Single Molecule Protein Segments Sequencing by a Plasmonic Nanopore.

Obtaining sequential and conformational information on proteins is vital to understand their functions. Although the nanopore-based electrical detection can sense single molecule (SM) protein and distinguish among different amino acids, this approach still faces difficulties in slowing down protein translocation and improving ionic current signal-to-noise ratio. Here, we observe the unfolding and multistep sequential translocation of SM cytochrome c (cyt c) through a surface enhanced Raman scattering (SERS) active conical gold nanopore. High bias voltage unfolds SM protein causing more exposure of amino acid residues to the nanopore, which slows down the protein translocation. Specific SERS traces of different SM cyt c segments are then recorded sequentially when they pass through the hotspot inside the gold nanopore. This study shows that the combination of SM SERS with a nanopore can provide a direct insight into protein segments and expedite the development of nanopore toward SM protein sequencing.

Single-Molecule Electrical and Spectroscopic Profiling Protein Allostery Using a Gold Plasmonic Nanopore.

Direct structural and dynamic characterization of protein conformers in solution is highly desirable but currently impractical. Herein, we developed a single molecule gold plasmonic nanopore system for observation of protein allostery, enabling us to monitor translocation dynamics and conformation transition of proteins by ion current detection and SERS spectrum measurement, respectively. Allosteric transition of calmodulin (CaM) was elaborately probed by the nanopore system. Two conformers of CaM were well-resolved at a single-molecule level using both the ion current blockage signal and the SERS spectra. The collected SERS spectra provided structural evidence to confirm the interaction between CaM and the gold plasmonic nanopore, which was responsible for the different translocation behaviors of the two conformers. SERS spectra revealed the amino acid residues involved in the conformational change of CaM upon calcium binding. The results demonstrated that the excellent spectral characterization furnishes a single-molecule nanopore technique with an advanced capability of direct structure analysis.

Collagen type V alpha 2 promotes the development of gastric cancer via M2 macrophage polarization.

Gastric cancer is a type of digestive tract cancer with a high morbidity and mortality, which leads to a major health burden worldwide. More research into the functions of the immune system will improve therapy and survival in gastric cancer patients. We attempted to identify potential biomarkers or targets in gastric cancer via bioinformatical analysis approaches. Three gene expression profile datasets (GSE79973, GSE103236, and GSE118916) of gastric tissue samples were obtained from the Gene Expression Omnibus database. There were 65 overlapping differentially expressed genes (DEGs) identified from three microarrays. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway were carried out for the key functions and pathways enriched in the DEGs. Then, ten hub genes were identified by protein-protein interaction network. In addition, we observed that collagen type V alpha 2 (COL5A2) was linked to gastric cancer prognosis as well as M2 macrophage infiltration. Furthermore, COL5A2 enhanced gastric cancer cell proliferation through the PI3K-AKT signaling pathway and polarized M2 macrophage cells. Therefore, in this study, we found that COL5A2 was associated with the development of gastric cancer which might function as a potential therapeutic target for the disease.

Single Molecule DNA Analysis Based on Atomic-Controllable Nanopores in Covalent Organic Frameworks.

We explored the application of two-dimensional covalent organic frameworks (2D COFs) in single molecule DNA analysis. Two ultrathin COF nanosheets were exfoliated with pore sizes of 1.1 nm (COF-1.1) and 1.3 nm (COF-1.3) and covered closely on a quartz nanopipette with an orifice of 20 ± 5 nm. COF nanopores exhibited high size selectivity for fluorescent dyes and DNA molecules. The transport of long (calf thymus DNA) and short (DNA-80) DNA molecules through the COF nanopores was studied. Because of the strong interaction between DNA bases and the organic backbones of COFs, the DNA-80 was transported through the COF-1.1 nanopore at a speed of 270 µs/base, which is the slowest speed ever observed compared with 2D inorganic nanomaterials. This study shows that the COF nanosheet can work individually as a nanopore monomer with controllable pore size like its biological counterparts.

High Spatial Resolution of Ultrathin Covalent Organic Framework Nanopores for Single-Molecule DNA Sensing.

Ultrathin nanosheets of two-dimensional covalent organic frameworks covered a quartz nanopipette and then acted as a nanopore device for single-molecule DNA sensing. Our results showed that a single DNA homopolymer as short as 6 bases could be detected. The dwell times of 30-mer DNA homopolymers were obviously longer than the times of 10- or 6-mer ones. For different bases, poly(dA)6 showed the slowest transport speed (∼595 µs/base) compared with cytosine (∼355 µs/base) in poly(dC)6 and thymine (∼220 µs/base) in poly(dT)6. Such translocation speeds are the slowest ever reported in two-dimensional material-based nanopores. Poly(dA)6 also showed the biggest current blockade (94.74 pA) compared with poly(dC)6 (79.54 pA) and poly(dT)6 (71.41 pA). However, the present difference in blockade current was not big enough to distinguish the four DNA bases. Our study exhibits the shortest single DNA molecules that can be detected by COF nanopores at the present stage and lights the way for DNA sequencing based on solid-state nanopores.

Degradation of MicroRNA miR-466d-3p by Japanese Encephalitis Virus NS3 Facilitates Viral Replication and Interleukin-1ß Expression.

Japanese encephalitis virus (JEV) infection alters microRNA (miRNA) expression in the central nervous system (CNS). However, the mechanism contributing to miRNA regulation in the CNS is not known. We discovered global degradation of mature miRNA in mouse brains and neuroblastoma (NA) cells after JEV infection. Integrative analysis of miRNAs and mRNAs suggested that several significantly downregulated miRNAs and their targeted mRNAs were clustered into an inflammation pathway. Transfection with miRNA 466d-3p (miR-466d-3p) decreased interleukin-1ß (IL-1ß) expression and inhibited JEV replication in NA cells. However, miR-466d-3p expression increased after JEV infection in the presence of cycloheximide, indicating that viral protein expression reduced miR-466d-3p expression. We generated all the JEV coding proteins and demonstrated NS3 helicase protein to be a potent miRNA suppressor. The NS3 proteins of Zika virus, West Nile virus, and dengue virus serotype 1 (DENV-1) and DENV-2 also decreased miR-466d-3p expression. Results from helicase-blocking assays and in vitro unwinding assays demonstrated that NS3 could unwind pre-miR-466d and induce miRNA dysfunction. Computational models and an RNA immunoprecipitation assay revealed arginine-rich domains of NS3 to be crucial for pre-miRNA binding and degradation of host miRNAs. Importantly, site-directed mutagenesis of conserved residues in NS3 revealed that R226G and R202W reduced the binding affinity and degradation of pre-miR-466d. These results expand the function of flavivirus helicases beyond unwinding duplex RNA to degrade pre-miRNAs. Hence, we revealed a new mechanism for NS3 in regulating miRNA pathways and promoting neuroinflammation.IMPORTANCE Host miRNAs have been reported to regulate JEV-induced inflammation in the CNS. We found that JEV infection could reduce expression of host miRNA. The helicase region of the NS3 protein bound specifically to miRNA precursors and could lead to incorrect unwinding of miRNA precursors, thereby reducing the expression of mature miRNAs. This observation led to two major findings. First, our results suggested that JEV NS3 protein induced miR-466d-3p degradation, which promoted IL-1ß expression and JEV replication. Second, arginine molecules on NS3 were the main miRNA-binding sites, because we demonstrated that miRNA degradation was abolished if arginines at R226 and R202 were mutated. Our study provides new insights into the molecular mechanism of JEV and reveals several amino acid sites that could be mutated for a JEV vaccine.

Photoelectrochemical analysis of the alkaline phosphatase activity in single living cells.

Conventional photoelectrochemical (PEC) analysis mostly utilizes photoactive material modified planar indium tin oxides (ITOs) to obtain photocurrent responses for the measurement of analytes in solution. In this work, a CdS quantum dot (QD) modified nanopipette was prepared for the PEC analysis of the alkaline phosphatase (ALP) activity in single MCF-7 cells. The nanopipette was filled with ascorbic acid 2-phosphate (AAP) that was egressed outside the nanopipette by electrochemical pumping. Next, AAP was catalyzed by ALP to generate ascorbic acid (AA), which is an efficient electron donor for CdS QDs under illumination. Based on the result that the nanopipette showed a linear photocurrent response to AA, a nearly linear correlation between the photocurrent and the activity of ALP was established. Accordingly, using these CdS QD modified nanopipettes, the ALP activity in single MCF-7 cells was determined to be 0.12 U mL-1 by PEC analysis. This work does not expand the application of PEC bioanalysis, but offers a new strategy for single cell analysis.

A Review of Clinical and Preclinical Studies on Therapeutic Strategies Using Interleukin-12 in Cancer Therapy and the Protective Role of Interleukin-12 in Hematological Recovery in Chemoradiotherapy.

Interleukin-12 (IL-12), a heterodimeric glycoprotein with alpha and ß subunits covalently bonded with a disulfide bond, is a potent anticancer agent. Its action is accomplished through a linkage of the adaptive and innate immune responses. IL-12 can promote the recovery of the hematopoietic system after cancer chemoradiotherapy by stimulating the physiological processes of stem cells, including cell proliferation and differentiation, reconstitution of hematopoietic function, and peripheral blood count recovery. We review therapeutic strategies using IL-12 in clinical studies, including single-agent and combination strategies in hematological tumors and solid tumors, and studies on the protective effects of IL-12 in chemoradiotherapy. This review highlights promising therapeutic strategies based on the anticancer role of IL-12 and the potential protective effects of IL-12 for cancer patients receiving chemoradiotherapy.

Conditional Lie-Bäcklund Symmetries and Differential Constraints of Radially Symmetric Nonlinear Convection-Diffusion Equations with Source.

A conditional Lie-Bäcklund symmetry method and differential constraint method are developed to study the radially symmetric nonlinear convection-diffusion equations with source. The equations and the admitted conditional Lie-Bäcklund symmetries (differential constraints) are identified. As a consequence, symmetry reductions to two-dimensional dynamical systems of the resulting equations are derived due to the compatibility of the original equation and the additional differential constraint corresponding to the invariant surface equation of the admitted conditional Lie-Bäcklund symmetry.

Surface-Enhanced Raman Scattering Probing the Translocation of DNA and Amino Acid through Plasmonic Nanopores.

DNA and amino acids are important biomolecules in living organisms. Probing such biomolecules with structural characters can provide valuable information for life study. Here, gold plasmonic nanopores (GPNs) with high SERS activity (a local enhancement factor higher than 109) are synthesized at the tip of a glass nanopipette. An electric field drives individual molecules to translocate through the GPNs, which enables in situ collection of the surface-enhanced Raman scattering (SERS). Nonresonant biomolecules, including nucleobases, amino acids, and oligonucleotides (DNA), with single nucleobase differences can be distinguished. The intensity of SERS is tunable by modulating the affinity between DNA and the GPNs. The present study shows the feasibility of applying a plasmonic nanopore to DNA and protein detection, which may also provide an easy way for tracking single molecule translocation by developing a well-defined single plasmonic nanopore.

Paeoniflorin inhibits activation of the IRAK1-NF-κB signaling pathway in peritoneal macrophages from lupus-prone MRL/lpr mice.

Systemic lupus erythematosus (SLE) is a chronic and multisystemic autoimmune disease. Interleukin-1 receptor-associated kinase 1 (IRAK1) is associated with the susceptibility of SLE in humans and paeoniflorin has recently been reported to exhibit immunosuppressive properties. The aim of this study was to determine the effect of paeoniflorin on lipopolysaccharide (LPS)-triggered macrophage activation and and its role in LPS-induced IRAK1-nuclear factor κB (NF-κB) signaling pathways. Peritoneal macrophages from lupus-prone MRL/lpr mice and ICR mice were isolated, prepared and cultured. Cells were treated with LPS alone or LPS with paeoniflorin, and macrophage proliferation was analyzed using the CCK8 assay. The expression of IRAK1 in cells was analyzed by immunofluorescence staining. The level of gene expression of IRAK1, NF-κB, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) was measured by RT-PCR, and TNF-α, IL-6 levels in the cell supernatant were determined by ELISA. The protein expression of IRAK1 and downstream molecules tumor necrosis factor receptor-associated factor 6 (TRAF6), inhibitor of nuclear factor kappa-B kinase (IKK), NF-kappa-B inhibitor alpha (IKBα), and NF-κB was detected by Western-blot analysis. Paeoniflorin was found to decrease the phosphorylation of IRAK1 and its downstream proteins induced by LPS and inhibit the expression of TNF-α and IL-6. Taken together, the data obtained indicate that paeoniflorin inhibits LPS-induced cell activation by inhibiting the IRAK1-NF-κB pathway in MRL/lpr mouse macrophages. Therefore, paeoniflorin may be a potential therapy for SLE.

Nanopipette-Based SERS Aptasensor for Subcellular Localization of Cancer Biomarker in Single Cells.

Single cell analysis is essential for understanding the heterogeneity, behaviors of cells, and diversity of target analyte in different subcellular regions. Nucleolin (NCL) is a multifunctional protein that is markedly overexpressed in most of the cancer cells. The variant expression levels of NCL in subcellular regions have a marked influence on cancer proliferation and treatments. However, the specificity of available methods to identify the cancer biomarkers is limited because of the high level of subcellular matrix effect. Herein, we proposed a novel technique to increase both the molecular and spectral specificity of cancer diagnosis by using aptamers affinity based portable nanopipette with distinctive surface-enhanced Raman scattering (SERS) activities. The aptamers-functionalized gold-coated nanopipette was used to capture target, while p-mercaptobenzonitrile (MBN) and complementary DNA modified Ag nanoparticles (AgNPs) worked as Raman reporter to produce SERS signal. The SERS signal of Raman nanotag was lost upon NCL capturing via modified DNA aptamers on nanoprobe, which further helped to verify the specificity of nanoprobe. For proof of concept, NCL protein was specifically extracted from different cell lines by aptamers modified SERS active nanoprobe. The nanoprobes manifested specifically good affinity for NCL with a dissociation constant Kd of 36 nM and provided a 1000-fold higher specificity against other competing proteins. Furthermore, the Raman reporter moiety has a vibrational frequency in the spectroscopically silent region (1800-2300 cm-1) with a negligible matrix effect from cell analysis. The subcellular localization and spatial distribution of NCL were successfully achieved in various types of cells, including MCF-7A, HeLa, and MCF-10A cells. This type of probing technique for single cell analysis could lead to the development of a new perspective in cancer diagnosis and treatment at the cellular level.

Expression, purification, and characterization of recombinant 8 kDa gelsolin fragment.

A mutation (D187N/Y) in human plasma gelsolin (GSN) leads to the generation of an 8 kDa GSN fragment (8 kDa-GSN), and consequently causes the familial amyloidosis of Finnish type. Because of its faster kinetics of amyloid formation under physiologically relevant conditions, 8 kDa-GSN is used to explore gelsolin amyloidosis and screen small molecules that can disaggregate amyloids. However, the synthetic 8 kDa-GSN is expensive, and substantial quantities of 8 kDa-GSN are needed for the screen. Here we report a study to obtain recombinant 8 kDa-GSN with high yield from Escherichia coli. Firstly, 8 kDa-GSN in fusion with Mxe GyrA intein was purified by Ni-affinity chromatography. Then 8 kDa-GSN was released by intein-mediated protein cleavage, and separated from intein by ion-exchange chromatography. The yield of 8 kDa-GSN was only 1.5 mg/L from bacterial culture in the previous report, while it was improved to 4.25 mg/L in our study. Finally, the amyloidogenic property of 8 kDa-GSN was validated by circular dichroism spectrometry and dynamic light scattering.

Expression, purification, and characterization of recombinant human L-chain ferritin.

Ferritins form nanocage architectures and demonstrate their potential to serve as functional nanomaterials with potential applications in medical imaging and therapy. In our study, the cDNA of human L-chain ferritin was cloned into plasmid pET-28a for its overexpression in Escherichia coli. However, the recombinant human L-chain ferritin (rLF) was prone to form inclusion bodies. Molecular chaperones were co-expressed with rLF to facilitate its correct folding. Our results showed that the solubility of rLF was increased about 3-fold in the presence of molecular chaperones, including GroEL, GroES and trigger factor. Taking advantage of its N-terminal His-tag, rLF was then purified with Ni-affinity chromatography. With a yield of 10 mg/L from bacterial culture, the purified rLF was analyzed by circular dichroism spectrometry for its secondary structure. Furthermore, the rLF nanocages were characterized using dynamic light scattering and transmission electron microscopy.

Expression, purification, and characterization of recombinant human H-chain ferritin.

Based on their nanocage architectures, ferritins show their potential applications in medical imaging and therapeutic delivery systems. However, the recombinant human H-chain ferritin (rHF) is prone to form inclusion bodies in Escherichia coli. In our study, the cDNA of rHF was cloned into plasmid pET28a under the control of a T7 promoter. Molecular chaperones, including GroES, GroEL, and trigger factor, were coexpressed with rHF to facilitate its correct folding. The results showed that the solubility of rHF was increased more than threefold with the help of molecular chaperones. Taking advantages of its N-terminal His-tag, rHF was then purified with Ni-affinity chromatography. With a yield of 15 mg/L from bacterial culture, the purified rHF was analyzed by circular dichroism spectrometry for its secondary structure. Moreover, the rHF nanocages were characterized by transmission electron microscopy and dynamic light scattering. Our results indicate that rHF is able to self-assemble into nanocages with a narrow size distribution.

[Study on UPLC fingerprint of red ginseng based on good separation and good purity of chromatographic peaks].

This study is to establish the UPLC fingerprint of red ginseng. The separation was performed on a Waters Acquity BEH C18 column (2.1 mm × 50 mm,1.7 µm), with the mobile phase consisting of acetonitrile and water for gradient elution. The detection wavelength was set at 203 nm. The UPLC fingerprint of red ginseng was established by using sample chromatography of 22 different purchase areas and 26 common peaks were found. Compared with the reference substances, 11 of the common peaks were identified as ginsenosides Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rh1, ginsenoside Rg2, ginsenoside Rb1, 20(S)-ginsenoside F1, ginsenoside Rb2, ginsenoside Rb3, 20(S)-ginsenoside Rg3 and 20(R)-ginsenoside Rg3, respectively. It is worth noting that 20(S)-ginsenoside Rg3 and 20(R)-ginsenoside Rg3 are the characteristic ingredients of red ginseng, and they could be used not only for distinguishing red ginseng and ginseng, but also for process controlling of the preparation of red ginseng. The similarity was analyzed with' Similarity Evaluation System for Chromatographic Fingerprint of Chinese Materia Medica, and the similarity of 18 batches samples is up to 0.9. Compared to the literature methods, the method is simple, time-saving,specific for the separation of ginsenosides from red ginseng. So, this method could be used for the species identification and quality control of ginseng, red ginseng and American ginseng, and it will alsoprovide a theoretical basis of raising quality standards of the above mentioned Chinese herb medicines.

[Quantitative analysis of nine ginsenosides in red ginseng using QAMS method based on high-resolution and reference chromatography of red ginseng].

Using reversed-phase high performance liquid chromatography, nine ginsenosides were simultaneously separated on an UltimateC18 column with high-resolution and high purity of each chromatographic peak. Adopting the QAMS quality evaluation model for traditional Chinese medicines, ginsenoside Rb1 was used as the internal reference substance, and the relative correction factors (RCFs) and the relative retention values (RTRs) of ginsenosides Rg1, Re, Rf, Rb1, Rc, Rb2, Rb3, Rd and 20 (S)-ginsenoside Rg3 to ginsenoside Rb1 were calculated individually. Through a series of methodology evaluations, and positioned by the red ginseng reference chromatograph and RTVs, nine ginsenosides in red ginseng were simultaneously assayed only by quantitative determined ginsenoside Rb1.

[A prospective, randomized, controlled clinical study of Huai Qi Huang granules in treatment of childhood primary nephrotic syndrome].

OBJECTIVE: To study the efficacy of Huai Qi Huang granules in the treatment of childhood primary nephrotic syndrome. METHODS: Between July 2009 and December 2011, patients who were admitted and diagnosed for the first time as childhood primary nephrotic syndrome were randomized into a treatment group (Huai Qi Huang granules plus glucocorticoid; n=23) and a control group (glucocorticoid alone; n=19) for a prospective study. The two groups were compared for regression time of edema, time to urinary protein clearance, relapse rate, incidence of infection, dosage of glucocorticoid, and humoral and cellular immunological indicators. RESULTS: There were no significant differences in regression time of edema, time to urinary protein clearance, and relapse rate between the treatment and control groups (P>0.05). The treatment group had significantly lower incidence of infection and daily dose of glucocorticoid (at month 6) than the control group (P<0.05). Humoral and cellular immunological indicators showed no significant differences between the two groups (P>0.05). No Huai Qi Huang-related adverse events were observed in this study. CONCLUSIONS: Huai Qi Huang granules treatment can reduce the dose of glucocorticoid and the incidence of infection in children with primary nephrotic syndrome and has a favourable safety.

Donnan potential caused by polyelectrolyte monolayers.

The Donnan potential is successfully isolated from ion pair potential on a ferrocene-labeled polyelectrolyte (DNA) monolayer. The isolated Donnan potential shifts negatively upon the increase in NaClO4 concentration with a slope of -58.8 mV/decade. With the salt concentration grown up to 1 M, the stretched DNA chains in low salt concentration are found to experience a gradual conformation relaxing process. At salt concentrations higher than 2 M, Donnan breakdown occurs where only the ion pair effect modulates the apparent potential. The apparent formal potential also shows strong dependence on solution pH, which reveals that the charge density in the polyelectrolyte monolayer plays an important role in the establishment of Donnan equilibrium.

Prospects for the Application of Transplantation With Human Amniotic Membrane Epithelial Stem Cells in Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a multi-organ and systemic autoimmune disease characterized by an imbalance of humoral and cellular immunity. The efficacy and side effects of traditional glucocorticoid and immunosuppressant therapy remain controversial. Recent studies have revealed abnormalities in mesenchymal stem cells (MSCs) in SLE, leading to the application of bone marrow-derived MSCs (BM-MSCs) transplantation technique for SLE treatment. However, autologous transplantation using BM-MSCs from SLE patients has shown suboptimal efficacy due to their dysfunction, while allogeneic mesenchymal stem cell transplantation (MSCT) still faces challenges, such as donor degeneration, genetic instability, and immune rejection. Therefore, exploring new sources of stem cells is crucial for overcoming these limitations in clinical applications. Human amniotic epithelial stem cells (hAESCs), derived from the eighth-day blastocyst, possess strong characteristics including good differentiation potential, immune tolerance with low antigen-presenting ability, and unique immune properties. Hence, hAESCs hold great promise for the treatment of not only SLE but also other autoimmune diseases.