Efficient gene targeting in soybean using Ochrobactrum haywardense-mediated delivery of a marker-free donor template.

Gene targeting (GT) for precise gene insertion or swap into pre-defined genomic location has been a bottleneck for expedited soybean precision breeding. We report a robust selectable marker-free GT system in soybean, one of the most economically important crops. An efficient Oh H1-8 (Ochrobactrum haywardense H1-8)-mediated embryonic axis transformation method was used for the delivery of CRISPR-Cas9 components and donor template to regenerate T0 plants 6-8 weeks after transformation. This approach generated up to 3.4% targeted insertion of the donor sequence into the target locus in T0 plants, with ∼ 90% mutation rate observed at the genomic target site. The GT was demonstrated in two genomic sites using two different donor DNA templates without the need for a selectable marker within the template. High-resolution Southern-by-Sequencing analysis identified T1 plants with precise targeted insertion and without unintended plasmid DNA. Unlike previous low-frequency GT reports in soybean that involved particle bombardment-mediated delivery and extensive selection, the method described here is fast, efficient, reproducible, does not require a selectable marker within the donor DNA, and generates nonchimeric plants with heritable GT.

Development of an efficient marker-free soybean transformation method using the novel bacterium Ochrobactrum haywardense H1.

We have discovered a novel bacterium, Ochrobactrum haywardense H1 (Oh H1), which is capable of efficient plant transformation. Ochrobactrum is a new host for Agrobacterium-derived vir and T-DNA-mediated transformation. Oh H1 is a unique, non-phytopathogenic species, categorized as a BSL-1 organism. We engineered Oh H1 with repurposed Agrobacterium virulence machinery and demonstrated Oh H1 can transform numerous dicot species and at least one monocot, sorghum. We generated a cysteine auxotrophic Oh H1-8 strain containing a binary vector system. Oh H1-8 produced transgenic soybean plants with an efficiency 1.6 times that of Agrobacterium strain AGL1 and 2.9 times that of LBA4404Thy-. Oh H1-8 successfully transformed several elite Corteva soybean varieties with T0 transformation frequency up to 35%. In addition to higher transformation efficiencies, Oh H1-8 generated high-quality, transgenic events with single-copy, plasmid backbone-free insertion at frequencies higher than AGL1. The SpcN selectable marker gene is excised using a heat shock-inducible excision system resulting in marker-free transgenic events. Approximately, 24.5% of the regenerated plants contained only a single copy of the transgene and contained no vector backbone. There were no statistically significant differences in yield comparing T3 null-segregant lines to wild-type controls. We have demonstrated that Oh H1-8, combined with spectinomycin selection, is an efficient, rapid, marker-free and yield-neutral transformation system for elite soybean.

Non-hypervascular Hypointense Nodules on Hepatocyte Phase Gadoxetic Acid-Enhanced MR Images: Transformation of MR Hepatobiliary Hypointense Nodules into Hypervascular Hepatocellular Carcinomas.

BACKGROUND/AIMS: The annual risk of transformation of non-hypervascular magnetic resonance (MR) hepatobiliary phase imaging (HBPI) hypointense nodules into hypervascular hepatocellular carcinomas (HCCs) was evaluated. METHODS: Cirrhotic patients with non-hypervascular HBPI hypointense nodules were retrospectively analyzed if they were diagnosed as HCC and MR followed up longer than 1 year during the period from January 2010 to October 2016 with regular intervals of 3 to 6 months. Risk factors for transformation into hypervascular HCCs were analyzed using the Cox proportional hazard model. RESULTS: Among the 103 non-hypervascular HBPI hypointense nodules meeting the inclusion criteria, transformation into hypervascular HCCs occurred in 44 tumors (42.7%). The median follow-up period was 24 months. Multivariate analysis revealed that hyperintensity on T2-weighted images (T2WI) and diffusion-weighted images (DWI) were the two independent predictors of transformation into hypervascular HCCs (p=0.036 and p=0.041, respectively). Most tumors with hyperintensity on T2WI or DWI on the initial or follow-up MR were transformed into hypervascular HCCs within the first year. Among the 22 nodules (21.3%) showing a new change in dynamic phases during follow-up, 14 nodules (13.6%) showed malignant transformations. CONCLUSIONS: The transformation rates of HBPI hypointense nodules into hypervascular HCCs could be predicted according to the initial or serial MRI findings.

Anti-cancer activity of Ginger (Zingiber officinale) leaf through the expression of activating transcription factor 3 in human colorectal cancer cells.

BACKGROUND: Ginger leaf (GL) has long been used as a vegetable, tea and herbal medicine. However, its pharmacological properties are still poorly understood. Thus, we performed in vitro studies to evaluate anti-cancer properties of ginger leaf and then elucidate the potential mechanisms involved. METHODS: Cell viability was measured by MTT assay. ATF3 expression level was evaluated by Western blot or RT-PCR and ATF3 transcriptional activity was determined using a dual-luciferase assay kit after the transfection of ATF3 promoter constructs. In addition, ATF3-dependent apoptosis was evaluated by Western blot after ATF3 knockdown using ATF3 siRNA. RESULTS: Exposure of GL to human colorectal cancer cells (HCT116, SW480 and LoVo cells) reduced the cell viability and induced apoptosis in a dose-dependent manner. In addition, GL reduced cell viability in MCF-7, MDA-MB-231 and HepG-2 cells. ATF3 knockdown attenuated GL-mediated apoptosis. GL increased activating transcription factor 3 (ATF3) expressions in both protein and mRNA level and activated ATF3 promoter activity, indicating transcriptional activation of ATF3 gene by GL. In addition, our data showed that GL-responsible sites might be between -318 and -85 region of the ATF3 promoter. We also observed that ERK1/2 inhibition by PD98059 attenuated GL-mediated ATF3 expression but not p38 inhibition by SB203580, indicating ERK1/2 pathway implicated in GL-induced ATF3 activation. CONCLUSIONS: These findings suggest that the reduction of cell viability and apoptosis by GL may be a result of ATF3 promoter activation and subsequent increase of ATF3 expression through ERK1/2 activation in human colorectal cancer cells.

Detecting hepatocellular carcinoma in gadoxetic-acid-enhanced hepatobiliary-phase MR imaging at 3T: comparing high and low flip angle.

PURPOSE: We evaluated the diagnostic performance of fat-suppressed 3D T1-weighted gradient-echo magnetic resonance imaging (MRI) sequences for the hepatobiliary phase of gadoxetic-acid-enhanced liver MRI between low and high flip angle (FA) at 3T. MATERIALS AND METHODS: Forty-six patients with 62 HCCs were enrolled in this retrospective study from among 267 consecutive patients who underwent 3T MRI with low and high FA (10° and 25°) sequences at the hepatobiliary phase. A radiologist measured signal intensities and standard deviations (SD) of lesion, liver, and spleen and calculated signal-to-noise ratio, liver-spleen contrast, and liver-lesion contrast. Two reviewers assessed both image sequences using a five-point rating scale focusing on detecting hypointense lesions. RESULTS: The high FA sequence showed significantly higher liver-spleen and liver-lesion contrast compared with those of low FA (p < 0.05, p < 0.05, respectively). Per-lesion sensitivities of high FA were higher than those of low FA (p < 0.05, p < 0.05, respectively), and per-person sensitivities were elevated on high FA (p < 0.05 in a reviewer). There were statistically significant differences for detecting HCCs larger than 1 cm (p < 0.05, p < 0.05, respectively). CONCLUSION: Increasing FA in T1-weighted hepatobiliary-phase liver MRI may help in detecting HCC at 3T.

Correlations of dynamic contrast-enhanced magnetic resonance imaging with morphologic, angiogenic, and molecular prognostic factors in rectal cancer.

PURPOSE: To investigate the correlations between parameters of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and prognostic factors in rectal cancer. MATERIALS AND METHODS: We studied 29 patients with rectal cancer who underwent gadolinium contrast-enhanced, T1-weighted DCE-MRI with a three Tesla scanner prior to surgery. Signal intensity on DCE-MRI was independently measured by two observers to examine reproducibility. A time-signal intensity curve was generated, from which four semiquantitative parameters were calculated: steepest slope (SLP), time to peak (Tp), relative enhancement during a rapid rise (Erise), and maximal enhancement (Emax). Morphologic prognostic factors including T stage, N stage, and histologic grade were identified. Tumor angiogenesis was evaluated in terms of microvessel count (MVC) and microvessel area (MVA) by morphometric study. As molecular factors, the mutation status of the K-ras oncogene and microsatellite instability were assessed. DCE-MRI parameters were correlated with each prognostic factor using bivariate correlation analysis. A p-value of <0.05 was considered significant. RESULTS: Erise was significantly correlated with N stage (r=-0.387 and -0.393, respectively, for two independent data), and Tp was significantly correlated with histologic grade (r=0.466 and 0.489, respectively). MVA was significantly correlated with SLP (r= -0.532 and -0.535, respectively) and Erise (r=-0.511 and -0.446, respectively). MVC was significantly correlated with Emax (r=-0.435 and -0.386, respectively). No significant correlations were found between DCE-MRI parameters and T stage, K-ras mutation, or microsatellite instability. CONCLUSION: DCE-MRI may provide useful prognostic information in terms of histologic differentiation and angiogenesis in rectal cancer.

Abdominal computed tomography findings of malaria infection with Plasmodium vivax.

Abdominal computed tomography (CT) findings of malaria are not well-known even though malaria is a serious infectious disease. To identify abdominal CT findings, we selected 34 of 405 patients who had a positive peripheral blood smear for Plasmodium vivax and had underwent abdominal CT as the malaria group. We also selected 80 patients who had fever and a negative peripheral blood smear as the control group and 120 healthy people as the normal group. We reviewed and analyzed their medical records and CT findings retrospectively. The mean spleen and liver length were significantly larger in the malaria group and the incidence of splenomegaly, splenic focal low attenuation, and spontaneous splenic rupture were much higher in the malaria group (P < 0.05). Although abdominal CT is not an indispensable tool for diagnosis, these CT findings will help in the diagnosis of malaria in patients with fever.

Findings from CT, MRI, and PET/CT of a primary malignant melanoma arising in a spinal nerve root.

Primary spinal malignant melanoma is an extremely rare condition. We here describe a case of a 71-year-old Asian female presenting with left upper extremity tingling sensation. Computed tomography (CT) showed a homogeneously enhanced mass occupying the left neural foramen at the C6-7 level. Magnetic resonance imaging revealed enhanced mass in intra- and extradural space compressing the spinal cord at this level. It also widened the neural foramen mimicking neurofibroma or schwannoma. Partial resection of the mass was performed. Pathologic diagnosis of the mass was malignant melanoma. Postoperative whole body positron emission tomography/CT scan demonstrated an intense (18)F-FDG uptake at the residual mass site without abnormal uptake at other sites in the body.

Differentiation of benign and malignant solid pseudopapillary neoplasms of the pancreas.

OBJECTIVE: The aim of this study was to investigate differential imaging features between benign and malignant solid pseudopapillary neoplasms (SPN) of the pancreas on computed tomographic and magnetic resonance imagings. METHODS: Between January 2001 and January 2007, we identified 30 patients with confirmed SPN by surgery. The computed tomographic and magnetic resonance images were reviewed by 3 radiologists in consensus. Each tumor was analyzed for the following categories: location of tumor, tumor margin, proportion of solid component, morphology of capsule, growth pattern, calcification, and presence of upstream pancreatic ductal dilatation. RESULTS: Benign SPN usually had oval/round or smoothly lobulated margins, and malignant SPN more commonly had focal lobulated margins (P = 0.027). Presence of complete encapsulation was more frequently seen in benign SPN, whereas focal discontinuity of capsule was more commonly seen in malignant SPN (P = 0.005). There was no statistical difference between benign and malignant tumors in other imaging findings. CONCLUSIONS: A focal lobulated margin and a focal discontinuity of the capsule may suggest malignant SPN, whereas a round or smoothly lobulated margin and a complete encapsulation were more commonly seen in benign SPN.

Varying appearances of cholangiocarcinoma: radiologic-pathologic correlation.

Intrahepatic cholangiocarcinoma is the second most common primary hepatic tumor. Various risk factors have been reported for intrahepatic cholangiocarcinoma, and the radiologic and pathologic findings of this disease entity may differ depending on the underlying risk factors. Intrahepatic cholangiocarcinoma can be classified into three types on the basis of gross morphologic features: mass-forming (the most common), periductal infiltrating, and intraductal growth. At computed tomography (CT), mass-forming intrahepatic cholangiocarcinoma usually appears as a homogeneous low-attenuation mass with irregular peripheral enhancement and can be accompanied by capsular retraction, satellite nodules, and peripheral intrahepatic duct dilatation. Periductal infiltrating cholangiocarcinoma is characterized by growth along the dilated or narrowed bile duct without mass formation. At CT and magnetic resonance imaging, diffuse periductal thickening and increased enhancement can be seen with a dilated or irregularly narrowed intrahepatic duct. Intraductal cholangiocarcinoma may manifest with various imaging patterns, including diffuse and marked ductectasia either with or without a grossly visible papillary mass, an intraductal polypoid mass within localized ductal dilatation, intraductal castlike lesions within a mildly dilated duct, and a focal stricture-like lesion with mild proximal ductal dilatation. Awareness of the underlying risk factors and morphologic characteristics of intrahepatic cholangiocarcinoma is important for accurate diagnosis and for differentiation from other hepatic tumorous and nontumorous lesions.

Leiomyosarcoma of the ovarian vein: a case report with radiological findings.

Leiomyosarcomas of the ovarian vein are very rare. Four cases have been reported in the English language clinical literature. We present a case of leiomyosarcomas where the use of multi-detector CT had a substantial role in the establishment of the preoperative diagnosis. The radiological images as well as intraoperative features are illustrated. We also discuss the radiological findings of the ovarian vein leiomyosarcoma in comparison with those of other venous or retroperitoneal leiomyosarcomas. We expect that the use of multi-detector CT will be the choice for the diagnostic work-up of vascular leiomyosarcomas.

Fitz-Hugh-Curtis syndrome: CT findings of three cases.

Clinical manifestations and computed tomography (CT) findings of Fitz-Hugh-Curtis syndrome (FHCS) are relatively well stabilized as right upper quadrant abdominal pain and hepatic capsular enhancement because of perihepatitis associated with pelvic inflammatory disease caused by N. gonorrhoeae and C. trachomatis. We encountered three patients with serial FHCS associated with pelvic inflammatory disease, who visited the emergency room with right upper quadrant abdominal pain. Abdominal CT revealed hepatic capsular or pericapsular enhancement along the anterior surface of the liver on the arterial phase. Recently, multi-detector CT has evolved as the first-line imaging modality of acute abdomen at the emergency room; we reemphasized the importance of the CT findings of this syndrome for differential diagnosis of right upper quadrant abdominal pain in sexually active young women. Physicians at the emergency room acknowledge the syndrome and should perform dynamic abdominopelvic CT including the arterial phase.

Evolution of a microbial acetyltransferase for modification of glyphosate: a novel tolerance strategy.

N-Acetylation is a modification of glyphosate that could potentially be used in transgenic crops, given a suitable acetyltransferase. Weak enzymatic activity (k(cat) = 5 min(-1), K(M) = 1 mM) for N-acetylation of glyphosate was discovered in several strains of Bacillus licheniformis (Weigmann) Chester by screening a microbial collection with a mass spectrometric assay. The parental enzyme conferred no tolerance to glyphosate in any host when expressed as a transgene. Eleven iterations of DNA shuffling resulted in a 7000-fold improvement in catalytic efficiency (k(cat)/K(M)), sufficient for conferring robust tolerance to field rates of glyphosate in transgenic tobacco and maize. In terms of k(cat)/K(M), the native enzyme exhibited weak activity (4-450% of that with glyphosate) with seven of the common amino acids. Evolution of the enzyme towards an improved k(cat)/K(M) for glyphosate resulted in increased activity toward aspartate (40-fold improved k(cat)), but activity with serine and phosphoserine almost completely vanished. No activity was observed among a broad sampling of nucleotides and antibiotics. Improved catalysis with glyphosate coincided with increased thermal stability.

Discovery and directed evolution of a glyphosate tolerance gene.

The herbicide glyphosate is effectively detoxified by N-acetylation. We screened a collection of microbial isolates and discovered enzymes exhibiting glyphosate N-acetyltransferase (GAT) activity. Kinetic properties of the discovered enzymes were insufficient to confer glyphosate tolerance to transgenic organisms. Eleven iterations of DNA shuffling improved enzyme efficiency by nearly four orders of magnitude from 0.87 mM-1 min-1 to 8320 mM-1 min-1. From the fifth iteration and beyond, GAT enzymes conferred increasing glyphosate tolerance to Escherichia coli, Arabidopsis, tobacco, and maize. Glyphosate acetylation provides an alternative strategy for supporting glyphosate use on crops.