[Survey of antibody levels of pertussis, diphtheria and tetanus in 495 pregnant women in Nanshan District of 2019, Shenzhen].

Objective: The purpose of this study was to investigate the IgG antibody levels of whooping cough, diphtheria, and tetanus in pregnant women in Nanshan District. Methods: From January to March 2019, 495 pregnant women who met the inclusion criteria in a hospital in Nanshan District, Shenzhen were selected as the survey subjects. The enzyme-linked immunosorbent assay was used to detect serum levels of pertussis, diphtheria, and tetanus IgG antibodies and we compared the differences in antibody levels of pregnant women with different characteristics. Results: The maternal age was (29.23±4.08) years old. The geometric mean concentration of pertussis antibody was 2.589 (1.172-4.953) IU/ml, 1.01% (5 cases) of pregnant women had pertussis antibody concentration ≥ 40 IU/ml, and 75.15% (372 cases) of pregnant women had pertussis antibody concentration<5 IU/ml. The GMC value and antibody positive rate of diphtheria in pregnant women were 0.024(0.009-0.065) IU/ml and 72.53% (359 cases), respectively. The GMC value and antibody positive rate of tetanus in pregnant women were 0.014 (0.006-0.034) IU/ml and 53.74% (266 cases), respectively. There was no statistical difference in the antibody level and antibody positive rate among pregnant women of diphtheria and tetanus, respectively. Conclusion: The concentration of antibodies against pertussis, diphtheria, and tetanus in pregnant women are all at a low level, which is not enough to protect themselves from disease infection.

[Amplitude of low-frequency fluctuations of resting-state functional MRI in colorectal cancer patients with depression].

Objective: To investigate the alterations of the cerebral resting-state spontaneous neural activity in colorectal cancer patients with depressive symptoms. Methods: Thirty-three colorectal cancer patients (patient group) with depression and 43 healthy subjects (control group) underwent the resting state functional magnetic resonance imaging (rs-fMRI). The amplitude of low-frequency fluctuations (ALFF) and fractional ALFF (fALFF) were calculated. Two independent samples t test were used to compare the ALFF and fALFF values between two groups by DPABI software, and then correlation analysis was performed between ALFF and fALFF with statistical significance and Patient Health Questionnaire (PHQ-9) scores and Generalized Anxiety Disorder (GAD-7) scores. Results: Compared with the control group, the patient group showed significantly lower ALFF and fALFF values in the bilateral precuneus, calcarine gyrus, lingual gyrus, left cuneus, superior, middle, inferior occipital gyrus and right fusiform gyrus (t=-5.730, P<0.05; t=-4.872, P<0.05). There were no significant correlations between the ALFF and fALFF values in these regions and PHQ-9 or GAD-7 scores (P>0.05). Conclusion: Spontaneous decrease of neural activity in occipital and parietal lobes exists in colorectal cancer patients with depression at resting-sate, which may be a potential neurobiological marker.

[Interpretation for the group standards of the Isolation and Identification of Campylobacter jejuni and Campylobacter coli].

Foodborne Campylobacter is recognized as the leading causes of the bacterial diarrheal illness in both developing and developed countries. C. jejuni and C. coli caused 95% of the human campylobacterisosis. Bacteria culture has been recognized as the "Gold standard" for the diagnosis of the Campylobacter infection. The National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention coordinated the experienced researchers from China National Center for Food Safety Risk Assessment and other local Centers for Disease Control and Prevention to write up the standards for entitled Isolation and Identification of Campylobacter jejuni and Campylobacter coli (T/CPMA 006-2019). The standard is drafted with principles of emphasizing the scientific, normative, applicability and feasible nature. This group standard recommended the procedures and steps for the isolation and identification of C. jejuni and C.coli from variant samples. The standard aims to improve the capacity for Campylobacter identification in China.

The identification of up-regulated ebv-miR-BHRF1-2-5p targeting MALT1 and ebv-miR-BHRF1-3 in the circulation of patients with multiple sclerosis.

Epstein-Barr virus (EBV) is a well-documented aetiological factor for multiple sclerosis (MS). EBV encodes at least 44 microRNAs (miRNAs) that are readily detectable in the circulation of human. Previous studies have demonstrated that EBV-encoded miRNAs regulate host immune response and may serve as biomarkers for EBV-associated diseases. However, the roles of EBV miRNAs in MS are still unknown. To fill the gap, we conducted a comprehensive profiling of 44 mature EBV miRNAs in 30 relapsing-remitting MS (RRMS) patients at relapse and 30 matched healthy controls. Expression levels of ebv-miR-BHRF1-2-5p and ebv-miR-BHRF1-3 were elevated significantly in the circulation and correlated positively with the expanded disability status scale (EDSS) scores of MS patients. Receiver operating characteristic (ROC) analyses confirmed that the expression of these two miRNAs distinguished MS patients clearly from healthy controls. Luciferase assays revealed that ebv-miR-BHRF1-2-5p may directly target MALT1 (mucosa-associated lymphoid tissue lymphoma transport protein 1), a key regulator of immune homeostasis. In conclusion, we described the expression of EBV miRNAs in MS and preliminarily validated the potential target genes of significantly altered EBV miRNAs. The findings may pave the way for prospective study about the pathogenesis of MS.

Mitochondrial tRNA mutations may be infrequent in hepatocellular carcinoma patients.

Mitochondrial DNA mutations have been shown to play important roles in the pathogenesis of hepatocellular carcinoma (HCC). In particular, genes encoding mitochondrial tRNA (mt-tRNA) are hotspots for pathogenic mutations associated with HCC. Recently, an increasing number of studies have reported the involvement of such mutations in this disease. As a result, several mt-tRNA mutations associated with HCC have been described. Some of these are neutral polymorphisms and may not cause mitochondrial dysfunction. Moreover, the molecular mechanisms by which these pathogenic mutations result in HCC remain unclear. To address this problem, we evaluated five mt-tRNA variants (tRNA(Val) T1659C, tRNA(Ala) G5650A, tRNA(Arg) T10463C, tRNA(Glu) A14679G, and tRNA(Pro) C15975T) implicated in the clinical manifestation of HCC in humans. We performed evolutionary conservation analysis and used a bioinformatic tool to predict the secondary structure of the mt-tRNAs carrying these mutations. Using an established pathogenicity scoring system, we classified T10463C and A14679G as neutral polymorphisms, and determined that the T1659C, G5650A, and C15975T variants should be regarded as pathogenic mutations. To the best of our knowledge, this is the first report to establish the pathogenicity of HCC-associated mt-tRNA mutations.

First Report of Phomopsis longicolla Causing Leaf Spot on Soybean in China.

During July 2012, leaf spots affecting 60% of the leaves were observed on soybean cultivar He Feng 60 in fields near Shenyang City, Liaoning Province, leading to 5 to 10% yield loss. The leaf spots were associated with the leaf margins and were irregularly shaped, with brown to black margins and surrounded by a thin, yellow halo. Often, several spots merged to form large necrotic areas, which contained numerous pycnidia on the underside of the leaf. Small pieces (5 mm2) were excised from the margin of diseased and healthy tissue, surface-sterilized in 70% ethanol solution for 30 s and 0.1% mercuric chloride solution for 1 min, washed in three changes of sterile distilled water, and transferred to plates containing potato dextrose agar (PDA). Cultures were maintained in an incubator at 25°C with a 12 h dark/light photoperiod for 5 to 7 days. On PDA, colonies were white with yellow areas, floccose, dense, and moderately fast growing, attaining a diameter of 3.9 mm after 5 days and 9.0 mm after 14 days. Finally, large black stromata appeared after 28 days at 25°C. The conidiomata pycnidia were black, stomatic, globose, length 83.6 to 232 µm, width 37.9 to 146.3 µm and produced α-conidia that were unicellular, hyaline, sometimes two-guttulate, length 4.75 to 8.25 µm, width 1.50 to 3.00 µm. ß-Conidia were not observed. To confirm the morphological identification, the ribosomal internal transcribed spacers (ITS1-5.8S-ITS2) from isolates were sequenced (GenBank Accession No. KC460334). The PCR products were cloned into a pMD-19T Cloning Vector (Sangon Biotech, Shanghai, China). The clones were purified with TIANprep Mini Plasmid Kit (Tiangen Biotech, Beijing, China) to get the full-length ITS sequence. BLAST analysis of the isolates showed 100% nucleotide sequence identity with Phomopsis longicolla (AY745021). Four additional primer pairs-large subunit (NL1/NL4), beta-tublin gene (Bt2a/Bt2b), translation elongation factor 1α gene(EF1-728F/EF1-986R), and act gene(ACT-512F/ACT-783R) (1,2)-were amplified and sequenced as described above. The large subunit gene, ß-tubulin gene, and translation elongation factor 1α gene from isolates were sequenced (Sangon Biotech). BLAST analysis indicated that the isolates had 100% nucleotide sequence identity with P. longicolla (AB107259, HQ333514, and AF398896). Because the act gene sequence of P. longicolla was not in the NCBI database, this sequence had 94% nucleotide sequence identity with P. cuppatea (JN230389). To fulfill Koch's postulates, five leaves on five healthy soybean plants were inoculated with a conidial suspension (106/ml). Plants inoculated with sterile water served as the noninoculated controls. Plants were incubated in the greenhouse at 25°C. All the inoculated leaves developed pinhead spots on the leaves, gradually increasing to large brown spots. Spots were irregularly shaped, brown and necrotic in the center, and surrounded by a yellow halo. Black pycnidia appeared after 10 days, whereas the noninoculated control plants remained asymptomatic. P. longicolla was consistently recovered from all inoculated plants, except the control. Morphological description of isolates was similar to that of Hobbs (3). However, as described by Hobbs and others, P. longicolla conidiomata pycnidia have prominent necks more than 200 µm long, opening by apical ostioles; locules are uniostiolate or multiostiolate, globose, up to 500 µm wide. The pycnidia size of isolates by frozen section method was smaller than that of Hobbs. Based on morphological and sequence comparisons, the pathogen of leaf spot disease is caused by P. longicolla. This is the first reported leaf spot caused by P. longicolla on soybean in China. References: (1) T. Boekhou et al. Stud. Mycol. 38:75, 1995. (2) P. W. Crous et al. Stud. Mycol. 75:37, 2013. (3) T. W. Hobbs et al. Mycologia 77:535, 1985.

DNA methylation regulates expression of VEGF-C, and S-adenosylmethionine is effective for VEGF-C methylation and for inhibiting cancer growth

DNA hypomethylation may activate oncogene transcription, thus promoting carcinogenesis and tumor development. S-adenosylmethionine (SAM) is a methyl donor in numerous methylation reactions and acts as an inhibitor of intracellular demethylase activity, which results in hypermethylation of DNA. The main objectives of this study were to determine whether DNA hypomethylation correlated with vascular endothelial growth factor-C (VEGF-C) expression, and the effect of SAM on VEGF-C methylation and gastric cancer growth inhibition. VEGF-C expression was assayed by Western blotting and RT-qPCR in gastric cancer cells, and by immunohistochemistry in tumor xenografts. VEGF-C methylation was assayed by bisulfite DNA sequencing. The effect of SAM on cell apoptosis was assayed by flow cytometry analyses and its effect on cancer growth was assessed in nude mice. The VEGF-C promoters of MGC-803, BGC-823, and SGC-7901 gastric cancer cells, which normally express VEGF-C, were nearly unmethylated. After SAM treatment, the VEGF-C promoters in these cells were highly methylated and VEGF-C expression was downregulated. SAM also significantly inhibited tumor growth in vitro and in vivo. DNA methylation regulates expression of VEGF-C. SAM can effectively induce VEGF-C methylation, reduce the expression of VEGF-C, and inhibit tumor growth. SAM has potential as a drug therapy to silence oncogenes and block the progression of gastric cancer.

DNA methylation regulates expression of VEGF-C, and S-adenosylmethionine is effective for VEGF-C methylation and for inhibiting cancer growth.

DNA hypomethylation may activate oncogene transcription, thus promoting carcinogenesis and tumor development. S-adenosylmethionine (SAM) is a methyl donor in numerous methylation reactions and acts as an inhibitor of intracellular demethylase activity, which results in hypermethylation of DNA. The main objectives of this study were to determine whether DNA hypomethylation correlated with vascular endothelial growth factor-C (VEGF-C) expression, and the effect of SAM on VEGF-C methylation and gastric cancer growth inhibition. VEGF-C expression was assayed by Western blotting and RT-qPCR in gastric cancer cells, and by immunohistochemistry in tumor xenografts. VEGF-C methylation was assayed by bisulfite DNA sequencing. The effect of SAM on cell apoptosis was assayed by flow cytometry analyses and its effect on cancer growth was assessed in nude mice. The VEGF-C promoters of MGC-803, BGC-823, and SGC-7901 gastric cancer cells, which normally express VEGF-C, were nearly unmethylated. After SAM treatment, the VEGF-C promoters in these cells were highly methylated and VEGF-C expression was downregulated. SAM also significantly inhibited tumor growth in vitro and in vivo. DNA methylation regulates expression of VEGF-C. SAM can effectively induce VEGF-C methylation, reduce the expression of VEGF-C, and inhibit tumor growth. SAM has potential as a drug therapy to silence oncogenes and block the progression of gastric cancer.

A Reassessment of Virulence Phenotypes of Soybean Cyst Nematode (Heterodera glycines) in China with HG Typing Method.

Soybean cyst nematode, Heterodera glycines Ichinohe, is the most destructive pest of soybean (Glycine max) worldwide, including China. H. glycines virulence phenotypes can be described in two ways. One is the race determination test that uses four soybean lines to categorize H. glycines into 16 "races" (3). The HG type designation is similar, but avoids the implications of the term "race" and includes seven soybean lines rather than four (2). In China, previous data revealed the occurrence of nine H. glycines races including races 1 through 7, 9, and 14, whereas HG types have not been recorded (1). The objective of this study was to reassess virulence phenotypes of H. glycines in China by means of HG types. In 2011 and 2012, 10 SCN populations from the primary soybean production regions of China were identified as six races (races 1, 2, 3, 4, 5, 6, and 14) with the race test scheme (3) and were cultured on a H. glycines-susceptible soybean cultivar. In 2013, seven indicator soybean lines (PI 548402, PI 88788, PI 90763, PI 437654, PI 209332, PI 89772, and PI 548316) plus the susceptible standard soybean "Lee74" were used to determine the HG types of these populations following standardized procedures (2) with some modification in a growth chamber set at 28°C under 16-h days. After 30 days, females were extracted from roots and collected, and a female index was calculated for each indicator line (2,3). The average number of females on Lee74 was more than 100 in all the tests. Eight HG types were identified in the populations tested: HG type 0 (race 3), HG type 7 (race 3 or 6), HG type 2.7 (race 1 or 5), HG type 5.7 (race 3), HG type 1.3.7 (race 14), HG type 2.5.7 (race 1), HG type 1.2.5.7 (race 2), and HG type 1.2.3.5.7 (race 4). To our knowledge, this is the first report of H. glycines HG types in China, which will contribute to development of management strategies implementing the use of resistant cultivars. This work was supported by the Special Fund for Agro-scientific Research in the Public Interest 200903040-03 and the China Agriculture Research System CARS-04. References: (1) W. G. Lu et al. Agr. Sci. China 5:615, 2006. (2) T. L. Niblack et al. J. Nematol. 34:279, 2002. (3) R. D. Riggs and D. P. Schmitt. J. Nematol. 20:392, 1988.

Calycosin induces apoptosis by upregulation of RASD1 in human breast cancer cells MCF-7.

Breast cancer is a leading cause of cancer death among women, and the failure of normal apoptosis has been proved in the development of breast cancer. The phytoestrogen, calycosin, is extracted from Chinese medical herb Radix astragali. We recently reported that calycosin successfully stimulated proliferation of ER-positive MCF-7 human breast cancer cells at low concentration. In the present study, we assessed the proapoptotic function of calycosin in MCF-7 cells at high concentration in vitro, as well as the possible mechanism of its effect. MCF-7 cells were treated with different concentrations of calycosin, and then detected by MTT assay for cellular viability, Hoechst assay, and flow cytometry for apoptosis. RASD1 is identified as a Ras-family member and a regulator in MAPK-mediated cascade leading to cell proliferation or apoptosis. To provide insight into the functions of RASD1 signaling pathway in calycosin-induced apoptosis, the expression of Bcl-2, Bax, and RASD1 in calycosin-treated cells were determined by Western blot assay. The results showed that high concentrations of calycosin significantly suppressed the proliferation of MCF-7 cells and promoted cell apoptosis. Moreover, compared with control group, the expression of Bcl-2 decreased with calycosin in MCF-7 cells, while Bax increased, which was significantly correlated with elevated expression of RASD1. Together, we present evidence that at relatively high concentration calycosin triggered cell apoptosis through the mitochondrial apoptotic pathway by upregulating RASD1. And for the first time, this study revealed that calycosin may have potential as a therapeutic agent for the treatment of breast cancer.

First Report of the Cactus Cyst Nematode, Cactodera cacti, on Cactus in Northern China.

During a survey for cyst nematodes from August to September 2011, nearly 15% of cactus (Cereus jamacaru) plants exhibited yellowing and wilting symptoms in greenhouses from Shenyang Botanical Garden, Liaoning Province, China. Cysts (averaging 50 per 100 g of samples) were detected by the sieving-decanting method on the roots and in rhizospheric soil. Second stage juveniles and eggs were isolated directly from cysts. Cysts, juveniles, and eggs were identified by morphology. Cysts (n = 12) were rounded to lemon-shaped with a protruding neck and vulva. The cyst wall had a zig-zag pattern. The vulval cone was circumfenestrate without underbridge and bullae but generally with vulval denticles. The cysts were characterized by body length excluding neck (range = 399.5 to 622.0 µm, mean = 524.9), body width (300.4 to 469.9 µm, 383.4), length to width ratio (1.1 to 1.7, 1.4), neck length (41.0 to 130.9 µm, 61.5) and width (55.0 to 98.7 µm, 76), and circumfenestral diameter length (24.6 to 30.2 µm, 28.4). Measurements of second-stage juveniles (n = 20) included length of body (range = 467.3 to 542.5 µm, mean = 513.8), stylet (23.0 to 25.8 µm, 24.6) with knobs rounded to slightly projecting anteriorly and concave on anterior surface, tail (45.9 to 59.5 µm, 52.2), and hyaline tail terminal (16.3 to 23.2 µm, 19.1). Eggs (n = 20) had heavy punctations on the shell surface. All morphological data and characteristics were consistent with Cactodera cacti (3). Molecular evidence confirmed the identification. DNA from a single cyst was extracted by using the protocol described by Subbotin et al. (2), the rDNA-internal transcribed spacer (ITS) and D2-D3 fragments of the 28S rDNA were amplified with universal primers TW81 (5'-GTTTCCGTAGGTGAACCTGC-3') and AB28 (5'-ATATGCTTAAGTTCAGCGGGT-3'), D2A(5'-ACAAGTACCGTGAGGGAAAGTTG-3') and D3B(5'-TCGGAAGGAACCAGCTACTA-3'), respectively. The ITS PCR product was digested with eight restriction enzymes (AluI, AvaI, Bsh1236I, BsuRI, CfoI, MvaI, PstI, and RsaI) to obtain restriction fragment length polymorphism profiles (4). The ITS and D2D3 sequences were cloned and assayed using an ABI-PRISM 3730 Genetic Analyzer (Applied Sangon, Shanghai, China) and were subjected to a database search using BLAST (National Centre for Biotechnology Information). The 980-bp ITS sequence exhibited 99% similarity with that of a C. cacti isolate from Iran (GenBank Accession No. AF498393.1) and the 787-bp D2D3 sequence exhibited 99% similarity with a C. cacti isolate from Germany (GenBank Accession No. DQ328702.1). Cactus cyst nematode has been mainly reported on ornamental plants of the family Cactaceae grown in greenhouses. Infested plants become reddish brown to yellow in color, wilted, stunted, with reduced flower production and shortening flower period. With high population densities of C. cacti, death of plants may occur (1). To the best of our knowledge, this is the first report of C. cacti in northern China. References: (1) R. P. Esser. Division of Plant Industry, 197:3, 1992. (2) S. A. Subbotin et al. Nematology, 2:153, 2000. (3) S. A. Subbotin et al. Systematics of Cyst Nematodes (Nematoda: Heteroderinae). Volume 8 Part A. Brill, Leiden, the Netherlands, 2010. (4) M. Z. Tanha et al. Nematology, 5:99, 2003.

First Report of Leaf Spot of Soybean Caused by Aristastoma guttulosum in China.

In fall, 2008, leaf spots were observed during the flowering stage of the Zhong Huang 13 cultivar of soybean in the fields of Anhui Province, China. The leaf spots were irregularly shaped, necrotic, brown-black, and surrounded by yellow halos. Often, on a given leaf, several spots joined one another to form a large blighted area. Finally, those leaves turned yellow followed by defoliation. Damaged leaves showed scattered black spots (i.e., numerous pycnidia) on the lower side. Fresh material was collected from infected plants and a single spore of the putative causal pathogen was isolated on potato dextrose agar (PDA) and incubated at 25°C during a 12-h dark/light cycle. The isolate produced a white fungal colony and black pycnidia after 30 days. The pycnidia are characterized as globose, dark brown-black, and distinctly papillate, with ostiolar setae, and are more or less straight, unbranched, and tapered at the apex. The conidia are clavate, hyaline, mostly with three transverse septa per cell; conidia are either straight or slightly bent, obviously guttulate, and 16 to 29 × 2.5 to 3.5 µm. This pathogen is similar to other Aristastoma guttulosum Sutton (1964), but with the following differences: (a) it has more than 10 versus 4 to 9 setae; (b) conidia are 16 to 29 × 2.5 to 3.5 µm versus 32 to 42 × 3.9 to 4.6 µm as reported for A. guttulosum (1). Conidia of the Chinese isolate were used to inoculate leaves of soybean. Five soybean leaves from potted plants, 1 month old, were sprayed with a suspension of conidia in water. Conidia were harvested from PDA cultures and the suspension was adjusted to 3 × 105 conidia/ml with a hemocytometer. Five leaves were sprayed with sterile distilled water as controls. Inoculated plants were kept in the greenhouse. All five of the inoculated leaves displayed the same symptoms observed in the fields. The symptoms developed initially as brown pinhead spots on the upper side of the leaves, gradually increasing to large brown spots. These spots were irregularly shaped, brown and necrotic in the center and surrounded by a yellow halo. Black pycnidia appeared after 1 week whereas the controls remained asymptomatic. The pathogen was reisolated from the inoculated soybean leaves according to standard Koch's postulates. Primers ITS1 and ITS4 were used in PCR reactions to amplify the internal transcribed spacer region (ITS) (3). Sequencing was performed using the same primers. The ITS sequence (GenBank Accession No. JF825548.1) for this pathogen (587 bp) was submitted to a BLAST search in GenBank. Since the ITS sequence of the genus Aristastoma has never been previously submitted, results did not show high similarity with any extant GenBank sequences. The genus Aristastoma Tehon (1933) was described by Tehon (2). Five of the species in this genus were described by Sutton (1). The number of septate conidium and lack of obvious guttulate within the conidium are the morphological basis to separate these five species. Morphological features of the pathogen from soybean leaves in China were slightly different from those of A. guttulosum. To our knowledge, this is the first report of leaf spot caused by A. guttulosum on soybean in China. References: (1) B. C. Sutton. Mycological Papers. 97:10, 1964. (2) L. R. Tehon. Mycologia XXV. 25:249, 1933. (3) T. J. White et al. Academic Press, San Diego, 1990.