ALBERT

Description: Different genetic backgrounds can modify the effect of mutated genes. Human α-synuclein (SNCA) gene encodes α-synuclein, and its oligomeric complexes accumulate with age and mediate the disruption of cellular homeostasis, resulting in the neuronal death that is characteristic of Parkinson’s Disease. Polymorphic variants modulate this complex pathologic mechanism. Previously, we constructed five transgenic introgression lines of a Caenorhabditis elegans model of α-synuclein using genetic backgrounds that are genetically diverse from the canonical wild-type Bristol N2. A gene expression analysis revealed that the α-synuclein transgene differentially affects genome-wide transcription due to background modifiers. To further investigate how complex traits are affected in these transgenic lines, we measured the α-synuclein transgene expression, the overall accumulation of the fusion protein of α-synuclein and yellow fluorescent protein (YFP), the lysosome-related organelles, and the body size. By using quantitative PCR (qPCR), we demonstrated stable and similar expression levels of the α-synuclein transgene in different genetic backgrounds. Strikingly, we observed that the levels of the a-synuclein:YFP fusion protein vary in different genetic backgrounds by using the COPAS™ biosorter. The quantification of the Nile Red staining assay demonstrates that α-synuclein also affects lysosome-related organelles and body size. Our results show that the same α-synuclein introgression in different C. elegans backgrounds can produces differing effects on complex traits due to background modifiers.

Description: Here, we present empirical data documenting the siRNA-mediated protection of cells after Zika virus (ZIKV) infection. siRNAs were designed to target well-conserved sequences across the ZIKV genome. Several delivery technologies were utilized. After the electroporation of 100 nM siRNA into human hepatocyte-derived carcinoma (Huh7) cells, the Feron Zv-2 sequence (specific to the ZIKV NS3 gene) yielded a cell viability of 150.3% ± 7.4% (SEM: n = 4) (p = 0.0004) relative to the cells treated only with the virus (33.9% ± 12%, SEM: n = 4). Furthermore, 100 nM siRNA Feron Zv-4 (specific to ZIKV 3’UTR) resulted in 119.1% ± 11.2% cell viability (SEM: n = 4) relative to the control cells treated with ZIKV (p = 0.0021). The cells were electroporated with siRNA prior to ZIKV infection and viability was monitored four days after this. Additionally, two novel siRNA delivery systems were tested. The first utilized recombinant Bacillus anthracis PA83 (octomer-forming mutants), co-incubated with the N-terminal 255 amino acids of B. anthracis lethal factor (LFn) fused in-frame with the RNA binding domain for human protein kinase R (LFn-PKR) at a concentration of 50 µg/mL (each). Here, baby hamster kidney (BHK) cells, treated with 100 nM siRNA Feron Zv-1, yielded 79.0% ± 4.0% viability relative to the control (50.2% ± 1.7%, SEM: n = 3) three days after exposure to ZIKV (p = 0.0096). Finally, HeLa exosomes loaded with siRNA Feron-Zv2 were incubated with Huh7 cells prior to ZIKV infection. For the siRNA-exosome treated cells, a viability of 123% ± 46% (SEM: n = 18), relative to 8% ± 16% (SEM: n = 18) for the same concentration of control HeLa exosomes, was recorded (p = 0.0416). In each instance, 0.3 moI was used and cell viability monitored using the PierceTM Firefly Luciferase Glow Assay Kit by Thermo ScientificTM. Here, we show for the first time that siRNA can significantly reduce ZIKV-induced cell killing. Future work will require quantitating ZIKV mRNA in relation to siRNA treatment, as well as testing the siRNAs and delivery systems within more complex models.

Description: The remediation of contaminated sites using plant-based techniques has gained increasing attention in recent decades. However, information on the effects of contaminant imbibition on seed viability and germination rates are often lacking in the literature. To this end, our research investigated, by means of an event-time model, the effect of diesel fuel imbibition on the seed viability and germination rate of Medicago sativa, a plant species with great potential for remediation of organic contaminants. The event-time model provided an accurate and biologically relevant method for analysing germination data. Our results reveal that the direct imbibition of diesel fuel by M. sativa seeds for ≤48 h, or their exposure to soil diesel fuel concentrations of 0–10 g/kg diesel fuel, affects their germination rates, as shown by increasing t50 values from 90.6 (±2.78) to 114.2 (±2.67) hours, without significantly affecting seed viability. On the other hand, diesel fuel imbibition of longer duration, or the exposure of M. sativa seeds to ≥20 g/kg diesel fuel-contaminated soils, leads to no further effect on time to seed emergence. Instead, these conditions compromise seed viability, resulting in a decrease in the proportion of germinated seeds from 0.91 (±0.03) in 10 g/kg diesel fuel contaminated soil to 0.84 (±0.04) and 0.70 (±0.05) in 20 and 30 g/kg diesel fuel-contaminated soils, respectively. The fact that low concentrations of diesel fuel and 0–48 h of direct imbibition delayed seed emergence without adversely affecting the percentage of viable seeds suggests that this inhibitory effect on germination at low diesel fuel exposure could be attributed more to physical constraints rather than biological damage on the seeds. The models used in this study provide an accurate and biologically relevant method for the analyses of germination data. This is vital since expensive germination experiments, be it in the field of toxicology or agriculture, deserve to be accurately analysed.

Description: Cyanobacterial blooms are becoming more common in freshwater systems, causing ecological degradation and human health risks through exposure to cyanotoxins. The role of phosphorus and nitrogen in cyanobacterial bloom formation is well documented and these are regularly the focus of management plans. There is also strong evidence that trace metals are required for a wide range of cellular processes, however their importance as a limiting factor of cyanobacterial growth in ecological systems is unclear. Furthermore, some studies have suggested a direct link between cyanotoxin production and some trace metals. This review synthesises current knowledge on the following: (1) the biochemical role of trace metals (particularly iron, cobalt, copper, manganese, molybdenum and zinc), (2) the growth limitation of cyanobacteria by trace metals, (3) the trace metal regulation of the phytoplankton community structure and (4) the role of trace metals in cyanotoxin production. Iron dominated the literature and regularly influenced bloom formation, with 15 of 18 studies indicating limitation or colimitation of cyanobacterial growth. A range of other trace metals were found to have a demonstrated capacity to limit cyanobacterial growth, and these metals require further study. The effect of trace metals on cyanotoxin production is equivocal and highly variable. Better understanding the role of trace metals in cyanobacterial growth and bloom formation is an essential component of freshwater management and a direction for future research.

Description: Injectable, thermoresponsive hydrogels are promising candidates for the delivery, maintenance and controlled release of adoptive cell therapies. Therefore, there is significant interest in the development of cytocompatible and biodegradable thermoresponsive hydrogels with appropriate gelling characteristics. Towards this end, a series of thermoresponsive copolymers consisting of poly(caprolactone) (PCL), poly(ethylene glycol) (PEG) and poly(propylene glycol) (PPG) segments, with various PEG:PPG ratios, were synthesised via ring-opening polymerisation (ROP) of ε-caprolactone and epoxy-functionalised PEG and PPG derivatives. The resultant PCL–PEG–PPG copolymers were characterised via proton nuclear magnetic resonance (1H NMR) spectroscopy, gel permeation chromatography (GPC) and differential scanning calorimetry (DSC). The thermoresponsive characteristics of the aqueous copolymer solutions at various concentrations was investigated using the inversion method. Whilst all of the copolymers displayed thermoresponsive properties, the copolymer with a ratio of 1:2 PEG:PPG exhibited an appropriate sol–gel transition (28 °C) at a relatively low concentration (10 wt%), and remained a gel at 37 °C. Furthermore, the copolymers were shown to be enzymatically degradable in the presence of lipases and could be used for the encapsulation of CD4+ T-cell lymphocytes. These results demonstrate that the thermoresponsive PCL–PEG–PPG hydrogels may be suitable for use as an adoptive cell therapy (ACT) delivery vehicle.

Description: Liver ischaemia–reperfusion injury (IRI) is an intrinsic part of the transplantation process and damages the parenchymal cells of the liver including hepatocytes, endothelial cells and cholangiocytes. Many biomarkers of IRI have been described over the past two decades that have attempted to quantify the extent of IRI involving different hepatic cellular compartments, with the aim to allow clinicians to predict the suitability of donor livers for transplantation. The advent of machine perfusion has added an additional layer of complexity to this field and has forced researchers to re-evaluate the utility of IRI biomarkers in different machine preservation techniques. In this review, we summarise the current understanding of liver IRI biomarkers and discuss them in the context of machine perfusion.

Description: We previously demonstrated that clinical administration of mobilized CD133+ bone marrow stem cells (BMSC) accelerates hepatic regeneration. Here, we investigated the potential of platelets to modulate CD133+BMSC homing to hepatic endothelial cells and sequestration to warm ischemic livers. Modulatory effects of platelets on the adhesion of CD133+BMSC to human and mouse liver-sinusoidal- and micro- endothelial cells (EC) respectively were evaluated in in vitro co-culture systems. CD133+BMSC adhesion to all types of EC were increased in the presence of platelets under shear stress. This platelet effect was mostly diminished by antagonization of P-selectin and its ligand P-Selectin-Glyco-Ligand-1 (PSGL-1). Inhibition of PECAM-1 as well as SDF-1 receptor CXCR4 had no such effect. In a model of the isolated reperfused rat liver subsequent to warm ischemia, the co-infusion of platelets augmented CD133+BMSC homing to the injured liver with heightened transmigration towards the extra sinusoidal space when compared to perfusion conditions without platelets. Extravascular co-localization of CD133+BMSC with hepatocytes was confirmed by confocal microscopy. We demonstrated an enhancing effect of platelets on CD133+BMSC homing to and transmigrating along hepatic EC putatively depending on PSGL-1 and P-selectin. Our insights suggest a new mechanism of platelets to augment stem cell dependent hepatic repair.

Description: Ecto-nucleotidase triphosphate diphosphohydrolase-2 (NTPDase2) is an ecto-enzyme that is expressed on portal fibroblasts in the liver that modulates P2 receptor signaling by regulating local concentrations of extracellular ATP and ADP. NTPDase2 has protective properties in liver fibrosis and may impact bile duct epithelial turnover. Here, we study the role of NTPDase2 in acute liver injury using an experimental model of acetaminophen (APAP) intoxication in mice with global deletion of NTPDase2. Acute liver toxicity was caused by administration of acetaminophen in wild type (WT) and NTPDase2-deficient (Entpd2 null) mice. The extent of liver injury was compared by histology and serum alanine transaminase (ALT). Markers of inflammation, regeneration and fibrosis were determined by qPCR). We found that Entpd2 expression is significantly upregulated after acetaminophen-induced hepatotoxicity. Entpd2 null mice showed significantly more necrosis and higher serum ALT compared to WT. Hepatic expression of IL-6 and PDGF-B are higher in Entpd2 null mice. Our data suggest inducible and protective roles of portal fibroblast-expressed NTPDase2 in acute necrotizing liver injury. Further studies should investigate the relevance of these purinergic pathways in hepatic periportal and sinusoidal biology as such advances in understanding might provide possible therapeutic targets.

Description: The tripeptide NH2–Gly–His–Lys–COOH (GHK), cis-urocanic acid (cis-UCA) and Cu(II) ions are physiological constituents of the human body and they co-occur (e.g., in the skin and the plasma). While GHK is known as Cu(II)-binding molecule, we found that urocanic acid also coordinates Cu(II) ions. Furthermore, both ligands create ternary Cu(II) complex being probably physiologically functional species. Regarding the natural concentrations of the studied molecules in some human tissues, together with the affinities reported here, we conclude that the ternary complex [GHK][Cu(II)][cis-urocanic acid] may be partly responsible for biological effects of GHK and urocanic acid described in the literature.

Description: Escape routes keep firefighters safe by providing efficient evacuation pathways from the fire line to safety zones. Effectively utilizing escape routes requires a precise understanding of how much time it will take firefighters to traverse them. To improve this understanding, we collected GPS-tracked travel rate data from US Interagency Hotshot “Type 1” Crews during training in 2019. Firefighters were tracked while hiking, carrying standard loads (e.g., packs, tools, etc.) along trails with a precisely-measured terrain slope derived from airborne lidar. The effects of the slope on the instantaneous travel rate were assessed by three models generated using non-linear quantile regression, representing low (bottom third), moderate (middle third), and high (upper third) rates of travel, which were validated using k-fold cross-validation. The models peak at about a −3° (downhill) slope, similar to previous slope-dependent travel rate functions. The moderate firefighter travel rate model mostly predicts faster movement than previous slope-dependent travel rate functions, suggesting that firefighters generally move faster than non-firefighting personnel while hiking. Steepness was also found to have a smaller effect on firefighter travel rates than previously predicted. The travel rate functions produced by this study provide guidelines for firefighter escape route travel rates and allow for more accurate and flexible wildland firefighting safety planning.