Cell-free Conditions Research Articles

Synthesis and Biological Activity Assessment of 2-Styrylbenzothiazoles as Potential Multifunctional Therapeutic Agents.

A current trend in healthcare research is to discover multifunctional compounds, able to interact with multiple biological targets, in order to simplify multi-drug therapies and improve patient compliance. The aim of this work was to outline the growing demand for innovative multifunctional compounds, achieved through the synthesis, characterisation and SAR evaluation of a series of 2-styrylbenzothiazole derivatives. The six synthesised compounds were studied for their potential as photoprotective, antioxidant, antiproliferative, and anti-inflammatory agents. In order to profile antioxidant activity against various radical species, in vitro DPPH, FRAP and ORAC assays were performed. UV-filtering activity was studied, first in solution and then in formulation (standard O/W sunscreen containing 3% synthesised molecules) before and after irradiation. Compound BZTst6 proved to be photostable, suitable for broad-spectrum criteria, and is an excellent UVA filter. In terms of antioxidant activity, only compound BZTst4 can be considered a promising candidate, due to the potential of the catechol moiety. Both also showed exceptional inhibitory action against the pro-inflammatory enzyme 5-lipoxygenase (LO), with IC50 values in the sub-micromolar range in both activated neutrophils and under cell-free conditions. The results showed that the compounds under investigation are suitable for multifunctional application purposes, underlining the importance of their chemical scaffolding in terms of different biological behaviours.

Differential Rates of Glycation Following Exposure to Unique Monosaccharides.

A complication of reducing sugars is that they can undergo Maillard chemical reactions, forming advanced glycation end-products (AGEs) that can induce oxidative stress and inflammation via engagements with the main receptor for AGEs (RAGE) in various tissues. Certain sugars, such as glucose and fructose, are well known to cause AGE formation. Recently, allulose has emerged as a rare natural sugar that is an epimer of fructose and which is of low caloric content that is minimally metabolized, leading to it being introduced as a low-calorie sugar alternative. However, the relative ability of allulose to generate AGEs compared to glucose and fructose is not known. Here we assess the accumulation of AGEs in cell-free, in vitro, and in vivo conditions in response to allulose and compare it to glycation mediated by glucose or fructose. AGEs were quantified in cell-free samples, cell culture media and lysates, and rat serum with glycation-specific ELISAs. In cell-free conditions, we observed concentration and time-dependent increases in AGEs when bovine serum albumin (BSA) was incubated with glucose or fructose and significantly less glycation when incubated with allulose. AGEs were significantly elevated when pulmonary alveolar type II-like cells were co-incubated with glucose or fructose; however, significantly less AGEs were detected when cells were exposed to allulose. AGE quantification in serum obtained from rats fed a high-fat, low-carb (HFLC) Western diet for 2 weeks revealed significantly less glycation in animals co-administered allulose compared to those exposed to stevia. These results suggest allulose is associated with less AGE formation compared to fructose or glucose, and support its safety as a low-calorie sugar alternative.

Mapping the structural landscape of the yeast Ty3 retrotransposon RNA genome.

Long terminal repeat (LTR)-retrotransposons are significant contributors to the evolution and diversity of eukaryotic genomes. Their RNA genomes (gRNA) serve as a template for protein synthesis and reverse transcription to a DNA copy, which can integrate into the host genome. Here, we used the SHAPE-MaP strategy to explore Ty3 retrotransposon gRNA structure in yeast and under cell-free conditions. Our study reveals the structural dynamics of Ty3 gRNA and the well-folded core, formed independently of the cellular environment. Based on the detailed map of Ty3 gRNA structure, we characterized the structural context of cis-acting sequences involved in reverse transcription and frameshifting. We also identified a novel functional sequence as a potential initiator for Ty3 gRNA dimerization. Our data indicate that the dimer is maintained by direct interaction between short palindromic sequences at the 5' ends of the two Ty3 gRNAs, resembling the model characteristic for other retroelements like HIV-1 and Ty1. This work points out a range of cell-dependent and -independent Ty3 gRNA structural changes that provide a solid background for studies on RNA structure-function relationships important for retroelement biology.

Abstract 5768: Proximity labeling with molecular signal sensing of oncogenic PIK3CA

Abstract PIK3CA mutations are major oncogenic drivers for oncogenesis. Mutant PIK3CA continuously activates the PI3K pathway, transmitting oncogenic signals within the transformed cells. Despite decades of efforts focused on targeting oncogenic PIK3CA for cancer therapy, the precise molecular and functional machinery remains incomplete due to the molecular complexity of the PI3K pathway. Mapping of proximity proteins in the oncogenic PI3K pathway and identifying mutant signals will signify a deep understanding of oncogenesis and a broad range of therapeutic applications. In this study, we hypothesize that PIK3CA mutant cancer cells establish a unique molecular program networking with distinct proteins, which differs from that of cancer cells with wild-type (WT) PIK3CA. To test this hypothesis, we have developed a novel bioengineering approach to decode protein-protein interactions of oncogenic PIK3CA and identify key molecular partners, potentially providing therapeutic vulnerabilities in cancer cells. First, we applied the proximity labeling technique, based on the fusion of a promiscuous protein ligase to a targeting protein of interest (POI), featuring proximity-dependent biotinylation of interacting and neighboring proteins. The promiscuous biotin ligases, BioID and BioID2, were utilized to integrate WT PIK3CA or gain-of-function hotspot mutations in at E545K or H1047R residues. To optimize the labeling protocol and biotinylation conditions, the BioID and BioID2 plasmids were fused with each POI, incorporating multiple combinations of protein tags, including myc, HA, and red fluorescence protein. Lamin A was used as the positive control, and a total of 15 plasmids were constructed in pcDNA3.1 mammalian expression vector. The sizes of the plasmids and the gene sequences were validated by PCR amplification and Sanger sequencing. Next, we employed a programmable nuclease, the prokaryotic Argonaute (pAgo), assisted by fluorescence resonance energy transfer technology, to detect oncogenic signals derived from mutant PIK3CA in cell-free conditions. To test our methods, we engineered two cancer cell lines (DHSA-1426 and COSB) derived from naturally occurring hemangiosarcoma in dogs, to induce PIK3CA H1047R mutations using CRISPR/Cas9. Our technical approach enables temporal and spatial molecular sensing of oncogenic PIK3CA and its functional interactome in living cancer cells by integrating high-resolution molecular mapping with a pAgo-based detection system. Our ongoing work involves the standardization and optimization of proximity labeling for oncogenic PIK3CA across ontogenetically distinct cancer cells. Furthermore, we will determine the sensitivity and specificity of the integrated approach to achieve proximity labeling with programmable detection of molecular signals from oncogenic PIK3CA. Citation Format: Rong Li, Donghee Lee, Md Abdullah, Jong Hyuk Kim. Proximity labeling with molecular signal sensing of oncogenic PIK3CA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5768.

Abstract 4583: Development of selective HDAC6 inhibitors to improve cancer immunotherapy

Abstract Histone Deacetylases (HDACs) are enzymes that modify histones and non-histone proteins. Our group has previously demonstrated that HDAC6 inhibitors (HDAC6is) modulate multiple immune pathways, including those that suppress the antitumoral M2 phenotype, leading to improved antitumor immunity. Despite the potential of HDAC6is, current inhibitors have poor selectivity and off-target effects and only work at micromolar concentrations. In this work, we designed a screening pipeline of HDAC6i, considering selectivity, potency, capacity to prevent the protumoral phenotype of macrophages, and low cytotoxicity on macrophages and other immune cells. This approach has not been tested before, as the standard screening method for antitumoral HDACis has focused on its cytotoxic effect on cancer cells. We initially designed in silico 980 compound derivatives from previously reported HDAC6i, such as SS208, Nexturastat A, and Suprastat. Twenty-four compounds with a high capacity to bind HDAC6 and chemical stability were screened by their ability to inhibit HDAC6 in cell-free conditions. Sixteen compounds demonstrated high potency and were further evaluated in murine macrophages by their cytotoxic effect using CellTox. The capacity to impede tubulin deacetylation was assessed by western blot and deacetylase activity by HDAC-Glo. The top six candidates were then evaluated by their selectivity compared to other HDAC members and by their effect on repressing the expression of protumoral M2 macrophages Arginase 1 and Fizz1, and activation of antitumoral markers TNFa and iNOS by western blot and qRT-PCR. The best three candidates were then evaluated at nanomolar concentrations to explore their immunomodulatory potential of M1 against M2 by performing antigen presentation (SIINFEKL presentation) and phagocytic capacity using microscopy. Two of these compounds, SM-06-09 and SM-05-947, will be further tested in syngeneic murine melanoma models for their ability in tumor regression and antitumor immunity. This study has yielded important insights into developing new and improved HDAC6is with minimal cytotoxicity, and we believe our work will help advance cancer research and ultimately lead to better patient outcomes. Citation Format: Nithya Gajendran, Manasa Suresh, David Quiceno, Xintang Li, Marie Durr, Karen Tan, Satish Kumar Reddy Noonepalle, Alejandro Villagra. Development of selective HDAC6 inhibitors to improve cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4583.

A genetic circuit on a single DNA molecule as an autonomous dissipative nanodevice

Realizing genetic circuits on single DNA molecules as self-encoded dissipative nanodevices is a major step toward miniaturization of autonomous biological systems. A circuit operating on a single DNA implies that genetically encoded proteins localize during coupled transcription-translation to DNA, but a single-molecule measurement demonstrating this has remained a challenge. Here, we use a genetically encoded fluorescent reporter system with improved temporal resolution and observe the synthesis of individual proteins tethered to a DNA molecule by transient complexes of RNA polymerase, messenger RNA, and ribosome. Against expectations in dilute cell-free conditions where equilibrium considerations favor dispersion, these nascent proteins linger long enough to regulate cascaded reactions on the same DNA. We rationally design a pulsatile genetic circuit by encoding an activator and repressor in feedback on the same DNA molecule. Driven by the local synthesis of only several proteins per hour and gene, the circuit dynamics exhibit enhanced variability between individual DNA molecules, and fluctuations with a broad power spectrum. Our results demonstrate that co-expressional localization, as a nonequilibrium process, facilitates single-DNA genetic circuits as dissipative nanodevices, with implications for nanobiotechnology applications and artificial cell design.

'Dark' CO2 fixation in succinate fermentations enabled by direct CO2 delivery via hollow fiber membrane carbonation.

Anaerobic succinate fermentations can achieve high-titer, high-yield performance while fixing CO2 through the reductive branch of the tricarboxylic acid cycle. To provide the needed CO2, conventional media is supplemented with significant (up to 60g/L) bicarbonate (HCO3-), and/or carbonate (CO32-) salts. However, producing these salts from CO2 and natural ores is thermodynamically unfavorable and, thus, energetically costly, which reduces the overall sustainability of the process. Here, a series of composite hollow fiber membranes (HFMs) were first fabricated, after which comprehensive CO2 mass transfer measurements were performed under cell-free conditions using a novel, constant-pH method. Lumen pressure and total HFM surface area were found to be linearly correlated with the flux and volumetric rate of CO2 delivery, respectively. Novel HFM bioreactors were then constructed and used to comprehensively investigate the effects of modulating the CO2 delivery rate on succinate fermentations by engineered Escherichia coli. Through appropriate tuning of the design and operating conditions, it was ultimately possible to produce up to 64.5g/L succinate at a glucose yield of 0.68g/g; performance approaching that of control fermentations with directly added HCO3-/CO32- salts and on par with prior studies. HFMs were further found to demonstrate a high potential for repeated reuse. Overall, HFM-based CO2 delivery represents a viable alternative to the addition of HCO3-/CO32- salts to succinate fermentations, and likely other 'dark' CO2-fixing fermentations.

Virtual Screening of Protein Data Bank via Docking Simulation Identified the Role of Integrins in Growth Factor Signaling, the Allosteric Activation of Integrins, and P-Selectin as a New Integrin Ligand.

Integrins were originally identified as receptors for extracellular matrix (ECM) and cell-surface molecules (e.g., VCAM-1 and ICAM-1). Later, we discovered that many soluble growth factors/cytokines bind to integrins and play a critical role in growth factor/cytokine signaling (growth factor-integrin crosstalk). We performed a virtual screening of protein data bank (PDB) using docking simulations with the integrin headpiece as a target. We showed that several growth factors (e.g., FGF1 and IGF1) induce a integrin-growth factor-cognate receptor ternary complex on the surface. Growth factor/cytokine mutants defective in integrin binding were defective in signaling functions and act as antagonists of growth factor signaling. Unexpectedly, several growth factor/cytokines activated integrins by binding to the allosteric site (site 2) in the integrin headpiece, which is distinct from the classical ligand (RGD)-binding site (site 1). Since 25-hydroxycholesterol, a major inflammatory mediator, binds to site 2, activates integrins, and induces inflammatory signaling (e.g., IL-6 and TNFα secretion), it has been proposed that site 2 is involved in inflammatory signaling. We showed that several inflammatory factors (CX3CL1, CXCL12, CCL5, sPLA2-IIA, and P-selectin) bind to site 2 and activate integrins. We propose that site 2 is involved in the pro-inflammatory action of these proteins and a potential therapeutic target. It has been well-established that platelet integrin αIIbβ3 is activated by signals from the inside of platelets induced by platelet agonists (inside-out signaling). In addition to the canonical inside-out signaling, we showed that αIIbβ3 can be allosterically activated by inflammatory cytokines/chemokines that are stored in platelet granules (e.g., CCL5, CXCL12) in the absence of inside-out signaling (e.g., soluble integrins in cell-free conditions). Thus, the allosteric activation may be involved in αIIbβ3 activation, platelet aggregation, and thrombosis. Inhibitory chemokine PF4 (CXCL4) binds to site 2 but did not activate integrins, Unexpectedly, we found that PF4/anti-PF4 complex was able to activate integrins, indicating that the anti-PF4 antibody changed the phenotype of PF4 from inhibitory to inflammatory. Since autoantibodies to PF4 are detected in vaccine-induced thrombocytopenic thrombosis (VIPP) and autoimmune diseases (e.g., SLE, and rheumatoid arthritis), we propose that this phenomenon is related to the pathogenesis of these diseases. P-selectin is known to bind exclusively to glycans (e.g., sLex) and involved in cell-cell interaction by binding to PSGL-1 (CD62P glycoprotein ligand-1). Unexpectedly, through docking simulation, we discovered that the P-selectin C-type lectin domain functions as an integrin ligand. It is interesting that no one has studied whether P-selectin binds to integrins in the last few decades. The integrin-binding site and glycan-binding site were close but distinct. Also, P-selectin lectin domain bound to site 2 and allosterically activated integrins.

Axenic Long-Term Cultivation of Pneumocystis jirovecii.

Pneumocystis jirovecii, a fungus causing severe Pneumocystis pneumonia (PCP) in humans, has long been described as non-culturable. Only isolated short-term experiments with P. jirovecii and a small number of experiments involving animal-derived Pneumocystis species have been published to date. However, P. jirovecii culture conditions may differ significantly from those of animal-derived Pneumocystis, as there are major genotypic and phenotypic differences between them. Establishing a well-performing P. jirovecii cultivation is crucial to understanding PCP and its pathophysiological processes. The aim of this study, therefore, was to develop an axenic culture for Pneumocystis jirovecii. To identify promising approaches for cultivation, a literature survey encompassing animal-derived Pneumocystis cultures was carried out. The variables identified, such as incubation time, pH value, vitamins, amino acids, and other components, were trialed and adjusted to find the optimum conditions for P. jirovecii culture. This allowed us to develop a medium that produced a 42.6-fold increase in P. jirovecii qPCR copy numbers after a 48-day culture. Growth was confirmed microscopically by the increasing number and size of actively growing Pneumocystis clusters in the final medium, DMEM-O3. P. jirovecii doubling time was 8.9 days (range 6.9 to 13.6 days). In conclusion, we successfully cultivated P. jirovecii under optimized cell-free conditions in a 70-day long-term culture for the first time. However, further optimization of the culture conditions for this slow grower is indispensable.

Effects of truncations in the N- and C-terminal domains of filensin on filament formation with phakinin in cell-free conditions and cultured cells.

Filensin and phakinin are lens fiber cell-specific proteins that constitute the beaded filaments that are critical for maintaining lens transparency. In the Shumiya cataract rat, filensin 94 kDa undergoes N- and C-terminal proteolytic processing to give a transient 50 kDa fragment and a final 38 kDa fragment, just before opacification. To characterize effects of this processing on filensin function, recombinant proteins representing the two filensin fragments, termed Fil(30-416) and Fil(30-369), respectively, were examined. Fil(30-416) lacks the N-terminal 29 amino acids and the C-terminal 248 amino acids. Fil(30-369) lacks the N-terminal 29 residues and the C-terminal 295 residues. In cell-free assembly characterized by electron microscopy, filensin and Fil(30-416) co-polymerized with phakinin and formed rugged, entangled filaments, whereas Fil(30-369) formed only aggregates. In cultured SW-13 and MCF-7 cells expressing fluorescent fusion proteins, filensin and Fil(30-416) co-polymerized with phakinin and formed cytoplasmic sinuous filaments with different widths, while Fil(30-369) gave aggregates. Therefore, while truncation of the N-terminal 29 amino acids did not affect filament formation, truncation of the C-terminal 295 but not the 248 residues resulted in failure of filament formation. These results indicate that the tail B region (residues 370-416) of rat filensin is essential for filament formation with phakinin. Truncation of the tail B region by proteolytic processing in the cataract rat lens might interfere with beaded filament formation and thereby contribute to opacification.

Anti-PD-L1 antibodies promote cellular proliferation by activating the PD-L1-AXL signal relay in liver cancer cells.

Immune checkpoint inhibitors (ICIs) are emerging treatments for advanced hepatocellular carcinoma (HCC); however, evidence has shown they may induce hyperprogressive disease via unexplained mechanisms. In this study, we investigated the possible stimulative effect of ICIs on programmed cell death-ligand 1 (PD-L1)-harboring liver cancer cells under immunocompetent cell-free conditions. The sarcomatous HAK-5 cell line displayed the highest expression of PD-L1 among 11 human liver cancer cell lines used in this study. HLF showed moderate expression, while HepG2, Hep3B, and HuH-7 did not show any. Moreover, sarcomatous HCC tissues expressed high levels of PD-L1. We observed approximately 20% increase in cell proliferation in HAK-5 cells treated with anti-PD-L1 antibodies, such as durvalumab and atezolizumab, for 48h compared with that of those treated with the control IgG and the anti-PD-1 antibody pembrolizumab. No response to durvalumab or atezolizumab was shown in PD-L1-nonexpressing cells. Loss-of-function and gain-of-function experiments for PD-L1 in HAK-5 and HepG2 cells resulted in a significant decrease and increase in cell proliferation, respectively. Phosphorylated receptor tyrosine kinase array and immunoprecipitation revealed direct interactions between PD-L1 and AXL in tumor cells. This was stabilized by extrinsic anti-PD-L1 antibodies in a glycosylated PD-L1-dependent manner. Activation of AXL, triggering signal relay to the Akt and Erk pathways, boosted tumor cell proliferation both in vitro and in xenografted tumors in NOD/SCID mice. Collectively, this suggests that anti-PD-L1 antibodies stimulate cell proliferation via stabilization of the PD-L1-AXL complex in specific types of liver cancer, including in HCC with mesenchymal components. Therapeutic anti-PD-L1 antibodies promote cell proliferation by stabilizing the PD-L1-AXL complex in PD-L1-abundant neoplasms, including in HCC with mesenchymal components. Such a mechanism may contribute to the development of hyperprogressive disease.

Cyanine- and Rhodamine-Derived Alkynes for the Selective Targeting of Cancerous Mitochondria through Radical Thiol-Yne Coupling in Live Cells.

Despite their long history and their synthetic potential underlined by various recent advances, radical thiol-yne coupling reactions have so far only rarely been exploited for the functionalization of biomolecules, and no examples yet exist for their application in live cells - although natural thiols show widespread occurrence therein. By taking advantage of the particular cellular conditions of mitochondria in cancer cells, we have demonstrated that radical thiol-yne coupling represents a powerful reaction principle for the selective targeting of these organelles. Within our studies, fluorescently labeled reactive alkyne probes were investigated, for which the fluorescent moiety was chosen to enable both mitochondria accumulation as well as highly sensitive detection. After preliminary studies under cell-free conditions, the most promising alkyne-dye conjugates were evaluated in various cellular experiments comprising analysis by flow cytometry and microscopy. All in all, these results pave the way for improved future therapeutic strategies relying on live-cell compatibility and selectivity among cellular compartments.

A color-tunable single-benzene fluorophore-based sensor for sensitive detection of palladium in solution and living cells.

Single-benzene fluorophores are bright and the smallest fluorochromes known so far. In single-benzene fluorophores, the fluorescence is mediated by the push/pull effect of substituting groups. Despite a plethora of advantageous properties, this group of molecules has not been extensively studied for design of high-performance fluorescent sensors of catalytic or enzymatic activities. Thus, herein, new fluorescent probes based on the Tsuji-Trost reaction were developed for the selective detection of palladium and other transition metals (platinum and gold) in an aqueous/organic mixed solvent with the sensitivity down to 2.5 nM (for palladium). The relative flexibility in the synthesis of these probes allows for facile color tuning of the emitted fluorescence. In this study, we have successfully utilized a yellow emission variant for sensitive detection of palladium under cell-free conditions and in living cells, validating its possible applicability for high-throughput optical sensing of catalysts for bioorthogonal chemistry under physiological conditions.

Effects of Two Soluble ACE2-Fc Variants on Blood Pressure and Albuminuria in Hypertensive Mice: Research Letter.

Angiotensin-converting enzyme 2 (ACE2) hydrolyzes angiotensin (Ang) II to Ang-(1-7), promoting vasodilatation, and inhibiting oxidative stress and inflammation. Plasma membrane ACE2 is the receptor for all known SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) viral variants. In COVID-19 infection, soluble ACE2 variants may act as decoys to bind and neutralize the coronavirus, reducing its tissue infectivity. Furthermore, soluble ACE2 variants have been proposed as potential therapeutics for kidney disease and hypertensive disorders. Soluble ACE2 variants conjugated to human Fc domains and selected for high-potency viral SARS-CoV-2 neutralization were prepared and evaluated for ACE2 activity in vitro. Lead candidates were then tested for systemic ACE2 activity, stability, and effects on blood pressure and albuminuria in mice with Ang II-induced hypertension. ACE2 activity of 10 soluble ACE2 variants was first assessed in cell-free conditions using a fluorogenic substrate, or by Ang II hydrolysis to Ang-(1-7). Hypertension was induced in male or female mice by implantation of osmotic minipumps containing Ang II. Two lead ACE2 variants were injected intravenously (i.v.) into hypertensive mice, followed by measurements of blood pressure (tail-cuff plethysmography), albuminuria, and tissue ACE2 activity and protein (immunoblots). Soluble ACE2-Fc variants demonstrated significant ACE2 enzymatic activity, with kinetics comparable with human recombinant ACE2. In hypertensive mice, single dose i.v. injection of ACE2-Fc variant K (10 mg/kg) significantly decreased systolic blood pressure at 24 hours, with partial lowering sustained to 48 hours, and tendency to reduce albuminuria at 72 hours. By contrast, ACE2-Fc variant I had no effect on blood pressure or albuminuria in hypertensive mice; ACE2-Fc variant K was detected by immunoblotting in plasma, kidney, heart, lung, liver, and spleen lysates 72 hours after injection, associated with significantly increased ACE2 activity in all tissues except kidney and spleen. Angiotensin-converting enzyme 2-Fc variant I had no effect on plasma ACE2 activity. Soluble ACE2-Fc variant K reduces blood pressure and tends to lower albuminuria in hypertensive mice. Furthermore, soluble ACE2-Fc variant K has prolonged tissue retention, associated with increased tissue ACE2 activity. The results support further studies directed at the therapeutic potential of soluble ACE2-Fc variant K for cardiovascular and kidney protection.

Targeting MYC Regulation with Polypurine Reverse Hoogsteen Oligonucleotides.

The oncogene MYC has key roles in transcription, proliferation, deregulating cellular energetics, and more. Modulating the expression or function of the MYC protein is a viable therapeutic goal in an array of cancer types, and potential inhibitors of MYC with high specificity and selectivity are of great interest. In cancer cells addicted to their aberrant MYC function, suppression can lead to apoptosis, with minimal effects on non-addicted, non-oncogenic cells, providing a wide therapeutic window for specific and efficacious anti-tumor treatment. Within the promoter of MYC lies a GC-rich, G-quadruplex (G4)-forming region, wherein G4 formation is capable of mediating transcriptional downregulation of MYC. Such GC-rich regions of DNA are prime targets for regulation with Polypurine Reverse Hoogsteen hairpins (PPRHs). The current study designed and examined PPRHs targeting the G4-forming and four other GC-rich regions of DNA within the promoter or intronic regions. Six total PPRHs were designed, examined in cell-free conditions for target engagement and in cells for transcriptional modulation, and correlating cytotoxic activity in pancreatic, prostate, neuroblastoma, colorectal, ovarian, and breast cancer cells. Two lead PPRHs, one targeting the promoter G4 and one targeting Intron 1, were identified with high potential for further development as an innovative approach to both G4 stabilization and MYC modulation.

Identification of Potential Artefacts in In Vitro Measurement of Vanadium-Induced Reactive Oxygen Species (ROS) Production.

We investigated vanadium, i.e., a redox-active heavy metal widely known for the generation of oxidative stress in cultured mammalian cells, to determine its ability to interfere with common oxidative stress-related bioassays in cell-free conditions. We first assessed the prooxidant abilities (H2O2 level, oxidation of DHR 123, and DCFH-DA dyes) and antioxidant capacity (ABTS, RP, OH, and DPPH methods) of popular mammalian cell culture media, i.e., Minimal Essential Medium (MEM), Dulbecco's Minimal Essential Medium (DMEM), Dulbecco's Minimal Essential Medium-F12 (DMEM/F12), and RPMI 1640. Out of the four media studied, DMEM has the highest prooxidant and antioxidant properties, which is associated with the highest concentration of prooxidant and antioxidant nutrients in its formulation. The studied vanadium compounds, vanadyl sulphate (VOSO4), or sodium metavanadate (NaVO3) (100, 500, and 1000 µM), either slightly increased or decreased the level of H2O2 in the studied culture media. However, these changes were in the range of a few micromoles, and they should rather not interfere with the cytotoxic effect of vanadium on cells. However, the tested vanadium compounds significantly stimulated the oxidation of DCFH-DA and DHR123 in a cell-independent manner. The type of the culture media and their pro-oxidant and antioxidant abilities did not affect the intensity of oxidation of these dyes by vanadium, whereas the vanadium compound type was important, as VOSO4 stimulated DCFH-DA and DHR oxidation much more potently than NaVO3. Such interactions of vanadium with these probes may artefactually contribute to the oxidation of these dyes by reactive oxygen species induced by vanadium in cells.

A BAK subdomain that binds mitochondrial lipids selectively and releases cytochrome C.

How BAK and BAX induce mitochondrial outer membrane (MOM) permeabilization (MOMP) during apoptosis is incompletely understood. Here we have used molecular dynamics simulations, surface plasmon resonance, and assays for membrane permeabilization in vitro and in vivo to assess the structure and function of selected BAK subdomains and their derivatives. Results of these studies demonstrate that BAK helical regions α5 and α6 bind the MOM lipid cardiolipin. While individual peptides corresponding to these helical regions lack the full biological activity of BAK, tandem peptides corresponding to α4-α5, α5-α6, or α6-α7/8 can localize exogenous proteins to mitochondria, permeabilize liposomes composed of MOM lipids, and cause MOMP in the absence of the remainder of the BAK protein. Importantly, the ability of these tandem helices to induce MOMP under cell-free conditions is diminished by mutations that disrupt the U-shaped helix-turn-helix structure of the tandem peptides or decrease their lipid binding. Likewise, BAK-induced apoptosis in intact cells is diminished by CLS1 gene interruption, which decreases mitochondrial cardiolipin content, or by BAK mutations that disrupt the U-shaped tandem peptide structure or diminish lipid binding. Collectively, these results suggest that BAK structural rearrangements during apoptosis might mobilize helices involved in specific protein-lipid interactions that are critical for MOMP.

Activation of nano-photosensitizers by Y-90 microspheres to enhance oxidative stress and cell death in hepatocellular carcinoma

While radioembolization with yttrium-90 (Y-90) microspheres is a promising treatment for hepatocellular carcinoma (HCC), lower responses in advanced and high-grade tumors present an urgent need to augment its tumoricidal efficacy. The purpose of this study was to determine whether clinically used Y-90 microspheres activate light-responsive nano-photosensitizers to enhance hepatocellular carcinoma (HCC) cell oxidative stress and cytotoxicity over Y-90 alone in vitro. Singlet oxygen and hydroxyl radical production was enhanced when Y-90 microspheres were in the presence of several nano-photosensitizers compared to either alone in cell-free conditions. Both the SNU-387 and HepG2 human HCC cells demonstrated significantly lower viability when treated with low activity Y-90 microspheres (0.1–0.2 MBq/0.2 mL) and a nano-photosensitizer consisting of both titanium dioxide (TiO2) and titanocene (TC) labelled with transferrin (TiO2-Tf-TC) compared to Y-90 microspheres alone or untreated cells. Cellular oxidative stress and cell death demonstrated a linear dependence on Y-90 at higher activities (up to 0.75 MBq/0.2 mL), but was significantly more accentuated in the presence of increasing TiO2-Tf-TC concentrations in the poorly differentiated SNU-387 HCC cell line (p < 0.0001 and p = 0.0002 respectively) but not the well-differentiated HepG2 cell line. Addition of TiO2-Tf-TC to normal human hepatocyte THLE-2 cells did not increase cellular oxidative stress or cell death in the presence of Y-90. The enhanced tumoricidal activity of nano-photosensitizers with Y-90 microspheres is a potentially promising adjunctive treatment strategy for certain patient subsets. Applications in clinically relevant in vivo HCC models are underway.

Characterizing a New Fluorescent Protein for a Low Limit of Detection Sensing in the Cell-Free System.

Cell-free protein synthesis-based biosensors have been developed as highly accurate, low-cost biosensors. However, since most biomarkers exist at low concentrations in various types of biopsies, the biosensor’s dynamic range must be increased in the system to achieve low limits of detection necessary while deciphering from higher background signals. Many attempts to increase the dynamic range have relied on amplifying the input signal from the analyte, which can lead to complications of false positives. In this study, we aimed to increase the protein synthesis capability of the cell-free protein synthesis system and the output signal of the reporter protein to achieve a lower limit of detection. We utilized a new fluorescent protein, mNeonGreen, which produces a higher output than those commonly used in cell-free biosensors. Optimizations of DNA sequence and the subsequent cell-free protein synthesis reaction conditions allowed characterizing protein expression variability by given DNA template types, reaction environment, and storage additives that cause the greatest time constraint on designing the cell-free biosensor. Finally, we characterized the fluorescence kinetics of mNeonGreen compared to the commonly used reporter protein, superfolder green fluorescent protein. We expect that this finely tuned cell-free protein synthesis platform with the new reporter protein can be used with sophisticated synthetic gene circuitry networks to increase the dynamic range of a cell-free biosensor to reach lower detection limits and reduce the false-positive proportion.

A mouse infection model and long-term lymphatic endothelium co-culture system to evaluate drugs against adult Brugia malayi.

The development of new drugs targeting adult-stage lymphatic filarial nematodes is hindered by the lack of a robust long-term in vitro culture model. Testing potential direct-acting and anti-Wolbachia therapeutic candidates against adult lymphatic filariae in vitro requires their propagation via chronic infection of gerbils. We evaluated Brugia malayi parasite burden data from male Mongolian gerbils compared with two immune-deficient mouse strains highly susceptible to B. malayi: CB.17 Severe-Combined Immmuno-Deficient (SCID) and interleukin-4 receptor alpha, interleukin-5 double knockout (IL-4Rα-/-IL-5-/-) mice. Adult worms generated in IL-4Rα-/-IL-5-/- mice were tested with different feeder cells (human embryonic kidney cells, human adult dermal lymphatic endothelial cells and human THP-1 monocyte differentiated macrophages) and comparative cell-free conditions to optimise and validate a long-term in vitro culture system. Cultured parasites were compared against those isolated from mice using motility scoring, metabolic viability assay (MTT), ex vivo microfilariae release assay and Wolbachia content by qPCR. A selected culture system was validated as a drug screen using reference anti-Wolbachia (doxycycline, ABBV-4083 / flubentylosin) or direct-acting compounds (flubendazole, suramin). BALB/c IL-4Rα-/-IL-5-/- or CB.17 SCID mice were superior to Mongolian gerbils in generating adult worms and supporting in vivo persistence for periods of up to 52 weeks. Adult females retrieved from BALB/c IL-4Rα-/-IL-5-/- mice could be cultured for up to 21 days in the presence of a lymphatic endothelial cell co-culture system with comparable motility, metabolic activity and Wolbachia titres to those maintained in vivo. Drug studies confirmed significant Wolbachia depletions or direct macrofilaricidal activities could be discerned when female B. malayi were cultured for 14 days. We therefore demonstrate a novel methodology to generate adult B. malayi in vivo and accurately evaluate drug efficacy ex vivo which may be adopted for drug screening with the dual benefit of reducing overall animal use and improving anti-filarial drug development.