Phosphoglycerate Kinase 1: An Effective Therapeutic Target in Cancer.

Phosphoglycerate kinase 1 (PGK1) serves as a pivotal enzyme in the cellular glycolysis pathway, facilitating adenosine-triphosphate (ATP) production in tumor cells and driving the Warburg effect. PGK1 generates ATP through the reversible phosphorylation reaction of 1,3-bisphosphoglycerate (1,3-BPG) to Mg-adenosine-5'-diphosphate (Mg-ADP). In addition to its role in regulating cellular metabolism, PGK1 plays a pivotal role in autophagy induction, regulation of the tricarboxylic acid cycle (TCA), and various mechanisms including tumor cell drug resistance, and so on. Given its multifaceted functions within cells, the involvement of PGK1 in many types of cancer, including breast cancer, astrocytoma, metastatic colon cancer, and pancreatic ductal adenocarcinoma, is intricate. Notably, PGK1 can function as an intracellular protein kinase to coordinate tumor growth, migration, and invasion via posttranslational modifications (PTMs). Furthermore, elevated expression levels of PGK1 have been observed in cancer tissues, indicating its association with unfavorable treatment outcomes and prognosis. This review provides a comprehensive summary of PGK1's expression pattern, structural features, functional properties, involvement in PTMs, and interaction with tumors. Additionally highlighted are the prospects for developing and applying related inhibitors that confirm the indispensable value of PGK1 in tumor progression.

Advances in MiRNAs Involved in Endometrial Carcinoma.

Endometrial carcinoma (EC) is a common malignancy worldwide. Existing evidence has revealed that EC could be associated with abnormal gene expression. Meantime, evidence supports that miRNAs act as critical regulators in gene expression through the binding to the 3'- untranslated region (3'-UTR). Accordingly, this review concludes some recent studies focusing on miRNAs that influence EC, aiming at understanding the association between miRNAs and EC more clearly and providing a reference for further studies on miRNA-related drugs treating EC.

Black phosphorus-based nanoparticles induce liver cancer cell mitochondrial apoptosis and immune cell tumor infiltration for enhancing dendritic cell therapy.

Cellular immunotherapy is a crucial aspect of current tumor immunotherapy, though it presents several challenges such as immune cell dysfunction, limited recognition of neoantigens, and inadequate lymphocyte infiltration into the tumor microenvironment. This study proposes a novel approach utilizing a combination of dendritic cell (DC)-based cellular immunotherapy and a photothermal nanoadjuvant black phosphorus (BP) nanoparticles to overcome these challenges. A new platform called PLGA@BP-R848, which consists of modifying poly-(lactic-co-glycolic acid) (PLGA) onto BP nanosheets loading the immune adjuvant R848. The PLGA@BP-R848 nanoparticles demonstrated exceptional drug delivery and release capabilities, as well as a photothermal effect, biocompatibility, and the ability to activate the mitochondrial apoptotic pathway Blc-2-Bax-Cytochrome c-caspase-3 and inhibit the PI3K-AKT-mTOR signaling pathway. In a hepatocellular carcinoma mouse model, the binding of PLGA@BP-R848 nanoparticles and dendritic cells primed with GPC3 peptides, successfully induced a systemic anti-tumor immune response. PLGA@BP-R848 nanoparticles bolster immune cell infiltration into tumors and induce cancer cell apoptosis. The synergistic therapy involving dendritic cells and photothermal nanoadjuvant effectively suppressed tumor growth, and facilitated the formation of tertiary lymphatic structures (TLS) in tumors. This study presents a novel approach in using photothermal nanoadjuvants to advance antitumor effect of cellular immunotherapy, such as DCs therapy.

Promotion of ICD via Nanotechnology.

Immunotherapy represents the most promising treatment strategy for cancer, but suffers from compromised therapeutic efficiency due to low immune activity of tumor cells and an immunosuppressive microenvironment, which significantly hampers the clinical translations of this treatment strategy. To promote immunotherapy with desired therapeutic efficiency, immunogenic cell death (ICD), a particular type of death capable of reshaping body's antitumor immune activity, has drawn considerable attention due to the potential to stimulate a potent immune response. Still, the potential of ICD effect remains unsatisfactory because of the intricate tumor microenvironment and multiple drawbacks of the used inducing agents. ICD has been thoroughly reviewed so far with a general classification of ICD as a kind of immunotherapy strategy and repeated discussion of the related mechanism. However, there are no published reviews, to the authors' knowledge, providing a systematic summarization on the enhancement of ICD via nanotechnology. For this purpose, this review first discusses the four stages of ICD according to the development mechanisms, followed by a comprehensive description on the use of nanotechnology to enhance ICD in the corresponding four stages. The challenges of ICD inducers and possible solutions are finally summarized for future ICD-based enhanced immunotherapy.

Regulation of progesterone receptor expression in endometriosis, endometrial cancer, and breast cancer by estrogen, polymorphisms, transcription factors, epigenetic alterations, and ubiquitin-proteasome system.

The uterus and breasts are hormone-responsive tissues. Progesterone and estradiol regulate gonadotropin secretion, prepare the endometrium for implantation, maintain pregnancy, and regulate the differentiation of breast tissue. Dysregulation of these hormones causes endometriosis, endometrial cancer, and breast cancer, damaging the physical and mental health of women. Emerging evidence has shown that progesterone resistance or elevated progesterone activity is the primary hormonal substrate of these diseases. Since progesterone acts through its specific nuclear receptor, the abnormal expression of the progesterone receptor (PR) dysregulates progesterone function. This review discusses the regulatory mechanisms of PR expression in patients with endometriosis, and endometrial or breast cancer, including estrogen, polymorphisms, transcription factors, epigenetics, and the ubiquitin-proteasome system. (1) Estrogen promotes the expression of PRA (a PR isoform) mRNA and protein through the interaction of estrogen receptors (ERs) and Sp1 with half-ERE/Sp1 binding sites. ERs also affect the binding of Sp1 and Sp1 sites to promote the expression of PRB (another PR isoform)(2) PR polymorphisms, mainly PROGINS and + 331 G/A polymorphism, regulate PR expression by affecting DNA methylation and transcription factor binding. (3) The influence of epigenetic alterations on PR expression occurs through DNA methylation, histone modification, and microRNA. (4) As one of the main protein degradation pathways in vivo, the ubiquitin-proteasome system (UPS) regulates PR expression by participating in protein degradation. These mechanisms may provide new molecular targets for diagnosing and treating endometriosis, endometrial, and breast cancer.

Circulating Tumor DNA-A Novel Biomarker of Tumor Progression and Its Favorable Detection Techniques.

Cancer is the second leading cause of death in the world and seriously affects the quality of life of patients. The diagnostic techniques for tumors mainly include tumor biomarker detection, instrumental examination, and tissue biopsy. In recent years, liquid technology represented by circulating tumor DNA (ctDNA) has gradually replaced traditional technology with its advantages of being non-invasive and accurate, its high specificity, and its high sensitivity. ctDNA may carry throughout the circulatory system through tumor cell necrosis, apoptosis, circulating exosome secretion, etc., carrying the characteristic changes in tumors, such as mutation, methylation, microsatellite instability, gene rearrangement, etc. In this paper, ctDNA mutation and methylation, as the objects to describe the preparation process before ctDNA analysis, and the detection methods of two gene-level changes, including a series of enrichment detection techniques derived from PCR, sequencing-based detection techniques, and comprehensive detection techniques, are combined with new materials. In addition, the role of ctDNA in various stages of cancer development is summarized, such as early screening, diagnosis, molecular typing, prognosis prediction, recurrence monitoring, and drug guidance. In summary, ctDNA is an ideal biomarker involved in the whole process of tumor development.

Mediation of synergistic chemotherapy and gene therapy via nanoparticles based on chitosan and ionic polysaccharides.

Nanoparticles (NPs)-based on various ionic polysaccharides, including chitosan, hyaluronic acid, and alginate have been frequently summarized for controlled release applications, however, most of the published reviews, to our knowledge, focused on the delivery of a single therapeutic agent. A comprehensive summarization of the co-delivery of multiple therapeutic agents by the ionic polysaccharides-based NPs, especially on the optimization of the polysaccharide structure for overcoming various extracellular and intracellular barriers toward maximized synergistic effects, to our knowledge, has been rarely explored so far. For this purpose, the strategies used for overcoming various extracellular and intracellular barriers in vivo were introduced first to provide guidance for the rational design of ionic polysaccharides-based NPs with desired features, including long-term circulation, enhanced cellular internalization, controllable drug/gene release, endosomal escape and improved nucleus localization. Next, four preparation strategies were summarized including three physical methods of polyelectrolyte complexation, ionic crosslinking, and self-assembly and a chemical conjugation approach. The challenges and future trends of this rapidly developing field were finally discussed in the concluding remarks. The important guidelines on the rational design of ionic polysaccharides-based NPs for maximized synergistic efficiency drawn in this review will promote the future generation and clinical translation of polysaccharides-based NPs for cancer therapy.

Association of the PROGINS PgR polymorphism with susceptibility to female reproductive cancer: A meta-analysis of 30 studies.

AIMS: The progesterone response of the nuclear progesterone receptor plays an important role in the female reproductive system. Changes in the function of the progesterone receptor gene may increase the risk of reproductive cancer. The present study performed a meta-analysis to examine whether the progesterone receptor gene PROGINS polymorphism was a susceptibility factor for female reproductive cancer. MATERIALS AND METHODS: We searched the PubMed, Cochrane Library, Web of Science and EMBASE databases for literature on PROGINS polymorphisms and female reproductive cancer published before September 2020. We evaluated the risk using odds ratios [ORs] and 95% confidence intervals via fixed effects models and random-effects models, which were calculated for all five genetic models. We grouped the analyses by race, cancer, and HWE. RESULTS: Thirty studies comprised of 25405 controls and 19253 female reproductive cancer cases were included in this meta-analysis. We observed that the Alu insertion polymorphism and the V660L polymorphism were significantly associated with female reproductive cancer in the allele and dominant genetic models. The allele genetic model and (Alu-insertion polymorphism: OR = 1.22, 95% CI = 1.02-1.45; V660L polymorphism: OR = 1.02, 95% CI = 1.00-1.13) dominant genetic model (Alu-insertion polymorphism: OR = 1.27, 95% CI = 1.03-1.58; V660L polymorphism: OR = 1.10, 95% CI = 1.011.19) demonstrated a significantly increased risk of female reproductive cancer. A subgroup analysis according to ethnicity found that the Alu insertion was associated with female reproductive cancer incidence in white (Allele model: OR = 1.21, 95% CI = 1.00-1.45; Heterozygous model: OR = 3.44, 95% CI = 1.30-9.09) and Asian (Dominant model: OR = 3.12, 95% CI = 1.25-7.79) populations, but the association disappeared for African and mixed racial groups. However, the V660L polymorphism was significantly associated with female reproductive cancer in the African (Allele model: OR = 2.52, 95% CI = 1.14-5.56; Heterozygous model: OR = 2.83, 95% CI = 1.26-6.35) and mixed racial groups (Dominant model: OR = 1.28, 95% CI = 1.01-1.62). Subgroup analysis by cancer showed that the PROGINS polymorphism increased the risk of cancer in the allele model, dominant mode and heterozygous model, but the confidence interval for this result spanned 1 and was not statistically significant. This sensitivity was verified in studies with HWE greater than 0.5. CONCLUSION: Our meta-analysis showed that the progesterone receptor gene Alu insertion and the V660L polymorphism contained in the PROGINS polymorphism were susceptibility factors for female reproductive cancer.

Correction: Enhanced transglycosylation activity of an Endo-F3 mutant by ligand-directed localization.

Correction for 'Enhanced transglycosylation activity of an Endo-F3 mutant by ligand-directed localization' by Xiangman Zou et al., Org. Biomol. Chem., 2022, 20, 3086-3095, https://doi.org/10.1039/D2OB00030J.

Application of rational emotive behavior therapy in patients with colorectal cancer undergoing adjuvant chemotherapy.

Objective: This study aimed to explore the effects of our rational emotive behavior therapy (REBT) program on symptoms, anxiety, depression, and sleep state in patients with colorectal cancer (CRC) undergoing chemotherapy. Methods: From October 2020 to May 2021, fifty-six patients with CRC in a hospital in the Hunan Province were randomly divided into an intervention group (n = 28) and a control group (n = 28). The patients in the intervention group completed a 6-week REBT program based on routine nursing care, including four courses: 1) establish a relationship and formulate health files; 2) group communications and study symptom management; 3) continuously provide health knowledge and strengthen healthy behavior; and 4) review the treatment and summary. The control group maintained routine nursing care. The simplified Chinese version of the Memorial Symptom Assessment Scale Short Form (MSAS-SF-SC), the Hospital Anxiety and Depression Scale (HADS), and the Pittsburgh Sleep Quality Index (PSQI) scale were used to investigate and compare the intervention effects of the two groups at baseline (T1, before the intervention), four weeks (T2), and six weeks (T3) after the intervention. Results: The intervention group was significantly improved in symptoms, anxiety, depression, and sleep state, compared with the control group. At T2, MSAS-SF-SC (24.43 ± 4.26 vs.28.07 ± 3.91), symptom distress (17.29 ± 4.04 vs. 19.39 ± 3.59), symptom frequency (7.14 ± 1.51 vs. 8.68 ± 1.42), HADS (13.68 ± 3.38 vs. 15.86 ± 3.79), anxiety (3.89 ± 1.85 vs. 5.18 ± 2.18), and depression (9.79 ± 2.06 vs. 10.68 ± 2.23), showed that the difference between the two groups was statistically significant (P < 0.05). At T3, MSAS-SF-SC (23.89 ± 3.54 vs. 30.14 ± 3.94), symptom distress (17.61 ± 3.52 vs. 21.32 ± 3.57), symptom frequency (6.29 ± 1.49 vs. 8.82 ± 1.47), HADS (11.82 ± 2.57 vs. 16.29 ± 3.13), anxiety (3.21 ± 1.64 vs. 5.61 ± 1.77), and depression (8.61 ± 1.52 vs. 10.68 ± 1.81), showed that the difference between the two groups was statistically significant (P < 0.05). The sleep state of the intervention group was better than the control group at T3, with decreased score of PSQI [4.00 (3.00,8.00) vs. 9.00 (7.00,12.50), Z = -3.706, P < 0.001]. Conclusion: The 6-week REBT program can effectively improve the symptom, anxiety, depression, and sleep state of patients with CRC undergoing chemotherapy, which could as a care plan for patients with CRC who are repeatedly admitted to the hospital for chemotherapy.

Enhanced transglycosylation activity of an Endo-F3 mutant by ligand-directed localization.

At present, numerous studies have been reported to remodel the N-glycans of therapeutic antibodies for the gain of functions. Among the ways of remodeling antibody N-glycans, the chemoenzymatic glycoengineering approach by endoglycosidase (ENGase) has been deeply investigated and provided a significant tool for IgG glycoengineering. Among these cases, the transglycosylation activity of Endo-F3, compared to Endo-S and S2, is insufficient and limits its power in remodeling IgG glycosylation. Herein, we chemically conjugated the Endo-F3 mutant D165A with an Fc binding peptide (FcBP), aiming to improve the affinity of Endo-F3 D165A to IgGs, and therefore enhance the transglycosylation activity of D165A. In this report, we investigated the conjugation site of FcBP to D165A and the linkers between them and found that the conjugation indeed significantly increases the transglycosylation activity of D165A. Meanwhile, we optimized the FcBP-D165A catalyzed transglycosylation process, including the enzyme quantity, oxazoline concentration, and so on. Finally, by this method, we remodeled the N-glycans of rituximab and trastuzumab into homogeneous S2G2F, G2F, GN2M3, and M3 types with decreased enzyme quantity, oxazoline ratio, and catalyzing time. This method not only provides an enhanced ENGase for IgG glycoengineering but also suggests that ligand-directed localization of enzymes is a potential strategy to enhance the activity of enzymes towards the targeted substrate.

Icariin Stimulates hFOB 1.19 Osteoblast Proliferation and Differentiation via OPG/RANKL Mediated by the Estrogen Receptor.

BACKGROUND: Icariin (ICA), one of the main effective components isolated from the traditional Chinese herb Epimedium brevicornu Maxim., has been reported to possess extensive pharmacological actions, including enhanced sexual function, immune regulation, anti-inflammation, and antiosteoporosis. METHODS: Our study was designed to investigate the effect of ICA on cell proliferation and differentiation and the molecular mechanism of OPG/RANKL mediated by the Estrogen Receptor (ER) in hFOB1.19 human osteoblast cells. RESULTS: The experimental results show that ICA can stimulate cell proliferation and increase the activity of Alkaline Phosphatase (ALP), Osteocalcin (BGP) and I Collagen (Col I) and a number of calcified nodules. Furthermore, the mRNA and protein expression of OPG and RANKL and the OPG/ RANKL mRNA and protein expression ratios were upregulated by ICA. The above-mentioned results indicated that the optimal concentration of ICA for stimulating osteogenesis was 50ng/mL. Subsequent mechanistic studies comparing 50ng/mL ICA with an estrogen receptor antagonist demonstrated that the effect of the upregulated expression is connected with the estrogen receptor. In conclusion, ICA can regulate bone formation by promoting cell proliferation and differentiation and upregulating the OPG/RANKL expression ratio by the ER in hFOB1.19 human osteoblast cells.

A novel genotyping technique for discriminating LVAS-associated high-frequency variants in SLC26A4 gene.

An increasing number of biological and epidemiological evidence suggests that c.919-2A > G and c.2168A > G variants of solute carrier family 26, member 4 (SLC26A4) gene play a critical role in the development of large vestibular aqueduct syndrome (LVAS). In this study, we developed a rapid genotyping method for discriminating LVAS-associated high-frequency variants in SLC26A4 gene. The genotyping technique consists of 3' terminal exonuclease-resistant phosphorothioate-modified allele specific primer extension mediated by exo+ polymerase. In PCR amplification by Pfu polymerase, allelic specific primers perfectly matching wild type allele were extended while no specific products were yielded from primers targeting variant allele. Similarly, allelic specific primers perfectly matching variant allele were extended and no specific products were observed from primers targeting wild type allele. The clinical application of 3' terminal phosphorothioate-modified allele specific primer extension mediated by Pfu polymerase identified both homozygous for SLC26A4 gene c.919-2A > G variant in two patients clinically diagnosed as LVAS by temporal bone CT scan. The genetic results from this method are consistent with that of DNA sequencing. The data suggest that exo+ polymerase-mediated 3' terminal phosphorothioate-modified primer extension is reliable in the identification of SLC26A4 gene high-frequency variant prior to high-resolution CT scan. The method is extremely suitable for quickly molecular etiologic screening and early diagnosis and aggressive prevention therapy of LVAS.

[Association of EGFR gene G719S and T790M mutations with cervical cancer].

OBJECTIVE: To establish a rapid and accurate "on/off" switch technique consisted of 3'-phosphorothioate-modified allele-specific primers and exo+ polymerase to screen the G719S and T790M mutations of epidermal growth factor receptor (EGFR) gene. The switch was used to identify cervical cancer patients who are sensitive to tyrosine kinase inhibitor (TKI). METHODS: Allele-specific primers targeting recombinant wild-type and mutation-type templates were designed with 3' terminal phosphorothioate modification. Two-directional primer extension was carried out using Pfu polymerase. The G719S and T790M mutations were detected by the technique among cervical cancer tissues. The results were verified by Sanger sequencing. RESULTS: No mutation was detected among the 80 cervical cancer cases, and the results were consistent with that of Sanger sequencing. No significant difference was found between the frequencies of the G719S and T790M mutations between the patient and the control groups (P> 0.05). CONCLUSION: A sensitive "on/off" switch technique for detecting the two EGFR mutations was established. The G719S and T790M mutations are not associated with cervical cancer.

A Novel Assay Coupling Dephosphorylation and Blue/White Colony Screening for the G > A Hotspot Mutation at Codon 13 of KRAS Gene.

Development of sensitive assay for detection of hotspot mutations of cancer driving gene is crucial for circulating tumor DNA analysis. This study tested the possibilities of applying restriction enzyme digestion and dephosphorylation coupled with blue/white screening technology for analyzing a hotspot point mutation in codon 13 of KRAS gene. The present study has documented that the combination of PCR with restriction digestion, dephosphorylation, blue/white screening and Sanger's sequencing can identify rare mutations with sensitivities at 0.003%. This novel assay with high sensitivity may have application in the diagnosis of early cancer targeting ctDNAs.

Curcumin enhances vascular contractility via induction of myocardin in mouse smooth muscle cells.

A variety of cardiovascular diseases is accompanied by the loss of vascular contractility. This study sought to investigate the effects of curcumin, a natural polyphenolic compound present in turmeric, on mouse vascular contractility and the underlying mechanisms. After mice were administered curcumin (100 mg·kg-1·d-1, ig) for 6 weeks, the contractile responses of the thoracic aorta to KCl and phenylephrine were significantly enhanced compared with the control group. Furthermore, the contractility of vascular smooth muscle (SM) was significantly enhanced after incubation in curcumin (25 µmol/L) for 4 days, which was accompanied by upregulated expression of SM marker contractile proteins SM22α and SM α-actin. In cultured vascular smooth muscle cells (VSMCs), curcumin (10, 25, 50 µmol/L) significantly increased the expression of myocardin, a "master regulator" of SM gene expression. Curcumin treatment also significantly increased the levels of caveolin-1 in VSMCs. We found that as a result of the upregulation of caveolin-1, curcumin blocked the activation of notch1 and thereby abolished Notch1-inhibited myocardin expression. Knockdown of caveolin-1 or activation of Notch1 signaling with Jagged1 (2 µg/mL) diminished these effects of curcumin in VSMCs. These findings suggest that curcumin induces the expression of myocardin in mouse smooth muscle cells via a variety of mechanisms, including caveolin-1-mediated inhibition of notch1 activation and Notch1-mediated repression of myocardin expression. This may represent a novel pathway, through which curcumin protects blood vessels via the beneficial regulation of SM contractility.

Progesterone levels on the hCG day and outcomes in vitro fertilization in women with polycystic ovary syndrome.

BACKGROUND: The purpose of this study was to determine the incidence of premature luteinization in patients with polycystic ovary syndrome and compared the main determinants of success in in-vitro fertilization in PCOS patients with and without premature luteinization. METHODS: Retrospective analysis of 180 PCOS women of Chinese Han origin with infertility who underwent controlled ovarian hyperstimulation (COH) with an exogenous gonadotropin/GnRH antagonist protocol. Hormone levels on the hCG day and IVF outcomes were assessed. RESULTS: The incidence of premature luteinization was 23.3 %. Compared with PCOS patients without premature luteinization, PCOS patients with premature luteinization(PL) had a higher number of oocytes retrieved (18.20 ± 6.6 vs 15.08 ± 7.3, p = 0.037) and a higher fertilization rate (72.9 ± 1.9 vs63.1 ± 2.3, p = 0.033), but clinical pregnancy rates were no statistical significance (53.3 vs 56.0, p = 0.836). Though the implantation rate was higher in no premature luteinization patients, but the difference was not statistically significant (37.7 vs 30.3, p = 0.115). CONCLUSION: The PCOS patients with premature luteinization had a higher fertilization rate and high number of oocytes retrieved, and the similar implantation rate and clinical PRs as PCOS patients without premature luteinization.

Discrimination of A1555G and C1494T point mutations in the mitochondrial 12S rRNA gene by on/off switch.

The objective of this study was to apply the "on/off" switch consisting of 3' phosphorothioate-modified allele specific primers and exo(+) polymerase in single base discrimination of A1555G and C1494T mutations in the highly conserved sites of the mitochondrial 12S rRNA. The two point mutations are the hotspot mutations associated with either aminoglycoside antibiotics induced deafness or inherited nonsyndromic hearing loss. The PCR products of mitochondrial DNA (mtDNA) 12S rRNA gene were inserted into the pMD19-T vector for transformation into Escherichia coli JM109 competent cells for preparing wild-type pMD19-T/mt vector. Inverse PCR was carried out for mtDNA 12S rRNA gene C1494T and A1555G mutagenesis and DpnI endonuclease degradating methylated pMD19-T/mt vector existing in the inverse PCR products was carried out to construct the mutation-type pMD19-T/mtM vector. These constructed vectors were confirmed by DNA sequencing. Allelic specific primers targeting wild-type and mutation-type templates were designed with 3' terminal phosphorothioate modification. Two-directional primer extension was performed using Pfu polymerases. Amplified by exo(+) polymerase, allelic specific primers perfectly matching wild-type allele were extended while no products were produced from primers targeting point-mutated deafness-related allele. Similarly, allelic specific primers perfectly matching point-mutated deafness-related mutation-type allele were extended and no products were yielded from primers targeting wild-type allele. No specific product was observed in the primer extension reaction mediated by on/off switch in screening the mtDNA 12S rRNA gene harboring either C1494T or A1555G mutation in 40 healthy volunteers tested. These data suggest that the "off switch" mediated by exo(+) polymerase is highly reliable in the diagnosis of monogenic diseases and the novel "on/off" switch has enormous applications in systematic and extended screening of the12S rRNA gene A1555G and C1494T mutations. The established assay can be widely used not only for hearing loss patients but also for normal subjects before the use of aminoglycoside antibiotics.

[Preparation and identification of monoclonal antibody against human thrombomodulin.].

To produce specific monoclonal antibody (McAb) against human thrombomodulin (hTM), the full-length hTM cDNA-expressing plasmid pThr402 was transfected into CHO cells by Lipofectamine 2000 reagent. The hTM-expressing CHO cells, which was confirmed by flow cytometry and Western blot, were obtained by G418 selection. Then the McAb against hTM was prepared with classic hybridoma technique. A cell line of CHO-TM5 with high level of hTM was used to immunize female Balb/c mice 3 times at an interval of 4 weeks. On the third day after the third immunization, mice were sacrificed and spleen cells were harvested to prepare hybridoma cells with SP2/0 cells at the ratio of 10 to 1. Hybridoma cells were then cultured at 96-well plates for screening. Cellular enzyme-linked immunoabsorbent assay (CELISA) was applied twice. The first CELISA was done with polythene ELISA plate with a monolayer of CHO-TM5 cells. The positive clones from the first screen were then selected by reacting with similar screening ELISA plate but with CHO cell monolayer instead. Only clones that were positive for the first screening and negative for the second screening were kept, and called as CHO-TM5(+)CHO(-) hybridoma cells. Balb/c mice were intraperitoneally injected with the selected hybridoma cells. Ascites were collected and monoclonal antibodies were purified using FPLC, and its Ig class, subclass, and titer were then determined respectively. The specificity of the yielded McAb was identified with CELISA, flow cytometry, ABC immunohistochemistry and immunoblotting. One line of hybridoma cells with high expression of specific McAb against hTM, NH-1, was obtained. The Ig subclass of the McAb was IgG1 and the titer of ascitic McAb was 1x10(-6). Flow cytometry, CELISA and Western blot assays demonstrated that McAb NH-1 could specifically recognize hTM expressed in CHO-TM5 cells and human umbilical vascular endothelial cells. Meanwhile, the tissue specificity of antigen recognized by McAb NH-1 was identified by immunohistochemical ABC staining. NH-1 can specifically recognize the natural hTM expressed mainly in vascular endothelial cells, which will potentially be useful for investigation of the functions and clinic values of hTM.

On/off switch mediated by Exo+ polymerases: experimental analysis for its physiological and technological implications.

The potential physiological role and technological application of the premature termination of DNA polymerization through the off-switch of exo+ polymerases were studied using 3' phosphorothioate-modified or unmodified primers with single base mismatch distal to the 3' terminus. With exonuclease-digestible unmodified primers, a gradient premature termination of DNA polymerization was observed when amplified with exo+ polymerases. With 3' allele specific phosphorothioate-modified primers, an efficient off-switch effect occurred in the discrimination of a single nucleotide polymorphism when directly using genomic DNA. Clearly, the off-switch of exo+ polymerases is useful in biomedical research.