Sustainable Sources of Biomass for Bioremediation of Heavy Metals in Waste Water Derived from Coal-Fired Power Generation

Biosorption of heavy metals using dried algal biomass has been extensively described but rarely implemented. We contend this is because available algal biomass is a valuable product with a ready market. Therefore, we considered an alternative and practical approach to algal bioremediation in which algae were cultured directly in the waste water stream. We cultured three species of algae with and without nutrient addition in water that was contaminated with heavy metals from an Ash Dam associated with coal-fired power generation and tested metal uptake and bioremediation potential. All species achieved high concentrations of heavy metals (to 8% dry mass). Two key elements, V and As, reached concentrations in the biomass of 1543 mg.kg−1 DW and 137 mg.kg−1 DW. Growth rates were reduced by more than half in neat Ash Dam water than when nutrients were supplied in excess. Growth rate and bioconcentration were positively correlated for most elements, but some elements (e.g. Cd, Zn) were concentrated more when growth rates were lower, indicating the potential to tailor bioremediation depending on the pollutant. The cosmopolitan nature of the macroalgae studied, and their ability to grow and concentrate a suite of heavy metals from industrial wastes, highlights a clear benefit in the practical application of waste water bioremediation

Remote ischemic conditioning: from experimental observation to clinical application: report from the 8th Biennial Hatter Cardiovascular Institute Workshop

In 1993, Przyklenk and colleagues made the intriguing experimental observation that 'brief ischemia in one vascular bed also protects remote, virgin myocardium from subsequent sustained coronary artery occlusion' and that this effect '.... may be mediated by factor(s) activated, produced, or transported throughout the heart during brief ischemia/reperfusion'. This seminal study laid the foundation for the discovery of 'remote ischemic conditioning' (RIC), a phenomenon in which the heart is protected from the detrimental effects of acute ischemia/reperfusion injury (IRI), by applying cycles of brief ischemia and reperfusion to an organ or tissue remote from the heart. The concept of RIC quickly evolved to extend beyond the heart, encompassing inter-organ protection against acute IRI. The crucial discovery that the protective RIC stimulus could be applied non-invasively, by simply inflating and deflating a blood pressure cuff placed on the upper arm to induce cycles of brief ischemia and reperfusion, has facilitated the translation of RIC into the clinical setting. Despite intensive investigation over the last 20 years, the underlying mechanisms continue to elude researchers. In the 8th Biennial Hatter Cardiovascular Institute Workshop, recent developments in the field of RIC were discussed with a focus on new insights into the underlying mechanisms, the diversity of non-cardiac protection, new clinical applications, and large outcome studies. The scientific advances made in this field of research highlight the journey that RIC has made from being an intriguing experimental observation to a clinical application with patient benefit

Expression of human tissue plasminogen activator using the Bombyx mori nuclear polyhedrosis virus system

In this study, we utilized the BmNPV to express active human tissue plasminogen activator, a potent fibrinolytic agent, in BmN cells and the silkworm Bombyx mori. Two expression vectors, one containing the original t-PA cDNA and the other a mutated t-PA cDNA, were constructed. The BmLSP signal peptide was used to replace the original t-PA signal peptide, and a short sequence containing 7 bases, TATAAAT, was added immediately before the signal peptide. Recombinant viruses were generated from these vectors, and were used to infect BmN cells. Media from a titne course experiment of 5 days were studied. The radial caseinolysis assay was employed to check the activities of the expressed products. This study gave positive indication that human t-PA could be expressed by the BmNPV system. It seemed that the mutation introduced into the t-PA gene did enhance the level of expression and secretion to a certain extent. However, more work need to be done on the characterization and the purification of the expressed products

T4 BacteriophageDominatesT7 Bacteriophage During Co- infection of Escherichia coli C600

Co-infection is the simultaneous infection of a host cell by multiple viruses. It can result in complex competitive interactions that have significant consequences on virulence and disease dynamics. Our study investigated the intracellular mechanisms utilized for viral competition and identified a dominant virus during co-infection with a T4 and T7 bacteriophage model in E. coli strain C600. The co-infection was performed under two different input multiplicity of infection (MOI) conditions, each favouring one phage type. The amount of infectious progeny from coinfected samples was assessed with plaque assays. Results indicated that T4 dominated in both conditions, whereas PCR data showed T7 amplification from samples that produced only T4 plaques. We speculate that the dominance exhibited by T4 is due to its ability to modify the host RNA polymerase to only transcribe its own genes, thereby inhibiting T7 early gene transcription. In conclusion, bacteriophage T4 exhibited viral dominance over T7 during co-infection of E. coli C600, via mechanisms that interfered with T7 maturation. It has been observed in nature that multiple virions can coinfect a single host cell. Co-infection remains very important in virus evolution because of processes such as genetic reassortment, recombination, and complementation that can lead to increased virulence(1). However, coinfection also comes at a cost due to the sharingof host resources, which can be further limited with greater numbers of infecting virions per cell. Thus, many viruses have evolved mechanisms to inhibit viral competitors that coinfect the same host cell (2). These mechanisms can involve manipulation of host enzymes and repression of viral replication (2). In 2012, Brewster et al. (2) studied the co-infection of Escherichia coliC3000 by bacteriophages MS2, ϕX174, and T7. It was observed that co-infections resulted in decreased host cell lysis time compared to the component mono-infections, thus indicating that viral competition played a significant effect on viral outcome (1). However, they did not identify the dominant bacteriophage during co-infection or adequately address the specific mechanisms utilized for competition. Previous bacteriophage studies show mechanisms for viral competition affect intracellular stages of the phage life cycle (2). Therefore, our study explored the intracellular aspects of viral competition using a single cell for coinfection experiments. It was determined that E. coli C600 was a susceptible host for the genetically similar T-even and T-odd bacteriophages, T4 and T7, and could be utilized as a co-infection model. T4 and T7 are lytic phages, which utilize a tail component with tail fibers to facilitate attachment and entry into host cells using the LPS receptor (3, 4, 5). Bacteriophage T4 has a 169 kb genome consisting of early, middle, and late genes that are transcribed by the host RNA polymerase which becomes modified at each stage to allow progression into the phage replication cycle (6). Bacteriophage T7 has a 40 kb genome divided into early genes transcribed by the host RNA polymerase and late genes, which are transcribed by the T7 bacteriophageencoded RNA polymerase (4). In this experiment we defined viral dominance as the ability of one virus to limit the production of competitive infectious progeny. The results indicated that T4 exhibited dominance over T7 during co-infection, through mechanisms that affect T7 maturation. Our experiment provides greater insight into dominance during viral coinfection via intracellular mechanisms involving the viral life cycle. MATERIALS AND METHODS Strains and growth conditions. Escherichia coli C600 and bacteriophages T4 and T7 were obtained from the Microbiology 421 culture collection from the Department of Microbiology and Immunology, University of British Columbia. E. colicultures were grown at 30°C in L-Media described in Overby et al. Plaque assays. Phage samples were enumerated using the overlay agar plaque assay method described by Fortier and Moineau(8). A 4% soft-overlay agar was used. Infection with T7 phages produced characteristically larger plaques than with T4. Co-infection of T4 and T7 in E. coli C600.Two samples of 4x10 8 cells/ml E. coli C600were coinfected with T4 and T7. The first was infected with MOI of 0.5 and 1.2, respectively, while the second was infected with MOI of 1.2 and 0.5, respectively. The infected cultures were incubated for 5 minutes before centrifugation at 8000 x g for 10 minutes and disposal of the supernatant to remove non-adsorbed bacteriophages. Each pellet was resuspended, diluted and separated into 25 aliquots of 0.8 cells per tube with a total volume of 200 µl per tube. The aliquots were incubated at 37°C for 60 min to allow lysis of any infected cells. Plaque assays were performed to detect the presence of phages

Efficient in vivo bone formation by BMP-2 engineered human mesenchymal stem cells encapsulated in a projection stereolithographically fabricated hydrogel scaffold

Background: Stem cell-based bone tissue engineering shows promise for bone repair but faces some challenges, such as insufficient osteogenesis and limited architecture flexibility of the cell-delivery scaffold. Methods: In this study, we first used lentiviral constructs to transduce ex vivo human bone marrow-derived stem cells with human bone morphogenetic protein-2 (BMP-2) gene (BMP-hBMSCs). We then introduced these cells into a hydrogel scaffold using an advanced visible light-based projection stereolithography (VL-PSL) technology, which is compatible with concomitant cell encapsulation and amenable to computer-aided architectural design, to fabricate scaffolds fitting local physical and structural variations in different bones and defects. Results: The results showed that the BMP-hBMSCs encapsulated within the scaffolds had high viability with sustained BMP-2 gene expression and differentiated toward an osteogenic lineage without the supplement of dditional BMP-2 protein. In vivo bone formation efficacy was further assessed using an intramuscular implantation model in severe combined immunodeficiency (SCID) mice. Microcomputed tomography (micro-CT) imaging indicated rapid bone formation by the BMP-hBMSC-laden constructs as early as 14 days post-implantation. Histological examination revealed a mature trabecular bone structure with considerable vascularization. Through tracking of the implanted cells, we also found that BMP-hBMSC were directly involved in the new bone formation. Conclusions: The robust, self-driven osteogenic capability and computer-designed architecture of the construct developed in this study should have potential applications for customized clinical repair of large bone defects or non-unions. Keywords: Osteogenesis, Bone tissue engineering, Bone formation, 3D bioprinting, Gene therapy, Ex vivo gen

One-Step Fabrication of Bone Morphogenetic Protein-2 Gene-Activated Porous Poly-L-Lactide Scaffold for Bone Induction

Bone morphogenetic protein 2 (BMP2) is an efficacious inducer for the osteogenesis of mesenchymal stem cells (MSCs). Conventional applications of BMP2 have involved either the direct incorporation of BMP2 protein or ex vivo BMP2 gene transfer into stem cells prior to their transplantation. These approaches are able to promote bone formation to some extent; however, they are hampered by either the lack of stability and sustainability of BMP2 protein or the time-consuming and cost-prohibitive in vitro cell culture procedure. To overcome these limitations, we have developed a gene-activated poly-L-lactide acid (PLLA) scaffold with the encapsulation of recombinant adeno-associated viral (AAV) vector encoding a full-length cDNA of human BMP2 using an ice-based microparticle porogenization method that was recently developed. Results showed continuous release of AAV particles from the micropores of scaffolds for up to 1 week, subsequently transducing embedded human MSCs and producing functional BMP2. MSCs within scaffolds underwent efficacious osteogenesis, on the basis of osteoinductive gene expression and osteogenic differentiation, which resulted in robust new bone formation in vivo at 4 weeks. These findings show the potential of the technology toward developing clinical applications of a rapid, cost-effective, and potentially point-of-care approach for the repair of bone defects

Projection Stereolithographic Fabrication of BMP-2 Gene-activated Matrix for Bone Tissue Engineering

Abstract Currently, sustained in vivo delivery of active bone morphogenetic protein-2 (BMP-2) protein to responsive target cells, such as bone marrow-derived mesenchymal stem cells (BMSCs), remains challenging. Ex vivo gene transfer method, while efficient, requires additional operation for cell culture and therefore, is not compatible with point-of-care treatment. In this study, two lentiviral gene constructs – (1) Lv-BMP/GFP, containing human BMP-2 and green fluorescent protein (GFP) gene (BMP group); or (2) Lv-GFP, containing GFP gene (GFP group) – were incorporated with human BMSCs into a solution of photocrosslinkable gelatin, which was then subjected to visible light-based projection stereolithographic printing to form a scaffold with desired architectures. Upon in vitro culture, compared to the GFP group, cells from BMP group showed >1,000-fold higher BMP-2 release, and the majority of them stained intensely for alkaline phosphatase activity. Real-time RT-PCR also showed dramatically increased expression of osteogenesis marker genes only in the BMP group. 3.5 months post-implantation into SCID mice, the micro-computed tomography imaging showed detectable mineralized areas only in the BMP group, which was restricted within the scaffolds. Alizarin red staining and immunohistochemistry of GFP and osteocalcin further indicated that the grafted hBMSCs, not host cells, contributed primarily to the newly formed bone

3’ UTR structural elements in CYP24A1 are associated with infantile hypercalcaemia type 1

Loss-of-function mutations in the CYP24A1 protein-coding region causing reduced 25OHD and 1,25(OH)2D catabolism have been observed in some cases of Infantile hypercalcaemia type 1 (HCINF1), which can manifest as nephrocalcinosis, hypercalcaemia and adult-onset hypercalciuria and renal stone formation. Some cases present with apparent CYP24A1 phenotypes but do not exhibit pathogenic mutations. Here, we assessed the molecular mechanisms driving apparent HCINF1 where there was a lack of CYP24A1 mutation. We obtained blood samples from 47 patients with either a single abnormality of no obvious cause or a combination of hypercalcemia, hypercalciuria and nephrolithiasis as part of our metabolic and stone clinics. We used liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine serum vitamin D metabolites and direct sequencing to confirm CYP24A1 genotype. Six patients presented with profiles characteristic of altered CYP24A1 function but lacked protein-coding mutations in CYP24A1. Analysis up- and downstream of the coding sequence showed single nucleotide variants (SNVs) in the CYP24A1 3’ untranslated region (UTR). Bioinformatics approaches revealed that these 3’ UTR abnormalities did not result in microRNA silencing but altered the CYP24A1 messenger RNA (mRNA) secondary structure, which negatively impacted translation. Our experiments showed that mRNA misfolding driven by these 3’ UTR sequence-dependent structural elements was associated with normal 25OHD but abnormal 1,25(OH)2D catabolism. Using CRISPR-Cas9, we developed an in vitro mutant model for future CYP24A1 studies. Our results form a basis for future studies investigating structure-function relationships and novel CYP24A1 mutations producing a semi-functional protein