Virus-Mediated Transient Expression Techniques Enable Functional Genomics Studies and Modulations of Betalain Biosynthesis and Plant Height in Quinoa.

Quinoa (Chenopodium quinoa), native to the Andean region of South America, has been recognized as a potentially important crop in terms of global food and nutrition security since it can thrive in harsh environments and has an excellent nutritional profile. Even though challenges of analyzing the complex and heterogeneous allotetraploid genome of quinoa have recently been overcome, with the whole genome-sequencing of quinoa and the creation of genotyped inbred lines, the lack of technology to analyze gene function in planta is a major limiting factor in quinoa research. Here, we demonstrate that two virus-mediated transient expression techniques, virus-induced gene silencing (VIGS) and virus-mediated overexpression (VOX), can be used in quinoa. We show that apple latent spherical virus (ALSV) can induce gene silencing of quinoa phytoene desaturase (CqPDS1) in a broad range of quinoa inbred lines derived from the northern and southern highland and lowland sub-populations. In addition, we show that ALSV can be used as a VOX vector in roots. Our data also indicate that silencing a quinoa 3,4-dihydroxyphenylalanine 4,5-dioxygenase gene (CqDODA1) or a cytochrome P450 enzyme gene (CqCYP76AD1) inhibits betalain production and that knockdown of a reduced-height gene homolog (CqRHT1) causes an overgrowth phenotype in quinoa. Moreover, we show that ALSV can be transmitted to the progeny of quinoa plants. Thus, our findings enable functional genomics in quinoa, ushering in a new era of quinoa research.

The genotype-dependent phenotypic landscape of quinoa in salt tolerance and key growth traits.

Cultivation of quinoa (Chenopodium quinoa), an annual pseudocereal crop that originated in the Andes, is spreading globally. Because quinoa is highly nutritious and resistant to multiple abiotic stresses, it is emerging as a valuable crop to provide food and nutrition security worldwide. However, molecular analyses have been hindered by the genetic heterogeneity resulting from partial outcrossing. In this study, we generated 136 inbred quinoa lines as a basis for the molecular identification and characterization of gene functions in quinoa through genotyping and phenotyping. Following genotyping-by-sequencing analysis of the inbred lines, we selected 5,753 single-nucleotide polymorphisms (SNPs) in the quinoa genome. Based on these SNPs, we show that our quinoa inbred lines fall into three genetic sub-populations. Moreover, we measured phenotypes, such as salt tolerance and key growth traits in the inbred quinoa lines and generated a heatmap that provides a succinct overview of the genotype-phenotype relationship between inbred quinoa lines. We also demonstrate that, in contrast to northern highland lines, most lowland and southern highland lines can germinate even under high salinity conditions. These findings provide a basis for the molecular elucidation and genetic improvement of quinoa and improve our understanding of the evolutionary process underlying quinoa domestication.

Draft genome sequence of an inbred line of Chenopodium quinoa, an allotetraploid crop with great environmental adaptability and outstanding nutritional properties.

Chenopodium quinoa Willd. (quinoa) originated from the Andean region of South America, and is a pseudocereal crop of the Amaranthaceae family. Quinoa is emerging as an important crop with the potential to contribute to food security worldwide and is considered to be an optimal food source for astronauts, due to its outstanding nutritional profile and ability to tolerate stressful environments. Furthermore, plant pathologists use quinoa as a representative diagnostic host to identify virus species. However, molecular analysis of quinoa is limited by its genetic heterogeneity due to outcrossing and its genome complexity derived from allotetraploidy. To overcome these obstacles, we established the inbred and standard quinoa accession Kd that enables rigorous molecular analysis, and presented the draft genome sequence of Kd, using an optimized combination of high-throughput next generation sequencing on the Illumina Hiseq 2500 and PacBio RS II sequencers. The de novo genome assembly contained 25 k scaffolds consisting of 1 Gbp with N50 length of 86 kbp. Based on these data, we constructed the free-access Quinoa Genome DataBase (QGDB). Thus, these findings provide insights into the mechanisms underlying agronomically important traits of quinoa and the effect of allotetraploidy on genome evolution.

Determinants of local progression after computed tomography-guided percutaneous radiofrequency ablation for unresectable lung tumors: 9-year experience in a single institution.

The purpose of this study was to retrospectively determine the local control rate and contributing factors to local progression after computed tomography (CT)-guided radiofrequency ablation (RFA) for unresectable lung tumor. This study included 138 lung tumors in 72 patients (56 men and 16 women; age 70.0 +/- 11.6 years (range 31-94); mean tumor size 2.1 +/- 1.2 cm [range 0.2-9]) who underwent lung RFA between June 2000 and May 2009. Mean follow-up periods for patients and tumors were 14 and 12 months, respectively. The local progression-free rate and survival rate were calculated to determine the contributing factors to local progression. During follow-up, 44 of 138 (32%) lung tumors showed local progression. The 1-, 2-, 3-, and 5-year overall local control rates were 61, 57, 57, and 38%, respectively. The risk factors for local progression were age (>or=70 years), tumor size (>or=2 cm), sex (male), and no achievement of roll-off during RFA (P < 0.05). Multivariate analysis identified tumor size >or=2 cm as the only independent factor for local progression (P = 0.003). For tumors <2 cm, 17 of 68 (25%) showed local progression, and the 1-, 2-, and 3-year overall local control rates were 77, 73, and 73%, respectively. Multivariate analysis identified that age >or=70 years was an independent determinant of local progression for tumors <2 cm in diameter (P = 0.011). The present study showed that 32% of lung tumors developed local progression after CT-guided RFA. The significant risk factor for local progression after RFA for lung tumors was tumor size >or=2 cm.

Frequency and risk factors of various complications after computed tomography-guided radiofrequency ablation of lung tumors.

OBJECTIVE: To retrospectively determine the frequency and risk factors of various side effects and complications after percutaneous computed tomography-guided radiofrequency (RF) ablation of lung tumors. METHODS: We reviewed and analyzed records of 112 treatment sessions in 57 of our patients (45 men and 12 women) with unresectable lung tumors treated by ablation. Risk factors, including sex, age, tumor diameter, tumor location, history of surgery, presence of pulmonary emphysema, electrode gauge, array diameter, patient position, maximum power output, ablation time, and minimum impedance during ablation, were analyzed using univariate and multivariate analyses. RESULTS: Total rates of side effects and minor and major complications occurred in 17%, 50%, and 8% of treatment sessions, respectively. Side effects, including pain during ablation (46% of sessions) and pleural effusion (13% of sessions), occurred with RF ablation. Minor complications, including pneumothorax not requiring chest tube drainage (30% of sessions), subcutaneous emphysema (16% of sessions), and hemoptysis (9% of sessions) also occurred after the procedure. Regarding major complications, three patients developed fever >38.5 degrees C; three patients developed abscesses; two patients developed pneumothorax requiring chest tube insertion; and one patient had air embolism and was discharged without neurologic deficit. Univariate and multivariate analyses suggested that a lesion located < or =1 cm of the chest wall was significantly related to pain (p < 0.01, hazard index 5.76). Risk factors for pneumothorax increased significantly with previous pulmonary surgery (p < 0.05, hazard index 6.1) and presence of emphysema (p <0.01, hazard index 13.6). CONCLUSION: The total complication rate for all treatment sessions was 58%, and 25% of patients did not have any complications after RF ablation. Although major complications can occur, RF ablation of lung tumors can be considered a safe and minimally invasive procedure.

Angiosarcoma of the breast - specific findings of MRI.

We present a case of low-grade angiosarcoma of the breast. A 26-year old woman presented with a lump in the left breast. An elastic hard and ill-defined tumor, 80 x 50 mm in size, was palpated in the upper region of her left breast. Mammography showed a dense lesion with poorly defined border. Ultrasonography showed a hyper-and hypo-echoic lesion with an unclear border, but no definite tumor. Fine needle aspiration cytology showed no evidence of malignancy. Therefore, she was followed with a diagnosis of mastopathy. Six months later, the lump got enlarged. A contrast-enhanced MRI of the breast was performed. It showed a 100 x 60 mm enhancing vascular mass. Most parts of the tumor enhanced remarkably at the early phase, and prolonged enhancement was recognized at the late phase. Core needle biopsy was performed, and a possible angiosarcoma was diagnosed. It is not easy to diagnose the mammary angiosarcoma. MRI may contribute to the accurate diagnosis and play an important role regarding this entity.

Fluorine-18-fluorodeoxyglucose positron emission tomography for assessment of patients with unresectable recurrent or metastatic lung cancers after CT-guided radiofrequency ablation: preliminary results.

OBJECTIVES: We compared the diagnostic value of fluorine-18-fluorodeoxyglucose positron emission tomography (FDG-PET) with that of computed tomography (CT) following radiofrequency ablation (RFA) of inoperable recurrent or metastatic cancers in the lung. METHODS: Twelve patients (9 males and 3 females; 5 had recurrent lung cancer and the other 7 had metastatic nodules from a variety of primary cancers) were treated by RFA for 17 pulmonary nodules. FDG-PET was performed before and 2 months after RFA, and the mean standardized uptake value (SUV) was calculated. The response evaluation was based on the percent reduction relative to the baseline and the absolute values of SUV on FDG-PET performed at 2 months after RFA. We compared the response evaluations made based on findings of FDG-PET and CT at 2 and > or =6 months (mean 10.2) after RFA. RESULTS: The percent reduction in uptake at 2 months was significantly lower in nodules considered progressive (69.6 +/- 18.6%) than nonprogressive disease (38.7 +/- 12.5%; p < 0.01) based on CT findings at > or =6 months after RFA. The absolute SUV at 2 months was significantly higher in nodules considered progressive (2.61 +/- 0.75) than nonprogressive disease (1.05 +/- 0.67; p < 0.01) based on CT findings at > or =6 months post-RFA. CONCLUSION: Although our pilot study comprised few cases of various histopathological types of cancers in the lung, the results suggest that FDG-PET could predict regrowth on subsequent follow-up CT. Regrowth could be diagnosed earlier by FDG-PET than by CT, and nodules with residual uptake and with <60% reduction of uptake relative to baseline on FDG-PET at 2 months after ablation might require additional therapy.

Radiofrequency ablation in a porcine lung model: correlation between CT and histopathologic findings.

OBJECTIVE: The objective of our study was to investigate the time course changes of the ablated lesion after radiofrequency ablation in the porcine lung and the correlation between CT and histopathologic findings. CONCLUSION: Ground-glass attenuation on CT led to overestimation of the size of necrotic lesions. The layered structural findings on CT were consistent with the histopathologic findings. Although CT findings reflect the histopathologic findings, attention should be paid to the dissociation of ablated lesions and high-density areas in clinical interpretation of CT images.

Radiofrequency ablated lesion in the normal porcine lung: long-term follow-up with MRI and pathology.

PURPOSE: To evaluate the effectiveness of magnetic resonance imaging (MRI) in reflecting histopathologic changes after radiofrequency ablation (RFA) to the lung and to assess accurately the extent of tissue necrosis for evaluating untreated lesions. METHODS: Percutaneous RFA was performed on 72 lung regions in 12 pigs under computed tomographic guidance. After performing MRI, the animals were divided into three experimental phases: in the acute phase, 4 pigs were killed immediately after the procedure; in the subacute phase, 4 pigs were killed at 1 week; and in the chronic phases, 2 pigs were killed at 4 and 8 weeks after the procedure, respectively. MRI-histopathologic correlation was performed. RESULTS: In the acute phase, the inner zone showed hypointensity on T2-weighted images and isointensity on T1-weighted images, with a lack of enhancement on contrast-enhanced T1-weighted images, representing early changes in coagulative necrosis on histopathologic examination. The outer zone showed hyperintensity on T2-weighted images and isointensity on T1-weighted images, with ring-like enhancement on contrast-enhanced T1-weighted images. The histopathologic section showed alveolar fluid collections and congestion. In the subacute phase the MR zone pattern was essentially similar to that of the acute phase, but the ablated lesion showed extensive coagulative necrosis with a fibrovascular rim on histopathologic examination. In the chronic phase, there was no change in the zone pattern on MRI. The lesions showed gradual resorption of coagulative necrosis. The area of coagulative necrosis correlated closely with the histopathologic size (p < 0.05). CONCLUSIONS: MRI effectively visualized the histopathologic changes after RFA and accurately determined the extent of the necrotic lesion. MRI is potentially a useful modality for evaluating therapeutic efficacy after RFA to the lung.

Assessment of cerebral microembolism during percutaneous radiofrequency ablation of lung tumors using diffusion-weighted imaging.

OBJECTIVE: It is well known that radiofrequency ablation generates microbubbles in the liver. We hypothesized that microbubbles generated during percutaneous radiofrequency ablation of lung tumors flow into the pulmonary veins and are distributed to the systemic arteries, as with radiofrequency ablation of liver tumors. To assess the risk of cerebral infarction during radiofrequency ablation of lung tumors, we performed diffusion-weighted imaging and, if possible, monitored microemboli in the carotid artery during radiofrequency ablation. SUBJECTS AND METHODS: We prospectively studied 20 patients (19 men and one woman) who underwent radiofrequency ablation of lung tumors. Pre- and postoperative MRI examinations were performed in all 20 patients, and during 17 radiofrequency ablation sessions, sonography was used to monitor whether microemboli were generated. RESULTS: Radiofrequency ablation was technically feasible for the treatment of selected pulmonary tumors. Microemboli, which were believed to represent microbubbles, were seen on sonography during three of the 17 radiofrequency ablation sessions. They were rarely observed when a lung tumor was small, the treatment session was brief, and the radiofrequency emission power was low. No new area of abnormal intensity was seen on postoperative MRI in all 20 patients. Although the microemboli were observed, MRI could not confirm infarction. CONCLUSION: We concluded that cerebral infarction as a result of microbubbles generated during radiofrequency ablation of lung tumors has a low possibility of becoming a clinical problem.

[Radiofrequency ablation of pulmonary malignancies].

Radiofrequency ablation (RFA) with a maximum output of < or = 70 W was used under CT guidance to treat 14 tumors (median diameter 1.5 cm, range 0.8 to 2.4 cm) in 6 patients. Primary lung cancer was treated in 2 patients, and metastatic lung tumors were treated in 4 patients. Good response was confirmed in 6 tumors on CT images, in one tumor on PET images, and in 2 tumors by histologic examination. In the other 5 tumors, curative effect was not determined because consolidation shadows surrounded the tumors. Complications were mild (pneumothoraces, 5; sputum cruentum, 4; subcutaneous emphysema, 3) except for one case of pleurisy. RFA of pulmonary malignancies appears to be a safe, effective treatment if a low output of less than 70 W is applied.