[Effects of pretreatment HIV drug resistance on the virological response of HIV-infected patients after 3-year antiretroviral therapy].

Objective: To investigate the impact of pretreatment drug resistance (PDR) on virological effect among HIV-infected patients having received antiretroviral therapy (ART) after three years. Methods: The baseline survey of PDR among HIV-infected patients was conducted in 2018, with a three-year follow up study. The clinic data and virological laboratory test variables were statistically analyzed. Results: Of the 2 433 participants, 41.6% (1 012/2 433) were aged between 18 and 34, 82.8% (2 015/2 433) were males, 46.9% (1 142/2 433) had education of high school or above, 22.4% (544/2 433) were farmers, 33.8% (823/2 433) were unmarried, 48.1% (1 169/2 433) were infected heterosexually and 41.3% (1 004/2 433) were with CRF07_BC. The prevalence of PDR was 4.5% (109/2 433). The prevalence of virological suppression failure (viral load ≥50 copies/ml) and drug resistance at three years follow up after ART was 8.1%(196/2 433) and 2.5%(60/2 433) respectively. The prevalence of virological suppression failure and drug resistance at three years follow up after ART were 18.3% (20/109) and 7.6% (176/2 324), and 4.6% (5/109) and 2.4% (55/2 324) among participants with PDR and non-PDR, respectively. The results of multivariate logistic regression model showed that illiteracy (aOR=3.26, 95%CI: 1.82-5.86), primary and junior high school education (aOR=1.54, 95%CI: 1.09-2.18), CD4+T lymphocyte count <200/µl (aOR=2.77, 95%CI: 1.75-4.37) and CD4+T lymphocyte count 200-499/µl (aOR=1.55, 95%CI: 1.10-2.18) at a three year follow up visit after ART, missed drugs in the past month (aOR=4.24, 95%CI: 2.92-6.17), and PDR (aOR=2.84, 95%CI: 1.67-4.85) were statistically significant with virological suppression failure on treatment. Conclusions: The prevalence of PDR in China at a low level currently, and the virological suppression failure rate is low after three years of ART. It is necessary to strengthen drug resistance monitoring of HIV-infected patients and pay attention to the influence of PDR on treatment effect.

[Global guidelines of colorectal cancer screening in high-risk population with family history of colorectal cancer: a systematic review].

Objective: To systematically summarize and evaluate the development of update and detailed recommendations of the existing global screening guidelines in high-risk population with a family history of colorectal cancer. Methods: The words "colorectal cancer", "screening", "guideline", "consensus", "recommendations" and "family history" in Chinese and English were used as MESH terms for literature retrieval, as well as entry terms. The retrieval was performed based on China National Knowledge Infrastructure, Wanfang Data Knowledge Service Platform, PubMed, Embase, Cochrane Library and Web of Science, as well as official websites. The languages of literatures were limited in Chinese and English. As of May 24, 2022, a total of 20 valid literatures had been retrieved. The basic information of the literatures and the recommendations of colorectal cancer screening for people with family history were collected and analyzed. Results: The analysis on the 20 literatures indicated that most countries/regions/institutions recommended age range of screening, screening modalities and intervals for people with family history of colorectal cancer. For the individuals who have one first-degree relative diagnosed with colorectal cancer before 60 years of age,most guidelines recommended the screening to be started at 40 years or 10 years earlier than the age when the youngest first-degree relative was diagnosed. The most commonly recommended screening modality was colonoscopy. Conclusions: Most current screening guidelines for high-risk people with family history of colorectal cancer recommend colonoscopy as the main modality. This review will provide reference for the update of screening strategies in high-risk people with family history of colorectal cancer in China, and further improve the practices of screening, early diagnosis and treatment of colorectal cancer.

[Progress in genomic DNA methylation of esophageal squamous cell carcinoma].

Abnormal genomic DNA methylation is an important epigenetic change in malignant tumors. Esophageal squamous cell cancer is one of common malignant tumors in our country. In this paper summarized and discussed the progress of genomic DNA methylation in the esophageal squamouscell cancer, including the level of genomic DNA methylation, frequent abnormally methylated genes, methylation markers and potential targets, etc. This paper might provide candidate biomarkers and targets for further studies on the mechanism of the tumorigenesis and development of the esophagealsquamouscell cancer, as well as for the clinical application of esophageal cancer.

Neuropilin-1 contributes to esophageal squamous cancer progression via promoting P65-dependent cell proliferation.

Neuropilin-1 (NRP1) is a non-kinase receptor recently implicated in tumor progression. Here we revealed that over-expression of NRP1 correlates with poor prognosis in esophageal squamous cell carcinoma (ESCC). NRP1-knockdown suppressed ESCC cell proliferation and xenograft tumor growth. Reduced NRP1 expression downregulated P65 mRNA and protein expression, and ectopic expression of P65-restored cell proliferation in NRP1-silenced cells. NRP1 regulates P65 transcription by activating cAMP responsive element binding protein (CREB). NRP1 interacted with and activated epidermal growth factor receptor (EGFR), and b1/b2 domain of NRP1 is responsible for the activation of EGFR. We also found that EGFR regulated CREB transcriptional activity via AKT. These data suggest that NRP1 is an upstream regulator in the P65-dependent proliferation signaling pathway and a candidate therapeutic target for ESCC.

Expression and characterization of a codon-optimized blood coagulation factor VIII.

Essentials Recombinant factor VIII (FVIII) is known to be expressed at a low level in cell culture. To increase expression, we used codon-optimization of a B-domain deleted FVIII (BDD-FVIII). This resulted in 7-fold increase of the expression level in cell culture. The biochemical properties of codon-optimized BDD-FVIII were similar to the wild-type protein. SUMMARY: Background Production of recombinant factor VIII (FVIII) is challenging because of its low expression. It was previously shown that codon-optimization of a B-domain-deleted FVIII (BDD-FVIII) cDNA resulted in increased protein expression. However, it is well recognized that synonymous mutations may affect the protein structure and function. Objectives To compare biochemical properties of a BDD-FVIII variants expressed from codon-optimized and wild-type cDNAs (CO and WT, respectively). Methods Each variant of the BDD-FVIII was expressed in several independent Chinese hamster ovary (CHO) cell lines, generated using a lentiviral platform. The proteins were purified by two-step affinity chromatography and analyzed in parallel by PAGE-western blot, mass spectrometry, circular dichroism, surface plasmon resonance, and chromogenic, clotting and thrombin generation assays. Results and conclusion The average yield of the CO was 7-fold higher than WT, whereas both proteins were identical in the amino acid sequences (99% coverage) and very similar in patterns of the molecular fragments (before and after thrombin cleavage), glycosylation and tyrosine sulfation, secondary structures and binding to von Willebrand factor and to a fragment of the low-density lipoprotein receptor-related protein 1. The CO preparations had on average 1.5-fold higher FVIII specific activity (activity normalized to protein mass) than WT preparations, which was attributed to better preservation of the CO structure as a result of considerably higher protein concentrations during the production. We concluded that the codon-optimization of the BDD-FVIII resulted in significant increase of its expression and did not affect the structure-function properties.

[Expression and intratumoral heterogeneity of LN-5γ2 in esophageal squamous cell carcinoma].

OBJECTIVE: The present study aimed to evaluate the expression and intratumoral heterogeneity of LN-5γ2 in esophageal squamous cell carcinoma (ESCC). METHODS: The expression of LN-5γ2 protein was examined in 135 ESCC cases by immunohistochemistry, and to analyze its relationship with the clinical relevance of patients. The protein expressions in different regions in the same tumor as well as different nests in the same region were compared. RESULTS: Moderate and high expression of LN-5γ2 protein was detected in 40.0% (54/135) of tumor tissues. Positive immunohistochemical staining was observed in 31.1% (23/74) of early stage (stages Ⅰ/Ⅱ) cases and 50.8% (31/61) of late stage (stage Ⅲ) cases, with a significant difference between these two groups (P=0.023). There was no statistical association of LN-5γ2 expression with age, sex of patients, PT sage, lymph node metastasis and degree of tumor differentiation (P>0.05). However, differential expression of LN-5γ2 protein was found at different sampling sites in the same tumor and the same sampling site in different carcinomas. CONCLUSION: High expression of LN-5γ2 is positively correlated with tumor clinical stages and there existed intratumoral heterogeneity of LN-5γ2 expression in ESCC tissues.

Microtubule-associated protein 4 is an important regulator of cell invasion/migration and a potential therapeutic target in esophageal squamous cell carcinoma.

Cell invasion and migration significantly contribute to tumor metastasis. Microtubule-associated protein 4 (MAP4) protein is one member of microtubule-associate proteins family. It is responsible for stabilization of microtubules by modulation of microtubule dynamics. However, there is little information about the involvement of MAP4 in human cancer. Here we show that MAP4 serves as a regulator of invasion and migration in esophageal squamous cancer cells. By activating the ERK-c-Jun-vascular endothelial growth factor A signaling pathway, MAP4 promotes cell invasion and migration in vitro, tumor growth and metastasis in mouse models. Immunohistochemical staining of operative tissues indicated that MAP4 expression was associated with tumor stage, lymph node metastasis and shorter survival of the patients with esophageal squamous cell carcinoma (ESCC). Multivariate Cox regression analysis showed that MAP4 is an independent prognostic indicator. In the serial sections of ESCC tissues, there was a positive correlation between MAP4 and vascular endothelial growth factor A expression. Notably, an intratumoral injection of MAP4-small interfering RNA (siRNA) remarkably inhibited the growth of the tumors that formed by the MAP4-expressing ESCC cells in nude mice, and a combination of MAP4-siRNA and Bevacizumab significantly enhanced the inhibition effect. Our data suggest that MAP4 is probably a useful prognostic biomarker and a potential therapeutic target for the disease.

Association between the LXRα polymorphism and stroke in a Chinese population.

We examined the relationship between the liver X receptor α gene (LXRα) polymorphism and the susceptibility to stroke. We utilized the single fluorescent-labeled probe technique to detect the genotype of rs12221497 in the LXRα gene in 400 stroke patients and 400 healthy control subjects. The difference in genotype distribution between the 2 groups was analyzed using the chi-square test. Serum lipids and glucose levels between the different genotypes were also compared. We found that the risk of stroke in carriers with the AA + GA genotype was 2.02-fold higher than that in GG genotype carriers (odds ratio = 2.02, 95% confidence interval = 1.18-2.87, P < 0.05), and that the risk of stroke in carriers with the A allele increased by 0.606-fold compared to that in G allele carriers (odds ratio = 1.606, 95% confidence interval = 1.158-2.228). Logistic regression analysis showed that after adjusting for other confounding factors, the A allele was an independent risk for stroke. However, there were no differences in serum lipids and glucose levels between each genotype. We conclude that the rs12221497 polymorphism in the LXRα gene was associated with the susceptibility to stroke in a Han Chinese population.

First Report of Race Composition and Distribution of Sunflower Broomrape, Orobanche cumana, in China.

Sunflower broomrape (Orobanche cumana Wallr.) is a holoparasitic plant that penetrates the vascular system of sunflower roots, absorbs plant nutrients and water, and thus causes stunting, reduced growth, and severe yield losses (3). To date, seven races of sunflower broomrape (O. cumana) have been identified by using international standard race differential hosts in Bulgaria, Serbia, Romania, Turkey, and Russia (4). However, the race types present in China are unknown. To identify the race composition of sunflower broomrape in China, race differential hosts of sunflower broomrape were received from Dr. Dragan Skoric (Serbian Academy of Sciences and Arts, Novi Sad, Serbia): Line AD66 has no resistant genes; Kruglik-41 contains resistant gene Or1; B-RO-02A has Or2; Record has Or3; LC1002B has Or4; LC1003B has Or5; LC-1093 has Or6, and Race-G-2 has Or7 (1). Eighteen sunflower broomrape samples were collected in August of 2011, 2012, and 2013 from different provinces/locations in China, including Xinjiang (Xinyuan, Shihezi, Tekesi, Beitun, Urumqi, and Yining), Inner Mongolia (Linhe, Xixiaozhao, Wuqianqi, Tuzuoqi, Keyouqianqi, and Aohanqi), Shanxi (Hunyuan, Shilou, Mizhi, and Dingbian), Jilin (Tongyu), and Hebei (Xuanhua). The differential hosts were each inoculated with the seeds of each broomrape isolate that was recovered, as described by Pancenko with minor modification (2). Briefly, two parts of field soil and one part of vermiculite were mixed together and used as potting mix. The mix was inoculated with broomrape seeds at 10 mg of seeds per 100 g of potting mix. The inoculated mix was placed in a 7-cm (diameter) × 11-cm (height) plastic pot to fill two-thirds of the pot volume. Three sunflower seeds were placed on the surface of the mix at an even distance from each other and covered with additional mix. The pots were kept in a greenhouse under a 16-h photoperiod at 10,000 lux of illumination intensity, temperature of 20-25°C, and 40% relative humidity. Forty days after incubation, sunflower seedlings were taken out from the pot and the roots washed with tap water. The number of tubercles was recorded on the root of each differential host. Race types were determined based on the reaction (tubercule formation on roots) of all the standard differential hosts to the test isolate. The results showed that races A, D, E, and G of O. cumana were present among the isolates. Race G was found in Wuqianqi, Xixiaozhao, and Linhe in the western part of Inner Mongolia. Race E was found only in Shihezi of Xinjiang. Race D was found in Aohanqi and Keyouqianqi (eastern part of Inner Mongolia); Xinyuan, Tekesi, Beitun, and Urumqi (northern part of Xinjiang); and Tongyu (northern part of Jilin). Race A was found in Mizhi, Shilou, and Hunyuan of Shanxi province and Xuanhua in Hebei province. Additionally, race A was also found in Tuzuoqi, the middle region of Inner Mongolia. Thus, races A, D, E, and G were the main race types of O. cumana in China. Race D was the predominant race type and had the widest distribution. Race G was the highest level race type in this study but was mainly limited to the western part of Inner Mongolia. This is the first report of race composition and distribution of sunflower broomrape (O. cumana) in China. References: (1) Y. Kaya et al. Helia 40:211, 2004. (2) A. N. Pancenko, Zbirnik VNIIMK. Page 107, 1973. (3) C. Parker. Page 17 in: Proc. 3rd Int. Workshop on Orobanche and Related Striga Research, 1994. (4) P. Shindrova et al. Helia 35:87, 2012.

Identification of putative target genes for amplification within 11q13.2 and 3q27.1 in esophageal squamous cell carcinoma.

BACKGROUND: Genomic aberration is a common feature of human cancers and also is one of the basic mechanisms that lead to overexpression of oncogenes and underexpression of tumor suppressor genes. Our study aims to identify frequent genomic changes and candidate copy number driving genes in esophageal squamous cell carcinoma (ESCC). METHODS: We used array comparative genomic hybridization to identify recurrent genomic alterations and screened the candidate targets of selected amplification regions by quantitative and semi-quantitative RT-PCR. RESULTS: Thirty-four gains and 16 losses occurred in more than 50 % of ESCCs. High-level amplifications at 7p11.2, 8p12, 8q24.21, 11q13.2-q13.3, 12p11.21, 12q12 and homozygous deletions at 2q22.1, 8p23.1-p21.2, 9p21.3 and 14q11.2 were also identified. 11q13.2 was a frequent amplification region, in which five genes including CHKA, GAL, KIAA1394, LRP5 and PTPRCAP were overexpressed in tumor tissues than paracancerous normal tissues. The expression of ALG3 at 3q27.1 was higher in ESCCs, especially in patients with lymph node metastasis. CONCLUSIONS: Target gene identification of amplifications or homozygous deletions will help to reveal the mechanism of tumor formation and explore new therapy method.

First Report of Fusarium oxysporum and F. solani Causing Fusarium Dry Rot of Carrot in China.

Carrot (Daucus carota L.) is an economically important vegetable crop in China. In August 2008, a disease was observed on carrot in Inner Mongolia. The symptoms appeared as dry rot lesions on root surface, expressing light brown cankers with defined rounded or irregular shapes (1,3). The average disease incidence was up to 80% in Tuo Ke Tuo County. The disease has been a serious problem in these two counties since then, especially where consecutive carrot cropping was practiced. Carrot roots with typical dry rot symptoms were washed with tap water. Root tissues near the margin of necrotic lesions were excised, surface sterilized with 1% NaOCl for 3 min, and rinsed with sterile distilled water three times. The disinfected tissue was placed on potato dextrose agar (PDA) in a petri dish. Plates were incubated at 25 ± 1°C in the dark for 4 days. Fusarium single spore isolates were obtained from characteristic colonies (1). Three isolates (CF1, CF2, and CF3) were used for further study. The isolates were identified as Fusarium spp. on the basis of microscopic morphology on PDA. CF1 produced pink pigment, abundant falciform macroconidia of 14.7 to 38.2 × 4.5 to 5.7 µm with 2 to 3 septates, and elliptic microconidia of 7.5 to 15.1 × 3.3 to 5.4 µm with none or one septate. CF2 and CF3 produced light blue pigment, abundant falciform macroconidia of 16.4 to 34.4 × 4.0 to 6.1 µm with 2 to 3 septates, and elliptic microconidia of 6.7 to 10.7 × 3.0 to 4.9 µm with none or one septate. They were further identified and confirmed by PCR. The PCR involved amplifying the internal transcribed spacer (ITS) region of ribosomal DNA using genomic DNA as the template with universal primers ITS1 and ITS4 (2). The PCR products were sequenced. BLAST analysis of these sequences against the GenBank database determined the taxonomy of the isolates. The sequence of CF1 was 99% identical to F. oxysporum (Accession No. KC594035); sequences of CF2 and CF3 were 99% identical to F. solani (KC215123). To confirm the pathogenicity of the isolates, mature carrot roots (cv. Hong Ying 2) were inoculated with mycelial plugs (5 mm in diameter) cut from the margin of actively growing colonies on PDA plates. One mycelial plug was placed on each carrot root, with the mycelial side facing the root. PDA plugs were used for controls. Each treatment had five replicates. The inoculated roots were incubated in a humid chamber (90% RH) at 25°C. Four days after incubation, mycelia of the isolates developed and covered most of the surface of carrot roots, and brown rot lesions were observed on all inoculated roots, while the controls remained symptomless. This experiment was repeated. In another trial, carrot seeds (cv. Hong Ying 2) were sown in sterilized soil in pots (30 × 25 cm opening) with 15 seeds per pot. The soil was infested with either CF1, CF2, or CF3 by adding spore suspension to make the final concentration of 1 × 104 CFU/g soil. Plants grown in non-infested soil served as controls. There were three replicates per treatment. All the treated pots were placed in a field. After 13 weeks, the same symptoms of dry rot were observed as previously described. No symptoms were observed on the control plants. The trial was repeated. Symptomatic tissues from the inoculated roots were sampled and the pathogen was re-isolated, and identified using PCR. To our knowledge, this is the first report of F. oxysporum and F. solani causing dry rot of carrot in China. References: (1) H. Abe et al. Annual Report of the Society of Plant Protection of North Japan, 48:106-108, 1997. (2) X. Lu. Plant Dis. 97:991, 2013. (3) A. F. Sherf and A. MacNab. Pages 138-139 in: Vegetable Diseases and Their Control. John Wiley & Sons, Inc., 1986.

Distribution of Lettuce Big-Vein Incidence Under Three Irrigation Systems.

Spatial patterns of lettuce big-vein (LBV) incidence under furrow, sprinkler, and subsurface drip irrigation systems were determined. Because LBV pathogen is a virus and is vectored by the soilborne chytrid Olpidium brassicae, different irrigation systems likely affect the movement of the vector and were hypothesized to result in different distribution patterns and levels of the disease. Lettuce plants were mapped by recording the location of each LBV-infected or healthy plant in arbitrarily selected plots of sizes 16 by 30, 20 by 30, and 18 by 50 m in Salinas, Gonzales, and Santa Maria in California. Data were arrayed into different quadrat sizes by rearrangement, and disease incidence was calculated for each quadrat. Frequency distribution analysis and spatial autocorrelation analyses were performed on this data. LBV incidence was aggregated in all furrow-irrigated fields, four of five subsurface drip-irrigated fields, and two of three sprinkler-irrigated fields. The remaining fields had a random distribution. As the quadrat size increased, index of aggregation decreased, and vice versa. In fields under sprinkler irrigation, regardless of whether the spatial pattern of LBV was random or aggregated, no directional orientation occurred. However, under furrow or subsurface drip irrigation, the aggregation mostly occurred across the rows. Although irrigation type influenced LBV distribution pattern and incidence in lettuce fields, the differential effects of irrigation type on vector O. brassicae could not be discerned in this study. The sprinkler irrigation practiced in lettuce production until thinning may influence the vector distribution and the subsequent irrigation methods adapted for the remainder of the season in individual fields may play a significant role in disease incidence.

Complete mitochondrial genomes of the Bright Sunbeam Curetis bulis and the Small Copper Lycaena phlaeas (Lepidoptera: Lycaenidae) and their phylogenetic implications.

In this study, the complete mitochondrial genomes of Curetis bulis and Lycaena phlaeas were determined and analyzed. The circular genomes are 15,162 bp long for C. bulis and 15,280 bp long for L. phlaeas, with a total A+T content of 82.6 and 83.1%, respectively. Both mitogenomes contain 37 genes, and their gene orders are similar to those of other lepidopterans. All protein-coding genes (PCGs) are initiated by ATN codons, except for cox1, which is started with the CGA codon; all PCGs terminate in the typical stop codon TAA, except for cox1, cox2, and nad4, which end with a single T. The codons TTA (Leu), ATT (Ile), TTT (Phe), ATA (Met), and AAT (Asn) appear the most frequently. Both of the mitogenome A+T-rich regions harbor the motif ATAGA, followed by a 19-bp poly(T) stretch, with C. bulis containing a microsatellite-like (AT)5 element next to the ATTTA motif, and L. phlaeas containing a microsatellite-like (TA)6 (AT) element next to the ATTTA motif. The phylogenetic trees of the 17 representative butterfly species, including the two species of this study, were reconstructed with the maximum likelihood and Bayesian inference methods, based on the 13 PCG nucleotide sequence data. The results of the phylogenetic analyses strongly supported the relationships of ((((Lycaenidae + Pieridae) + Nymphalidae) + Hesperiidae) + Papilionidae), which was markedly different from the traditional morphological view of the Lycaenidae and Nymphalidae considered to be sisters of each other.

First Report of Pythium recalcitrans Causing Cavity Spot of Carrot in Michigan.

During a survey of carrot (Daucus carota L.) cavity spot in Michigan in September 2010, carrot roots with typical cavity spot symptoms were collected from production fields in Fremont Co. The lesions were excised from infected roots, surface-disinfested with 0.62% NaClO for 3 min, rinsed in sterilized, distilled water three times, cut into 0.5 cm long pieces, and then plated on water agar (WA) amended with carbendazim (10 µg/ml), ampicillin (50 µg/ml), rifampicin (50 µg/ml), and pentachloronitrobenzene (10 µg/ml) (cumulatively referred to as CARP). Plates were incubated at 22 ± 1°C in the dark for 3 days. Pure cultures of the isolates were obtained by transferring a single hyphal tip of each colony to potato dextrose agar (PDA) amended with CARP. Among the 33 isolates obtained, M2-05 was identified as a Pythium sp. that differed from the known cavity spot pathogens of carrot. The isolate has spherical hyphal swellings but no other distinguishing morphological characteristics. M2-05 was further classified by analyzing the partial sequences of four genes: the internal transcribed spacer (ITS) region of ribosomal DNA, beta-tubulin (ß-tub), cytochrome c oxidase subunit 2 (cox 2), and NADH dehydrogenase subunit 1 (nadh 1) (1,3). A BLAST search of these sequences for M2-05 was conducted using the nucleotide database of GenBank, resulting in 100% similarity to all four sequenced genes of P. recalcitrans (2). The DNA sequences of M2-05 were deposited in GenBank (JQ734349, JQ734229, JQ734289, and JQ734409 for ITS, ß-tub, cox 2, and nadh 1, respectively). Koch's postulates were conducted by inoculating mature carrot roots (cv. Nantindo) with mycelial plugs (4 mm in diameter) cut from the margin of actively growing colonies of M2-05 on PDA plates. Two mycelial plugs were placed on each carrot root at 3-cm intervals, with the mycelial side facing the root; and two non-colonized agar plugs were placed similarly for the non-inoculated control treatment. In comparison, carrot roots also were inoculated with an isolate of each of P. sulcatum and P. violae using the same method. There were four replicate carrot roots inoculated for each isolate and each of the control treatments. The inoculated roots were placed on a plastic grid (7 mm in height) in a closed plastic container, with moist paper towels underneath the grids. The container was incubated in the dark at 22 ± 1°C, and the roots were sprayed gently daily with sterilized, distilled water to maintain high humidity. Brown lesions were observed on all inoculated carrot roots 5 days after inoculation. The lesions measured 0.68 ± 0.48, 1.20 ± 0.71, and 0.56 ± 0.31 mm2 averaged over all eight lesions for the isolates of P. recalcitrans, P. sulcatum, and P. violae, respectively. Symptomatic tissues from the inoculated roots were excised and incubated on WA-CARP plates, and the culture from each lesion confirmed as the isolates inoculated using the same molecular methods described above. The carrot tissue under the control agar plugs remained symptomless, and no Pythium was recovered from the control roots. P. recalcitrans was described in 2008 as infecting roots of Beta vulgaris and Vitis vinifera (2). To our knowledge, this is the first published report of P. recalcitrans naturally infecting carrot roots, not only in Michigan, but anywhere in the world. References: (1) L. Kroon et al. Fungal. Genet. Biol. 41:766, 2004. (2) E. Moralejo et al. Mycologia 100:310, 2008. (3) N. O. Villa et al. Mycologia 98:410, 2006.

Characterizing a novel strain of Bacillus amyloliquefaciens BAC03 for potential biological control application.

AIMS: To identify and characterize a bacterial strain BAC03, evaluate its biological control activity against potato common scab (Streptomyces spp.) and characterize an antimicrobial substance produced by BAC03. METHODS AND RESULTS: Bacterial strain BAC03, isolated from potato common scab suppressive soil, was identified as Bacillus amyloliquefaciens by analysing sequences of fragments of the recA, recN, cheA and gyrA genes. BAC03 displayed an antagonistic activity against Streptomyces spp. on agar plates using a co-culture method. In glasshouse assays, BAC03 applied in potting mix significantly reduced common scab severity (P < 0·05) and potentially increased the growth of potato plants (P < 0·05). An antimicrobial substance extracted from BAC03 by ammonium sulfate precipitation was identified as an LCI protein using liquid chromatography-mass spectrometry. The antimicrobial activity of either a BAC03 liquid culture or the ammonium sulfate precipitate fraction was stable under a wide range of temperatures, and pH levels, as well as following incubation with several chemicals, but was reduced by all proteinases tested. CONCLUSIONS: Bacillus amyloliquefaciens strain BAC03 displayed a strong antimicrobial activity, that is, the suppression of potato common scab, and may potentially enhance the plant growth. LCI protein is associated with some of the antimicrobial activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial strain BAC03 has the potential to be developed as a commercial biological control agent for potato common scab.

Fusarium spp. Causing Dry Rot of Seed Potato Tubers in Michigan and Their Sensitivity to Fungicides.

A survey of seed potato tubers in Michigan seed production storage facilities was carried out during 2009 and 2010. Fusarium spp. associated with tuber dry rot symptoms were identified to species and tested for sensitivity to difenoconazole, fludioxonil, and thiabendazole. Symptomatic tubers (n = 370) were collected from a total of 51 seed lots, from which 228 isolates of Fusarium were recovered and identified to 11 species. Fusarium oxysporum was the most commonly isolated species (30.3%), followed by F. equiseti (19.3%). F. sambucinum and F. avenaceum were third most prevalent (each at 13.6%). Less prevalent species (each at 4 to 10%) included F. cerealis, F. solani, and F. acuminatum; and species present at ≤3% included F. sporotrichioides, F. torulosum, F. tricinctum, and F. graminearum. Representative isolates of all species were pathogenic when inoculated onto seed tubers ('Dark Red Norland'). Isolates of F. sambucinum were the most virulent. All 228 isolates of Fusarium were sensitive to difenoconazole (effective fungicide concentration that caused 50% inhibition of mycelial growth [EC50] < 5 mg/liter). Insensitivity to fludioxonil (EC50 > 100 mg/liter) was detected only for F. sambucinum and F. oxysporum isolates at 8.9 and 20.4%, respectively. All isolates were sensitive to thiabendazole (EC50 < 5 mg/liter), except for those of F. sambucinum (EC50 > 100 mg/liter). Therefore, knowledge of what Fusarium spp. are present in seed potato storage facilities in Michigan may be important if using fludioxonil or thiabendazole for seed piece treatment but not when using difenoconazole.

First Report of Streptomyces stelliscabiei Causing Potato Common Scab in Michigan.

Potato (Solanum tuberosum L.) common scab can be caused by multiple Streptomyces spp., with S. scabies as a predominant species (2,3). However, according to our survey in August 2007, many symptomatic potato tubers did not have S. scabies in Michigan. To identify the pathogen, potato tubers with scab symptoms were collected from two fields in Michigan, and Streptomyces spp. were isolated using Streptomyces selective medium (STR) (2). Pure cultures of the isolates were obtained by transferring single colonies and incubation at 28°C on STR. Three isolates, designated HER21, HER24, and HER26, were identified as Streptomyces stelliscabiei based on morphological and physiological characterization (1). Bacterial cultures were prepared in liquid yeast malt extract at 28°C on an incubator shaker at 150 rpm. Genomic DNA was extracted from the cultures and used as a template for PCR with species-specific primers for Streptomyces spp. (4). The isolates gave a positive PCR reaction with primers Stel3 and T2st2 for S. stelliscabiei, but negative for any other Streptomyces spp. reported as pathogenic to potato. The 16S rRNA genes were amplified using primers previously reported (4) and amplicons were sequenced and submitted to GenBank (Accession Nos. HM018115, HM018116, and HM018117 for the three isolates, respectively). BLAST analysis of these sequences against the NCBI GenBank database determined these sequences to have 99 to 100% sequence identity with S. stelliscabiei sequences such as Accession No. FJ546728 (4). These isolates were all confirmed by PCR, using the same conditions described above, to have txtAB, nec1, and tomA genes (4), which are associated with pathogenicity of scab-causing Streptomyces spp. To complete Koch's postulates, cell suspensions of the isolates were mixed in vermiculate media (3) at a final concentration of 106 colony-forming units/ml, which were used as inocula. Potato (cv Snowden) tubers were incubated in sterilized potting mix in a growth chamber at 25°C until the seed germinated. Each potato seedling was transferred to a new pot in the greenhouse. Two weeks later, the potting mix was infested with the bacterial spore suspensions of either HER21, HER24, or HER26, with five pots per isolate. Potting mix with only media or media with S. scabies isolate 49173 were used as negative and positive controls, respectively. Three months later, potato tubers were harvested and evaluated for scab symptoms (3). The experiment was done twice. Potato tubers inoculated with either S. stelliscabiei or S. scabies exhibited superficial, raised, or pitted scabby symptoms, and no symptoms were observed on tubers grown in noninfested potting mix. Disease index values from the combined trials averaged 0, 37.8, 26.5, 11.1, and 30.5% for negative control and isolates HER21, HER24, HER26, and 49173, respectively. The pathogen was reisolated from the lesions and confirmed identical to the original isolate by DNA sequences. To our knowledge, this is the first report of S. stelliscabiei causing potato common scab in Michigan (4). References: (1) K. Bouchek-Mechiche et al. Int. J. Syst. Evol. Microbiol. 50:91, 2000. (2) Conn et al. Plant Dis. 82:631, 1998. (3) Hao et al. Plant Dis. 93:1329, 2009. (4) L. A. Wanner. Am. J. Potato Res. 86:247, 2009.

Calreticulin promotes cell motility and enhances resistance to anoikis through STAT3-CTTN-Akt pathway in esophageal squamous cell carcinoma.

We have shown earlier that overexpression of Calreticulin (CRT) contributed to a poor prognosis for patients with esophageal squamous cell carcinoma (ESCC). Here, we have shown an important role of CRT in tumorigenesis through enhancing cell motility and anoikis resistance. SiRNA-mediated knockdown of CRT caused impaired cell migration, invasion and resistance to anoikis. Notably, CRT downregulation decreased the expression of Cortactin (CTTN), which has been previously reported as a candidate oncogene associated with anoikis through the PI3K-Akt pathway. In addition, Akt phosphorylation was abolished after CRT downregulation and its activation can be refreshed by CRT upregulation, suggesting that CRT-enhanced cell resistance to anoikis through the CRT-CTTN-PI3K-Akt pathway. Moreover, the CTTN mRNA level was decreased in CRT-siRNA cells, coupled with the inactivation of STAT3. Expression of both CTTN and p-STAT3 was reduced in tumor cells following incubation with the JAK-specific inhibitor, AG490. Chromatin immunoprecipitation assay showed direct binding of p-STAT3 to the conservative STAT3-binding sequences in CTTN promoter. Furthermore, overexpression of CTTN in CRT-downregulated ESCC cells restored its motility and resistance to anoikis. This study not only reveals a role of CRT in motility promotion and anoikis resistance in ESCC cells, but also identifies CRT as an upstream regulator in the CRT-STAT3-CTTN-Akt pathway.

Characterization of a New Streptomyces Strain, DS3024, That Causes Potato Common Scab.

A novel strain of Streptomyces (named DS3024) was isolated from a potato field in Michigan in 2006. The taxonomy of the organism was determined by morphology, biochemistry, and genetic analysis. Analysis of the 16S ribosomal RNA gene sequence indicated that the organism was most similar to an isolate of Streptomyces sp., ME02-6979.3a, which is not pathogenic to potato tubers but is distinct from other known pathogenic Streptomyces spp. Strain DS3024 has genes that encode thaxtomin synthetase (txtAB), which is required for pathogenicity and virulence, and tomatinase (tomA), which is a common marker for many pathogenic Streptomyces spp. However, the nec1 gene (associated with virulence in most pathogenic Streptomyces spp.) was not detected. The new strain was capable of growth at pH 4.5, caused necrosis on potato tuber slices, and produced thaxtomin A. In greenhouse experiments, DS3024 caused scab symptoms on potato tubers similar to those caused by Streptomyces scabies on tubers of potato cv. Atlantic, which is scab susceptible. We propose that DS3024 is a new strain of Streptomyces capable of causing common scab on potato tubers. The prevalence of this strain of Streptomyces in potato-producing areas in the north-central United States has not been determined.

Efficacy of Germination Stimulants of Sclerotia of Sclerotium cepivorum for Management of White Rot of Garlic.

The ability of soil-applied garlic powder and diallyl disulfide to stimulate germination of sclerotia of Sclerotium cepivorum, the cause of white rot of onion and garlic, was evaluated in four field trials. Because sclerotia germinate in response to exudation of specific volatile sulfides and thiols from allium roots, sulfides applied to the ground in the absence of an allium crop cause death of the sclerotia after they germinate and exhaust nutrient reserves. In this study, garlic powder and a synthetic garlic oil, diallyl disulfide, were incorporated into the soil in commercial fields naturally infested with S. cepivorum. Methyl bromide was included as a chemical control. Within 3 months after treatment, over 90% of the sclerotia died in the plots treated with the germinationstimulants, which was similar to the reduction of viable sclerotia achieved with an application of methyl bromide. The degree of sclerotial mortality in plots treated with garlic powder at 112 kg/ha or more was almost equal to that achieved by diallyl disulfide at 0.5 ml/m2 or methyl bromide at 448 kg/ha. Despite the efficacy of the stimulants and methyl bromide to reduce populations of sclerotia, the pathogen caused substantial root rot and yield losses in subsequent garlic crops planted about a year after soil treatment. However, germination stimulants have utility because the reduction of the vast majority of sclerotia in a field reduces the risk of spread of the pathogen to neighboring fields.