Significant enhancement of fatty acid composition in seeds of the allohexaploid, Camelina sativa, using CRISPR/Cas9 gene editing.

The CRISPR/Cas9 nuclease system is a powerful and flexible tool for genome editing, and novel applications of this system are being developed rapidly. Here, we used CRISPR/Cas9 to target the FAD2 gene in Arabidopsis thaliana and in the closely related emerging oil seed plant, Camelina sativa, with the goal of improving seed oil composition. We successfully obtained Camelina seeds in which oleic acid content was increased from 16% to over 50% of the fatty acid composition. These increases were associated with significant decreases in the less desirable polyunsaturated fatty acids, linoleic acid (i.e. a decrease from ~16% to <4%) and linolenic acid (a decrease from ~35% to <10%). These changes result in oils that are superior on multiple levels: they are healthier, more oxidatively stable and better suited for production of certain commercial chemicals, including biofuels. As expected, A. thaliana T2 and T3 generation seeds exhibiting these types of altered fatty acid profiles were homozygous for disrupted FAD2 alleles. In the allohexaploid, Camelina, guide RNAs were designed that simultaneously targeted all three homoeologous FAD2 genes. This strategy that significantly enhanced oil composition in T3 and T4 generation Camelina seeds was associated with a combination of germ-line mutations and somatic cell mutations in FAD2 genes in each of the three Camelina subgenomes.

A small-scale, inexpensive method for detecting formaldehyde or methanol in biochemical reactions containing interfering substances.

A simple, inexpensive microdistillation device is described for capturing methanol or formaldehyde as end products of biochemical reactions or in environmental samples. We demonstrate that the microdistillation protocol, coupled with the use of alcohol oxidase and the formaldehyde-sensitive reagent Purpald (4-amino-3-hydrazino-5-mercapto-1,2,4-triazole), serves as a quick and inexpensive alternative to chromatographic and mass spectrometer analyses for determining if formaldehyde or methanol is a product of reactions that contain substances that interfere with the Purpald reaction. These techniques were used to affirm formaldehyde as the end product of the dicamba monooxygenase-catalyzed O-demethylation of the herbicide dicamba (2-methoxy-3,6-dichlorobenzoic acid).

A role for BELLRINGER in cell wall development is supported by loss-of-function phenotypes.

BACKGROUND: Homeodomain transcription factors play critical roles in metazoan development. BELLRINGER (BLR), one such transcription factor, is involved in diverse developmental processes in Arabidopsis, acting in vascular differentiation, phyllotaxy, flower and fruit development. BLR also has a redundant role in meristem maintenance. Cell wall remodelling underpins many of these processes, and BLR has recently been shown to regulate expression of PECTIN METHYL-ESTERASE 5 (PME5), a cell wall modifying enzyme in control of phyllotaxy. We have further explored the role of BLR in plant development by analysing phenotypes and gene expression in a series of plants over-expressing BLR, and generating combinatorial mutants with blr, brevipedicellus (bp), a member of the KNOX1 family of transcription factors that has previously been shown to interact with blr, and the homeodomain transcription factor revoluta (rev), required for radial patterning of the stem. RESULTS: Plants over-expressing BLR exhibited a wide range of phenotypes. Some were defective in cell size and demonstrated misregulation of genes predominantly affecting cell wall development. Other lines with more extreme phenotypes failed to generate lateral organs, consistent with BLR repressing transcription in the shoot apex. Cell wall dynamics are also affected in blr mutant plants, and BLR has previously been shown to regulate vascular development in conjunction with BP. We found that when bp and blr were combined with rev, a set of defects was observed that were distinct from those of bp blr lines. In these triple mutants xylem development was most strikingly affected, resulting in an almost complete lack of vessels and xylem parenchyma with secondary thickening. CONCLUSIONS: Our data support a role for BLR in ordering the shoot apex and, in conjunction with BP and REV, playing a part in determining the composition and organisation of the vascular system. Microarray analysis strongly indicates that the striking vascular phenotypes of blr bp rev triple mutants and plants over-expressing BLR result from the misregulation of a suite of genes, targets of BLR in wild type plants, that determine cell size and structure in the developing vasculature.

Identification of the biomarkers for the prediction of efficacy in first-line chemotherapy of metastatic colorectal cancer patients using SELDI-TOF-MS and artificial neural networks.

BACKGROUND/AIMS: For patients with metastatic colorectal cancer, both FOLFOX regimen and FOLFIRI regimen are considered as first-line choices. There are no clinically useful markers that could predict the response to these regimens respectively. We aimed at identifying serum protein patterns which could predict the efficacy of chemotherapy. METHODOLOGY: Serum from 70 patients diagnosed as metastatic colorectal cancer before first-line chemotherapy were collected and analyzed for protein patterns using ANN analysis of SELDI-TOF-MS. Among the 70 cases, 44 patients received FOLFOX chemotherapy, while the other 26 patients received FOLFIRI chemotherapy. After four cycles of the treatment, RECIST criteria were used to define the responders (R) and non-responders (NR). RESULTS: A potential predicting pattern consisting of 6 biomarkers was identified in the patients receiving FOLFOX chemotherapy. Using this predicting pattern, the responders could be separated from the non-responders with a sensitivity of 92.9% and a specificity of 81.3%. Another potential predicting pattern that consisted of 7 bio-markers was identified in the patients who have received FOLFIRI chemotherapy. The sensitivity and the specificity of this predicting pattern were 92.3% and 92.3% respectively. CONCLUSIONS: Two potential pat-terns for the prediction of efficacy of FOLFOX or FOLFIRI chemotherapy were established in this preliminary study.

TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain.

DNA double-strand breaks enhance homologous recombination in cells and have been exploited for targeted genome editing through use of engineered endonucleases. Here we report the creation and initial characterization of a group of rare-cutting, site-specific DNA nucleases produced by fusion of the restriction enzyme FokI endonuclease domain (FN) with the high-specificity DNA-binding domains of AvrXa7 and PthXo1. AvrXa7 and PthXo1 are members of the transcription activator-like (TAL) effector family whose central repeat units dictate target DNA recognition and can be modularly constructed to create novel DNA specificity. The hybrid FN-AvrXa7, AvrXa7-FN and PthXo1-FN proteins retain both recognition specificity for their target DNA (a 26 bp sequence for AvrXa7 and 24 bp for PthXo1) and the double-stranded DNA cleaving activity of FokI and, thus, are called TAL nucleases (TALNs). With all three TALNs, DNA is cleaved adjacent to the TAL-binding site under optimal conditions in vitro. When expressed in yeast, the TALNs promote DNA homologous recombination of a LacZ gene containing paired AvrXa7 or asymmetric AvrXa7/PthXo1 target sequences. Our results demonstrate the feasibility of creating a tool box of novel TALNs with potential for targeted genome modification in organisms lacking facile mechanisms for targeted gene knockout and homologous recombination.

Identification biomarkers of eutopic endometrium in endometriosis using artificial neural networks and protein fingerprinting.

Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) protein chip array technology was used to detect biomarkers of eutopic endometrium in endometriosis patients. Five potential biomarkers (6,898 m/z, 5,891 m/z, 5,385 m/z, 6,448 m/z, and 5,425 m/z) were found.

Crystal structure of dicamba monooxygenase: a Rieske nonheme oxygenase that catalyzes oxidative demethylation.

Dicamba (3,6-dichloro-2-methoxybenzoic acid) is a widely used herbicide that is efficiently degraded by soil microbes. These microbes use a novel Rieske nonheme oxygenase, dicamba monooxygenase (DMO), to catalyze the oxidative demethylation of dicamba to 3,6-dichlorosalicylic acid (DCSA) and formaldehyde. We have determined the crystal structures of DMO in the free state, bound to its substrate dicamba, and bound to the product DCSA at 2.10-1.75 A resolution. The structures show that the DMO active site uses a combination of extensive hydrogen bonding and steric interactions to correctly orient chlorinated, ortho-substituted benzoic-acid-like substrates for catalysis. Unlike other Rieske aromatic oxygenases, DMO oxygenates the exocyclic methyl group, rather than the aromatic ring, of its substrate. This first crystal structure of a Rieske demethylase shows that the Rieske oxygenase structural scaffold can be co-opted to perform varied types of reactions on xenobiotic substrates.

[Application of serum protein markers to distinguish familial adenomatous polyposis (FAP) and sporadic colorectal adenomas].

OBJECTIVE: To screen out specifically-expressed serum protein markers in familial adenomatous polyposis (FAP) and to establish a serum protein fingerprint diagnostic model for distinguishing FAP from sporadic colorectal adenomas. METHODS: Serum samples were collected from 19 FAP cases and 16 sporadic colorectal adenomas with informed consent. Serum protein fingerprint profiles were detected by SELDI-TOF-MS with CM 10 protein chip to screen out FAP adenoma-related serum protein markers, and support vector machine (SVG) technique was used to establish the diagnostic model to distinguish FAP from sporadic colorectal adenomas. RESULTS: Six differently-expressed protein peaks (P < 0.01) were detected. Among them proteins of 5640, 3160, 4180 and 4290 m/z were highly expressed in FAP adenomas, and proteins of 3940 and 3400 m/z were highly expressed in sporadic colorectal adenomas. The accuracy of diagnostic model established with SVG to distinguish FAP adenomas and sporadic colorectal adenomas was 94.7% and 93.7%, respectively. CONCLUSION: SELDI-TOF-MS can be effectively used to screen out the differentially expressed serum protein markers in FAP adenomas and sporadic colorectal adenomas, and a diagnostic model build by SVG to distinguish them has been successfully established. Therefore, a useful breakthrough point for research on molecular mechanisms of FAP pathogenesis is provided.

Comparison of responses to Mn deficiency between the UK wheat genotypes Maris Butler, Paragon and the Australian wheat genotype C8MM.

Wheat grown in Mn-deficient soil has been widely observed to produce much reduced yields. Breeding for Mn-efficient wheat genotypes adapted to Mn-deficient soils would represent a long-term solution for wheat agronomy. To characterize the physiological basis of Mn efficiency in wheat genotypes would facilitate the breeding programs for producing Mn-efficient wheat. Using a solution culture and a soil culture system in the present study, a Mn-efficient UK wheat genotype Maris Butler and a Mn-inefficient UK wheat genotype Paragon have been compared with a Mn-efficient Australian wheat genotype C8MM in the responses to Mn deficiency in order to characterize the Mn efficiency in these wheat genotypes. Results showed that in solution culture, Maris Butler grown under Mn deficiency had 77% relative dry matter yield of control plants that were grown under Mn sufficiency, whereas C8MM and Paragon had 60% and 58% relative dry matter yield of their respective controls. Results from the soil culture demonstrated that relative dry matter yield remained high for Maris Butler and C8MM (53% and 56%, respectively), whereas the value for Paragon dropped to 33%. In terms of dry matter yield and photosynthetic efficiency, Maris Butler demonstrated Mn efficiency in both solution culture and soil culture, whereas C8MM showed Mn efficiency only in soil culture. Results also demonstrated that under Mn-depleted supply in soil, plants of C8MM had a significantly higher ability in Mn uptake, whereas plants of Maris Butler showed a higher internal Mn use efficiency in comparison with plants of Paragon. Results from the present study indicate that the ability of C8MM to accumulate higher amounts of Mn is the basis of the improved Mn efficiency of this genotype in comparison with Paragon, and in Maris Butler there is a higher internal use of Mn expressed as an improved photosynthetic efficiency in conferring its Mn efficiency. It is suggested that more than one mechanism has arisen in wheat to confer tolerance to Mn deficiency.

Artificial neural networks combined with surface-enhanced laser desorption/ionization mass spectra distinguish endometriosis from healthy population.

Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry protein chip array technology was used to detect the serum proteomic patterns in patients with endometriosis. Four potential biomarkers (8,141 m/z, 6,096 m/z, 5,894 m/z, and 3,269 m/z) were found. This method showed great potential in screening better biomarkers for endometriosis.

Dicamba resistance: enlarging and preserving biotechnology-based weed management strategies.

The advent of biotechnology-derived, herbicide-resistant crops has revolutionized farming practices in many countries. Facile, highly effective, environmentally sound, and profitable weed control methods have been rapidly adopted by crop producers who value the benefits associated with biotechnology-derived weed management traits. But a rapid rise in the populations of several troublesome weeds that are tolerant or resistant to herbicides currently used in conjunction with herbicide-resistant crops may signify that the useful lifetime of these economically important weed management traits will be cut short. We describe the development of soybean and other broadleaf plant species resistant to dicamba, a widely used, inexpensive, and environmentally safe herbicide. The dicamba resistance technology will augment current herbicide resistance technologies and extend their effective lifetime. Attributes of both nuclear- and chloroplast-encoded dicamba resistance genes that affect the potency and expected durability of the herbicide resistance trait are examined.

Quantitative real-time RT-PCR detection for CEA, CK20 and CK19 mRNA in peripheral blood of colorectal cancer patients.

This study is aimed at establishing a sensitive approach to detect disseminated tumor cells in peripheral blood and evaluate its clinical significance. A total of 198 blood samples including 168 from colorectal carcinoma (CRC) patients and 30 from healthy volunteers were examined by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) to evaluate the expression of carcinoembryonic antigen (CEA), cytokeratin 20 (CK20) and cytokeratin 19 (CK19) mRNA. CEA mRNA was detected in 35.8% of patients and 3.3% of controls, CK20 mRNA in 28.3% of patients and 6.7% of controls, and CK19 mRNA in 41.9% of patients and 3.3% of controls. CEA and CK20 mRNA positive ratio increased with the advancing Dukes stages, but there was no significant difference in positive ratio between any two stages (P>0.05). Also, relatively high positive ratio of CEA, CK20 and CK19 mRNA expression was observed in some CRC patients with earlier Dukes stages. A higher positive ratio was obtained when two or three detection markers were combined compared to a single marker. Our study indicates that quantitative real-time RT-PCR detection for CEA, CK20 and CK19 mRNA in peripheral blood is a valuable tool for monitoring early stage dissemination of CRC cells in blood circulation.

[Significance of CK20 mRNA expression in peripheral blood of colorectal cancer patients by real-time fluorescent quantitative RT-PCR].

OBJECTIVE: To detect the expression of cytokeratin 20 (CK20) mRNA in peripheral blood of colorectal carcinoma and to discuss its clinical value. METHODS: Real-time fluorescent quantitative RT-PCR was used to detect the CK20 mRNA expression in the peripheral blood of 51 patients with colorectal carcinoma and 30 healthy volunteers. RESULTS: 27.45% of the patients showed CK20 mRNA expression, while it was 6.67% for the control group (P<0.025). With the progress of Dukes' stages, the expression level of CK20 mRNA increased, but there was no statistic significance (P<0.05). More samples in Dukes'C and D than in Dukes'A and B stages showed >10 copies/ml. CONCLUSION: The detection of CK20 mRNA expression in peripheral blood of patients with colorectal carcinoma may be helpful to identify early shedding tumor cells. It is also useful to monitor the progression of the disease and observe the effect of clinical treatment.