China Initiative for Multi-Domain Intervention (CHINA-IN-MUDI) to Prevent Cognitive Decline: Study Design and Progress.

BACKGROUND: Alzheimer's disease (AD), the most common type of irreversible dementia, is predicted to affect 152 million people by 2050. Evidence from large-scale preventive randomized controlled trials (RCTs) on modifiable risk variables in Europe has shown that multi-domain lifestyle treatments for older persons at high risk of dementia may be practical and effective. Given the substantial differences between the Chinese and European populations in terms of demographics and living conditions, direct adoption of the European program in China remains unfeasible. Although a RCT has been conducted in China previously, its participants were mainly from rural areas in northern China and, thus, are not representative of the entire nation.There is an urgent need to establish cohorts that represent different economic, cultural, and geographical situations in order to explore implementation strategies and evaluate the effects of early multi-domain interventions more comprehensively and accurately. MEDTODS: We developed an integrated intervention procedure implemented in urban neighborhood settings, namely China Initiative for Multi-Domain Intervention (CHINA-IN-MUDI). CHINA-IN-MUDI is a 2-year multicenter open-label cluster-randomised controlled trial centered around a Chinese-style multi-domain intervention to prevent cognitive decline. Participants aged 60-80 years were recruited from a nationally representative study, i.e. China Healthy Aging and Dementia Study cohort. An external harmonization process was carried out to preserve the original FINGER design. Subsequently, we standardized a series of Chinese-style intervention programs to align with cultural and socioeconomic status. Additionally, we expanded the secondary outcome list to include genomic and proteomic analyses. To enhance adherence and facilitate implementation, we leveraged an e-health application. RESULTS: Screening commenced in July 2022. Currently, 1,965 participants have been randomized into lifestyle intervention (n = 772) and control groups (n = 1,193). Both the intervention and control groups exhibited similar baseline characteristics. Several lifestyle and vascular risk factors were present, indicating a potential window of opportunity for intervention. The intervention will be completed by 2025. CONCLUSIONS: This project will contribute to the evaluation of the effectiveness and safety of intervention strategies in controlling AD risk and reducing clinical events, providing a basis for public health decision-making in China.

The relationship between famine exposure in early life and left atrial enlargement in adulthood.

BACKGROUND: Increased left atrium diameter (LAD) is associated with an elevated risk of cardiovascular diseases. The relationship between nutrition status and left atrial enlargement (LAE) is still unclear. The present study aimed to investigate the association of famine exposure in early life with LAE in adulthood. METHODS: Participants were divided into non-exposed, fetal, early, middle and late childhood exposed groups according to birth data. LAE was defined when LAD was ≥3.9 cm in women and ≥4.1 cm in men, or ≥2.3 cm m-2 by a sex-independent cut-off normalised for body surface area. Multivariate logistic regression was performed to calculate the odds ratio (OR) and confidence interval (CI) between famine exposure and LAE. RESULTS: In total, 2522 [905 male, mean (SD) age 59.1 (3.65) years] subjects were enrolled, including 392 (15.5%) LAE subjects. The prevalence of LAE in non-exposed, fetal, early, middle and late childhood exposed groups was 55 (10.8%), 38 (11.2%), 88 (18.1%), 102 (16.7%) and 109 (19.0%), respectively. Compared to the non-exposed group, the ORs for LAE were in fetal (OR = 0.956, 95% CI = 0.605-1.500, P = 0.847), late (OR = 1.748, 95% CI = 1.208-2.555, P = 0.003), middle (OR = 1.647, 95% CI = 1.140-2.403, P = 0.008) and early (OR = 1.630, 95% CI = 1.116-2.399, P = 0.012) childhood exposed groups after adjusting potential cofounders. When stratified by gender, smoking, body mass index, hypertension and diabetes, we found that the effect of famine exposure on LAE was only modified by diabetes (Pinteraction  = 0.007). CONCLUSIONS: Famine exposure during childhood stage might increase the risk of LAE in adults, and this effect interacts with diabetes.

Nervous system complications of COVID-19 with a focus on stroke.

As a severe and highly contagious infection, coronavirus disease (COVID-19) affects all aspects of society and has become a global public health problem. Because of the complexity of the pathology of COVID-19, it is difficult to treat. An increasing number of reports have indicated that COVID-19 may have neurological complications, including stroke. The nervous system complications of COVID-19 have gradually attracted research attention. In this review, we summarize the latest findings related to COVID 19, elaborate on the possible mechanism of COVID 19 related onset of stroke, and summarize current treatment options because an improved understanding and appropriate treatments may improve the prognosis of patients with COVID-19-related stroke.

MicroRNA-95-3p promoted the development of prostatic cancer via regulating DKK3 and activating Wnt/ß-catenin pathway.

OBJECTIVE: Previous studies have shown that microRNA-95-3p (miR-95-3p) plays a crucial role in multiple human cancers except for prostatic cancer (PCa). Therefore, the function of miR-95-3p was investigated in PCa in the present work. PATIENTS AND METHODS: The expression of miR-95-3p was measured by Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) assay. Western blot assay was used to examine the protein expression of epithelial-mesenchymal transition (EMT) markers. In addition, the function of miR-95-3p was detected through MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and transwell assays. Dual Luciferase assay was applied to confirm the relationship between miR-95-3p and dickkopf-3 (DKK3). The tumor growth was observed through xenograft tumor formation assay. RESULTS: The upregulation of miR-95-3p was detected in PCa tissues and cell lines, which predicted poor prognosis of PCa patients. Moreover, miR-95-3p promoted cell proliferation, migration and invasion in PCa by targeting DKK3 and activating the Wnt/ß-catenin pathway. MiR-95-3p also promoted the tumor growth of PCa in vivo. Besides that, downregulation of DKK3 was identified in PCa and low DKK3 expression predicted poor prognosis of PCa patients. CONCLUSIONS: MiR-95-3p promoted the development of PCa via targeting DKK3 and activating the Wnt/ß-catenin pathway.

Regulation of geminin by neuropeptide Y in vascular smooth muscle cell proliferation : A current review.

Geminin, a key regulator of DNA replication licensing in the cell cycle, plays an essential role in determining the fate of cells via suppression of cell proliferation and cellular differentiation. Neuropeptide Y (NPY) intensifies the proliferation of vascular smooth muscle cells (VSMCs) directly by binding with Y1 receptors. In vitro experiments have shown that stimulation of NPY on VSMCs via regulation of geminin is a double-edged sword. Given that the proliferation and the phenotypic transformation of VSMCs increase the risk for progression of atherosclerosis, we focus on the role of geminin interference in determining the fate of VSMCs. Furthermore, we discuss the therapeutic potential of peripheral neurotransmitter interference, thus pointing toward future research directions in the treatment of atherosclerosis.

The safety profile of subcutaneous allergen immunotherapy in children with asthma in Hangzhou, East China

Background: The aim of the current study is to evaluate the prevalence, severity and possible risk factors of systemic reactions (SRs) to subcutaneous allergen immunotherapy (SCIT) in children and adolescents with asthma in Hangzhou, east China's Zhejiang province. Methods: From January 2011 to December 2016, this survey analysed the SCIT-related SRs involving 429 patients (265 children and 134 adolescents) affected by allergic asthma. Recorded data included demographics, diagnosis, patient statuses, pulmonary function testing results before and after each injection, allergen dosage, and details of SRs. Results: All patients finished the initial phase and six patients withdrew during the maintenance phase. There were 2.59% (328/12,655) SRs in all injections (3.28% in children and 1.47% in adolescents); 15.62% (67/429) patients experienced SRs (18.49% children and 10.98% adolescents). There were 54.57% SRs of grade 1; 42.37% SRs of grade 2; 3.05% SRs of grade 3; and no grades 4 or grade 5 SRs occurred in patients. Most reactions were mild, and were readily controlled by immediate emergency treatment. There was no need for hospitalisation. The occurrence of SRs was significantly higher in children than that in adolescents (p < 0.01). A higher ratio of SRs was found among patients with moderate asthma. Conclusion: This retrospective survey showed that properly-conducted SCIT was a safe treatment for children and adolescents with asthma in Hangzhou, East China. Children and patients with moderate asthma may be prone to develop SRs (AU)

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MiR-214 inhibits human mesenchymal stem cells differentiating into osteoblasts through targeting ß-catenin.

OBJECTIVE: Wnt/ß-catenin signaling pathway promotes osteoblasts (OBs) differentiation through up-regulating osteoblast-specific gene runt-related transcription factor 2 (RUNX2) expression. It was showed that microRNA-214 (miR-214) was abnormally increased in bone tissue from osteoporosis patients, suggesting its role in osteogenesis. Bioinformatics analysis revealed the complementary binding site between miR-214 and 3'-UTR of ß-catenin. This study investigated the effects of miR-214 in regulating ß-catenin expression and bone marrow mesenchymal stem cells (BMSCs) differentiating into OB. MATERIALS AND METHODS: BMSCs were induced to differentiate to OB in a specific medium. MiR-214, ß-catenin, and RUNX2 expressions were detected. The regulatory relationship between miR-214 and ß-catenin was confirmed by dual luciferase reporter gene assay. BMSCs were divided into five groups, including agomir-control, miR-214 agomir, pGPU6-normal control group (pGPU6-NC), pGPU6-ß-catenin, and miR-214 agomir + pGPU6-ß-catenin groups. ß-catenin and RUNX2 levels were tested after 21 days' induction. OB differentiation degree was evaluated by alizarin red staining. RESULTS: MiR-214 was down-regulated, while ß-catenin and RUNX2 were enhanced in the process of BMSCs differentiating into OBs. MiR-214 agomir and/or ß-catenin shRNA pGPU6-ß-catenin transfection significantly reduced ß-catenin expression, declined RUNX2 level, and attenuated OB differentiation in BMSCs. CONCLUSIONS: Wnt/ß-catenin signaling pathway was enhanced, while the miR-214 level was decreased in the process of BMSCs differentiating into OBs. Up-regulation of miR-214 inhibited the OB differentiation of BMSCs through targeted suppressing ß-catenin expression and attenuating Wnt/ß-catenin signaling pathway activity.

The safety profile of subcutaneous allergen immunotherapy in children with asthma in Hangzhou, East China.

BACKGROUND: The aim of the current study is to evaluate the prevalence, severity and possible risk factors of systemic reactions (SRs) to subcutaneous allergen immunotherapy (SCIT) in children and adolescents with asthma in Hangzhou, east China's Zhejiang province. METHODS: From January 2011 to December 2016, this survey analysed the SCIT-related SRs involving 429 patients (265 children and 134 adolescents) affected by allergic asthma. Recorded data included demographics, diagnosis, patient statuses, pulmonary function testing results before and after each injection, allergen dosage, and details of SRs. RESULTS: All patients finished the initial phase and six patients withdrew during the maintenance phase. There were 2.59% (328/12,655) SRs in all injections (3.28% in children and 1.47% in adolescents); 15.62% (67/429) patients experienced SRs (18.49% children and 10.98% adolescents). There were 54.57% SRs of grade 1; 42.37% SRs of grade 2; 3.05% SRs of grade 3; and no grades 4 or grade 5 SRs occurred in patients. Most reactions were mild, and were readily controlled by immediate emergency treatment. There was no need for hospitalisation. The occurrence of SRs was significantly higher in children than that in adolescents (p<0.01). A higher ratio of SRs was found among patients with moderate asthma. CONCLUSION: This retrospective survey showed that properly-conducted SCIT was a safe treatment for children and adolescents with asthma in Hangzhou, East China. Children and patients with moderate asthma may be prone to develop SRs.

Insulin resistance in Alzheimer's disease (AD) mouse intestinal macrophages is mediated by activation of JNK.

OBJECTIVE: Alzheimer's disease (AD) has been considered as a metabolic disorder disease, which closely related to insulin signaling impairment. Therefore, identifying the potential mechanism of insulin resistance is important for AD treatment. MATERIALS AND METHODS: An APP/PS1 double transgenic AD mouse model was introduced to study insulin resistance in gut. The expressions of AD markers and key elements of insulin signaling were detected in ileum and intestinal macrophages of AD mice by immunohistochemistry. Furthermore, mouse intestinal macrophage cell line RAW264.7 was treated by Aß25-35 or Aß25-35 + insulin to explore the mechanism of insulin resistance in vitro. The expression of IR-ß and the activation of cell signaling related proteins (Insulin receptor substrate 1 (IRS1), protein kinase B (AKT) and c-Jun N-terminal kinase (JNK)) in Aß25-35-stimulated macrophages were performed via Western blotting. RESULTS: The expressions of IRS1, Aß and Tuj in AD mice ileum were significantly different from WT mice (p<0.05). Also, there were significant discrepancies in the expressions of ß2AR and eNOS in intestinal macrophages of two groups (p<0.05). After exposure to Aß25-35, cell proliferation rate (p<0.01) of macrophage and the levels of TNF-α (p<0.01) and Il-6 (p<0.01) was significant elevated and treatment with insulin could reverse these changes (p<0.05). The amount of IR-ß and the p-AKT/AKT ratio significantly decreased in Aß25-35-treated macrophages (p<0.01), while the ratios of p-IRS1/IRS1 and p-JNK/JNK significantly enlarged (p<0.01). Furthermore, all the changes caused by Aß25-35 treatment were attenuated by insulin addition. CONCLUSIONS: Activation of JNK pathway played an important role in insulin resistance of AD mice, suggesting that inhibition of JNK pathway might be a new strategy toward resolving insulin resistance related diseases, such as AD.

WWOX inhibits the invasion of lung cancer cells by downregulating RUNX2.

The WW domain-containing oxidoreductase (WWOX) is a tumor suppressor that is lost or decreased in most human tumors. The role of WWOX in human lung carcinoma invasion is still not clear. This study aimed to elucidate the potential role of WWOX in lung cancer cell invasion. WWOX mRNA levels in human lung cancers and lung cancer cell lines were assayed by quantitative real-time PCR. WWOX in lung cancer cell lines was manipulated by transfection of expression vector or small interfering RNA. Cell migration and invasion were assessed by wound healing and/or transwell migration and invasion assays. The protein levels of WWOX, E-cadherin and RUNX2 were analyzed by western blot or immunofluorescence. WWOX expression is inversely correlated to invasiveness of lung cancer. WWOX overexpression in highly invasive H1299 cells reduced cell motility and invasiveness, and inhibited the expression of RUNX2 and its target gene matrix metalloproteinase-9 (MMP-9). Silencing WWOX in less invasive NL9980 cells resulted in opposite effects. Overexpressing RUNX2 in H1299 or silencing RUNX2 in NL9980 cells reversed the effects of WWOX. These results suggested that WWOX inhibited the invasive phenotype of lung cancer through downregulating the expression of RUNX2.

Serum human epididymis protein 4 is associated with the treatment response of concurrent chemoradiotherapy and prognosis in patients with locally advanced non-small cell lung cancer

Aim: To investigate the role of human epididymis protein 4 (HE4) in the diagnosis and prognosis of patients with locally advanced non-small cell lung cancer (LA-NSCLC) receiving concurrent chemoradiotherapy (CRT). Methods: A total of 218 patients with LA-NSCLC were enrolled. All patients underwent CRT. The treatment response to CRT was evaluated. The prognosis analysis was performed using relapse-free survival (RFS) and overall survival [1]. Results: Our data show that the serum HE4 can discriminate patients who respond well to CRT from those who respond poorly. Higher serum HE4 had dramatically increased risk of being non-responders to CRT. Serum HE4 level is also associated with prognosis of patients after CRT. Patients with high HE4 level had shorter RFS and OS compared to those with low HE4 level. Conclusion: Our data suggest that serum HE4 may be a useful prognostic biomarker for LA-NSCLC patients who underwent CRT (AU)

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Serum human epididymis protein 4 is associated with the treatment response of concurrent chemoradiotherapy and prognosis in patients with locally advanced non-small cell lung cancer.

AIM: To investigate the role of human epididymis protein 4 (HE4) in the diagnosis and prognosis of patients with locally advanced non-small cell lung cancer (LA-NSCLC) receiving concurrent chemoradiotherapy (CRT). METHODS: A total of 218 patients with LA-NSCLC were enrolled. All patients underwent CRT. The treatment response to CRT was evaluated. The prognosis analysis was performed using relapse-free survival (RFS) and overall survival [1]. RESULTS: Our data show that the serum HE4 can discriminate patients who respond well to CRT from those who respond poorly. Higher serum HE4 had dramatically increased risk of being non-responders to CRT. Serum HE4 level is also associated with prognosis of patients after CRT. Patients with high HE4 level had shorter RFS and OS compared to those with low HE4 level. CONCLUSION: Our data suggest that serum HE4 may be a useful prognostic biomarker for LA-NSCLC patients who underwent CRT.

Expression of multidrug resistance 1 and multidrug resistance-related protein 1 in C57BL/6 mice treated with benzene.

ATP-binding cassette super family (ABC) proteins are considered key to oncology and pharmacology studies. We examined the effect of benzene on ABC pump protein levels in C57BL/6 mouse bone marrow mononuclear cells. After a 2-week gavage (200 mg/kg, 5 days per week), the number of peripheral leukocytes, lymphocytes and basophils dropped significantly; there was also a significant decrease in MDR1 and MRP1 gene expression. A significant reduction in expression of P-gp was found; however, there was no significant decrease in the expression of MRP1 and NF-κB p65. We conclude that regulation of membrane efflux transport protein could be a factor in benzene hematotoxicity.

Genetically elevated levels of circulating triglycerides and brachial-ankle pulse wave velocity in a Chinese population.

Elevated levels of circulating triglycerides and increased arterial stiffness are associated with cardiovascular disease. Numerous studies have reported an association between levels of circulating triglycerides and arterial stiffness. We used Mendelian randomization to test whether this association is causal. We investigated the association between circulating triglyceride levels, the apolipoprotein A-V (ApoA5) -1131T>C single nucleotide polymorphism and brachial-ankle pulse wave velocity (baPWV) by examining data from 4421 subjects aged 18-74 years who were recruited from the Chinese population. baPWV was significantly associated with the levels of circulating triglycerides after adjusting for age, sex, body mass index (BMI), systolic blood pressure, heart rate, waist-to-hip ratio, antihypertensive treatment and diabetes mellitus status. The -1131C allele was associated with a 5% (95% confidence interval 3-8%) increase in circulating triglycerides (adjusted for age, sex, BMI, waist-to-hip ratio, diabetes mellitus and antihypertensive treatment). Instrumental variable analysis showed that genetically elevated levels of circulating triglycerides were not associated with increased baPWV. These results do not support the hypothesis that levels of circulating triglycerides have a causal role in the development of arterial stiffness.

Meta-analysis: silymarin and its combination therapy for the treatment of chronic hepatitis B.

Silymarin is used by many patients with chronic hepatitis B, but its efficacy remains unknown. The aim of this investigation was to conduct a meta-analysis to determine the efficacy and safety of silymarin and its combination therapy for the treatment of chronic hepatitis B. We searched Chinese and English reports from January 1966 to December 2011, using 12 databases. Two reviewers independently screened all reports to identify randomized placebo-controlled trials that evaluated silymarin and its combination therapy for the treatment of chronic hepatitis B. Twelve trials satisfied the eligibility criteria for this meta-analysis. Silymarin was equivalent to antiviral drug or protection liver drugs in serum transaminases, viral load and hepatic fibrosis markers. But silymarin combined with antiviral drug or antiviral drug and protection liver drugs significantly reduced the level of serum transaminases, hepatic fibrosis markers and serum TGF-ß1, TNF-α, IL-6 versus antiviral drug or protection liver drugs. Silymarin combined with protection liver drugs significantly reduced the level of serum transaminases, TBIL, hepatic fibrosis markers and was equivalent to protection liver drugs in the normalisation rates of serum transaminases, TBIL, but protection liver drugs significantly increased the improvement rates of hepatic fibrosis markers. Silymarin combined with antiviral drug or antiviral drug and protection liver drugs may have potential therapeutic value. Treatment with silymarin appears to be safe and well tolerated. The data are too limited to exclude a substantial benefit or harm of silymarin and its combination therapy on serum transaminases, and also to support recommending this herbal compound for the treatment of chronic hepatitis B.

Co-inherited ß-thalassemia trait and HbH disease: clinical characteristics and interference in diagnosis of thalassemia by high-performance liquid chromatography.

INTRODUCTION: To identify the clinical and hematological characteristics in a large group of patients with combined HbH disease and ß-thalassemia trait. METHODS: Hemoglobinopathy analysis and full genotyping identified a cohort of patients with HbH disease, ß-thalassemia trait, or combined HbH disease and ß-thalassemia trait. RESULTS: Co-inheritance of ß-thalassemia trait and HbH disease significantly decreased the mean corpuscular volume (MCV) in 27 patients when compared to 287 patients with HbH disease alone. The combined condition also alleviated anemia in nondeletional HbH disease but not in the deletional cases. Beta-thalassemia trait also significantly decreased the expression of HbH, Hb Constant Spring when present, and HbA(2) , with levels as low as 3.6% on high-performance liquid chromatography (HPLC). CONCLUSION: These cases, although relatively common in the South Chinese population, may be difficult do diagnose correctly when only examined on HPLC. Therefore, molecular analysis of the α and ß globin genes should be done in all cases with hemolytic anemia and low MCV without clear HbH disease or ß-thalassemia parameters.

Beta-cypermethrin impairs reproductive function in male mice by inducing oxidative stress.

Cypermethrin (CYP), an insecticide, has deleterious effects on male reproductive function. The objective was to identify whether the effects of beta-CYP on male reproductive organs were associated with oxidative stress. Three doses of beta-CYP (1, 10, and 20mg/kg) were administered to male mice for 35 d, with or without vitamin E (20mg/kg). The moderate (10mg/kg) and high (20mg/kg) doses of beta-CYP not only decreased body weight and the weight of the testes, epididymides, seminal vesicles, and prostate (P<0.05) but also reduced serum testosterone concentration and the expression of steroidogenic acute regulatory protein (P<0.05), in addition to damaging the seminiferous tubules and sperm development. Furthermore, moderate and high doses of beta-CYP administration decreased sperm number, sperm motility, and intact acrosome rate (P<0.05). Based on ultrastructural analyses, high doses of beta-CYP produced swelling and degeneration of mitochondria and the smooth endoplasmic reticulum of Leydig cells and caused the formation of concentric circles. These toxic effects of beta-CYP may be mediated by increasing oxidative stress, as the moderate and high doses of this compound increased malondialdehyde and nitric oxide in testes (P<0.05); reduced the activity of catalase, glutathione peroxidase (GSH-Px), and superoxide dismutase (P<0.05); and activated ERK1/2 (P<0.05). Vitamin E reversed the effects of beta-CYP on testosterone production and testis damage (P<0.05 vs. the high-dose group). Therefore, we inferred that beta-CYP damaged the structure of testes and decreased sperm output by inducing oxidative stress.

T-helper and T-cytotoxic cell subsets monitoring during active cytomegalovirus infection in liver transplantation.

OBJECTIVE: The objectives of this study was to analyze the peripheral blood T-lymphocyte subsets of the Th1-related versus Th2-related cytokines of CD4+ cells, and the Tc1 versus Tc2 cytokines of CD8+ cells liver transplant recipients with versus without active cytomegalovirus (CMV) infection. METHODS: Isolated peripheral blood mononuclear cells (PBMC) were stimulated using PMA/Ionomycin/Monensin. Interleukin (IL)-4 and interferon gamma (IFN-gamma) production by CD4+ and CD8+T cells were determined using fluorescence-activated cell sorter (FACS) analysis. RESULTS: The ratios of CD4/CD8 were significantly lower among active CMV-infected patients. The levels of Th2 and Tc2 cytokines (IL-4) were similar between CMV-infected and uninfected patients. However, the levels of Th1-type and Tc1-type cytokines (IFN-r) were significantly lower among active CMV-infected patients. CONCLUSIONS: Low levels of Th1-type cytokines seem to correlate with active CMV infection in liver transplant recipients.

Plant regeneration from in vitro-cultured seedling leaf protoplasts of Actinidia eriantha Benth.

Newly expanded in vitro leaves of Actinidia eriantha were used for protoplast isolation. Protoplasts were cultured in liquid MS medium (lacking NH4NO3) supplemented with 2,4-D (2,4-dichlorophenoxyacetic acid) and 0.4 M glucose. The plating efficiency after 3 weeks of culture was 19.4%, and calli were recovered without addition of fresh medium. These calli regenerated shoots on transfer to MS medium containing 2.28 µM zeatin and 0.57 µM IAA (indole-3-acetic acid). Regenerated shoots were rooted by immersion in 20 ppm IBA (indole-3-butyric acid) solution before culturing on half-strength MS medium lacking growth regulators. Somaclonal variation, in terms of chromosome number and nuclei per cell of protoplast-derived plants, was estimated.