and region) did not directly correlate to the subtypes of HT, i.e., gross or microscopic hemorrhage. Contrast enhancement MRI using Gd-DTPA provides a method to detect gross and microscopic HT after stroke in rats.