Low intensity pulsed ultrasound ameliorates Adriamycin-induced chronic renal injury by inhibiting ferroptosis.

OBJECTIVE: It is very important to develop a new therapeutic strategy to cope with the increasing morbidity and mortality of chronic kidney disease (CKD). As a kind of physical therapy, low intensity pulsed ultrasound (LIPUS) has remarkable anti-inflammatory and repair-promoting effects and is expected to become a new therapeutic method for CKD. This study aims to clarify the treatment effect of LIPUS on CKD-related renal inflammation and fibrosis, and to further explore the potential signal network of LIPUS treatment for ameliorating chronic renal injury. METHODS: A rat model simulating the progress of CKD was established by twice tail-vein injection of Adriamycin (ADR). Under anesthesia, bilateral kidneys of CKD rats were continuously stimulated by LIPUS for four weeks. The parameters of LIPUS were 1.0 MHz, 60 mW/cm2, 50% duty cycle and 20 min/d. RESULTS: LIPUS treatment effectively inhibited ADR-induced renal inflammation and fibrosis, and improved CKD-related to oxidative stress and ferroptosis. In addition, the therapeutic effect of LIPUS is closely related to the regulation of TGF-ß1/Smad and Nrf2/keap1/HO-1 signalling pathways. DISCUSSION: This study provides a new direction for further mechanism research and lays an important foundation for clinical trials.

Transthoracic, thoracoabdominal, and transabdominal surgical approaches for gastric cardia adenocarcinomas: a survival evaluation based on a cohort of 7103 patients.

BACKGROUND: This study compared the survival outcomes of different surgical approaches to determine the optimal approach for gastric cardia adenocarcinoma (GCA) and aimed to standardize the surgical treatment guidelines for GCA. METHODS: A total of 7103 patients with GCA were enrolled from our previously established gastric cardia and esophageal carcinoma databases. In our database, when the epicenter of the tumor was at or within 2 cm distally from the esophagogastric junction, the adenocarcinoma was considered to originate from the cardia and was considered a Siewert type 2 cancer. The main criteria for the enrolled patients included treatment with radical surgery, no radio- or chemotherapy before the operation, and detailed clinicopathological information. Follow-up was mainly performed by telephone or through home interviews. According to the medical records, the surgical approaches included transthoracic, thoracoabdominal, and transabdominal approaches. Kaplan-Meier and Cox proportional hazards regression models were applied to correlate the surgical approach with survival in patients with GCA. RESULTS: There were marked differences in age and tumor stage among the patients who underwent the three surgical approaches (P < 0.001). Univariate analysis showed that survival was related to sex, age, tumor stage, and N stage (P < 0.001 for all). Cox regression model analysis revealed that thoracoabdominal approach (P < 0.001) and transabdominal approach (P < 0.001) were significant risk factors for poor survival. GCA patients treated with the transthoracic approach had the best survival (5-year survival rate of 53.7%), and survival varied among the different surgical approaches for different tumor stages. CONCLUSION: Thoracoabdominal approach and transabdominal approach were shown to be poor prognostic factors. Patients with (locally advanced) GCA may benefit from the transthoracic approach. Further prospective randomized clinical trials are necessary.

B7-H4, a promising target for immunotherapy.

The coinhibitory molecule B7-H4, an important member of the B7 family, is abnormally expressed in tumors, inflammation and autoimmune diseases. B7-H4 negatively regulates T cell immune response and promotes immune escape by inhibiting the proliferation, cytokine secretion, and cell cycle of T cells. Moreover, B7-H4 plays an extremely important role in tumorigenesis and tumor development including cell proliferation, invasion, metastasis, anti-apoptosis, etc. In addition, B7-H4 has the other biological functions, such as protection against type 1 diabetes (T1D) and islet cell transplantation. Therefore, B7-H4 has been identified as a novel marker or a therapeutic target for the treatment of tumors, inflammation, autoimmune diseases, and organ transplantation. Here, we summarized the expression profiles, physiological and pathological functions, and regulatory mechanisms of B7-H4, the signaling pathways involved, as well as B7-H4-based immunotherapy.

Recyclable three-dimensional Ag nanorod arrays decorated with O-g-C3N4 for highly sensitive SERS sensing of organic pollutants.

A three-dimensional (3D) substrate was developed by assembling a monolayer of graphitic carbon nitride (O-g-C3N4) on Ag nanorod arrays (Ag NRs) for sensitive and recyclable surface enhanced Raman scattering (SERS) detection. The prepared Ag NRs/O-g-C3N4 substrate not only generated a significant Raman enhancement effect as a result of the strong π-π stacking interaction between O-g-C3N4 and the analytes but also possessed excellent self-cleaning property via visible-light irradiation that was attributed to its outstanding catalytic performance. Highly sensitive SERS detection could be achieved with a LOD of 8.2 × 10-10 M for R6 G, and the substrate could be used repeatedly for at least four cycles with tolerable intensity attenuation. In addition, the 3D substrate exhibited long-term stability originating from the electron-donor effect of O-g-C3N4 and high reproducibility due to the uniform decoration of O-g-C3N4 on the Ag NRs through the strong interaction. Furthermore, using Ag NRs/O-g-C3N4, the recyclable detection of antibiotics in a water sample was demonstrated with high sensitivity, which indicates that the 3D Ag NRs/O-g-C3N4 substrate is a promising candidate for eliminating the challenges of single-use SERS substrates and building a portable SERS platform to sense organic molecular species.

A polymorphism in the promoter region of PD-L1 serves as a binding-site for SP1 and is associated with PD-L1 overexpression and increased occurrence of gastric cancer.

PD-L1 is a member of the B7 family co-inhibitory molecules and plays a critical role in tumor immune escape. In this study, we found a polymorphism rs10815225 in the PD-L1 promoter region was significantly associated with the occurrence of gastric cancer. The GG homozygous frequency was higher in the cancer patients than that in the precancerous lesions, which was higher than that in the health controls. This polymorphism locates in the binding-site of Sp1 transcription factor (SP1). The expression level of PD-L1 mRNA in the GG homozygous cancer patients was apparently higher than that in the GC heterozygotes. Luciferase reporter results showed that SP1 bonded to rs10815225 G-allelic PD-L1 promoter instead of C-allelic. Upregulation and knockdown of SP1 resulted in elevation and attenuation of PD-L1 in SGC-7901 cells, respectively. The chromatin immunoprecipitation results further confirmed the binding of SP1 to the promoter of PD-L1. Additionally, rs10815225 was found to be in disequilibrium with a functional polymorphism rs4143815 in the PD-L1 3'-UTR, and the haplotypes of these two polymorphisms were also markedly related to gastric cancer risk. These results revealed a novel mechanism underlying genetic polymorphisms influencing PD-L1 expression modify gastric cancer susceptibility.

Genetically engineered pre-microRNA-34a prodrug suppresses orthotopic osteosarcoma xenograft tumor growth via the induction of apoptosis and cell cycle arrest.

Osteosarcoma (OS) is the most common primary malignant bone tumor in children, and microRNA-34a (miR-34a) replacement therapy represents a new treatment strategy. This study was to define the effectiveness and safety profiles of a novel bioengineered miR-34a prodrug in orthotopic OS xenograft tumor mouse model. Highly purified pre-miR-34a prodrug significantly inhibited the proliferation of human 143B and MG-63 cells in a dose dependent manner and to much greater degrees than controls, which was attributed to induction of apoptosis and G2 cell cycle arrest. Inhibition of OS cell growth and invasion were associated with release of high levels of mature miR-34a from pre-miR-34a prodrug and consequently reduction of protein levels of many miR-34a target genes including SIRT1, BCL2, c-MET, and CDK6. Furthermore, intravenous administration of in vivo-jetPEI formulated miR-34a prodrug significantly reduced OS tumor growth in orthotopic xenograft mouse models. In addition, mouse blood chemistry profiles indicated that therapeutic doses of bioengineered miR-34a prodrug were well tolerated in these animals. The results demonstrated that bioengineered miR-34a prodrug was effective to control OS tumor growth which involved the induction of apoptosis and cell cycle arrest, supporting the development of bioengineered RNAs as a novel class of large molecule therapeutic agents.

Combination therapy with bioengineered miR-34a prodrug and doxorubicin synergistically suppresses osteosarcoma growth.

Osteosarcoma (OS) is the most common form of primary malignant bone tumor and prevalent among children and young adults. Recently we have established a novel approach to bioengineering large quantity of microRNA-34a (miR-34a) prodrug for miRNA replacement therapy. This study is to evaluate combination treatment with miR-34a prodrug and doxorubicin, which may synergistically suppress human OS cell growth via RNA interference and DNA intercalation. Synergistic effects were indeed obvious between miR-34a prodrug and doxorubicin for the suppression of OS cell proliferation, as defined by Chou-Talalay method. The strongest antiproliferative synergism was achieved when both agents were administered simultaneously to the cells at early stage, which was associated with much greater degrees of late apoptosis, necrosis, and G2 cell cycle arrest. Alteration of OS cellular processes and invasion capacity was linked to the reduction of protein levels of miR-34a targeted (proto-)oncogenes including SIRT1, c-MET, and CDK6. Moreover, orthotopic OS xenograft tumor growth was repressed to a significantly greater degree in mouse models when miR-34a prodrug and doxorubicin were co-administered intravenously. In addition, multiple doses of miR-34a prodrug and doxorubicin had no or minimal effects on mouse blood chemistry profiles. The results demonstrate that combination of doxorubicin chemotherapy and miR-34a replacement therapy produces synergistic antiproliferative effects and it is more effective than monotherapy in suppressing OS xenograft tumor growth. These findings support the development of mechanism-based combination therapy to combat OS and bioengineered miR-34a prodrug represents a new natural miRNA agent.

Bioengineering Novel Chimeric microRNA-34a for Prodrug Cancer Therapy: High-Yield Expression and Purification, and Structural and Functional Characterization.

Development of anticancer treatments based on microRNA (miRNA/miR) such as miR-34a replacement therapy is limited to the use of synthetic RNAs with artificial modifications. Herein, we present a new approach to a high-yield and large-scale biosynthesis, in Escherichia coli using transfer RNA (tRNA) scaffold, of chimeric miR-34a agent, which may act as a prodrug for anticancer therapy. The recombinant tRNA fusion pre-miR-34a (tRNA/mir-34a) was quickly purified to a high degree of homogeneity (>98%) using anion-exchange fast protein liquid chromatography, whose primary sequence and post-transcriptional modifications were directly characterized by mass spectrometric analyses. Chimeric tRNA/mir-34a showed a favorable cellular stability while it was degradable by several ribonucleases. Deep sequencing and quantitative real-time polymerase chain reaction studies revealed that tRNA-carried pre-miR-34a was precisely processed to mature miR-34a within human carcinoma cells, and the same tRNA fragments were produced from tRNA/mir-34a and the control tRNA scaffold (tRNA/MSA). Consequently, tRNA/mir-34a inhibited the proliferation of various types of human carcinoma cells in a dose-dependent manner and to a much greater degree than the control tRNA/MSA, which was mechanistically attributable to the reduction of miR-34a target genes. Furthermore, tRNA/mir-34a significantly suppressed the growth of human non-small-cell lung cancer A549 and hepatocarcinoma HepG2 xenograft tumors in mice, compared with the same dose of tRNA/MSA. In addition, recombinant tRNA/mir-34a had no or minimal effect on blood chemistry and interleukin-6 level in mouse models, suggesting that recombinant RNAs were well tolerated. These findings provoke a conversation on producing biologic miRNAs to perform miRNA actions, and point toward a new direction in developing miRNA-based therapies.

Chimeric MicroRNA-1291 Biosynthesized Efficiently in Escherichia coli Is Effective to Reduce Target Gene Expression in Human Carcinoma Cells and Improve Chemosensitivity.

In contrast to the growing interests in studying noncoding RNAs (ncRNAs) such as microRNA (miRNA or miR) pharmacoepigenetics, there is a lack of efficient means to cost effectively produce large quantities of natural miRNA agents. Our recent efforts led to a successful production of chimeric pre-miR-27b in bacteria using a transfer RNA (tRNA)-based recombinant RNA technology, but at very low expression levels. Herein, we present a high-yield expression of chimeric pre-miR-1291 in common Escherichia coli strains using the same tRNA scaffold. The tRNA fusion pre-miR-1291 (tRNA/mir-1291) was then purified to high homogeneity using affinity chromatography, whose primary sequence and post-transcriptional modifications were directly characterized by mass spectrometric analyses. Chimeric tRNA/mir-1291 was readily processed to mature miR-1291 in human carcinoma MCF-7 and PANC-1 cells. Consequently, recombinant tRNA/mir-1291 reduced the protein levels of miR-1291 target genes, including ABCC1, FOXA2, and MeCP2, as compared with cells transfected with the same doses of control methionyl-tRNA scaffold with a sephadex aptamer (tRNA/MSA). In addition, tRNA-carried pre-miR-1291 suppressed the growth of MCF-7 and PANC-1 cells in a dose-dependent manner, and significantly enhanced the sensitivity of ABCC1-overexpressing PANC-1 cells to doxorubicin. These results indicate that recombinant miR-1291 agent is effective in the modulation of target gene expression and chemosensitivity, which may provide insights into high-yield bioengineering of new ncRNA agents for pharmacoepigenetics research.

A general approach to high-yield biosynthesis of chimeric RNAs bearing various types of functional small RNAs for broad applications.

RNA research and therapy relies primarily on synthetic RNAs. We employed recombinant RNA technology toward large-scale production of pre-miRNA agents in bacteria, but found the majority of target RNAs were not or negligibly expressed. We thus developed a novel strategy to achieve consistent high-yield biosynthesis of chimeric RNAs carrying various small RNAs (e.g. miRNAs, siRNAs and RNA aptamers), which was based upon an optimal noncoding RNA scaffold (OnRS) derived from tRNA fusion pre-miR-34a (tRNA/mir-34a). Multi-milligrams of chimeric RNAs (e.g. OnRS/miR-124, OnRS/GFP-siRNA, OnRS/Neg (scrambled RNA) and OnRS/MGA (malachite green aptamer)) were readily obtained from 1 l bacterial culture. Deep sequencing analyses revealed that mature miR-124 and target GFP-siRNA were selectively released from chimeric RNAs in human cells. Consequently, OnRS/miR-124 was active in suppressing miR-124 target gene expression and controlling cellular processes, and OnRS/GFP-siRNA was effective in knocking down GFP mRNA levels and fluorescent intensity in ES-2/GFP cells and GFP-transgenic mice. Furthermore, the OnRS/MGA sensor offered a specific strong fluorescence upon binding MG, which was utilized as label-free substrate to accurately determine serum RNase activities in pancreatic cancer patients. These results demonstrate that OnRS-based bioengineering is a common, robust and versatile strategy to assemble various types of small RNAs for broad applications.

Rapid production of novel pre-microRNA agent hsa-mir-27b in Escherichia coli using recombinant RNA technology for functional studies in mammalian cells.

Noncoding microRNAs (miRNAs or miRs) have been revealed as critical epigenetic factors in the regulation of various cellular processes, including drug metabolism and disposition. However, research on miRNA functions is limited to the use of synthetic RNA and recombinant DNA agents. Herein, we show that novel pre-miRNA-27b (miR-27b) agents can be biosynthesized in Escherichia coli using recombinant RNA technology, and recombinant transfer RNA (tRNA)/mir-27b chimera was readily purified to a high degree of homogeneity (>95%) using anion-exchange fast protein liquid chromatography. The tRNA-fusion miR-27b was revealed to be processed to mature miRNA miR-27b in human carcinoma LS-180 cells in a dose- and time-dependent manner. Moreover, recombinant tRNA/miR-27b agents were biologically active in reducing the mRNA and protein expression levels of cytochrome P450 3A4 (CYP3A4), which consequently led to lower midazolam 1'-hydroxylase activity. These findings demonstrate that pre-miRNA agents can be produced by recombinant RNA technology for functional studies.

Clinical significance of costimulatory molecule B7-H3 expression on CD3(+) T cells in colorectal carcinoma.

BACKGROUND: B7-H3 has been widely studied in the context of tumor progression in recent years, and behaves as a tumor cell marker in a variety of tumors including colorectal carcinoma. The mechanism of B7-H3 in tumor progression is complicated and not clear yet. Studies have revealed that B7 family molecules are expressed on infiltrated lymphocytes as well as tumor cells in tumor microenvironment, which indicates that different expression pattern may lead to different clinical outcomes. METHODS: The expression of B7-H3 was detected in tissues of 98 colorectal carcinoma patients by using immunohistochemistry. Then the expression of B7-H3 on CD3(+) T lymphocytes isolated from fresh cancer tissues of 12 colorectal carcinoma patients was analyzed by flow cytometry assay. The relationship between the expression of B7-H3 on CD3(+) T lymphocytes and patients' clinical pathological parameters was demonstrated with statistical analysis. RESULTS: Patients with more CD3(+) T cell infiltration survived much longer than patients with less CD3(+) T cell infiltration (P < 0.05); B7-H3 was highly expressed by infiltrating CD3(+) T lymphocytes in colorectal carcinoma tissues. The expression of B7-H3 was found to be significantly related with lymph node metastasis status (P < 0.05), but not with the patient's gender, age, tumor size, differentiation degree, depth of tumor invasion, Dukes' stage, distant metastasis and whether or not mucinous adenocarcinoma was present (P > 0.05). Moreover, the survival time of patients with low expression of B7-H3 was obviously longer than those of high B7-H3 expression patients, but the seven-year survival rate showed no difference between the high and low B7-H3 expression patients (P > 0.05). CONCLUSION: The negative costimulatory molecule B7-H3 on infiltrating CD3(+) T lymphocytes in colorectal carcinoma bears importance in the clinical pathological progress and prognosis of colorectal carcinoma.

Screening and test of CD40 related signal transduction pathway in AGS cells and construction of gene silencing vector.

This study is designed to screen the CD40 related signal transduction pathway in AGS cells and construction of gene silencing vector. Analysis results showed 414 differential genes expression, including upregulation of 209 genes and downregulation of 205 genes. Basing on the ratio of signal in experimental group to signal in control group, 45 genes (38 genes upregulation and seven genes downregulation) with significant (P<0.01) change in expression levels were screened according to the screening standard (signal log ratio≥1 or ≤-1). These genes involved into metabolism, cell cycle and apoptosis, signal transduction and stress response. Furthermore, PI3K mRNA expression level in PI3K siRNA transfected AGS cells was 0.2335±0.0116 72 h after transfection. This value was significantly (P<0.05) lower than that in blank and negative control groups. PI3K protein expression in PI3K siRNA transfected AGS cells was significantly (P<0.05) lower than that in blank and PI3K siRNA/N transfected groups. Therefore, PI3K siRNA gene silencing vector can significantly inhibit PI3K mRNA and protein expression in AGS cells.

Effect of PI3K gene silencing on growth, migration and related proteins expression of CD40 signal-mediated gastric cancer cells.

In this study, we investigate effect of PI3K gene silencing on growth, migration and related proteins expression of CD40 signal-mediated gastric cancer cells. We observed that combination of sCD40L with PI3K siRNA could significantly inhibit AGS cells growth, block cells in G1 phase, and promote tumour cells apoptosis after 24 h treatment. Transwell test showed that numbers of cells per visual field in group PI3K siRNA or group sCD40L (after 24 h PI3K siRNA or sCD40L alone treatment) were fewer than that (32.54 ± 4.22) in control group. Numbers of cells per visual field in (after 24 h combination treatment of PI3K siRNA with sCD40L) were significantly fewer than that in group PI3K siRNA or group sCD40L. Compared with group sCD40L, expression level of Fas protein in group sCD40L + PI3K siRNA was significantly increased. The findings suggest that PI3K siRNA may strengthen CD40-induced specific antitumour effect via blocking PI3K/Akt signal pathway, resisting tumour immunoediting regulated by CD40 signal. Combination of sCD40L and PI3K siRNA is an important mechanism of gastric cancer therapy.

Combined effect of sCD40L and PI3K siRNA on transplanted tumours growth and microenvironment in nude mice with gastric cancer.

The objective of the paper is to investigate the combined effect of sCD40L and phosphatidylinositol 3-kinase (PI3K) siRNA on transplanted tumours growth and microenvironment in nude mice with gastric cancer. 48 h after modeling, the animals were randomly divided into saline + AGS group (A), sCD40L + AGS group (B), saline + PI3K siRNA group (C) and sCD40L + PI3K siRNA group (D), six mice in each group. The mouse in the groups was inoculated with exponential phase AGS cell or PI3K gene silencing cells (100 µl, 5 × 10(6)). After tumour size reaches 0.2-0.3 cm, Tumours in animals of groups were injected with sCD40L (100 µl, 10 mg/kg) or equal volume of saline, thrice each day, respectively. Microvessel density (MVD), apoptosis index, and expression levels of PI3K, Survivin and vascular endothelial growth factor (VEGF) proteins in transplanted tumor cells in gastric cancer nude mice were analyzed by utilizing Immunohistochemistry, western blot, terminal deoxynucleotidyl transferase dUTP nick end labeling assays. Results showed that combination of sCD40L with PI3K siRNA could significantly decrease tumour size, MVD, expression levels of PI3K, Survivin and VEGF proteins, and increase apoptosis index. It can be concluded that combination of sCD40L with PI3K siRNA provides a promising future for gastric cancer therapy.

CD40 signal expression in gastric cancer tissue and its correlation with prognosis of gastric cancer patients.

CD40 signaling plays a critical role in the survival rate of gastric cancer patients. Tumour samples were collected from 73 patients with who were diagnosed as gastric cancer in general surgery department in the 1st affiliated hospital of Suzhou University between September 2002 and July 2003. All patients had not received radiotherapy and chemotherapy before operation. These patients include 46 male and 27 female. Here we show that CD40 is constitutively expressed in the human gastric carcinoma tissues, and CD40 protein and mRNA positive expression in gastric cancer tissues closely correlated with lymph node metastasis and tumour TNM stage. CD40 positive expression in gastric cancer patients with lymph node metastasis was markedly higher than that in gastric cancer patients without lymph node metastasis. CD40 positive expression in stage III-IV gastric cancer patients was markedly higher than that in stage I-II gastric cancer patients. Moreover, CD40 expression closely correlated with prognosis of gastric cancer patients. Therefore, CD40 was taken as grouping variable, and lymph node metastasis and clinical staging were taken as stratification variables, respectively, further analysis showed that prognosis in gastric cancer patients with lymph node metastasis and CD40 positive expression was markedly worse than that in gastric cancer patients without lymph node metastasis and CD40 negative expression (P = 0.0076). These results suggest that CD40 signaling plays a critical role in the survival of gastric cancer patients.

Genotypic variants at 2q33 and risk of esophageal squamous cell carcinoma in China: a meta-analysis of genome-wide association studies.

Genome-wide association studies have identified susceptibility loci for esophageal squamous cell carcinoma (ESCC). We conducted a meta-analysis of all single-nucleotide polymorphisms (SNPs) that showed nominally significant P-values in two previously published genome-wide scans that included a total of 2961 ESCC cases and 3400 controls. The meta-analysis revealed five SNPs at 2q33 with P< 5 × 10(-8), and the strongest signal was rs13016963, with a combined odds ratio (95% confidence interval) of 1.29 (1.19-1.40) and P= 7.63 × 10(-10). An imputation analysis of 4304 SNPs at 2q33 suggested a single association signal, and the strongest imputed SNP associations were similar to those from the genotyped SNPs. We conducted an ancestral recombination graph analysis with 53 SNPs to identify one or more haplotypes that harbor the variants directly responsible for the detected association signal. This showed that the five SNPs exist in a single haplotype along with 45 imputed SNPs in strong linkage disequilibrium, and the strongest candidate was rs10201587, one of the genotyped SNPs. Our meta-analysis found genome-wide significant SNPs at 2q33 that map to the CASP8/ALS2CR12/TRAK2 gene region. Variants in CASP8 have been extensively studied across a spectrum of cancers with mixed results. The locus we identified appears to be distinct from the widely studied rs3834129 and rs1045485 SNPs in CASP8. Future studies of esophageal and other cancers should focus on comprehensive sequencing of this 2q33 locus and functional analysis of rs13016963 and rs10201587 and other strongly correlated variants.

Newborn screening for inborn errors of metabolism in mainland china: 30 years of experience.

The history of the Newborn Screening Program in Mainland China begins in 1981, when a pilot plan was developed that demonstrated the feasibility of its implementation. It has so far focused on the detection of congenital hypothyroidism (CH) and phenylketonuria (PKU) to prevent or reduce mental and physical developmental retardation in children. Throughout this period, a total of 35,795,550 dried blood samples (DBS) of newborns (NB) have been analyzed for PKU, and 35,715,988 for CH. During this period, 3,082 cases with PKU have been diagnosed, resulting in an incidence of 1 case per 11,614 (95% confidence interval 11,218-12,039) live births. In relation to CH, 17,556 cases have been confirmed, arriving at an incidence of 1 case per 2,034(95% confidence interval 2,005-2,065) live births. The biggest challenge for universal newborn screening is still to increase coverage to mid-western area. In Mainland China, MS/MS newborn screening started in 2004. In a pilot study, 371,942 neonates were screened, and 98 cases were detected with one of the metabolic disorders, and the collective estimated prevalence amounted to 1 in 3795 (95% confidence interval 3,168-4,732) live births, with a sensitivity of 98.99%, a specificity of 99.83%, and a positive predictive value of 13.57%. The most important is to get the government's policy and financial support for expanded screening.

Quantitative determination of allele frequency in pooled DNA by using sequencing method.

Quantitative determination of the allele frequency of single-nucleotide polymorphism (SNP) in pooled DNA samples is a promising approach to clarify the relationships between SNPs and diseases. Here, we present such a simple, accurate, and inexpensive method for quantitative determining the allele frequency in pooled DNA samples. Three steps of DNA pooling, PCR amplification and sequencing are involved in this assay. Although direct determination of the allele frequency from the two allele-specific fluorescence intensities is possible, correction for differential response of alleles is important. We explored the effect of differential response of alleles on test statistics and provide a solution to this problem based on heterozygous fluorescence intensities. We demonstrate the accuracy and reliability of this assay on pooled DNA samples with pre-determined allele frequencies from 7.1% to 53.9%. The accuracy of allele frequency measurements is high, with a correlation coefficient of r² = 0.997 between measured and known frequencies. We believe that by providing a means for SNP genotyping up to hundreds of samples simultaneously, inexpensively, and reproducibly, this method is a powerful strategy for detecting meaningful polymorphic differences in candidate gene association studies.