[HIV infection rate, high-risk behavior and pre-exposure prophylaxis/post-exposure prophylaxis in men who have sex with men in Beijing].

Objective: To understand the incidence of HIV infection, high risk behaviors and pre-exposure prophylaxis/post-exposure prophylaxis (PrEP/PEP) utilization in men who have sex with men (MSM) in Beijing. Methods: Sample size was estimated to be 1 227 persons and 207 person year respectively in the survey and the cohort by using Epi Info 7.0 software. Using convenient sampling method, MSM were recruited by using Wechat app. Questionnaire was completed online to collect the information about demographic characteristics, high risk behavior, and utilization of PrEP/PEP of the MSM. MSM collected dry blood spot (DBS) samples by themselves, and mailed the DBS samples to laboratory for HIV nucleic acid testing. Open cohort was established and those with HIV negative nucleic acid testing results were followed up. Non-conditional binary logistic regression method was used to identify the associated factors for high risk anal sex in the last month and having multiple homosexual partners in the last month. Results: A total of 1 147 MSM were recruited, and follow up for 236 person years was conducted in 956 MSM with negative HIV nucleic acid testing results. The detection rate of new HIV infection was 1.3 per 100 person-years (3/236). During the last month, the proportions of consistent condom use in anal sex and oral sex were 50.7% (238/469) and 4.9% (23/469). In the MSM, 5.9% (43/723) had sex with HIV positive partners in the last month. 9.8% (103/1 049) used PrEP, and 8.7% (91/1 049) used PEP. The proportion of consistent condom use in PrEP and PEP were 34.3% (24/70) and 72.2% (39/54) respectively. Logistic regression analysis revealed that compared with those who used no PrEP/PEP, those who used PrEP/PEP were more likely to have unprotected anal sex in the last month (aOR=3.16, 95%CI:1.45-7.18), and more likely to have multiple homosexual partners in the last month (aOR=2.64, 95%CI:1.19-6.30), and compared with those who used no Rush Popper or drugs in the last month, those who used Rush Popper or drugs in the last month were more likely to have unprotected anal sex in the last month (aOR=2.34, 95%CI:1.67-3.30), and more likely to have multiple homosexual partners (aOR=2.42,95%CI:1.76-3.33). Conclusions: It is necessary to strengthen the health education to promote condom use and introduce the harm of drug use in MSM. In PrEP and PEP services, it is still necessary to suggest consistent condom use for MSM.

[Focusing on timing selection and whole-course management of liver transplantation treatment for patients with acute-on-chronic liver failure].

Acute-on-chronic liver failure (ACLF) is a clinical syndrome of acute decompensation accompanied by organ failure that occurs on the basis of chronic liver disease and has a high short-term mortality rate. Currently, there are still differences in relation to the definition of ACLF; thus, baseline characteristics and dynamic changes are important bases for clinical decision-making in patients with liver transplantation and others. The basic strategies for treating ACLF currently include internal medicine treatment, artificial liver support systems, and liver transplantation. Multidisciplinary active collaborative management throughout the whole course is of great significance for further improving the survival rate in patients with ACLF.

[Progress in research of influencing factors of prophylaxis drug use after non-occupational exposure to HIV].

Non-occupational post-exposure prophylaxis (nPEP), a biological means to block the transmission of HIV, is recommended by European countries, USA and WHO to use in HIV high-risk groups, but its utilization rate is still very low. The information-motivation-behavioral skills model (IMB) can accurately explain the prevalence and change of health behaviors. Based on this model, this paper summarizes the progress in research of the influencing factors for nPEP use to provide a basis for further research to promote the use of nPEP.

[Establishment and validation of a predictive nomogram model for advanced gastric cancer with perineural invasion].

Objective: Peripheral nerve invasion (PNI) is associated with local recurrence and poor prognosis in patients with advanced gastric cancer. A risk-assessment model based on preoperative indicators for predicting PNI of gastric cancer may help to formulate a more reasonable and accurate individualized diagnosis and treatment plan. Methods: Inclusion criteria: (1) electronic gastroscopy and enhanced CT examination of the upper abdomen were performed before surgery; (2) radical gastric cancer surgery (D2 lymph node dissection, R0 resection) was performed; (3) no distant metastasis was confirmed before and during operation; (4) postoperative pathology showed an advanced gastric cancer (T2-4aN0-3M0), and the clinical data was complete. Those who had other malignant tumors at the same time or in the past, and received neoadjuvant radiochemotherapy or immunotherapy before surgery were excluded. In this retrospective case-control study, 550 patients with advanced gastric cancer who underwent curative gastrectomy between September 2017 and June 2019 were selected from the Affiliated Hospital of Qingdao University for modeling and internal verification, including 262 (47.6%) PNI positive and 288 (52.4%) PNI negative patients. According to the same standard, clinical data of 50 patients with advanced gastric cancer who underwent radical surgery from July to November 2019 in Qingdao Municipal Hospital were selected for external verification of the model. There were no statistically significant differences between the clinical data of internal verification and external verification (all P>0.05). Univariate analysis and multivariate logistic regression analysis were used to determine the independent risk factors for PNI in advanced gastric cancer, and the clinical indicators with statistically significant difference were used to establish a preoperative nomogram model through R software. The Bootstrap method was applied as internal verification to show the robustness of the model. The discrimination of the nomogram was determined by calculating the average consistency index (C-index). The calibration curve was used to evaluate the consistency of the predicted results with the actual results. The Hosmer-Lemeshow test was used to examine the goodness of fit of the discriminant model. During external verification, the corresponding C-index index was also calculated. The area under ROC curve (AUC) was used to evaluate the predictive ability of the nomogram in the internal verification and external verification groups. Results: A total of 550 patients were identified in this study, 262 (47.6%) of which had PNI. Multivariate logistic regression analysis revealed that carcinoembryonic antigen level ≥ 5 µg/L (OR=5.870, 95% CI: 3.281-10.502, P<0.001), tumor length ≥5 cm (OR=5.539,95% CI: 3.165-9.694, P<0.001), mixed Lauren classification (OR=2.611, 95%CI: 1.272-5.360, P=0.009), cT3 stage (OR=13.053, 95% CI: 5.612-30.361, P<0.001) and the presence of lymph node metastasis (OR=4.826, 95% CI: 2.729-8.533, P<0.001) were significant independent risk factors of PNI in advanced gastric cancer (all P<0.05). Based on these results, diffused Lauren classification and cT4 stage were included to establish a predictive nomogram model. CEA ≥ 5 µg/L was for 68 points, tumor length ≥ 5 cm was for 67 points, mixed Lauren classification was for 21 points, diffused Lauren classification was for 38 points, cT3 stage was for 75 points, cT4 stage was for 100 points, and lymph node metastasis was for 62 points. Adding the scores of all risk factors was total score, and the probability corresponding to the total score was the probability that the model predicted PNI in advanced gastric cancer before surgery. The internal verification result revealed that the AUC of nomogram was 0.935, which was superior than that of any single variable, such as CEA, Lauren classification, cT stage, tumor length and lymph node metastasis (AUC: 0.731, 0.595, 0.838, 0.757 and 0.802, respectively). The external verification result revealed the AUC of nomogram was 0.828. The C-ndex was 0.931 after internal verification. External verification showed a C-index of 0.828 from the model. The calibration curve showed that the predictive results were good in accordance with the actual results (P=0.415). Conclusion: A nomogram model constructed by CEA, tumor length, Lauren classification (mixed, diffuse), cT stage, and lymph node metastasis can predict the PNI of advanced gastric cancer before surgery.

[Integration of production-university-research based on artificial intelligence for technological innovation and transformation in gastrointestinal surgery].

Medical artificial intelligence is a deeply integrated field of production-university-research. The collaborative innovation system of "industry, education and research" has broken down the barriers between various disciplines and has shown great impetus in scientific and technological innovation and achievement transformation. The Affiliated Hospital of Qingdao University where the authors work, relying on Shandong Key Laboratory of Digital Medicine and Computer-Assisted Surgery and Institute of Digital Medicine and Computer-Assisted Surgery of Qingdao University, has jointly established a close "industry-university-research" cooperation mechanism with the State Key Laboratory of Virtual Reality Technology and Systems of Beijing University of Aeronautics and Astronautics and Qingdao Hisense Medical Equipment Co., Ltd., which lasted for 10 years and carried out a series of useful explorations in scientific and technological innovation and achievement transformation in the field of gastrointestinal surgery of artificial intelligence with fruitful results. It mainly comprises of the following: (1) The research and development (R&D) of Hisense computer-assisted surgery (CAS) system has important clinical significance for laparoscopic gastrointestinal surgery guided by surrounding blood vessels that grasps globally the movement and variation of blood vessels in order to accurately select surgical strategies. (2) R&D of an automatic identification system for gastrointestinal diseases (namely image-assisted diagnosis system of artificial intelligence) initially applies deep neural network of artificial intelligence to the identification of lymph node metastases in rectal cancer, which has achieved accurate clinical diagnosis. (3) The virtual endoscopy platform for the gastrointestinal tract developed can successfully provide a new virtual endoscopy view of the intestinal wall eversion and realize the one-way navigation of the intestinal wall. (4) A localized platform of laparoscopic surgery simulation training is established with completely independent intellectual property rights. We realize that clinical requirement is the source of research and development, clinical practice is the source of innovation, and clinicians are practitioners of transformation of achievements. The establishment of artificial intelligence models often requires the support of massive and high-quality clinical data. Therefore, the effectiveness of its promotion and application can only be ensured by adopting a multi-center research approach. In the whole process of scientific and technological innovation and achievement transformation, the issues of medical ethics and intellectual property rights cannot be ignored.

[Application of convolutional neural network to risk evaluation of positive circumferential resection margin of rectal cancer by magnetic resonance imaging].

Objective: To explore the feasibility of using faster regional convolutional neural network (Faster R-CNN) to evaluate the status of circumferential resection margin (CRM) of rectal cancer in the magnetic resonance imaging (MRI). Methods: This study was registered in the Chinese Clinical Trial Registry (ChiCTR-1800017410). Case inclusion criteria: (1) the positive area of CRM was located between the plane of the levator ani, anal canal and peritoneal reflection; (2) rectal malignancy was confirmed by electronic colonoscopy and histopathological examination; (3) positive CRM was confirmed by postoperative pathology or preoperative high-resolution MRI. Exclusion criteria: patients after neoadjuvant therapy, recurrent cancer after surgery, poor quality images, giant tumor with extensive necrosis and tissue degeneration, and rectal tissue construction changes in previous pelvic surgery. According to the above criteria, MRI plain scan images of 350 patients with rectal cancer and positive CRM in The Affiliated Hospital of Qingdao University from July 2016 to June 2019 were collected. The patients were classified by gender and tumor position, and randomly assigned to the training group (300 cases) and the validation group (50 cases) at a ratio of 6:1 by computer random number method. The CRM positive region was identified on the T2WI image using the LabelImg software. The identified training group images were used to iteratively train and optimize parameters of the Faster R-CNN model until the network converged to obtain the best deep learning model. The test set data were used to evaluate the recognition performance of the artificial intelligence platform. The selected indicators included accuracy, sensitivity, positive predictive value, receiver operating characteristic (ROC) curves, areas under the ROC curves (AUC), and the time taken to identify a single image. Results: The accuracy, sensitivity, specificity, positive predictive value, and negative predictive value of the CRM status determined by the trained Faster R-CNN artificial intelligence approach were 0.884, 0.857, 0.898, 0.807, and 0.926, respectively; the AUC was 0.934 (95% CI: 91.3% to 95.4%). The Faster R-CNN model's automatic recognition time for a single image was 0.2 s. Conclusion: The artificial intelligence model based on Faster R-CNN for the identification and segmentation of CRM-positive MRI images of rectal cancer is established, which can complete the risk assessment of CRM-positive areas caused by in-situ tumor invasion and has the application value of preliminary screening.

[Feasibility on the Internet-based HIV nucleic acid testing with dry blood spots and risk factors associated with HIV infection in men having sex with men in Beijing].

Objectives: To understand the prevalence of HIV nucleic acid using internet-based dry blood spots HIV testing strategy in men who had sex with men (MSM) and to probe the factors associated with HIV infection. Methods: Using convenient sampling method, 1 375 MSM were recruited and their dry blood spots samples were collected before being mailed to the laboratories for HIV nucleic acid testing. Results were showed to these MSM on a specific website by inputting their codes to it. Non-conditional binary logistic regression method was used to identify the associated factors on HIV infection. Results: The overall proportions of HIV nucleic acid positives appeared as 9.7% (131/1 349) and HIV antibody positives as 8.3% (112/1 349). Fresh infections accounted for 14.5% (19/131) among the newly-identified HIV nucleic acid positives, and the interval was ranging from 6 to 120 days, between the laboratory testings and the closest date that experiencing high risk behavior. Risk factors that related to HIV infection would include: 30 to 39 years of age (comparing to those under the age of 30, OR=1.88, 95%CI: 1.07-3.29), ≥8 000 Yuan of monthly income (comparing to those without income, OR=0.42, 95%CI: 0.19-0.96), inconsistent condom use during anal sexual contacts in the last six months (compared with those who had not anal sex or used condoms consistently in anal sex in the past six months, OR=2.22, 95%CI: 1.45-3.40), ever use of Rush Poppers (compared with those who never used Rush Poppers, OR=2.33, 95%CI: 1.49-3.64), addictive drug abuse (compared with those who never abused addictive drugs, OR=5.43, 95%CI: 2.32-12.69), and not having regular sexual partners (compared with having regular sexual partners, OR=1.74, 95%CI: 1.13-2.68) etc.. Conclusions: Dry blood spots HIV nucleic acid testing could help to identify the fresh HIV infections at an early stage, so as to prevent further transmission in the MSM population, among which fresh HIV infections accounted for a fairly large proportion. It is necessary to set up programs in reducing the abuse of drugs or Rush Poppers, and to promote condom use and advocate on stable sexual partnership etc., among the MSM population.

[Clinical application of convolutional neural network in pathological diagnosis of metastatic lymph nodes of gastric cancer].

Objective: To examine the value and clinical application of convolutional neural network in pathological diagnosis of metastatic lymph nodes of gastric cancer. Methods: Totally 124 patients with advanced gastric cancer who underwent radical gastrectomy plus D2 lymphadenectomy at Affiliated Hospital of Qingdao University from July 2016 to December 2018 were selected in the study. According to the chronological order, the first 80 cases were served as learning group. The remaining 44 cases were served as verification group. There were 45 males and 35 females in the study group, with average age of 57.6 years. There were 29 males and 15 females in the validation group, with average age of 9.2 years. The pre-training convolutional neural network architecture Resnet50 was trained and fine-tuned by 21 352 patches with cancer areas and 14 997 patches without cancer areas in the training group. A total of 78 whole-slide image served as a test dataset including positive (n=38) and negative (n=40) lymph nodes. The convolutional neural network computer-aided detection (CNN-CAD) system was used to analyze the ability of convolutional neural network system to screen metastatic lymph nodes at the level of slice by setting threshold, and evaluate the system's classification accuracy by calculating its sensitivity, specificity, positive predictive value, negative predictive value and area under the receiver operating characteristic curve (AUC). Results: The classification accuracy of CNN-CAD system at slice level was 100%.The AUC for the CNN-CAD system was 0.89. The sensitivity was 0.778, specificity was 0.995, overall accuracy was 0.989. Positive and negative predictive values were 0.822 and 0.994, respectively. The CNN-CAD system achieved the same classification results as pathologists. Conclusions: The CNN-CAD system has been constructed to distinguished benign and malignant lymph node slides with high accuracy and specificity. It could achieve the similar classification results as pathologists.

[Establish a predictive modeling under antiviral therapy for hepatitis B e antigen seroconversion in chronic hepatitis B].

Objective: To explore the predictive factors by demonstrating a predictive modeling under antiviral therapy for hepatitis B e antigen seroconversion in HBeAg-positive chronic hepatitis B patients. Methods: 198 cases with HBeAg-positive chronic hepatitis B were enrolled. Fatty liver, family history of hepatitis B, age, sex, drinking history, HBsAg, HBeAg, HBV-DNA levels, total bilirubin (TBil), CD4/CD8, albumin (ALB), alanine amino transferase (ALT) levels were used as a predictor variables of HBeAg seroconversion. Serological seroconversion of HBeAg was observed at 144 weeks of antiviral therapy. Predictive factors of HBeAg seroconversion was analyzed by logistic regression analysis, and the receiver operating characteristic curve was plotted. Results: HBeAg seroconversion rate was 36.87%. Univariate analysis demonstrated that fatty liver (χ(2) = 35.377; P < 0.001), family history of hepatitis B (χ(2) = 15.687; P < 0.001), the levels of HBeAg (t = 5.034; P < 0.001), HBsAg (t = 3.454; P < 0.001) and HBV-DNA levels (Z = 4.651; P < 0.001) were predictor variables of HBeAg seroconversion. Multivariate analysis showed that family history of hepatitis B, fatty liver, HBV-DNA levels and HBeAg were independent predictors of HBeAg seroconversion. The established logistic regression model for HBeAg through regression analysis was logit P = 9.623-1.228 × family history of hepatitis B - 1.726 × fatty liver - 0.764 × HBV-DNA levels - 0.146 × HBeAg and area under curve was 0.875. When the cut-off value was -0.9350, the sensitivity and specificity were 92.70%, 75.50%, 83.22%, respectively. Conclusion: Family history of hepatitis B, fatty liver, HBV-DNA levels and HBeAg may be independent predictors of HBeAg seroconversion at 144 weeks of antiviral therapy in HBeAg-positive chronic hepatitis B patients.

[Measurement of morphological parameters of internal acousticmeatus using thin-section CT].

Objective: This study aimed to measure the morphological parameters of the internal acoustic meatus(IAM) and its adjacent structures using temporal-bone thin-section CT(computed tomography). Methods: CT images were obtained from 50 Chinese adult patients (25 males and 25 females, 100 sides) which had no visible lesion in the petrous part of the temporal bone and inner ear, the morphological parameters of all inner ear parts were sectionally measured on the specified plane using SPSS 22.0 software for statistical analysis. Results: The integral morphological characteristics of the IAM were observed. These results revealed that anterior-posterior diameter of the internal acoustic poer(IAP)(CD) was (6.93±1.85)mm, the superior-inferior diameter of the IAP(EF) was (4.40±0.86)mm, the length of the IAM(AB) was (9.30±1.60)mm, the superior-inferior diameter of the IAM(the intersection of inner 1/3 section and middle 1/3 section) was (4.13±0.83)mm, the superior-inferior diameter of the IAM(the intersection of middle 1/3 section and outer 1/3 section) was (4.61±1.02)mm, the anterior-posterior diameter of the IAM(the intersection of inner 1/3 section and middle 1/3 section) was (6.62±1.92)mm, the anterior-posterior diameter of the IAM(the intersection of middle 1/3 section and outer 1/3 section) was (6.28±1.65)mm, the depth of transverse crest (superior wall) was (3.10±0.75)mm, the depth of transverse crest (interior wall)the was (1.46±0.59)mm, the distance from transverse crest vertex A to the superior wall of the IAM was (2.05±0.42)mm, the distance from transverse crest vertex A to the interior wall of the IAM was (2.93±0.41)mm, the thickness of the superior bone wall of the IAM (the intersection of inner 1/3 section and middle 1/3 section) was (4.45±1.34)mm, the thickness of the superior bone wall of the IAM (the intersection of middle 1/3 section and outer 1/3 section) was (4.32±1.12)mm, the thickness of the superior bone wall of the IAM (the intersection of outer 1/3 section and transverse crest vertex) was (4.37±1.28)mm, and the appearance ratio of the cells in the whole IAM superior wall was 32%.The whole IAM assumed the shape of short cylinder, inclining about 1 cm outward, with the upper-lower diameter and anterior-posterior diameter about 5 mm. Conclusion: It is necessary for carrying out preoperative the temporal-bone thin-section CT to obtain the morphological parameters of the IAM, determine its basic morphology, and provide references to avoid damaging the other important structures during IAM surgeries.

Presence, distribution and molecular epidemiology of multi-drug-resistant Gram-negative bacilli from medical personnel of intensive care units in Tianjin, China, 2007-2015.

BACKGROUND: Multi-drug-resistant Gram-negative bacteria (MDRGNB) have become an important cause of nosocomial infection in intensive care units (ICUs). AIMS: To investigate the molecular epidemiology of MDRGNB isolated from medical personnel (MP) and non-medical personnel (NMP) at 69 ICUs in Tianjin, China. METHODS: From April 2007 to October 2015, 2636 nasal and hand swab samples from 1185 MP and 133 NMP were cultured for GNB (including MDRGNB), meticillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). The susceptibilities of GNB to 14 antimicrobial agents were determined, and 80 MDRGNB were characterized using pulsed-field gel electrophoresis (PFGE) and dendrogram analysis. FINDINGS: In total, 301 GNB were identified in 269 MP, including 109 MDRGNB isolates in 104 MP. Forty-two GNB were isolated from 39 NMP, which included 20 NMP with MDRGNB. Overall, 8.8% of MP were colonized with MDRGNB, which greatly exceeded colonization rates with MRSA (0.9%) and VRE (0.1%). Three pairs of Klebsiella pneumoniae and one pair of Enterobacter aerogenes were indistinguishable from each other, but the majority of isolate tests had distinct PFGE profiles. CONCLUSIONS: The prevalence of MDRGNB was high among ICU MP in Tianjin, and greatly exceeded that of VRE and MRSA. There was no difference in the rates of nasal carriage of MDRGNB between MP and NMP, but NMP were significantly more likely to have hand colonization with MDRGNB. PFGE profiles showed that there was only limited sharing of strains of MDR E. aerogenes and K. pneumoniae between personnel.

An association between the level of oxidative stress and the concentrations of NEFA and BHBA in the plasma of ketotic dairy cows.

The aim of this study was to evaluate the oxidative status in ketotic cows. We observed changes in the oxidative status and correlations between the oxidative and metabolic status in non-ketotic (n = 10), subclinical ketotic (n = 10) and ketotic cows (n = 10). Plasma samples were analysed by standard biochemical techniques and ELISA to determine traditional metabolic parameters: triglyceride (TG), phosphonium (P), calcium (Ca), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total protein (TP), albumin (ALB), immune globulin (Ig), total cholesterol (TC), high-density lipoprotein (HDL), very low-density lipoprotein (VLDL) and lactate dehydrogenase (LDH); energy metabolism indices: glucose, ß-hydroxybutyrate (BHBA) and non-esterified fatty acids (NEFA); and indices of oxidative status: malondialdehyde (MDA), hydrogen peroxide (H2 O2 ), vitamin C, vitamin E, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), xanthine oxidase (XOD) and total antioxidant capacity (TAOC). The results of this study showed that plasma glucose levels were lower in ketotic and subclinical ketotic cows than in non-ketotic cows; however, the plasma NEFA and BHBA concentrations were higher. In addition, significant decreases in TC, HDL and VLDL and significant increases in AST, ALT and LDH were observed in the plasma of the ketotic cows. The ketotic cows showed decreased plasma SOD, CAT, vitamin C and vitamin E, inhibited hydroxyl radical capacity and increased plasma H2 O2 and MDA. There were positive correlations between the plasma NEFA and ALT, AST, LDH and MDA and negative correlations between the plasma NEFA and TC, HDL, VLDL, SOD, vitamin C, vitamin E, 1542280 uric acid and inhibited hydroxyl radical capacity. In addition, there were positive correlations between BHBA concentrations and ALT, AST and LDH and negative correlations between plasma BHBA concentrations and TC, HDL, VLDL, vitamin E and inhibited hydroxyl radical capacity. Overall, ketotic dairy cows experience oxidative stress, which is presumably associated with hyperketonemia and higher NEFA.

Effects of the acid-tolerant engineered bacterial strain Megasphaera elsdenii H6F32 on ruminal pH and the lactic acid concentration of simulated rumen acidosis in vitro.

The aim of this study was to examine the effects of the acid-tolerant engineered bacterial strain Megasphaera elsdenii H6F32 (M. elsdenii H6F32) on ruminal pH and the lactic acid concentrations in simulated rumen acidosis conditions in vitro. A mixed culture of ruminal bacteria, buffer, and primarily degradable substrates was inoculated with equal numbers of M. elsdenii H6 or M. elsdenii H6F32. The pH and lactic acid concentrations in the mixed culture were determined at 0, 2, 4, 6, 8, 10, 12, 14, 16, and 18 h of incubation. Acid-tolerant M. elsdenii H6F32 reduced the accumulation of lactic acid and increased the pH value. These results indicate that acid-tolerant M. elsdenii H6F32 could be a potential candidate for preventing rumen acidosis.

Andrographolide suppresses RANKL-induced osteoclastogenesis in vitro and prevents inflammatory bone loss in vivo.

BACKGROUND AND PURPOSE: Osteoclasts play a pivotal role in diseases such as osteoporosis, rheumatoid arthritis and tumour bone metastasis. Thus, searching for natural compounds that may suppress osteoclast formation and/or function is promising for the treatment of osteoclast-related diseases. Here, we examined changes in osteoclastogenesis and LPS-induced osteolysis in response to andrographolide (AP), a diterpenoid lactone isolated from the traditional Chinese and Indian medicinal plant Andrographis paniculata. EXPERIMENTAL APPROACH: Effects of AP on osteoclast differentiation and bone resorption were measured in vitro. Western blots and RT-PCR techniques were used to examine the underlying molecular mechanisms. The bone protective activity of AP in vivo was assessed in a mouse model of osteolysis. KEY RESULTS: AP concentration-dependently suppressed RANKL-mediated osteoclast differentiation and bone resorption in vitro and reduced the expression of osteoclast-specific markers, including tartrate-resistant acid phosphatase, calcitonin receptors and cathepsin K. Further molecular analysis revealed that AP impaired RANKL-induced NF-κB signalling by inhibiting the phosphorylation of TGF-ß-activated kinase 1, suppressing the phosphorylation and degradation of IκBα, and subsequently preventing the nuclear translocation of the NF-κB p65 subunit. AP also inhibited the ERK/MAPK signalling pathway without affecting p38 or JNK signalling. CONCLUSIONS AND IMPLICATIONS: AP suppressed RANKL-induced osteoclastogenesis through attenuating NF-κB and ERK/MAPK signalling pathways in vitro, thus preventing bone loss in vivo. These data indicated that AP is a promising natural compound for the treatment of osteoclast-related bone diseases.

Short communication: high insulin concentrations inhibit fatty acid oxidation-related gene expression in calf hepatocytes cultured in vitro.

In dairy cows, ketosis is an important disease associated with negative energy balance, which leads to low blood glucose levels and high blood nonesterified fatty acid levels. The liver is the most active organ in cows for the metabolism of nonesterified fatty acids. Insulin is an anabolic hormone that plays numerous roles in the metabolism of carbohydrates, lipids, and proteins, as well as being a potent regulator of fatty acid oxidation. In this study, using fluorescent quantitative reverse-transcription PCR, ELISA, and primary hepatocytes cultured in vitro, we examined the effect of insulin (0, 5, 10, 20, 50, and 100 nmol/L) on fatty acid oxidation by monitoring mRNA and protein expression levels of key enzymes: long-chain acyl-coenzyme A synthetase, carnitine palmitoyltransferase I, and long-chain acyl-coenzyme A dehydrogenase. The results showed that the mRNA and protein expression of long-chain acyl-coenzyme A synthetase, carnitine palmitoyltransferase I, and long-chain acyl-coenzyme A dehydrogenase was markedly decreased when the concentration of insulin in the media was increased. These findings indicate that high levels of insulin significantly inhibit the expression of genes related to fatty acid oxidation and consequently results in a decreased level of fatty acid oxidation in calf hepatocytes.

Alterations of fatty acid ß-oxidation capability in the liver of ketotic cows.

Dairy cows are highly susceptible to ketosis after parturition. In the present study, we evaluated the expression of fatty acid ß-oxidation-related enzymes in the liver of ketotic (n=6) and nonketotic (n=6) cows. Serum levels of nonesterified fatty acids (NEFA), ß-hydroxybutyrate (BHBA), and glucose were determined by using standard biochemical techniques. The mRNA abundance and protein content of acyl-CoA synthetase long-chain (ACSL), carnitine palmitoyltransferase I (CPT I), carnitine palmitoyltransferase II (CPT II), acyl-CoA dehydrogenase long chain (ACADL), 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS), and acetyl-CoA carboxylase (ACC) were evaluated by real-time PCR and ELISA. We found that serum glucose levels were lower in ketotic cows than in nonketotic cows, but serum BHBA and NEFA concentrations were higher. Messenger RNA and protein levels of ACSL were significantly higher in livers of ketotic cows than those in nonketotic cows. In contrast, mRNA levels of CPT I and mRNA and protein levels of CPT II, ACADL, HMGCS, and ACC were decreased in the liver of ketotic cows. Serum NEFA concentration positively correlated with ACSL protein levels and negatively correlated with protein levels of CPT II, HMGCS, ACADL, and ACC. In addition, serum BHBA concentration negatively correlated with protein levels of CPT II, HMGCS, and ACADL. Overall, fatty acid ß-oxidation capability was altered in the liver of ketotic compared with nonketotic cows. Furthermore, high serum NEFA and BHBA concentrations play key roles in affecting pathways of fatty acid metabolism in the liver.

Preparation of specific monoclonal antibodies against chelated copper ions.

Copper ions are too small to elicit an immune response. Therefore, copper was conjugated to carrier proteins using S-2-(4-isothiocyanatobenzyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid, a bifunctional chelator, to make artificial antigens for copper. Several mice were immunized, and monoclonal antibodies (MAbs) against chelated copper were produced. Spleen cells of immunized mice were fused with myeloma cells. The resulting hybridomas were screened using protein conjugates which were covalently bound to metal-free 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA) or Cu-DOTA. Two hybridoma cell lines (F4 and B2) that produced MAbs with high selectivity and sensitivity were expanded for further study. Cross-reactivities with other metals were below 1%. These antibodies were used to construct competitive ELISAs for copper ions. The IC(50) for F4 and B2 were 0.39 and 1.66 mg/l, respectively. The detection range and the lowest detection limit for copper using the antibody F4 was 0.019-8.22 and 0.003 mg/l, respectively. Spike recovery studies in tap water showed that the most sensitive antibody could be used for copper detection in drinking water.