Management of anastomotic biliary stricture through utilizing percutaneous transhepatic cholangioscopy.

AIM: Occurrence of anastomotic biliary stricture (AS) remains an essential issue following hepatobiliary surgeries, and percutaneous transhepatic cholangioscopy (PTCS) has great therapeutic significance in handling refractory AS for patients with altered gastrointestinal anatomy after cholangio-jejunostomy. This present study aimed to investigate feasibility of PTCS procedures in AS patients for therapeutic indications. MATERIALS AND METHODS: This study was a single-center, retrospective cohort study with a total number of 124 consecutive patients who received therapeutic PTCS due to AS. Clinical success rate, required number, and adverse events of therapeutic PTCS procedures as well as patients survival state were reviewed. RESULTS: These 124 patients previously underwent choledochojejunostomy or hepatico-jejunostomy, and there was post-surgical altered gastrointestinal anatomy. Overall, 366 therapeutic PTCS procedures were performed for these patients through applying rigid choledochoscope, and the median time of PTCS procedures was 3 (1-11). Among these patients, there were 34 cases (27.32%) accompanied by biliary strictures and 100 cases (80.65%) were also combined with biliary calculi. After therapeutic PTCS, most patients presented with relieved clinical manifestations and improved liver functions. The median time of follow-up was 26 months (2-86 months), and AS was successfully managed through PTCS procedures in 104 patients (83.87%). During the follow-up period, adverse events occurred in 81 cases (65.32%), most of which were tackled through supportive treatment. CONCLUSION: PTCS was a feasible, safe and effective therapeutic modality for refractory AS, which may be a promising alternative approach in clinical cases where the gastrointestinal anatomy was changed after cholangio-jejunostomy.

IL-33/ST2 Signaling and its Correlation with Macrophage Heterogeneity and Clinicopathologic Features in Human Intrahepatic Cholangiocarcinoma.

BACKGROUND: IL-33/ST2 signaling plays crucial roles in the development and progression of various human malignancies. However, its significance in intrahepatic cholangiocarcinoma (ICC) still remains unclear. OBJECTIVE: This study aimed to investigate the expression of IL-33/ST2 signaling and its correlations with macrophage heterogeneity and ICC patients' clinicopathologic features. METHODS: The expression of different phenotype macrophage markers and IL-33/ST2 signalingrelated markers was detected. The correlation between L-33/ST2 signaling and different phenotype macrophage markers as well as ICC patients' clinicopathologic data was evaluated. RESULTS: Massive heterogeneous cancer cells and PAS-positive cells were observed in tumor tissues. CD68-positive cells accumulated in tumor tissues and expression of both M1 phenotype markers and M2 phenotype macrophage markers was higher in tumor samples than para-carcinoma samples. However, M2 phenotype macrophages represented the dominant macrophage population in ICC tissues. Plasma levels of IL-33, ST2, and MIF were evidently enhanced in ICC patients compared to healthy controls. IL-33/ST2 signaling-related markers exhibited a massive increase in tumor samples than para-carcinoma samples. IL-33 and ST2 expression in ICC tissues was positively associated with M1 and M2 phenotype macrophages. Plasma levels of IL-33, ST2, and MIF were correlated with the diameter of tumor lesions, lymph node metastasis, TNM stage, and tumor differentiation degree. Multivariate analysis demonstrated IL-33 expression to exhibit a correlation with the diameter of tumor lesions, lymph node metastasis, and TNM stage. Additionally, there was a relationship observed between ST2, MIF expression, and diameter of tumor lesions plus TNM stage. CONCLUSION: IL-33/ST2 signaling exhibited a positive relationship with macrophage heterogeneity in ICC tissues, and upregulated levels of IL-33, ST2, and MIF were associated with aggressive clinicopathologic characteristics. These findings may provide promising diagnostic biomarkers and potential therapeutic strategies for ICC patients targeting IL-33/ST2 signaling.

Effects of continuous monoculture on rhizosphere soil nutrients, growth, physiological characteristics, hormone metabolome of Casuarina equisetifolia and their interaction analysis.

Continuous planting is unavoidable in agricultural production, but continuous planting affects plant growth and physiological characteristics. In this study, we analyzed rhizosphere soil nutrients, physiological characteristics, hormone metabolome changes and their interactions of Casuarina equisetifolia (C. equisetifolia) with the increase of continuous planting number. The results found that C. equisetifolia root was significantly inhibited, the plant height was dwarfed and the biomass was significantly reduced as continuous planting number increased. Secondly, continuous planting caused a decrease in the rhizosphere soil nutrient transformation capacity, and a significant decrease in the total soil nutrient and available nutrient content. Analysis of physiological indexes showed that continuous planting resulted in a decrease in nitrogen, phosphorus, and potassium content, a decrease in the activity of physiological indexes of resistance, and a decrease in photosynthetic capacity of C. equisetifolia leaves. Hormone metabolome analysis showed that continuous planting critically affected the accumulation of five characteristic hormones in C. equisetifolia leaves, in which salicylic acid 2-O-ß-glucoside (SAG), 2-oxindole-3-acetic acid (OxIAA), trans-zeatin-O-glucoside (tZOG) and gibberellin A3 (GA3) content decreased significantly while abscisic acid (ABA) content increased significantly. In conclusion, continuous planting lowered the rhizosphere soil nutrient transformation capacity of C. equisetifolia, lowered the soil available nutrient content, inhibited their root growth, and hindered the nutrient uptake and transportation by the root, thus led to the decrease of the nutrient accumulation capacity in the leaves of C. equisetifolia, and the decrease of SAG, OxIAA, and tZOG, GA3 synthesis ability decreased, ABA accumulated in large quantities, C. equisetifolia resistance and photosynthesis ability decreased, and their growth was impeded. This study provides insights for the effective management of continuous planting in the cultivation of C. equisetifolia.

N6-Methyladenosine Reader YTHDF1 Promotes Stemness and Therapeutic Resistance in Hepatocellular Carcinoma by Enhancing NOTCH1 Expression.

N6-methyladenosine (m6A) RNA modification is the most common and conserved epigenetic modification in mRNA and has been shown to play important roles in cancer biology. As the m6A reader YTHDF1 has been reported to promote progression of hepatocellular carcinoma (HCC), it represents a potential therapeutic target. In this study, we evaluated the clinical significance of YTHDF1 using human HCC samples and found that YTHDF1 was significantly upregulated in HCCs with high stemness scores and was positively associated with recurrence and poor prognosis. Analysis of HCC spheroids revealed that YTHDF1 was highly expressed in liver cancer stem cells (CSC). Stem cell-specific conditional Ythdf1 knockin (CKI) mice treated with diethylnitrosamine showed elevated tumor burden as compared with wild-type mice. YTHDF1 promoted CSCs renewal and resistance to the multiple tyrosine kinase inhibitors lenvatinib and sorafenib in patient-derived organoids and HCC cell lines, which could be abolished by catalytically inactive mutant YTHDF1. Multiomic analysis, including RNA immunoprecipitation sequencing, m6A methylated RNA immunoprecipitation sequencing, ribosome profiling, and RNA sequencing identified NOTCH1 as a direct downstream of YTHDF1. YTHDF1 bound to m6A modified NOTCH1 mRNA to enhance its stability and translation, which led to increased NOTCH1 target genes expression. NOTCH1 overexpression rescued HCC stemness in YTHDF1-deficient cells in vitro and in vivo. Lipid nanoparticles targeting YTHDF1 significantly enhanced the efficacy of lenvatinib and sorafenib in HCC in vivo. Taken together, YTHDF1 drives HCC stemness and drug resistance through an YTHDF1-m6A-NOTCH1 epitranscriptomic axis, and YTHDF1 is a potential therapeutic target for treating HCC. SIGNIFICANCE: Inhibition of YTHDF1 expression suppresses stemness of hepatocellular carcinoma cells and enhances sensitivity to targeted therapies, indicating that targeting YTHDF1 may be a promising therapeutic strategy for liver cancer.

Metagenomics-based exploration of key soil microorganisms contributing to continuously planted Casuarina equisetifolia growth inhibition and their interactions with soil nutrient transformation.

Casuarina equisetifolia (C. equisetifolia) is an economically important forest tree species, often cultivated in continuous monoculture as a coastal protection forest. Continuous planting has gradually affected growth and severely restricted the sustainable development of the C. equisetifolia industry. In this study, we analyzed the effects of continuous planting on C. equisetifolia growth and explored the rhizosphere soil microecological mechanism from a metagenomic perspective. The results showed that continuous planting resulted in dwarfing, shorter root length, and reduced C. equisetifolia seedling root system. Metagenomics analysis showed that 10 key characteristic microorganisms, mainly Actinoallomurus, Actinomadura, and Mycobacterium, were responsible for continuously planted C. equisetifolia trees. Quantitative analysis showed that the number of microorganisms in these three genera decreased significantly with the increase of continuous planting. Gene function analysis showed that continuous planting led to the weakening of the environmental information processing-signal transduction ability of soil characteristic microorganisms, and the decrease of C. equisetifolia trees against stress. Reduced capacity for metabolism, genetic information processing-replication and repair resulted in reduced microbial propagation and reduced microbial quantity in the rhizosphere soil of C. equisetifolia trees. Secondly, amino acid metabolism, carbohydrate metabolism, glycan biosynthesis and metabolism, lipid metabolism, metabolism of cofactors and vitamins were all significantly reduced, resulting in a decrease in the ability of the soil to synthesize and metabolize carbon and nitrogen. These reduced capacities further led to reduced soil microbial quantity, microbial carbon and nitrogen, microbial respiration intensity, reduced soil enzyme nutrient cycling and resistance-related enzyme activities, a significant reduction in available nutrient content of rhizosphere soils, a reduction in the ion exchange capacity, and an impediment to C. equisetifolia growth. This study provides an important basis for the management of continuously planted C. equisetifolia plantations.

Effect of continuous planting on Casuarina equisetifolia rhizosphere soil physicochemical indexes, microbial functional diversity and metabolites.

Continuous planting has a severe impact on the growth of Casuarina equisetifolia. In this study, the effects of three different long-term monocultures (one, two and three replanting) on the physicochemical indexes, microbial functional diversity, and soil metabolomics were analyzed in C. equisetifolia rhizosphere soil. The results showed that rhizosphere soil organic matter content, cation exchange capacity, total and available nitrogen, total and available phosphorus, and total and available potassium contents significantly decreased with the increasing number of continuous plantings. The evaluation of microbial functional diversity revealed a reduction in the number of soil microorganisms that rely on carbohydrates for carbon sources and an increase in soil microorganisms that used phenolic acid, carboxylic acid, fatty acid, and amines as carbon sources. Soil metabolomics analysis showed a significant decrease in soil carbohydrate content and a significant accumulation of autotoxic acid, amine, and lipid in the C. equisetifolia rhizosphere soil. Consequently, the growth of C. equisetifolia could hinder total nutrient content and their availability. Thus, valuable insights for managing the cultivation of C. equisetifolia and soil remediation were provided.

A new method for evaluating the heat leak of cryogenic vessels without exhaust gas.

The thermal insulation performance of cryogenic vessels is evaluated by the boil-off gas (BOG) rate and heat leak as a reference standard; however, it consumes considerable testing time and working medium. In this study, we proposed a method of evaluating the thermal insulation performance of cryogenic vessels from the heat leak in the self-pressurization process, which can minimize the testing time and preserve the working medium. To improve the accuracy of heat leak calculation, the vapor temperature distribution of cryogenic vessels is analyzed and considered in the calculation of heat leak. However, it is ignored in the current standard test method. In this study, the energy equations of cryogenic vessels in the self-pressurization process were established by considering the vapor temperature distribution. Then, based on the experimental data, the heat leak was calculated for demonstrating the thermal insulation performance of cryogenic vessels depending on the new method. The results indicate that the vapor temperature should be seriously considered when the liquid level ratio is less than 50 %.

Exosomes derived from TGF-ß1-pretreated mesenchymal stem cells alleviate biliary ischemia-reperfusion injury through Jagged1/Notch1/SOX9 pathway.

BACKGROUND: This study aimed to evaluate the efficacy of exosomes (EXO) derived from TGF-ß1-pretreated mesenchymal stem cells (MSCs) on biliary ischemia reperfusion injury (IRI) and further reveal the possible mechanisms. METHODS: Bone marrow-derived MSCs were treated with exogenous TGF-ß1, Jagged1/Notch1/SOX9 pathway inhibitor LY450139, or their combination. Then, EXO were isolated from the culture supernatants and further characterized. After establishing IRI model of biliary epithelial cells (EpiCs), EXO derived from differently-treated MSCs were applied to detect their protective effects on EpiCs, and LY450139 was applied in EpiCs to detect the possible mechanisms after treatment with MSCs-EXO. EXO derived from differently-treated MSCs were further injected into the hepatic artery immediately after establishment of intrahepatic biliary IRI for animal studies. RESULTS: Pretreatment with TGF-ß1 significantly enhanced MSCs-EXO production and elevated the levels of massive miRNAs associated with anti-apoptosis and tissue repair, which were evidently decreased after TGF-ß1 plus LY450139 cotreatment. Notable improvement was observed in EpiCs after MSCs-EXO treatment, evidenced by reduced cellular apoptosis, increased cellular proliferation and declined oxidative stress, which were more evident in EpiCs that were treated with EXO derived from TGF-ß1-pretreated MSCs. However, application of EXO derived from TGF-ß1 plus LY450139-cotreated MSCs reversely enhanced cellular apoptosis, decreased cellular proliferation and anti-oxidants production. Interestingly, LY450139 application in EpiCs after treatment with MSCs-EXO also reversed the declined cellular apoptosis and enhanced oxidative stress induced by TGF-ß1 pretreatment. In animal studies, administration of EXO derived from TGF-ß1-pretreated MSCs more effectively attenuated biliary IRI through reducing oxidative stress, apoptosis, inflammation and enhancing the expression levels of TGF-ß1 and Jagged1/Notch1/SOX9 pathway-related markers, which were reversed after administration of EXO derived from TGF-ß1 plus LY450139-cotreated MSCs. CONCLUSION: Our results provided a vital insight that TGF-ß1 pretreatment endowed MSCs-EXO with stronger protective effects to improve biliary IRI via Jagged1/Notch1/SOX9 pathway.

Multiomic Analysis Reveals Comprehensive Tumor Heterogeneity and Distinct Immune Subtypes in Multifocal Intrahepatic Cholangiocarcinoma.

PURPOSE: Targeted therapy and immunotherapy are transforming the treatment approach for intrahepatic cholangiocarcinoma (ICC). However, little is known about the intertumor heterogeneity (ITH) of multifocal ICC and its impacts on patient response to these treatments. We aimed to characterize the immunogenomic and epigenomic heterogeneity of multifocal ICC to guide treatment decision making. EXPERIMENTAL DESIGN: We obtained 66 tumor samples from 16 patients with multifocal ICC and characterized the tumor and immune heterogeneity using whole-exome sequencing, bulk and single-cell RNA sequencing, methylation microarray, and multiplex immunostaining. Patients were divided into high- or low-ITH groups according to the median ITH index. Two independent cohorts were used to validate findings. Responses to anti-PD-1 therapy were assessed. RESULTS: Multifocal ICC presented considerable intertumor genomic, transcriptional, and epigenomic heterogeneity within a patient in high ITH group. The immune profile among multiple tumors within a patient was relatively less heterogeneous in high- or low-ITH group, and consistent responses of multiple tumors to anti-PD-1 immunotherapy were observed. Unsupervised clustering of immune markers identified one low and one high immune subtype, with higher immune cell infiltration, closer tumor-immune cell interactions, and upregulated IFN-signature expression in high-immune subtype. Determining expression levels of CD8B and ICOS facilitated this immune classification and prediction of patient prognosis. Finally, promoter DNA methylation contributed to different immune profiles of two subtypes by regulating immune-gene expression. CONCLUSIONS: There is comprehensive heterogeneity in the genome, transcriptome, and epigenome of multifocal ICC. On the basis of the less heterogeneous immune profile of ICC, we suggest an immune classification that stratifies patients' prognosis and may support personalized immunotherapy.

Insufficient Radiofrequency Ablation Promotes Hepatocellular Carcinoma Metastasis Through N6-Methyladenosine mRNA Methylation-Dependent Mechanism.

BACKGROUND AND AIMS: The dynamic N6-methyladenosine (m6 A) mRNA modification is essential for acute stress response and cancer progression. Sublethal heat stress from insufficient radiofrequency ablation (IRFA) has been confirmed to promote HCC progression; however, whether m6 A machinery is involved in IRFA-induced HCC recurrence remains open for study. APPROACH AND RESULTS: Using an IRFA HCC orthotopic mouse model, we detected a higher level of m6 A reader YTH N6-methyladenosine RNA binding protein 1-3 (YTHDF1) in the sublethal-heat-exposed transitional zone close to the ablation center than that in the farther area. In addition, we validated the increased m6 A modification and elevated YTHDF1 protein level in sublethal-heat-treated HCC cell lines, HCC patient-derived xenograft (PDX) mouse model, and patients' HCC tissues. Functionally, gain-of-function/loss-of-function assays showed that YTHDF1 promotes HCC cell viability and metastasis. Knockdown of YTHDF1 drastically restrains the tumor metastasis evoked by sublethal heat treatment in tail vein injection lung metastasis and orthotopic HCC mouse models. Mechanistically, we found that sublethal heat treatment increases epidermal factor growth receptor (EGFR) m6 A modification in the vicinity of the 5' untranslated region and promotes its binding with YTHDF1, which enhances the translation of EGFR mRNA. The sublethal-heat-induced up-regulation of EGFR level was further confirmed in the IRFA HCC PDX mouse model and patients' tissues. Combination of YTHDF1 silencing and EGFR inhibition suppressed the malignancies of HCC cells synergically. CONCLUSIONS: The m6 A-YTHDF1-EGFR axis promotes HCC progression after IRFA, supporting the rationale for targeting m6 A machinery combined with EGFR inhibitors to suppress HCC metastasis after RFA.

Long Non-Coding RNA LUCAT1 Promotes Proliferation and Invasion in Clear Cell Renal Cell Carcinoma Through AKT/GSK-3ß Signaling Pathway.

BACKGROUND/AIMS: Long non-coding RNAs (lncRNAs) have emerged as new regulators and biomarkers in several cancers. However, few lncRNAs have been well characterized in clear cell renal cell carcinoma (ccRCC). METHODS: We investigated the lncRNA expression profile by microarray analysis in 5 corresponding ccRCC tissues and adjacent normal tissues. Lung cancer-associated transcript 1 (LUCAT1) expression was examined in 90 paired ccRCC tissues by real-time PCR and validated by The Cancer Genome Atlas (TCGA) database. Kaplan-Meier analysis was used to examine the prognostic value of LUCAT1 and CXCL2 in ccRCC patients. Loss and gain of function were performed to explore the effect of LUCAT1 on proliferation and invasion in ccRCC cells. Western blotting was performed to evaluate the underlying mechanisms of LUCAT1 in ccRCC progression. Chemokine stimulation assay was performed to investigate possible mechanisms controlling LUCAT1 expression in ccRCC cells. Enzyme-linked immunosorbent assays were performed to determine serum CXCL2 in ccRCC patients and healthy volunteers. Receiver operating characteristic curve analysis was performed to examine the clinical diagnostic value of serum CXCL2 in ccRCC. RESULTS: We found that LUCAT1 was significantly upregulated in both clinical ccRCC tissues (n = 90) and TCGA ccRCC tissues (n = 448) compared with normal tissues. Statistical analysis revealed that the LUCAT1 expression level positively correlated with tumor T stage (P < 0.01), M stage (P < 0.01), and TNM stage (P < 0.01). Overall survival and disease-free survival time were significantly shorter in the high-LUCAT1-expression group than in the low-LUCAT1-expression group (log-rank P < 0.01). LUCAT1 knockdown inhibited ccRCC cell proliferation and colony formation, induced cell cycle arrest at G1 phase, and inhibited cell migration and invasion. Overexpression of LUCAT1 promoted proliferation, migration, and invasion of ccRCC cells. Mechanistic investigations showed that LUCAT1 induced cell cycle G1 arrest by regulating the expression of cyclin D1, cyclin-dependent kinase 4, and phosphorylated retinoblastoma transcriptional corepressor 1. Moreover, LUCAT1 promoted proliferation and invasion in ccRCC cells partly through inducing the phosphorylation of AKT and suppressing the phosphorylation of GSK-3ß. We also revealed that chemokine CXCL2, upregulated in ccRCC, induced LUCAT1 expression and might be a diagnostic and prognostic biomarker in ccRCC. CONCLUSIONS: LUCAT1 was upregulated in ccRCC tissues and renal cancer cell lines, and significantly correlated with malignant stage and poor prognosis in ccRCC. LUCAT1 promoted proliferation and invasion in ccRCC cells through the AKT/GSK-3ß signaling pathway. We also revealed that LUCAT1 overexpression was induced by chemokine CXCL2. These findings indicate that the CXCL2/LUCAT1/AKT/GSK-3ß axis is a potential therapeutic target and molecular biomarker for ccRCC.

Upregulated GAPLINC predicts a poor prognosis in bladder cancer patients and promotes tumor proliferation and invasion.

Previous studies have demonstrated that long noncoding RNAs (lncRNAs) exhibit critical regulatory roles in cancer biology. However, few lncRNAs have been well characterized in bladder cancer. In the previous study, we demonstrated that gastric adenocarcinoma associated, positive CD44 regulator, long intergenic noncoding RNA (GAPLINC) was significantly upregulated in bladder cancer tissues compared with normal tissues in The Cancer Genome Atlas (TCGA) cohort (P=0.039) and a validated cohort of 80 patients with bladder cancer (P=0.021). Statistical analysis revealed that GAPLINC expression level was associated with tumor stage in the validated cohort (P=0.017). Kaplan-Meier analysis demonstrated that patients in the high GAPLINC expression group had a worse overall survival (P=0.0386), indicating that GAPLINC may be a sensitive prognostic biomarker for patients with bladder cancer. Furthermore, knockdown of GAPLINC inhibited cell proliferation and colony formation, promoted cells cycle arrest at G1 phase and suppressed cells migration and invasion. The findings of the present study suggest that GAPLINC exhibits an oncogenic role in bladder cancer and may be a potential prognostic biomarker and therapeutic target.

Knockdown of long non-coding RNA PVT1 induces apoptosis and cell cycle arrest in clear cell renal cell carcinoma through the epidermal growth factor receptor pathway.

Previous years have witnessed the importance of long non-coding RNAs (lncRNAs) in cancer research. The lncRNA Pvt1 oncogene (non-protein coding) (PVT1) was revealed to be upregulated in various cancer types. The aim of the present study was to investigate the function of PVT1 in clear cell renal cell carcinoma (ccRCC). The expression of PVT1 in ccRCC was analyzed using reverse transcription-quantitative polymerase chain reaction, and it was revealed that PVT1 expression was upregulated in ccRCC tissues compared with that in normal adjacent tissues. Next, PVT1 expression from The Cancer Genome Atlas datasets was validated, and it was also revealed that the high expression of PVT1 was associated with advanced disease stage and a poor prognosis. Furthermore, the knockdown of PVT1 induced apoptosis by increasing the expression of poly ADP ribose polymerase and Bcl-2-associated X protein, and promoted cell cycle arrest at the G1 phase by decreasing the expression of cyclin D1. Study of the mechanism involved indicated that PVT1 promoted the progression of ccRCC partly through activation of the epidermal growth factor receptor pathway. Altogether, the results of the present study suggested that PVT1 serves oncogenic functions and may be a biomarker and therapeutic target in ccRCC.

CXCL13/CXCR5 Axis Predicts Poor Prognosis and Promotes Progression Through PI3K/AKT/mTOR Pathway in Clear Cell Renal Cell Carcinoma.

The chemokine ligands and their receptors play critical roles in cancer progression and patients outcomes. We found that CXCL13 was significantly upregulated in ccRCC tissues compared with normal tissues in both The Cancer Genome Atlas (TCGA) cohort and a validated cohort of 90 pairs ccRCC tissues. Statistical analysis showed that high CXCL13 expression related to advanced disease stage and poor prognosis in ccRCC. We also revealed that serum CXCL13 levels in ccRCC patients (n = 50) were significantly higher than in healthy controls (n = 40). Receiver operating characteristic (ROC) curve revealed that tissue and serum CXCL13 expression might be a diagnostic biomarker for ccRCC with an area under curve (AUC) of 0.809 and 0.704, respectively. CXCL13 was significantly associated with its receptor, CXCR5, in ccRCC tissues, and ccRCC patients in high CXCL13 high CXCR5 expression group have a worst prognosis. Functional and mechanistic study revealed that CXCL13 promoted the proliferation and migration of ccRCC cells by binding to CXCR5 and activated PI3K/AKT/mTOR signaling pathway. These results suggested that CXCL13/CXCR5 axis played a significant role in ccRCC and might be a therapeutic target and prognostic biomarker.

Stable Expression of Basic Fibroblast Growth Factor in Chloroplasts of Tobacco.

Basic fibroblast growth factor (bFGF) is a multifunctional factor in acceleration of cell proliferation, differentiation and transference, and therefore widely used in clinical applications. In this study, expression vector pWX-Nt03 harboring a codon-optimized bFGF gene was constructed and introduced into the tobacco chloroplasts by particle bombardment. After four rounds of selection, bFGF was proved to integrate into the chloroplast genome of regenerated plants and two of four transgenic plants were confirmed to be homoplastomic by PCR and Southern hybridization. ELISA assay indicated that bFGF represented approximately 0.1% of total soluble protein in the leaves of transplastomic tobacco plants. This is the first report of bFGF expression via chloroplast transformation in model plant, providing an additional option for the production of chloroplast-produced therapeutic proteins.

A new approach for obtaining rapid uniformity in rice (Oryza sativa L.) via a 3x x 2x cross.

A triploid (2n = 3x = 36) rice plant was obtained by screening a twin seedling population in which each seed germinated to two or three sprouts that were then crossed with diploid plants. One diploid plant was chosen among the various F(1) progenies and developed into an F (2) population via self-pollination. Compared with the control variety Shanyou 63, this F (2) population had a stable agronomical performance in field trials, as confirmed by the F-test. The stability of the F (2) population was further substantiated by molecular analysis with simple sequence repeat markers. Specifically, of 160 markers assayed, 37 (covering all 12 chromosomes) were polymorphic between the parental lines. Testing the F (1) hybrid individually with these markers showed that each PCR product had only a single band instead of two bands from each parent. The bands were identical to either maternal (23 markers) or paternal (eight markers) bands or distinct from both parents (six markers). The amplified bands of all 60 randomly selected F (2) plants were uniform and identical to those of the F (1) hybrid. These results suggest that the F (1) plant is a non-segregating hybrid and that a stable F (2) population was obtained. This novel system provides an efficient means for shortening the cycle of hybrid rice seed production.

A new approach for obtaining rapid uniformity in rice (Oryza sativa L. ) via a 3x x 2x cross

A triploid (2n = 3x = 36) rice plant was obtained by screening a twin seedling population in which each seed germinated to two or three sprouts that were then crossed with diploid plants. One diploid plant was chosen among the various F1 progenies and developed into an F2 population via self-pollination. Compared with the control variety Shanyou 63, this F2 population had a stable agronomical performance in field trials, as confirmed by the F-test. The stability of the F2 population was further substantiated by molecular analysis with simple sequence repeat markers. Specifically, of 160 markers assayed, 37 (covering all 12 chromosomes) were polymorphic between the parental lines. Testing the F1 hybrid individually with these markers showed that each PCR product had only a single band instead of two bands from each parent. The bands were identical to either maternal (23 markers) or paternal (eight markers) bands or distinct from both parents (six markers). The amplified bands of all 60 randomly selected F2 plants were uniform and identical to those of the F1 hybrid. These results suggest that the F1 plant is a non-segregating hybrid and that a stable F2 population was obtained. This novel system provides an efficient means for shortening the cycle of hybrid rice seed production.