Applications of Genome Editing Technologies in CAD Research and Therapy with a Focus on Atherosclerosis.

Cardiovascular diseases, particularly coronary artery disease (CAD), remain the leading cause of death worldwide in recent years, with myocardial infarction (MI) being the most common form of CAD. Atherosclerosis has been highlighted as one of the drivers of CAD, and much research has been carried out to understand and treat this disease. However, there remains much to be better understood and developed in treating this disease. Genome editing technologies have been widely used to establish models of disease as well as to treat various genetic disorders at their root. In this review, we aim to highlight the various ways genome editing technologies can be applied to establish models of atherosclerosis, as well as their therapeutic roles in both atherosclerosis and the clinical implications of CAD.

Capsicum Waste as a Sustainable Source of Capsaicinoids for Metabolic Diseases.

Capsaicinoids are pungent alkaloid compounds enriched with antioxidants, anti-microbial, anti-inflammatory, analgesics, anti-carcinogenic, anti-obesity and anti-diabetic properties. These compounds are primarily synthesised in the placenta of the fruit and then transported to other vegetative parts. Different varieties of capsicum and chillies contain different capsaicinoid concentrations. As capsicums and chillies are grown extensively throughout the world, their agricultural and horticultural production leads to significant amount of waste generation, in the form of fruits and plant biomass. Fruit wastes (placenta, seeds and unused fruits) and plant biowaste (stems and leaves) can serve as sources of capsaicinoids which can provide opportunities to extract these compounds for development of nutraceutical products using conventional or advanced extraction techniques. Capsaicin and dihydrocapsaicin are two most abundantly found pungent compounds. Considering the health benefits of capsaicinoids, these compounds can help in reducing metabolic disease complications. The development of an advanced encapsulation therapy of safe and clinically effective oral capsaicinoid/capsaicin formulation seem to require evaluation of strategies to address challenges related to the dosage, limited half-life and bioavailability, adverse effects and pungency, and the impacts of other ligands antagonising the major capsaicinoid receptor.

Molecular Regulation and Evolution of Cytokinin Signaling in Plant Abiotic Stresses.

The sustainable production of crops faces increasing challenges from global climate change and human activities, which leads to increasing instances of many abiotic stressors to plants. Among the abiotic stressors, drought, salinity and excessive levels of toxic metals cause reductions in global agricultural productivity and serious health risks for humans. Cytokinins (CKs) are key phytohormones functioning in both normal development and stress responses in plants. Here, we summarize the molecular mechanisms on the biosynthesis, metabolism, transport and signaling transduction pathways of CKs. CKs act as negative regulators of both root system architecture plasticity and root sodium exclusion in response to salt stress. The functions of CKs in mineral-toxicity tolerance and their detoxification in plants are reviewed. Comparative genomic analyses were performed to trace the origin, evolution and diversification of the critical regulatory networks linking CK signaling and abiotic stress. We found that the production of CKs and their derivatives, pathways of signal transduction and drought-response root growth regulation are evolutionarily conserved in land plants. In addition, the mechanisms of CK-mediated sodium exclusion under salt stress are suggested for further investigations. In summary, we propose that the manipulation of CK levels and their signaling pathways is important for plant abiotic stress and is, therefore, a potential strategy for meeting the increasing demand for global food production under changing climatic conditions.

Molecular Regulation and Evolution of Redox Homeostasis in Photosynthetic Machinery.

The recent advances in plant biology have significantly improved our understanding of reactive oxygen species (ROS) as signaling molecules in the redox regulation of complex cellular processes. In plants, free radicals and non-radicals are prevalent intra- and inter-cellular ROS, catalyzing complex metabolic processes such as photosynthesis. Photosynthesis homeostasis is maintained by thiol-based systems and antioxidative enzymes, which belong to some of the evolutionarily conserved protein families. The molecular and biological functions of redox regulation in photosynthesis are usually to balance the electron transport chain, photosystem II, photosystem I, mesophyll and bundle sheath signaling, and photo-protection regulating plant growth and productivity. Here, we review the recent progress of ROS signaling in photosynthesis. We present a comprehensive comparative bioinformatic analysis of redox regulation in evolutionary distinct photosynthetic cells. Gene expression, phylogenies, sequence alignments, and 3D protein structures in representative algal and plant species revealed conserved key features including functional domains catalyzing oxidation and reduction reactions. We then discuss the antioxidant-related ROS signaling and important pathways for achieving homeostasis of photosynthesis. Finally, we highlight the importance of plant responses to stress cues and genetic manipulation of disturbed redox status for balanced and enhanced photosynthetic efficiency and plant productivity.

Proto Kranz-like leaf traits and cellular ionic regulation are associated with salinity tolerance in a halophytic wild rice.

Species of wild rice (Oryza spp.) possess a wide range of stress tolerance traits that can be potentially utilized in breeding climate-resilient cultivated rice cultivars (Oryza sativa) thereby aiding global food security. In this study, we conducted a greenhouse trial to evaluate the salinity tolerance of six wild rice species, one cultivated rice cultivar (IR64) and one landrace (Pokkali) using a range of electrophysiological, imaging, and whole-plant physiological techniques. Three wild species (O. latifolia, O. officinalis and O. coarctata) were found to possess superior salinity stress tolerance. The underlying mechanisms, however, were strikingly different. Na+ accumulation in leaves of O. latifolia, O. officinalis and O. coarctata were significantly higher than the tolerant landrace, Pokkali. Na+ accumulation in mesophyll cells was only observed in O. coarctata, suggesting that O. officinalis and O. latifolia avoid Na+ accumulation in mesophyll by allocating Na+ to other parts of the leaf. The finding also suggests that O. coarctata might be able to employ Na+ as osmolyte without affecting its growth. Further study of Na+ allocation in leaves will be helpful to understand the mechanisms of Na+ accumulation in these species. In addition, O. coarctata showed Proto Kranz-like leaf anatomy (enlarged bundle sheath cells and lower numbers of mesophyll cells), and higher expression of C4-related genes (e.g., NADPME, PPDK) and was a clear outlier with respect to salinity tolerance among the studied wild and cultivated Oryza species. The unique phylogenetic relationship of O. coarctata with C4 grasses suggests the potential of this species for breeding rice with high photosynthetic rate under salinity stress in the future.

Molecular Interaction and Evolution of Jasmonate Signaling With Transport and Detoxification of Heavy Metals and Metalloids in Plants.

An increase in environmental pollution resulting from toxic heavy metals and metalloids [e.g., cadmium (Cd), arsenic (As), and lead (Pb)] causes serious health risks to humans and animals. Mitigation strategies need to be developed to reduce the accumulation of the toxic elements in plant-derived foods. Natural and genetically-engineered plants with hyper-tolerant and hyper-accumulating capacity of toxic minerals are valuable for phytoremediation. However, the molecular mechanisms of detoxification and accumulation in plants have only been demonstrated in very few plant species such as Arabidopsis and rice. Here, we review the physiological and molecular aspects of jasmonic acid and the jasmonate derivatives (JAs) in response to toxic heavy metals and metalloids. Jasmonates have been identified in, limiting the accumulation and enhancing the tolerance to the toxic elements, by coordinating the ion transport system, the activity of antioxidant enzymes, and the chelating capacity in plants. We also propose the potential involvement of Ca2+ signaling in the stress-induced production of jasmonates. Comparative transcriptomics analyses using the public datasets reveal the key gene families involved in the JA-responsive routes. Furthermore, we show that JAs may function as a fundamental phytohormone that protects plants from heavy metals and metalloids as demonstrated by the evolutionary conservation and diversity of these gene families in a large number of species of the major green plant lineages. Using ATP-Binding Cassette G (ABCG) transporter subfamily of six representative green plant species, we propose that JA transporters in Subgroup 4 of ABCGs may also have roles in heavy metal detoxification. Our paper may provide guidance toward the selection and development of suitable plant and crop species that are tolerant to toxic heavy metals and metalloids.

Triangulation of methods using insect cell lines to investigate insecticidal mode-of-action.

BACKGROUND: This study investigated three in vitro models to assist in elucidating possible mode-of-action, which could be adopted to evaluate insecticidal activity of complex, unknown, or multi-constituent formulations. We used a combination of absorbance spectrometry, confocal scanning laser microscopy and microelectrode ion flux estimation (MIFE) to provide insight into potential target sites for insecticides. This study used two insect cell lines and evaluated three pyrethroid insecticides. RESULTS: We observed that the two cell lines produced distinctly different responses. Drosophila melanogaster D.mel-S2 cell line was a useful model to monitor ion flux changes, resulting from insecticides with neural toxicity; however, it was less useful to determine some metabolic pathway indicators of toxic stress. Conversely, the Spodoptera frugiperda Sf9 cell line produced acute reactive oxygen species (ROS) in response to insecticide treatments, but was not highly responsive in electrophysiological experiments. We also showed that the natural, multi-constituent botanical extract of pyrethrum elicited different Na+ , Cl- and Ca2+ ion fluxes than its synthetic, single constituent analogues, α-cypermethrin and esfenvalerate. These two methods used in combination with absorbance spectrometry measuring cell growth inhibition plus cell mortality assays shed some light on cytotoxic responses in differing model cell lines. CONCLUSION: This research highlights the importance of using multiple cell types and interdisciplinary methods to provide a better insight into mode of insecticidal action. This is especially pertinent to novel biopesticide discovery, as the underlying mechanisms for toxicity in initial screening processes are likely to be unknown.

QTLs for stomatal and photosynthetic traits related to salinity tolerance in barley.

BACKGROUND: Stomata regulate photosynthesis and transpiration, and these processes are critical for plant responses to abiotic stresses such as salinity. A barley double haploid population with 108 lines derived from a cross between CM72 (salt-tolerant) and Gairdner (salt-sensitive) was used to detect quantitative trait loci (QTLs) associated with stomatal and photosynthetic traits related to salinity tolerance. RESULTS: A total of 11 significant QTLs (LOD > 3.0) and 11 tentative QTLs (2.5 < LOD < 3.0) were identified. These QTLs are distributed on all the seven chromosomes, except 5H and explain 9.5-17.3% of the phenotypic variation. QTLs for biomass, intercellular CO2 concentration, transpiration rate and stomatal conductance under control conditions co-localised together. A QTL for biomass also co-located with one for transpiration rate under salinity stress. A linkage was found between stomatal pore area and gas exchange. A QTL for salinity tolerance also co-localised with QTLs for grain yield and biomass on chromosome 3H. Based on the draft barley genome, the candidate genes for salinity tolerance at this locus are proposed. CONCLUSIONS: The lack of major QTLs for gas exchange and stomatal traits under control and saline conditions indicates a complex relationship between salinity and leaf gas exchange due to the fact that these complex quantitative traits are under the control of multiple genes.

Nitrate reductase mutation alters potassium nutrition as well as nitric oxide-mediated control of guard cell ion channels in Arabidopsis.

Maintaining potassium (K(+) ) nutrition and a robust guard cell K(+) inward channel activity is considered critical for plants' adaptation to fluctuating and challenging growth environment. ABA induces stomatal closure through hydrogen peroxide and nitric oxide (NO) along with subsequent ion channel-mediated loss of K(+) and anions. However, the interactions of NO synthesis and signalling with K(+) nutrition and guard cell K(+) channel activities have not been fully explored in Arabidopsis. Physiological and molecular techniques were employed to dissect the interaction of nitrogen and potassium nutrition in regulating stomatal opening, CO2 assimilation and ion channel activity. These data, gene expression and ABA signalling transduction were compared in wild-type Columbia-0 (Col-0) and the nitrate reductase mutant nia1nia2. Growth and K(+) nutrition were impaired along with stomatal behaviour, membrane transport, and expression of genes associated with ABA signalling in the nia1nia2 mutant. ABA-inhibited K(+) in current and ABA-enhanced slow anion current were absent in nia1nia2. Exogenous NO restored regulation of these channels for complete stomatal closure in nia1nia2. While NO is an important signalling component in ABA-induced stomatal closure in Arabidopsis, our findings demonstrate a more complex interaction associating potassium nutrition and nitrogen metabolism in the nia1nia2 mutant that affects stomatal function.

Linking stomatal traits and expression of slow anion channel genes HvSLAH1 and HvSLAC1 with grain yield for increasing salinity tolerance in barley.

Soil salinity is an environmental and agricultural problem in many parts of the world. One of the keys to breeding barley for adaptation to salinity lies in a better understanding of the genetic control of stomatal regulation. We have employed a range of physiological (stomata assay, gas exchange, phylogenetic analysis, QTL analysis), and molecular techniques (RT-PCR and qPCR) to investigate stomatal behavior and genotypic variation in barley cultivars and a genetic population in four experimental trials. A set of relatively efficient and reliable methods were developed for the characterization of stomatal behavior of a large number of varieties and genetic lines. Furthermore, we found a large genetic variation of gas exchange and stomatal traits in barley in response to salinity stress. Salt-tolerant cultivar CM72 showed significantly larger stomatal aperture under 200 mM NaCl treatment than that of salt-sensitive cultivar Gairdner. Stomatal traits such as aperture width/length were found to significantly correlate with grain yield under salt treatment. Phenotypic characterization and QTL analysis of a segregating double haploid population of the CM72/Gairdner resulted in the identification of significant stomatal traits-related QTLs for salt tolerance. Moreover, expression analysis of the slow anion channel genes HvSLAH1 and HvSLAC1 demonstrated that their up-regulation is linked to higher barley grain yield in the field.

Allosteric regulation of rhomboid intramembrane proteolysis.

Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Förster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii). We demonstrate significant differences in catalytic efficiency (kcat/K0.5) values for transmembrane substrate psTatA (TatA from Providencia stuartii) cleavage for three rhomboids: AarA from P. stuartii, ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite-mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases.

Analysis of gas exchange, stomatal behaviour and micronutrients uncovers dynamic response and adaptation of tomato plants to monochromatic light treatments.

Light spectrum affects the yield and quality of greenhouse tomato, especially over a prolonged period of monochromatic light treatments. Physiological and chemical analysis was employed to investigate the influence of light spectral (blue, green and red) changes on growth, photosynthesis, stomatal behaviour, leaf pigment, and micronutrient levels. We found that plants are less affected under blue light treatment, which was evident by the maintenance of higher A, gs, Tr, and stomatal parameters and significantly lower VPD and Tleaf as compared to those plants grown in green and red light treatments. Green and red light treatments led to significantly larger increase in the accumulation of Fe, B, Zn, and Cu than blue light. Moreover, guard cell length, width, and volume all showed highly significant positive correlations to gs, Tr and negative links to VPD. There was negative impact of monochromatic lights-induced accumulation of Mn, Cu, and Zn on photosynthesis, leaf pigments and plant growth. Furthermore, most of the light-induced significant changes of the physiological traits were partially recovered at the end of experiment. A high degree of morphological and physiological plasticity to blue, green and red light treatments suggested that tomato plants may have developed mechanisms to adapt to the light treatments. Thus, understanding the optimization of light spectrum for photosynthesis and growth is one of the key components for greenhouse tomato production.

Oncogenic ERBB3 mutations in human cancers.

The human epidermal growth factor receptor (HER) family of tyrosine kinases is deregulated in multiple cancers either through amplification, overexpression, or mutation. ERBB3/HER3, the only member with an impaired kinase domain, although amplified or overexpressed in some cancers, has not been reported to carry oncogenic mutations. Here, we report the identification of ERBB3 somatic mutations in ~11% of colon and gastric cancers. We found that the ERBB3 mutants transformed colonic and breast epithelial cells in a ligand-independent manner. However, the mutant ERBB3 oncogenic activity was dependent on kinase-active ERBB2. Furthermore, we found that anti-ERBB antibodies and small molecule inhibitors effectively blocked mutant ERBB3-mediated oncogenic signaling and disease progression in vivo.

Oligomeric state study of prokaryotic rhomboid proteases.

Rhomboid peptidases (proteases) play key roles in signaling events at the membrane bilayer. Understanding the regulation of rhomboid function is crucial for insight into its mechanism of action. Here we examine the oligomeric state of three different rhomboid proteases. We subjected Haemophilus influenzae, (hiGlpG), Escherichia coli GlpG (ecGlpG) and Bacillus subtilis (YqgP) to sedimentation equilibrium analysis in detergent-solubilized dodecylmaltoside (DDM) solution. For hiGlpG and ecGlpG, rhomboids consisting of the core 6 transmembrane domains without and with soluble domains respectively, and YqgP, predicted to have 7 transmembrane domains with larger soluble domains at the termini, the predominant species was dimeric with low amounts of monomer and tetramers observed. To examine the effect of the membrane domain alone on oligomeric state of rhomboid, hiGlpG, the simplest form from the rhomboid class of intramembrane proteases representing the canonical rhomboid core of six transmembrane domains, was studied further. Using gel filtration and crosslinking we demonstrate that hiGlpG is dimeric and functional in DDM detergent solution. More importantly co-immunoprecipitation studies demonstrate that the dimer is present in the lipid bilayer suggesting a physiological dimer. Overall these results indicate that rhomboids form oligomers which are facilitated by the membrane domain. For hiGlpG we have shown that these oligomers exist in the lipid bilayer. This is the first detailed oligomeric state characterization of the rhomboid family of peptidases.

Crystal structure of the N-lobe of lactoferrin binding protein B from Moraxella bovis.

Lactoferrin (Lf) is a bi-lobed, iron-binding protein found on mucosal surfaces and at sites of inflammation. Gram-negative pathogens from the Neisseriaceae and Moraxellaceae families are capable of using Lf as a source of iron for growth through a process mediated by a bacterial surface receptor that directly binds host Lf. This receptor consists of an integral outer membrane protein, lactoferrin binding protein A (LbpA), and a surface lipoprotein, lactoferrin binding protein B (LbpB). The N-lobe of the homologous transferrin binding protein B, TbpB, has been shown to facilitate transferrin binding in the process of iron acquisition. Currently there is little known about the role of LbpB in iron acquisition or how Lf interacts with the bacterial receptor proteins. No structural information on any LbpB or domain is available. In this study, we express and purify from Escherichia coli the full-length LbpB and the N-lobe of LbpB from the bovine pathogen Moraxella bovis for crystallization trials. We demonstrate that M. bovis LbpB binds to bovine but not human Lf. We also report the crystal structure of the N-terminal lobe of LbpB from M. bovis and compare it with the published structures of TbpB to speculate on the process of Lf mediated iron acquisition.

Insights into substrate gating in H. influenzae rhomboid.

Rhomboids are a remarkable class of serine proteases that are embedded in lipid membranes. These membrane-bound enzymes play key roles in cellular signaling events, and disruptions in these events can result in numerous disease pathologies, including hereditary blindness, type 2 diabetes, Parkinson's disease, and epithelial cancers. Recent crystal structures of rhomboids from Escherichia coli have focused on how membrane-bound substrates gain access to a buried active site. In E. coli, it has been shown that movements of loop 5, with smaller movements in helix 5 and loop 4, act as substrate gate, facilitating inhibitor access to rhomboid catalytic residues. Herein we present a new structure of the Haemophilus influenzae rhomboid hiGlpG, which reveals disorder in loop 5, helix 5, and loop 4, indicating that, together, they represent mobile elements of the substrate gate. Substrate cleavage assays by hiGlpG with amino acid substitutions in these mobile regions demonstrate that the flexibilities of both loop 5 and helix 5 are important for access of the substrates to the catalytic residues. Mutagenesis indicates that less mobility by loop 4 is required for substrate cleavage. A reexamination of the reaction mechanism of rhomboid substrates, whereby cleavage of the scissile bond occurs on the si-face of the peptide bond, is discussed.

Antiviral activity of various 1-(2'-deoxy-ß-D-lyxofuranosyl), 1-(2'-fluoro-ß-D-xylofuranosyl), 1-(3'-fluoro-ß-D-arabinofuranosyl), and 2'-fluoro-2',3'-didehydro-2',3'-dideoxyribose pyrimidine nucleoside analogues against duck hepatitis B virus (DHBV) and human hepatitis B virus (HBV) replication.

Despite the existence of successful vaccine and antiviral therapies, infection with hepatitis B virus (HBV) continues to be a major global cause of acute and chronic liver disease and high mortality. We synthesized and evaluated several lyxofuranosyl, 2'-fluoroxylofuranosyl, 3'-fluoroarabinofuranosyl, and 2'-fluoro-2',3'-didehydro-2',3'-dideoxyribose pyrimidine nucleoside analogues for antiviral activities against hepatitis B virus. Among the compounds examined, 1-(2-deoxy-ß-d-lyxofuranosyl)thymine (23), 1-(2-deoxy-ß-d-lyxofuranosyl)-5-trifluoromethyluracil (25), 1-(2-deoxy-2-fluoro-ß-d-xylofuranosyl)uracil (38), 1-(2-deoxy-2-fluoro-ß-d-xylofuranosyl)thymine (39), 2',3'-dideoxy-2',3'-didehydro-2'-fluorothymidine (48), and 2',3'-dideoxy-2',3'-didehydro-2'-fluoro-5-ethyluridine (49) were found to possess significant anti-HBV activity against DHBV in primary duck hepatocytes with EC(50) values of 4.1, 3.3, 40.6, 3.8, 0.2, and 39.0 µM, respectively. Compounds 23, 25, 39, 48, and 49 (EC(50) = 41.3, 33.7, 19.2, 2.0-4.1, and 39.0 µM, respectively) exhibited significant activity against wild-type human HBV in 2.2.15 cells. Intriguingly, 25, 39, 48, and 49 retained sensitivity against lamivudine-resistant HBV containing a single mutation (M204I) and 48 emerged as an effective inhibitor of drug-resistant HBV with an EC(50) of 4.1 µM. In contrast, 50% inhibition could not be achieved by lamivudine at 44 µM concentration in the drug-resistant strain. The compounds investigated did not show cytotoxicity to host cells up to the highest concentrations tested.

Antiviral activity of 2,3'-anhydro and related pyrimidine nucleosides against hepatitis B virus.

Various 2,3'-anhydro analogs of 5-substituted 1-(2-deoxy-ß-d-lyxofuranosyl)uracils (10-15) and a related 1-(3-O-mesyl-2-deoxy-ß-d-lyxofuranosyl) pyrimidine nucleoside analog (18) have been synthesized for evaluation as a new class of potential anti-HBV agents. The compounds 10, 12, and 15 demonstrated most potent anti-HBV activities against duck HBV (DHBV) and human HBV with EC(50) values in the range of 2.5-10 and 5-10 µg/mL, respectively, at non-toxic concentrations (CC(50) = > 200 µg/mL). The nucleoside 18 also demonstrated significant anti-HBV activity against DHBV with an EC(50) value of 2.5 µg/mL, however, it was less active against HBV in 2.2.15 cells (EC(50) = > 10 µg/mL).

Synthesis and in vitro antiviral activities of 3'-fluoro (or chloro) and 2',3'-difluoro 2',3'-dideoxynucleoside analogs against hepatitis B and C viruses.

Chronic infections with hepatitis B virus (HBV) and hepatitis C virus (HCV) lead to serious liver diseases worldwide. Co-infection with HBV and HCV is very common and is associated with increased risk of liver pathogenesis, liver cancer, and liver failure. Several 5-substituted 3'-fluoro (or chloro) (1-4, 6, 7, 17-19) and 2',3'-difluoro 2',3'-dideoxynucleosides (15 and 16) were synthesized and evaluated for in vitro antiviral activities against duck hepatitis B virus (DHBV), human hepatitis B virus, and hepatitis C virus. Of these compounds 4, 7, 17, and 19 demonstrated moderate anti-HBV activity, and 2, 4, 7, 8, and 19 were weak inhibitors of HCV. Although 5-iodo derivative (7) was most inhibitory against HCV, it exhibited a reduction in cellular RNA levels in Huh-7 cells. The 5-hydroxymethyl-3'-fluoro-2',3'-dideoxyuridine (4) and 1-(3-chloro-2,3-dideoxy-ß-d-erythro-pentofuranosyl)-5-fluorouracil (19) provided the most inhibition of both viruses without cytotoxicity.