Comparative study of methotrexate and human umbilical cord mesenchymal stem cell transplantation in the treatment of rheumatoid arthritis.

In this study, a collagen-induced arthritis (CIA) model was established to simulate rheumatoid arthritis (RA) using two intradermal injections of bovine type II collagen and Freund’s complete adjuvant mixture given at two-week intervals. Subsequently, the transplantation of human umbilical cord mesenchymal stem cells (hUC-MSCs) was used to treat RA and the treatment efficacy, as well as the possible regulatory mechanism underlying hUC-MSC transplantation, was observed. During the study, forty rats were randomly divided into four groups and their blood samples were collected at different time points to measure levels of serum cartilage oligomeric matrix protein (COMP). Based on the symptoms and pathological features of the rats, a total success rate of 83% was achieved by the treatment. Furthermore, the improvement of joint symptoms was more obvious when methotrexate and MSC transplantation were used. In summary, it was concluded that MSC transplantation relieved the symptoms of arthritis by down-regulating the expression of COMP on the synovial membrane and in the serum of CIA rats.

[Association between polymorphism of ADAM33 gene and bronchial asthma in Mongolian population].

Objective: To investigate the different genotype and allele frequency distribution of ADAM33 gene T1, T2, V4, S2 sites Mongolian population, and discuss the relationship between ADAM33 gene polymorphism and bronchial asthma. Methods: From January 2014 to December 2015, a total of 180 cases of Mongolian patients with asthma were detected, compared with 186 cases of healthy Mongolian as controled and screening significant genes.Selected restriction fragment length polymorphism (PCR-RFLP) method to detected ADAM33 gene polymorphism.According to condition , the asthma group was divided into mild(n=83), medium group(n=47)and severe group(n=50). The distribution difference of different genotype and every genotype of V4 FEV1, eosinophils, IgE comparison were compared , and analysis their correlation. Results: In ADAM33 , the distribution of T1 sites (AA and AG genotypes) had statistical significance compared asthma group with control group(χ2=8.810, 8.294, P<0.05, OR=1.983, 0.500). The OR value of G allele was 0.580.The distribution of S2 site(CC genotype) had statistical significance(χ2=4.277, P<0.05), the OR value of G allele was 1.423.the distribution of V4 sites (GC and GG genotypes) had statistical significance between the two groups (χ2=7.880, 10.313, P<0.05), OR value was 0.459, 2.130, G allele OR value was 1.496.The distribution frequency difference of each genotype in V4 sites in mild, medium and severe group was statistically significant (χ2=16.049, P<0.05), and compared various genotypes of FEV1, IgE, the difference was statistically significant (P<0.05), for each genotype of T2 site in asthma group and the control group there was no statistically significant in the distribution (χ2=1.218, 0.248, 1.287, P>0.05). Conclusions: T1, V4, S2 locus polymorphism of ADAM33 gene may play a role in the Mongolian asthma population, and T2 locus polymorphism may has no relationship with Mongolian asthma patients.And the genotype polymorphism of V4 sites may be associated with asthma severity.

First Report of Cherry mottle leaf virus Infecting Cherry in China.

Cherry mottle leaf virus (CMLV) is a member of the genus Trichovirus (family Betaflexiviridae). CMLV infects several species of the genus Prunus including cherry (Prunus avium) and peach (P. persica) (2,3). It is spread via budding and grafting with infected wood and can be transmitted from infected bitter cherry (P. emarginata), or infected but symptomless peach trees to healthy sweet cherry trees by the bud mite (Eriophyes inaequalis) (1). On susceptible sweet cherry cultivars, CMLV causes symptoms such as chlorotic mottle-leaf pattern, distortion, puckering of younger leaves, and small fruits that ripen late (1), which may lead to severe economic losses in some cultivars. Cherry is one of the most important fruit tree species in North China, and Shandong Province is one of the major cherry production areas. In June 2013, a survey of possible CMLV presence was conducted in a cherry orchard planted in 1996 in Zoucheng city, Shandong. The sweet cherry cultivars in this orchard included Black Tartarian, Bing, Hongdeng (a hybrid between cvs. Napoleon and Huangyu), and others; the rootstock cultivar utilized to graft these cultivars was mountain cherry (P. tomentosa). During the survey, characteristic symptoms on leaves such as leaf mottling, distortion, and puckering similar to those caused by CMLV were observed on some trees of the cv. Hongdeng, and the symptomatic trees accounted for ~10% of the total trees of this cultivar. Five symptomatic cherry leaf samples and three healthy-looking cherry leaf samples of cv. Hongdeng were collected. Total RNA extracted from the leaf samples using RNeasy plant mini kit (Qiagen Inc., Valencia, CA) was subjected to first strand cDNA synthesis with the reverse primer CMLV-3R (5'-CTCGAGAACACAGAGATTTGTCGAGAC-3', sequence in italics indicates restriction site XhoI) and M-MLV reverse transcriptase (Promega, Madison, WI) according to the manufacturer's instruction. The cDNA was then used as template in the PCR assay using primers CMLV-5F (5'-GGATCCATGTCGGCGCGATTGAATC-3', sequence in italics indicates restriction site BamHI) and CMLV-3R, which amplify the genome fragment including the capsid protein gene of CMLV. The expected PCR product ~590 bp was amplified from all five symptomatic samples, while no such PCR product was amplified from the symptomless samples. The PCR products were cloned into pMD18-T vector (TaKaRa, Dalian, China). Three positive clones for each of the five amplicons were sequenced in both directions. Sequence alignment and nucleotide BLAST analysis of the sequences revealed that they were 99% to 100% identical to the corresponding capsid protein gene sequence of a cherry isolate of CMLV (GenBank Accession No. AF170028) and 85% identical with that of the peach wart strain of CMLV (KC207480). Our results confirm the infection of cherry trees by CMLV in Shandong. To our knowledge, this is the first report of CMLV on cherry in China. As the spread of CMLV by mite vector in the field is rare (1), and no bud mite outbreak had occurred in this orchard in the past years, so it is possible that virus-infected propagation materials are largely responsible for the spread of this virus. Considering the importance of cherry cultivation in China, this report prompts the need to survey the occurrence of this virus in Shandong and other provinces, and the need to develop more effective management strategies such as the use of certified virus-free nursery stocks to reduce the impact of CMLV. References: (1) J. E. Adaskaveg et al. Diseases. Page 61 in: UC IPM Pest Management Guidelines: Cherry. University of California ANR Publication 3444, 2014. (2) D. James et al. Arch. Virol. 145:995, 2000. (3) T. A. Mekuria et al. Arch. Virol. 158:2201, 2013.

First Report of Broad bean wilt virus 2 in Echinacea purpurea in China.

Field-grown Echinacea purpurea plants showing necrosis, leaf roll, yellow mosaic, and mosaic symptoms in leaves were collected in June 2010 in Huairou, Beijing, China. ELISAs of extracts of four samples showed that one sample with mosaic symptoms had a positive reaction with Broad bean wilt virus 2 (BBWV-2) monoclonal antibody provided by Professor X. P. Zhou (1). The monoclonal antibody recognized the 44.7 kD coat protein subunit of BBWV-2. We used Chenopodium quinoa as an assay species to isolate the virus by sap transmissions and to maintain the virus strain. Sap from infected C. quinoa, when inoculated onto indicator plant species, induced the following symptoms: C. quinoa: local lesions in inoculated leaves, systemic chlorotic mottle in upper leaves, deformation, and apical necrosis; C. amaranticolor: chlorotic local lesions, systemic mosaic and leaf distortion; Nicotiana benthamiana: systemic mosaic; Gomphrena globosa: local purple spots in inoculated leaves and systemic infection in upper leaves; Tetragonia expansa: local lesions, but no symptoms of systemic infection; Physalis floridana: systemic mosaic. No symptoms were observed on Capsicum annuum, Datura stramonium, N. glutinosa, or N. tabacum cv. White Burley. To confirm BBWV-2 infection, total RNAs extracted from infected C. quinoa leaves were reverse transcripted to cDNA using oligo-dT primer (T17V). The primer pair Fab5'R1F (5'-AAATATTAAAACAAACAGCTTTCGTT-3') and Fab5'R1R (5'-TTCAAAGCTCGTGCCATNTYATTKGC-3') for specific detection of the Fabavirus genus (2) was used for PCR analysis. The amplified fragment is between the 5'-terminal non-translatable region (NTR) and the beginning of the coding region of RNA1. Amplicons of approximately the expected size (~391 bp) were produced from the virus-infected C. quinoa and a BBWV-2 positive control (ATCC PV131, PV0537). Amplicons of approximately the expected size (~350 bp) were produced from the BBWV-1 positive control (ATCC PV132). However, no such amplicons were observed from healthy C. quinoa plants and water control. The 391-bp amplicons of RNA1 obtained from the infected C. quinoa were cloned and sequenced. Comparison with sequences of other BBWV-2 isolates showed that the isolate we obtained (No. JX070674) had approximately 99% nt identity (98% amino acid identity) with Chinese BBWV-2 isolate BC (No. FJ485686.1) (3). As an ornamental and medicinal plant, E. purpurea is widely cultivated in northern China. Up until now, Tomato ring spot virus, Tobacco rattle virus, Cucumber mosaic virus, and Tomato spotted wilt virus have been detected or isolated from E. purpurea in the world (4). To our knowledge, this is the first report of BBWV-2 infecting E. purpurea in China. BBWV-2-infected E. purpurea may have less secondary metabolites, which could influence the quality and therapeutic efficacy of this herbal medicine. References: (1) L. Qing et al. Acta Microbiologica Sinica 40:166, 2000. (2) R. M. Ferrer et al. J. Virol. Methods 144:156, 2007. (3) C. Sui et al. Plant Dis. 93:844, 2009. (4) B. Dikova. Bulgarian J. Agric. Sci. 17:306, 2011.

Identification of WA-type three-line hybrid rice with real-time polymerase chain reaction (PCR) method.

A real-time fluorescent PCR (RTF-PCR) was developed to detect and quantify wild abortive (WA)-type three-line hybrid rice (Oryza sativa L.). The mitochondrial R2₋630 WA gene was reported to be closely related to male sterility in plants, and developed as a molecular maker to identify the cytoplasmic male sterility system of hybrid rice. First, we got the DNA sequence of R2₋630 WA gene in 17 rice species with traditional PCR. Then, a pair of specific primers (P3, P4) and TaqMan fluorescence probe (P3₋14) were designed based on the R2₋630 DNA sequence. The following RTF-PCR was performed on the 17 rice species finally. The results indicate that the probes used here are specific for three-line hybrid rice F1 and male sterile lines. We can even identify a single hybrid seed using the probes, which confirmed that the probes can be applied to the identification and quantification of the WA-type three-line hybrid rice. In addition, the RFT-PCR system can be optimized when the annealing temperature is 60 °C and the Mg²âº concentration is 3.5 mmol/L.

First Report of a Soft Rot of Banana in Mainland China Caused by a Dickeya sp. (Pectobacterium chrysanthemi).

Banana is one of the most important fruit crops grown in China (2). A severe outbreak of a soft rot of banana occurred in Guangzhou, China from 2009 to 2010. The disease was characterized by an odorous soft rot of the center of the rhizome. The rot progressed up the pseudostem, destroying the growing point and causing internal decay and often accompanied by vascular discoloration. Yellowing and wilting of the leaves were also characteristic symptoms. A survey of three areas of production of Musa sapientum (cv. ABB) covering 10 ha in Guangzhou revealed that 82% of the fields were affected at an incidence ranging from 20 to 40%. Forty-five bacterial isolates were obtained from lesions on plants sampled from these fields by surface-sterilizing symptomatic tissue in 0.3% NaOCl for 10 min, rinsing the tissue sections three times in sterile water, and plating the sections on nutrient agar. Three representative isolates selected randomly were all gram negative, caused a soft rot of potato disks, utilized malonate, tested positive for phosphatase production, and tested negative for acid production from palatinose, glucopyranoside, and trehalose. A Biolog similarity index of 0.803 indicated that the three isolates had a high similarity to the Biolog reference strain of Pectobacterium chrysanthemi (Version 4.2, Biolog Inc., Hayward, CA). The 16S rDNA sequence (GenBank Accession No. 1321085) of each of the three isolates was determined (1). When compared with sequences in GenBank, the highest degree of sequence similarity was with P. chrysanthemi AF373199. On the basis of a phylogenetic tree of the sequences, the three bacterial isolates are related to Pectobacterium (100% bootstrap values). On the basis of two diagnostic methods, the three isolates were identified as P. chrysanthemi. However, according to Samson et al. (3), they are a Dickeya sp. Additional genetic comparisons with type strains will be needed for the strains to be assigned to a known species of Dickeya. Pathogenicity of each of the three strains on M. sapientum (cv. ABB) was confirmed by injecting 60 40-day-old seedlings each with 5 ml of a suspension of the isolate (108 CFU/ml) into the rhizome. Sixty plants of the same cultivar injected with sterile water served as the control treatment. After 48 h, yellowing and wilting of the leaves, similar to symptoms observed on field plants, were observed on all inoculated seedlings for each of the three bacterial strains. There were no symptoms on the control plants. Koch's postulates were fulfilled by reisolating bacteria from lesions on the leaves of inoculated seedlings. The reisolates were identical to the inoculated strains in biochemical characteristics. Bacteria characteristic of the inoculated strains were not reisolated from the control plants. To our knowledge, this is the first report of a Dickeya sp. causing soft rot of banana in mainland China. References: (1) W. S. Kaneshiro et al. Plant Dis. 92:1444, 2008. (2) Y. P. Ke et al. China Trop. Agric. 1:14, 2008. (3) R. Samson et al. Evol. Microbiol. 55:1415, 2005.

Detection of the Pinewood Nematode, Bursaphelenchus xylophilus, Using a Real-Time Polymerase Chain Reaction Assay.

ABSTRACT The pinewood nematode, Bursaphelenchus xylophilus, has caused significant damage to pine plantations both in East Asia and North America and is an important quarantine organism. A real-time polymerase chain reaction (PCR) assay was developed to detect B. xylophilus. A set of primers and probe specific for B. xylophilus was designed to target the ribosomal DNA internal transcribed spacer region. Optimal primer concentration, Mg(2+) concentration, and extension temperature were 400 nM, 3.0 mM, and 60 degrees C, respectively. The assay was highly specific and sensitive, detecting as little as 0.01 ng of B. xylophilus DNA. The real-time PCR assay also successfully detected B. xylophilus in field samples, and it should be very useful for quarantine purposes.