Complex Pattern Formation in Solutions of Protein and Mixed Salts Using Dehydrating Sessile Droplets.

A sessile droplet of a complex fluid exhibits several stages of drying leading to the formation of a final pattern on the substrate. We report such pattern formation in dehydrating droplets of protein (BSA) and salts (MgCl2 and KCl) at various concentrations of the two components (protein and salts) as part of a parametric study for the understanding of complex patterns of dehydrating biofluid droplets (blood and urine), which will eventually be used for diagnosis of bladder cancer. The exact analysis of the biofluid patterns will require a rigorous parametric study; however, the current work provides an initial understanding of the effect of the basic components present in a biofluid droplet. Arrangement of the protein and the salts, due to evaporation, leads to the formation of some very distinctive final structures at the end of the droplet lifetime. Furthermore, these structures can be manipulated by varying the initial ratio of the two components in the solution. MgCl2 forms chains of crystals beyond a threshold initial concentration of protein (>3 wt %). However, the formation of such a crystal is also limited by the maximum concentration of the salt initially present in the droplet (≤1 wt %). On the other hand, KCl forms dendritic and rectangular crystals in the presence of BSA. The formation of these crystals also depends on the relative concentration of salt and protein in the droplet. We also investigated the dried-out patterns in dehydrating droplets of mixed salts (MgCl2 + KCl) and protein. The patterns can be tuned from a continuous dendritic structure to a snow-flake type structure just by altering the initial ratio of the two salts in the mixture, keeping all other parameters constant.

Treatment of breast cancer with autophagy inhibitory microRNAs carried by AGO2-conjugated nanoparticles.

Nanoparticle based gene delivery systems holds great promise. Superparamagnetic iron oxide nanoparticles (SPIONs) are being heavily investigated due to good biocompatibility and added diagnostic potential, rendering such nanoparticles theranostic. Yet, commonly used cationic coatings for efficient delivery of such anionic cargos, results in significant toxicity limiting translation of the technology to the clinic. Here, we describe a highly biocompatible, small and non-cationic SPION-based theranostic nanoparticles as novel gene therapy agents. We propose for the first-time, the usage of the microRNA machinery RISC complex component Argonaute 2 (AGO2) protein as a microRNA stabilizing agent and a delivery vehicle. In this study, AGO2 protein-conjugated, anti-HER2 antibody-linked and fluorophore-tagged SPION nanoparticles were developed (SP-AH nanoparticles) and used as a carrier for an autophagy inhibitory microRNA, MIR376B. These functionalized nanoparticles selectively delivered an effective amount of the microRNA into HER2-positive breast cancer cell lines in vitro and in a xenograft nude mice model of breast cancer in vivo, and successfully blocked autophagy. Furthermore, combination of the chemotherapy agent cisplatin with MIR376B-loaded SP-AH nanoparticles increased the efficacy of the anti-cancer treatment both in vitro in cells and in vivo in the nude mice. Therefore, we propose that AGO2 protein conjugated SPIONs are a new class of theranostic nanoparticles and can be efficiently used as innovative, non-cationic, non-toxic gene therapy tools for targeted therapy of cancer.

The in vitro effects of a novel estradiol analog on cell proliferation and morphology in human epithelial cervical carcinoma.

BACKGROUND: The majority of novel chemotherapeutics target the cell cycle, aiming to effect arrest and cause apoptosis. One such agent, 2-methoxyestradiol (2ME), has been shown to possess anticancer properties against numerous cancer types, both in vitro and in vivo. Despite its promise, 2ME has exhibited limitations, including low oral bioavailability and rapid hepatic enzymatic inactivation in vivo. A novel sulphamoylated estrogen analog, 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16), was in silico-designed in our laboratory to overcome these issues. It was then synthesized by a pharmaceutical company and used in an in vitro antiproliferative effect study on a human cervical carcinoma (HeLa) cell line. RESULTS: Cell proliferation data obtained from the crystal violet assay and real-time cell analysis demonstrated that 0.2 µM of ESE-16 had a significant inhibitory effect on the HeLa cells 24 h post-exposure. Immunofluorescence showed that ESE-16 is a microtubule disruptor that causes cells to undergo a mitotic block. Qualitative morphological studies using polarization-optical transmitted light differential interference contrast (PlasDIC) and light microscopy revealed a decrease in cell density and an increase in the number of cells arrested in metaphase. After ESE-16 exposure, hallmarks of apoptosis were also observed, including membrane blebbing, chromatin condensation and the presence of apoptotic bodies. Flow cytometry provided quantitative results from cell cycle progression analysis, indicating cells undergoing apoptosis and cells in the G2/M phase of the cell cycle, confirming cell cycle arrest in metaphase after ESE-16 treatment. Quantification of the ESE-16-mediated upregulation of cyclin B in HeLa cells and spectrophotometric and flow cytometric confirmation of cell death via apoptosis further confirmed the substance's impact. CONCLUSION: ESE-16 exerts its antiproliferative effects through microtubule disruption, which induces a mitotic block culminating in apoptosis. This research provided information on ESE-16 as a potential antitumor agent and on cellular targets that could aid in the design of prospective microtubule-disrupting compounds. Further in vitro and in vivo investigations of this novel compound are needed.

RACK1 Is an Interaction Partner of ATG5 and a Novel Regulator of Autophagy.

Autophagy is biological mechanism allowing recycling of long-lived proteins, abnormal protein aggregates, and damaged organelles under cellular stress conditions. Following sequestration in double- or multimembrane autophagic vesicles, the cargo is delivered to lysosomes for degradation. ATG5 is a key component of an E3-like ATG12-ATG5-ATG16 protein complex that catalyzes conjugation of the MAP1LC3 protein to lipids, thus controlling autophagic vesicle formation and expansion. Accumulating data indicate that ATG5 is a convergence point for autophagy regulation. Here, we describe the scaffold protein RACK1 (receptor activated C-kinase 1, GNB2L1) as a novel ATG5 interactor and an autophagy protein. Using several independent techniques, we showed that RACK1 interacted with ATG5. Importantly, classical autophagy inducers (starvation or mammalian target of rapamycin blockage) stimulated RACK1-ATG5 interaction. Knockdown of RACK1 or prevention of its binding to ATG5 using mutagenesis blocked autophagy activation. Therefore, the scaffold protein RACK1 is a new ATG5-interacting protein and an important and novel component of the autophagy pathways.

Lipid Droplets in Health and Disease.

Lipids are essential building blocks synthesized by complex molecular pathways and deposited as lipid droplets (LDs) in cells. LDs are evolutionary conserved organelles found in almost all organisms, from bacteria to mammals. They are composed of a hydrophobic neutral lipid core surrounding by a phospholipid monolayer membrane with various decorating proteins. Degradation of LDs provide metabolic energy for divergent cellular processes such as membrane synthesis and molecular signaling. Lipolysis and autophagy are two main catabolic pathways of LDs, which regulate lipid metabolism and, thereby, closely engaged in many pathological conditons. In this review, we first provide an overview of the current knowledge on the structural properties and the biogenesis of LDs. We further focus on the recent findings of their catabolic mechanism by lipolysis and autophagy as well as their connection ragarding the regulation and function. Moreover, we discuss the relevance of LDs and their catabolism-dependent pathophysiological conditions.

3D bioprinting of biomimetic aortic vascular constructs with self-supporting cells.

Cardiovascular diseases are the leading cause of deaths throughout the world. Vascular diseases are mostly treated with autografts and blood vessel transplantations. However, traditional grafting methods have several problems including lack of suitable harvest sites, additional surgical costs for harvesting procedure, pain, infection, lack of donors, and even no substitutes at all. Recently, tissue engineering and regenerative medicine approaches are used to regenerate damaged or diseased tissues. Most of the tissue engineering investigations have been based on the cell seeding into scaffolds by providing a suitable environment for cell attachment, proliferation, and differentiation. Because of the challenges such as difficulties in seeding cells spatially, rejection, and inflammation of biomaterials used, the recent tissue engineering studies focus on scaffold-free techniques. In this paper, the development of novel computer aided algorithms and methods are developed for 3D bioprinting of scaffold-free biomimetic macrovascular structures. Computer model mimicking a real human aorta is generated using imaging techniques and the proposed computational algorithms. An optimized three-dimensional bioprinting path planning are developed with the proposed self-supported model. Mouse embryonic fibroblast (MEF) cell aggregates and support structures (hydrogels) are 3D bioprinted layer-by-layer according to the proposed self-supported method to form an aortic tissue construct.

Artificial intelligence assisted patient blood and urine droplet pattern analysis for non-invasive and accurate diagnosis of bladder cancer.

Bladder cancer is one of the most common cancer types in the urinary system. Yet, current bladder cancer diagnosis and follow-up techniques are time-consuming, expensive, and invasive. In the clinical practice, the gold standard for diagnosis remains invasive biopsy followed by histopathological analysis. In recent years, costly diagnostic tests involving the use of bladder cancer biomarkers have been developed, however these tests have high false-positive and false-negative rates limiting their reliability. Hence, there is an urgent need for the development of cost-effective, and non-invasive novel diagnosis methods. To address this gap, here we propose a quick, cheap, and reliable diagnostic method. Our approach relies on an artificial intelligence (AI) model to analyze droplet patterns of blood and urine samples obtained from patients and comparing them to cancer-free control subjects. The AI-assisted model in this study uses a deep neural network, a ResNet network, pre-trained on ImageNet datasets. Recognition and classification of complex patterns formed by dried urine or blood droplets under different conditions resulted in cancer diagnosis with a high specificity and sensitivity. Our approach can be systematically applied across droplets, enabling comparisons to reveal shared spatial behaviors and underlying morphological patterns. Our results support the fact that AI-based models have a great potential for non-invasive and accurate diagnosis of malignancies, including bladder cancer.

Role of autophagy in cancer-associated fibroblast activation, signaling and metabolic reprograming.

Tumors not only consist of cancerous cells, but they also harbor several normal-like cell types and non-cellular components. cancer-associated fibroblasts (CAFs) are one of these cellular components that are found predominantly in the tumor stroma. Autophagy is an intracellular degradation and quality control mechanism, and recent studies provided evidence that autophagy played a critical role in CAF formation, metabolic reprograming and tumor-stroma crosstalk. Therefore, shedding light on the autophagy and its role in CAF biology might help us better understand the roles of CAFs and the TME in cancer progression and may facilitate the exploitation of more efficient cancer diagnosis and treatment. Here, we provide an overview about the involvement of autophagy in CAF-related pathways, including transdifferentiation and activation of CAFs, and further discuss the implications of targeting tumor stroma as a treatment option.

Tumor-derived CTF1 (cardiotrophin 1) is a critical mediator of stroma-assisted and autophagy-dependent breast cancer cell migration, invasion and metastasis.

Macroautophagy/autophagy is an evolutionarily conserved cellular stress response mechanism. Autophagy induction in the tumor microenvironment (stroma) has been shown to support tumor metabolism. However, cancer cell-derived secreted factors that initiate communication with surrounding cells and stimulate autophagy in the tumor microenvironment are not fully documented. We identified CTF1/CT-1 (cardiotrophin 1) as an activator of autophagy in fibroblasts and breast cancer-derived carcinoma-associated fibroblasts (CAFs). We showed that CTF1 stimulated phosphorylation and nuclear translocation of STAT3, initiating transcriptional activation of key autophagy proteins. Additionally, following CTF1 treatment, AMPK and ULK1 activation was observed. We provided evidence that autophagy was important for CTF1-dependent ACTA2/α-SMA accumulation, stress fiber formation and fibroblast activation. Moreover, promotion of breast cancer cell migration and invasion by activated fibroblasts depended on CTF1 and autophagy. Analysis of the expression levels of CTF1 in patient-derived breast cancer samples led us to establish a correlation between CTF1 expression and autophagy in the tumor stroma. In line with our in vitro data on cancer migration and invasion, higher levels of CTF1 expression in breast tumors was significantly associated with lymph node metastasis in patients. Therefore, CTF1 is an important mediator of tumor-stroma interactions, fibroblast activation and cancer metastasis, and autophagy plays a key role in all these cancer-related events.Abbreviations: ACTA2/α-SMA: actin, alpha 2, smooth muscle CAFs: cancer- or carcinoma-associated fibroblasts CNT Ab.: control antibody CNTF: ciliary neurotrophic factor CTF1: cardiotrophin 1 CTF1 Neut. Ab.: CTF1-specific neutralizing antibody GFP-LC3 MEF: GFP-fused to MAP1LC3 protein transgenic MEF LIF: leukemia inhibitory factor IL6: interleukin 6 MEFs: mouse embryonic fibroblasts MEF-WT: wild-type MEFs OSM: oncostatin M TGFB/TGFß: transforming growth factor beta.

Cleavage of Atg3 protein by caspase-8 regulates autophagy during receptor-activated cell death.

Autophagy is an evolutionarily conserved mechanism contributing to cell survival under stress conditions including nutrient and growth factor deprivation. Connections and cross-talk between cell death mechanisms and autophagy is under investigation. Here, we describe Atg3, an essential regulatory component of autophagosome biogenesis, as a new substrate of caspase-8 during receptor-mediated cell death. Both, tumor necrosis factor α and tumor necrosis factor-related apoptosis inducing ligand induced cell death was accompanied by Atg3 cleavage and this event was inhibited by a pan-caspase inhibitor (zVAD) or a caspase-8-specific inhibitor (zIETD). Indeed, caspase-8 overexpression led to Atg3 degradation and this event depended on caspase-8 enzymatic activity. Mutation of the caspase-8 cleavage site on Atg3 abolished its cleavage both in vitro and in vivo, demonstrating that Atg3 was a direct target of caspase-8. Autophagy was inactive during apoptosis and blockage of caspases or overexpression of a non-cleavable Atg3 protein reestablished autophagic activity upon death receptor stimulation. In this system, autophagy was important for cell survival since inhibition of autophagy increased cell death. Therefore, Atg3 provides a novel link between apoptosis and autophagy during receptor-activated cell death.

Autophagy-related gene, TdAtg8, in wild emmer wheat plays a role in drought and osmotic stress response.

An autophagy-related gene Atg8 was cloned for the first time from wild emmer wheat, named as TdAtg8, and its role on autophagy under abiotic stress conditions was investigated. Examination of TdAtg8 expression patterns indicated that Atg8 expression was strongly upregulated under drought stress, especially in the roots when compared to leaves. LysoTracker(®) red marker, utilized to observe autophagosomes, revealed that autophagy is constitutively active in Triticum dicoccoides. Moreover, autophagy was determined to be induced in plants exposed to osmotic stress when compared to plants grown under normal conditions. Functional studies were executed in yeast to confirm that the TdATG8 protein is functional, and showed that the TdAtg8 gene complements the atg8∆::kan MX yeast mutant strain grown under nitrogen deficiency. For further functional analysis, TdATG8 protein was expressed in yeast and analyzed using Western immunoblotting. Atg8-silenced plants were exposed to drought stress and chlorophyll and malondialdehyde (MDA) content measurements demonstrated that Atg8 plays a key role on drought stress tolerance. In addition, Atg8-silenced plants exposed to osmotic stress were found to have decreased Atg8 expression level in comparison to controls. Hence, Atg8 is a positive regulator in osmotic and drought stress response.

Glutamate Scavenging as a Neuroreparative Strategy in Ischemic Stroke.

Stroke is the second highest reason of death in the world and the leading cause of disability. The ischemic stroke makes up the majority of stroke cases that occur due to the blockage of blood vessels. Therapeutic applications for ischemic stroke include thrombolytic treatments that are in limited usage and only applicable to less than 10% of the total stroke patients, but there are promising new approaches. The main cause of ischemic neuronal death is glutamate excitotoxicity. There have been multiple studies focusing on neuroprotection via reduction of glutamate both in ischemic stroke and other neurodegenerative diseases that ultimately failed due to the obstacles in delivery. At that point, systemic glutamate grabbing, or scavenging is an ingenious way of decreasing glutamate levels upon ischemic stroke. The main advantage of this new therapeutic method is the scavengers working in the circulating blood so that there is no interference with the natural brain neurophysiology. In this review, we explain the molecular mechanisms of ischemic stroke, provide brief information about existing drugs and approaches, and present novel systemic glutamate scavenging methods. This review hopefully will elucidate the potential usage of the introduced therapeutic approaches in stroke patients.

Cancer Recurrence and Omics: Metabolic Signatures of Cancer Dormancy Revealed by Transcriptome Mapping of Genome-Scale Networks.

A major problem in medicine and oncology is cancer recurrence through the activation of dormant cancer cells. A system scale examination of metabolic dysregulations associated with the cancer dormancy offers promise for the discovery of novel molecular targets for cancer precision medicine, and importantly, for the prevention of cancer recurrence. In this study, we mapped the total mRNA sequencing-based transcriptomic data from dormant cancer cell lines and nondormant cancer controls onto a human genome-scale metabolic network by using a graph-based approach, and two mass balance-based approaches with one based on reaction activity/inactivity and the other one on flux changes. The gene expression datasets were accessed from Gene Expression Omnibus (GSE83142 and GSE114012). This analysis included two diverse cancer types, a liquid and a solid cancer, namely, acute lymphoblastic leukemia and colorectal cancer. For the dormant cancer state, we observed changes in major adenosine triphosphate-producing pathways, including the citric acid cycle, oxidative phosphorylation, and glycolysis/gluconeogenesis, indicating a reprogramming in the metabolism of dormant cells away from Warburg-based energy metabolism. All three computational approaches unanimously predicted that folate metabolism, pyruvate metabolism, and glutamate metabolism, as well as valine/leucine/isoleucine metabolism are likely dysregulated in cancer dormancy. These findings provide new insights and molecular pathway targets on cancer dormancy, comprehensively catalog dormancy-associated metabolic pathways, and inform future research aimed at prevention of cancer recurrence in particular.

Novel protein complexes containing autophagy and UPS components regulate proteasome-dependent PARK2 recruitment onto mitochondria and PARK2-PARK6 activity during mitophagy.

Autophagy is an evolutionarily conserved eukaryotic cellular mechanism through which cytosolic fragments, misfolded/aggregated proteins and organelles are degraded and recycled. Priming of mitochondria through ubiquitylation is required for the clearance the organelle by autophagy (mitophagy). Familial Parkinson's Disease-related proteins, including the E3-ligase PARK2 (PARKIN) and the serine/threonine kinase PARK6 (PINK1) control these ubiquitylation reactions and contribute to the regulation of mitophagy. Here we describe, novel protein complexes containing autophagy protein ATG5 and ubiquitin-proteasome system (UPS) components. We discovered that ATG5 interacts with PSMA7 and PARK2 upon mitochondrial stress. Results suggest that all three proteins translocate mitochondria and involve in protein complexes containing autophagy, UPS and mitophagy proteins. Interestingly, PARK2 and ATG5 recruitment onto mitochondria requires proteasome components PSMA7 and PSMB5. Strikingly, we discovered that subunit of 20 S proteasome, PSMA7, is required for the progression of PARK2-PARK6-mediated mitophagy and the proteasome activity following mitochondrial stress. Our results demonstrate direct, dynamic and functional interactions between autophagy and UPS components that contribute to the regulation of mitophagy.

A novel ATG5 interaction with Ku70 potentiates DNA repair upon genotoxic stress.

The maintenance of cellular homeostasis in living organisms requires a balance between anabolic and catabolic reactions. Macroautophagy (autophagy herein) is determined as one of the major catabolic reactions. Autophagy is an evolutionarily conserved stress response pathway that is activated by various insults including DNA damage. All sorts of damage to DNA potentially cause loss of genetic information and trigger genomic instability. Most of these lesions are repaired by the activation of DNA damage response following DNA repair mechanisms. Here we describe, a novel protein complex containing the autophagy protein ATG5 and the non-homologous end-joining repair system proteins. We discovered for the first time that ATG5 interacted with both Ku80 (XRCC5) and Ku70 (XRCC6). This novel interaction is facilitated mainly via Ku70. Our results suggest that this interaction is dynamic and enhanced upon genotoxic stresses. Strikingly, we identified that ATG5-Ku70 interaction is necessary for DNA repair and effective recovery from genotoxic stress. Therefore, our results are demonstrating a novel, direct, dynamic, and functional interaction between ATG5 and Ku70 proteins that plays a crucial role in DNA repair under genotoxic stress conditions.

Autophagy and Hepatic Tumor Microenvironment Associated Dormancy.

The goal of successful cancer treatment is targeting the eradication of cancer cells. Although surgical removal of the primary tumors and several rounds of chemo- and radiotherapy reduce the disease burden, in some cases, asymptomatic dormant cancer cells may still exist in the body. Dormant cells arise from the disseminated tumor cells (DTCs) from the primary lesion. DTCs escape from immune system and cancer therapy and reside at the secondary organ without showing no sign of proliferation. However, under some conditions. dormant cells can be re-activated and enter a proliferative state even after decades. As a stress response mechanism, autophagy may help the adaptation of DTCs at this futile foreign microenvironment and may control the survival and re-activation of dormant cells. Studies indicate that hepatic microenvironment serves a favorable condition for cancer cell dormancy. Although, no direct study was pointing out the role of autophagy in liver-assisted dormancy, involvement of autophagy in both liver microenvironment, health, and disease conditions has been indicated. Therefore, in this review article, we will summarize cancer dormancy and discuss the role and importance of autophagy and hepatic microenvironment in this context.

Autophagy and Cancer Dormancy.

Metastasis and relapse account for the great majority of cancer-related deaths. Most metastatic lesions are micro metastases that have the capacity to remain in a non-dividing state called "dormancy" for months or even years. Commonly used anticancer drugs generally target actively dividing cancer cells. Therefore, cancer cells that remain in a dormant state evade conventional therapies and contribute to cancer recurrence. Cellular and molecular mechanisms of cancer dormancy are not fully understood. Recent studies indicate that a major cellular stress response mechanism, autophagy, plays an important role in the adaptation, survival and reactivation of dormant cells. In this review article, we will summarize accumulating knowledge about cellular and molecular mechanisms of cancer dormancy, and discuss the role and importance of autophagy in this context.

Crosstalk between autophagy and DNA repair systems.

Autophagy and DNA repair are two essential biological mechanisms that maintain cellular homeostasis. Impairment of these mechanisms was associated with several pathologies such as premature aging, neurodegenerative diseases, and cancer. Intrinsic or extrinsic stress stimuli (e.g., reactive oxygen species or ionizing radiation) cause DNA damage. As a biological stress response, autophagy is activated following insults that threaten DNA integrity. Hence, in collaboration with DNA damage repair and response mechanisms, autophagy contributes to the maintenance of genomic stability and integrity. Yet, connections and interactions between these two systems are not fully understood. In this review article, current status of the associations and crosstalk between autophagy and DNA repair systems is documented and discussed.

Transcriptional landscape of cellular networks reveal interactions driving the dormancy mechanisms in cancer.

Primary cancer cells exert unique capacity to disseminate and nestle in distant organs. Once seeded in secondary sites, cancer cells may enter a dormant state, becoming resistant to current treatment approaches, and they remain silent until they reactivate and cause overt metastases. To illuminate the complex mechanisms of cancer dormancy, 10 transcriptomic datasets from the literature enabling 21 dormancy-cancer comparisons were mapped on protein-protein interaction networks and gene-regulatory networks to extract subnetworks that are enriched in significantly deregulated genes. The genes appearing in the subnetworks and significantly upregulated in dormancy with respect to proliferative state were scored and filtered across all comparisons, leading to a dormancy-interaction network for the first time in the literature, which includes 139 genes and 1974 interactions. The dormancy interaction network will contribute to the elucidation of cellular mechanisms orchestrating cancer dormancy, paving the way for improvements in the diagnosis and treatment of metastatic cancer.

Antitumor Efficacy of Ceranib-2 with Nano-Formulation of PEG and Rosin Esters.

Ceranib-2 is a recently discovered, poorly water-soluble potent ceramidase inhibitor, with the ability to suppress cancer cell proliferation and delay tumor growth. However, its poor water solubility and weak cellular bioavailability hinder its use as a therapeutic agent for cancer. PEGylated rosin esters are an excellent platform as a natural polymer for drug delivery applications, especially for controlling drug release due to their degradability, biocompatibility, capability to improve solubility, and pharmacokinetics of potent drugs. In this study, stable aqueous amphiphilic submicron-sized PEG400-rosin ester-ceranib-2 (PREC-2) particles, ranging between 100 and 350 nm in a 1:1 mixture, were successfully synthesized by solvent evaporation mediated by sonication.Conclusion: Stable aqueous PEGylated rosin ester nanocarriers might present a significant solution to improve solubility, pharmacokinetic, and bioavailability of ceranib-2, and hold promises for use as an anticancer adjacent drug after further investigations.