A rapid detection of tomato yellow leaf curl virus using recombinase polymerase amplification-lateral flow dipstick assay.

Tomato yellow leaf curl disease which is caused by Tomato yellow leaf curl virus (TYLCV) is economically important and a widely spread tomato disease in China. Rapid and accurate detection methods are important in the control TYLCV. Here, a rapid method was developed to identify TYLCV on the basis of recombinase polymerase amplification (RPA) that can be visualized in 5 min using lateral flow dipsticks. The sensitivity and the specificity of this method were evaluated. This method can detect 0·5 pg DNA after 30 min at 37°C without any expensive instrumentation. In addition, it showed higher sensitivity than a PCR method when purified DNA was used. Moreover, the TYLCV was specifically detected, whereas other viruses infecting tomato produced negative results. The crude tomato extracts used in this assay has potential application in minimally equipped plant clinic laboratories. This method will facilitate the early and rapid detection of TYLCV for the timely application of control measures.

LncRNA PART1 modulates chondrocyte proliferation, apoptosis, and extracellular matrix degradation in osteoarthritis via regulating miR-373-3p/SOX4 axis.

OBJECTIVE: Osteoarthritis (OA) is a common disease in articular cartilages. It has been reported that long non-coding RNAs (lncRNAs) play an important role in various pathological processes of OA. However, the role of PART1 in OA development is unclear. PATIENTS AND METHODS: The expression levels of PART1 and miR-373-3p were detected in cartilage tissues and chondrocytes using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Cell proliferation was assessed by Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis was determined by flow cytometry and Western blot assay. The expression of extracellular matrix (ECM)-related proteins was examined by Western blot assay. Expression of SRY-related high-mobility group box 4 (SOX4) was measured by qRT-PCR or Western blot assay. The interactions among PART1, miR-373-3p, and SOX4 were predicted using starBase v2.0 database and confirmed by Dual-Luciferase reporter assay and RNA immunoprecipitation (RIP) assay. RESULTS: PART1 and SOX4 were up-regulated while miR-373-3p was down-regulated in OA cartilage tissues and chondrocytes. PART1 silencing hindered cell proliferation and ECM degradation, and triggered cell apoptosis in OA chondrocytes. PART1 modulated SOX4 expression by targeting miR-373-3p. Inhibition of miR-373-3p restored the regulation of cell proliferation, ECM degradation, and apoptosis induced by interfering PART1. Upregulation of SOX4 restored the effects on OA progression induced by inhibiting PART1. CONCLUSIONS: PART1 promoted OA progression by regulating miR-373-3p/SOX4 axis, providing an effective therapeutic target for osteoarthritis.

MicroRNA-29b sensitizes osteosarcoma cells to doxorubicin by targeting matrix metalloproteinase 9 (MMP-9) in osteosarcoma.

OBJECTIVE: To discover the effect and mechanism of exogenous microRNA-29b (miR-29b) on proliferation, apoptosis and the sensitivity to chemotherapy of osteosarcoma (OS) cells. PATIENTS AND METHODS: We assessed the expression of microRNA-29b in osteosarcoma tissues evaluating the regulation of in on the OS cell growth and drug sensitivity in human osteosarcoma MG-63 cell model. Firstly, quantitative RT-PCR (reverse transcription-PCR, RT-PCR) was used to measure the expression of miR-29b and matrix metallopeptidase 9(MMP-9) in primary osteosarcoma samples, and to evaluate the correlation between the two molecules. Secondly, miR-29b mimics or mimics were used to modify its expression in MG-63 cells. Luciferase reporter assay, Western blotting, cell viability, colony forming assay and apoptosis examination were performed to assess the regulation by manipulated miR-29b in the osteosarcoma-derived cells. RESULTS: We found that miR-29b is down-expressed, whereas the MMP-9 level was markedly higher in primary osteosarcoma tissues and osteosarcoma-derived cells. We also found that exogenous miR-29b reduces the proliferation, promotes the apoptosis and upregulates the sensitivity to chemotherapy (doxorubicin) of osteosarcoma cells via direct targeting of the MMP-9. CONCLUSIONS: Our data suggest that the reduced miRNA-29b may serve as a predictor of response to chemotherapy and as a therapeutic target in human osteosarcomas.

Investigation of the role of VEGF gene polymorphisms in the risk of osteosarcoma.

We conducted a case-control study in a Chinese population, and assessed whether the VEGF -634G/C, +936C/T, and +1612G/A polymorphisms could affect the risk of osteosarcoma and its association with environmental factors. A total of 180 consecutive osteosarcoma patients and 360 controls were included in our study. The genotypes of VEGF -634G/C, +936C/T, and +1612G/A were determined by the polymerase chain reaction-restriction fragment length polymorphism assay. Conditional logistic regression analyses showed that subjects carrying the GG genotype and the G allele of VEGF -634G/C were significantly associated with increased risk of osteosarcoma compared to those with the CC genotype; the ORs (95%CIs) were 2.28 (1.31-3.96) and 1.51 (1.16-1.97) for the GG genotype and G allele, respectively. We found that the GG genotype of VEGF -634G/C was associated with a significantly increased risk of osteosarcoma in patients of either gender with younger age and a family history of cancer. In summary, this study found that the VEGF -634G/C gene polymorphism was associated with an increased risk of osteosarcoma.

Clinical outcomes of elderly patients receiving neoadjuvant chemoradiation for locally advanced rectal cancer.

BACKGROUND: Studies of clinical outcomes of elderly patients treated with neoadjuvant chemoradiation (nCRT) for locally advanced rectal cancer (LARC) are limited. Our aim was to assess the impact of age on clinical outcomes in a large multi-institutional database. PATIENTS AND METHODS: Data for patients diagnosed with LARC who received nCRT and curative-intent surgery between 2005 and 2012 were collected from five major Canadian cancer centers. Age was analyzed as a continuous and dichotomous variable (< 70 versus ≥ 70 years) and correlated with disease-free survival (DFS), cancer-specific survival (CSS) and overall survival (OS). Cox regression models were used to adjust for important prognostic factors. RESULTS: Of 1172 patients included, 295 (25%) were ≥ 70 years, and they were less likely to receive adjuvant chemotherapy (ACT; 60% versus 79%, P < 0.0001), oxaliplatin-based ACT (12% versus 31%, P < 0.0001), less likely to complete nCT (76% versus 86%, P < 0.001), and more likely to be anemic at initiation of nCRT (42% versus 30%, P = 0.0004). In multivariate analyses, age ≥ 70 years was associated with similar DFS [hazard ratio (HR) 0.93, 95% confidence interval (CI) 0.68-1.26, P = 0.63], similar CSS (HR 0.81, 95% CI 0.46-1.41, P = 0.45), and similar OS (HR 1.28, 95% CI 0.88-1.86, P = 0.20), compared with the younger age group. As a continuous variable, increasing age was not predictive of DFS (HR 1.00, 95% CI 0.99-1.02, P = 0.49) or CSS (HR 1.002, 95% CI 0.98-1.02, P = 0.88); however, it correlated with an inferior OS (HR 1.02, 95% CI 1.00-1.03, P = 0.04). CONCLUSIONS: Elderly patients (≥ 70 years) who receive nCRT followed by surgery appear to have similar outcomes compared with younger patients. Decisions regarding eligibility for nCRT and surgery should not be based on age alone.

Effects of musk ketone on nerve recovery after spinal cord injury.

The present study aimed to determine the effects of musk ketone on nerve recovery in rats after spinal cord injury. A total of 105 SD female rats were used to establish the rat with dorsal spinal cord injury model (modified Allen's method). The rats weighed from 200 to 250 g and were provided by the Experimental Animal Center of Chongqing Medical University. They were randomly divided into five treatment groups: saline (NS group), methylprednisolone (MP group), and musk ketone groups (MO1, MO2, and MO3 groups). The Swash plate test and BBB behavioral score were used to determine neurological function recovery after spinal cord injury. Hematoxylin-eosin (HE) staining was used to detect general structural changes in spinal cord tissue. The enzyme-linked immunosorbent assay was used for the determination of interleukin 10 (IL-10) in spinal cord tissue. We found that compared with the NS control group, critical angle, BBB score and IL-10 levels in rat spinal cord tissue significantly increased in the MP group and MO groups 7 and 14 days after the operation. HE staining showed that in the NS group, there was hemorrhage, edema, necrosis, axonal demyelination, inflammatory cell infiltration and glial cell response in spinal cord tissue. After 7 days, spinal cord edema and inflammation were reduced and neuronal degeneration and necrosis were not evident in the MP and MO groups. We conclude that musk ketone can reduce secondary damage after spinal cord injury and promote nerve recovery in rats.

PPAR-γ agonist pioglitazone affects rat gouty arthritis by regulating cytokines.

The objective was to study peroxisome proliferator-activated receptor gamma (PPARγ) agonist pioglitazone regulation effect and its mechanism of expression of cytokines on acute gouty arthritis synovial in rats. Rats with unilateral ankle were injected with artificial monosodium urate (MSU) crystals to make the acute gouty arthritis model. Taking the synovium 48 h after the injection of MSU and using RT-PCR, we assessed the effect of pioglitazone (20 mg·kg(-1)·day(-1), oral administration) on synovial expression, by detecting tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interferon-γ (IFN-γ). The pioglitazone treatment group showed synovial expression of TNF-α, and IFN-γ was significantly lower than in the control group; the inhibition rates were 78.5 and 60.4%. The IL-1 expression difference was not statistically significant between the two groups. Pioglitazone has anti-inflammatory effects on acute gouty arthritis by inhibiting the expression of TNF-α and IFN-γ.

Identification crucial genes in peripheral neuropathic pain induced by spared nerve injury.

AIM: Peripheral neuropathic pain (PNP) is a kind of neuropathic pain caused by damage or disease that affects the peripheral nervous system. This study aimed to investigate the molecular mechanism of PNP and identify therapy targets for treating PNP in a spared nerve injury (SNI) model. MATERIALS AND METHODS: Gene expression data with accession number of GSE18803 were downloaded from Gene Expression Omnibus (GEO). This dataset included microarray data of four kinds of rat samples (adult rats with SNI, adult rats with sham injury, neonate rats with SNI, and neonate rats with sham injury). Differentially expressed genes (DEGs) were identified by using Limma software package, and further, Gene Ontology (GO) function and pathway analysis of DEGs were performed through the DAVID online tools. Protein-protein interaction (PPI) network of DEGs were constructed by STRING online database, and co-expression networks were constructed through Cytoscape. RESULTS: Totally 111 DEGs which were specially differentially expressed in adult rats with SNI were identified. Functional enrichment analysis suggest the majority of DEGs were related with immune functions. By comparing the three lists of genes got from GO and KEGG enrichment analysis, PPI network, and co-expression network analysis, 15 crucial genes were identified. CONCLUSIONS: Pathological nerve pain might be associated with immune dysfunctions and the 15 crucial genes might play an important role in the development of pathological nerve pain and have potential to be therapy targets.

Gene expression profiling of melatonin receptor subtypes in the ovarian hierarchical follicles of the Sichuan white goose.

Melatonin (N-acetyl-5-methoxytryptamine), an indole hormone, regulates a variety of important central and peripheral actions related to circadian rhythms and reproduction through its specific receptor subtypes (Mel-1a, Mel-1b and Mel-1c). However, the expression profile of melatonin receptor genes (MTNR1A, MTNR1B and MTNR1C) in ovarian hierarchical follicles of geese remains to be clarified. In this study, the expression level of melatonin receptors in small white follicle (SWF), small yellow follicle (SYF), the largest follicle (F1), second largest (F2), third largest (F3), fourth largest (F4), fifth largest (F5), and postovulatory follicle (POF) in the Sichuan white goose were examined using quantitative real-time PCR (qRT-PCR). The results showed that the expression levels of MTNR1A, MTNR1B and MTNR1C initially increased and later decreased. The highest levels of gene expression of these receptor subtypes were observed in F5 or F4 in all examined follicles. Furthermore, the expression of MTNR1A and MTNR1B mRNA was significantly greater in SYF compared with SWF (P<0.05), but MTNR1C was absent in SWF. The expression of MTNR1A, MTNR1B and MTNR1C mRNA was significantly greater in F5 compared with SYF (P<0.05), and the expression of MTNR1A and MTNR1C mRNA was higher in F1 compared with POF (P<0.05). In addition, the oestrogen concentration in SWF, SYF, F4, F3, F2, F1 and POF was measured using ELISA. The oestrogen concentration and melatonin receptor expression both were initially observed to increase and subsequently decrease. The oestrogen concentration in F4 and F3 was highest in all examined samples and was 1318.2pg/g and 1318.1pg/g, respectively. These results suggest that the melatonin receptor may be involved in the activation of the SWF and SYF to allow the SWF and SYF to develop into the subsequent follicles. Furthermore, follicles and the expression of the melatonin receptors may be regulated by the secretion of the oestrogen.

First Report of Coleus blumei viroid 5 from Coleus blumei in India and Indonesia.

Coleus blumei, which was found originally in Indonesia, is an ornamental plant grown worldwide. It can be infected by several viroids of the genus Coleviroid, family Pospiviroidae. Six main viroids that infect coleus have been reported: Coleus blumei viroid 1 through 6 (CbVd-1 ~ CbVd-6). Although CbVd-1 was first reported in a commercial coleus in Brazil in 1989 (1), and then in Germany, Japan, Canada, Korea, China, and India, CbVd-5 was reported only in China in 2009 (2). Symptoms caused by CbVd-5 varied depending on different cultivars, and in case of an unknown cultivar of "Red with dark green edge," are very clear albino symptoms. From 2010 to 2011, 60 and 3 leaf samples of coleus were collected from Hyderabad, India, and Java, Indonesia, respectively, and subjected to low molecular weight RNA extraction according to Li et al. (3). The results of dot-blot hybridization using CbVd-5 cRNA probes and RT-PCR using CbVd-5 specific primers (CbVd-5-PF: 5'-TGACTAGAACAGTAGTAAAG-3' / CbVd-5-PR: 5'-AATTGAGGTCAAACCTCTTT-3') demonstrated that 28 out of the 60 samples from India and all three samples from Indonesia were positive for CbVd-5. The resulting RT-PCR fragments from one sample selected randomly from each country were cloned into the pMD18-T vector (Takara) and transformed into E. coli DH5α competent cells. Five positive clones of each sample were sequenced. The result of sequence analysis revealed that the similarities of CbVd-5 between the sequences we obtained and the reference sequence (GenBank Accession No. NC003683) were 97.8 to 100%. Bioassay using nine viroid-free coleus plants from three cultivars (three from each cultivar), inoculated with CbVd-5 infectious clones by stem slashing, demonstrated that CbVd-5 could induce albino symptom on the leaves of the unknown cultivar "Red with dark green edge" 2 months after inoculation. To our knowledge, this is the first report of CbVd-5 from India and Indonesia, and the second report of CbVd-5 in the world. Considering the effect of CbVd-5 on the appearance of coleus and its recombination ability, a certification program may be needed to control the spread of this viroid. References: (1) M. E. N. Fonseca et al. Fitopatol. Bras. 14:94, 1989. (2) W. Y. Hou et al. Arch. Virol. 154:315, 2009. (3) S. F. Li et al. Ann. Phytopathol. Soc. Jpn. 61:381, 1995.

Additive effect of tetramethylpyrazine and deferoxamine in the treatment of spinal cord injury caused by aortic cross-clamping in rats.

STUDY DESIGN: Tetramethylpyrazine and deferoxamine are effective agents for nerve injury. Experiments in a Sprague-Dawley rat model of spinal cord injury were performed. OBJECTIVES: This study investigated the effects of tetramethylpyrazine and deferoxamine on neurological outcome and spinal cord hisotpathology after transient spinal cord ischemia in rats. SETTING: Chongqing Medical University and Third Military Medical University, Chongqing, China. METHODS: Spinal cord ischemia was induced in Sprague-Dawley rats by infrarenal aortic occlusion for 30 min followed by 72 h of reperfusion. Animals were divided into a sham group with a sham procedure; control group with aortic occlusion, but no pharmacological intervention applied; and treatment group with aortic occlusion, treated with tetramethylpyrazine and deferoxamine. After 72 h of reperfusion, neurological status was evaluated in the animals. A histopathological study of spinal cords was performed, and glutamate level and metabotropic glutamate receptor-1 (mGluR1) mRNA expression were determined. RESULTS: All animals in the control group were completely paraplegic with 0% recovery. Tarlov criteria were significantly better in the animals treated with tetramethylpyrazine and deferoxamine than that in the control group (P<0.05). Functional parameters were fully correlated with the morphological findings. Glutamate level was elevated in the control group, whereas it was significantly supressed in animals treated with tetramethylpyrazine and deferoxamine treatment. The infrarenal artery occlusion significantly elevated the expression of mGluR-1 mRNA, whereas tetramethylpyrazine and deferoxamine greatly supressed the expression of mGluR-1 mRNA. CONCLUSION: The combination of tetramethylpyrazine and deferoxamine significantly reduced the incidence of paraplegia induced by spinal cord ischemia/reperfusion.

First Report of Coleus blumei viroid 2 from Commercial Coleus in China.

Coleus (Coleus blumei) is an ornamental plant that is susceptible to infection by several viroids of the genus Coleviroid, which is a member of the family Pospiviroidae. Coleus blumei viroid (CbVd) -1 was first reported in commercial yellow coleus fields in Brazil in 1989 (1). In addition, CbVd-2, CbVd-3, and CbVd-4 have only been detected from coleus in Germany in 1996 (4). CbVd-5 and CbVd-6 were recently identified in China (2). In March 2010, leaves were collected from 50 symptomless coleus plants from a commercial nursery in Hainan Province, China. Total RNA was extracted from the leaves (3). Reverse transcription (RT)-PCR using CbVd-2 specific primers (forward: 5'-AGCTTACCTGGGTTCCCT-3' and reverse: 5'-CTCTCCTCTATTTACTCTCTTCTC-3') corresponding to positions 76 to 93 and 52 to 75 on the CbVd-2 reference sequence, respectively (GenBank Accession No. NC003682). Amplification of a 301-bp product was obtained from one sample. This PCR product was then cloned into pMD18-T (Takara, Dalian, China). Twelve positive clones were sequenced and the results were subjected to BLAST analysis. Sequence analysis showed that two sequences (GenBank Accessions Nos. HQ727542 and HQ727544) shared 99% identity with the reference sequence of CbVd-2 (NC003682), and four sequences (HQ727541, HQ727543, HQ727545 and HQ727547) had 99.34% identity with the reference sequence of CbVd-2 (NC003682). The proposed secondary structures of these variants have approximately 75% paired nucleotides. Results suggested the presence of CbVd-2, which is a member of the Coleviroid genus, Pospoviroidae family. To our knowledge, this is the first report of CbVd-2 from commercial coleus in China. References: (1) M. E. N. Fonseca et al. Fitopatol. Bras. 14:94, 1989. (2) W. Y. Hou et al. Arch. Virol. 154:993, 2009. (3) S. F. Li et al. Ann. Phytopathol. Soc. Jpn. 61:381, 1995. (4) R. L. Spieker et al. J. Gen. Virol. 77:2839, 1996.

Insulin with chondroitinase ABC treats the rat model of acute spinal cord injury.

Traumatic brain injury is often associated with acute spinal cord injury (ASCI). Insulin and chondroitinase ABC (ChABC) are both therapeutically effective, but the combined therapeutic effect of insulin and ChABC is still not clear. A combination of insulin and ChABC were used to treat a rat model of ASCI. This combination therapy prevented neuronal cell death by improving motor function, increasing cell growth and inhibiting cell apoptosis in ASCI rats. Expression of growth-associated protein 43, a marker of axonal re-growth, increased after combined treatment with insulin and ChABC. These results may provide a basis for a future method of treating ASCI.

Immunological study of allogeneic mesenchymal stem cells during bone formation.

Autologous mesenchymal stem cells (MSCs) are limited in their clinical application because tissue-engineered bone cannot be pre-fabricated. Allogeneic MSCs are readily available but carry the risk of transplant rejection. It is not yet clear whether allogeneic MSCs can induce a rejection response during bone formation. In this study, two strains of genetically unmatched mini-pigs were used as experimental animals to study the immunological changes in MSCs in vitro and in vivo when generating bone. Mini-pig MSCs showed low immunogenicity during osteogenesis both in vitro and in vivo, indicating that allogeneic MSCs had little or no immunogenicity in osteosis. In conclusion, allogeneic MSCs are an important source of seed cells for the tissue engineering of bone. This favours the clinical application of pre-constructed tissue-engineered bone.

Approximation of flammability region for natural gas-air-diluent mixture.

The growing implementation of exhaust gas recirculation (EGR) in reducing NO(x) emissions of engine is of paramount motivation to perform a fundamental research on the flammability characteristics of fuel-air-diluent mixtures. In this work, the influences of EGR on the flammability region of natural gas-air-diluent flames were experimentally studied in a constant volume bomb. An assumption of critical burning velocity at flammability limit is proposed to approximately determine the flammability region of these mixtures. Based on this assumption, an estimation of the flammability map for natural gas-air-diluent mixtures was obtained by using the empirical formula of burning velocity data. The flammability regions of natural gas-air mixtures with EGR are plotted versus the EGR rate. From the comparison of estimated results and experimental measurements, it is suggested that the accuracy of prediction is largely dependent upon the formula of burning velocity used. Meanwhile, the influence of pressure on the critical burning velocity at flammability limit is also investigated. On the basis of the pressure dependence criterion, the estimation was performed for the circumstance of high temperature and pressure, and the prediction results still agree well with those of experiments.

Experimental study of flammability limits of natural gas-air mixture.

Flammability limits data are essential for a quantitative risk assessment of explosion hazard associated with the use of combustible gas. The present work is to obtain the fundamental flammability data for prevention of the hazards in the practical applications. Experiments have been conducted in a constant volume combustion bomb, and the fuel considered here is natural gas (NG). The pressure histories in the combustion bomb are recorded and a criterion of 7% pressure rise has been used to judge a flammable mixture. The effects of ethane on NG-air flammability limits have been investigated. By adding diluent (carbon dioxide, nitrogen or their mixture) into NG-air mixture, the dilution effects on the flammability limits have been explored as well, and the results are plotted as functions of diluent ratio.

[Comparative study on osteogenesis of three types growth factors recombination artificial bones].

OBJECTIVE: To compare the osteogenesis of recombination artificial bones, which are bovine deproteined bone (bDPB) and bovine bone morphogenetic protein (bBMP), combined with tumor necrosis factor alpha (TNF alpha), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) respectively. METHODS: One hundred trephined skull bone defects in fifty rabbits were divided into four groups, which implanted with bDPB/bBMP/TNF alpha, bDPB/bBMP/bFGF, bDPB/bBMP/EGF, and bDPB/bBMP respectively. X-ray and histological changes were observed in the 1st, 2nd, 4th, 6th, 8th weeks after implantation. The content of 35S and 45Ca and ash weight were measured at 10 and 42 days after operation. RESULTS: The osteogenesis of bDPB/bBMP/TNF alpha group was stronger than that of bDPB/bBMP/bFGF group(P < 0.01), while bDPB/bBMP/bFGF group was stronger than that of bDPB/bBMP/EGF(P < 0.01). No significant statistical difference were found between bDPB/bBMP/EGF and bDPB/bBMP(P > 0.05). CONCLUSION: TNF alpha combined with bBMP and carrier can stimulate bone formation and increase the volume of new bone in vivo. It suggests that bDPB/bBMP/TNF alpha is a valuable biomaterial of bone graft.

[Mismatch negativity (MMN) to very short interval between regular tones].

The Mismatch Negativity (MMN) component of the auditory event-related potential (AERP) is elicited by infrequent, physically "deviant" stimuli in a sequence of frequent homogeneous stimuli ("standard"), for instance, by a change in frequency, intensity or duration etc. When the inter-stimulus interval (ISI) of tone pips was occasionally shortened either to 300, 150, 75, 60 and 52 ms from regular ISI 600 ms, which resulted in the elicitation of MMN. The amplitude of MMN elicited by occasional shorter ISI was not reduced as a function of ISI shortening. This indicates that the MMN is not just due to activation of new afferent elements by deviant stimulus. Thus, by no means the elicitation of the MMN can be explained on the basis of the refractoriness.