Gold nanoparticles (AuNPs) have been extensively used as nanomaterials for theranostic applications due to their multifunctional characteristics in therapeutics, imaging, and surface modification. In ...this study, the unique functionalities of exosome-derived membranes were combined with synthetic AuNPs for targeted delivery to brain cells. Here, we report the surface modification of AuNPs with brain-targeted exosomes derived from genetically engineered mammalian cells by using the mechanical method or extrusion to create these novel nanomaterials. The unique targeting properties of the AuNPs after fabrication with the brain-targeted exosomes was demonstrated by their binding to brain cells under laminar flow conditions as well as their enhanced transport across the blood brain barrier. In a further demonstration of their ability to target brain cells, in vivo bioluminescence imaging revealed that targeted-exosome coated AuNPs accumulated in the mouse brain after intravenous injection. The surface modification of synthetic AuNPs with the brain-targeted exosome demonstrated in this work represents a highly novel and effective strategy to provide efficient brain targeting and shows promise for the future in using modified AuNPs to penetrate the brain.
Mineralization is a ubiquitous process in the animal kingdom and is fundamental to human development and health. Dysfunctional or aberrant mineralization leads to a variety of medical problems, and ...so an understanding of these processes is essential to their mitigation. Osteoblasts create the nano-composite structure of bone by secreting a collagenous extracellular matrix (ECM) on which apatite crystals subsequently form. However, despite their requisite function in building bone and decades of observations describing intracellular calcium phosphate, the precise role osteoblasts play in mediating bone apatite formation remains largely unknown. To better understand the relationship between intracellular and extracellular mineralization, we combined a sample-preparation method that simultaneously preserved mineral, ions, and ECM with nano-analytical electron microscopy techniques to examine osteoblasts in an in vitro model of bone formation. We identified calcium phosphate both within osteoblast mitochondrial granules and intracellular vesicles that transported material to the ECM. Moreover, we observed calcium-containing vesicles conjoining mitochondria, which also contained calcium, suggesting a storage and transport mechanism. Our observations further highlight the important relationship between intracellular calcium phosphate in osteoblasts and their role in mineralizing the ECM. These observations may have important implications in deciphering both how normal bone forms and in understanding pathological mineralization.
Hepatitis B virus (HBV) is a human pathogen of global concern, while a high diversity of viruses related to HBV have been discovered in other animals during the last decade. Recently, the novel ...mammalian hepadnavirus, tentatively named domestic cat hepadnavirus (DCH), was detected in an immunocompromised cat. Herein, a collection of 209 cat sera and 15 hepato-diseased cats were screened for DCH using PCR, resulting in 12.4% and 20% positivity in the tested sera and necropsied cats, respectively. Among the DCH-positive sera, a significantly high level of co-detection with retroviral infection was found, with the highest proportion being co-detection with feline immunodeficiency virus (FIV). Full-length genome characterization of DCH revealed the genetic diversity between the nine Thai DCH sequences obtained, and that they phylogenetically formed three distinct monophyletic clades. A putative DCH recombinant strain was found, suggesting a possible role of recombination in DCH evolution. Additionally, quantitative PCR was used to determine the viral copy number in various organs of the DCH-moribund cats, while the pathological findings were compared to the viral localization in hepatocytes, adjacent to areas of hepatic fibrosis, by immunohistochemical (IHC) and western blot analysis. In addition to the liver, positive-DCH immunoreactivity was found in various other organs, including kidneys, lung, heart, intestine, brain, and lymph nodes, providing evidence of systemic infection. Ultrastructure of infected cells revealed electron-dense particles in the nucleus and cytoplasm of hepatocytes, bronchial epithelial cells, and fibroblasts. We propose the intracellular development mechanism of this virus. Although the definitive roles of pathogenicity of DCH remains undetermined, a contributory role of the virus associated with systemic diseases is possible.
The innate immune response is a first-line defense mechanism triggered by rabies virus (RABV). Interferon (IFN) signaling and ISG products have been shown to confer resistance to RABV at various ...stages of the virus's life cycle. Human tetherin, also known as bone marrow stromal cell antigen 2 (hBST2), is a multifunctional transmembrane glycoprotein induced by IFN that has been shown to effectively counteract many viruses through diverse mechanisms. Here, we demonstrate that hBST2 inhibits RABV budding by tethering new virions to the cell surface. It was observed that release of virus-like particles (VLPs) formed by RABV G (RABV-G VLPs), but not RABV M (RABV-G VLPs), were suppressed by hBST2, indicating that RABV-G has a specific effect on the hBST2-mediated restriction of RABV. The ability of hBST2 to prevent the release of RABV-G VLPs and impede RABV growth kinetics is retained even when hBST2 has mutations at dimerization and/or glycosylation sites, making hBST2 an antagonist to RABV, with multiple mechanisms possibly contributing to the hBST2-mediated suppression of RABV. Our findings expand the knowledge of host antiviral mechanisms that control RABV infection.
Abstract
Reptilian ferlavirus, a pathogen of serious concern in snakes, has been reported in Western countries, but little is known about its prevalence in Thailand, where many snake breeding farms ...are located. In this study, we investigated the reptilian ferlavirus via swab samples derived from 49 diseased snakes and 77 healthy snakes as well as tissue samples taken from nine dead snakes from five independent snake farms. Using molecular detection, we found the ferlavirus in 8.16% of diseased snakes, but not in healthy snakes. Out of nine farmed snakes, eight snakes derived from four farms were found to be positive. Four complete genome sequences of the ferlavirus were successfully obtained and phylogenetically clustered to the highly pathogenic ferlavirus. Tissue tropism of the ferlavirus was identified in various epithelial cell types using the in situ hybridization technique. Interestingly, the hybridization signals were strongly labeled in the male genital tract. Transmission electron microscopy was used to support the ferlaviral localization in the male genital tract. This study provides the first evidence of ferlavirus localization in the male genital tract and contributes to the knowledge about ferlavirus epidemiology, indicating that there needs to be further awareness and elucidation regarding vertical transmission of reptilian ferlavirus.
Carnivore protoparvovirus-1 (CPPV-1), a viral species containing feline panleukopenia virus (FPV) and canine parvovirus (CPV) variants, are widely spread among domestic and wild carnivores causing ...systemic fatal diseases. Wild fishing cats (Prionailurus viverrinus), a globally vulnerable species, have been found dead. Postmortem examination of the carcasses revealed lesions in intestine, spleen and kidney. CPPV-1 antigen identification in these tissues, using polymerase chain reaction (PCR) and immunohistochemistry (IHC), supported the infection by the virus. PCR- and IHC-positivity in kidney tissues revealed atypical localization of the virus while in situ hybridization (ISH) and transmission electron microscopy (TEM) with the pop-off technique confirmed the first description of viral localization in kidneys. Complete genome characterization and deduced amino acid analysis of the obtained CPPV-1 from the fishing cats revealed FPV as a causative agent. The detected FPV sequences showed amino acid mutations at I566M and M569R in the capsid protein. Phylogenetic and evolutionary analyses of complete coding genome sequences revealed that the fishing cat CPPV-1 genomes are genetically clustered to the FPV genomes isolated from domestic cats in Thailand. Since the 1970s, these genomes have also been shown to share a genetic evolution with Chinese FPV strains. This study is the first evidence of CPPV-1 infection in fishing cats and it is the first to show its localization in the kidneys. These findings support the multi-host range of this parvovirus and suggest fatal CPPV-1 infections may result in other vulnerable wild carnivores.
Canine bocaviruses (CBoVs) have been recognized as pathogens associated with intestinal diseases. Hematogenous spreading caused by CBoV has been documented and may potentiate the virus entry across ...the blood-brain barrier to initiate a brain infection. This study focused attention on CBoV detection in cases of encepahlopathy and attempted to determine its viral localization. A total of 107 dog brains that histologically exhibited encephalopathy (ED) were investigated for the presence of CBoVs using polymerase chain reaction (PCR). Thirty-three histologically normal brain samples from dogs were used as a control group (CD). CBoV-2 was detected in 15 ED dogs (14.02%) but not in CD dogs (p = 0.02), while no CBoV-1 and -3 were detected. Among the CBoV-2 positive dogs, brain histological changes were characterized by nonsuppurative encephalitis, with inclusion body-like materials in some brains. In situ hybridization (ISH) and transmission electron microscopy (TEM) confirmed the presence of CBoV-2 viral particles in glial cells, supporting neurotropism of this virus. ISH signals were also detected in the intestines, lymphoid organs, and the heart, suggesting both enteral and parenteral infections of this virus. Whole genome characterization and evolutionary analysis revealed genetic diversity of CBoV-2 sequences and it was varying among the different countries where the virus was detected. This study points to a possible association of CBoV-2 with encephalopathy in dogs. It also highlights the genetic diversity and cellular tropism of this virus.
An important aim of regenerative medicine is to restore tissue function with implantable, laboratory-grown constructs that contain tissue-specific cells that replicate the function of their ...counterparts in the healthy native tissue. It remains unclear, however, whether cells used in bone regeneration applications produce a material that mimics the structural and compositional complexity of native bone. By applying multivariate analysis techniques to micro-Raman spectra of mineralized nodules formed in vitro, we reveal cell-source-dependent differences in interactions between multiple bone-like mineral environments. Although osteoblasts and adult stem cells exhibited bone-specific biological activities and created a material with many of the hallmarks of native bone, the 'bone nodules' formed from embryonic stem cells were an order of magnitude less stiff, and lacked the distinctive nanolevel architecture and complex biomolecular and mineral composition noted in the native tissue. Understanding the biological mechanisms of bone formation in vitro that contribute to cell-source-specific materials differences may facilitate the development of clinically successful engineered bone.
Fenugreek, or
L. (family Leguminosae) seeds, are typically used as food supplements to increase postnatal lactation. Fenugreek extract displays antioxidative and anti-inflammatory properties, but its ...mechanisms against skin aging have not been exploited. In this research, we are the first to define an in vitro collagenase inhibitory activity of fenugreek extract (IC
= 0.57 ± 0.02 mg/mL), which is 2.6 times more potent than vitamin C (IC
= 1.46 mg/mL). Nanoencapsulation has been applied to improve the extract stability, and subsequently enhanced its bioactivities. Liponiosome encapsulating fenugreek extract (LNF) was prepared using a high-speed homogenizer, resulting in homogeneous spherical nanoparticles with sizes in the range of 174.7 ± 49.2 nm, 0.26 ± 0.04 in PdI, and 46.6 ± 7.4% of entrapment efficiency. LNF formulation significantly facilitated a sustained release and significantly enhanced skin penetration over the extracts, suggesting a potential use of LNF for transdermal delivery. The formulated LNF was highly stable, not toxic to human fibroblast, and was able to enhance cell viability, collagen production, and inhibit MMP1, MMP9, IL-6, and IL-8 secretions compared to the extract in the co-cultured skin model. Therefore, ethanolic fenugreek extract and its developed LNF display molecular mechanisms against skin aging and could potentially be used as an innovative ingredient for the prevention of skin aging.
Valve interstitial cells populate aortic valve cusps and have been implicated in aortic valve calcification. Here we investigate a common in vitro model for aortic valve calcification by ...characterizing nodule formation in porcine aortic valve interstitial cells (PAVICs) cultured in osteogenic (OST) medium supplemented with transforming growth factor beta 1 (TGF-β1). Using a combination of materials science and biological techniques, we investigate the relevance of PAVICs nodules in modeling the mineralised material produced in calcified aortic valve disease. PAVICs were grown in OST medium supplemented with TGF-β1 (OST+TGF-β1) or basal (CTL) medium for up to 21 days. Murine calvarial osteoblasts (MOBs) were grown in OST medium for 28 days as a known mineralizing model for comparison. PAVICs grown in OST+TGF-β1 produced nodular structures staining positive for calcium content; however, micro-Raman spectroscopy allowed live, noninvasive imaging that showed an absence of mineralized material, which was readily identified in nodules formed by MOBs and has been identified in human valves. Gene expression analysis, immunostaining, and transmission electron microscopy imaging revealed that PAVICs grown in OST+TGF-β1 medium produced abundant extracellular matrix via the upregulation of the gene for Type I Collagen. PAVICs, nevertheless, did not appear to further transdifferentiate to osteoblasts. Our results demonstrate that 'calcified' nodules formed from PAVICs grown in OST+TGF-β1 medium do not mineralize after 21 days in culture, but rather they express a myofibroblast-like phenotype and produce a collagen-rich extracellular matrix. This study clarifies further the role of PAVICs as a model of calcification of the human aortic valve.
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