Biomedical subjects

Zhen Zhang

Publications and source records attributed to Zhen Zhang.

At least 19 recordsLinked to original sources

PubMed2026

Clinical Validation of a Multiplex Urine Biomarker Assay for Surveillance of Recurrent Bladder Cancer.

PURPOSE: More than 50% of patients with non-muscle-invasive bladder cancer experience recurrence, requiring lifelong surveillance with repeated cystoscopy. Given the invasive nature and cost of cystoscopy, accurate noninvasive tools are needed to support risk-adapted monitoring. We evaluated the ability of Oncuria-Monitor, a multiplex urine biomarker assay, to detect recurrent bladder cancer during surveillance. PATIENTS AND METHODS: Between February 2017 and August 2020, six medical centers in the United States and Japan prospectively enrolled 300 patients with a history of bladder cancer, generating 1,248 serial urine samples. Participants were divided into training and validation cohorts. At each surveillance visit over 2 years, urine samples were analyzed in a blinded manner using Oncuria-Monitor and BladderChek, alongside urine cytology. Test performance was compared with cystoscopy and histopathology-confirmed recurrence. RESULTS: Recurrent bladder cancer was identified in 31% (93/300) of participants, with 143 total recurrences during follow-up, including 90 tumors in the validation cohort. In the validation cohort, Oncuria-Monitor achieved a sensitivity of 85.6% [95% confidence interval (CI), 78.1%-92.2%] and a negative predictive value (NPV) of 93% (95% CI, 89.2%-96.4%). In comparison, BladderChek demonstrated a sensitivity of 20.0% and an NPV of 88%, whereas urine cytology showed a sensitivity of 36.9% and an NPV of 91.6%. The number needed to evaluate to detect one recurrence was 3 for both cystoscopy and Oncuria-Monitor, compared with 15 for BladderChek and 8 for cytology. CONCLUSIONS: In this large prospective longitudinal study, Oncuria-Monitor demonstrated clinically actionable performance, enabling a rule-out strategy that could safely reduce cystoscopy in approximately 25% of surveillance visits. These findings support a paradigm shift toward biomarker-guided, risk-adapted surveillance in bladder cancer that reduces unnecessary invasive procedures while maintaining oncologic safety.

Humans↗

PubMed2026

Integrating clinical and genomic features to predict response to neoadjuvant therapy in microsatellite-stable rectal cancer.

BACKGROUND: Neoadjuvant therapy (NAT) has shifted rectal cancer management toward organ preservation. However, achieving a complete response (CR) for "watch-and-wait" strategies is hindered by high response heterogeneity. Although immunotherapy-combined NAT has expanded the candidate pools, the predictive significance of molecular alterations remains unclear. OBJECTIVES: This study aimed to evaluate clinical and genomic profiles of rectal cancer patients undergoing NAT to identify response predictors and to develop a nomogram for estimating CR probability. DESIGN: Retrospective, single-center cohort study. METHODS: This study included 437 patients with rectal adenocarcinoma at Fudan University Shanghai Cancer Center between December 2019 and March 2023. Patients underwent paired tumor and germline genomic sequencing (887-gene panel) before NAT. Logistic and Cox regression analyses were performed to identify clinical and genetic risk factors associated with tumor response and long-term survival. RESULTS: Of the 437 patients, 96.6% had microsatellite-stable (MSS) tumors. In the MSS locally advanced rectal cancer cohort (N = 307), the CR rate was 35.5%. Multivariate analysis identified immunotherapy-combined NAT (iTNT) (OR 4.41, 95% CI: 2.42-8.27), SYNE1 mutation (OR 2.12, 95% CI: 1.06-4.26), negative mesorectal fascia (MRF) status (OR 0.34, 95% CI: 0.17-0.66), and lower tumor location (OR 0.48, 95% CI: 0.27-0.84) as independent predictors of CR. KRAS mutation was the sole independent predictor of reduced disease-free survival (DFS; HR 1.93, 95% CI: (1.11-3.36), p = 0.020). KRAS G12D subtype was associated with the worst 2-year distant metastasis-free survival (71.3%) and exhibited a distinct predilection for lung metastasis. The clinical-genomic nomogram yielded strong discrimination (AUC = 0.705) and calibration, with favorable DCA net benefit. CONCLUSION: Clinical and genomic features jointly determine outcomes in MSS rectal cancer. SYNE1 mutation serves as a novel biomarker for CR, while KRAS mutations, especially the G12D subtype, identify patients at high risk for systemic relapse. The clinical-genomic nomogram facilitates individualized selection for organ-preservation strategies.

biomarker↗

PubMed2026

Characterization of ZIC5 expression in esophageal squamous cell carcinoma and its association with patient survival.

Esophageal squamous cell carcinoma (ESCC) is a prevalent malignancy known for its aggressive nature and poor prognosis. The present study aimed to investigate the expression levels and clinical importance of the Zic family member 5 (ZIC5) gene in ESCC. Gene expression data and survival information obtained from The Cancer Genome Atlas and Gene Expression Omnibus were utilized. In 176 patients with surgically resected ESCC, immunohistochemical analysis was conducted to validate the expression of ZIC5 protein in cancerous and adjacent tissues. The findings of the present study revealed a significant upregulation of ZIC5 in ESCC compared with normal tissues (P<0.05), which was further corroborated by immunohistochemistry exhibiting a notable association between ZIC5 expression and clinical parameters such as tumor size, invasion depth, lymph node metastasis and TNM staging (P<0.05). Survival analysis further indicated that high ZIC5 expression was an independent prognostic factor for poor outcomes in patients with ESCC (hazard ratio=1.519; 95% CI: 1.017-2.269; P<0.05). In addition, bioinformatic analyses predicted that hsa-microRNA-212-5p may regulate ZIC5 mRNA and gene enrichment analysis suggested that ZIC5 may facilitate ESCC progression through involvement in the cell cycle and DNA repair pathways. In conclusion, ZIC5 is highly expressed in ESCC and associated with a poor prognosis, indicating its potential as a therapeutic target and biomarker for ESCC management. Further studies are warranted to elucidate the precise mechanisms underlying the role of ZIC5 in ESCC progression.

ESCC↗

PubMed2026

From detection to action: ctDNA-MRD surveillance and translational strategies in early breast cancer.

Recurrence remains a major cause of mortality in early breast cancer (EBC), and conventional follow-up often identifies relapse only after clinically detectable disease has emerged. Circulating tumor DNA-based minimal residual disease (ctDNA-MRD) testing offers the possibility of detecting molecular relapse earlier and refining recurrence-risk assessment during follow-up. This narrative review examines the evolving role of ctDNA-MRD in EBC, focusing on assay interpretation, longitudinal surveillance, MRD-guided trial design, and clinical implementation. Prospective studies consistently show that postoperative or surveillance ctDNA positivity is associated with an increased risk of recurrence. However, test performance and interpretation vary with assay characteristics and sampling strategies, and whether treatment initiated solely on the basis of MRD positivity can improve patient outcomes remains unresolved. The central challenge is no longer simply to detect residual disease earlier, but to determine when and how that information should influence care. Further prospective validation, assay standardization, clear pathways for uncertain findings, and patient-centered implementation will be needed before ctDNA-MRD can be integrated into routine management of EBC.

circulating tumor DNA↗

PubMed2026

Loss of chromosome Y in hematopoietic cells: mechanisms and implications for human disease.

The chromosome Y, once thought to function primarily in male reproduction, is now recognized to have broader biological roles. Hematopoietic loss of chromosome Y (LOY) is one of the most frequent somatic genomic alterations in male blood, with prevalence increasing markedly with age. Advances in technology have enabled robust detection of LOY in blood at both the population scale and the single-cell level. Hematopoietic LOY arises from mitotic chromosome mis-segregation and is influenced by inherited genetic variation, environmental exposures, and aging. Population-based genome-wide association study (GWAS) analyses have identified robust epidemiological associations between hematopoietic LOY and cardiovascular disease, brain disease, immune disorders, and cancer. Mechanistic studies demonstrate that LOY has functional consequences, including altered gene expression, immune dysregulation, and clonal expansion. Some findings are strongly supported by CRISPR-based LOY mouse and cellular models, which recapitulate key disease-related phenotypes. Collectively, these findings establish hematopoietic LOY as a biologically meaningful form of somatic mosaicism with important implications for disease susceptibility.

Humans↗

PubMed2026

Exploring Causal Links Between 91 Circulating Inflammatory Proteins and Hashimoto's Thyroiditis: A Bidirectional Mendelian Randomization Study.

BACKGROUND: Increasing evidence has linked inflammation to Hashimoto's thyroiditis (HT) etiology. However, the causal role of circulating inflammatory proteins in HT remains uncertain. To investigate this, we conducted a bidirectional Mendelian randomization (MR) study. METHODS: Genetic data for 91 inflammatory proteins and HT were sourced from publicly available GWAS databases. The PhenoScanner database was then searched for pleiotropic SNPs associated with potential confounders. Inverse variance weighted (IVW) analysis was used as the primary analysis, simultaneously supplemented by five sensitivity analyses to strengthen the results. RESULTS: The results revealed that, after false discovery rate (FDR) correction, interleukin (IL)-12p40 was causally associated with increased risk of HT (OR [95% CI] = 1.295 [1.172, 1.431], p = 3.66 × 10-7, PFDR = 3.33×10-5). Conversely, none of the inflammatory proteins was a consequence of HT. CONCLUSION: This study suggests that IL-12p40 is probably one of the factors correlated with HT etiology, contributing to a better understanding of the pathogenesis of HT and underscoring the potential for therapeutic interventions targeting inflammatory proteins.

Humans↗

PubMed2025

Identification of marginal zone B cells in head and neck cancer with immunomodulatory characteristics.

INTRODUCTION: Recently we observed high numbers of marginal zone B cells (MZBs) within murine head and neck squamous cell carcinoma (HNSCC) with immunosuppressive potential. To date, MZBs have not been linked to tumor development or tumor prevention. OBJECTIVES: Based on our previous findings the present study aimed to validate the presence of MZB in HNSCC and to investigate their possible implications in tumorigenesis and prognosis. METHODS: Flow cytometry was used to uncover MZB within tumors and blood of HNSCC patients. A single-cell RNA sequencing cohort of 118 HNSCC patients across different disease stages and 6 healthy donors (HDs) was compiled. Comparative transcriptomic profiling of B lymphocytes between HNSCC and HDs were performed. Downstream analysis, such as pathway enrichment, cell-cell communication, pseudotime trajectory inference, survival correlation, and spatial transcriptomics were applied. RESULTS: Two MZB subsets were revealed in tissues and blood of HNSCC patients and HDs. The tumor-associated MZBs were featured with hypoxia stress and viral-related hallmark genes. MZB-2, characterized by elevated expression of activation markers and immune-regulatory genes, displayed strong interactions with CD4+ T cells and antigen-presenting cells. These interactions were supported by costimulatory signals in HDs but were absent in HNSCC patients. Co-localization of MZB-2, germinal center B cell (GCB), and CD4+ follicular helper T cell (Tfh) was detected in HNSCC, suggesting the presence of an intratumoral MZB-Tfh-GCB axis. Clinically, MZB-2 abundance was associated with favorable prognosis in early-stage HNSCC, but not in advanced disease. Immunosuppressive gene signatures were not exclusive to MZBs, indicating that they do not represent a purely regulatory B cell phenotype. CONCLUSION: Our findings demonstrate an immunomodulatory role of MZBs in tumor immunity, balancing antigen presentation, cytokine signaling, and immune suppression. The association of MZB-2 with improved prognosis in early-stage HNSCC highlights its potential as a beneficial regulator of antitumor immunity during early tumor progression.

Humans↗

PubMed2025

Identification of four novel ACADVL variants in eight Chinese unrelated patients with very long-chain acyl-CoA dehydrogenase deficiency.

BACKGROUND: Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) is a disorder of mitochondrial fatty acid oxidation with an autosomal recessive manner and is due to the VLCAD enzyme deficiency which is encoded by the ACADVL gene. The purpose of this study was to elucidate the clinical manifestations and analyze the molecular findings of eight Chinese patients with VLCADD. METHODS: We investigated eight Chinese VLCADD patients (three males, five females) from eight unrelated families. Molecular analysis was performed under the application of next-generation sequencing (NGS) in combination with Sanger sequencing validation to confirm the likely pathogenic variants in these patients. RESULTS: Patient 1 (P1) exhibited the most severe clinical features and passed away 3 h after admission on the second day of life. Unfortunately, P2 succumbed to hypoketotic hypoglycemia at 5 months of age. Except for asymptomatic P7, the remaining patients also developed clinical presentations of varying severity at different ages. Molecular data revealed that all affected individuals were compound heterozygotes for ACADVL variants. A total of 14 variants (4 novel and 10 known) were identified and the pathogenicity was evaluated based on the American College of Medical Genetics and Genomics (ACMG) criteria and different in silico prediction tools. CONCLUSIONS: The analysis of genotype-phenotype relationships preliminarily suggests that the compound heterozygous variants identified in these patients are likely the primary cause of VLCADD. Our study expands the mutation spectrum of ACADVL and highlights the significance of genetic analysis in early diagnosis and therapeutic intervention of monogenic hereditary diseases especially those with rapid disease progression.

Female↗

PubMed2025

An elegant co-transformation strategy for recalcitrant wheat using morphogenic regulators.

Common wheat (Triticum aestivum L.) is a vital global crop, but many elite cultivars remain recalcitrant to genetic transformation, hindering functional genomics and crop improvement. Here, we developed an efficient co-transformation strategy for recalcitrant wheat varieties (e.g., Aikang58 and Xinong979) using the morphogenic gene mTaGRF4-TaGIF1. This approach entails mixing Agrobacterium tumefaciens cultures carrying two separate vectors: a standard gene-of-interest (GOI) vector (containing a selectable marker) and a gene-of-co-transformation vector (GOC, expressing mTaGRF4-TaGIF1 without a selectable marker). Co-transformation enhanced regeneration efficiency to ~37.38% in AK58, a marked improvement over conventional methods, enabling consistent recovery of transgenic plants. Among regenerants, ~63.25% carried both GOI and GOC (GOI&GOC), while ~11.92% contained only the GOI. Only-GOI plants could also be obtained through progeny segregation from GOI&GOC lines. We successfully generated GUS- and RUBY-expressing transgenic lines, as well as CRISPR-Cas9-edited mutants targeting Q and Ph1 genes, confirming the method's efficacy for both gain-of-function and genome editing application. Furthermore, the strategy was successfully extended to another recalcitrant variety Xinong979, demonstrating its potential for broad applicability. Unlike existing methods dependent on complex excision systems or tissue-specific promoters, our co-transformation methodology significantly simplifies both vector design and procedural workflow while maintaining high efficiency. Collectively, these findings establish a technically advanced yet operationally simplified transformation platform that addresses the long-standing challenge of genetic transformation in recalcitrant wheat varieties, providing researchers with a powerful tool for functional genomics studies and accelerating precision breeding programs in elite wheat cultivars.

Triticum↗

PubMed2007

Time-resolved fluoroimmunoassay for ractopamine in swine tissue.

This study describes the development and validation of a time-resolved fluoroimmunoassay (TR-FIA) for screening ractopamine (RAC) in swine tissue. The method is based on the direct competitive-type immunoassay using europium-labeled anti-RAC monoclonal antibody as a tracer and RAC-ovalbumin as a solid-phase antigen. When RAC was spiked at levels of 1-10 microg kg(-1), recoveries ranged from 88.2 to 118.5% for swine liver and muscle with coefficients of variation from 7.1 to 20.5%. The detection limit was 0.1 microg kg(-1). The proposed TR-FIA method was applied to the determination of RAC in an actual residue study and the applicability was confirmed by liquid chromatography-tandem mass spectrometry.

Animals↗

PubMed2007

Tbx1 regulation of myogenic differentiation in the limb and cranial mesoderm.

The T-box transcription factor Tbx1 has been implicated in DiGeorge syndrome, the most frequent syndrome due to a chromosomal deletion. Gene inactivation of Tbx1 in mice results in craniofacial and branchial arch defects, including myogenic defects in the first and second branchial arches. A T-box binding site has been identified in the Xenopus Myf5 promoter, and in other species, T-box genes have been implicated in myogenic fate. Here we analyze Tbx1 expression in the developing chick embryo relating its expression to the onset of myogenic differentiation and cellular fate within the craniofacial mesoderm. We show that Tbx1 is expressed before capsulin, the first known marker of branchial arch 1 and 2 muscles. We also show that, as in the mouse, Tbx1 is expressed in endothelial cells, another mesodermal derivative, and, therefore, Tbx1 alone cannot specify the myogenic lineage. In addition, Tbx1 expression was identified in both chick and mouse limb myogenic cells, initially being restricted to the dorsal muscle mass, but in contrast, to the head, here Tbx1 is expressed after the onset of myogenic commitment. Functional studies revealed that loss of Tbx1 function reduces the number of myocytes in the head and limb, whereas increasing Tbx1 activity has the converse effect. Finally, analysis of the Tbx1-mesoderm-specific knockout mouse demonstrated the cell autonomous requirement for Tbx1 during myocyte development in the cranial mesoderm.

Animals↗

PubMed2007

The radiation-induced block in spermatogonial differentiation is due to damage to the somatic environment, not the germ cells.

Radiation and chemotherapeutic drugs cause permanent sterility in male rats, not by killing most of the spermatogonial stem cells, but by blocking their differentiation in a testosterone-dependent manner. However, it is not known whether radiation induces this block by altering the germ or the somatic cells. To address this question, we transplanted populations of rat testicular cells containing stem spermatogonia and expressing the green fluorescent protein (GFP) transgene into various hosts. Transplantation of the stem spermatogonia from irradiated adult rats into the testes of irradiated nude mice, which do not show the differentiation block of their own spermatogonia, permitted differentiation of the rat spermatogonia into spermatozoa. Conversely transplantation of spermatogonial stem cells from untreated prepubertal rats into irradiated rat testes showed that the donor spermatogonia were able to colonize along the basement membrane of the seminiferous tubules but could not differentiate. Finally, suppression of testosterone in the recipient irradiated rats allowed the differentiation of the transplanted spermatogonia. These results conclusively show that the defect caused by radiation in the rat testes that results in the block of spermatogonial differentiation is due to injury to the somatic compartment. We also observed colonization of tubules by transplanted Sertoli cells from immature rats. The present results suggest that transplantation of spermatogonia, harvested from prepubertal testes to adult testes that have been exposed to cytotoxic therapy might be limited by the somatic damage and may require hormonal treatments or transplantation of somatic elements to restore the ability of the tissue to support spermatogenesis.

Animals↗

PubMed2006

Multiple Coordination of CO on Molybdenum Nanoparticles: Evidence for Intermediate Mox(CO)y Species by XPS and UPS.

CO chemisorption on the metallic molybdenum nanoparticles supported on the thin alumina film was investigated by X-ray photoelectron spectroscopy (XPS) and ultraviolet photoelectron spectroscopy (UPS). A binary compound of molybdenum and CO is found to be formed on the surface upon CO dose, accompanied with a positive binding energy shift of the Mo 3d doublet and a localized Mo 4d valence band. A loose packing of the metallic molybdenum favors the formation of this intermediate Mox(CO)y species. The formation of the Mox(CO)y species implies that the property of the metallic molybdenum nanoparticles on the thin alumina film is much different from that of the bulk molybdenum, indicating a significant nanometer size effect.

Journal Article↗

PubMed2006

Isolated and characterization of a cDNA encoding ethylene-responsive element binding protein (EREBP)/AP2-type protein, RCBF2, in Oryza sativa L.

A transcription factor RCBF2 which interacts with C-repeat/DRE was isolated from Oryza sativa L. by a yeast one-hybrid method. Analysis of the deduced RCBF2 amino acid sequence revealed that RCBF2 contained a conserved ethylene-responsive element binding protein (EREBP)/AP2 domain of 59 amino acids and a potential nuclear localization sequence. RCBF2 showed a high level of homology with other CBF family members only in AP2 domain. Phylogenetic analysis showed that RCBF2 might be different from other eight DRE-binding proteins on evolutionary relationship. The semi-quantitative RT-PCR (s-Q RT-PCR) analysis indicated the expression of RCBF2 gene was induced by cold, dehydration and high-salinity, but not by abscisic acid, and the transcription of RCBF2 gene accumulated primarily in rice immature seeds, growing point and shoots.

Base Sequence↗

PubMed2006

Gene expression signatures differentiate ovarian/peritoneal serous carcinoma from diffuse malignant peritoneal mesothelioma.

PURPOSE: Ovarian/primary peritoneal serous carcinoma (OC/PPC) and diffuse peritoneal malignant mesothelioma (DMPM) are highly aggressive tumors that are closely related morphologically and histogenetically. It remains unclear whether both tumors are molecularly distinct neoplasms. The current study compared global gene expression patterns in OC/PPC and DMPM. EXPERIMENTAL DESIGN: Ten OC/PPC and five DMPM effusions were analyzed for gene expression profiles using the Affymetrix U133 Plus 2 arrays and the dCHIP analysis program. Differentially expressed candidate genes were validated using quantitative real-time PCR and immunohistochemistry. RESULTS: Unsupervised hierarchical clustering using all 54,675 genes in the array classified the samples into two groups: DMPM specimens versus OC/PPC specimens. A total of 189 genes that were differentially expressed in these two groups were selected based on statistical significance. Genes overexpressed in DMPM (n = 68) included calretinin, vitronectin, claudin 15, alpha4 laminin, hyaluronan synthase 1, cadherin 11, RAB7, v-maf, and the epidermal growth factor-containing fibulin-like extracellular matrix protein 1. Genes overexpressed in OC/PPC (n = 121) included insulin-like growth factor II (IGF-II); IGF-II binding protein 3; cyclin E1; folate receptors 1 and 3; RAB25; MUC4; endothelin-1; CD24; kallikreins 6, 7, and 8; claudins 3, 4, and 6; Notch3; and MMP-7. Quantitative real-time PCR validated the differential expression of 13 genes, and immunohistochemistry confirmed the differences for four gene products. CONCLUSIONS: Expression profiling separates OC/PPC from DMPM and identifies a number of genes that are differentially expressed in these tumors. The molecular signatures unique to OC/PPC and DMPM should provide a molecular basis to study both tumors and new potential markers for facilitating their differential diagnosis.

Carcinoma↗

PubMed2006

Determination of the in vivo stoichiometry of tyrosyl radical per betabeta' in Saccharomyces cerevisiae ribonucleotide reductase.

The class I ribonucleotide reductases catalyze the conversion of nucleotides to deoxynucleotides and are composed of two subunits: R1 and R2. R1 contains the site for nucleotide reduction and the sites that control substrate specificity and the rate of reduction. R2 houses the essential diferric-tyrosyl radical (Y(*)) cofactor. In Saccharomyces cerevisiae, two R1s, alpha(n) and , have been identified, while R2 is a heterodimer (betabeta'). beta' cannot bind iron and generate the Y(*); consequently, the maximum amount of Y(*) per betabeta' is 1. To determine the cofactor stoichiometry in vivo, a FLAG-tagged beta ((FLAG)beta) was constructed and integrated into the genome of Y300 (MHY343). This strain facilitated the rapid isolation of endogenous levels of (FLAG)betabeta' by immunoaffinity chromatography, which was found to have 0.45 +/- 0.08 Y(*)/(FLAG)betabeta' and a specific activity of 2.3 +/- 0.5 micromol min(-1) mg(-1). (FLAG)betabeta' isolated from MMS-treated MHY343 cells or cells containing a deletion of the transcriptional repressor gene CRT1 also gave a Y(*)/(FLAG)betabeta' ratio of 0.5. To determine the Y(*)/betabeta' ratio without R2 isolation, whole cell EPR and quantitative Western blots of beta were performed using different strains and growth conditions. The wild-type (wt) strains gave a Y(*)/betabeta' ratio of 0.83-0.89. The same strains either treated with MMS or containing a crt1Delta gave ratios between 0.49 and 0.72. Nucleotide reduction assays and quantitative Western blots from the same strains provided an independent measure and confirmation of the Y(*)/betabeta' ratios. Thus, under normal growth conditions, the cell assembles stoichiometric amounts of Y(*) and modulation of Y(*) concentration is not involved in the regulation of RNR activity.

Blotting, Western↗

PubMed2006

Tbx1 regulates population, proliferation and cell fate determination of otic epithelial cells.

The T-box transcription factor Tbx1 is required for inner ear morphogenesis. Tbx1 null mutants have a small otocyst that fails to grow and remodel and does not give rise to the vestibular and cochlear apparata. Here we show that Tbx1 expression-driven cell tracing identifies a population of otic epithelial cells that contributes to most of the otocyst. Tbx1 is essential for the contribution of this population to the inner ear. Ablation of Tbx1 after this cell population has established itself in the otocyst, restores marker expression lost in germ line mutants, but causes severe reduction in mitotic activity, cell autonomously. Furthermore, timed cell fate mapping demonstrates that loss of Tbx1 switches the fate of some members of the Tbx1-dependent cell population, from non-neurogenic to neurogenic, an event associated with activation of the Delta-Notch pathway. Finally, tissue-specific ablation of Tbx1 demonstrates that, while the abovementioned phenotypic abnormalities are due to loss of epithelial expression of Tbx1, cochlear morphogenesis requires mesodermal Tbx1 expression. We conclude that the main functions of Tbx1 in the inner ear are to control, cell-autonomously, contribution, size and fate of a large population of otic epithelial cells, and, cell non-autonomously, cochlear morphogenesis.

Animals↗

PubMed2006

High efficiency and throughput system in directed evolution in vitro of reporter gene.

In vitro directed evolution, especially with DNA shuffling, is a powerful means in biological studies of protein structure and function, and consequently for industrial applications. Escherichia coli beta-glucuronidase (gusA) gene, a versatile and efficient reporter gene, was the model for studying in vitro directed evolution because of its stability, easy analysis of the enzyme properties and conveniently visible phenotype. We developed a high efficiency, throughput system for in vitro directed evolution using gusA reporter gene as the model. The system consisted mainly of three aspects: a prokaryotic expression vector pYPX251, an easy method for obtaining the mutated gene from DNA shuffling and a suitable selected strategy. The vector pYPX251 carried the moderately strong aacC1 gene promoter and T1T2 transcription terminator that allowed expression in E. coli. Over 10,000 individuals could be selected individually in a 9 cm Petri dish after colonies were absorbed on a nitrocellulose filter. A library, which contained 100,000 individuals was screened by incubating ten filter papers with X-Glu. The polymerase chain reaction products of the gusA gene, the fragments of 50-100 bp, with high mutation rates were purified using a dialysis bag from 10% PAGE after electrophoresis. The possibility of obtaining desirable mutations was increased dramatically as the size of the library expanded. A GUS variant, named GUS-TR, was obtained through this system, which is significantly more resistant to high temperature than the wild type enzyme. GUS-TR maintained its high activity even when the nitrocellulose filter containing the variant colony was heated at 100 degrees C for 30 min.

Biotechnology↗