Digital Detection of Multiple Minority Mutants and Expression Levels of Multiple Colorectal Cancer-Related Genes Using Digital-PCR Coupled with Bead-Array

<div><p>To simultaneously analyze mutations and expression levels of multiple genes on one detection platform, we proposed a method termed “multiplex ligation-dependent probe amplification–digital amplification coupled with hydrogel bead-array” (MLPA–DABA) and applied it to diagnose colorectal cancer (CRC). CRC cells and tissues were sampled to extract nucleic acid, perform MLPA with sequence-tagged probes, perform digital emulsion polymerase chain reaction (PCR), and produce a hydrogel bead-array to immobilize beads and form a single bead layer on the array. After hybridization with fluorescent probes, the number of colored beads, which reflects the abundance of expressed genes and the mutation rate, was counted for diagnosis. Only red or green beads occurred on the chips in the mixed samples, indicating the success of single-molecule PCR. When a one-source sample was analyzed using mixed MLPA probes, beads of only one color occurred, suggesting the high specificity of the method in analyzing CRC mutation and gene expression. In gene expression analysis of a CRC tissue from one CRC patient, the mutant percentage was 3.1%, and the expression levels of CRC-related genes were much higher than those of normal tissue. The highly sensitive MLPA–DABA succeeds in the relative quantification of mutations and gene expressions of exfoliated cells in stool samples of CRC patients on the same chip platform. MLPA–DABA coupled with hydrogel bead-array is a promising method in the non-invasive diagnosis of CRC.</p></div

Specificity of hydrogel bead-array for detecting targets, including targets of <i>β-actin</i> ligated with multiplex ligation-dependent probe amplification (MLPA) probes containing the sequence tag for coding Cy5-labeled probes (A) and targets of colorectal cancer (CRC)-related genes ligated with MLPA probes containing the sequence tag for coding Cy3-labeled probes (B).

<p>Both the Cy5-labeled and Cy3-labeled probes were added to the bead-array for hybridization, and electrophoresis was performed to remove the mismatched and free probes. Finally, the three photographs of panel A or panel B are taken by using three kinds of fluorescence channels, both Cy3 and Cy5 channels, only Cy3 channel, and only Cy5 channel, respectively.</p

Sequences of MLPA probes for gene expression analysis.

<p>Note. The bold bases represent the sequence of common primer. The italic letter bases are source-specific sequence for hybridization with fluorescent probes. The bases with no mark indicate gene-specific sequence.</p><p>Sequences of MLPA probes for gene expression analysis.</p

Sequences of MLPA probes for mutation detection.

<p>Note. The bold bases represent the sequence of common primer. The italic letter bases are source-specific sequence for hybridization with fluorescent probes. The first base at the 5’ end of probe 2 and 3 are a mutation site of interest. The bases with no mark indicate gene-specific sequence.</p><p>Sequences of MLPA probes for mutation detection.</p

Detection results of stool samples from a colorectal cancer (CRC) patient and a healthy volunteer using multiplex ligation-dependent probe amplification-digital amplification coupled with hydrogel bead-array.

<p>(A) Mutation detection of the stool sample from the CRC patient; (B) analysis of CRC-related gene expression of the stool sample from the CRC patient; (C) Mutation detection of the stool sample from the healthy volunteer; (D) analysis of CRC-related gene expression of the stool sample from the healthy volunteer.</p

Selective Synthesis of Fullerenol Derivatives with Terminal Alkyne and Crown Ether Addends

A series of isomerically pure alkynyl-substituted fullerenol derivatives such as C₆₀(OH)₆(O­(CH₂)₃CCH)₂ were synthesized through Lewis acid catalyzed epoxy ring opening and/or S_N1 replacement reactions starting from the fullerene–mixed peroxide C₆₀(O)­(<i>t</i>-BuOO)₄. Copper-catalyzed azide–alkyne cycloaddition readily converted the terminal alkynyl groups into triazole groups. Intramolecular oxidative alkyne coupling afforded a fullerenyl crown ether derivative

Divalent Folate Modification on PEG: An Effective Strategy for Improving the Cellular Uptake and Targetability of PEGylated Polyamidoamine–Polyethylenimine Copolymer

The stability and targeting ability of nanocarrier gene delivery systems are necessary conditions to ensure the good therapeutic effect and low nonspecific toxicity of cancer treatment. Poly­(ethylene glycol) (PEG) has been widely applied for improving stability and as a spacer for linking ligands and nanocarriers to improve targetability. However, the cellular uptake and endosomal escape capacity of nanocarriers has been seriously harmed due to the introduction of PEG. In the present study, we synthesized a new gene delivery vector by coupling divalent folate-PEG (PEG_3.4k-FA₂) onto polyamidoamine–polyethylenimine (PME) copolymer (PME–(PEG_3.4k-FA₂)_1.72). Both PEG and monovalent folate-PEG (PEG_3.4k-FA₁) modified PME were prepared as control polymers, which were named as PME–(PEG_3.5k)_1.69 and PME–(PEG_3.4k-FA₁)_1.66, respectively. PME–(PEG_3.4k-FA₂)_1.72 exhibited strong DNA condensation capacity like parent polymer PME which was not significantly influenced by PEG. PME–(PEG_3.4k-FA₂)_1.72/DNA complexes at N/P = 10 had a diameter ∼143 nm and zeta potential ∼13 mV and showed the lowest cytotoxicity and hemolysis and the highest transfection efficiency among all tested polymers. In folate receptor positive (FR-positive) cells, the cellular uptake and transfection efficiency were increased with the increase in the number of folates coupled on PEG; the order was PME–(PEG_3.4k-FA₂)_1.72 > PME–(PEG_3.4k-FA₁)_1.66 > PME–(PEG_3.5k)_1.69. Folate competition assays showed that PME–(PEG_3.4k-FA₂)_1.72 complexes had stronger targeting ability than PME–(PEG_3.5k)_1.69 and PME–(PEG_3.4k-FA₁)_1.66 complexes due to their higher folate density per PEG molecule. Cellular uptake mechanism study showed that the folate density on PEG could change the endocytosis pathway of PME–(PEG_3.5k)_1.69 from clathrin-mediated endocytosis to caveolae-mediated endocytosis, leading to less lysosomal degradation. Distribution and uptake in 3D multicellular spheroid assays showed that divalent folate could offer PME–(PEG_3.4k-FA₂)_1.72 complexes stronger penetrating ability and higher cellular uptake. With these advantages, PME–(PEG_3.4k-FA₂)_1.72 may be a promising nonviral vector candidate for efficient gene delivery. This study also indicates that divalent folate modification on PEG can serve as an efficient strategy to improve the cellular uptake and targeting ability of PEGylated cationic polymers for gene delivery

Fluorine Functionalized Graphene Quantum Dots as Inhibitor against hIAPP Amyloid Aggregation

Fibrillar deposits of the human islet amyloid polypeptide (hIAPP) are considered as a root of Type II diabetes mellitus. Fluorinated graphene quantum dots (FGQDs) are new carbon nanomaterials with unique physicochemical properties containing highly electronegative F atoms. Herein we report a single step synthesis method of FGQDs with an inhibitory effect on aggregation and cytotoxicity of hIAPP in vitro. Highly fluorescent and water dispersible FGQDs, less than 3 nm in size, were synthesized by the microwave-assisted hydrothermal method. Efficient inhibition capability of FGQDs to amyloid aggregation was demonstrated. The morphologies of hIAPP aggregates were observed to change from the entangled long fibrils to short thin fibrils and amorphous aggregates in the presence of FGQDs. In thioflavin T fluorescence analysis, inhibited aggregation with prolonged lag time and reduced fluorescence intensity at equilibrium were observed when hIAPP was incubated together with FGQDs. Circular dichroism spectrum results reveal that FGQDs could inhibit conformational transition of the peptide from native structure to β-sheets. FGQDs could also rescue the cytotoxicity of INS-1 cells induced by hIAPP in a dose dependent manner. This study could be beneficial for design and preparation of inhibitors for amyloids, which is important for prevention and treatment of amyloidosis

Additional file 1 of Pooling sputum samples for the Xpert MTB/RIF assay: a practical screening strategy for highly infectious tuberculosis cases

Supplementary Material

Burden of chemotherapy-induced myelosuppression among patients with ES-SCLC in US community oncology settings Supplementary materials

Burden of chemotherapy-induced myelosuppression among patients with ES-SCLC in US community oncology settings Supplementary materials</p