Reference gene selection for quantitative real-time RT-PCR normalization in Iris. lactea var. chinensis roots under cadmium, lead, and salt stress conditions.

Quantitative real time PCR (RT-qPCR) has emerged as an accurate and sensitive method to measure the gene expression. However, obtaining reliable result depends on the selection of reference genes which normalize differences among samples. In this study, we assessed the expression stability of seven reference genes, namely, ubiquitin-protein ligase UBC9 (UBC), tubulin alpha-5 (TUBLIN), eukaryotic translation initiation factor (EIF-5A), translation elongation factor EF1A (EF1 α ), translation elongation factor EF1B (EF1b), actin11 (ACTIN), and histone H3 (HIS), in Iris. lactea var. chinensis (I. lactea var. chinensis) root when the plants were subjected to cadmium (Cd), lead (Pb), and salt stress conditions. All seven reference genes showed a relatively wide range of threshold cycles (C t ) values in different samples. GeNorm and NormFinder algorithms were used to assess the suitable reference genes. The results from the two software units showed that EIF-5A and UBC were the most stable reference genes across all of the tested samples, while TUBLIN was unsuitable as internal controls. I. lactea var. chinensis is tolerant to Cd, Pb, and salt. Our results will benefit future research on gene expression in response to the three abiotic stresses.

Base substitution mutations in uridinediphosphate-dependent glycosyltransferase 76G1 gene of Stevia rebaudiana causes the low levels of rebaudioside A: mutations in UGT76G1, a key gene of steviol glycosides synthesis.

Steviol glycosides, extracted from the leaves of Stevia rebaudiana (Bert) Bertoni, are calorie-free sugar substitute of natural origin with intensely sweet (Boileau et al., 2012). Stevioside and rebaudioside A are the two main kinds of the diterpenic glycosides. We analyzed the concentration of stevioside and rebaudioside A in Stevia leaves of about 500 samples (hybrid progenies) and discovered a mutation plant "Z05" with very low levels of rebaudioside A. Because UGT76G1, a uridinediphosphate-dependent glycosyltransferases, is responsible for the conversion from stevioside to rebaudioside A (Richman et al., 2005), so mutation identification was done by sequencing the candidate gene, UGT76G1. In this study molecular analysis of two strains revealed a heterozygotic nonsense mutation of c.389T > G (p.L121X) in UGT76G1. Meanwhile, we found some amino acid substitutions significant change the protein structure. And the difference of enzyme activity between two strains proved the lack of functionality of UGT76G1 of the mutation "Z05". So the nonsense mutation and amino acid substitution mutation resulted in the low levels of rebaudioside A.

Overexpression of Iris. lactea var. chinensis metallothionein llMT2a enhances cadmium tolerance in Arabidopsis thaliana.

Metallothioneins (MTs) are cysteine-rich, low molecular weight, heavy metal-binding protein molecules. Here, a full-length cDNA homologue of MT2a (type 2 metallothionein) was isolated from the cadmium-tolerant species Iris. lactea var. chinensis (I. lactea var. chinensis). Expression of IlMT2a in I. lactea var. chinensis roots and leaves was up-regulated in response to cadmium stress. When the gene was constitutively expressed in Arabidopsis thaliana (A. thaliana), root length of transgenic lines was longer than that of wild-type under 50µM or 100µM cadmium stress. However, there was no difference of cadmium absorption between wild-type and trangenic lines. Histochemical staining by 3,3-diaminobenzidine (DAB) and nitroblue tetrazoliu (NBT) clearly demonstrated that transgenic lines accumulated remarkably less H2O2 and O2(-) than wild-type. Together, IlMT2a may be a promising gene for the cadmium tolerance improvement.

Salvage treatment improved survival of patients with relapsed extranodal natural killer/t-cell lymphoma, nasal type.

PURPOSE: To evaluate the clinical outcome of salvage treatment for patients with relapsed natural killer (NK)/T-cell lymphoma, nasal type. METHODS AND MATERIALS: Forty-four patients who had achieved complete response during initial treatment and experienced histologically proven relapse were reviewed. Twenty-nine of them received salvage treatment with radiotherapy (RT) alone (n = 7), chemotherapy (CT) alone (n = 10), or both RT and CT (n = 12); the other 15 patients received best supportive care alone. RESULTS: The estimated 5-year overall survival (OS) rate for patients with or without salvage treatment was 37.8% vs. 0 (p < 0.0001), respectively. Salvage CT did not improve survival of relapsed Stage IE and IIE patients. Among relapsed Stage IIIE and IVE patients who received salvage treatment, RT developed significantly better survival when compared with that of non-RT (1-year OS, 62.5% vs. 0, p = 0.006). Relapsed Ann Arbor stage and receiving salvage treatment were found to be significant factors influencing OS at both univariate and multivariate levels. CONCLUSIONS: Salvage treatment improved survival in patients with relapsed NK/T-cell lymphoma, nasal type. Salvage RT may play an important role in salvage treatment of relapsed extranodal NK/T-cell lymphoma.

[Correlation study of physical and biologic parameters to acute radiation pneumonitis].

OBJECTIVE: To study the relationship between the level of plasma transform growth factor-betal (TGF-betal), interleukin-6 (IL-6), thrombomodulin (TM) dose-volume factors and acute radiation pneumonitis (ARP). METHODS: Three dimensional conformal radiation therapy and chemotherapy were applied to 27 lung cancer patients, 15 esophageal carcinoma, and 1 thymoma patients. 19 patients received adjuvant radiochemotherapy, and 25 patients received concurrent radio-chemotherapy. Enzyme-linked immunosorbent assay (ELISA) was applied to detect the serum IL-6, TGF-betal and TM levels of patients before radiotherapy (B-RT) and at 30 Gy(M-RT). ARP was graded according to NCI common toxicity criteria (CTCAE v 3.0), Grade 2 or more ARP was taken as the main end point. The relationship between the levels of serum TGF-betal, IL-6, TM, parameters of lung function and dose-volume factors, and the incidence rate of ARP were analyzed. Mean lung dose (MLD) and the Vx were considered as the dose-volume factors. RESULTS: Among 44 patients, 15 of them had ARP. 9 got grade 2 ARP and 6 had grade 3. After received a dose of 30 Gy, TGF-betal value in M-RT was (396 +/- 338) and (866 +/- 270) pg/mL in non-ARP and ARP group (P = 0.000). The ARP incidence rate in <60 and > or =60 age group were 46.15% (12/26) and 16.67% (3/18) (P = 0.042), and 45.45% (15/33), 0% (0/11) (P = 0.017) in had smoking history and nonsmoking history group, respectively. 1 out of 13 patients (7.69%) who had increased M-RT TM value suffered of ARP, while 14 out of 31 patients (45.16%) whose M-RT TM value lower than pr-RT suffered of ARP (P = 0.044). The results of multivariate analysis implied that the MLD and the value of TGF-betal in M-RT were associated with severe ARP. CONCLUSION: TGF-beta1 and MLD are significant indicators of ARP. FEV1 actual value/predicted value% might have predicting effect for the severity of ARP.