Activation of c-Jun N-terminal kinase is required for ultraviolet B-induced apoptosis of murine peritoneal macrophages in vitro

The mechanisms of ultraviolet B (UVB)-induced apoptosis and the role of c-Jun N-terminal kinase (JNK) mitogen activated protein kinase (MAPK) in murine peritoneal macrophages, the terminally differentiated non-dividing cells were investigated. Exposure of macrophages to UVB 100 mJ/cm2 induced rapid apoptosis concurrent with activation of JNK and mitochondrial cytochrome c release leading to procaspase-3 activation. Late into the UVB-induced apoptosis, a caspase-mediated cleavage of Bid was observed. Caspase inhibitors N-Benzylocarbonyl-Val-Asp-fluoromethyl ketone and N-Acetyl-Asp-Glu-Val-Asp-aldehyde inhibited the UVB-induced apoptosis without preventing the release of cytochrome c and JNK activation. The inhibition of JNK MAPK prevented UVB-induced apoptosis, concomitant with inhibition in cytochrome c release and procaspase-3 activation. However, it had no effect on procaspase-8 activation. These results indicate that activation of JNK MAPK upstream of caspases might play an important role in the apoptotic process of macrophages exposed to UVB irradiation.     

High throughput screening for neurodegeneration and complex disease phenotypes

High throughput screening (HTS) for complex diseases is challenging. This stems from the fact that complex phenotypes are difficult to adapt to rapid, high throughput assays. We describe the recent development of high throughput and high-content screens (HCS) for neurodegenerative diseases, with a focus on inherited neurodegenerative disorders, such as Huntington's disease. We describe, among others, HTS assays based on protein aggregation, neuronal death, caspase activation and mutant protein clearance. Furthermore, we describe high-content screens that are being used to prioritize hits identified in such HTS assays. These assays and screening approaches should accelerate drug discovery for neurodegenerative disorders and guide the development of screening approaches for other complex disease phenotypes.     

A Fluorescence-Polarization-Based Lipopolysaccharide–Caspase-4 Interaction Assay for the Development of Inhibitors

Recognition of intracellular lipopolysaccharide (LPS) by Caspase-4 (Casp-4) is critical for host defense against Gram-negative pathogens. LPS binds to the N-terminal caspase activation and recruitment domain (CARD) of procaspase-4, leading to auto-proteolytic activation followed by pro-inflammatory cytokine release and pyroptotic cell death. Aberrant hyper-activation of Casp-4 leads to amplification of the inflammatory response linked to sepsis. While the active site of a caspase has been targeted with peptide inhibitors, inhibition of LPS–Casp-4 interaction is an emerging strategy for the development of selective inhibitors with a new mode of action for treating infectious diseases and sepsis induced by LPS. In this study, a high-throughput screening (HTS) system based on fluorescence polarization (FP) was devised to identify inhibitors of the LPS and Casp-4 interaction. Using HTS and IC₅₀ determination and subsequently showing inhibited Casp-4 activity, we demonstrated that the LPS–Casp-4 interaction is a druggable target for Casp-4 inhibition and possibly a non-canonical inflammatory pathway.     

Managing missing measurements in small-molecule screens

In a typical high-throughput screening (HTS) campaign, less than 1 % of the small-molecule library is characterized by confirmatory experiments. As much as 99 % of the library’s molecules are set aside—and not included in downstream analysis—although some of these molecules would prove active were they sent for confirmatory testing. These missing experimental measurements prevent active molecules from being identified by screeners. In this study, we propose managing missing measurements using imputation—a powerful technique from the machine learning community—to fill in accurate guesses where measurements are missing. We then use these imputed measurements to construct an imputed visualization of HTS results, based on the scaffold tree visualization from the literature. This imputed visualization identifies almost all groups of active molecules from a HTS, even those that would otherwise be missed. We validate our methodology by simulating HTS experiments using the data from eight quantitative HTS campaigns, and the implications for drug discovery are discussed. In particular, this method can rapidly and economically identify novel active molecules, each of which could have novel function in either binding or selectivity in addition to representing new intellectual property.     

Cytotoxicity tests of cellulose nanofibril-based structures

Cellulose nanofibrils based on wood pulp fibres are most promising for biomedical applications. Bacterial cellulose has been suggested for some medical applications and is presently used as wound dressing. However, cost-efficient processes for mass production of bacterial cellulose are lacking. Hence, fibrillation of cellulose wood fibres is most interesting, as the cellulose nanofibrils can efficiently be produced in large quantities. However, the utilization of cellulose nanofibrils from wood requires a thorough verification of its biocompatibility, especially with fibroblast cells which are important in regenerative tissue and particularly in wound healing. The cellulose nanofibril structures used in this study were based on Eucalyptus and Pinus radiata pulp fibres. The nanofibrillated materials were manufactured using a homogenizer without pre-treatment and with 2,2,6,6-tetramethylpiperidine-1-oxy radical as pre-treatment, thus yielding nanofibrils low and high level of anionic charge, respectively. From these materials, two types of nanofibril-based structures were formed; (1) thin and dense structures and (2) open and porous structures. Cytotoxicity tests were applied on the samples, which demonstrated that the nanofibrils do not exert acute toxic phenomena on the tested fibroblast cells (3T3 cells). The cell membrane, cell mitochondrial activity and the DNA proliferation remained unchanged during the tests, which involved direct and indirect contact between the nano-structured materials and the 3T3 cells. Some samples were modified using the crosslinking agent polyethyleneimine (PEI) or the surfactant cetyl trimethylammonium bromide (CTAB). The sample modified with CTAB showed a clear toxic behaviour, having negative effects on cell survival, viability and proliferation. CTAB is an antimicrobial component, and thus this result was as expected. The sample crosslinked with PEI also had a significant reduction in cell viability indicating a reduction in DNA proliferation. We conclude that the neat cellulose nanostructured materials tested in this study are not toxic against fibroblasts cells. This is most important as nano-structured materials based on nanofibrils from wood pulp fibres are promising as substrate for regenerative medicine and wound healing.     

HPV E6 antisense induces apoptosis in CaSki cells via suppression of E6 splicing

Cervical cancer is known to be highly associated with viral oncogene E6 and E7 of human papilloma virus. Down-regulation of oncogene expression by antisense-based gene therapy has been extensively studied. To investigate the effect of HPV 16 E6 antisense nucleic acid (AS) on cervical cancer cells, human cervical cancer cell lines, CaSki and SiHa cells harboring HPV 16 genome were transfected with plasmid containing E6(AS). The decreased viability and the apoptotic morphology were observed in E6(AS)-transfected cervical cancer cell lines. By 6 h after transfection, inhibition of E6 splicing, rapid upregulations of p53 and a p53-responsive protein, GADD45, were displayed in E6(AS)-transfected CaSki cells. Furthermore, E6(AS) induced loss of mitochondrial transmembrane potential, release of mitochondrial cytochrome c into the cytoplasm, and subsequent activation of caspase-9 and caspase-3. These results indicate that HPV 16 E6(AS) induces apoptosis in CaSki cells via upregulation of p53 and release of cytochrome c into cytoplasm, consequently activating procaspase-9 and procaspase-3.     

Inhibitory Role of Berberine,an Isoquinoline Alkaloid,on NLRP3 Inflammasome Activation for the Treatment of Inflammatory Diseases

The pyrin domain-containing multiprotein complex NLRP3 inflammasome, consisting of the NLRP3 protein, ASC adaptor, and procaspase-1, plays a vital role in the pathophysiology of several inflammatory disorders, including neurological and metabolic disorders, chronic inflammatory diseases, and cancer. Several phytochemicals act as promising anti-inflammatory agents and are usually regarded to have potential applications as complementary or alternative therapeutic agents against chronic inflammatory disorders. Various in vitro and in vivo studies have reported the anti-inflammatory role of berberine (BRB), an organic heteropentacyclic phytochemical and natural isoquinoline, in inhibiting NLRP3 inflammasome-dependent inflammation against many disorders. This review summarizes the mechanism and regulation of NLRP3 inflammasome activation and its involvement in inflammatory diseases, and discusses the current scientific evidence on the repressive role of BRB on NLRP3 inflammasome pathways along with the possible mechanism(s) and their potential in counteracting various inflammatory diseases.     

Beta galactosidase enzyme fragment complementation as a novel technology for high throughput screening

In this review, the applications of beta galactosidase complementation are described. alpha Complementation is a naturally occurring process in bacteria and in engineered cells, and can also occur in eukaryotic cells. Two forms of alpha complementation have been used in high throughput screening (HTS), in which interacting fragments complement with either low or high affinity. Low affinity complementation is used to monitor protein protein interactions, such as those occurring in homodimerization of the epidermal growth factor receptor (EGFR), and provides a robust screen for detection of EGFR inhibitors. High affinity complementation provides the basis for several HTS assays, in which analytes, such as cAMP or IP(3), are detected in crude cell lysates. A development of the latter approach is protein labeling, providing for measurement of cell protein expression and trafficking. Collectively, the use of beta galactosidase complementation provides a novel and flexible technology for highly sensitive, homogeneous HTS assay development.     

Artificial Peptide-Protein Necrosomes Promote Cell Death

The presence of disordered region or large interacting surface within proteins significantly challenges the development of targeted drugs, commonly known as the “undruggable” issue. Here, we report a heterogeneous peptide-protein assembling strategy to selectively phosphorylate proteins, thereby activating the necroptotic signaling pathway and promoting cell necroptosis. Inspired by the structures of natural necrosomes formed by receptor interacting protein kinases (RIPK) 1 and 3, the kinase-biomimetic peptides are rationally designed by incorporating natural or _D-amino acids, or connecting _D-amino acids in a retro-inverso (DRI) manner, leading to one RIPK3-biomimetic peptide PR3 and three RIPK1-biomimetic peptides. Individual peptides undergo self-assembly into nanofibrils, whereas mixing RIPK1-biomimetic peptides with PR3 accelerates and enhances assembly of PR3 . In particular, RIPK1-biomimetic peptide DRI-PR1 exhibits reliable binding affinity with protein RIPK3, resulting in specific cytotoxicity to colon cancer cells that overexpress RIPK3. Mechanistic studies reveal the increased phosphorylation of RIPK3 induced by RIPK1-biomimetic peptides, elucidating the activation of the necroptotic signaling pathway responsible for cell death without an obvious increase in secretion of inflammatory cytokines. Our findings highlight the potential of peptide-protein hybrid aggregation as a promising approach to address the “undruggable” issue and provide alternative strategies for overcoming cancer resistance in the future.     

An empirical process for the design of high-throughput screening deck filters

A process for objective identification and filtering of undesirable compounds that contribute to high-throughput screening (HTS) deck promiscuity is described. Two methods of mapping hit promiscuity have been developed linking SMARTS-based structural queries with historical primary HTS data. The first compares an expected assay hit rate to actual hit rates. The second examines the propensity of an individual compound to hit multiple assays. Statistical evaluation of the data indicates a correlation between the resultant functional group filters and compound promiscuity. These data corroborate a number of commonly applied filters as well as producing some unexpected results. Application of these models to HTS collection triage reduced the number of in-house compounds considered for screening by 12%. The implications of these findings are further discussed in the context of the HTS screening set and combinatorial library design as well as compound acquisition.     

Chip-based high-throughput screening of herbal medicines

At present, high-throughput screening (HTS) programs in drug discovery rely mainly on compound libraries from combinational chemistry. Similarly, natural flora has been used as a prominent origin for new and potent herbal drugs. Herbal medicines have been used worldwide for thousands of years to cure many diseases. As such, herbal secondary metabolites show a remarkable structural diversity that supplements chemically synthesized compound analogs in drug discovery screening. Unfortunately, there is often a considerable deterioration in the quality of herbal drugs in such screening programs as there are time-consuming manual processes involved in the isolation of active ingredients from the highly complex mixtures of herbal plant products. The quality and quantity of herbal samples are critical for the success of HTS programs. In the recent past, there have been substantial improvements in HTS due to the miniaturization and integration of microchip (e.g., Herbochip(?), DNA chip, protein chip, cell chip, etc.)-based technologies so as to design herbal drugs that compete with synthetic drug analogs. Here we will review various technologies used for HTS of herbal medicines. Finally, we will summarize our efforts to develop a novel chip-based HTS assay to explore the antioxidant and radioprotective properties of herbal plants.     

Electrospinning of composite nanofibers based on chitosan,poly(ethylene oxide), and chitin nanofibrils

Composite chitosan nanofibers containing 20 wt % chitin nanofibrils and 10 wt % PEO are obtained via the electrospinning method. Additions of 0.5–20.0 wt % chitin nanofibrils into chitosan solutions with concentrations of 3–7 wt % in acetic acid (70 vol %) insignificantly increase the electrical conductivity, surface-tension coefficient, and viscosity of these mixed solutions. Decreases in the viscosities of chitosan solutions containing chitin nanofibrils with increases in shear rate provide evidence for the structuring of solutions and the orientation of chitosan macromolecules and chitin nanofibrils in the shear flow. The effects of shear stress and a high-voltage electric field on chitosan solutions containing chitin nanofibrils and PEO result in a decrease in the imperfection of composite nanofibers. The introduction of chitin nanofibrils allows the content of PEO in the composite nanofibers to be reduced.     

P3HT stripe structure with oriented nanofibrils enabled by controlled inclining evaporation

The preparation of the poly(3-hexylthiophene) (P3HT) stripe structure with oriented nanofibrils prepared by controlled inclining evaporative technique is reported. The distance of the adjacent stripes could be controlled from 40 μm to 100 μm by decreasing the inclining angle. The oriented nanofibrils in the stripes can be obtained because the P3HT lamellae diffuse directionally and form 1D crystals at the three-phase contact line of the drop. In order to get the oriented P3HT stripes, the proper solvent evaporation rate which is controlled by the inclining angle and the wettability of the substrate must be carefully chosen to match the P3HT 1D crystallization rate. It is found that large inclining angle and the hydrophilic substrate (for example: glass and PEDOT) are beneficial to get P3HT stripe structure with oriented nanofibrils.     

Nucleation and Growth of Amino Acid and Peptide Supramolecular Polymers through Liquid–Liquid Phase Separation

The transition of peptides and proteins from the solution phase into fibrillar structures is a general phenomenon encountered in functional and aberrant biology and is increasingly exploited in soft materials science. However, the fundamental molecular events underpinning the early stages of their assembly and subsequent growth have remained challenging to elucidate. Here, we show that liquid–liquid phase separation into solute‐rich and solute‐poor phases is a fundamental step leading to the nucleation of supramolecular nanofibrils from molecular building blocks, including peptides and even amphiphilic amino acids. The solute‐rich liquid droplets act as nucleation sites, allowing the formation of thermodynamically favorable nanofibrils following Ostwald's step rule. The transition from solution to liquid droplets is entropy driven while the transition from liquid droplets to nanofibrils is mediated by enthalpic interactions and characterized by structural reorganization. These findings shed light on how the nucleation barrier toward the formation of solid phases can be lowered through a kinetic mechanism which proceeds through a metastable liquid phase.     

Human telomere sequence DNA in water-free and high-viscosity solvents: g-quadruplex folding governed by kramers rate theory

Structures formed by human telomere sequence (HTS) DNA are of interest due to the implication of telomeres in the aging process and cancer. We present studies of HTS DNA folding in an anhydrous, high viscosity deep eutectic solvent (DES) comprised of choline choride and urea. In this solvent, the HTS DNA forms a G-quadruplex with the parallel-stranded ("propeller") fold, consistent with observations that reduced water activity favors the parallel fold, whereas alternative folds are favored at high water activity. Surprisingly, adoption of the parallel structure by HTS DNA in the DES, after thermal denaturation and quick cooling to room temperature, requires several months, as opposed to less than 2 min in an aqueous solution. This extended folding time in the DES is, in part, due to HTS DNA becoming kinetically trapped in a folded state that is apparently not accessed in lower viscosity solvents. A comparison of times required for the G-quadruplex to convert from its aqueous-preferred folded state to its parallel fold also reveals a dependence on solvent viscosity that is consistent with Kramers rate theory, which predicts that diffusion-controlled transitions will slow proportionally with solvent friction. These results provide an enhanced view of a G-quadruplex folding funnel and highlight the necessity to consider solvent viscosity in studies of G-quadruplex formation in vitro and in vivo. Additionally, the solvents and analyses presented here should prove valuable for understanding the folding of many other nucleic acids and potentially have applications in DNA-based nanotechnology where time-dependent structures are desired.     

Oriented poly(3-hexylthiophene) nanofibril with the π-π stacking growth direction by solvent directional evaporation

In this article, the uniaxial alignment of poly(3-hexylthiophene) (P3HT) nanofibrils with a π-π stacking growth direction in which P3HT chains adopt a flat-on conformation was obtained by solvent directional evaporation using a glass cover slide and a poly(dimethylsiloxane) (PDMS) sheet to press the P3HT film in a carbon disulfide (CS(2)) atmosphere. By controlling the CS(2) vapor pressure during the film-forming process, we got a well-oriented P3HT film whose order parameter reached as high as 0.97. The orientation of the film was induced by the crystallization nucleation of P3HT and the directional evaporation of the solvent. Under a CS(2) vapor atmosphere, P3HT crystals preferred to adopt the form II modification, which started by nucleation. Owing to the solvent directional evaporation from the center to the margin, P3HT at the center of the sample would precipitate first to induce nucleation. Then the peripheral P3HT would directly diffuse, precipitate, and then adhere to the nucleus to form the uniaxial alignment of P3HT nanofibrils along the direction of solvent evaporation. Furthermore, in the P3HT nanofibrils, the π-π stacking direction of P3HT lamellae was parallel to the crystal growth direction, which would provide an effective path for charge transport.     

Growth of Long Triisopropylsilylethynyl Pentacene (TIPS‐PEN) Nanofibrils in a Polymer Thin Film During Spin‐Coating

This study demonstrates the growth of long triisopropylsilyethynyl pentacene (TIPS‐PEN) nanofibrils in a thin film of a crystalline polymer, poly(ε‐caprolactone) (PCL). During spin‐coating, TIPS‐PEN molecules are locally extracted around the PCL grain boundaries and they crystallize into [010] direction forming long nanofibrils. Molecular weight of PCL and weight fraction (α) of TIPS‐PEN in PCL matrix are key factors to the growth of nanofibrils. Long high‐quality TIPS‐PEN nanofibrils are obtained with high‐molecular‐weight PCL and at the α values in the range of 0.03–0.1. The long nanofibrils are used as an active layer in a field‐effect organic transistor.

     

Luminescent proteins from Aequorea victoria: applications in drug discovery and in high throughput analysis

Recent progress in generating a vast number of drug targets through genomics and large compound libraries through combinatorial chemistry have stimulated advancements in drug discovery through the development of new high throughput screening (HTS) methods. Automation and HTS techniques are also highly desired in fields such as clinical diagnostics. Luminescence-based assays have emerged as an alternative to radiolabel-based assays in HTS as they approach the sensitivity of radioactive detection along with ease of operation, which makes them amenable to miniaturization. Luminescent proteins provide the advantage of reduced reagent and operating costs because they can be produced in unlimited amounts through the use of genetic engineering tools. In that regard, the use of two naturally occurring and recombinantly produced luminescent proteins from the jellyfish Aequorea victoria, namely, aequorin and the green fluorescent protein (GFP), has attracted attention in a number of analytical applications in diverse research areas. Aequorin is naturally bioluminescent and has therefore, virtually no associated background signal, which allows its detection down to attomole levels. GFP has become the reporter of choice in a variety of applications given that it is an autofluorescent protein that does not require addition of any co-factors for fluorescence emission. Furthermore, the generation of various mutants of GFP with differing luminescent and spectral properties has spurred additional interest in this protein. In this review, we focus on the use of aequorin and GFP in the development of highly sensitive assays that find applications in drug discovery and in high throughput analysis.     

Luminescent proteins from Aequorea victoria: applications in drug discovery and in high throughput analysis

Recent progress in generating a vast number of drug targets through genomics and large compound libraries through combinatorial chemistry have stimulated advancements in drug discovery through the development of new high throughput screening (HTS) methods. Automation and HTS techniques are also highly desired in fields such as clinical diagnostics. Luminescence-based assays have emerged as an alternative to radiolabel-based assays in HTS as they approach the sensitivity of radioactive detection along with ease of operation, which makes them amenable to miniaturization. Luminescent proteins provide the advantage of reduced reagent and operating costs because they can be produced in unlimited amounts through the use of genetic engineering tools. In that regard, the use of two naturally occurring and recombinantly produced luminescent proteins from the jellyfish Aequorea victoria, namely, aequorin and the green fluorescent protein (GFP), has attracted attention in a number of analytical applications in diverse research areas. Aequorin is naturally bioluminescent and has therefore, virtually no associated background signal, which allows its detection down to attomole levels. GFP has become the reporter of choice in a variety of applications given that it is an autofluorescent protein that does not require addition of any co-factors for fluorescence emission. Furthermore, the generation of various mutants of GFP with differing luminescent and spectral properties has spurred additional interest in this protein. In this review, we focus on the use of aequorin and GFP in the development of highly sensitive assays that find applications in drug discovery and in high throughput analysis.     

Combined use of NMR relaxation measurements and hydrodynamic calculations to study protein association. Evidence for tetramers of low molecular weight protein tyrosine phosphatase in solution

We describe a novel method for determining weak association constants of oligomeric protein complexes formed transiently under equilibrium conditions. This type of equilibrium process is recognized as being biologically important, but generally hard to study. Heteronuclear spin relaxation rates measured at multiple protein concentrations are analyzed using relaxation rates predicted from hydrodynamic calculations, yielding equilibrium constants and structural characterization of the protein complexes. The method was used to study the oligomerization equilibrium of bovine low molecular weight protein tyrosine phosphatase. X-ray structures of monomeric and dimeric forms of the protein have been reported previously. Using longitudinal and transverse (15)N relaxation rates measured at four different protein concentrations, we detected the monomer, dimer, and a previously unknown tetramer and measured the dissociation constants of the equilibria involving these species. A comparison of experimental and predicted relaxation rates for individual backbone amide (15)N spins enabled delineation of the tetramerization interface. The results suggest a novel concept for substrate modulation of enzymatic activity based on a "supramolecular proenzyme". The fast and reversible switching of the "supramolecular proenzyme" would have obvious advantages for the regulation of enzymes involved in cell signaling pathways.