Facile and sensitive detection of mercury ions based on fluorescent structure-switching aptamer probe and exonuclease â ¢-assisted signal amplification.

Hg2+ is highly toxic to human health and ecosystem. In this work, based on the unique fluorescent property of 2-Aminopurine (2-AP), the formation of T-Hg2+-T mismatch structure and the signal amplification of exonuclease III (Exo III) assisted target cycle, a fluorescent probe for facile and sensitive detection of Hg2+ is constructed. The hairpin-looped DNA probe is rationally designed with 2-AP embedded in the stem and thymine-rich recognition overhangs extended at the termini. The cleavage of the double stranded DNA stem with stable T-Hg2+-T pairs catalyzed by Exo III is prompted to happen upon recognition of trace Hg2+. Under the optimal reaction conditions, there is an excellent linear relationship between Hg2+ concentration and fluorescence intensity in the range of 7.5-200 nM with a detection limit of 0.38 nM. In addition, the detection results of Hg2+ in Songhua River water and fish samples are satisfactory. The fluorescent probe avoids labeling additional quenchers or quenching materials and has strong anti-interference ability. Thus, the fluorescent probe has a broad prospect in practical application.

Gold nanoclusters-manganese dioxide composite-based fluorescence immunoassay for sensitive monitoring of fenitrothion degradation in Chinese cabbage.

Understanding the residues and degradation of organophosphorus pesticides (OPs) in crops has attracted increasing attention. Herein, we designed a sensitive fluorescence immunoassay (FIA) by employing nanobody-linked alkaline phosphatase (Nb-ALP) and gold nanoclusters anchored manganese dioxide (AuNCs-MnO2) composite. In immunoassay protocol, Nb-ALP is used to competitively recognize the coating antigen and pesticide. After competitive immunoreaction, alkaline phosphatase catalyzes l-ascorbic acid-2-phosphate to produce ascorbic acid that can trigger the decomposition of the AuNCs-MnO2 composite, regulating the fluorescence response. As a proof-of-concept, fenitrothion (FNT) is chosen as the target analyte. As a result, the developed FIA exhibits high detection sensitivity (IC10 = 5.78 pg/mL), which is about 56-times higher than that of the conventional enzyme-linked immunosorbent assay. The developed FIA has been successfully applied for precisely monitoring the degradation of FNT in Chinese cabbage with excellent anti-interference ability and reproducibility, paving the way for the determination of pesticide residues in real food samples.

Protein tyrosine phosphatase PTPN2 regulates TGF-ß signaling through Smad4 dephosphorylation.

Transforming Growth Factor beta (TGF-ß) is a multifunctional cytokine that regulates cell proliferation, differentiation, and apoptosis. Dysregulation of the TGF-ß signaling is one of the major mechanisms underlying tumor progression. We have previously reported that anaplastic lymphoma kinase (ALK) phosphorylates Smad4 at Tyr95, which compromises the DNA-binding ability of Smad4 and thus renders ALK-positive cancer cells resistant to TGF-ß tumor-suppressive action. In this study, we demonstrated that tyrosine phosphatase PTPN2 positively regulated TGF-ß signaling through dephosphorylating Smad4 at the Tyr95 site. Both in vitro and cell-based assays revealed that PTPN2 bound to and dephosphorylated Smad4, thereby preserving the DNA-binding ability of Smad4. Furthermore, overexpression of PTPN2 restored TGF-ß transcriptional and growth inhibitory responses in ALK-positive cancer cells. Consistently, Spermidine, an activator of PTPN2, also promoted TGF-ß-induced gene expression, apoptosis, and anti-proliferation effect. Taken together, we revealed that PTPN2 functioned as a tumor suppressor to antagonize the inhibitory effect of tyrosine phosphorylation of Smad4 and to ensure the proper TGF-ß growth inhibitory signaling in cancer cells.

Nuclear receptors NHR-49 and NHR-79 promote peroxisome proliferation to compensate for aldehyde dehydrogenase deficiency in C. elegans.

The intracellular level of fatty aldehydes is tightly regulated by aldehyde dehydrogenases to minimize the formation of toxic lipid and protein adducts. Importantly, the dysregulation of aldehyde dehydrogenases has been implicated in neurologic disorder and cancer in humans. However, cellular responses to unresolved, elevated fatty aldehyde levels are poorly understood. Here, we report that ALH-4 is a C. elegans aldehyde dehydrogenase that specifically associates with the endoplasmic reticulum, mitochondria and peroxisomes. Based on lipidomic and imaging analysis, we show that the loss of ALH-4 increases fatty aldehyde levels and reduces fat storage. ALH-4 deficiency in the intestine, cell-nonautonomously induces NHR-49/NHR-79-dependent hypodermal peroxisome proliferation. This is accompanied by the upregulation of catalases and fatty acid catabolic enzymes, as indicated by RNA sequencing. Such a response is required to counteract ALH-4 deficiency since alh-4; nhr-49 double mutant animals are sterile. Our work reveals unexpected inter-tissue communication of fatty aldehyde levels and suggests pharmacological modulation of peroxisome proliferation as a therapeutic strategy to tackle pathology related to excess fatty aldehydes.

PTPN3 acts as a tumor suppressor and boosts TGF-ß signaling independent of its phosphatase activity.

TGF-ß controls a variety of cellular functions during development. Abnormal TGF-ß responses are commonly found in human diseases such as cancer, suggesting that TGF-ß signaling must be tightly regulated. Here, we report that protein tyrosine phosphatase non-receptor 3 (PTPN3) profoundly potentiates TGF-ß signaling independent of its phosphatase activity. PTPN3 stabilizes TGF-ß type I receptor (TßRI) through attenuating the interaction between Smurf2 and TßRI. Consequently, PTPN3 facilitates TGF-ß-induced R-Smad phosphorylation, transcriptional responses, and subsequent physiological responses. Importantly, the leucine-to-arginine substitution at amino acid residue 232 (L232R) of PTPN3, a frequent mutation found in intrahepatic cholangiocarcinoma (ICC), disables its role in enhancing TGF-ß signaling and abolishes its tumor-suppressive function. Our findings have revealed a vital role of PTPN3 in regulating TGF-ß signaling during normal physiology and pathogenesis.

LECT2 drives haematopoietic stem cell expansion and mobilization via regulating the macrophages and osteolineage cells.

Haematopoietic stem cells (HSCs) can differentiate into cells of all lineages in the blood. However, the mechanisms by which cytokines in the blood affect HSC homeostasis remain largely unknown. Here we show that leukocyte cell-derived chemotaxin 2 (LECT2), a multifunctional cytokine, induces HSC expansion and mobilization. Recombinant LECT2 administration results in HSC expansion in the bone marrow and mobilization to the blood via CD209a. The effect of LECT2 on HSCs is reduced after specific depletion of macrophages or reduction of osteolineage cells. LECT2 treatment reduces the tumour necrosis factor (TNF) expression in macrophages and osteolineage cells. In TNF knockout mice, the effect of LECT2 on HSCs is reduced. Moreover, LECT2 induces HSC mobilization in irradiated mice, while granulocyte colony-stimulating factor does not. Our results illustrate that LECT2 is an extramedullar cytokine that contributes to HSC homeostasis and may be useful to induce HSC mobilization.

Identification of lipid droplet structure-like/resident proteins in Caenorhabditis elegans.

The lipid droplet (LD) is a cellular organelle that stores neutral lipids in cells and has been linked with metabolic disorders. Caenorhabditis elegans has many characteristics which make it an excellent animal model for studying LDs. However, unlike in mammalian cells, no LD structure-like/resident proteins have been identified in C. elegans, which has limited the utility of this model for the study of lipid storage and metabolism. Herein based on three lines of evidence, we identified that MDT-28 and DHS-3 previously identified in C. elegans LD proteome were two LD structure-like/resident proteins. First, MDT-28 and DHS-3 were found to be the two most abundant LD proteins in the worm. Second, the proteins were specifically localized to LDs and we identified the domains responsible for this targeting in both proteins. Third and most importantly, the depletion of MDT-28 induced LD clustering while DHS-3 deletion reduced triacylglycerol content (TAG). We further characterized the proteins finding that MDT-28 was ubiquitously expressed in the intestine, muscle, hypodermis, and embryos, whereas DHS-3 was expressed mainly in intestinal cells. Together, these two LD structure-like/resident proteins provide a basis for future mechanistic studies into the dynamics and functions of LDs in C. elegans.

Specific control of BMP signaling and mesenchymal differentiation by cytoplasmic phosphatase PPM1H.

Bone morphogenetic proteins (BMPs) belong to the TGF-ß superfamily of structurally related signaling proteins that regulate a wide array of cellular functions. The key step in BMP signal transduction is the BMP receptor-mediated phosphorylation of transcription factors Smad1, 5, and 8 (collectively Smad1/5/8), which leads to the subsequent activation of BMP-induced gene transcription in the nucleus. In this study, we describe the identification and characterization of PPM1H as a novel cytoplasm-localized Smad1/5/8-specific phosphatase. PPM1H directly interacts with Smad1/5/8 through its Smad-binding domain, and dephosphorylates phospho-Smad1/5/8 (P-Smad1/5/8) in the cytoplasm. Ectopic expression of PPM1H attenuates BMP signaling, whereas loss of PPM1H activity or expression greatly enhances BMP-dependent gene regulation and mesenchymal differentiation. In conclusion, this study suggests that PPM1H acts as a gatekeeper to prevent excessive BMP signaling through dephosphorylation and subsequent nuclear exclusion of P-Smad1/5/8 proteins.

E2F1 is a novel fibrogenic gene that regulates cholestatic liver fibrosis through the Egr-1/SHP/EID1 network.

UNLABELLED: E2F transcription factor 1 (E2F1) is an important regulator of metabolic diseases; however, its role in liver function remains elusive. This study unraveled a regulatory cascade involving E2F1, early growth response-1 (Egr-1), nuclear receptor small heterodimer partner (SHP, NR0B2), and EIA-like inhibitor of differentiation 1 (EID1) in cholestatic liver fibrosis. Liver E2F1 messenger RNA (mRNA) and protein expression was strongly up-regulated in human nonalcoholic steatohepatitis (NASH) and alcohol cirrhosis; the latter was inversely correlated with diminished SHP expression. E2F1 was also highly induced by 3,5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) feeding and bile-duct ligation (BDL) in mice. E2F1-/- mice exhibited reduced biliary fibrosis by DDC as determined by Masson Trichrome and Picro Sirius red staining, and decreased serum bile acid (BA), BA pool size, and fecal BA excretion. In addition, cholestatic liver fibrosis induced by BDL, as determined by immunohistochemistry analysis of a1 collagen expression, was increased in SHP-/- mice but attenuated in hepatocyte SHP-overexpressed transgenic (STG) mice. Egr-1 exhibited marked induction in livers of SHP-/- mice compared to the wild-type mice in both sham and BDL groups, and reduction in STG livers. Egr-1 promoter was activated by E2F1, and the activation was abrogated by expression of SHP and its co-repressor EID1 in hepatoma cells Huh7, Hepa1, and stellate cells LX2. Chromatin immunoprecipitation assays further confirmed the association of E2F1, SHP, and EID1 proteins with the Egr-1 promoter, and their direct protein interactions were determined by glutathione S-transferase pull-down assays. Interestingly, E2F1 activated Egr-1 expression in a biphasic fashion as described in both human and mouse hepatocytes. CONCLUSION: E2F1 is a fibrogenic gene and could serve as a potential new diagnostic marker for nonalcoholic and alcoholic liver fibrosis/cirrhosis.

A hybrid CPU-GPU accelerated framework for fast mapping of high-resolution human brain connectome.

Recently, a combination of non-invasive neuroimaging techniques and graph theoretical approaches has provided a unique opportunity for understanding the patterns of the structural and functional connectivity of the human brain (referred to as the human brain connectome). Currently, there is a very large amount of brain imaging data that have been collected, and there are very high requirements for the computational capabilities that are used in high-resolution connectome research. In this paper, we propose a hybrid CPU-GPU framework to accelerate the computation of the human brain connectome. We applied this framework to a publicly available resting-state functional MRI dataset from 197 participants. For each subject, we first computed Pearson's Correlation coefficient between any pairs of the time series of gray-matter voxels, and then we constructed unweighted undirected brain networks with 58 k nodes and a sparsity range from 0.02% to 0.17%. Next, graphic properties of the functional brain networks were quantified, analyzed and compared with those of 15 corresponding random networks. With our proposed accelerating framework, the above process for each network cost 80∼150 minutes, depending on the network sparsity. Further analyses revealed that high-resolution functional brain networks have efficient small-world properties, significant modular structure, a power law degree distribution and highly connected nodes in the medial frontal and parietal cortical regions. These results are largely compatible with previous human brain network studies. Taken together, our proposed framework can substantially enhance the applicability and efficacy of high-resolution (voxel-based) brain network analysis, and have the potential to accelerate the mapping of the human brain connectome in normal and disease states.

The FATP1-DGAT2 complex facilitates lipid droplet expansion at the ER-lipid droplet interface.

At the subcellular level, fat storage is confined to the evolutionarily conserved compartments termed lipid droplets (LDs), which are closely associated with the endoplasmic reticulum (ER). However, the molecular mechanisms that enable ER-LD interaction and facilitate neutral lipid loading into LDs are poorly understood. In this paper, we present evidence that FATP1/acyl-CoA synthetase and DGAT2/diacylglycerol acyltransferase are components of a triglyceride synthesis complex that facilitates LD expansion. A loss of FATP1 or DGAT2 function blocked LD expansion in Caenorhabditis elegans. FATP1 preferentially associated with DGAT2, and they acted synergistically to promote LD expansion in mammalian cells. Live imaging indicated that FATP1 and DGAT2 are ER and LD resident proteins, respectively, and electron microscopy revealed FATP1 and DGAT2 foci close to the LD surface. Furthermore, DGAT2 that was retained in the ER failed to support LD expansion. We propose that the evolutionarily conserved FATP1-DGAT2 complex acts at the ER-LD interface and couples the synthesis and deposition of triglycerides into LDs both physically and functionally.

Nuclear cGMP-dependent kinase regulates gene expression via activity-dependent recruitment of a conserved histone deacetylase complex.

Elevation of the second messenger cGMP by nitric oxide (NO) activates the cGMP-dependent protein kinase PKG, which is key in regulating cardiovascular, intestinal, and neuronal functions in mammals. The NO-cGMP-PKG signaling pathway is also a major therapeutic target for cardiovascular and male reproductive diseases. Despite widespread effects of PKG activation, few molecular targets of PKG are known. We study how EGL-4, the Caenorhabditis elegans PKG ortholog, modulates foraging behavior and egg-laying and seeks the downstream effectors of EGL-4 activity. Using a combination of unbiased forward genetic screen and proteomic analysis, we have identified a conserved SAEG-1/SAEG-2/HDA-2 histone deacetylase complex that is specifically recruited by activated nuclear EGL-4. Gene expression profiling by microarrays revealed >40 genes that are sensitive to EGL-4 activity in a SAEG-1-dependent manner. We present evidence that EGL-4 controls egg laying via one of these genes, Y45F10C.2, which encodes a novel protein that is expressed exclusively in the uterine epithelium. Our results indicate that, in addition to cytoplasmic functions, active EGL-4/PKG acts in the nucleus via a conserved Class I histone deacetylase complex to regulate gene expression pertinent to behavioral and physiological responses to cGMP. We also identify transcriptional targets of EGL-4 that carry out discrete components of the physiological response.

Genetic and dietary regulation of lipid droplet expansion in Caenorhabditis elegans.

Dietary fat accumulates in lipid droplets or endolysosomal compartments that undergo selective expansion under normal or pathophysiological conditions. We find that genetic defects in a peroxisomal beta-oxidation pathway cause size expansion in lipid droplets that are distinct from the lysosome-related organelles in Caenorhabditis elegans. Expansion of lipid droplets is accompanied by an increase in triglycerides (TAG) that are resistant to fasting- or TAG lipase-triggered lipolysis. Nevertheless, in mutant animals, a diet poor in vaccenic acid reduced the TAG level and lipid droplet size. Our results implicate peroxisomal dysfunction in pathologic lipid droplet expansion in animals and illustrate how dietary factors modulate the phenotype of such genetic defects.

Intra-specific genetic relationship analyses of Elaeagnus angustifolia based on RP-HPLC biochemical markers.

Elaeagnus angustifolia Linn. has various ecological, medicinal and economical uses. An approach was established using RP-HPLC (reversed-phase high-performance liquid chromatography) to classify and analyse the intra-specific genetic relationships of seventeen populations of E. angustifolia, collected from the Xinjiang areas of China. Chromatograms of alcohol-soluble proteins produced by seventeen populations of E. angustifolia, were compared. Each chromatogram of alcohol-soluble proteins came from a single seed of one wild plant only. The results showed that when using a Waters Delta Pak. C18, 5 microm particle size reversed phase column (150 mm x 3.9 mm), a linear gradient of 25%-60% solvent B with flow rate of 1 ml/min and run time of 67 min, the chromatography yielded optimum separation of E. angustifolia alcohol-soluble proteins. Representative peaks in each population were chosen according to peak area and occurrence in every seed. The converted data on the elution peaks of each population were different and could be used to represent those populations. GSC (genetic similarity coefficients) of 41% to 62% showed a medium degree of genetic diversity among the populations in these eco-areas. Cluster analysis showed that the seventeen populations of E. angustifolia could be divided into six clusters at the GSC=0.535 level and indicated the general and unique biochemical markers of these clusters. We suggest that E. angustifolia distribution in these eco-areas could be classified into six variable species. RP-HPLC was shown to be a rapid, repeatable and reliable method for E. angustifolia classification and identification and for analysis of genetic diversity.

Corticotropin-releasing factor receptor subtype 1 and somatostatin modulating hypoxia-caused downregulated mRNA of pituitary growth hormone and upregulated mRNA of hepatic insulin-like growth factor-I of rats.

The study aims to examine the effects of restraint, cold, and in combination of hypoxia on pituitary GH mRNA and hepatic IGF-I mRNA and its protein in rats, and the potential involvement of corticotropin-releasing factor receptor subtype 1 (CRFR1) and SS in mediating the effects of continual hypoxia. Continual or intermittent hypoxia of 5 km (10.8% O2) was simulated in a hypobaric chamber. The mRNAs and peptides were determined using RT-PCR and Elisa or histochemistry. Continual hypoxia of 5 km markedly enhanced immunostaining pituitary GH and hepatic IGF-I for 1 and 2 days restoring afterward. The hypoxia for 5 days significantly reduced the pituitary GH mRNA and increased the hepatic IGF-I mRNA. Intermittent hypoxia of 5 km 4 h/day for 2 days, cold (4 degrees C) 4h/day for 2 days, and restraint 4 h/day for 2 days alone or in combination significantly enhanced immunostaining pituitary GH and hepatic IGF-I (except cold). The combined stresses had greater effects than single stresses alone. CRFR1 antagonist (CP154526) or SS antagonist (cysteamine) markedly blocked hypoxia-reduced pituitary GH mRNA and hypoxia-activated hepatic IGF-I mRNA, and further reduced hypoxia-reduced plasma IGF. In conclusion, hypoxia (continually or intermittently), restraint, cold alone or in combination modulate pituitary GH and hepatic IGF-I. The pituitary GH/GH mRNA and hepatic IGF-I/IGF-I mRNA, and plasma IGF-I are modified by hypoxia through SS and CRFR1 mediation.

Intermittent hypoxia causes a suppressed pituitary growth hormone through somatostatin.

OBJECTIVES: The aim of this study was to investigate the response of the growth hormone (GH) in rat anterior pituitary to intermittent hypoxia (IH) and its modulation by hypothalamic somatostatin (SS). SETTING AND DESIGN: To observe the hypoxic response, rats were exposed to simulated altitude hypoxia (2 km or 5 km) in a hypobaric chamber for various days (4 h/d); to clarify SS-involvement, rats were pretreated with SS antagonist (cysteamine, CSH, 200 mg/kg/d, s.c.) then exposed to IH (5 km) for 2d. The GH mRNA and immunostaining GH in pituitary as well as immunostaining SS in median eminence (ME) of hypothalamus were assayed by RT-PCR and immuno-histochemistry, respectively. RESULTS: IH of 5 km altitude (IH5) markedly suppressed the body weight gain (BWG) of rats from 1d to 10d, and it was returned to control level henceforth, while no significant influence was showed in the group of IH of 2 km altitude (IH2). IH5 for 2 and 5d significantly decreased GH mRNA expression in the pituitary. The pituitary immunostaining GH was remarkably increased in groups of IH2 for 5, 10, and 15 d, and in groups of IH5 for 2, 5, and 10d. Immunostaining SS in ME was significantly reduced in group of IH2 for 5d, and in groups of IH5 for 2d and 5d. Pretreatments (s.c.) with SS antagonist (CSH) significantly reversed IH5-caused increase of immunostaining GH and reduction of mRNA levels in pituitary. CONCLUSIONS: IH may cause a short-term and recoverable suppression of GH release, and reduce GH mRNA expression in anterior pituitary, which may depend on the intensity and duration of the hypoxia. This suppression may be due to a modulation of hypoxia-activated SS.