Femoral Tunnel Malposition, Increased Lateral Tibial Slope, and Decreased Notch Width Index Are Risk Factors for Non-Traumatic Anterior Cruciate Ligament Reconstruction Failure.

PURPOSE: To identify risk factors for patients who sustain nontraumatic anterior cruciate ligament reconstruction (ACLR) failure. METHODS: A retrospective analysis was performed on patients undergoing primary or revision ACLR in our institution between 2010 and 2018. Patients sustaining insidious-onset knee instability without history of trauma were identified as nontraumatic ACLR failure and assigned to the study group. The control group of subjects who showed no evidence of ACLR failure with minimum 48-month follow-up were matched in a 1:1 ratio based on age, sex, and body mass index. Anatomic parameters including tibial slope (lateral [LTS], medial [MTS]); tibial plateau subluxation (lateral [LTPsublx], medial [MTPsublx]); notch width index (NWI); and lateral femoral condyle ratio were measured with magnetic resonance imaging or radiography. Graft tunnel position was assessed using 3-dimensional computed tomography and reported in 4 dimensions: deep-shallow ratio (DS ratio) and high-low ratio for femoral tunnel, anterior-posterior ratio and medial-lateral ratio for tibial tunnel. Interobserver and intraobserver reliability were evaluated by the intraclass correlation coefficient (ICC). Patients' demographic data, surgical factors, anatomic parameters, and tunnel placements were compared between the groups. Multivariate logistic regression and receiver operating characteristic curve analysis was used to discriminate and assess the identified risk factors. RESULTS: A total of 52 patients who sustained nontraumatic ACLR failure were included and matched with 52 control subjects. Compared to patients with intact ACLR, those who sustained nontraumatic ACLR failure showed significantly increased LTS, LTPsublx, MTS, and deceased NWI (all P < .001). Moreover, the average tunnel position in the study group was significantly more anterior (P < .001) and superior (P = .014) at the femoral side and more lateral (P = .002) at the tibial side. Multivariate regression analysis identified LTS (odds ratio [OR] = 1.313; P = .028), DS ratio (OR = 1.091; P = .002), and NWI (OR = 0.813; P = .040) as independent predictors of nontraumatic ACLR failure. LTS appeared to be the best independent predictive factor (area under the curve [AUC] = 0.804; 95% confidence interval [CI], 0.721-0.887), followed by DS ratio (AUC = 0.803; 95% CI, 0.717-0.890), and NWI (AUC = 0.756; 95% CI, 0.664-0.847). The optimal cutoff values were 6.7° for increased LTS (sensitivity = 0.615, specificity = 0.923); 37.4% for increased DS ratio (sensitivity = 0.673, specificity = 0.885); and 26.4% for decreased NWI (sensitivity = 0.827, specificity = 0.596). Intraobserver and interobserver reliability was good to excellent, with ICCs ranging from 0.754 to 0.938 for all radiographical measurements. CONCLUSIONS: Increased LTS, decreased NWI, and femoral tunnel malposition are predictive risk factors for nontraumatic ACLR failure. LEVEL OF EVIDENCE: Level III, retrospective comparative study.

What Is the Best Way for Patients to Take Photographs of Medical Images (Radiographs, CT, and MRI) Using a Smartphone?

BACKGROUND: Teleradiology has become one of the most important approaches to virtual clinical diagnosis; its importance has only grown during the coronavirus 2019 pandemic. In developing countries, asking patients to take photographs of their images using a smartphone can facilitate the process and help keep its costs down. However, the images taken by patients with smartphones often are of poor quality, and there is no regulation or standard instruction about how to use smartphones to take photographs of medical examination images effectively. These problems limit the use of smartphones in remote diagnosis and treatment. QUESTIONS/PURPOSES: To formulate a set of guidelines for the most appropriate and effective use of smartphones to capture images (radiographs, CT images, and MR images), and to determine whether these guidelines are more effectively adopted by patients of differing ages and genders. METHODS: In this prospective study, a set of step-by-step instructions was created with the goal of helping patients take better smartphone photographs of orthopaedic diagnostic images for transfer to telemedicine services. Following the advice of surgeons, experts in smartphone technology, imaging experts, and suggestions from patients, the instructions were modified based on clinical experience and finalized with the goals of simplicity, clarity, and convenience. Potentially eligible patients were older than 18 years, had no cognitive impairment, and used smart phones. Based on that, 256 participants (patients or their relatives and friends) who visited the orthopaedic department of our hospital from June to October 2020 potentially qualified for this study. A total of 11% (29) declined to participate, leaving 89% (227) for analysis here. Their mean age was 36 ± 11 years, 50% were women (113 of 227), and the patient himself/herself represented in 34% (78 of 227) of participants while relatives or friends of patients made up 66% (149 of 227) of the group. In this study, the diagnoses included spinal stenosis (47% [107 of 227]), disc herniation without spinal stenosis (31% [71 of 227]), vertebral fractures (14% [32 of 227]), and other (7% [17 of 227]). Each study participant first took photographs of their original medical images based on their own knowledge of how to use the smartphone camera function; each participant then took pictures of their original images again after receiving our instructional guidance. Three senior spine surgeons (YZ, TQL, TCM) in our hospital analyzed, in a blinded manner, the instructed and uninstructed imaging files based on image clarity (the content of the image is complete, the text information in the image is clearly visible, there is neither reflection nor shadow in the image) and image position (it is not tilted, curled, inverted, or reversed). If either of these conditions was not satisfied, the picture quality was deemed unacceptable; two of three judges' votes determined the outcome. Interobserver reliability with kappa values for the three judges were 0.89 (YZ versus TQL), 0.92 (YZ versus TCM), and 0.90 (TQL versus TCM). RESULTS: In this study, the overall proportion of smartphone medical images deemed satisfactory increased from 40% (91 of 227) for uninstructed participants to 86% (196 of 227) for instructed participants (risk ratio 2.15 [95% CI 1.82 to 2.55]; p<0.001). The proportion of acceptable-quality images in different age groups improved after instruction, except for in patients aged 51 years or older (3 of 17 uninstructed participants versus 8 of 17 instructed participants; RR 2.67 [95% CI 0.85 to 8.37]; p = 0.07). The proportion of acceptable-quality images in both genders improved after instruction, but there was no difference between the genders. CONCLUSION: We believe our guidelines for patients who wish to take smartphone photographs of their medical images will decrease image transmission cost and facilitate orthopaedic telemedicine consultations. However, it appears that patients older than 50 years are more likely to have difficulty with this approach, and if so, they may benefit from more hands-on assistance from clinic staff or younger relatives or friends. The degree to which our findings are culture-specific should be verified by other studies in other settings, but on the face of it, there is little reason to believe our findings would not generalize to a reasonable degree. Other studies in more heterogeneous populations should also evaluate factors related to levels of educational attainment and wealth differences, but in the meantime, our findings can give clinical teams an idea of which patients may need a little extra assistance. LEVEL OF EVIDENCE: Level II, therapeutic study.

Cathodal tDCS exerts neuroprotective effect in rat brain after acute ischemic stroke.

BACKGROUND: Transcranial direct current stimulation (tDCS) is a non-invasive brain modulation technique that has been proved to exert beneficial effects in the acute phase of stroke. To explore the underlying mechanism, we investigated the neuroprotective effects of cathodal tDCS on brain injury caused by middle cerebral artery occlusion (MCAO). RESULTS: We established the MCAO model and sham MCAO model with an epicranial electrode implanted adult male Sprague-Dawley rats, and then they were randomly divided into four groups (MCAO + tDCS, MCAO + sham tDCS (Sham), Control + tDCS and Control + Sham group). In this study, the severity degree of neurological deficit, the morphology of brain damage, the apoptosis, the level of neuron-specific enolase and inflammatory factors, the activation of glial cells was detected. The results showed that cathodal tDCS significantly improved the level of neurological deficit and the brain morphology, reduced the brain damage area and apoptotic index, and increased the number of Nissl body in MCAO rats, compared with MCAO + Sham group. Meanwhile, the high level of NSE, inflammatory factors, Caspase 3 and Bax/Bcl2 ratio in MCAO rats was reduced by cathodal tDCS. Additionally, cathodal tDCS inhibited the activation of astrocyte and microglia induced by MCAO. No difference was found in two Control groups. CONCLUSION: Our results suggested that cathodal tDCS could accelerate the recovery of neurologic deficit and brain damage caused by MCAO. The inhibition of neuroinflammation and apoptosis resulted from cathodal tDCS may be involved in the neuroprotective process.

Effects of 1.8 GHz radiofrequency field on microstructure and bone metabolism of femur in mice.

To investigate the effects of 1.8 GHz radiofrequency (RF) field on bone microstructure and metabolism of femur in mice, C57BL/6 mice (male, age 4 weeks) were whole-body exposed or sham exposed to 1.8 GHz RF field. Specific absorption rates of whole body and bone were approximately 2.70 and 1.14 W/kg (6 h/day for 28 days). After exposure, microstructure and morphology of femur were observed by microcomputed tomography (micro-CT), Hematoxylin and Eosin (HE) and Masson staining. Subsequently, bone parameters were calculated directly from the reconstructed images, including structure model index, bone mineral density, trabecular bone volume/total volume, connectivity density, trabecular number, trabecular thickness, and trabecular separation. Biomarkers that reflect bone metabolism, such as serum total alkaline phosphatase (ALP), bone-specific alkaline phosphatase (BALP), and tartrate-resistant acid phosphatase 5b (TRACP-5b), were determined by biochemical assay methods. Micro-CT and histology results showed that there was no significant change in bone microstructure and the above parameters in RF group, compared with sham group. The activity of serum ALP and BALP increased 29.47% and 16.82%, respectively, in RF group, compared with sham group (P < 0.05). In addition, there were no significant differences in the activity of serum TRACP-5b between RF group and sham group. In brief, under present experimental conditions, we did not find support for an effect of 1.8 GHz RF field on bone microstructure; however, it might promote metabolic function of osteoblasts in mice. Bioelectromagnetics. 39:386-393, 2018. © 2018 Wiley Periodicals, Inc.

Involvement of P38 and ERK1/2 in mitochondrial pathways independent cell apoptosis in oviduct magnum epithelial cells of layers challenged with vanadium.

Vanadium (V) can induce cell apoptosis in layers' oviduct resulting in egg quality reduction. In this study, we investigated the relationship between the mitogen-activated protein kinase (MAPK)-signaling pathway and V-induced apoptosis in poultry oviduct magnum epithelial cells (OMECs). Cultured OMECs were divided into 8 treatment groups: 0 µmol/L V (control), 100 µmol/L V (V100), V100 + P38MAPK inhibitor (SB203580), SB203580, V100 + extracellular signal-regulated kinases 1 and 2 (ERK1/2) inhibitor (U0126), U0126, V100 + c-JUN NH2 -terminal kinase (JNK) inhibitor (SP600125), and SP600125. The OMECs were pretreated with the MAPK inhibitors before their treatment with V100 for 12 h. V100 increased the apoptosis of OMECs (P < .05), while 3 MAPK inhibitors suppressed V100-induced apoptosis P < .05); V100 enhanced the depolarization of △ψm (P < .05), and SB203580 and U0126 alleviated the V100-induced △ψm decrease (P < .05); V100 downregulated B-cell lymphoma-2 (Bcl-2) and poly [Adenosine diphosphate ribose] polymerase 1 (PARP1) mRNA expression (P < .05), meanwhile it upregulated Bcl-2 associated x (Bax), Apaf1, cytochrome C (CytC) and cysteine aspartase (caspase) 3, 8, 9 mRNA expression (P < .05). All MAPKs inhibitors alleviated the up-regulation of V100 for Bax and caspase 3 mRNA expression and down-regulation of V100 for Bcl-2 expression (P < .05). SB203580 and U0126 upregulated CytC expression treated by V100 (P < .05), except SP600125, while SB203580 administration resulted in a similar upregulation of PARP1 expression (P < .05). SP600125 can alleviated V triggered p-P38MAPK (phosphor-P38), p-ERK1/2 (phosphor-ERK1/2), p-JNK (phosphor-JNK) increase on OME cells, and SB203580 and U0126 had a similar response to phosphor-P38 and p-JNK (P < .05). It concluded that V-induced apoptosis in OMECs through the activation of P38 and ERK1/2, and by increasing the ratio of Bax/Bcl-2, which resulted in △ψm decrease, CytC release into the cytosol; consequently caspase 3 is recruited and activated, PARP1 is cleaved, eventually leading to apoptosis.

Metabolomics analysis to interpret changes in physiological and metabolic responses to chronic heat stress in Pekin ducks.

Heat stress (HS) is a major environmental threat that affects duck production in subtropical and tropical regions, especially in summer. This study aimed to evaluate the physiological and metabolic responses of Pekin ducks to chronic HS conditions via liquid chromatography-mass spectrometry (LC-MS) using a paired-fed (PF) experimental design. On the basis of equivalent feed intake (HS vs. PF), HS significantly reduced growth performance and the percentage of leg and breast muscles, however, markedly increased the percentage of abdominal fat and breast skin fat. Serum metabolomics results revealed that heat-stressed ducks showed enhanced glycolysis and pentose phosphate pathways, as demonstrated by higher glucose 6-phosphate and 6-phogluconic acid levels in the PF vs. HS comparison. HS decreased hepatic mRNA levels of mitochondrial fatty acid ß-oxidation-related genes (MCAD and SCAD) compared to the PF group, resulting in acetylcarnitine accumulation in serum. Moreover, HS elevated the concentrations of serum amino acids and mRNA levels of ubiquitination-related genes (MuRF1 and MAFbx) in the skeletal muscle and amino acid transporter-related genes (SLC1A1 and SLC7A1) and gluconeogenesis-related genes (PCK1 and PCase) in the liver compared to the PF group. When compared to the normal control group (NC), HS further decreased growth performance, but it elevated the abdominal fat rate. However, increased mRNA levels of ubiquitination-related genes and serum amino acid accumulation were not observed in the HS group compared to the NC group, implying that reduced feed intake masked the effect of HS on skeletal muscle breakdown and is a form of protection for the organism. These results suggest that chronic HS induces protein degradation in the skeletal muscle to provide amino acids for hepatic gluconeogenesis to provide sufficient energy, as Pekin ducks under HS conditions failed to efficiently oxidise fatty acids and ketones in the mitochondria, leading to poor growth performance and slaughter characteristics.

Broilers fed dietary vitamins harbor higher diversity of cecal bacteria and higher ratio of Clostridium, Faecalibacterium, and Lactobacillus than broilers with no dietary vitamins revealed by 16S rRNA gene clone libraries.

Research on the interaction between dietary vitamins and intestinal bacteria is poorly understood. To investigate the effect of dietary vitamins on the cecal bacterial communities, 2 bacterial 16S rRNA gene clone libraries were constructed from pooled PCR products obtained from the cecal digesta of 28-d broilers fed diets with vitamins (V) at the NRC level or with no vitamins (NV). The results showed that BW gain and average feed intake of V broilers was significantly higher (P < 0.01) than NV broilers, whereas the feed/gain ratio was significantly lower (P < 0.01) in V broilers. A total of 188 and 185 clones were sequenced for the NV and V broilers, respectively. Sequence identity criterion of 98% was used to assign sequences to operational taxonomic units (OTU). Clones from the NV group broilers were assigned to 14 OTU, with 33% clones affiliated with the genus Clostridium, 19% affiliated with the genera Escherichia/Shigella, 14% affiliated with the genus Bacteroides, and the remaining clones (34%) affiliated with 5 other bacterial genera (Faecalibacterium, Parasporobacterium, Ruminococcus, Streptococcus, and Subdoligranulum). Clones from the V group broilers were assigned to 23 OTU, with 46% of the clones affiliated with the genus Clostridium, 11% affiliated with the genus Fecalibacterium, and the remaining clones (43%) affiliated with 8 other genera (Anaerofilum, Lactobacillus, Anaerotruncus, Oscillibacter, Alistipes, Gracilibacter, Acetivibrio, and Haloplasma). Three OTU assigned to Clostridium, Faecalibacterium, and Ruminoccus were shared between the 2 libraries. Shannon diversity index showed the V broilers exhibited significantly higher bacterial diversity (P = 0.05), and Libshuff analysis indicated that the community structure between the 2 groups was significantly different (P < 0.0001). These results suggest that lack of dietary vitamins can increase the ratio of facultative pathogenic bacteria and decrease the diversity of bacteria in the cecum of broilers. Our results provide new leads for further investigations on the interaction between dietary vitamin additives and the gut health of broilers.

Decarboxylated osteocalcin, a possible drug for type 2 diabetes, triggers glucose uptake in MG63 cells.

BACKGROUND: Uncarboxylated osteocalcin (GluOC) has been reported to improve glucose metabolism, prevent type 2 diabetes, and decrease the severity of obesity in mice with type 2 diabetes. GluOC can increase glucose uptake in a variety of cells. Glucose metabolism is the main source of energy for osteoblast proliferation and differentiation. We hypothesized that decarboxylated osteocalcin (dcOC), a kind of GluOC, can increase glucose uptake in MG63 cells (osteoblast-like osteosarcoma cells) and influence their proliferation and differentiation. AIM: To investigate the effects of dcOC on glucose uptake in human osteoblast-like osteosarcoma cells and the possible signaling pathways involved. METHODS: MG63 cells (human osteoblast-like osteosarcoma cells) were treated with dcOC (0, 0.3, 3, 10, or 30 ng/mL) for 1 and 72 h, and glucose uptake was measured by flow cytometry. The effect of dcOC on cell proliferation was measured with a CCK-8 assay, and alkaline phosphatase (ALP) enzyme activity was measured. PI3K was inhibited with LY294002, and hypoxia-inducible factor 1 alpha (HIF-1α) was silenced with siRNA. Then, GPRC6A (G protein-coupled receptor family C group 6 subtype A), total Akt, phosphorylated Akt, HIF-1α, and glucose transporter 1 (GLUT1) levels were measured by Western blot to elucidate the possible pathways by which dcOC modulates glucose uptake. RESULTS: The glucose uptake of MG63 cells was significantly increased compared with that of the paired control cells after short-term (1 h) treatment with dcOC at different concentrations (0.3, 3, and 10 ng/mL groups, P < 0.01; 30 ng/mL group, P < 0.05). Glucose uptake of MG63 cells was significantly increased compared with that of the paired control cells after long-term (72 h) treatment with dcOC at different concentrations (0.3, 3, and 10 ng/mL groups, P < 0.01; 30 ng/mL group, P < 0.05). DcOC triggered Akt phosphorylation in a dose-dependent manner, and the most effective stimulatory concentration of dcOC for short-term (1 h) was 3 ng/mL (P < 0.01). LY294002 abolished the dcOC-mediated (1 h) promotion of Akt phosphorylation and glucose uptake without affecting GLUT1 protein expression. Long-term dcOC stimulation triggered Akt phosphorylation and increased the protein levels of HIF-1α, GLUT1, and Runx2 in a dose-dependent manner. Inhibition of HIF-1α with siRNA abolished the dcOC-mediated glucose uptake and substantially decreased GLUT1 protein expression. DcOC intervention promoted cell proliferation in a time- and dose-dependent manner as determined by the CCK-8 assay. Treatment with both 3 ng/mL and 10 ng/mL dcOC affected the ALP activity in MG63 cells after 72 h (P < 0.01). CONCLUSION: Short- and long-term dcOC treatment can increase glucose uptake and affect proliferation and ALP activity in MG63 cells. This effect may occur through the PI3K/Akt, HIF-1α, and GLUT1 signaling factors.

Vanadium in high-fat diets sourced from egg yolk decreases growth and antioxidative status of Wistar rats.

The objective of this paper was to evaluate the effect of vanadium (V) in high-fat diets sourced from egg yolk on body weight gain, feed intake, blood characteristics and antioxidative status of Wistar rats. A total of 72 female Wistar rats were allocated according to a 2 × 4 factorial design throughout a 5-wk trial, including 2 levels of dietary fat (normal and high; ether extract 40.3 and 301.2 g/kg; fat sourced from egg yolk) and 4 levels of dietary V (0, 3, 15 and 30 mg/kg). Vanadium decreased (P ≤ 0.05) body weight gain (V at 30mg/kg during wk 1 and 2; V at 15 and 30 mg/kg during the overall phase), feed intake (V at 30 mg/kg during wk 3 and the overall phase; V at 15 and 30 mg/kg during wk 4), but increased the relative weight of liver (V at 30 mg/kg, P ≤ 0.05). Moreover, increasing dietary V significantly increased (P ≤ 0.05) plasma aspartate aminotransferase, alanine aminotransferase and malondialdehyde levels and decreased triglyceride level, and V at 30 mg/kg in high-fat treatment had the highest or lowest values (interaction, P ≤ 0.05). Under the same dietary V dose, V residual content in liver (dietary V at 15 and 30 mg/kg) and kidney (dietary V at 15 mg/kg) was higher in high-fat diet treatment compared with normal-fat diet treatment (P ≤ 0.05). In conclusion, it is suggested that V could decrease the body weight together with the feed intake, and the high fat could enhance oxidative stress induced by V of Wistar rats.

Effects of 220 MHz Pulsed Modulated Radiofrequency Field on the Sperm Quality in Rats.

Under some occupational conditions, workers are inevitably exposed to high-intensity radiofrequency (RF) fields. In this study, we investigated the effects of one-month exposure to a 220 MHz pulsed modulated RF field at the power density of 50 W/m² on the sperm quality in male adult rats. The sperm quality was evaluated by measuring the number, abnormality and survival rate of sperm cells. The morphology of testis was examined by hematoxylin-eosin (HE) staining. The levels of secreting factors by Sertoli cells (SCs) and Leydig cells (LCs) were determined by enzyme linked immunosorbent assay (ELISA). The level of cleaved caspase 3 in the testis was detected by immunofluorescence staining. Finally, the expression levels of the apoptosis-related protein (caspase 3, BAX and BCL2) in the testis were assessed by Western blotting. Compared with the sham group, the sperm quality in the RF group decreased significantly. The levels of secreting factors of SCs and the morphology of the testis showed an obvious change after RF exposure. The level of the secreting factor of LCs decreased significantly after RF exposure. The levels of cleaved caspase 3, caspase 3, and the BAX/BCL2 ratio in the testis increased markedly after RF exposure. These data collectively suggested that under the present experimental conditions, 220 MHz pulsed modulated RF exposure could impair sperm quality in rats, and the disruption of the secreting function of LCs and increased apoptosis of testis cells induced by the RF field might be accounted for by this damaging effect.

Merging " Anti-Baldwin" 3- Exo-Dig Cyclization with 1,2-Alkynyl Migration for Radical Alkylalkynylation of Unactivated Olefins.

A new combination of " anti-Baldwin" 3- exo-dig cyclization with 1,2-alkynyl migration of 1,4-enynes with simple cycloalkanes was established, enabling C-C breaking and reconstruction to access a wide range of α-alkynyl ketones with generally good yields by FeCl2/di- tert-butyl peroxide (DTBP) as a catalytic oxidation system. Radical-induced C(sp3)-H functionalization of cycloalkanes was realized, leading to the direct formation of C(sp3)-C(sp3) and C(sp)-C(sp3) bonds. The mechanism for forming α-alkynyl ketones was proposed.

Effects of 1.8 GHz Radiofrequency Fields on the Emotional Behavior and Spatial Memory of Adolescent Mice.

The increasing use of mobile phones by teenagers has raised concern about the cognitive effects of radiofrequency (RF) fields. In this study, we investigated the effects of 4-week exposure to a 1.8 GHz RF field on the emotional behavior and spatial memory of adolescent male mice. Anxiety-like behavior was evaluated by open field test (OFT) and elevated plus maze (EPM) test, while depression-like behavior was evaluated by sucrose preference test (SPT), tail suspension test (TST) and forced swim test (FST). The spatial learning and memory ability were evaluated by Morris water maze (MWM) experiments. The levels of amino acid neurotransmitters were determined by liquid chromatography-mass spectrometry (LC-MS). The histology of the brain was examined by hematoxylin-eosin (HE) staining. It was found that the depression-like behavior, spatial memory ability and histology of the brain did not change obviously after RF exposure. However, the anxiety-like behavior increased in mice, while, the levels of γ-aminobutyric acid (GABA) and aspartic acid (Asp) in cortex and hippocampus significantly decreased after RF exposure. These data suggested that RF exposure under these conditions do not affect the depression-like behavior, spatial memory and brain histology in adolescent male mice, but it may however increase the level of anxiety, and GABA and Asp were probably involved in this effect.

Enhancement of X-ray Induced Apoptosis by Mobile Phone-Like Radio-Frequency Electromagnetic Fields in Mouse Spermatocyte-Derived Cells.

To explore the combined effects of environmental radio-frequency (RF) field and X-ray, mouse spermatocyte-derived (GC-1) cells were exposed to 1950 MHz RF field at specific absorption rate (SAR) of 3 W/kg for 24 h combined with or without X-ray irradiation at 6 Gy. After treatment, the cell proliferation level was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) Assay and 5-Bromo-2-deoxy Uridine (BrdU) enzyme linked immunosorbent (ELISA) Assay. The apoptosis level was detected by annexin V flow cytometry assay, transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) Assay and Caspase-3 Activity Assay. It was found that the proliferation and apoptosis level did not change in GC-1 cells after RF exposure alone. However, compared with the X-ray group, the proliferation level significantly decreased and the apoptotic rate significantly increased in the RF+X-ray group. Moreover, a significant decrease in Bcl-2 protein expression and increase in Bax protein expression were observed. The findings suggested that RF exposure at SAR of 3 W/kg did not affect apoptosis and proliferation in GC-1 cells by itself, but that it did enhance the effects of X-ray induced proliferation inhibition and apoptosis, in which B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) might be involved.

Afaf, a novel vesicle membrane protein, is related to acrosome formation in murine testis.

As a cell-specific organelle, acrosome (Acr) and its formation are an important event for spermiogenesis. However, the Acr formation is far more complicated than has been proposed. In this study, we have cloned a novel membrane protein Afaf (Acr formation associated factor) that was expressed abundantly in the round spermatids, localized in the inner and outer membrane of forming Acrs, and declined in the maturing Acrs. In the transfected Hela cells, Afaf protein was localized in the plasma membrane, EEA1-positive early endosomes (EEs) and occasionally in the nuclei. Therefore, we propose that EEs and plasma membrane may be also directly involved in the Acr biogenesis.

An oligonucleotide microarray study on gene expression profile in mouse testis of experimental cryptorchidism.

To investigate the germ cell apoptosis under body temperature in testis, we analyzed the gene expression patterns on day 1, day 4, day 7, day 14, day 28 and normal control adult mouse testis after experimental cryptorchidism (EC) using Affymetrix MOE430A microarray. Our data showed that EC led to the oxidative stress and gene expression fluctuation in the first 28 days, both of which were highly coincident in timing. Cryptorchid testis showed more effective antioxidative capability in the first 4 days, and suddenly lowered the capability from day 5 on, then gradually restored the antioxidation from day 10 to day 14, and turned to worse on day 28 again. The extensive high gene expression on day 4 after EC and the up-rising of oxidative stress level on day 5 and the abrupt down-regulation of the gene on day 7 were closely related. From the chip data, we have found that the high level of reactive oxidative species (ROS) was not only related to the dysfunction or abnormality of the direct origin of ROS generation, but also related to the abnormality of the more upstream physiological events in energy metabolism, lipid metabolism. The selective regulation of metabolic substrate transporter in different cell population implied the existence of various regulation of the selective signal pathways among different cell populations by EC.

[Production of mature red blood cell by using peripheral blood mononuclear cells].

Most protocols for in vitro producing red blood cells (RBC) use the CD34(+) cells or embryonic stem cells from cord blood, bone marrow or peripheral blood as the start materials. This study was purposed to produce the mature RBC in vitro by using peripheral blood mononuclear cells as start material. The peripheral blood mononuclear cells (PBMNC) were isolated from buffy coat after blood leukapheresis, the mature red blood cells (RBC) were prepared by a 4-step culture protocol. The results showed that after culture by inducing with the different sets of cytokines and supporting by mouse MS-5 cell line, the expansion of PBMNC reached about 1000 folds at the end of the culture. About 90% of cultured RBC were enucleated mature cells which had the comparable morphological characteristics with normal RBC. Colony-forming assays showed that this culture system could stimulate the proliferation of progenitors in PBMNC and differentiate into erythroid cells. The structure and function analysis indicated that the mean cell volume of in vitro cultured RBC was 118 ± 4 fl, which was slight larger than that of normal RBC (80-100 fl); the mean cell hemoglobin was 36 ± 1.2 pg, which was slight higher than that of normal RBC (27-31 pg); the maximal deformation index was 0.46, which approachs level of normal RBC; the glucose-6-phosphate dehydrogenase and pyrurvate kinase levels was consistant with young RBC. It is concluded that PBMNC are feasble, convenient and low-cost source for producing cultured RBC and this culture system is suitable to generate the RBC from PBMNC.

Toxicity of melamine and cyanuric acid in broilers and residues in tissues.

The purpose of this study was to characterize the toxicity potential of melamine (MEL), cyanuric acid (CYA), and a combination of MEL and CYA in broilers. A total of 720 commercial 1-day-old COBB 500 male broilers were randomly allotted into 6 groups with 6 replicates each and 20 broilers in each replicate. The dietary treatments were as follows: group I was the control group, group II included 10 mg/kg MEL and 3.3 mg/kg CYA, group III included 30 mg/kg MEL and 10 mg/kg CYA, group IV included 100 mg/kg MEL and 33.3 mg/kg CYA, group V included 100 mg/kg MEL, and group VI included 33.3 mg/kg CYA. The trial lasted for 42 days. CYA alone and the combination of MEL and CYA had adverse effects on the performance, but MEL alone had no effects on the performance. On day 21, the uric acid (UA) content of group IV was increased in serum (p < 0.05); on day 42, the serum aspartate aminotransferase (AST) activity and the level of tumor necrosis factor (TNF)-α and interleukin (IL)-8 increased in group IV (p < 0.05); 100 mg/kg MEL alone increased the level of TNF-α and the rate of renal apoptosis (p < 0.05); and 33.3 mg/kg CYA alone increased the level of IL-8 and the rate of renal apoptosis (p < 0.05). The livers contained MEL concentrations of 17-125 µg/kg wet weight and CYA concentrations of 28-73 µg/kg, and the muscle contained MEL concentrations of 14-105 µg/kg wet weight. It was indicated that MEL alone, CYA alone, and a combination of MEL and CYA inhibit the growth and damage the kidney and liver.

Toxicity of cyanuric acid to broilers on hepatic and renal health with and without melamine.

The purpose of this study was to evaluate the toxicity potential of cyanuric acid (CYA) and a combination of melamine (MEL) and CYA in broilers. A total of 1200 male COBB 500 broilers were randomly allocated into 1 of 10 treatment groups by a 5 × 2 factorial design in a 42-d experiment. The dietary treatments were as follows: T(1) to T(5): basal diets with 0, 10, 20, 33.3, and 50 mg CYA per kg diet; T(6) to T(10): basal diet with CYA regimens similar to T(1) to T(5) but with 100 mg MEL per kg diet. There were 6 replication pens with 20 chicks per pen. No differences were observed in alanine transaminase (ALT) and aspartate aminotransferase (AST) activities. But on d 22, uric acid (UA) and creatinine (Crea) concentrations were significantly greater when birds were fed CYA at 33.3 mg/kg with MEL 100 mg/kg, and Crea concentration was also higher when birds were fed CYA at 50 mg/kg. No crystals were found in kidneys but dilated renal tubules and small blood vessel expansion were found in kidneys of birds fed CYA at 50 mg/kg and CYA at 33.3 mg/kg with MEL 100 mg/kg. The apoptosis rate (AR) of kidneys of all birds fed CYA and MEL contaminated diets were higher than the control group. These results indicated that the dietary addition of CYA and MEL could induce kidney damage, and the effects were harmful when the ratio of CYA/MEL was 1:3.

[Influence of cytokine combinations on proliferation and differentiation of umbilical cord blood CD34(+) cells into megakaryocytes/platelets in vitro].

In order to investigate the influence of cytokine combinations on proliferation and differentiation of human umbilical cord blood CD34(+) cells into megakaryocytes/platelets in vitro, the CD34(+) cells from human umbilical cord blood were amplified in serum-free medium StemSpan(SFEM) supplemented with several cytokine combinations by three-phase culture system. The effects of the cytokine combinations were compared. The results showed that at day 14 of the first culture phase, the CD34(+) cells cultured with cytokine combinations SCF + TPO + FL + IL-3 were amplified (11 000 ± 1 000) times, which were significantly higher than that of cells cultured with SCF + TPO + FL, but were not significantly different from that of cells cultured with SCF + TPO + IL-3 or SCF + TPO + FL + IL-3+ hydroxyl-corticosteroids. At day 7 of the second culture phase, the CD34(+) cells cultured with cytokine combination SCF + TPO + FL + IL-11 were amplified by (204666.7 ± 11718.9) times, which were significantly higher than that of cells cultured with SCF + TPO + FL + IL-3, but were not significantly different from that of cells cultured with SCF + TPO + FL + IL-11 + BMP4 + VEGF. At day 3 and day 6, the CD34(+) platelet-like cells accounted for about (39.8 ± 1.9)%, (39.7 ± 2.6)% and (25.5 ± 1.4)%, (23.1 ± 3.5)% cultured with SCF + TPO + FL + IL-11 and SCF + TPO + FL + IL-11 + BMP4 + VEGF, and significantly higher than that of the cells cultured with SCF + TPO + FL + IL-3. It is concluded that the cytokine combination of SCF + TPO + FL + IL-3 is most suitable cytokines combination for the amplification of CD34(+) hematopoietic progenitor cells. The cytokine combination of SCF + TPO + FL + IL-11 is preferred for the proliferation and differentiation of megakaryocytes, this study lays an experimental basis for investigating the proliferation and differentiation of CD34(+) into megakaryocytes/platelets in vitro.

Effects of active immunization against myostatin on carcass quality and expression of the myostatin gene in pigs.

The study was conducted to investigate the effects of active immunization against myostatin on the titer of myostatin antibody, carcass evaluation, activity of creatine kinase and the expression of the myostatin gene in pigs. Eighteen pigs were allotted into three groups (six pigs per group), and pigs in treatment 1, 2 and 3 were immunized with physiological saline, 1 mg or 4 mg myostatin per pig, respectively. Six pigs were killed by electrical stunning followed by exsanguination at BW of 100 kg. The results indicated that the titer of myostatin antibody was increased in treated groups compared to the control group on day 42 (P < 0.01) and d 84 (P < 0.01). The carcass lean percentage was significantly increased in the treatment groups compared to the control group (P < 0.01), and intramuscular fat was significantly decreased in the 4 mg group compared to the control group (P < 0.05). The muscle creatine kinase activity of pigs treated with 1 mg and 4 mg myostatin was lower than the control group. The immunization of myostatin significantly decreased the myostatin gene expression levels in muscle. It was concluded that optimal active immunization against myostatin could increase the content of myostatin antibody, suppress the activity of creatine kinase and the expression of myostatin gene, and therefore improve the carcass lean percentage for pigs.