A novel method for detecting nine hotspot mutations of deafness genes in one tube.

Deafness is a common sensory disorder. In China, approximately 70% of hereditary deafness originates from four common deafness-causing genes: GJB2, SLC26A4, GJB3, and MT-RNR1. A single-tube rapid detection method based on 2D-PCR technology was established for nine mutation sites in the aforementioned genes, and Sanger sequencing was used to verify its reliability and accuracy. The frequency of hotspot mutations in deafness genes was analysed in 116 deaf students. 2D-PCR identified 27 genotypes of nine loci according to the melting curve of the FAM, HEX, and Alexa568 fluorescence channels. Of the 116 deaf patients, 12.9% (15/116) carried SLC26A4 mutations, including c.919-2A > G and c.2168A > G (allele frequencies, 7.3% and 2.2%, respectively). The positivity rate (29.3%; 34/116) was highest for GJB2 (allele frequency, 15.9% for c.235delC, 6.0% for c.299_300delAT, and 2.6% for c.176-191del16). Sanger sequencing confirmed the consistency of results between the detection methods based on 2D-PCR and DNA sequencing. Common pathogenic mutations in patients with non-syndromic deafness in Changzhou were concentrated in GJB2 (c.235delC, c.299_300delAT, and c.176-191del16) and SLC26A4 (c.919-2A > G and c.2168 A > G). 2D-PCR is an effective method for accurately and rapidly identifying deafness-related genotypes using a single-tube reaction, and is superior to DNA sequencing, which has a high cost and long cycle.

Accurate identification of HLA-B*15:02 allele by two-dimensional polymerase chain reaction.

BACKGROUND: HLA-B*15:02 is highly associated with carbamazepine-induced SJS/TEN; however, there is no rapid and accurate detecting method. Here, we present a method to distinguish HLA-B*15:02 from 16 highly homologous HLA-B*15 alleles. METHODS: The high-throughput two-dimensional polymerase chain reaction (2D-PCR) technology was employed to identify HLA-B*15:02 in two-tube reaction. And, 2D-PCR accuracy was verified by PCR-sequence based typing (PCR-SBT). RESULTS: HLA-B*15:02 heterozygotes were identified by 14 melting valleys in the first tube reaction and none in the second, or by 13 melting valleys in the first tube reaction and one in the second. HLA-B*15:02 homozygote was identified by 13 melting valleys in the first tube reaction and none in the second. Three (0.16%) HLA-B*15:02 homozygotes and 84 (4.59%) HLA-B*15:02 heterozygotes were detected in 1830 samples of clinical general population without detecting 16 highly homologous alleles to HLA-B*15:02. The kappa test showed 100% coincidence between the 2D-PCR and PCR-SBT. CONCLUSIONS: 2D-PCR in two-tube reaction method for identifying HLA-B*15:02 was successfully established. Identification of HLA-B*15:02 is necessary prior to taking CBZ based on HLA-B*15:02 allele frequency.

Rapid Identification of Seven Common ABO Alleles by Two-Dimensional Polymerase Chain Reaction Technology.

Introduction: The molecular biology detection technology of the human ABO blood group system makes up for the limitations in many aspects compared with conventional serological typing technology. This study aimed to establish a new method to identify seven common ABO alleles (ABO*A1.01, ABO*A1.02, ABO*A2.01, ABO*B.01, ABO*O.01.01, ABO*O.01.02, and ABO*O.02.01) by two-dimensional polymerase chain reaction (2D PCR). 2D PCR can identify multiple target genes in a closed test tube by labeling specific primers with tags homologous to the sequence of fluorescently labeled probes, and melting curve analysis is performed after the fluorescent probes are hybridized with tag complementary sequences in PCR-specific products. In this study, 2D PCR and PCR sequence-specific primer (PCR-SSP) were combined to discriminate different alleles in a single reaction, which has the characteristics of high throughput, and compared with other typing techniques; the typing results can be obtained without additional operations. Methods: The ABO*A1.01 allele genetic sequence was used as the reference sequence. The specific sense and antisense primers for seven common ABO alleles were designed on exons 6 and 7 according to the principle of 2D PCR and PCR-SSP. Single nucleotide polymorphism sites for identifying seven alleles were detected in FAM and HEX channels, respectively. Two hundred sixty DNA samples were enrolled for rapid ABO genotyping by 2D PCR, and 95 of them were selected for Sanger sequencing. The Kappa test was used to analyze the agreement of the methodologies. Results: These 7 alleles each had four characteristic melting valleys at different single nucleotide polymorphism loci. A total of 15 genotypes were detected, including ABO*A1.01/A1.02, ABO*A1.01/O.01.01, ABO*A1.01/O.01.02, ABO*A1.02/A1.02, ABO*A1.02/O.01.01, ABO*A1.02/O.01.02, ABO*B.01/B.01, ABO*B.01/O.01.01, ABO*B.01/O.01.02, ABO*O.01.01/O.01.01, ABO*O.01.01/O.01.02, ABO*O.01.02/O.01.02, ABO*A1.01/B.01, ABO*A1.02/B.01, and ABO*B.01/O.01. v (containing a rare ABO*O allele, based on the sequencing results). The Kappa test showed completely consistent results for 2D PCR and Sanger sequencing (Kappa = 1). Conclusion: The 2D PCR technique could be used for molecular typing of the ABO blood group, which was efficient, rapid, accurate, and economical.

Apolipoprotein M promotes cholesterol uptake and efflux from mouse macrophages.

Apolipoprotein M (ApoM) exhibits various anti-atherosclerotic functions as a component of high-density lipoprotein (HDL) particles. Scavenger receptor class B type I (SR-BI) is a classic HDL receptor that mediates selective cholesterol uptake and enhances the efflux of cellular cholesterol to HDL. However, the effect of ApoM on cholesterol transport in macrophages remains unclear. In this study, we identified for the first time that ApoM is expressed in mouse macrophages and is involved in cholesterol uptake, similar to SR-BI. NBD-cholesterol uptake and efflux in cells were characterized using fluorescence spectrophotometry. The uptake ratios of cholesterol by macrophages from ApoM-/- SR-BI-/- mice were significantly lower than those from ApoM+/+ SR-BI-/- and ApoM-/- SR-BI+/+ mice. Real-time fluorescence quantitative PCR was used to analyze the expression of cholesterol transport-related genes involved in cholesterol uptake. ApoM-enriched HDL (ApoM+ HDL) facilitated more cholesterol efflux from murine macrophage Ana-1 cells than ApoM-free HDL (ApoM- HDL). However, recombinant human ApoM protein inhibited the ability of ApoM- HDL to induce cholesterol efflux. In conclusion, ApoM promotes cholesterol uptake and efflux in mouse macrophages. A better understanding of ApoM function may lead to the development of novel therapeutic strategies for treating atherosclerotic diseases.

Apolipoprotein M promotes the anti-inflammatory effect of high-density lipoprotein by binding to scavenger receptor BI.

BACKGROUND: Inflammation participates pivotally in the pathogenesis of atherosclerosis. Apolipoprotein M (apoM) is a high-density lipoprotein (HDL)-associated plasma protein that affects HDL metabolism and shows various anti-inflammatory functions in atherosclerosis. In this study, we aim to determine whether apoM is expressed in peripheral blood mononuclear cells (PBMCs) and promoted the anti-inflammatory effect of HDL by combing with scavenger receptor BI (SR-BI). METHODS: The expression of apoM in PBMCs is detected by a confocal fluorescence microscope and flow cytometry. The interactions between apoM and SR-BI are detected with co-immunoprecipitation. The multiplexed Luminex xMAP assay detects the inflammatory factors induced by apoM+ HDL and apoM- HDL in inflammatory cell model. RESULTS: ApoM is expressed on CD14+ monocytes, CD3+ T cells, and CD19+ B cells, CD16+ and CD56+ NK cells. CD14+ monocytes have the highest ratio of apoM+ cells. ApoM+ HDL, apoM- HDL, and recombinant apoM protein could be co-precipitated with SR-BI on the surface of human THP-1 monocytic leukemia cells. In vitro, apoM+ HDL induces significantly less expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1ß than apoM- HDL. CONCLUSIONS: ApoM was expressed on all PBMCs. ApoM interacted with SR-BI on THP-1. ApoM+ HDL has a more significant anti-inflammatory effect than apoM- HDL.

High-Throughput Two-Dimensional Polymerase Chain Reaction Technology.

Polymerase chain reaction (PCR) is a very powerful tool for clinical gene detection. Multiplex PCR especially improves the throughput of this technology. However, it is often necessary to employ techniques such as electrophoresis, mass spectrometry, or sequencing after multiplex PCR amplification for product identification, which requires additional equipment and has high risks of contamination. In this work, we developed a high-throughput two-dimensional (2D) PCR technology that can identify multiple target genes simultaneously in just one closed tube and within a relatively short time by using both fluorescence and the melting temperature (Tm). As an example, a method detecting 9 human papillomavirus (HPV) subtypes and reference genes in a single tube was successfully established using 2D PCR. If designed properly, 2D PCR is believed to have the capability to identify more than 30 genes in one closed tube at a time. This method is particularly suitable for distinguishing microorganisms, single-nucleotide polymorphisms, and the methylation of genes and will be of great help to clinical work.

Insulin Resistance in Apolipoprotein M Knockout Mice is Mediated by the Protein Kinase Akt Signaling Pathway.

BACKGROUND: Previous clinical studies have suggested that apolipoprotein M (apoM) is involved in glucose metabolism and plays a causative role in insulin sensitivity. OBJECTIVE: The potential mechanism of apoM on modulating glucose homeostasis is explored and differentially expressed genes are analyzed by employing ApoM deficient (ApoM-/- ) and wild type (WT) mice. METHODS: The metabolism of glucose in the hepatic tissues of high-fat diet ApoM-/- and WT mice was measured by a glycomics approach. Bioinformatic analysis was applied for analyzing the levels of differentially expressed mRNAs in the liver tissues of these mice. The insulin sensitivity of ApoM-/- and WT mice was compared using the insulin tolerance test and the phosphorylation levels of protein kinase Akt (AKT) and insulin stimulation in different tissues were examined by Western blot. RESULTS: The majority of the hepatic glucose metabolites exhibited lower concentration levels in the ApoM-/- mice compared with those of the WT mice. Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that ApoM deficiency affected the genes associated with the metabolism of glucose. The insulin tolerance test suggested that insulin sensitivity was impaired in ApoM-/- mice. The phosphorylation levels of AKT in muscle and adipose tissues of ApoM-/- mice were significantly diminished in response to insulin stimulation compared with those noted in WT mice. CONCLUSION: ApoM deficiency led to the disorders of glucose metabolism and altered genes related to glucose metabolism in mice liver. In vivo data indicated that apoM might augment insulin sensitivity by AKT-dependent mechanism.

DNA methylation detection methods used in colorectal cancer.

Colorectal cancer (CRC) remains a major contributor to the number of cancer-related deaths that occur annually worldwide. With the development of molecular biology methods, an increasing number of molecular biomarkers have been identified and investigated. CRC is believed to result from an accumulation of epigenetic changes, and detecting aberrant DNA methylation patterns is useful for both the early diagnosis and prognosis of CRC. Numerous studies are focusing on the development of DNA methylation detection methods or DNA methylation panels. Thus, this review will discuss the commonly used techniques and technologies to evaluate DNA methylation, their merits and deficiencies as well as the prospects for new methods.

Correction: The mechanism and regularity of quenching the effect of bases on fluorophores: the base-quenched probe method.

Correction for 'The mechanism and regularity of quenching the effect of bases on fluorophores: the base-quenched probe method' by Huihui Mao et al., Analyst, 2018, 143, 3292-3301.

Krüppel-like factor 4 expression in solid tumor prognosis: A meta-analysis.

BACKGROUND: Accumulating studies have demonstrated that Krüppel-like factor 4 (KLF4) can act as a tumor suppressor or oncogene in the carcinogenesis of diverse cancers. The prognostic value of KLF4 in various human solid cancers remains controversial. Thus, the present meta-analysis was conducted to evaluate the prognostic value of KLF4 in solid tumors. METHODS: Eligible literature was retrieved by searching the PubMed, Embase, and Cochrane Library. Combined hazard ratios (HRs) for overall survival (OS) and disease-free survival (DFS) were assessed using fixed-effects and random-effects models. Meta-regression and subgroup analyses were performed to identify the source of heterogeneity. In addition, publication bias was assessed using Begg's funnel plot and Egger's regression asymmetry test. RESULTS: The 22 eligible studies finally enrolled a total of 2988 patients to assess the prognostic value of KLF4 in solid tumors. Low KLF4 expression was clearly related to worse OS (HR = 1.71, 95% confidence interval [CI] = 1.30-2.24, P < 0.001) and DFS (HR = 1.74, 95% CI = 1.34-2.26, P < 0.001), indicating that low KLF4 expression could be an independent prognostic factor for poor survival in solid cancers. CONCLUSION: KLF4 might be a potential marker to predict prognosis in solid cancer patients.

The mechanism and regularity of quenching the effect of bases on fluorophores: the base-quenched probe method.

The base-quenched probe method for detecting single nucleotide polymorphisms (SNPs) relies on real-time PCR and melting-curve analysis, which might require only one pair of primers and one probe. At present, it has been successfully applied to detect SNPs of multiple genes. However, the mechanism of the base-quenched probe method remains unclear. Therefore, we investigated the possible mechanism of fluorescence quenching by DNA bases in aqueous solution using spectroscopic techniques. It showed that the possible mechanism might be photo-induced electron transfer. We next analyzed electron transfer or transmission between DNA bases and fluorophores. The data suggested that in single-stranded DNA, the electrons of the fluorophore are transferred to the orbital of pyrimidine bases (thymine (T) and cytosine (C)), or that the electron orbitals of the fluorophore are occupied by electrons from purine bases (guanine (G) and adenine (A)), which lead to fluorescence quenching. In addition, the electrons of a fluorophore excited by light can be transmitted along double-stranded DNA, which gives rise to stronger fluorescence quenching. Furthermore, we demonstrated that the quenching efficiency of bases is in the order of G > C ≥ A ≥ T and the capability of electron transmission of base-pairs in double-stranded DNA is in the order of CG[combining low line] ≥ GC[combining low line] > TA[combining low line] ≥ AT[combining low line] (letters representing bases on the complementary strand of the probe are bold and underlined), and the most common commercial fluorophores including FAM, HEX, TET, JOE, and TAMRA could be influenced by bases and are in line with this mechanism and regularity.

Prognostic role of long non-coding RNA XIST expression in patients with solid tumors: a meta-analysis.

BACKGROUND: The aberrant expression of long non-coding RNA (lncRNA) X inactivate-specific transcript (XIST) has been demonstrated to be involved in the tumourigenesis and the development of various cancers. Therefore, we conducted a meta-analysis to assess the prognostic role of lncRNA XIST expression in solid tumors. METHODS: The databases of PubMed, EMBase, Web of Science, Cochrane library (up to Dec 31, 2017) were searched for the related studies and identified 15 eligible studies containing 1209 patients to include in the meta-analysis. Hazards ratios (HRs) with corresponding 95% confidence intervals (CIs) were pooled to estimate the association between lncRNA XIST expression and survival of cancer patients from Asian. RESULTS: The result showed that higher lncRNA XIST expression in cancer tissue was related to a worse overall survival (OS) (HR = 1.54, 95% CI 1.07-2.23). In subgroup analysis, it revealed that lncRNA XIST overexpression was significantly associated with worse OS in digestive system tumors (HR = 1.67, 95% CI 1.11-2.51, p = 0.031). In addition, the association between high lncRNA XIST expression and poor OS was also statistically significant in other subgroups, including multivariate analysis (HR = 2.39, 95% CI 1.28-4.46, p = 0.006, random-effect), patients' number was greater than 65 (HR = 1.75, 95% CI 1.24-2.47, p = 0.001, random-effect), and reported in text (HR = 2.50, 95% CI 1.49-4.18, p = 0.000, random-effect). CONCLUSIONS: The expression of lncRNA XIST could be regarded as a poor prognostic biomarker for solid tumors, which might shed new light on epigenetic diagnostics and therapeutics in tumors.

Hematopoietic stem cell transplant in mice by intra-femoral injection.

In traditional bone marrow transplantation, the majority of peripherally introduced stem cells are trapped in peripheral organs, such as the lung and liver. The frequency of cells homed in bone marrow by such method is extremely low. This circumstance adds difficulty to the research of hematopoietic stem cell (HSC), a rare population to begin with. By introducing HSC directly into bone marrow cavity, the peripheral loss of HSC can be minimized. Thus, intra-femoral injection of HSC is a useful method for HSC study.

Purification and characterization of cellular proteins associated with histone H4 tails.

The histone H4 N-terminal tail has long been regarded as a major regulator in chromatin structure and function. Although the underlying mechanism has not been unraveled, an emerging body of evidence supports that H4 tail and its post-translational modification function as a recruitment motif for key factors required for proper regulation of chromatin transcription. To investigate these aspects, we have generated HeLa cell lines that constitutively express ectopic H4 tail domain for biochemical purification of proteins associated with H4 tail. We found that expressed H4 tails stably associate with sets of transcription regulatory factors and histone methyltransferases distinct from those that associate with histone H3 tails. Importantly, point mutations of four major lysine substrates to block cellular acetylation of ectopic H4 tail significantly inhibited the association of histone methyltransferases and sets of transcription-activating factors, supporting a major role of acetylation on recruitmentbased action of H4 tail during transcription. Further, our transcription analysis revealed that the proteins associated with wild-type/acetylated H4 tail, but not with mutant/unacetylated H4 tail, can enhance p300-dependent chromatin transcription. Taken together, these findings demonstrate novel roles for H4 tail and its acetylation in mediating recruitment of multiple regulatory factors that can change chromatin states for transcription regulation.

Isolation and characterization of proteins associated with histone H3 tails in vivo.

The histone H3 amino-terminal tails play an important role in regulating chromatin transcription. Although the mechanisms by which the H3 tail modulates transcription are not well understood, recent discoveries of specific interactions of regulatory factors with H3 tails suggest that H3 tails are a key player in the precise regulation of transcription activity. To investigate the recruitment-based action of H3 tails in chromatin transcription, we purified H3 tail-associated proteins from HeLa cells that stably express epitope-tagged H3 tails. This approach resulted in the identification of multiple histone methyltransferase activities and transcription regulatory factors that are specifically associated with expressed H3 tail domains. Point mutations of Lys-9 and Lys-27 to block cellular modifications of the tail domains completely abolished the association of specific factors, including HP1 and several repressors. Importantly, our transcription analysis revealed that the purified factors can significantly stimulate p300-mediated transcription from chromatin templates. These results implicate that the H3 tail, when accessible in relaxed chromatin, acts as a transcriptional regulator by mediating recruitment of specific sets of cofactors.

IL-12 facilitates both the recovery of endogenous hematopoiesis and the engraftment of stem cells after ionizing radiation.

OBJECTIVE: Severe myelosuppression is a common side effect of radiotherapy or chemotherapy. Methods have been developed to protect patients by stimulating white blood cell or red blood cell recovery/production using growth factors such as G-CSF or EPO. However, there is no available means to stimulate the full-lineage blood cell recovery from severe myelosuppression. In this study, we used lethally or sublethally irradiated animal models to evaluate the hematopoiesis stimulating effect of IL-12. MATERIALS AND METHODS: IL-12-treated lethally or sublethally irradiated animals were examined for the survival/lifespan, the function assays (bone marrow transplantation, CFU-S(12), CFC) of bone marrow cell subsets, and apoptosis assay. RESULTS: Using a low dose of IL-12 (10 times lower than previously reported dose), 91.4% of lethally irradiated animals survived long term without adverse effects on the gastrointestinal (GI) system. The reconstituted hematopoietic system was derived from long-term reconstituting hematopoietic stem cells (LTR HSC), which reconstituted hematopoiesis both endogenously after lethal radiation and in secondary recipients by bone marrow transplantation (BMT). IL-12 significantly attenuated the decline of blood cell counts in sublethally irradiated animals. The IL-12-stimulated hematopoiesis recovery resulted in a full-lineage blood cell production, including white and red blood cells, and platelets. There was no detectable expression of IL-12 receptor on LTR HSC. In IL-12-treated animals, the number of Sca-1(+) cells was significantly higher than in animals without IL-12 treatment. CONCLUSION: In this study, we showed a low dose of IL-12 has hematopoietic-protecting effects, which can attenuate severe myelosuppresion caused by lethal or sublethal irradiation. This study, together with previous studies showing IL-12 is also an anti-tumor and anti-angiogenic agent, suggest IL-12 may have clinical significance in cancer treatment and BMT.

Involvement of Niemann-Pick type C2 protein in hematopoiesis regulation.

Niemann-Pick type C2 (NPC2) protein has been characterized as a cholesterol-binding protein. Its loss leads to NPC2 disease, an inherited neurodegenerative disorder. When analyzing gene expression profile, we noticed high expression of both NPC2 and its receptor, mannose 6-phosphate receptor (MPR), in murine hematopoietic stem cells. NPC2 protein, in the presence of thrombopoietin (TPO), causes an increase in CFU-GEMM (colony-forming unit-granulocyte-erythroid-macrophage-megakaryocyte) and a decrease in CFU-GM (colony-forming unit-granulocyte-macrophage) colony number in colony-forming cell (CFC) assays. This effect is independent of cholesterol binding but does require the presence of MPR. With M07e cells, a TPO-dependent hematopoietic leukemia cell line, NPC2 can inhibit TPO-induced differentiation and enhance TPO-mediated anti-apoptosis effects. Strikingly, these results are not observed under the standard 20% O(2) level of the standard incubator, but rather at 7% O(2), the physiological oxygen level of bone marrow. Furthermore, NPC2 protein upregulates hypoxia inducible factor 1-alpha protein level at 7% O(2), but not at 20% O(2). Our results demonstrate that NPC2 protein plays a role in hematopoiesis at the physiologic bone marrow level of O(2).

Soluble factor(s) from bone marrow cells can rescue lethally irradiated mice by protecting endogenous hematopoietic stem cells.

OBJECTIVE: Ionizing radiation-induced myeloablation can be rescued via bone marrow transplantation (BMT) or administration of cytokines if given within 2 hours after radiation exposure. There is no evidence for the existence of soluble factors that can rescue an animal after a lethal dose of radiation when administered several hours postradiation. We established a system that could test the possibility for the existence of soluble factors that could be used more than 2 hours postirradiation to rescue animals. MATERIALS AND METHODS: Animals with an implanted TheraCyte immunoisolation device (TID) received lethal-dose radiation and then normal bone marrow Lin- cells were loaded into the device (thereby preventing direct interaction between donor and recipient cells). Animal survival was evaluated and stem cell activity was tested with secondary bone marrow transplantation and flow cytometry analysis. Donor cell gene expression of five antiapoptotic cytokines was examined. RESULTS: Bone marrow Lin- cells rescued lethally irradiated animals via soluble factor(s). Bone marrow cells from the rescued animals can rescue and repopulate secondary lethally irradiated animals. Within the first 6 hours post-lethal-dose radiation, there is no significant change of gene expression of the known radioprotective factors TPO, SCF, IL-3, Flt-3 ligand, and SDF-1. CONCLUSION: Hematopoietic stem cells can be protected in lethally irradiated animals by soluble factors produced by bone marrow Lin- cells.

Gene expression profile of murine long-term reconstituting vs. short-term reconstituting hematopoietic stem cells.

The hematopoietic stem cell (HSC) compartment is composed of long-term reconstituting (LTR) and short-term reconstituting (STR) stem cells. LTR HSC can reconstitute the hematopoietic system for life, whereas STR HSC can sustain hematopoiesis for only a few weeks in the mouse. Several excellent gene expression profiles have been obtained of the total hematopoietic stem cell population. We have used five-color FACS sorting to isolate separate populations of LTR and STR stem cell subsets. The LTR HSC has the phenotype defined as Lin- Sca+ Kit+ 38+ 34-; two subsets of STR HSC were obtained with phenotypes of Lin- Sca+ Kit+ 38+ 34+ and Lin- Sca+ Kit+ 38- 34+. The microarray profiling study reported here was able to identify genes specific for LTR functions. In the interrogated genes (approximately 12,000 probe sets corresponding to 8,000 genes), 210 genes are differentially expressed, and 72 genes are associated with LTR activity, including membrane proteins, signal transduction molecules, and transcription factors. Hierarchical clustering of the 210 differentially expressed genes suggested that they are not bone marrow-specific but rather appear to be stem cell-specific. Transcription factor-binding site analysis suggested that GATA3 might play an important role in the biology of LTR HSC.