Deafness-Associated ADGRV1 Mutation Impairs USH2A Stability through Improper Phosphorylation of WHRN and WDSUB1 Recruitment.

The ankle-link complex (ALC) consists of USH2A, WHRN, PDZD7, and ADGRV1 and plays an important role in hair cell development. At present, its architectural organization and signaling role remain unclear. By establishing Adgrv1 Y6236fsX1 mutant mice as a model of the deafness-associated human Y6244fsX1 mutation, the authors show here that the Y6236fsX1 mutation disrupts the interaction between adhesion G protein-coupled receptor V subfamily member 1 (ADGRV1) and other ALC components, resulting in stereocilia disorganization and mechanoelectrical transduction (MET) deficits. Importantly, ADGRV1 inhibits WHRN phosphorylation through regional cAMP-PKA signaling, which in turn regulates the ubiquitination and stability of USH2A via local signaling compartmentalization, whereas ADGRV1 Y6236fsX1 does not. Yeast two-hybrid screening identified the E3 ligase WDSUB1 that binds to WHRN and regulates the ubiquitination of USH2A in a WHRN phosphorylation-dependent manner. Further FlAsH-BRET assay, NMR spectrometry, and mutagenesis analysis provided insights into the architectural organization of ALC and interaction motifs at single-residue resolution. In conclusion, the present data suggest that ALC organization and accompanying local signal transduction play important roles in regulating the stability of the ALC.

One-step facile preparation of carbon dots with high fluorescence quantum yield and application in rapid latent fingerprint detection.

The development of luminescent materials greatly affects the development of fluorescence imaging technology. The preparation of carbon dots (CDs) with high photoluminescence quantum yield (PLQY) in the solid-state is challenging due to excessive resonance energy transfer (RET) and direct π-π interactions. In this study, we synthesized carbon dots that exhibit green fluorescence (GCDs) with absolute PLQYs up to 35.65% in one step by a microwave-assisted method. In the solid-state, the absolute PLQY reached 19.25%. Then, the GCDs were mixed with soluble starch in appropriate proportions, which improved the adsorption and dispersion of the GCDs and greatly reduced the cost of the fingerprint powder, and increased the absolute PLQY of the fingerprint powder to 41.75%. Finally, we prepared GCDs for preliminary fabrication of luminescent films, and the GCD-starch powder was successfully applied to high-quality latent fingerprint (LFP) imaging. The related properties of GCDs and the LFP detection performance of fingerprint detection powders prepared by GCDs were studied in detail. The results showed that the LFP system developed with GCDs-starch powder visualized LFPs with high definition and contrast under different conditions, and GCDs had potential for application in light-emitting devices. This study developed a new type of solid-state luminescent CDs and demonstrated that these GCDs have great application potential for LFP detection. This study may also provide inspiration for other applications based on efficient solid-state fluorescence.

Regulation of the sensitivity of hepatocarcinoma cells by ORMDL3, to sorafenib by autophagy.

Serum orosomucoid1-like protein 3 (ORMDL3) is a membrane protein in the endoplasmic reticulum, known to regulate many important signal transduction processes and autophagy regulation, but it is unclear whether it is involved in the intratumoral microenvironment and cancer drug resistance. Our present study found that silencing ORMDL3 increases the inhibitory effect of sorafenib on the viability and proliferation in HCC cells, and increases the sensitivity of HCC cells to sorafenib. In addition, silencing ORMDL3 can increase ROS levels by inhibiting autophagy, thereby increasing sorafenib-induced apoptosis of HCC cells. Further, our study also found that ORMDL3 silencing inhibits autophagy through the PERK-ATF4-Beclin1 pathway, thus affecting sorafenib sensitivity. The in vivo effects of sorafenib were tested by xenografting using nude mice. It showed that silencing ORMDL3 in HCC cells could increase the inhibitory effect of sorafenib on the growth of tumors. This is the first report to describe the relationships among ORMDL3, autophagy, and sorafenib resistance. This study provides available targets that might have a synergetic effect with sorafenib.

Biodegradation of 4-hydroxybenzoic acid by Acinetobacter johnsonii FZ-5 and Klebsiella oxytoca FZ-8 under anaerobic conditions.

4-Hydroxybenzoic acid (4-HBA) is a common organic compound that is prevalent in the environment, and the persistence of 4-HBA residues results in exertion of pollution-related detrimental effects. Bioremediation is an effective method for the removal of 4-HBA from the environment. In this study, two bacterial strains FZ-5 and FZ-8 capable of utilizing 4-HBA as the sole carbon and energy source under anaerobic conditions were isolated from marine sediment samples. Phylogenetic analysis identified the two strains FZ-5 and FZ-8 as Acinetobacter johnsonii and Klebsiella oxytoca, respectively. The strains FZ-5 and FZ-8 degraded 2000 mg·L-1 4-HBA in 72 h with degradation rates of 71.04% and 80.10%, respectively. The optimum culture conditions for degradation by the strains and crude enzymes were also investigated. The strains FZ-5 and FZ-8 also exhibited the ability to degrade other lignin-derived compounds, such as protocatechuic acid, cinnamic acid, and vanillic acid. Immobilization of the two strains showed that they could be used for the bioremediation of 4-HBA in an aqueous environment. Soils inoculated with the strains FZ-5 and FZ-8 showed higher degradation of 4-HBA than the uninoculated soil, and the strains could survive efficiently in anaerobic soil. This is the first report of 4-HBA-degrading bacteria, belonging to the two genera, which showed degradation ability under anaerobic conditions. This study expound the strains could efficiently degrade 4-HBA in anaerobic soil and will help in the development of 4-HBA anaerobic bioremediation systems.

Visual typing detection of brucellosis with a lateral flow immunoassay based on coloured latex microspheres.

AIMS: Treatment and preventive control strategies for Brucella melitensis (B. melitensis) and Brucella abortus (B. abortus) infection differ. A lateral flow immunoassay (LFIA) for the rapid typing and detection of brucellosis by using polychromatic dye-doped latex microspheres (LMs) as a labelling material was developed. METHODS AND RESULTS: This LFIA utilizes a double-antigen sandwich method in which the BP26 protein is used as the diagnostic antigen to detect brucellosis infection and the OMP31 protein is used as the identified antigen to distinguish between bovine and sheep brucellosis. Thus, people and animals infected with brucellosis can be diagnosed according to the different colours of the signals displayed on the detection lines. The results indicated that the accuracy of this assay was found to reach 98%, and the immunochromatographic test strip is highly accurate, shows good sensitivity and can facilitate typing diagnosis, among other features. CONCLUSIONS: The established LFIA can distinguish B. melitensis infection from B. abortus infection and produces results in a short period of time while retaining the advantages of LFIAs. SIGNIFICANCE AND IMPACT OF THE STUDY: This technology lays a foundation for the development of multi-disease test strips and the establishment of methods for rapid, multi-specimen quantitative detection and is thus of great importance for the development of medical diagnostic technologies.

Web-Based Open-Source Tool for Isotachophoresis.

We present the development of a client-side web-based simulator for complex electrophoresis phenomena, including isotachophoresis. The simulation tool is called Client-based Application for Fast Electrophoresis Simulation (CAFES). CAFES uses the broad cross-browser compatibility of JavaScript to provide a rapid and easy-to-use tool for coupled unsteady electromigration, diffusion, and equilibrium electrolyte reactions among multiple weak electrolytes. The code uses a stationary grid (for simplicity) and an adaptive time step to provide reliable estimates of ion concentration dynamics (including pH profile evolution), requiring no prior installation nor compilation. CAFES also offers a large database of commonly used species and their relevant physicochemical properties. We present a validation of predictions from CAFES by comparing them to experimental data of peak- and plateau-mode isotachophoresis experiments. The code yields accurate estimates of interface velocity, plateau length and relative intensity, and pH variations while significantly reducing the computation time compared to existing codes. The tool is open-source and available for free at https://microfluidics.stanford.edu/cafes.

The Rho GTPase Cell Division Cycle 42 Regulates Stereocilia Development in Cochlear Hair Cells.

Stereocilia are actin-based cell protrusions on the apical surface of inner ear hair cells, playing a pivotal role in hearing and balancing sensation. The development and maintenance of stereocilia is tightly regulated and deficits in this process usually lead to hearing or balancing disorders. The Rho GTPase cell division cycle 42 (CDC42) is a key regulator of the actin cytoskeleton. It has been reported to localize in the hair cell stereocilia and play important roles in stereocilia maintenance. In the present work, we utilized hair cell-specific Cdc42 knockout mice and CDC42 inhibitor ML141 to explore the role of CDC42 in stereocilia development. Our data show that stereocilia height and width as well as stereocilia resorption are affected in Cdc42-deficient cochlear hair cells when examined at postnatal day 8 (P8). Moreover, ML141 treatment leads to planar cell polarity (PCP) deficits in neonatal hair cells. We also show that overexpression of a constitutively active mutant CDC42 in cochlear hair cells leads to enhanced stereocilia developmental deficits. In conclusion, the present data suggest that CDC42 plays a pivotal role in regulating hair cell stereocilia development.

Flower-like gold nanoparticles labeled and silver deposition rapid vertical flow technology for highly sensitive detection of Brucella antibodies.

To prevent the transmission of brucellosis, rapid vertical flow technology (RVFT) was developed to detect brucellosis antibodies. To improve the sensitivity of the technique, lipopolysaccharides (LPS) were purified and used to detect brucellosis antibodies. To improve the sensitivity of serum antibody detection, a single multifunctional buffer was established in whole blood and other biological samples, and the advantages of the lateral flow immunoassay were retained. Flower-like gold nanoparticles were applied to RVFT for the first time. In this study, silver ions were catalyzed by flower-like gold nanoparticles into metal silver deposited on the surface of gold nanoparticles for the first time, which not only increased the particle size of gold nanoparticles, but also showed a more distinguishable black color on the test zone, further improving the sensitivity of RVFT. Standard Brucella-positive serum (containing Brucella antibody at 4000 IU mL-1) could be detected in this system even for a dilution factor of 2 × 10-3. The detection limit was 2 IU mL-1. RVFT can effectively avoid the false negative phenomenon in lateral flow immunoassay. RVFT is simple to operate, with a short reaction time, 2-3 minutes visible to the naked eye, without any equipment. Because it is very important to control the brucellosis epidemic, this approach has great application prospects in basic medical units and for veterinarians.

Green synthesis and characterization of gold nanoparticles and their application for the rapid detection of glycyrrhizin with immunochromatographic strips.

In this study, a facile and environmentally friendly synthesis process was proposed without regular chemical additives. We successfully synthesized spherical gold nanoparticles (AuNPs) coated with glycyrrhizin (GL) by using GL as both a reductant and a stabilizer to reduce chloroauric acid. The obtained NPs were approximately 35 nm in size. The formation of these GL-AuNPs was verified by the presence of a surface plasmon resonance band at 526 nm. We also experimentally determined that in terms of chemical structure, GL can be used as a reducing agent to obtain colloidal gold. The d-glucuronic acid structure, rather than glycyrrhetinic acid (GA), plays an important reducing role in colloidal gold production. From this, we hypothesized that other compounds with sugar structures in Glycyrrhiza may also have the ability to reduce chloroauric acid. To mitigate the high cost and low efficiency of current GL detection methods, we applied AuNPs to the immunochromatographic system. Then, a colloidal gold immunochromatographic test strip based on the indirect competition method was developed for the rapid detection of GL, and the detection limit of this strip was 25 ng mL-1. The cross-test showed that the strip has high specificity. The test results are consistent with the data obtained by high-performance liquid chromatography (HPLC) with a coincidence rate of up to 100%. The rapid test strip is simple, fast, highly efficient and inexpensive, making it suitable for large-scale, rapid glycyrrhizin content determination.

Development and application of a colloidal carbon test strip for the detection of antibodies against Mycoplasma bovis.

Mycoplasma bovis (M. bovis) is an important bovine mycoplasma implicated in economically important clinical diseases, such as respiratory diseases, otitis media, and mastitis. The prevalence of M. bovis-associated mastitis in both cattle and buffaloes has been increasingly recognized as a global problem. High morbidity rates and consequential economic losses have been devastating to the affected cattle and buffalo farms, especially those in developing countries. Therefore, a rapid and accurate method is urgently needed to detect M. bovis. In this study, a rapid and simple lateral flow strip for detecting antibodies against M. bovis was established that used carbon nanoparticles (CNPs) as the labelled materials. The results from the test strip were highly consistent with those from ELISA. The test showed high specificity (100%) and no cross-reaction with other bovine pathogens. The detection sensitivity of the test was also relatively high (97.67%). All the results indicated that the colloidal carbon test strip could serve as a simple, rapid, sensitive, and specific diagnostic method for detecting antibodies against M. bovis at cattle farms.

Design, synthesis and anti-inflammatory activity of 3-amino acid derivatives of ocotillol-type sapogenins.

Ocotillol-type sapogenins (OTS) are major ginsenoside metabolites in human hepatic tissue. In order to better utilize OTS and derivatives thereof as anti-inflammatory compounds, present study produced multiple novel 3-amino acid OTS derivatives and evaluated their anti-inflammatory activity in vitro. The nitric oxide (NO) inhibitory activity of these compounds was used for OTS structure-activity relationship (SAR) evaluations, revealing that both R/S stereochemistry at C-24 and the amino acid type at C-3 influence such NO inhibitory activity. This activity was highest for an N-Boc-protected neutral aliphatic amino acid derivative of 24R-OTS (5a), which performed better than even hydrocortisone sodium succinate in vitro. Compound 5a was also able to markedly suppress the LPS-induced upregulation of TNF-α, IL-6, iNOS, and COX-2 via the NF-κB and MAPK pathways. This suggests that OTS derivatives may be valuable anti-inflammatory compounds worthy of further preclinical evaluation.

Synthesis and Structure-Activity Relationship of Pyxinol Derivatives as Novel Anti-Inflammatory Agents.

Pyxinol, the main metabolite of 20S-protopanaxadiol in human liver, was chosen as a novel skeleton for the development of anti-inflammatory agents. Pyxinol derivatives modified at C-3, C-12, or C-25 and selected stereoisomers were designed, prepared, and investigated for in vitro anti-inflammatory activities. Structure-activity relationship (SAR), focused on skeleton, was analyzed based on their ability to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) synthesis. The preliminary SAR results signified that the biological activity of the pyxinol derivatives is largely dependent on the R/S stereochemistry of pyxinol skeleton and the hydroxy at C-3 is a modifiable position. Among the tested compounds, the 3-oximinopyxinol (4a) exhibited the most potent NO-inhibitory activity and was even comparable to the steroid drug. Furthermore, compound 4a also significantly decreased LPS-induced TNF-α and IL-6 synthesis and iNOS and COX-2 expressions via the NF-κB pathway. This study proves that pyxinol is an interesting skeleton for anti-inflammatory drug discovery.

Design, synthesis, and discovery of ocotillol-type amide derivatives as orally available modulators of P-glycoprotein-mediated multidrug resistance.

Multidrug resistance (MDR) is a major cause of failure in cancer treatment, in which the overexpression of P-glycoprotein (Pgp) plays a crucial role. Herein, a novel class of ocotillol-type amide derivatives has been designed, synthesized, and evaluated for their ability to reverse MDR. The structure-activity relationship of the reversal activity was analyzed. Ten compounds showed promising chemo-reversal ability, among which the 24R-ocotillol-type amide derivative 6c with an N-Boc-hexanoyl unit exhibited the most potency in reversing paclitaxel resistance in KBV cells. Compound 6c could inhibit Pgp-mediated rhodamine123 efflux function via stimulating Pgp-ATPase activity and exhibited high binding affinity with Pgp in molecular docking studies. Importantly, compound 6c enhanced the efficacy of paclitaxel against KBV cancer cell-derived xenograft tumors in nude mice after oral administration. These results indicate that ocotillol-type amide derivatives are promising lead compounds for overcoming MDR in cancer.

Cre-estrogen receptor-mediated hepatitis C virus structural protein expression in mice.

Hepatocyte apoptosis is an important feature of liver injury in hepatitis C virus (HCV) infection. However, the mechanism of apoptosis and consequences on disease progression in vivo have not been investigated fully in part due to the lack of adequate small animal models. In this study, transgenic (tg) mice were produced that express conditionally HCV structural proteins (core, E1, E2 and p7) in the liver following Cre-mediated DNA recombination. Using a novel Cre-estrogen receptor fusion protein (Cre-ER) induction strategy, tamoxifen was injected intraperitoneally (i.p.), which induced Cre nuclear translocation, transgene recombination and HCV protein expression in the liver. Hepatic expression of HCV core and envelope proteins resulted in increased hepatocyte apoptosis, detected by the TUNEL assay, between 7 and 33 days after induction. These results were confirmed by the presence of increased levels of apoptosis-associated cytokeratin 18 (CK-18) in the sera of the same animals. The presence of cleaved caspase-3 and elevated levels of CHOP/GADD153 in the liver suggests an endoplasmic reticulum (ER) stress-associated apoptosis mechanism. This study suggests an in vivo correlation between HCV structural protein expression, ER stress and hepatocyte apoptosis, implicating a potentially important mechanism of HCV pathogenesis.