Highly sensitive protein detection using recombinant spores and lateral flow immunoassay.

Lateral flow immunoassays (LFIs) can be used to detect intact bacteria or spores; when gold nanoparticles (AuNPs) are used as the signal reporters, the detection limits are very low. Spore-based surface display has been widely studied for enzyme immobilization and live-nontoxic oral vaccines. In this study, recombinant spores were used to improve the sensitivity of a LFI. We developed a test kit that combines streptavidin-displayed spores with a LFI assay for rapid protein detection. The recombinant spores served as a signal amplifier and AuNPs were used as the signal reporters. For detection of ß-galactosidase, which was used as the model protein, the detection limit was about 10-15 mol, while that of the conventional LFI is about 10-12 mol. In both methods, nanogold was used as the colorimetric signal and could be observed with the naked eye. This method improved LFI sensitivity without sacrificing its advantages. Furthermore, enhanced green fluorescent protein (eGFP) was also displayed on the surface of the streptavidin-displayed spores. Without AuNPs, the fluorescent recombinant spores acted as the signal, which could be detected by a fluorescence detector, such as a fluorescence microscope. The detection limit was 10-16 mol under fluorescence microscopy whose magnification was 25-fold. Therefore, in conclusion, in this proof of concept study, the detection limits of both proposed methods were far superior to those of traditional LFI assay.

Polymeric Gelatin Scaffolds Affect Mesenchymal Stem Cell Differentiation and Its Diverse Applications in Tissue Engineering.

Studies using polymeric scaffolds for various biomedical applications, such as tissue engineering, implants and medical substitutes, and drug delivery systems, have attempted to identify suitable material for tissue regeneration. This study aimed to investigate the biocompatibility and effectiveness of a gelatin scaffold seeded with human adipose stem cells (hASCs), including physical characteristics, multilineage differentiation in vitro, and osteogenic potential, in a rat model of a calvarial bone defect and to optimize its design. This functionalized scaffold comprised gelatin-hASCs layers to improve their efficacy in various biomedical applications. The gelatin scaffold exhibited excellent biocompatibility in vitro after two weeks of implantation. Furthermore, the gelatin scaffold supported and specifically regulated the proliferation and osteogenic and chondrogenic differentiation of hASCs, respectively. After 12 weeks of implantation, upon treatment with the gelatin-hASCs scaffold, the calvarial bone harboring the critical defect regenerated better and displayed greater osteogenic potential without any damage to the surrounding tissues compared to the untreated bone defect. These findings suggest that the present gelatin scaffold is a good potential carrier for stem cells in various tissue engineering applications.

The CCL5/CCR5 Axis Promotes Vascular Smooth Muscle Cell Proliferation and Atherogenic Phenotype Switching.

BACKGROUND/AIMS: Hyperlipidemia induces dysfunction in the smooth muscle cells (SMCs) of the blood vessels, and the vascular remodeling that ensues is a key proatherogenic factor contributing to cardiovascular events. Chemokines and chemokine receptors play crucial roles in vascular remodeling. Here, we examined whether the hyperlipidemia-derived chemokine CCL5 and its receptor CCR5 influence vascular SMC proliferation, phenotypic switching, and explored the underlying mechanisms. METHODS: Thoracoabdominal aorta were isolated from wild-type, CCL5 and CCR5 double-knockout mice (CCL5-/-CCR5-/-) fed a high-fat diet (HFD) for 12 weeks. Expression of the contractile, synthetic, and proliferation markers were assayed using immunohistochemical and western blotting. The effects of CCL5 and palmitic acid on cultured SMC proliferation and phenotypic modulation were evaluated using flow cytometry, bromodeoxyuridine (BrdU), and western blotting. RESULTS: Wild-type mice fed an HFD showed markedly increased total cholesterol, triglyceride, and CCL5 serum levels, as well as significantly increased CCL5 and CCR5 expression in the thoracoabdominal aorta vs. normal-diet-fed controls. HFD-fed CCL5-/-CCR5-/- mice showed significantly decreased expression of the synthetic phenotype marker osteopontin and the proliferation marker proliferating cell nuclear antigen, and increased expression of the contractile phenotype marker smooth muscle α-actin in the thoracoabdominal aorta vs. wild-type HFD-fed mice. Human aorta-derived SMCs stimulated with palmitic acid showed significantly increased expression of CCL5, CCR5, and synthetic phenotype markers, as well as increased proliferation. CCL5-treated SMCs showed increased cell cycle regulatory protein expression, paralleling increased synthetic and decreased contractile phenotype marker expression. Inhibition of CCR5 activity by the specific antagonist maraviroc or its expression using small interfering RNA significantly inhibited human aortic SMC proliferation and synthetic phenotype formation. Therefore, CCL5 induces SMC proliferation and phenotypic switching from a contractile to synthetic phenotype via CCR5. CCL5-mediated SMC stimulation activated ERK1/2, Akt/p70S6K, p38 MAPK, and NF-κB signaling. NF-κB inhibition significantly reduced CCR5 expression along with CCR5-induced SMC proliferation and synthetic phenotype formation. CONCLUSIONS: Hyperlipidemia-induced CCL5/CCR5 axis activation serves as a pivotal mediator of vascular remodeling, indicating that CCL5 and CCR5 are key chemokine-related factors in atherogenesis. SMC proliferation and synthetic phenotype transformation attenuation by CCR5 pharmacological inhibition may offer a new approach to treatment or prevention of atherosclerotic diseases associated with hyperlipidemia.

Dichroic Behavior of Gold Nanoparticles Synthesized in Aqueous Solution with Insufficient Reducing Agent.

The spherical gold nanoparticles (AuNPs) typically are red in solution. However, in this study, the dichroic and spherical AuNPs were synthesized using a modified seeding growth method under reducing agent insufficiency in an aqueous solution. This particular AuNP solution is orange in reflected light and red in transmitted light. The reflectance curves confirm that the dichroic AuNPs are different from the classic AuNPs. With particle assembling, the AuNP solution is fainter orange in reflected light, but purple in transmitted light when the color of classic spherical AuNP solution is purple in both lights. Furthermore, the aggregated-nanogold solutions were added to HAuCl4 solutions with the addition of an insufficient amount reducing agent. The solution changed from faint orange to bright orange in reflected light and from purple to blue in transmitted light. It indicates that the gold assembling under a reducing agent insufficiency, not the shape of AuNP, causes the dichroic phenomenon. To the best of our knowing, this is the first study to report how the AuNP is synthesized, not the shape, affects the color of the AuNP.

Combination of resveratrol-containing collagen with adipose stem cells for craniofacial tissue-engineering applications.

Repair and regeneration of craniofacial tissues is particularly challenging because they comprise a complex structure of hard and soft tissues involved in intricate functions. This study combined collagen scaffolds and human adipose stem cells (hASCs) for oral mucosal and calvarial bone regeneration by using resveratrol (RSV), which affects the differentiation of mesenchymal stem cells. We have evaluated the effect of collagen scaffold-containing RSV (collagen/RSV) scaffolds both in vitro and in vivo for their wound healing and bone regeneration potential. Scanning electron microscopy and immunostaining results reveal that hASCs adhere well to and proliferate on both collagen scaffolds and collagen/RSV scaffolds. Oral mucosal lesion experiments demonstrated that the collagen/RSV scaffold is more effective in wound closure and contraction than the collagen scaffold. The micro-computed tomography (µCT) images of calvarial bone display regenerating bone in defects covered with hASCs on collagen/RSV scaffolds that are more visible than that in defects covered with hASCs on a collagen scaffolds. RSV was more effective at inducing hASC differentiation on the collagen scaffold, suggesting that collagen/RSV scaffolds can provide useful biological cues that stimulate craniofacial tissue formation.

Rapid detection of Yersinia pestis recombinant fraction 1 capsular antigen.

Yersinia pestis, an infectious bacterium that is a causative agent of plague, a disease which has been shown to be one of the most feared in history and which has caused millions of deaths. The capsule-like fraction 1 (F1) antigen expressed by Y. pestis is a known specific marker for the identification of the bacteria; therefore, the detection of F1 is important for Y. pestis recognition. In this study, a rapid, sensitive, and specific technique, the lateral flow assay (LFA), was successfully developed to detect Y. pestis by the recombinant F1 antigen. The assay that utilized an anti-F1 polyclonal antibody (Pab) to identify the bacteria was based on a double-antibody sandwich format on a nitrocellulose membrane. With the LFA method, 50 ng/ml of recombinant F1 protein and 10(5) CFU/mL of Y. pestis could be detected in less than 10 min. This assay also showed no cross-reaction with other Yersinia spp. or with some selected capsule-producing Enterobacteriaceae strains. Furthermore, detection of Y. pestis in simulated samples has been evaluated. The detection sensitivity of Y. pestis in various matrices was 10(5) CFU/mL, which was identical to that in PBS buffer. The results obtained suggest that LFA is an excellent tool for detection of Y. pestis contamination in an environment and hence can be used to monitor plague diseases when they emerge.

Hemostasis and Anti-Inflammatory Abilities of AuNPs-Coated Chitosan Dressing for Burn Wounds.

Burn injuries are a common hazard in the military, as fire is likely to be weaponized. Thus, it is important to find an effective substance to accelerate burn wound healing. This study used chitosan and gold nanoparticles (AuNPs) as wound dressings and investigated their effectiveness in femoral artery hemorrhage swine and rat burn models. Chitosan dressing has significant hemostatic properties compared with gauze. Histological results showed that burn wounds treated with chitosan or AuNP-coated chitosan dressings exhibited more cells and a continuous structure of the epidermis and dermis than those of the control and untreated lesion groups. Furthermore, both chitosan dressings have been shown to positively regulate the expression of genes- and cytokines/chemokines-related to the wound healing process; AuNP-coated chitosan significantly lessened severe sepsis and inflammation, balanced the activities of pro-fibrotic and anti-fibrotic ligands for tissue homeostasis, regulated angiogenesis, and inhibited apoptosis activity, thereby being beneficial for the burn microenvironment. Hence, chitosan alone or in combination with AuNPs represents a prospective therapeutic substance as a burn dressing which might be helpful for burn wound care. This study provides a novel hemostasis dressing for modern warfare that is simple to use by most medical and paramedical personnel handling for burn treatment.

Development of a Nucleic Acid Lateral Flow Immunoassay for the Detection of Human Polyomavirus BK.

The BK virus (BKV) is an emerging pathogen in immunocompromised individuals and widespread in the human population. Polymerase chain reaction is a simple and highly sensitive method for detecting BKV, but it is time consuming and requires expensive instruments and expert judgment. The lateral flow assay, a rapid, low-cost, minimal-labor, and easy-to-use diagnostic method, was successfully applied for pathogen detection. In this study, we used oligonucleotide probes to develop a simple and rapid sandwich-type lateral flow immunoassay for detecting BKV DNA within 45 minutes. The detection limit for the synthetic single-stranded DNA was 5 nM. The specificity study showed no cross-reactivity with other polyomaviruses, such as JC virus and simian virus 40. For the Escherichia coli containing BKV plasmid cultured samples, the sensitivity was determined to be 107 copies/mL. The approach offers great potential for BKV detection of various target analytes in point-of-care settings.

Thermally stable and uniform DNA amplification with picosecond laser ablated graphene rapid thermal cycling device.

Rapid thermal cycling (RTC) in an on-chip device can perform DNA amplification in vitro through precise thermal control at each step of the polymerase chain reaction (PCR). This study reports a straightforward fabrication technique for patterning an on-chip graphene-based device with hole arrays, in which the mechanism of surface structures can achieve stable and uniform thermal control for the amplification of DNA fragments. A thin-film based PCR device was fabricated using picosecond laser (PS-laser) ablation of the multilayer graphene (MLG). Under the optimal fluence of 4.72 J/cm2 with a pulse overlap of 66%, the MLG can be patterned with arrays of 250 µm2 hole surface structures. A 354-bp DNA fragment of VP1, an effective marker for diagnosing the BK virus, was amplified on an on-chip device in less than 60 min. A thin-film electrode with the aforementioned MLG as the heater was demonstrated to significantly enhance temperature stability for each stage of the thermal cycle. The temperature control of the heater was performed by means of a developed programmable PCR apparatus. Our results demonstrated that the proposed integration of a graphene-based device and a laser-pulse ablation process to form a thin-film PCR device has cost benefits in a small-volume reagent and holds great promise for practical medical use of DNA amplification.

A competitive immunoassay for biotin detection using magnetic beads and gold nanoparticle probes.

Haptens are small molecules with low molecular weight that include biotin and many toxins in food. In this study, we used biotin as a model molecule for hapten detection. In this competitive immunoassay anti-biotin antibody-modified magnetic beads (Ab-MBs) and biotinylated thiol-DNA gold nanoparticles (biotin-GNPs) were used. The assay contains three reactions, the mixing of the sample and Ab-MBs, the capture of biotin-GNPs by Ab-MBs and the magnetic attraction. When biotin molecules were absent, the solution was transparent because biotin-GNPs bound to Ab-MBs which were caught by an external magnetic field. When biotin was present, the supernatant was red because the Ab-MBs bound to the analytes and the gold nanoparticles were still in solution. It was possible to complete all the operating steps in 15 min. The limit of detection (LOD) was 2 pmol. This rapid competitive-immunoassay has potential for application in detection of other haptens.

Biological Effects of Chitosan-Based Dressing on Hemostasis Mechanism.

There have been numerous recent advances in wound care management. Nevertheless, the assessment of hemostatic dressing is essential to enable surgeons and other physicians and healthcare professionals to make the correct decisions regarding the disposition of severe hemorrhage. Here, we investigated the relative efficacies of chitosan-based and conventional gauze dressings in a rat model of femoral artery hemorrhage and in patients with surgical wounds. Dressing effectiveness was evaluated based on hemostatic profiles, biocompatibility, antimicrobial activity, and blood factor responses in coagulation. Relative to standard gauze dressing, the chitosan fiber (CF) dressing treatment significantly shortened the time to hemostasis in injured rats. Moreover, the CF dressing significantly prolonged partial thromboplastin time, enhanced blood absorption, and reduced antithrombin production without altering the prothrombin ratio. Unlike regular gauze bandages, the CF dressing demonstrated remarkable antibacterial activity. The results of this study indicate the effectiveness of chitosan as a hemostatic dressing and elucidate its underlying mechanism. It is possible that chitosan surgical dressings could serve as first-line intervention in hospital emergency care for uncontrolled hemorrhage.

Wet-spinning-based Molding Process of Gelatin for Tissue Regeneration.

This article presents an inexpensive method to fabricate gelatin, as a natural polymer, into monofilament fibers or other appropriate forms. Through the wet spinning method, gelatin fibers are produced by smooth extrusion in a suitable coagulation medium. To increase the functional surface of these gelatin fibers and their ability to mimic the features of tissues, gelatin can be molded into a tube form by referring to this concept. Examined by in vitro and in vivo tests, the gelatin tubes demonstrate a great potential for application in tissue engineering. Acting as a suitable filling gap material, gelatin tubes can be used to substitute the tissue in the damaged area (e.g., in the nervous or cardiovascular system), as well as to promote regeneration by providing a direct replacement of stem cells and neural circuitry. This protocol provides a detailed procedure for creating a biomaterial based on a natural polymer, and its implementation is expected to greatly benefit the development of correlative natural polymers, which help to realize tissue regeneration strategies.

Bioapplications of Bacterial Cellulose Polymers Conjugated with Resveratrol for Epithelial Defect Regeneration.

Excellent wound dressing is essential for effective wound repair and regeneration. However, natural polymeric skin substitutes often lack mechanical strength and hydrophilicity. One way to overcome this limitation is to use biodegradable polymers with high mechanical strength and low skin-irritation induction in wet environments. Bacterial cellulose (BC) is an attractive polymer for medical applications; unlike synthetic polymers, it is biodegradable and renewable and has a strong affinity for materials containing hydroxyl groups. Therefore, we conjugated it with resveratrol (RSV), which has a 4'-hydroxyl group and exhibits good biocompatibility and no cytotoxicity. We synthesized BC scaffolds with immobilized RSV and characterized the resulting BC/RSV scaffold with scanning electron microscopy and Fourier-transform infrared spectroscopy. We found that RSV was released from the BC in vitro after ~10 min, and immunofluorescence staining showed that BC was highly biocompatible and regenerated epithelia. Additionally, Masson's trichrome staining showed that the scaffolds preserved the normal collagen-bundling pattern and induced re-epithelialization in defective rat epidermis. These results indicated that RSV-conjugated BC created a biocompatible environment for stem cell attachment and growth and promoted epithelial regeneration during wound healing.

Severe Burn Injury in a Swine Model for Clinical Dressing Assessment.

Wound healing is a dynamic repair process and is the most complex biological process in human life. In response to burn injury, alterations in biological pathways impair the inflammation response, resulting in delayed wound healing. Impaired wound healing frequently occurs in patients with diabetes leading to unfavorable outcomes such as amputation. Hence, dressings having beneficial effect in promoting burn wound repair are needed. However, studies on burn wound treatment are limited due to lack of proper animal models. Our previous study demonstrated wound-healing performance in rat and swine models using a minimally invasive surgical technique. This study aimed to demonstrate a swine model of severe burn injury that eliminates wound contraction and more closely approximates the human processes of re-epithelialization and new tissue formation. This protocol provides a detailed procedure for creating consistent burn wounds and examining the wound-healing performance under the treatment of an experimental dressing in a swine model. Six burn wounds were created symmetrically on the dorsum, which were covered with a clinical dressing composed of four layers: an inner contact layer of experimental materials, an inner intermediate layer of waterproof film, an outer intermediate layer of gauze, and an outer layer of adhesive plaster. Upon the completion of experiments, wound closure, wound area, and Vancouver Scar Scale score were examined. The samples of skin resected from each animal post-sacrifice were histologically prepared and stained using hematoxylin and eosin staining. Antibacterial activity of each dressing in the context of wound healing was also examined. The application of the clinical dressing to the wounds in swine model mimics the biological processes of human wound healing with respect to the processes of epithelialization, cellular proliferation, and angiogenesis. Therefore, this swine model provides an easy-to-learn, cost-effective, and robust method to assess the effect of clinical dressings in severe burn injury.

In vivo toxicologic study of larger silica nanoparticles in mice.

Silica nanoparticles (SiNPs) are being studied and used for medical purposes. As nanotechnology grows rapidly, its biosafety and toxicity have frequently raised concerns. However, diverse results have been reported about the safety of SiNPs; several studies reported that smaller particles might exhibit toxic effects to some cell lines, and larger particles of 100 nm were reported to be genotoxic to the cocultured cells. Here, we investigated the in vivo toxicity of SiNPs of 150 nm in various dosages via intravenous administration in mice. The mice were observed for 14 days before blood examination and histopathological assay. All the mice survived and behaved normally after the administration of nanoparticles. No significant weight change was noted. Blood examinations showed no definite systemic dysfunction of organ systems. Histopathological studies of vital organs confirmed no SiNP-related adverse effects. We concluded that 150 nm SiNPs were biocompatible and safe for in vivo use in mice.

Green synthesis of chondroitin sulfate-capped silver nanoparticles: characterization and surface modification.

A one-step route for the green synthesis of highly stable and nanosized silver metal particles with narrow distribution is reported. In this environmentally friendly synthetic method, silver nitrate was used as silver precursor and biocompatible chondroitin sulfate (ChS) was used as both reducing agent and stabilizing agent. The reaction was carried out in a stirring aqueous medium at the room temperature without any assisted by microwave, autoclave, laser irradiation, γ-ray irradiation or UV irradiation. The transparent colorless solution was converted to the characteristics light red then deep red-brown color as the reaction proceeds, indicating the formation of silver nanoparticles (Ag NPs). The Ag NPs were characterized by UV-visible spectroscopy (UV-vis), photon correlation spectroscopy, laser Doppler anemometry, transmission electron microscopy (TEM), and Fourier-transform infrared spectroscopy (FT-IR). The results demonstrated that the obtained metallic nanoparticles were Ag NPs capped with ChS. In this report, dynamic light scattering (DLS) was used as a routinely analytical tool for measuring size and distribution in a liquid environment. The effects of the reaction time, reaction temperature, concentration and the weight ratio of ChS/Ag+ on the particle size and zeta potential were investigated. The TEM image clearly shows the morphology of the well-dispersed ChS-capped Ag NPs are spherical in shape, and the average size (<20 nm) is much smaller than the Z-average value (76.7 nm) measured by DLS. Meanwhile, the ChS-capped Ag NPs coated with N-[(2-hydroxy-3-trimethylammonium) propyl] chitosan chloride (HTCC) were prepared by an ionic gelation method and the surface charge of Ag NPs was switched from negative to positive.

Salmonella detection using 16S ribosomal DNA/RNA probe-gold nanoparticles and lateral flow immunoassay.

An ultrasensitive, simple, and fast lateral flow immunoassay for Salmonella detection using gold nanoparticles conjugated with a DNA probe, which is complementary to the 16S ribosomal RNA and DNA of Salmonella, has been developed. The detection limit is 5 fmol for the synthetic single-stranded DNA. For the Salmonella cultured samples, the nucleic acids from 10(7) bacteria were rapidly detected in 30 min. After silver enhancement, the detection limit was as low as 10(4) cells which is lower than 10(5) bacteria cells, the human infective dose of food-borne Salmonella. Furthermore, the probes used in this study are specific to Salmonella compared to several other Enterobacteriaceae. This approach would be a useful tool for microbial detection regarding food safety or clinical diagnosis. It is also suitable for large-scale screening in developing countries because it is low-cost, sensitive, specific and convenient.