Identification of a novel amphioxus leucine-rich repeat receptor involved in phagocytosis reveals a role for Slit2-N-type LRR in bacterial elimination.

The basal chordate amphioxus is a model for tracing the origin and evolution of vertebrate immunity. To explore the evolution of immunoreceptor signaling pathways, we searched the associated receptors of the amphioxus Branchiostoma belcheri (Bb) homolog of immunoreceptor signaling adaptor protein Grb2. Mass-spectrum analysis of BbGrb2 immunoprecipitates from B. belcheri intestine lysates revealed a folate receptor (FR) domain- and leucine-rich repeat (LRR)-containing protein (FrLRR). Sequence and structural analysis showed that FrLRR is a membrane protein with a predicted curved solenoid structure. The N-terminal Fr domain contains very few folate-binding sites; the following LRR region is a Slit2-type LRR, and a GPI-anchored site was predicted at the C-terminus. RT-PCR analysis showed FrLRR is a transcription-mediated fusion gene of BbFR-like and BbSlit2-N-like genes. Genomic DNA structure analysis implied the B. belcheri FrLRR gene locus and the corresponding locus in Branchiostoma floridae might be generated by exon shuffling of a Slit2-N-like gene into an FR gene. RT-qPCR, immunostaining, and immunoblot results showed that FrLRR was primarily distributed in B. belcheri intestinal tissue. We further demonstrated that FrLRR localized to the cell membrane and lysosomes. Functionally, FrLRR mediated and promoted bacteria-binding and phagocytosis, and FrLRR antibody blocking or Grb2 knockdown inhibited FrLRR-mediated phagocytosis. Interestingly, we found that human Slit2-N (hSlit2-N) also mediated direct bacteria-binding and phagocytosis which was inhibited by Slit2-N antibody blocking or Grb2 knockdown. Together, these results indicate FrLRR and hSlit2-N may function as phagocytotic-receptors to promote phagocytosis through Grb2, implying the Slit2-N-type-LRR-containing proteins play a role in bacterial binding and elimination.

Isothermal exponential amplification reactions triggered by circular templates (cEXPAR) targeting miRNA.

BACKGROUND: Isothermal exponential amplification reaction (EXPAR) is an emerging amplification technique that is most frequently used to amplify microRNA (miRNA). However, EXPAR also exhibits non-specific background amplification in the absence of the targeted sequence, which limits the attainable assay sensitivity of EXPAR. METHODS AND RESULTS: A novel modified isothermal EXPAR based on circular amplification templates (cEXPAR) was developed in this study. The circular template consists of two same linear fragments that complement the target sequence, and these two linear fragments are separated by two nicking agent recognition sequences (NARS). Compared with the linear structure template, this circular template allows DNA or RNA fragments to be randomly paired with two repeated sequences and can be successfully amplified. This reaction system developed in this study could rapidly synthesize short oligonucleotide fragments (12-22 bp) through simultaneous nicking and displacement reactions. Highly sensitive chain reactions can be specifically triggered by as low as a single copy of target molecule, and non-specific amplification can be effectively eliminated in this optimized system. Moreover, the proposed approach applied to miRNA test can discriminate single-nucleotide variations between miRNAs. CONCLUSION: The newly developed cEXPAR assay provides a useful alternative tool for rapid, sensitive, and highly specific detection of miRNAs.

Association between triglyceride-glucose index and atrial fibrillation: A retrospective observational study.

Background: Insulin resistance is associated with atrial remodeling as well as atrial fibrillation (AF). However, there was limited evidence on the relationship of triglyceride-glucose index (TyG) index, a simple, valuable marker of insulin resistance, with AF. Thus, we aimed to investigate the association between TyG index and AF among hospitalized patients. Methods: A retrospective observational study was conducted in Daping Hospital, which included 356 hospitalized patients from the Department of Cardiology. Clinical and biochemical parameters were collected from electronic medical records and AF was diagnosed from electrocardiogram (ECG) findings. Results: We found that the TyG index was significantly higher in the AF group than in the group without AF. Multivariate logistic regression revealed that hypertension (OR = 1.756, 95%CI 1.135-2.717, P = 0.011) and TyG index (OR = 2.092, 95%CI 1.412-3.100, P<0.001) were positively associated with AF. The analysis of the area under the ROC curve was performed and revealed that area under curve (AUC) of TyG index was 0.600 (95%CI, 0.542-0.659, P = 0.001), the optimal critical value was 8.35, the sensitivity was 65.4%, and the specificity was 52.0%. Additional subgroup analyses of diabetic and non-diabetic subjects were also performed and found the TyG index was increased in non-diabetic subjects with AF. Furthermore, a logistic regression analysis showed TyG index was associated with AF (OR = 3.065, 95% CI, 1.819-5.166, P<0.001) in non-diabetic subjects. However, TyG index was not associated with AF in diabetic subjects. Conclusion: Elevated TyG index is an independent risk factor for AF among non-diabetic hospitalized patients.

Transient Receptor Potential Vanilloid Type 1 Protects Against Pressure Overload-Induced Cardiac Hypertrophy by Promoting Mitochondria-Associated Endoplasmic Reticulum Membranes.

ABSTRACT: Transient receptor potential vanilloid type 1 (TRPV1) is a nonselective cation channel that mediates the relationship between mitochondrial function and pathological myocardial hypertrophy. However, its underlying mechanisms remain unclear. This study aimed to investigate whether TRPV1 activation improves the morphology and function of intracellular mitochondria to protect cardiomyocytes after pressure overload-induced myocardial hypertrophy. The myocardial hypertrophy model was established by performing transverse aortic constriction surgery in C57BL/6 J male mice. The data revealed that TRPV1 activation significantly reduced myocardial hypertrophy, promoted ejection fraction% and fractional shortening%, and decreased the left ventricular internal diameter in end-diastole and left ventricular internal diameter in end-systole after transverse aortic constriction. Moreover, in vitro experiments revealed that TRPV1 reduces cardiomyocyte area and improves mitochondrial function by promoting mitochondria-associated endoplasmic reticulum membranes (MAMs) formation in a phenylephrine-treated cardiomyocyte hypertrophy model. TRPV1 up-regulates the phosphorylation levels of AMP-activated protein kinase and expression of mitofusin2 (MFN2). TRPV1 function is blocked by single-stranded RNA interfering with silent interfering MFN2. Activation of TRPV1 reduced mitochondrial reactive oxygen species caused by phenylephrine, whereas disruption of MAMs by siMFN2 abolished TRPV1-mediated mitochondrial protection. Our findings suggest that TRPV1 effectively protects against pressure overload-induced cardiac hypertrophy by promoting MAM formation and conserved mitochondrial function via the AMP-activated protein kinase/MFN2 pathway in cardiomyocytes.

Identification and Characterization of the Amphioxus Lck and Its Associated Tyrosine Phosphorylation-Dependent Inhibitory LRR Receptor.

Amphioxus (e.g., Branchiostoma belcheri, Bb) has recently emerged as a new model for studying the origin and evolution of vertebrate immunity. Mammalian lymphocyte-specific tyrosine kinase (Lck) plays crucial roles in T cell activation, differentiation and homeostasis, and is reported to phosphorylate both the ITIM and ITSM of PD-1 to induce the recruitment of phosphatases and thus the inhibitory function of PD-1. Here, we identified and cloned the amphioxus homolog of human Lck. By generating and using an antibody against BbLck, we found that BbLck is expressed in the amphioxus gut and gill. Through overexpression of BbLck in Jurkat T cells, we found that upon TCR stimulation, BbLck was subjected to tyrosine phosphorylation and could partially rescue Lck-dependent tyrosine phosphorylation in Lck-knockdown T cells. Mass spectrometric analysis of BbLck immunoprecipitates from immunostimulants-treated amphioxus, revealed a BbLck-associated membrane-bound receptor LRR (BbLcLRR). By overexpressing BbLcLRR in Jurkat T cells, we demonstrated that BbLcLRR was tyrosine phosphorylated upon TCR stimulation, which was inhibited by Lck knockdown and was rescued by overexpression of BbLck. By mutating single tyrosine to phenylalanine (Y-F), we identified three tyrosine residues (Y539, Y655, and Y690) (3Y) of BbLcLRR as the major Lck phosphorylation sites. Reporter gene assays showed that overexpression of BbLcLRR but not the BbLcLRR-3YF mutant inhibited TCR-induced NF-κB activation. In Lck-knockdown T cells, the decline of TCR-induced IL-2 production was reversed by overexpression of BbLck, and this reversion was inhibited by co-expression of BbLcLRR but not the BbLcLRR-3YF mutant. Sequence analysis showed that the three tyrosine-containing sequences were conserved with the tyrosine-based inhibition motifs (ITIMs) or ITIM-like motifs. And TCR stimulation induced the association of BbLcLRR with tyrosine phosphatases SHIP1 and to a lesser extent with SHP1/2. Moreover, overexpression of wild-type BbLcLRR but not its 3YF mutant inhibited TCR-induced tyrosine phosphorylation of multiple signaling proteins probably via recruiting SHIP1. Thus, we identified a novel immunoreceptor BbLcLRR, which is phosphorylated by Lck and then exerts a phosphorylation-dependent inhibitory role in TCR-mediated T-cell activation, implying a mechanism for the maintenance of self-tolerance and homeostasis of amphioxus immune system and the evolutionary conservatism of Lck-regulated inhibitory receptor pathway.

Concentration and integrity indexes of urine cell-free DNA as promising biomarkers for early lung cancer diagnosis.

Aim: To explore the role of urine cell-free DNA (ucfDNA) concentration and integrity indexes as potential biomarkers for lung cancer diagnosis. Materials & methods: Quantitative real-time PCR targeting Arthrobacter luteus (ALU) repeats at three size fragments (ALU-60, 115 and 247 bp) was performed in 55 lung cancer patients and 35 healthy individuals. Results: ucfDNA concentration and integrity indexes were significantly higher in lung cancer patients than in healthy controls. The area under the receiver operating characteristic curve for differentiating patients with stage I/II from healthy controls by ALU fragments concentration were 0.856, 0.909 and 0.932, respectively. In addition, the ucfDNA integrity indexes in patients with lymph node metastasis were significantly higher than in patients with non-metastatic. Conclusion: ucfDNA concentration and integrity indexes could serve as promising biomarkers for lung cancer diagnosis.

Scorpion primer PCR analysis for genotyping of allele variants of thiopurine s­methyltransferase*3.

Thiopurine S-methyltransferase (TPMT) plays an important role in the metabolism of thiopurines. Mutations in the TPMT gene can affect drug activity, which may have adverse effects in humans. Thus, genotyping can help elucidate genetic determinants of drug response to thiopurines and optimize the selection of drug therapies for individual patients, effectively avoiding palindromia during maintenance treatment caused by insufficient dosing and the serious side effects caused by excessive doses. The current available detection methods used for TPMT*3B and TPMT*3C are complex, costly and time­consuming. Therefore, innovative detection methods for TPMT genotyping are urgently required. The aim of the present study was to establish and optimize a simple, specific and timesaving TPMT genotyping method. Using the principles of Web­based Allele­Specific PCR and competitive real­time fluorescent allele­specific PCR (CRAS­PCR), two pairs of Scorpion primers were designed for the detection of TPMT*3B and *3C, respectively, and a mutation in TPMT*3A was inferred based on data from TPMT*3B and *3C. In total, 226 samples from volunteers living in Chongqing were used for CRAS­PCR to detect TPMT*3 mutations. Results showed that nine (3.98%) were mutant (MT) heterozygotes and none were MT homozygotes for TPMT*3C, and no TPMT*3A and TPMT*3B mutations were found. Three TPMT*3C MT heterozygotes were randomly selected for DNA sequencing, and CRAS­PCR results were consistent with the sequencing results. In conclusion, in order to improve simplicity, specificity and efficiency, the present study established and optimized CRAS­PCR assays for commonly found mutant alleles of TPMT*3A (G460A and A719G), TPMT*3B (G460A), and TPMT*3C (A719G).

Urine cell-free DNA as a promising biomarker for early detection of non-small cell lung cancer.

BACKGROUND: While blood-derived cell-free DNA has been shown to be a candidate biomarker able to provide diagnostic and prognostic insight in cancer patients, little is known regarding the potential application of urine cell-free DNA (ucfDNA) in diagnosis of cancer. Thus, the aim of this study was to investigate ucfDNA concentration and integrity index as potential biomarkers for early detection of non-small-cell lung cancer (NSCLC). METHODS: Urine samples were collected from 35 healthy controls and 55 NSCLC patients at various tumor node metastasis (TNM) stages. Two long interspersed nuclear element 1 (LINE1) fragments (LINE1-97 and 266 bp) were quantified via quantitative real-time PCR (qPCR). DNA integrity index was calculated as the ratio of LINE1-266/LINE-97. RESULTS: LINE1 fragments concentrations of ucfDNA (LINE1-97, 266 bp) were significantly higher in NSCLC patients with stage III/IV than in stage I/II and in healthy controls. The receiver operating characteristic (ROC) curves for discriminating patients with stage III/IV from healthy controls had areas under the curves (AUC) of 0.84 and 0.886, respectively. Moreover, ucfDNA integrity LINE1-266/97 was significantly higher in patients with stage III/IV than in stage I/II and in healthy controls. The AUC of ROC curve for discriminating patients with stage III/IV from healthy controls was 0.800. Furthermore, LINE1-266 fragment concentration was significantly higher in lymph node metastasis (LNM)-positive patients relative to LNM-negative patients. The ROC curve for discriminating LNM-positive from LNM-negative patients had an AUC of 0.822. CONCLUSION: UcfDNA could serve as a promising biomarker for early detection of NSCLC.

Sensitive detection of low-abundance in-frame deletions in EGFR exon 19 using novel wild-type blockers in real-time PCR.

Epidermal growth factor receptor (EGFR) mutations are associated with response of tyrosine kinase inhibitors (TKIs) for patients with advanced non-small cell lung cancer (NSCLC). However, the existing methods for detection of samples having rare mutations(i.e. ~0.01%) have limits in terms of specificity, time consumption or cost. In the current study, novel wild-type blocking (WTB) oligonucleotides modified with phosphorothioate or inverted dT at the 5'-termini were designed to precisely detect 11 common deletion mutations in exon 19 of EGFR gene (E19del) using a WTB-PCR assay. And internal competitive leptin amplifications were further applied to enhance the specificity of the WTB-PCR system. Our results showed that WTB-PCR could completely block amplification of wild-type EGFR when 200 ng of DNA was used as template. Furthermore, the current WTB-PCR assay facilitated the detection of E19del mutations with a selectivity of 0.01% and sensitivity as low as a single copy. And, the results showed that the current WTB-PCR system exceeded detection limits afforded by the ARMS-PCR assay. In conclusion, the current WTB-PCR strategy represents a simple and cost-effective method to precisely detect various low-abundance deletion mutations.

Development of duplex-crossed allele-specific PCR targeting of TPMT*3B and *3C using crossed allele-specific blockers to eliminate non-specific amplification.

Prospective testing for variants in the thiopurine S-methyltransferase (TPMT) is considered a key process in the development of thiopurine therapy. This testing is done to avoid toxicity and side effects in the management of diverse immunological and malignant conditions. Real-time fluorescent PCR techniques using duplex-crossed allele-specific primers in a single tube (DCAS-PCR) were developed in this study to genotype the common loss-of-function TPMT*3B c.460G > A (rs1800460) and TPMT*3C c.719A > G (rs1142345) usually occurring in individuals of Chinese ethnicity. In this method, several integrated strategies were used to completely eliminate the non-specific amplification that is commonly presented in traditional allele-specific (AS) PCR. These strategies include using AS-primers (ASP) that both are artificially mismatched in the penultimate positions and phosphorothioate modifications in the 5'-termini positions. In the assay, an AS-blocker was used, locus-specific TaqMan (LST) probes were used and we used at least two fragments were simultaneously amplified in a single tube which satisfy the thermodynamic characteristics of DNA polymerase to eliminate non-specific amplification. In a group of 200 unselected subjects, the results showed that 8 samples were heterozygous of TPMT*3C, and all samples possessed wild-type TPMT*3B. There was no non-specific amplification, and the genotypes were 100% consistent with Sanger sequencing.

Sensitive and selective detections of codon 12 and 13 KRAS mutations in a single tube using modified wild-type blocker.

It was hypothesized that in the WTB-PCR system, the greater number of cycles, associated with the thermodynamic driving force of DNA polymerase resulted in artificial introduction of mutant nucleotides in amplicons. In the current study, universal WTB-PCR was developed to overcome these limitations, in which two strategies were used: phosphorothioate modifications were made at the 5'-termini bases of the WTB oligonucleotides, and amplification of referenced internal positive controller (RIPC) fragments was performed. The results showed that universal WTB-PCR could detect single-copy KRAS mutant alleles with higher selectivity (i.e., 0.01%), and with greater ability to eliminate non-specific amplification of KRAS wild-type alleles in amounts up to 200 ng. Moreover, the introduction of referenced internal positive controller (RIPC) fragments prevented false-negative results caused by inadequate amounts of input sample DNA, and allowed for quantitative analysis of the mutation levels in each FFPE sample. In clinical application in 50 samples of FFPE tissue sections from mCRC patients, 70% (35/50) showed various mutations at codons 12 and 13 of KRAS genes; 30% (15/50) could be detected by traditional PCR without WTB oligonucleotides. In conclusion, universal WTB-PCR is a rapid, simple and low-cost method for detection of low-abundance KRAS mutations in mCRC patients.

Molecular mechanisms underlying the evolution of the slp76 signalosome.

The well-defined mammalian slp76-signalosome is crucial for T-cell immune response, yet whether slp76-signalosome exists in invertebrates and how it evolved remain unknown. Here we investigated slp76-signalosome from an evolutionary perspective in amphioxus Branchiostoma belcheri (bb). We proved slp76-signalosome components bbslp76, bbGADS and bbItk are present in amphioxus and bbslp76 interacts with bbGADS and bbItk, but differences exist between the interaction manners within slp76-signalosome components of amphioxus and human (h). Specifically, bbslp76 has a unique WW-domain that blocked its association with hItk and decreased TCR-induced tyrosine-phosphorylation and NFAT-activation. Deletion of WW-domain shifted the constitutive association between bbslp76 and hPLCγ1 to a TCR-enhanced association. Among slp76-signalosome, the interaction between slp76 and PLCγ1 is the most conserved and the binding between Itk and slp76 evolved from constitutive to stimulation-regulated. Sequence alignment and 3D structural analysis of slp76-signalosome molecules from keystone species indicated slp76 evolved into a more unfolded and flexible adaptor due to lack of WW-domain and several low-complexity-regions (LCRs) while GADS turned into a larger protein by a LCR gain, thus preparing more space for nucleating the coevolving slp76-signalosome. Altogether, through deletion of WW-domain and manipulation of LCRs, slp76-signalosome evolves from a rigid and stimulation-insensitive to a more flexible and stimulation-responding complex.

Over-expression of survivin is a factor responsible for differential responses of ovarian cancer cells to S-allylmercaptocysteine (SAMC).

While investigating the inhibitory effect of S-allylmercaptocysteine (SAMC), a garlic derivative, on ovarian cancer, we subjected three ovarian cancer cell lines, HO8910, HO8910PM, and SKOV3, to SAMC treatment. In vivo and in vitro experiments showed that only HO8910 and SKOV3 cells were highly sensitive to SAMC, whereas HO8910PM cells were resistant to SAMC. Subsequently, we examined the apoptosis-related genes in the three cell lines. We found that survivin gene was highly expressed in HO8910PM cells. Down regulation of survivin gene in HO8910PM cells with small interference RNA (siRNA), resulted in increased sensitivity to SAMC together with a decrease in invasiveness of tumor cells. We therefore concluded that the S-allylmercaptocysteine suppresses both the proliferation and distant metastasis of epithelial ovarian cancer cells, insensitivity of HO8910PM cells to SAMC was closely related to the high level of survivin expression and that combination of SAMC treatment together with survivin knockdown might be a potential strategy for treatment of certain variants of ovarian cancers.