Generating CRISPR/Cas9-mediated mutants in tomato ( Solanum lycopersicum L.) involves screening shoots regenerated from cultured cells transformed with a T-DNA harboring sequences encoding Cas9 and ...single guide RNAs (sgRNAs). Production of transformants can be inconsistent and obtaining transformants in large numbers may be difficult, resulting in a limited variety of mutations. Here, I report a method for generating various types of mutations from one transgenic plant harboring the CRISPR/Cas9 system. In this method, a wild-type plant was crossed with a T 0 biallelic mutant expressing two sgRNAs targeting the RIPENING INHIBITOR ( RIN ) gene, and the resulting F 1 seedlings were classified using a kanamycin resistance marker on the T-DNA. Genotyping of the RIN locus revealed that kanamycin-sensitive F 1 seedlings, which carried no T-DNA, always harbored the wild-type allele and a mutant allele from the transgenic parent. Kanamycin-resistant F 1 seedlings, which do carry the T-DNA, harbored a variety of novel mutant alleles, but not the wild-type allele, suggesting that it was mutated during crossing. The novel mutations included one-base insertions or short deletions at each target site, or large deletions across the two target sites. This method was also successfully applied to produce mutations in Geranylgeranyl pyrophosphate synthase 2 ( GGPS2 ). Because this method involves crossing rather than transformation, it can be readily scaled up to produce numerous novel mutations, even in plant species or cultivars for which transformation is inefficient. Therefore, when initial transgene experiments fail to induce the desired mutation, this method provides additional opportunities for generating mutants.
The fruit ripening developmental program is specific to plants bearing fleshy fruits and dramatically changes fruit characteristics, including color, aroma, and texture. The tomato (Solanum ...Iycopersicum) MADS box transcription factor RIPENING INHIBITOR (RIN), one of the earliest acting ripening regulators, is required for both ethylene-dependent and -independent ripening regulatory pathways. Recent studies have identified two dozen direct RIN targets, but many more RIN targets remain to be identified. Here, we report the large-scale identification of direct RIN targets by chromatin immunoprecipitation coupled with DNA microarray analysis (ChIP-chip) targeting the predicted promoters of tomato genes. Our combined ChIP-chip and transcriptome analysis identified 241 direct RIN target genes that contain a RIN binding site and exhibit RIN-dependent positive or negative regulation during fruit ripening, suggesting that RIN has both activator and repressor roles. Examination of the predicted functions of RIN targets revealed that RIN participates in the regulation of lycopene accumulation, ethylene production, chlorophyll degradation, and many other physiological processes. Analysis of the effect of ethylene using 1-methylcyclopropene revealed that the positively regulated subset of RIN targets includes ethylene-sensitive and -insensitive transcription factors. Intriguingly, ethylene is involved in the upregulation of RIN expression during ripening. These results suggest that tomato fruit ripening is regulated by the interaction between RIN and ethylene signaling.
We showed previously that subclinical low-risk papillary thyroid microcarcinoma (PTMC) could be observed without immediate surgery. Patient age is an important prognostic factor of clinical papillary ...thyroid carcinoma (PTC). In this study, we investigated how patient age influences the observation of low-risk PTMC.
Between 1993 and 2011, 1235 patients with low-risk PTMC chose observation without immediate surgery. They were followed periodically with ultrasound examinations. These patients were enrolled in this study. We divided them into three subsets based on age at the beginning of observation: young (<40 years), middle-aged (40-59 years), and old patients (≥60 years). Observation periods ranged from 18 to 227 months (average 75 months).
We set three parameters for the evaluation of PTMC progression: (i) size enlargement, (ii) novel appearance of lymph-node metastasis, and (iii) progression to clinical disease (tumor size reaching 12 mm or larger, or novel appearance of nodal metastasis). The proportion of patients with PTMC progression was lowest in the old patients and highest in the young patients. On multivariate analysis, young age was an independent predictor of PTMC progression. However, none of the 1235 patients showed distant metastasis or died of PTC during observation. Although only 51 patients (4%) underwent thyrotropin (TSH) suppression based on physician preference, the PTMC of all patients enrolled in this TSH suppression study, except one, were clinically stable. To date, 191 patients underwent surgery for various reasons after observation. None showed recurrence except for one in the residual thyroid, and none died of PTC after surgery.
Old patients with subclinical low-risk PTMC may be the best candidates for observation. Although PTMC in young patients may be more progressive than in older patients, it might not be too late to perform surgery after subclinical PTMC has progressed to clinical disease, regardless of patient age.
Introduction
Papillary thyroid carcinoma (PTC) generally shows an excellent prognosis except in cases with aggressive backgrounds or clinicopathological features. Although the cause-specific survival ...(CSS) of PTC patients has been extensively investigated, the overall survival (OS) of these patients is unclear. We herein investigated both the OS and CSS of a large PTC patient series.
Materials and methods
We enrolled 5897 PTC patients who underwent initial surgery between 1987 and 2005 (658 males and 5339 females; median age 51 years). Their median postoperative follow-up period was 177 months. Univariate and multivariate analyses for OS and CSS assessed the effects of gender, older age (≥55 years), distant metastasis at diagnosis (
M
1), significant extrathyroid extension, tumor size (cutoffs 2 and 4 cm), large node metastasis (
N
≥ 3 cm), and extranodal tumor extension.
Results
To date, 387 patients (7%) in this series have died from various causes, including 117 (2%) due to PTC. The 10-, 15-, and 20-year OS rates are 97, 95, and 90%, respectively. Older age and
M
1 were important prognostic factors for OS and CSS. Older age was a more significant factor than
M
1 for OS and vice versa for CSS. In the older patients,
M
1 was a prominent prognostic factor for both OS and CSS. In the young patients,
M
1 had less prognostic impact than in the older patients, and the prognostic values of
M
1 and
N
≥ 3 cm for OS and CSS were identical and similar, respectively.
Conclusions
The most important prognostic value for OS was patient age, indicating that PTC is generally indolent. However, the control of distant metastasis in older patients remains a future challenge in order to further improve their OS and CSS. PTC of ≥3 cm in young patients should be carefully followed, even in the absence of metastases, and these patients should undergo aggressive therapies for recurrent lesions and metastases.
The 2015 American Thyroid Association thyroid cancer management guidelines endorse an active surveillance management approach as an alternative to immediate biopsy and surgery in subcentimeter ...thyroid nodules with highly suspicious ultrasonographic characteristics and in cytologically confirmed very low risk papillary thyroid cancer (PTC). However, the guidelines provide no specific recommendations with regard to the optimal selection of patients for an active surveillance management approach. This article describes a risk-stratified clinical decision-making framework that was developed by the thyroid cancer disease management team at Memorial Sloan Kettering Cancer Center as the lessons learned from Kuma Hospital in Japan were applied to a cohort of patients with probable or proven papillary microcarcinoma (PMC) who were being evaluated for an active surveillance management approach in the United States.
A risk-stratified approach to the evaluation of patients with probable or proven PMC being considered for an active surveillance management approach requires an evaluation of three interrelated but distinct domains: (i) tumor/neck ultrasound characteristics (e.g., size of the primary tumor, the location of the tumor within the thyroid gland); (ii) patient characteristics (e.g., age, comorbidities, willingness to accept observation); and (iii) medical team characteristics (e.g., availability and experience of the multidisciplinary team). Based on an analysis of the critical factors within each of these domains, patients with probable or proven PTC can then be classified as ideal, appropriate, or inappropriate candidates for active surveillance.
Risk stratification utilizing the proposed decision-making framework will improve the ability of clinicians to recognize individual patients with proven or probable PMC who are most likely to benefit from an active surveillance management option while at the same time identifying patients with proven or probable PMC that would be better served with an upfront biopsy and surgical management approach.
Background:
The question of how to manage patients with low-risk papillary thyroid microcarcinoma (PTMC; T1aN0M0) has recently become an important clinical issue. Two Japanese centers have conducted ...prospective clinical trials of active surveillance (AS) for low-risk PTMC since the 1990s, reporting favorable outcomes. This policy has thus seen gradual adoption worldwide to avoid overtreatment. Not all PTMCs are suitable for AS, however, and many physicians still hesitate to apply the management policy in daily clinical practice. A task force on management for PTMC created by the Japan Association of Endocrine Surgery collected and analyzed bibliographic evidence and has produced the present consensus statements regarding indications and concrete strategies for AS to facilitate the management of adult patients diagnosed with low-risk PTMC.
Summary:
These statements provide indications for AS in adult patients with T1aN0M0 low-risk PTMC. PTMCs with clinical lymph node metastasis, distant metastasis, recurrent laryngeal nerve (RLN) paralysis due to carcinoma invasion, or protrusion into the tracheal lumen warrant immediate surgery. Tumors suspected of aggressive subtypes on cytology are recommended for immediate surgery. Immediate surgery is also recommended for tumors adherent to the trachea or located along the course of the RLN. Practical strategies include diagnosis, decision-making, follow-up, and monitoring related to the implementation of AS. The rate of low-risk PTMC progression is lower in older patients. However, we recommend continuing AS as long as circumstances permit. Future tasks in optimizing management for low-risk PTMC are also described, including molecular markers and patient-reported outcomes.
Conclusions:
An appropriate multidisciplinary team is necessary to accurately evaluate primary tumors and lymph nodes at the beginning of and during AS, and to adequately reach a shared-decision with individual patients. If appropriately applied, AS of low-risk PTMC is a safe management strategy offering favorable outcomes and preserves quality of life at low cost.
Differentiated thyroid carcinoma originates from thyroid follicular cells and is the most prominent malignancy of the endocrine organs. There are two histological types of differentiated carcinoma, ...namely, papillary and follicular carcinoma. According to reports from Western countries, papillary carcinoma comprises 85.3% of thyroid malignancies in whites, and 72.3% in blacks 1, 2. In Japan, a previous study showed that the prevalence of papillary carcinoma was 78.4% based on material registered between 1977 and 1986 3, but according to recent findings reported in 2004 by Japanese Society of Thyroid Surgeons (JSTS), papillary carcinoma accounted for as much as 93% of all thyroid carcinomas. Papillary carcinoma frequently metastasizes to the regional lymph node and shows multicentricity in the thyroid gland. It usually shows a typical ultrasonographic appearance and can be rather easily diagnosed by fine needle aspiration biopsy (FNAB) 4-6. Follicular carcinoma accounts for 10.9-20.5% of the patients in the United States 1, 2. In Japan, the prevalence of follicular carcinoma was reported to be 17.2% 3, but it decreased to 5% in a report by JSTS in 2004. This carcinoma is only occasionally diagnosed preoperatively, because it is hard to discriminate follicular carcinoma from benign adenoma on imaging studies and cytologic findings. In contrast to papillary carcinoma, follicular carcinoma more often metastasizes to distant organs than regional lymph nodes. In Japan, the prevalence of papillary carcinoma increased and that of follicular carcinoma decreased between reports from 1977 to 1986 and that in 2004, which may be because follicular variant of papillary carcinoma was classified into follicular carcinoma in the previous results. Generally, these carcinomas show an indolent character, but when the lesion dedifferentiates and becomes undifferentiated carcinoma, it displays very rapid growth with an adverse prognosis and is regarded even as the most aggressive malignancy among human solid carcinomas 7, 8. Furthermore, cases showing certain characteristics are likely to be constantly progressive and even become life-threatening. Such cases should be regarded as "high-risk" requiring careful and extensive surgical treatment and postoperative follow-up. Indeed, it is most important for physicians to correctly distinguish high-risk cases from those with an indolent character, although how to evaluate the biological characteristics of thyroid carcinoma and how to identify high-risk cases remains highly controversial. In this review, the methods of distinguishing high-risk cases and the appropriate therapeutic strategies for papillary and follicular carcinomas predominantly based on our experience are emphasized and our proposals for therapies including surgical treatment are demonstrated.
In plants, abscission removes senescent, injured, infected, or dispensable organs. Induced by auxin depletion and an ethylene burst, abscission requires pronounced changes in gene expression, ...including genes for cell separation enzymes and regulators of signal transduction and transcription. However, the understanding of the molecular basis of this regulation remains incomplete. To examine gene regulation in abscission, this study examined an ERF family transcription factor, tomato (Solanum lycopersicum) ETHYLENE-RESPONSIVE FACTOR 52 (SlERF52). SlERF52 is specifically expressed in pedicel abscission zones (AZs) and SlERF52 expression is suppressed in plants with impaired function of MACROCALYX and JOINTLESS, which regulate pedicel AZ development. RNA interference was used to knock down SlERF52 expression to show that SlERF52 functions in flower pedicel abscission. When treated with an abscission-inducing stimulus, the SlERF52-suppressed plants showed a significant delay in flower abscission compared with wild type. They also showed reduced upregulation of the genes for the abscission-associated enzymes cellulase and polygalacturonase. SlERF52 suppression also affected gene expression before the abscission stimulus, inhibiting the expression of pedicel AZ-specific transcription factor genes, such as the tomato WUSCHEL homologue, GOBLET, and Lateral suppressor, which may regulate meristematic activities in pedicel AZs. These results suggest that SlERF52 plays a pivotal role in transcriptional regulation in pedicel AZs at both pre-abscission and abscission stages.
Background During ripening, climacteric fruits increase their ethylene level and subsequently undergo various physiological changes, such as softening, pigmentation and development of aroma and ...flavor. These changes occur simultaneously and are caused by the highly synchronized expression of numerous genes at the onset of ripening. In tomatoes, the MADS-box transcription factor RIN has been regarded as a key regulator responsible for the onset of ripening by acting upstream of both ethylene- and non-ethylene-mediated controls. However, except for LeACS2, direct targets of RIN have not been clarified, and little is known about the transcriptional cascade for ripening. Results Using immunoprecipitated (IPed) DNA fragments recovered by chromatin immunoprecipitation (ChIP) with anti-RIN antibody from ripening tomato fruit, we analyzed potential binding sites for RIN (CArG-box sites) in the promoters of representative ripening-induced genes by quantitative PCR. Results revealed nearly a 5- to 20-fold enrichment of CArG boxes in the promoters of LeACS2, LeACS4, PG, TBG4, LeEXP1, and LeMAN4 and of RIN itself, indicating direct interaction of RIN with their promoters in vivo. Moreover, sequence analysis and genome mapping of 51 cloned IPed DNAs revealed potential RIN binding sites. Quantitative PCR revealed that four of the potential binding sites were enriched 4- to 17-fold in the IPed DNA pools compared with the controls, indicating direct interaction of RIN with these sites in vivo. Near one of the four CArG boxes we found a gene encoding a protein similar to thioredoxin y1. An increase in the transcript level of this gene was observed with ripening in normal fruit but not in the rin mutant, suggesting that RIN possibly induces its expression. Conclusions The presented results suggest that RIN controls fruit softening and ethylene production by the direct transcriptional regulation of cell-wall-modifying genes and ethylene biosynthesis genes during ripening. Moreover, the binding of RIN to its own promoter suggests the presence of autoregulation for RIN expression. ChIP-based analyses identified a novel RIN-binding CArG-box site that harbors a gene associated with RIN expression in its flanking region. These findings clarify the crucial role of RIN in the transcriptional regulation of ripening initiation and progression.
PDF
