Microwave synthesized novel biomass carbon dots applied in the fluorescent detection of crystal violet.

To establish a new method for detecting crystal violet (CV), a harmful dye, herein, a genre of novel biomass carbon dots (CDs) was synthesized via a microwave method and employed as a fluorescent probe, in which water spinach and polyethylene glycol (PEG) performed as raw materials. Based on the inner filter effect (IFE) between the luminescent CDs and CV, the blue emission of this probe at 430 nm could be quenched by CV. Hence, a new strategy was proposed to selectively determine CV in aquaculture ambient. Moreover, under the optimal experiment conditions, this method showed a good linearity between the concentration of CV (c) and fluorescence quenching rate (ΔF/F0) in the concentration range of 4-200 µmol/L with the corresponding correlation coefficient (r) and the detection limit of 0.997 and 710 nmol/L, respectively. With advantages of environmental protectivity, sensitivity, affordability, and user-friendliness, the facilely fabricated CDs could be successfully applied in detecting CV in aquaculture samples, providing a technical foundation for monitoring the pollution of CV and ensuring the quality and safety of aquatic products.

Safe and Efficacious Diphtheria Toxin-Based Treatment for Melanoma: Combination of a Light-On Gene-Expression System and Nanotechnology.

The controversy surrounding the use of diphtheria toxin (DT) as a therapeutic agent against tumor cells arises mainly from its unexpected harmfulness to healthy tissues. We encoded the cytotoxic fragment A of DT (DTA) as an objective gene in the Light-On gene-expression system to construct plasmids pGAVPO (pG) and pU5-DTA (pDTA). Meanwhile, a cRGD-modified ternary complex comprising plasmids, chitosan, and liposome (pG&pDTA@cRGD-CL) was prepared as a nanocarrier to ensure transfection efficiency. Benefiting from spatiotemporal control of this light-switchable transgene system and the superior tumor targeting of the carrier, toxins were designed to be expressed selectively in illuminated lesions. In vitro studies suggested that pG&pDTA@cRGD-CL exerted arrest of the S phase in B16F10 cells upon blue light irradiation and, ultimately, induced the apoptosis and necrosis of tumor cells. Such DTA-based treatment exerted enhanced antitumor activity in mice bearing B16F10 xenografts and displayed prolonged survival time with minimal side effects. Hence, we described novel DTA-based therapy combined with nanotechnology and the Light-On gene-expression system: such treatment could be a promising strategy against melanoma.

Polyvinyl Alcohol/Chitosan/Polyhexamethylene Biguanide Phase Separation System: A Potential Topical Antibacterial Formulation with Enhanced Antimicrobial Effect.

An aqueous polyvinyl alcohol (PVA)/chitosan (CHT)/polyhexamethylene biguanide (PHMB) blends (PVA/CHT/PHMB blends) has been developed as a potential low dose topical antibacterial formulation with enhanced antimicrobial effect. The preparation of PVA/CHT/PHMB blends was quite facilely, with just dissolved PVA, CHT, PHMB in water in order. There was the aggregates with 100 nm size around induced by phase separation in the blends and an aqueous two-phase system (ATPS) formed, as non-ionic polymer PVA formed a continuous phase and cationic polymer CHT and PHMB formed dispersed phases. The minimum inhibitory concentration (MIC) of PHMB in the PVA/CHT/PHMB blends was 0.5µg/mL, which was four times lower than the MIC of PHMB individually. A phase separation increased zeta potential mechanism was proposed to explain the enhanced antibacterial activities. In addition, the blends could easily form film on the skin surface with good water vapor permeability and be used as a liquid bandage to accelerate the scratch wound healing process of nude mouse. These findings provide experimental evidence that the PHMB-functionalized blends could be further explored as low-dose topical antibacterial formulations, and the nano-sized phase separation strategy could be used to design novel low-dose topical antibacterial formulations with an enhanced antimicrobial effect.

Alterations of the oral microbiome in patients treated with the Invisalign system or with fixed appliances.

INTRODUCTION: Although the Invisalign system has been used widely in recent years, the influences of this treatment on the oral microbiome and whether or not this influence is different from that of fixed appliances is still unknown. In this study, we investigated the changes in the oral microbiome in patients treated with the Invisalign system or with fixed appliances. METHODS: Fifteen subjects were enrolled, comprising 5 fixed appliance patients, 5 Invisalign patient, and 5 healthy controls. Saliva samples were collected, and high-throughput pyrosequencing was performed based on the 16S rRNA gene. RESULTS: Both fixed and Invisalign orthodontic treatments resulted in dysbiosis of the oral microbiome. Firmicutes and TM7 at the phyla level and Neisseria at the genus level displayed statistically significant differences between the 2 orthodontic groups. The effect of these changes with microbiome on oral health was inconsistent. The inferred microbial function of the Invisalign group suggested this group was more predisposed to periodontal diseases. CONCLUSION: The influence of the Invisalign system on the oral microbiome was no better for oral health compared with fixed appliances. The convenience of maintaining oral hygiene rather than changes in the oral microbiome may be the underlying reason for the performance of the Invisalign system on oral health.

Inhibition of phagocytic recognition of anti-D opsonized Rh D+ RBC by polymer-mediated immunocamouflage.

The Rh D antigen posed both a significant clinical risk and inventory supply issue in transfusion medicine. The successful development of the immunocamouflaged RBC has the potential to address both the risk of acute anti-D transfusion reactions and to improve D- blood inventory in geographic locations where D- blood is rare (e.g., China). The immunocamouflage of RBC was mediated by the covalent grafting of methoxy(polyethylene glycol) to the cell membrane thereby obscuring the D protein from the immune system. To determine the potential efficacy of mPEG-D+ RBC in D- recipients, anti-D alloantibodies from previously alloimmunized individuals were utilized. The effects of polymer chain size (2-30 kDa) and grafting concentration (0-4 mM) on antibody binding and erythrophagocytosis were determined using the clinically validated monocyte monolayer assay (MMA) and flow cytometry. The immunocamouflage of D was polymer size and grafting concentration dependent as determined using human anti-D alloantibodies (both pooled [RhoGAM] and single donors). Importantly, the 20 kDa polymer provided excellent immunocamouflage of D and reached a clinically significant level of protection, as measured by the MMA, at grafting concentrations of ≥1.5 mM. These findings further support the potential use of immunocamouflaged RBC to reduce the risk of acute transfusion reactions following administration of D+ blood to D- recipients in situations where D- units are unavailable or supply is geographically constrained.

Clinical application of reserved gastric tube in neuroendoscopic endonasal surgery for pituitary tumor.

BACKGROUND: The neuroendoscopic approach has the advantages of a clear operative field, convenient tumor removal, and less damage, and is the development direction of modern neurosurgery. At present, transnasal surgery for sphenoidal pituitary tumor is widely used. But it has been found in clinical practice that some patients with this type of surgery may experience post-operative nausea and vomiting and other discomforts. AIM: To explore the effect of reserved gastric tube application in the neuroendoscopic endonasal resection of pituitary tumors. METHODS: A total of 60 patients who underwent pituitary adenoma resection via the endoscopic endonasal approach were selected and randomly divided into the experimental and control groups, with 30 in each group. Experimental group: After anesthesia, a gastric tube was placed through the mouth under direct vision using a visual laryngoscope, and the fluid accumulated in the oropharynx was suctioned intermittently with low negative pressure throughout the whole process after nasal disinfection, during the operation, and when the patient recovered from anesthesia. Control group: Given the routine intraoperative care, no gastric tube was left. The number of cases of nausea/vomiting/aspiration within 24 h post-operation was counted and compared between the two groups; the scores of pharyngalgia after waking up, 6 h post-operation, and 24 h post-operation. The frequency of postoperative cerebrospinal fluid leakage and intracranial infection were compared. The hospitalization days of the two groups were statistically compared. RESULTS: The times of postoperative nausea and vomiting in the experimental group were lower than that in the control group, and the difference in the incidence of nausea was statistically significant (P < 0.05). After the patient woke up, the scores of sore throat 6 h after the operation and 24 h after operation were lower than those in the control group, and the difference was statistically significant (P < 0.05). The number of cases of postoperative cerebrospinal fluid leakage and intracranial infection was higher than that of the control group, but there was no statistically significant difference from the control group (P > 0.05). The hospitalization days of the experimental group was lower than that of the control group, and the difference was statistically significant (P < 0.05). CONCLUSION: Reserving a gastric tube in the endoscopic endonasal resection of pituitary tumors, combined with intraoperative and postoperative gastrointestinal decompression, can effectively reduce the incidence of nausea, reduce the number of vomiting and aspiration in patients, and reduce the complications of sore throat The incidence rate shortened the hospitalization days of the patients.

Thermosensitive curcumin/silver/montmorillonite-F127 hydrogels with synergistic photodynamic/photothermal/silver ions antibacterial activity.

Bacterial infections and the emergence of super-resistant bacteria pose a significant risk to human health. Effective sterilization to prevent the development of bacterial drug resistance remains a challenge. Herein, curcumin/silver/montmorillonite (Cur/Ag/Mt) was prepared through a green chemical reduction method with montmorillonite as the carrier, curcumin as the reducing agent and the capping agent, and citric acid as the structure guide agent. Then, a novel dual light-responsive and thermosensitive Pluronic F127-based hydrogel (CAM-F) was prepared by encapsulating Cur/Ag/Mt within the F127 hydrogel. The Cur/Ag/Mt showed strong absorption in the near-infrared region that efficiently converts light into heat for photothermal therapy when the molar ratio of curcumin to silver nitrate was 2 : 1. Specifically, triangular silver nanoparticles reduced by curcumin were immobilized on the Mt layers, which could enhance photodynamic therapy by the metal-enhanced singlet oxygen and metal-enhanced fluorescence mechanisms. Upon combining 405 nm and 808 nm laser irradiation, the CAM-F hydrogel could simultaneously generate reactive oxygen species, increase the local temperature, and sustain the release of Ag+, thus displaying excellent bactericidal performance against Gram-negative and Gram-positive bacteria. The antibacterial rates of CAM-F hydrogels were 99.26 ± 0.95% and 99.95 ± 0.98% for Escherichia coli and Staphylococcus aureus, respectively. The findings suggest the potential of the CAM-F hydrogel as a stable, biologically safe, and broad-spectrum antimicrobial material. The thermosensitive CAM-F hydrogels for synergetic phototherapy may provide a promising strategy for solving clinical problems caused by pathogenic infections.

Microfluidic coaxial 3D bioprinting of cell-laden microfibers and microtubes for salivary gland tissue engineering.

Replacement therapy for the salivary gland (SG) remains an unmet clinical need. Xerostomia ("dry mouth") due to hyposalivation can result from injury or disease to the SG, such as salivary acinar death caused by radiation therapy (RT) for head and neck squamous cell carcinoma (HNSCC). Currently, only palliative treatments exist for xerostomia, and many patients endure deteriorated oral health and poor quality of life. Tissue engineering could offer a permanent solution for SG replacement by isolating healthy SG tissues prior to RT, expanding its cells in vitro, and recreating a functional salivary neogland for implantation post-RT. 3D bioprinting methods potentiate spatial cell deposition into defined hydrogel-based architectures, mimicking the thin epithelia developed during the complex branching morphogenesis of SG. By leveraging a microfluidics-based bioprinter with coaxial polymer and crosslinker streams, we fabricated thin, biocompatible, and reproducible hydrogel features that recapitulate the thin epithelia characteristics of SG. This flexible platform enabled two modes of printing: we produced solid hydrogel fibers, with diameters <100 µm, that could be rastered to create larger mm-scale structures. By a second method, we generated hollow tubes with wall thicknesses ranging 45-80 µm, total tube diameters spanning 0.6-2.2 mm, and confirmed tube patency. In both cases, SG cells could be printed within the thin hydrogel features, with preserved phenotype and high viability, even at high density (5.0 × 106 cells/mL). Our work demonstrates hydrogel feature control across multiple length scales, and a new paradigm for addressing SG restoration by creating microscale tissue engineered components.

Antibacterial properties and human gingival fibroblast cell compatibility of TiO2/Ag compound coatings and ZnO films on titanium-based material.

Titanium (Ti)-based materials are widely used in biomedical implant components and are applied successfully in various types of bone-anchored reconstructions. However, in dental implants the Ti materials contact not only bone but also gingival tissues, and are partially exposed to the oral cavity that includes bacteria. This study used titania and silver (TiO(2)/Ag) compound coatings and zinc oxide (ZnO) films to enhance the antibacterial activity of the Ti-based implant. The hydrophobicity of each sample was examined by measuring the contact angle. Streptococcus mutans and human gingival fibroblast (HGF) was cultured on the coated samples, and the antibacterial effects and cell compatibility were determined using a Syto9 fluorescence staining and MTT methods. For the TiO(2)/Ag samples, depositing Ag on the plate at a higher power (which increased the proportion of Ag) increased the contact angle and the hydrophobicity. The bacterial count was lowest for the 50 W TiO(2)/Ag sample, which contained 5.9% Ag. The contact angles of the ZnO samples did not show the same tendency. The antibacterial effect was higher on ZnO-coated samples since bacterial count was threefold lower on ZnO samples as compared to control samples (Ti plate). From the MTT assay test, the mean optical density values for TiO(2)/Ag-coated samples after 72 h of HGF adhesion were similar to the value obtained from the uncoated Ti. However, biocompatibility was lower on ZnO films than in control samples. Conclusively, the antibacterial activity was higher but the cell compatibility was lower on ZnO films than on TiO(2)/Ag coatings.

The shelf-life of chestnut rose beverage packaged in PEN/PET bottles under long term storage: A comparison to packaging in ordinary PET bottles.

The shelf life of chestnut rose beverage is largely dependent on packaging method and storage temperature. In this study, we investigated the effects of packaging beverages in bottles made of either polyethylene terephthalate (PET) or PEN (polyethylene naphthalate)/PET and storage temperature (4, 25, 37, and 55 ℃) on the shelf life of chestnut rose beverage. The physicochemical parameters and enzyme activity of beverages were evaluated, and we found that at 4 °C, the vitamin C, superoxide dismutase, and total polyphenol contents of beverages stored in PEN/PET bottles increased by 9.95 ± 0.49%, 2.86 ± 0.13%, and 3.23 ± 0.09% respectively, compared to beverages in ordinary PET bottles. In addition, other characteristic indicators including total soluble solids, browning index, and color value were also significantly improved. A shelf-life model was established based on the Arrhenius equation, and it will help distributors and consumers to determine the storage time and optimal shelf life of chestnut rose beverage.

Design and development of novel fluorescence sensing material for exosome recognition.

Exosomes are extracellular vesicles with relatively specific expression of CD63 transmembrane protein. In this study, We designed and constructed a multisite-targeting polymer which has both fluorescence and targeting recognition. It can bond to the hydrophilic group of CD63 by connecting with hydrogen. The chemical structure and the ability to combine with CD63 of fluorescent monomer and polymer were characterized and confirmed by FTIR and 1H NMR. MTT assay was performed to detect the cytotoxicity and biocompatibility of this polymer. Then we found the cell viability was 80.64% and the hemolysis rate of erythrocyte was only 0.101% even at F concentration of 20 µM. In vitro, the proposed polymer showed better ability to enter cells after linking exosomes via CD63; in vivo, it showed the ability to bind stably to exosomes and target tumor implants.

Initial stability and bone strain evaluation of the immediately loaded dental implant: an in vitro model study.

OBJECTIVES: The aim of this study was to evaluate the effects of cortical bone thickness and trabecular bone elastic modulus on the strain in the bone surrounding an immediately loaded implant. We also examined the correlations between bone structure and the following indices of primary implant stability: insertion torque value (ITV), Periotest value (PTV), and implant stability quotient (ISQ). MATERIAL AND METHODS: The ITV, PTV, and ISQ were measured in 24 artificial jaw bone models representing cortical bone with four thicknesses (0, 1, 2, and 3 mm) and trabecular bone with four elastic moduli (137, 47.5, 23, and 12.4 MPa). Two loading conditions were applied (force of 130 N applied vertically and at 45° laterally), and the strains in the crestal region were measured by rosette strain gauges with a data acquisition system. RESULTS: When the cortical bone thickness and the elastic modulus of trabecular bone decreased, the bone strains increased by 10.3-52.1% and 39-73.1%, respectively, for vertical loading and by 35-62% and 42.4-56.2% for lateral loading. The cortical bone thickness has a stronger correlation (R(2) =0.95-0.71) with ITV, PTV, and ISQ than the elastic modulus of trabecular bone (R(2) =0.89-0.59). CONCLUSIONS: The initial stability at the time of implant placement is influenced by both the cortical bone thickness and the elastic modulus of trabecular bone; however, these parameters are not totally linearly correlated with ITV, PTV, and ISQ. The placement of an immediately loaded implant in cases with thin cortical bone and/or weak trabecular bone can induce extreme bone strains and may increase the risk of implant failure.

Biocompatibility and Microstructure-Based Stress Analyses of TiNbZrTa Composite Films.

BACKGROUND: the clinical application of orthopedic or dental implants improves the quality of the lives of patients. However, the long-term use of implants may lead to implant loosening and related complications. The purpose of this study is to deposit titanium (Ti)-niobium (Nb)-zirconium (Zr)-tantalum (Ta) alloys on the surface of Ti-6Al-4V to increase structural strength and biocompatibility for the possible future application of implants. MATERIALS AND METHODS: Ti, Nb, Zr, and Ta served as the materials for the surface modification of the titanium alloy. TiNbZr and TiNbZrTa coatings were produced using cathodic arc evaporation, and a small amount of nitrogen was added to produce TiNbZrTa(N) film. Annealing and oxidation were then conducted to produce TiNbZrTa-O and TiNbZrTa(N)-O coatings. In this study, biological tests and finite element analyses of those five alloy films, as well as uncoated Ti-6Al-4V, were performed. Human osteosarcoma cells (MG-63) and mouse fibroblast cells (L-929) were used to analyze cytotoxicity, cell viability, and cell morphology, and the bone differentiation of MG-63 was evaluated in an alkaline phosphatase experiment. Furthermore, for measuring the gene expression level of L-929, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was conducted. The three-dimensional (3D) computational models of the coated and uncoated sample films were constructed using images of transmission electron microscopy and computer-aided design software and, then, the stress distributions of all models were evaluated by finite element analysis. RESULT: the cytotoxicity test revealed that the surface treatment had no significant cytotoxic effects on MG-63 and L-929 cells. According to the results of the cell viability of L-929, more cell activity was observed in the surface-treated experimental group than in the control group; for MG-63, the cell viability of the coated samples was similar to that of the uncoated samples. In the cell morphology analysis, both MG-63 and L-929 exhibited attached filopodia and lamellipodia, verifying that the cells were well attached. The alkaline phosphatase experiment demonstrated that the surface treatment did not affect the characteristics of early osteogenic differentiation, whereas RT-qPCR analysis showed that surface treatment can promote better performance of L-929 cells in collagen, type I, α1, and fibronectin 1. Finally, the results of the finite element analysis revealed that the coated TiNb interlayer can effectively reduce the stress concentration inside the layered coatings. CONCLUSIONS: TiNbZrTa series films deposited using cathodic arc evaporation had excellent biocompatibility with titanium alloys, particularly in regard to soft tissue cells, which exhibited an active performance. The finite element analysis verified that the TiNb interlayer can reduce the stress concentration inside TiNbZrTa series films, increasing their suitability for application in biomedical implants in the future.

Enrichment separation of recombinant human CCR3 using detergent/polymer two-phase system.

Studies on the structure and functions of membrane proteins are impeded by their lability, hydrophobicity, difficulty in purification and low yields. Human chemokine receptor 3 (CCR3) is a G protein-coupled receptor related to allergic diseases. A Triton X-100/PEG20000 two-phase system was employed for enrichment separation of CCR3 over-expressed in E. coli. Optimal CCR3 partitioning with partition coefficient around 8 was obtained at pH 7.0, ionic strength of 0.3 mol/kg and 3 h equilibration time. Total recovery of CCR3 reached 102 +/- 15%, which was much higher than 32 +/- 5% of the normally used ultracentrifugation method. The recovered CCR3 was finally purified by two chromatography steps giving a final protein of 87 kDa.

In vivo microscopy of arterial distribution embolic particles in rabbit mesenteric artery.

PURPOSE: To study the arterial distribution of embosphere microsphere (EM) and polyvinyl alcohol (PVA) particles in rabbit mesenteric artery using in vivo microscopy.To study the arterial distribution of embosphere microsphere (EM) and polyvinyl alcohol (PVA) particles in rabbit mesenteric artery using in vivo microscopy. METHODS: Sixteen New Zealand rabbits were divided into four groups, namely large PVA (560-710 µm), small PVA (150-350 µm), large EM (500-700 µm), and small EM (100-300 µm). The mesenteric arteries of the experimental animals were embolized under fluoroscopic guidance and visualized using in vivo microscopy. The embolized vessel diameter and arterial distribution of embolic agents were compared. RESULTS: The diameters of occluded vessels in large PVA, small PVA, large EM, and small EM groups were 430.60 ± 67.30, 200.95 ± 70.54, 387.79 ± 92.51, and 143.81 ± 39.65 µm, respectively. PVA occluded significantly larger vessels than EM when the particle size was similar (P < 0.001). The proportion of EM at the bifurcation of the artery was significantly higher than that of PVA particles (large PVA < large EM, χ2 = 4.325, P < 0.038; small PVA < small EM, χ2 = 6.68, P < 0.01). CONCLUSION: Both PVA and EM could occlude vessels smaller than the particle size, and EM resulted in deeper penetration. The location of embolic particles in the artery is mainly related to the shape of particles.

Effects of Ag, Cu or Ca addition on microstructure and comprehensive properties of biodegradable Zn-0.8Mn alloy.

Zn-0.8Mn (in wt%) alloy with good ductility is used for design of novel Zn-0.8Mn-0.4x (x = Ag, Cu or Ca) alloys. Hot extrusion not only eliminates brittleness of the as-cast alloys but also significantly improves their strengths. Among them, Zn-0.8Mn-0.4Ca exhibits the highest strength, Zn-0.8Mn-0.4Ag exhibits the highest ductility, but Zn-0.8Mn-0.4Cu exhibits the best combination of strength and ductility. The minor addition of Ag, Cu or Ca accelerates alloy degradation in simulated body fluid. However, Cu addition much improves in vitro biocompatibility and endows antibacterial ability of Escherichia coli. Overall, Zn-0.8Mn-0.4Cu alloy has the best comprehensive properties.

Hemocompatibility of biodegradable Zn-0.8 wt% (Cu, Mn, Li) alloys.

Zinc alloys have been explored as potential materials for biodegradable vascular stents due to their tolerable corrosion rates and tunable mechanical properties. However, the performances of Zn alloys were not supported with enough toxicity or biological compatibility evaluation, particularly hemocompatibility for vascular scaffolding application. In this work, the hemocompatibility of three zinc alloys (Zn-0.8Cu, Zn-0.8Mn and Zn-0.8Li) was evaluated with 316 L stainless steel and pure zinc as controls. The hemolysis ratios of 316 L stainless steel, pure Zn, Zn-0.8Cu, Zn-0.8Mn and Zn-0.8Li were 0.38 ±â€¯0.08%, 1.04 ±â€¯0.21%, 0.47 ±â€¯0.21%, 0.57 ±â€¯0.14% and 0.52 ±â€¯0.22%, respectively, for direct contact method. Platelets aggregation on the 316 L stainless steel was observed, while the adhered platelets on the Zn alloys exhibited round shape with few pseudopodia spreading. The number of adhered platelets on the three zinc alloys (Zn-0.8Cu, Zn-0.8Mn and Zn-0.8Li) had no statistically difference compared with 316 L stainless steel, while significant fewer than the pure Zn group. None remarkable platelet activation, hematocyte aggregation, coagulation or complement activation was observed in any Zn alloy group. Furthermore, the Zn alloys prolonged prothrombin time and partial thromboplastin time, demonstrating a potential function of anticoagulation. The results demonstrated that Zn alloys presented in this work are indeed meeting the hemocompatible requirements of implant and showing the promise for perspective application as biodegradable stent.

The role of gene variants in the pathogenesis of neurodegenerative disorders as revealed by next generation sequencing studies: a review.

The clinical diagnosis of neurodegenerative disorders based on phenotype is difficult in heterogeneous conditions with overlapping symptoms. It does not take into account the disease etiology or the highly variable clinical course even amongst patients diagnosed with the same disorder. The advent of next generation sequencing (NGS) has allowed for a system-wide, unbiased approach to identify all gene variants in the genome simultaneously. With the plethora of new genes being identified, genetic rather than phenotype-based classification of Mendelian diseases such as spinocerebellar ataxia (SCA), hereditary spastic paraplegia (HSP) and Charcot-Marie-Tooth disease (CMT) has become widely accepted. It has also become clear that gene variants play a role in common and predominantly sporadic neurodegenerative diseases such as Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS). The observation of pleiotropy has emerged, with mutations in the same gene giving rise to diverse phenotypes, which further increases the complexity of phenotype-genotype correlation. Possible mechanisms of pleiotropy include different downstream effects of different mutations in the same gene, presence of modifier genes, and oligogenic inheritance. Future directions include development of bioinformatics tools and establishment of more extensive public genotype/phenotype databases to better distinguish deleterious gene variants from benign polymorphisms, translation of genetic findings into pathogenic mechanisms through in-vitro and in-vivo studies, and ultimately finding disease-modifying therapies for neurodegenerative disorders.

Identification and biochemical characterization of DC07090 as a novel potent small molecule inhibitor against human enterovirus 71 3C protease by structure-based virtual screening.

Hand, foot and mouth disease (HFMD) is a serious, highly contagious disease. HFMD caused by Enterovirus 71 (EV71), results in severe complications and even death. The pivotal role of EV71 3Cpro in the viral life cycle makes it an attractive target for drug discovery and development to treat HFMD. In this study, we identified novel EV71 3Cpro inhibitors by docking-based virtual screening. Totally 50 compounds were selected to test their inhibitory activity against EV71 3Cpro. The best inhibitor DC07090 exhibited the inhibition potency with an IC50 value of 21.72 ± 0.95 µM without apparent toxicity (CC50 > 200 µM). To explore structure-activity relationship of DC07090, 15 new derivatives were designed, synthesized and evaluated in vitro enzyme assay accordingly. Interestingly, four compounds showed inhibitory activities against EV71 3Cpro and only DC07090 inhibited EV71 replication with an EC50 value of 22.09 ± 1.07 µM. Enzyme inhibition kinetic experiments showed that the compound was a reversible and competitive inhibitor. The Ki value was determined to be 23.29 ± 12.08 µM. Further molecular docking, MD simulation and mutagenesis studies confirmed the binding mode of DC07090 and EV71 3Cpro. Besides, DC07090 could also inhibit coxsackievirus A16 (CVA16) replication with an EC50 value of 27.76 ± 0.88 µM. Therefore, DC07090 represents a new non-peptidyl small molecule inhibitor for further development of antiviral therapy against EV71 or other picornaviruses.

Systemic tumor-targeted gene delivery by anti-transferrin receptor scFv-immunoliposomes.

An ideal therapeutic for cancer would be one that selectively targets to tumor cells, is nontoxic to normal cells, and that could be systemically delivered, thereby reaching metastases as well as primary tumor. Immunoliposomes directed by monoclonal antibody or its fragments are promising vehicles for tumor-targeted drug delivery. However, there is currently very limited data on gene delivery using these vehicles. We have recently described a cationic immunoliposome system directed by a lipid-tagged, single-chain antibody Fv fragment (scFv) against the human transferrin receptor (TfR) that shows promising efficacy for systemic p53 tumor suppressor gene therapy in a human breast cancer metastasis model. However, the extremely low yield of this lipid-tagged scFv limited further downstream development and studies. Here we report a different expression strategy for the anti-TfR scFv, which produces high levels of protein without any tags, and a different approach for complexing the targeting scFv to the liposomes. This approach entails covalently conjugating the scFv to the liposome via a cysteine at the 3'-end of the protein and a maleimide group on the liposome. Our results show that this conjugation does not impair the immunological activity or targeting ability of the scFv. The scFv-cys targets the cationic liposome-DNA complex (lipoplex) to tumor cells and enhances the transfection efficiencies both in vitro and in vivo in a variety of human tumor models. This scFv-immunoliposome can deliver the complexed gene systemically to tumors in vivo, where it is efficiently expressed. In comparison with the whole antibody or transferrin molecule itself, the scFv has a much smaller size for better penetration into solid tumors. It is also a recombinant protein rather than a blood product; thus, large scale production and strict quality control are feasible. This new approach provides a promising system for tumor-targeted gene delivery that may have potential for systemic gene therapy of various human cancers.