Development of New Biological Nanopores and Their Application for Biosensing and Disease Detection

Nanopore technology has recently emerged as a new real-time single molecule sensing method. The current dominant technologies, such as mass spectrometry and immunoassay, for protein analysis is still slow and complex, which can’t meet the urgent need and fields of use. Development of a highly simple, portable and sensitive detection system for pathogen detection, disease diagnosis, and environmental monitoring is in great need. Membrane embedded Phi29 connector nanopore, the first protein nanopore coming from bacteriophage, was mainly focusing on DNA and RNA translocation in previous studies. Here, Phi29 connector nanopore was first time established for antibody detection by engineering Epithelial Cell Adhesion Molecule peptide as a probe. The results demonstrate that the specific antibody can be detected in presence of diluted serum or non-specific antibody. To enable detecting more different types of analytes with high sensitivity, developing new nanopore with various properties, such as size, charge, hydrophilic/hydrophobic and physical dimension, is needed. In this work, besides Phi29 nanopore, several new protein nanopores that derived from T3, T4, and SPP1 bacteriophages were developed. A shared property of three step conformational change among these portal channel has been discovered. Elucidating the sequence and oligomeric states of proteins and peptides is critical for understanding their biological functions. Here, SPP1 nanopore was used to characterize the translocation of TAT peptide with dimer and monomer forms. Translocation of the peptide was confirmed by optical single molecule imaging for the first time, and analyzed quantitatively. The dynamics of peptide oligomeric states were clearly differentiated based on their characteristic electronic signatures. Main challenge for probing protein structure, folding, detection and sequencing using nanopore is the ultra-fast translocation speed which normally beyond electronic detection limit. In this work, the peptides translocation was slowed in SPP1 nanopore by changing the charge shielding of the channel. A 500-fold reduction was observed for TAT peptide translocation. By using this method, arginine chain peptide as short as two arginine can be detected first time. Further improving the bandwidth may lead to single amino acid detection and has the potential for protein sequencing. Compared with protein nanopore, de novo designed nanopore can provide numerous advantages, such as tunable size and functionality, ease of construction, scale up and modification. In the final study, an RNA-based biomimetic nanopore was first time constructed. The insertion of RNA nanopore into lipid bilayer and cell membrane were characterized and translocation of short amino acids through RNA nanopore was detected. This new artificial nanopore has the potential to be used for sensing, disease diagnosis, and even protein sequencing

Lipid Bilayer-Integrated SPP1 Connector Protein Nanopore and SPP1 Connector Protein Variants for Use as Lipid Bilayer-Integrated Nanopore

A conductive channel-containing membrane includes a membrane layer, and a SPPl connector polypeptide variant that is incorporated into the membrane layer to form an aperture through which conductance can occur when an electrical potential is applied across the membrane. A method of sensing a molecule, such as a polypeptide or nucleic acid molecule, makes use of the conductive channel- containing membrane. A method of DNA sequence makes use of the conductive channel-containing membrane

Differential RhoA Dynamics in Migratory and Stationary Cells Measured by FRET and Automated Image Analysis

Genetically-encoded biosensors based on fluorescence resonance energy transfer (FRET) have been widely applied to study the spatiotemporal regulation of molecular activity in live cells with high resolution. The efficient and accurate quantification of the large amount of imaging data from these single-cell FRET measurements demands robust and automated data analysis. However, the nonlinear movement of live cells presents tremendous challenge for this task. Based on image registration of the single-cell movement, we have developed automated image analysis methods to track and quantify the FRET signals within user-defined subcellular regions. In addition, the subcellular pixels were classified according to their associated FRET signals and the dynamics of the clusters analyzed. The results revealed that the EGF-induced reduction of RhoA activity in migratory HeLa cells is significantly less than that in stationary cells. Furthermore, the RhoA activity is polarized in the migratory cells, with the gradient of polarity oriented toward the opposite direction of cell migration. In contrast, there is a lack of consistent preference in RhoA polarity among stationary cells. Therefore, our image analysis methods can provide powerful tools for high-throughput and systematic investigation of the spatiotemporal molecular activities in regulating functions of live cells with their shapes and positions continuously changing in time

Refined Qingkailing Protects MCAO Mice from Endoplasmic Reticulum Stress-Induced Apoptosis with a Broad Time Window

In the current study, we are investigating effect of refined QKL on ischemia-reperfusion-induced brain injury in mice. Methods. Mice were employed to induce ischemia-reperfusion injury of brain by middle cerebral artery occlusion (MCAO). RQKL solution was administered with different doses (0, 1.5, 3, and 6 mL/kg body weight) at the same time of onset of ischemia, and with the dose of 1.5 mL/kg at different time points (0, 1.5, 3, 6, and 9 h after MCAO). Neurological function and brain infarction were examined and cell apoptosis and ROS at prefrontal cortex were evaluated 24 h after MCAO, and western blot and intracellular calcium were also researched, respectively. Results. RQKL of all doses can improve neurological function and decrease brain infarction, and it performed significant effect in 0, 1.5, 3, and 6 h groups. Moreover, RQKL was able to reduce apoptotic process by reduction of caspase-3 expression, or restraint of eIF2a phosphorylation and caspase-12 activation. It was also able to reduce ROS and modulate intracellular calcium in the brain. Conclusion. RQKL can prevent ischemic-induced brain injury with a time window of 6 h, and its mechanism might be related to suppress ER stress-mediated apoptotic signaling

Finding of widespread viral and bacterial revolution dsDNA translocation motors distinct from rotation motors by channel chirality and size

BACKGROUND: Double-stranded DNA translocation is ubiquitous in living systems. Cell mitosis, bacterial binary fission, DNA replication or repair, homologous recombination, Holliday junction resolution, viral genome packaging and cell entry all involve biomotor-driven dsDNA translocation. Previously, biomotors have been primarily classified into linear and rotational motors. We recently discovered a third class of dsDNA translocation motors in Phi29 utilizing revolution mechanism without rotation. Analogically, the Earth rotates around its own axis every 24 hours, but revolves around the Sun every 365 days. RESULTS: Single-channel DNA translocation conductance assay combined with structure inspections of motor channels on bacteriophages P22, SPP1, HK97, T7, T4, Phi29, and other dsDNA translocation motors such as bacterial FtsK and eukaryotic mimiviruses or vaccinia viruses showed that revolution motor is widespread. The force generation mechanism for revolution motors is elucidated. Revolution motors can be differentiated from rotation motors by their channel size and chirality. Crystal structure inspection revealed that revolution motors commonly exhibit channel diameters larger than 3 nm, while rotation motors that rotate around one of the two separated DNA strands feature a diameter smaller than 2 nm. Phi29 revolution motor translocated double- and tetra-stranded DNA that occupied 32% and 64% of the narrowest channel cross-section, respectively, evidencing that revolution motors exhibit channel diameters significantly wider than the dsDNA. Left-handed oriented channels found in revolution motors drive the right-handed dsDNA via anti-chiral interaction, while right-handed channels observed in rotation motors drive the right-handed dsDNA via parallel threads. Tethering both the motor and the dsDNA distal-end of the revolution motor does not block DNA packaging, indicating that no rotation is required for motors of dsDNA phages, while a small-angle left-handed twist of dsDNA that is aligned with the channel could occur due to the conformational change of the phage motor channels from a left-handed configuration for DNA entry to a right-handed configuration for DNA ejection for host cell infection. CONCLUSIONS: The revolution motor is widespread among biological systems, and can be distinguished from rotation motors by channel size and chirality. The revolution mechanism renders dsDNA void of coiling and torque during translocation of the lengthy helical chromosome, thus resulting in more efficient motor energy conversion

CD23 expression in mantle cell lymphoma is associated with CD200 expression, leukemic non-nodal form, and a better prognosis

Mantle cell lymphoma (MCL) is usually CD23 negative, a feature helpful in distinguishing MCL from chronic lymphocytic leukemia/small lymphocytic lymphoma. However, a subset of MCL cases can be CD23+. Limited data are available regarding the clinicopathological features and prognosis of patients with CD23+ MCL. In this study, we reviewed 798 cases of MCL and identified 103 (13%) that were CD23+ by flow cytometry, all of which were positive for cyclin D1 and/or associated with CCND1/IGH. In all cases of CD23+ MCL, CD23 expression was dim partial or dim, unlike moderate to bright CD23 expression observed in chronic lymphocytic leukemia/small lymphocytic lymphoma. The clinicopathological features and outcome of patients with CD23+ MCL were compared with 240 patients with typical MCL negative for CD23. Patients with CD23+ MCL more often had an elevated leukocyte count (33% versus 18%, P = .009), bone marrow involvement (89% versus 78%, P = .02), stage 4 disease (87% versus 77%, P = .03), and a leukemic presentation (42% versus 11%, P = .0001). CD23+ MCL was also more often positive for CD200 (17% versus. 4.6%, P = .0005) and less commonly positive for SOX11 (55% versus. 74%, P = .027). All other clinicopathological features were similar. With similar treatment regimens and observation times, patients with CD23+ MCL had a significant better overall survival (P = .02) and progression-free survival (P = .029). In conclusion, CD23 expression was observed in 13% of MCL cases and is associated with a better prognosis in patients with MCL. CD23 is associated with leukocytosis, a leukemic presentation, bone marrow involvement, CD200 expression, and a lower frequency of SOX11 positivity

Avian Tembusu virus infection effectively triggers host innate immune response through MDA5 and TLR3-dependent signaling pathways

Additional file 4 ATMUV infection causes significant up-regulation of TLR3 and MDA5. RT-PCR was performed to examine the mRNA expression of TLR3 and MDA5 in CEF (A), chickens (B) and 293T cells (C) at the indicated time after ATMUV infection, respectively

Case report: Successful and effective percutaneous closure of a deep femoral artery pseudoaneurysm using proglide device

A 61-year-old man developed severe swelling in the left lower extremity after interventional embolization of liver tumor. Ultrasound examination showed a pseudoaneurysm and thrombosis in the upper thigh on the left. To recognize the causes and determine the effective therapy, lower extremity arteriography was performed. The results revealed a pseudoaneurysm arised from deep femoral artery. Considering of the size of cavity and symptoms of patient, a new method was tried instead of traditional treatment using PROGLIDE device. Postoperative angiography showed a powerful blocking effect. This case study provide us a specific treatment for pseudoaneurysm, and this method provide us a new therapeutic strategy in clinical practice