T-type voltage-gated channels, Na+/Ca2+-exchanger, and calpain-2 promote photoreceptor cell death in inherited retinal degeneration.

Inherited retinal degenerations (IRDs) are a group of untreatable and commonly blinding diseases characterized by progressive photoreceptor loss. IRD pathology has been linked to an excessive activation of cyclic nucleotide-gated channels (CNGC) leading to Na+- and Ca2+-influx, subsequent activation of voltage-gated Ca2+-channels (VGCC), and further Ca2+ influx. However, a connection between excessive Ca2+ influx and photoreceptor loss has yet to be proven.Here, we used whole-retina and single-cell RNA-sequencing to compare gene expression between the rd1 mouse model for IRD and wild-type (wt) mice. Differentially expressed genes indicated links to several Ca2+-signalling related pathways. To explore these, rd1 and wt organotypic retinal explant cultures were treated with the intracellular Ca2+-chelator BAPTA-AM or inhibitors of different Ca2+-permeable channels, including CNGC, L-type VGCC, T-type VGCC, Ca2+-release-activated channel (CRAC), and Na+/Ca2+ exchanger (NCX). Moreover, we employed the novel compound NA-184 to selectively inhibit the Ca2+-dependent protease calpain-2. Effects on the retinal activity of poly(ADP-ribose) polymerase (PARP), sirtuin-type histone-deacetylase, calpains, as well as on activation of calpain-1, and - 2 were monitored, cell death was assessed via the TUNEL assay.While rd1 photoreceptor cell death was reduced by BAPTA-AM, Ca2+-channel blockers had divergent effects: While inhibition of T-type VGCC and NCX promoted survival, blocking CNGCs and CRACs did not. The treatment-related activity patterns of calpains and PARPs corresponded to the extent of cell death. Remarkably, sirtuin activity and calpain-1 activation were linked to photoreceptor protection, while calpain-2 activity was related to degeneration. In support of this finding, the calpain-2 inhibitor NA-184 protected rd1 photoreceptors.These results suggest that Ca2+ overload in rd1 photoreceptors may be triggered by T-type VGCCs and NCX. High Ca2+-levels likely suppress protective activity of calpain-1 and promote retinal degeneration via activation of calpain-2. Overall, our study details the complexity of Ca2+-signalling in photoreceptors and emphasizes the importance of targeting degenerative processes specifically to achieve a therapeutic benefit for IRDs. Video Abstract.

Image-guided metabolomics and transcriptomics reveal tumour heterogeneity in luminal A and B human breast cancer beyond glucose tracer uptake.

BACKGROUND: Breast cancer is a metabolically heterogeneous disease, and although the concept of heterogeneous cancer metabolism is known, its precise role in human breast cancer is yet to be fully elucidated. METHODS: We investigated in an explorative approach a cohort of 42 primary mamma carcinoma patients with positron emission tomography/magnetic resonance imaging (PET/MR) prior to surgery, followed by histopathology and molecular diagnosis. From a subset of patients, which showed high metabolic heterogeneity based on tracer uptake and pathology classification, tumour centre and periphery specimen tissue samples were further investigated by a targeted breast cancer gene expression panel and quantitative metabolomics by nuclear magnetic resonance (NMR) spectroscopy. All data were analysed in a combinatory approach. RESULTS: [18 F]FDG (2-deoxy-2-[fluorine-18]fluoro-d-glucose) tracer uptake confirmed dominance of glucose metabolism in the breast tumour centre, with lower levels in the periphery. Additionally, we observed differences in lipid and proliferation related genes between luminal A and B subtypes in the centre and periphery. Tumour periphery showed elevated acetate levels and enrichment in lipid metabolic pathways genes especially in luminal B. Furthermore, serine was increased in the periphery and higher expression of thymidylate synthase (TYMS) indicated one-carbon metabolism increased in tumour periphery. The overall metabolic activity based on [18 F]FDG uptake of luminal B subtype was higher than that of luminal A and the difference between the periphery and centre increased with tumour grade. CONCLUSION: Our analysis indicates variations in metabolism among different breast cancer subtypes and sampling locations which details the heterogeneity of the breast tumours. Correlation analysis of [18 F]FDG tracer uptake, transcriptome and tumour metabolites like acetate and serine facilitate the search for new candidates for metabolic tracers and permit distinguishing luminal A and B. This knowledge may help to differentiate subtypes preclinically or to provide patients guide for neoadjuvant therapy and optimised surgical protocols based on individual tumour metabolism.

Acidic ascites inhibits ovarian cancer cell proliferation and correlates with the metabolomic, lipidomic and inflammatory phenotype of human patients.

BACKGROUND: The poor prognosis of ovarian cancer patients is strongly related to peritoneal metastasis with the production of malignant ascites. However, it remains largely unclear how ascites in the peritoneal cavity influences tumor metabolism and recurrence. This study is an explorative approach aimed at for a deeper molecular and physical-chemical characterization of malignant ascites and to investigate their effect on in vitro ovarian cancer cell proliferation. METHODS: This study included 10 malignant ascites specimens from patients undergoing ovarian cancer resection. Ascites samples were deeply phenotyped by 1H-NMR based metabolomics, blood-gas analyzer based gas flow analysis and flow cytomertry based a 13-plex cytokine panel. Characteristics of tumor cells were investigated in a 3D spheroid model by SEM and metabolic activity, adhesion, anti-apoptosis, migratory ability evaluated by MTT assay, adhesion assay, flowcytometry and scratch assay. The effect of different pH values was assessed by adding 10% malignant ascites to the test samples. RESULTS:  The overall extracellular (peritoneal) environment was alkaline, with pH of ascites at stage II-III = 7.51 ± 0.16, and stage IV = 7.78 ± 0.16. Ovarian cancer spheroids grew rapidly in a slightly alkaline environment. Decreasing pH of the cell culture medium suppressed tumor features, metabolic activity, adhesion, anti-apoptosis, and migratory ability. However, 10% ascites could prevent tumor cells from being affected by acidic pH. Metabolomics analysis identified stage IV patients had significantly higher concentrations of alanine, isoleucine, phenylalanine, and glutamine than stage II-III patients, while stage II-III patients had significantly higher concentrations of 3-hydroxybutyrate. pH was positively correlated with acetate, and acetate positively correlated with lipid compounds. IL-8 was positively correlated with lipid metabolites and acetate. Glutathione and carnitine were negatively correlated with cytokines IL-6 and chemokines (IL-8 & MCP-1). CONCLUSION: Alkaline malignant ascites facilitated ovarian cancer progression. Additionally, deep ascites phenotyping by metabolomics and cytokine investigations allows for a refined stratification of ovarian cancer patients. These findings contribute to the understanding of ascites pathology in ovarian cancer.

mTOR contributes to endothelium-dependent vasorelaxation by promoting eNOS expression and preventing eNOS uncoupling.

Clinically used inhibitors of mammalian target of rapamycin (mTOR) negatively impacts endothelial-dependent vasodilatation (EDD) through unidentified mechanisms. Here we show that either the endothelium-specific deletion of Mtor to inhibit both mTOR complexes, or depletion of Raptor or Rictor to disrupt mTORC1 or mTORC2, causes impaired EDD, accompanied by reduced NO in the serum of mice. Consistently, inhibition of mTOR decreases NO production by human and mouse EC. Specifically, inhibition of mTORC1 suppresses eNOS gene expression, due to impairment in p70S6K-mediated posttranscriptional regulation of the transcription factor KLF2 expression. In contrast to mTORC1 inhibition, a positive-feedback between MAPK (p38 and JNK) activation and Nox2 upregulation contributes to the excessive generation of reactive oxygen species (ROS), which causes eNOS uncoupling and decreased NO bioavailability in mTORC2-inhibited EC. Adeno-associated virus-mediated EC-specific overexpression of KLF2 or suppression of Nox2 restores EDD function in endothelial mTORC1- or mTORC2-inhibited mice.

mTOR Inhibition Promotes Pneumonitis through Inducing Endothelial Contraction and Hyperpermeability.

Compromised endothelial-cell (EC) barrier function is a hallmark of inflammatory diseases. mTOR inhibitors, widely applied as clinical therapies, cause pneumonitis through mechanisms that are not yet fully understood. This study aimed to elucidate the EC mechanisms underlying the pathogenesis of pneumonitis caused by mTOR inhibition (mTORi). Mice with EC-specific deletion of mTOR complex components (Mtor, Rptor or Rictor) were administered LPS to induce pulmonary injury. Cultured ECs were treated with pharmacologic inhibitors, siRNA, or overexpression plasmids. EC barrier function was evaluated in vivo with Evans blue assay and in vitro by measurement of transendothelial electrical resistance and albumin flux. mTORi increased basal and TNFα-induced EC permeability, which was caused by myosin light chain (MLC) phosphorylation-dependent cell contraction. Inactivation of mTOR kinase activity by mTORi triggered PKCδ/p38/NF-κB signaling that significantly upregulated TNFα-induced MLCK (MLC kinase) expression, whereas Raptor promoted the phosphorylation of PKCα/MYPT1 independently of its interaction with mTOR, leading to suppression of MLCP (MLC phosphatase) activity. EC-specific deficiency in mTOR, Raptor or Rictor aggravated lung inflammation in LPS-treated mice. These findings reveal that mTORi induces PKC-dependent endothelial MLC phosphorylation, contraction, and hyperpermeability that promote pneumonitis.

Enhancement of the photoluminescence efficiency of hybrid manganese halides through rational structural design.

Five new zero-dimensional hybrid manganese halides based on discrete [MnCl4]2- tetrahedrons were prepared and used as highly efficient green-light emitters. Through rational management of organic cations to tailor the MnMn separation distances between neighboring [MnCl4]2- tetrahedrons, the photoluminescence quantum yield increased significantly from 7.98% to 81.11%.

LINC00355 induces gastric cancer proliferation and invasion through promoting ubiquitination of P53.

Long noncoding RNAs (LncRNAs) have been reported to play critical roles in gastric cancer, but true biomarkers remain unknown. In this study, we found a new lncRNA LINC00355 that was involved in malignant progression of gastric cancer (GC) and further revealed its role and mechanism. Differentially expressed lncRNAs were identified through bioinformatics, and qRT-PCR was used to validate the expression of LINC00355 in gastric cancer tissues and cells. The biological role of LINC00355 in GC was detected by gene overexpression and knockdown experiments. Subcellular fractionation, qRT-PCR, and FISH were performed to detect the subcellular localization. Co-IP and western blotting were used to study the ubiquitination-mediated regulation of P53 and the expression of the E3 ligases RAD18 and UBE3C. The results showed that LINC00355 was significantly increased in gastric cancer cell lines and patient tissues and closely correlated with late stages, distant metastasis, and poor prognosis of patients. High expression of LINC00355 promoted the proliferation and invasion of gastric cancer cells in vivo and in vitro. Mechanistic studies found that LINC00355 that mainly located in the nucleus, acting as a transcriptional activator, promoted transcription of RAD18 and UBE3C, which both bind to P53 and mediate the ubiquitination and degradation of P53. Furthermore, LINC00355 overexpression enhanced the ubiquitination process, and LINC00355 knockdown alleviated it. These results indicated that LINC00355 induces gastric cancer cell proliferation and invasion by promoting transcription of RAD18 and UBE3C, which mediates ubiquitination of P53 and thereby plays a critical role in survival and tumorigenicity of gastric cancer cells. LINC00355 may represent a new mechanism for GC progression and provide a potential marker for GC diagnosis and treatment.

Mgs1 protein supports genome stability via recognition of G-quadruplex DNA structures.

The integrity of the genetic material is crucial for every organism. One intrinsic attack to genome stability is stalling of the replication fork which can result in DNA breakage. Several factors, such as DNA lesions or the formation of stable secondary structures (eg, G-quadruplexes) can lead to replication fork stalling. G-quadruplexes (G4s) are well-characterized stable secondary DNA structures that can form within specific single-stranded DNA sequence motifs and have been shown to block/pause the replication machinery. In most genomes several helicases have been described to regulate G4 unfolding to preserve genome integrity, however, different experiments raise the hypothesis that processing of G4s during DNA replication is more complex and requires additional, so far unknown, proteins. Here, we show that the Saccharomyces cerevisiae Mgs1 protein robustly binds to G4 structures in vitro and preferentially acts at regions with a strong potential to form G4 structures in vivo. Our results suggest that Mgs1 binds to G4-forming sites and has a role in the maintenance of genome integrity.

Platelet-driven formation of interface peptide nano-network biosensor enabling a non-invasive means for early detection of Alzheimer's disease.

Soft material fabricated with DNA origami or peptide cross-linking technique may be promising theranostic platforms in the future; however, their naturally occurring counterparts, such as the peptide aggregates in the neurodegenerative diseases, constitute an increasingly burdensome issue of public health. Thus, a design of artificial peptide nano-network biosensor is conceived, in an attempt to combat the natural pathological peptides, by mimicking their pathogenesis process. Specifically, periphery platelet can secrete A-beta and induce its cross-linking & aggregation to form a surface peptide nano-network, resulting in large numbers of poly-tyrosine strands being covalently trapped in the network to serve as an efficient signal amplifier, through the electrochemical oxidation of tyrosine. This method is sensitive and quantitative in the range of normal and pathological periphery platelet distribution and can effectively discriminate Alzheimer's disease (AD) patients based on the detected potential neurodegenerative activity of platelet. These results may point to some future perspective of this method in the early screening of AD.

An enzyme substrate binding aptamer complex based time-resolved fluorescence sensor for the adenosine deaminase detection.

In this work, we report an enzyme substrate binding aptamer complex based fluorescence sensor for an enzyme activity detection of adenosine deaminase (ADA). The sensor employs a DNA probe containing an adenosine aptamer region dually labeled with biotin and digoxigenin (DIG). The probe is immobilized in a streptavidin-modified 96-well micro plate via biotin-avidin bridge, and the DIG serves as an affinity tag for an Anti-DIG antibody conjugated with horseradish peroxidase (anti-DIG-HRP). The principle of our sensor is as follows: the aptamer forms a coiled structure making the DNA in a "closed" state in the presence of adenosine, which shields the DIG tag from the bulky anti-DIG-HRP due to a proper steric effect. After adding ADA in the test solution, adenosine will be converted to inosine regardless of the aptamer binding. Then, the inosine release causes the DNA to relax and consequently, the DIG becomes accessible to the bulky anti-DIG-HRP which will further conjugate a Eu³âº labeled anti-horseradish peroxidase (Eu-anti-HRP). The Eu-anti-HRP can give a fluorescence signal when an enhancement solution is added. In the result of the experiment, we found the sensor signal can reflect the enzyme activity accurately and the detection limit is lowered to 0.5 U L⁻¹ of ADA not only in buffer solution, but also in serum, and an enzyme inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride is studied. With a concentration of 0.01 nM it is enough to cause a distinct difference of the sensor response.

Electrochemical sensing telomere-bending motions caused by hTRF1.

It has been reported that human telomeric repeat binding factor 1 (hTRF1) may cause telomeric DNA bent; however there is no direct evidence, thus controversy still exists. In this work, the interaction between hTRF1 and a simulated telomeric DNA was investigated by using electrochemical method. While the telomeric DNA was immobilized on a gold electrode surface, a guanine-quadruplex-hemin complex was linked at the end of the DNA to serve as an electrochemical signal reporter. If hTRF1 made the telomeric tracts bent, electrochemical response from "off" to "on" could be observed. Therefore, this electrochemical method could give direct evidence whether hTRF1 binding to telomeric DNA would induce a shallow distortion of the DNA molecules, and a new way to explore the structural information of telomere was also proposed in this paper.

A novel electrochemical method to detect cell surface carbohydrates and target cells.

Glycosylation of cell surfaces is a critical factor in many biological processes; however, the lack of effective analytical tools for the detection of cell surface carbohydrates has been the bottleneck for probing into the processes. In this paper, a novel electrochemical method is presented for the analysis of cell surface carbohydrates, which can be also used to detect the target cells. Firstly, 5-hydroxy-3-hexanedithiol-1,4-naphthoquinone (JUG(thio)), the electrochemical reporter, and anti-selectin aptamer are successively modified onto the surface of a gold electrode. Different concentrations of intestinal human colon adenocarcinoma (LS180) cells are employed as the target cells for this study. Consequently, the specific carbohydrates on the surfaces of LS180 cells and anti-selectin aptamers will compete for combination with selectin in the system. As a result, the oxidation signal of JUG(thio) is changed and the detection of the cell surface carbohydrates can be achieved easily and sensitively. Furthermore, the proposed method can be used to specifically detect LS180 cells in a wide concentration range, from 10(3) to 10(7) cells/mL, with a good linear relationship and low detection limit, which might be promising for the diagnosis of cancer and some other diseases in the future.

Colorimetric study of the interaction between gold nanoparticles and a series of amino acids.

Study of the interaction between gold nanoparticles and a series of amino acids is reported in this paper. Amino acids with thiol, amine, or hydroxyl groups in their side chains are proven to make gold nanoparticles self-assemble under certain conditions. There is a progression of the effect on self-assembly of gold nanoparticles from hydroxyl < amine < thiol. Meanwhile, concentration of amino acids and the pH value of the solution have been found to be important for amino acids to exert the interesting effect on self-assembly of the nanoparticles.

Electrochemical study of photovoltaic effect of nano titanium dioxide on hemoglobin.

Nano titanium dioxide (TiO2) and hemoglobin (Hb) were co-modified on pyrolytic graphite (PG) electrode to study the photovoltaic effect of TiO2 nanoparticles (NPs) on the electron transfer reactivity and catalytic activity of the protein. By means of cyclic voltammetry (CV) and FTIR measurements, the study was characterized in both aerobic and anaerobic environments. Experimental results revealed that the factors which mainly interacted with Hb were electron/hole pairs and reactive oxygen species (ROS) generated by the photovoltaic effect when TiO2 NPs were irradiated under ultraviolet (UV) light. The electron/hole pairs generated on the surface of TiO2 would influence the structure of Hb gently, so the electron transfer reactivity and catalytic ability of the protein slightly changed. In contrast, ROS interacted with Hb intensively, which brought in much conformational change to Hb and its active centers, and even cause some damage. Consequently, the electron transfer reactivity and catalytic activity of Hb changed with a process of increasing initially and decreasing afterwards.

Effect of nano cadmium sulfide on the electron transfer reactivity and peroxidase activity of hemoglobin.

Hemoglobin (Hb) is immobilized with cadmium sulfide (CdS) nanoparticles (NPs) on pyrolytic graphite (PG) electrode to characterize the electrochemical reactivity and peroxidase activity of the protein. The result demonstrates that fine redox waves of Hb can be achieved after this protein is entrapped in CdS NPs. Meanwhile, the protein can exhibit nice catalytic activity towards hydrogen peroxide (H2O2). Linear relationship between the reductive peak current and the H2O2 concentration has been obtained from 5.0 x 10(-6) to 4.0 x 10(-4) mol/L, on the basis of which a new kind of H2O2 biosensor might be developed in the future.