Ligand-receptor Mechanism Research Articles

Computation and visualization of cell\u2013cell signaling topologies in single-cell systems data using Connectome

Single-cell RNA-sequencing data has revolutionized our ability to understand of the patterns of cell–cell and ligand–receptor connectivity that influence the function of tissues and organs. However, the quantification and visualization of these patterns in a way that informs tissue biology are major computational and epistemological challenges. Here, we present Connectome, a software package for R which facilitates rapid calculation and interactive exploration of cell–cell signaling network topologies contained in single-cell RNA-sequencing data. Connectome can be used with any reference set of known ligand–receptor mechanisms. It has built-in functionality to facilitate differential and comparative connectomics, in which signaling networks are compared between tissue systems. Connectome focuses on computational and graphical tools designed to analyze and explore cell–cell connectivity patterns across disparate single-cell datasets and reveal biologic insight. We present approaches to quantify focused network topologies and discuss some of the biologic theory leading to their design.

Fibroblast growth factor receptor 1 extracellular vesicle as novel therapy for osteoarthritis

Introduction Osteoarthritis (OA) is a joint condition that causes significant impairment of the chondrocyte. The gradual degradation of the cartilage lining of one or more freely moving joints, as well as persistent inflammation, are the causes of osteoarthritis. Current medication focuses on alleviating symptoms rather than curing the condition. Methods This review article was completed by searching for information with the keywords “Fibroblast Growth Factor Receptor-1”, “Extracellular Vesicle”, and “Osteoarthritis” in various journals in several search engines. Out of 102 publications found, 95 were suitable to be studied. Results The upregulated amount of fibroblast growth factor receptors (FGFR1) signaling suggesting the progression of degenerative cartilage that commonly seen in osteoarthritis (OA) patients. Several studies showed that the involvement of extracellular vesicles (EV) derived from MSCs could enhance cartilage repair and protect the cartilage from degradation. EVs have the potential to deliver effects to specific cell types through ligand-receptor interactions and several pathway mechanisms related with the FGFR1. EVs and FGFR1 have been postulated in recent years as possible therapeutic targets in human articular cartilage. Conclusions The protective benefits on both chondrocytes and synoviocytes in OA patients can be achieved by administering the MSC-EVs that may also stimulate chondrocyte proliferation and migration. EVs have a promising potential to become a novel therapy for treating patients with OA. However, further research is needed to discover possible application of this therapy.

Thinking Quantitatively of RNA-Based Information Transfer via Extracellular Vesicles: Lessons to Learn for the Design of RNA-Loaded EVs.

Extracellular vesicles (EVs) are 50–1000 nm vesicles secreted by virtually any cell type in the body. They are expected to transfer information from one cell or tissue to another in a short- or long-distance way. RNA-based transfer of information via EVs at long distances is an interesting well-worn hypothesis which is ~15 years old. We review from a quantitative point of view the different facets of this hypothesis, ranging from natural RNA loading in EVs, EV pharmacokinetic modeling, EV targeting, endosomal escape and RNA delivery efficiency. Despite the unique intracellular delivery properties endowed by EVs, we show that the transfer of RNA naturally present in EVs might be limited in a physiological context and discuss the lessons we can learn from this example to design efficient RNA-loaded engineered EVs for biotherapies. We also discuss other potential EV mediated information transfer mechanisms, among which are ligand–receptor mechanisms.

336-OR: Mandarin Fish Ranavirus Viral Insulin/IGF-Like Peptide Inhibits Human IGF-1 Receptor

Our recent studies published in PNAS and Molecular Metabolism showed for the very first time that viruses possess genes encoding viral insulin/IGF-1 like peptides (VILPs). These two studies provided several important insights on VILPs: 1. Identified VILPs in six different Iridoviridae viruses 2. Showed that chemically synthesized single chain (sc, IGF-1 like) and double chain (dc, insulin-like) VILPs can bind to human insulin and IGF-1 receptors and stimulate downstream signaling. 3. Demonstrated that VILPs can increase glucose uptake in vitro and in vivo. 4. Identified the unique tissue-specific effects of VILPs. 5. Found sequences of these viruses in the human gut and plasma virome. In the present study, we focused on a novel, Mandarin Fish Ranavirus (MFRV) VILP and characterized both sc and dc forms for the first time. MFRV scVILP binds to human IGF1R with an affinity 100x higher than insulin, making it a VILP with the highest affinity to this receptor so far discovered. MFRV scVILP also activates IGF1R phosphorylation and post-receptor signaling. Interestingly, although MFRV scVILP is a weak agonist of IGF1R, it inhibits IGF-1 signaling at higher concentrations. When we treat cells with both IGF-1 and MFRV VILP, MFRV scVILP inhibits IGF-1 (10nM) stimulated IGF1R phosphorylation and activation of PI3K and MAPK pathways at high concentrations (> 250 nM). Surprisingly, the dc version of MFRV VILP neither bind nor stimulate IGF1R. This suggests that the C-chain of MFRV VILP is essential for both binding to IGF1R and the IGF-1 inhibitory effects. Taken together, our results revealed that MFRV scVILP is a novel antagonist/inhibitor of IGF-1R. IGF1R signaling is overly activated in breast, prostate and colorectal cancers. Despite tremendous efforts to develop an effective inhibitor of IGF1R, these attempts failed. MFRV VILP has the potential to contribute development of new therapeutics in cancer therapy and to help us better understand ligand-receptor interactions and mechanisms of IGF1R inhibition. Disclosure M. Chrudinova: None. V. Gelfanov: Employee; Self; Novo Nordisk, Employee; Spouse/Partner; Eli Lilly and Company. L. Zakova: None. J. Cutone: None. A. Powis: None. K. Sevgi: None. J. Jiracek: None. R. Dimarchi: Employee; Self; Novo Nordisk. E. Altindis: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (K01DK117967)

Molecular Mechanisms of Druze Formation in the Retina in Age-Related Macular Degeneration

Age-related macular degeneration is the main cause of vision loss in aged people. A significant risk factor of disease is considered dysfunction of retinal pigment epithelium, which is accompanied by accumulation of lipids in Bruch membrane as drusen or linear deposits. As a result, there are disturbance of substances and oxygen transport from blood to retina cells of eye as well as elimination of toxic catabolites to choroid capillaries. The lack of a systematic analysis of pathogenetic mechanisms of drusen’s formation in age-related macular degeneration controls possibility of developing methods for treating and preventing development of early stage of age-related macular degeneration. There are the data of risk factors and pathogenesis of age-related macular degeneration in the article. It pays attention to the significance of oxidative stress in pigment epithelium of macula and triggering factors of inflammation development in eye retina. In context of differences in lipid composition and topographic location of cones and rods photoreceptor cells the localization of drusen, basal laminar and linear deposits in Bruch membrane is discussed. The possible mechanisms of drusen biogenesis associated with functioning of cholesterol carriers and lipid autophagy in retinal pigment epithelial cells as well as the role of macrophages in transport of local cholesterol depot to systemic circulation are presented. Analysis of literature allowed to identify several promising areas of development of targeted therapy for lipid metabolism disorders in retina. They include the reactivation of lipid carriers in RPE cells, ligand-receptor mechanisms that provide a reduction pH in lysosomes and maintaining functional activity of macrophages.

Up-regulation of voltage-gated sodium channels by peptides mimicking S4-S5 linkers reveals a variation of the ligand-receptor mechanism

Prokaryotic NaV channels are tetramers and eukaryotic NaV channels consist of a single subunit containing four domains. Each monomer/domain contains six transmembrane segments (S1-S6), S1-S4 being the voltage-sensor domain and S5-S6 the pore domain. A crystal structure of NaVMs, a prokaryotic NaV channel, suggests that the S4-S5 linker (S4-S5L) interacts with the C-terminus of S6 (S6T) to stabilize the gate in the open state. However, in several voltage-gated potassium channels, using specific S4-S5L-mimicking peptides, we previously demonstrated that S4-S5L/S6T interaction stabilizes the gate in the closed state. Here, we used the same strategy on another prokaryotic NaV channel, NaVSp1, to test whether equivalent peptides stabilize the channel in the open or closed state. A NaVSp1-specific S4-S5L peptide, containing the residues supposed to interact with S6T according to the NaVMs structure, induced both an increase in NaVSp1 current density and a negative shift in the activation curve, consistent with S4-S5L stabilizing the open state. Using this approach on a human NaV channel, hNaV1.4, and testing 12 hNaV1.4 S4-S5L peptides, we identified four activating S4-S5L peptides. These results suggest that, in eukaryotic NaV channels, the S4-S5L of DI, DII and DIII domains allosterically modulate the activation gate and stabilize its open state.

Computational Modeling of Trans-Zeatin as a Novel Target of Adenosine A2A Receptor: Insights into Molecular Interactions

Background: Adenosine A2A receptor (A2AR) is a G-protein-coupled receptor that is involved in various physiological functions. Zeatin, a plant cytokinin and a derivative of adenine, is recently identified as new ligand of A2AR. However, the ligand-receptor interaction mechanism is not fully revealed. Aims and Objectives: The main aim of this study is to reveal a model structure of A2AR in complex with zeatin for the first time to provide a better understanding of this novel interaction mechanism. Materials and Methods: The model structure of A2AR in complex with zeatin was created by docking and the structural dynamics of the complex were detected by molecular dynamic simulations during the study. A model structure of A2AR in complex with caffeine was used as a positive control. Result: Zeatin displayed the ability to stay more stable at the binding pocket compared with caffeine based on molecular dynamic simulations and the residues involved in the interaction are identified, leading a new sight for further studies on zeatin and A2AR interaction. Conclusion: We propose that zeatin is indeed a novel and promising target for A2AR.

Abstract 4332: Interactome between vascular endothelial cells and Proneural glioma stem cells protects seeds for GBM recurrence from radiation therapy

Abstract Glioblastoma (GBM) is the most aggressive form of intracranial tumors. Despite multimodal treatment strategies that include surgical resection, radiation and chemotherapy, most patients survive for less than two years. GBM cells tend to invade into adjacent normal brain tissues and therefore, cannot be completely resected by surgery. These remaining tumor cells are the “seeds” to escape post-surgical therapies, thereby giving rise to recurrent tumors during or after treatment. The exact mechanisms by which these seeds contain pre-existing resistant cells, and/or gain resistance to therapy remain elusive. Recent findings demonstrated that communication between normal and GBM cells play physiological roles in tumor initiation and progression. Here, we aim to determine how vascular endothelial (VE) cells affect GBM recurrence in response to radiotherapy. To this end, we first assessed the effect of secreted factors from VE cells on patient-derived GBM neurospheres in vitro by using their conditioned medium (CM). GBM neurospheres promoted their growth with CM from VE cells. qPCR and FACS exhibited that the CM treatment preferentially protected CD133+ population in GBM neurospheres that otherwise were depleted by irradiation treatment. Furthermore, RNA sequencing of VE cells and A2B5-expressing GBM stem-like cells directly isolated from GBM and epilepsy patients demonstrated that only tumor-derived VE cells and GBM stem-like cells express a high level of a secreted factor Endocan (ESM1) and its receptor CD11a, respectively. Preferential expression of CD11a in perivascular GBM cells was also confirmed by immunocytochemistry. Further, we found that recombinant Endocan is able to promote GBM neurosphere growth in vitro after irradiation and protected them from irradiation-induced aggressive mesenchymal transition of these cells. Taken together, these results demonstrate a potential role of a crosstalk between peri-vascular GBM stem-like cells and their adjacent VE cells, via the ligand-receptor interaction mechanism. This interactome network may help Proneural GBM stem cells to escape from radiation therapy, thereby reinitiating the tumor as recurrence via an Endocan-CD11a signaling. Hence, targeting Endocan in endothelial cells or CD11a in GBM cells may have potential as a novel efficacious strategy for preventing recurrence. Citation Format: Soniya Bastola, Marat S. Pavlyukov, Shinobu Yamaguchi, Jun Wang, Svetlana Komarova, Harley I. kornblum, Ichiro Nakano. Interactome between vascular endothelial cells and Proneural glioma stem cells protects seeds for GBM recurrence from radiation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4332. doi:10.1158/1538-7445.AM2017-4332

Wnt/TLR Dialog in Neuroinflammation, Relevance in Alzheimer's Disease.

The innate immune system (IIS) represents the first line of defense against exogenous and endogenous harmful stimuli. Different types of pathogens and diverse molecules can activate the IIS via a ligand–receptor mechanism. Cytokine release, recruitment of immunocompetent cells, and inflammation constitute the initial steps in an IIS-mediated response. While balanced IIS activity can resolve a harmful event, an altered response, such as deficient or persistent IIS activity, will have a critical effect on organism homeostasis. In this regard, chronic IIS activation has been associated with a wide range of diseases, including chronic inflammatory disorders (inflammatory bowel disease, arthritis, chronic obstructive pulmonary disease, among others), cancer and, more recently, neurodegenerative disorders. The relevance of the immune response, particularly inflammation, in the context of neurodegeneration has motivated rigorous research focused on unveiling the mechanisms underlying this response. Knowledge regarding the molecular hallmarks of the innate immune response and understanding signaling pathway cross talk are critical for developing new therapeutic strategies aimed at modulating the neuroinflammatory response within the brain. In the present review, we discuss the IIS in the central nervous system, particularly the cross talk between the toll-like receptor-signaling cascade and the wingless-related MMTV integration site (Wnt) signaling pathway and its relevance in neurodegenerative disorders such as Alzheimer’s disease.

Abstract 1199: A network biology screen reveals ligand-receptor pathway connections and resistance mechanisms to RTK-directed therapies in cancer cells

Abstract Purpose: Although there are many well-defined signaling pathways in tumor cells that provide druggable targets, emergence of growth factor-mediated resistance and parallel pathway compensation frequently occurs, limiting the effectiveness of these treatment strategies. The purpose of this study was to model cancer cell responses to combinations of growth factors (ligands) and targeted investigational therapies in order to identify which signaling pathways are mechanistically related. The results presented here demonstrate the use of network biology-based phenotypic screening and modeling to reveal unexpected behaviors, identify positive and negative biomarkers, and guide novel treatment strategies. Methods: To assess the effect of ligands and RTK-directed antibodies in combination with targeted investigational therapies in cancer cells, we conducted a high throughput viability screen in 3D cultures. A library of 87 small molecules targeting different components of signaling and metabolic pathways was used in combination with growth factors and therapeutic antibodies. The therapeutic antibodies included in the screen were MM-121 (anti-ErbB3), MM-131 (anti-Met/EpCAM), MM-141 (anti-IGF-1R/ErbB3), and MM-151 (anti-EGFR). Using these data, we inferred a pathway connectivity model to identify pathway crosstalk and novel and effective combination strategies, which were then further evaluated in vitro and in mouse xenograft models. Results: Overall, we found that ligand-mediated resistance varies depending on the cancer type. For example, HGF and EGF play particularly strong roles in gastric and colorectal cancer cells, respectively. Interestingly, a few cases were found in which a ligand reduced cell viability compared to control, but this had no effect on drug response. In most cases where a ligand rendered cells insensitive to a certain drug treatment, a combination with the appropriately matched therapeutic antibody re-sensitized cells to the drug, suggesting that a combination strategy could potentially be used to overcome ligand-mediated resistance. Among the findings that were observed in multiple cell lines, MM-131 combined well with EGFR-directed agents in HGF responsive tumor cells, and MM-141 combined well with metformin in colorectal and lung cancer cell lines. These findings were further validated in vivo. Conclusions: Growth factors for EGFR, Met, ErbB3, and IGF-1R decrease sensitivity to a wide range of targeted investigational agents in cancer cell lines. These effects, however, are not general, but instead are dependent on the type of cancer cell being treated. Insights from this screen may help guide the future clinical development of MM-121, MM-131, MM-141 and MM-151. Citation Format: Kristina Masson, Andreas Raue, Gavin MacBeath. A network biology screen reveals ligand-receptor pathway connections and resistance mechanisms to RTK-directed therapies in cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1199.

Low Doses of Traditional Nanophytomedicines for Clinical Treatment: Manufacturing Processes and Nonlinear Response Patterns.

The purpose of the present paper is to (a) summarize evidence for the nanoparticle nature and biological effects of traditional homeopathically-prepared medicines at low and ultralow doses; (b) provide details of historically-based homeopathic green manufacturing materials and methods, relating them to top-down mechanical attrition and plant-based biosynthetic processes in modern nanotechnology; (c) outline the potential roles of nonlinear dose-responses and dynamical interactions with complex adaptive systems in generating endogenous amplification processes during low dose treatment. Possible mechanisms of low dose effects, for which there is evidence involving nanoparticles and/or homeopathically-manufactured medicines, include hormesis, time-dependent sensitization, and stochastic resonance. All of the proposed mechanisms depend upon endogenous nonlinear amplification processes in the recipient organism in interaction with the salient, albeit weak signal properties of the medicine. Conventional ligand-receptor mechanisms relevant to higher doses are less likely involved. Effects, especially for homeopathically-prepared nanophytomedicines, include bidirectional host state-dependent changes in function. Homeopathic clinicians report successful treatment of serious infections and cancers. Preclinical biological evidence is consistent with such claims. Controlled biological data on homeopathically-prepared medicines indicate modulation of gene expression and biological signaling pathways regulating cell cycles, immune reactions, and central nervous system function from studies on cells, animals, and human subjects. As a 200-year old system of traditional medicine used by millions of people worldwide, homeopathy offers a pulsed low dose treatment strategy and strong safety record to facilitate progress in translational nanomedicine with plants and other natural products. In turn, modern nanotechnology methods can improve homeopathic manufacturing procedures, characterize nanoparticle end-products, and describe interactions of homeopathic nanophytomedicines with living systems at the nanoparticle and even individual organism level of detection. Faster progress toward safe and effective personalized nanophytomedicine treatments can result.

The chemokine (CCL2-CCR2) signaling axis mediates perineural invasion.

Perineural invasion is a form of cancer progression where cancer cells invade along nerves. This behavior is associated with poor clinical outcomes; therefore, it is critical to identify novel ligand-receptor interactions between nerves and cancer cells that support the process of perineural invasion. A proteomic profiler chemokine array was used to screen for nerve-derived factors secreted from tissue explants of dorsal root ganglion (DRG), and CCL2 was identified as a lead candidate. Prostate cancer cell line expression of CCR2, the receptor to CCL2, correlated closely with MAPK and Akt pathway activity and cell migration towards CCL2 and DRG. In vitro nerve and cancer coculture invasion assays of perineural invasion demonstrated that cancer cell CCR2 expression facilitates perineural invasion. Perineural invasion is significantly diminished in coculture assays when using DRG harvested from CCL2(-/-) knockout mice as compared with control CCL2(+/+) mice, indicating that CCR2 is required for perineural invasion in this murine model of perineural invasion. Furthermore, 20 of 21 (95%) patient specimens of prostate adenocarcinoma with perineural invasion exhibited CCR2 expression by immunohistochemistry, while just 3 of 13 (23%) lacking perineural invasion expressed CCR2. In summary, nerve-released CCL2 supports prostate cancer migration and perineural invasion though CCR2-mediated signaling. These results reveal CCL2-CCR2 signaling as a key ligand-receptor mechanism that mediates cancer cell communication with nerves during perineural invasion and highlight a potential future therapeutic target.

Amyotrophic lateral sclerosis as a spatiotemporal mislocalization disease: location, location, location.

Spatiotemporal localization of signals is a fundamental feature impacting cell survival and proper function. The cell needs to respond in an accurate manner in both space and time to both intra- and intercellular environment cues. The regulation of this comprehensive process involves the cytoskeleton and the trafficking machinery, as well as local protein synthesis and ligand-receptor mechanisms. Alterations in such mechanisms can lead to cell dysfunction and disease. Motor neurons that can extend over tens of centimeters are a classic example for the importance of such events. Changes in spatiotemporal localization mechanisms are thought to play a role in motor neuron degeneration that occurs in amyotrophic lateral sclerosis (ALS). In this review we will discuss these mechanisms and argue that possible misregulated factors can lead to motor neuron degeneration in ALS.

Direct Proteolytic Cleavage of NLRP1B Is Necessary and Sufficient for Inflammasome Activation by Anthrax Lethal Factor

Inflammasomes are multimeric protein complexes that respond to infection by recruitment and activation of the Caspase-1 (CASP1) protease. Activated CASP1 initiates immune defense by processing inflammatory cytokines and by causing a rapid and lytic cell death called pyroptosis. Inflammasome formation is orchestrated by members of the nucleotide-binding domain and leucine-rich repeat (NLR) or AIM2-like receptor (ALR) protein families. Certain NLRs and ALRs have been shown to function as direct receptors for specific microbial ligands, such as flagellin or DNA, but the molecular mechanism responsible for activation of most NLRs is still poorly understood. Here we determine the mechanism of activation of the NLRP1B inflammasome in mice. NLRP1B, and its ortholog in rats, is activated by the lethal factor (LF) protease that is a key virulence factor secreted by Bacillus anthracis, the causative agent of anthrax. LF was recently shown to cleave mouse and rat NLRP1 directly. However, it is unclear if cleavage is sufficient for NLRP1 activation. Indeed, other LF-induced cellular events have been suggested to play a role in NLRP1B activation. Surprisingly, we show that direct cleavage of NLRP1B is sufficient to induce inflammasome activation in the absence of LF. Our results therefore rule out the need for other LF-dependent cellular effects in activation of NLRP1B. We therefore propose that NLRP1 functions primarily as a sensor of protease activity and thus could conceivably detect a broader spectrum of pathogens than just B. anthracis. By adding proteolytic cleavage to the previously established ligand-receptor mechanism of NLR activation, our results illustrate the remarkable flexibility with which the NLR architecture can be deployed for the purpose of pathogen-detection and host defense.

T cell regulation mediated by interaction of soluble CD52 with the inhibitory receptor Siglec-10

Functionally diverse T cell populations interact to maintain homeostasis of the immune system. We found that human and mouse antigen-activated T cells with high expression of the lymphocyte surface marker CD52 suppressed other T cells. CD52(hi)CD4(+) T cells were distinct from CD4(+)CD25(+)Foxp3(+) regulatory T cells. Their suppression was mediated by soluble CD52 released by phospholipase C. Soluble CD52 bound to the inhibitory receptor Siglec-10 and impaired phosphorylation of the T cell receptor-associated kinases Lck and Zap70 and T cell activation. Humans with type 1 diabetes had a lower frequency and diminished function of CD52(hi)CD4(+) T cells responsive to the autoantigen GAD65. In diabetes-prone mice of the nonobese diabetic (NOD) strain, transfer of lymphocyte populations depleted of CD52(hi) cells resulted in a substantially accelerated onset of diabetes. Our studies identify a ligand-receptor mechanism of T cell regulation that may protect humans and mice from autoimmune disease.

Regulation of plant gravity sensing and signaling by the actin cytoskeleton

Gravitropism is a process by which plant organs readjust their growth toward or away from the gravity vector when the plant is reoriented. The actin cytoskeleton has often been a significant component of models explaining gravitropism, but its role in this process has become somewhat controversial in light of reports showing that actin inhibitors enhance the gravitropic response. The work with inhibitors implies that actin might function as a negative regulator of gravitropism. In this article, possibilities for how such a role might be accomplished are presented. First, the organization of actin in statocytes is revisited in an attempt to rationalize how compressive forces exerted by statoliths on membranes can lead to enhanced gravity sensing. Second, recent genetic work in the model plant Arabidopsis thaliana is discussed, focusing on the potential involvement of the protein degradation machinery in actin-mediated control of statolith dynamics and on the intriguing possibility that an actin-regulated, ligand-receptor mechanism for gravity signal transduction might operate in higher plants. Third, modifications in the trafficking of auxin efflux transporters are considered as possible mechanisms for the enhanced gravity responses observed in plant organs when the actin cytoskeleton is disrupted by chemical inhibitors. The various possibilities presented in this review emphasize the large amount of research that remains to be done before we can fully understand how the actin cytoskeleton modulates tropisms in higher plants.

Combination Treatment of Hypothermia and Mesenchymal Stromal Cells Amplifies Neuroprotection in Primary Rat Neurons Exposed to Hypoxic-Ischemic-Like Injury In Vitro: Role of the Opioid System

This study was designed to reveal the therapeutic regimen and mechanism of action underlying hypothermia treatment in combination with stem cell transplantation for ameliorating neonatal hypoxic-ischemic-like injury. Primary rat neurons were exposed to oxygen-glucose deprivation (OGD), which produced hypoxic-ischemic-like injury in vitro, then incubated at 25°C (severe hypothermia), 34°C (moderate hypothermia), and 37°C (normothermia) with or without subsequent co-culture with mesenchymal stromal cells (MSCs). Combination treatment of moderate hypothermia and MSCs significantly improved cell survival and mitochondrial activity after OGD exposure. The exposure of delta opioid human embryonic kidney cells (HEK293) to moderate hypothermia attenuated OGD-mediated cell alterations, which were much more pronounced in HEK293 cells overexpressing the delta opioid receptor. Further, the addition of delta opioid peptide to 34°C hypothermia and stem cell treatment in primary rat neurons showed synergistic neuroprotective effects against OGD which were significantly more robust than the dual combination of moderate hypothermia and MSCs, and were significantly reduced, but not completely abolished, by the opioid receptor antagonist naltrexone altogether implicating a ligand-receptor mechanism of neuroprotection. Further investigations into non-opioid therapeutic signaling pathways revealed growth factor mediation and anti-apoptotic function accompanying the observed therapeutic benefits. These results support combination therapy of hypothermia and stem cells for hypoxic-ischemic-like injury in vitro, which may have a direct impact on current clinical trials using stand-alone hypothermia or stem cells for treating neonatal encephalopathy.

Alert-QSAR. Implications for electrophilic theory of chemical carcinogenesis.

Given the modeling and predictive abilities of quantitative structure activity relationships (QSARs) for genotoxic carcinogens or mutagens that directly affect DNA, the present research investigates structural alert (SA) intermediate-predicted correlations ASA of electrophilic molecular structures with observed carcinogenic potencies in rats (observed activity, A = Log[1/TD50], i.e., ). The present method includes calculation of the recently developed residual correlation of the structural alert models, i.e., . We propose a specific electrophilic ligand-receptor mechanism that combines electronegativity with chemical hardness-associated frontier principles, equality of ligand-reagent electronegativities and ligand maximum chemical hardness for highly diverse toxic molecules against specific receptors in rats. The observed carcinogenic activity is influenced by the induced SA-mutagenic intermediate effect, alongside Hansch indices such as hydrophobicity (LogP), polarizability (POL) and total energy (Etot), which account for molecular membrane diffusion, ionic deformation, and stericity, respectively. A possible QSAR mechanistic interpretation of mutagenicity as the first step in genotoxic carcinogenesis development is discussed using the structural alert chemoinformation and in full accordance with the Organization for Economic Co-operation and Development QSAR guidance principles.

Effect of Functionalization of Colloidal Gold on Controlled Flocculation by the Ligand-Receptor Mechanism

Modification of colloidal gold particles with octadecanthiol and alkyl biotin, with the subsequent flocculation of these particles via introduction of a tetramer protein avidin, was studied.

Cryopreservation and thawing is associated with varying extent of activation of apoptotic machinery in subsets of ejaculated human spermatozoa.

We investigated the impact of cryopreservation and thawing on levels of caspases-3, -8, and -9 activity, intact mitochondrial membrane potential (Deltapsim), and DNA fragmentation in human spermatozoa. Eleven pools of cryopreserved and eight pools of fresh semen samples were examined. Mature and immature fractions were separated on a two-layer density gradient (47% and 90%) and further subdivided based on the externalization of phosphatidylserine and its binding to annexin V-labeled superparamagnetic microbeads (ANMB). Levels of activated caspases were assessed using fluorescein-labeled inhibitors of caspases (FLICA), Deltapsim using a lipophilic cationic dye, and DNA fragmentation by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Cryopreservation was significantly associated with activation of caspases-3, -8, and -9, as well as disruption of the mitochondrial membrane potential but no significant changes were observed in DNA fragmentation. In mature sperm, caspase activation was only detected in the ANMB+ fraction, whereas in immature sperm, both ANMB+ and ANMB- fractions showed activated caspase levels. In ANMB+ immature sperm, apoptosis seemed to be triggered by a surface ligand-receptor mechanism as well as by disruption of mitochondria, whereas in ANMB- immature sperm, apoptosis was induced by activation of caspase-9 following loss of intact Deltapsim. These results demonstrate that selection of annexin V-negative mature spermatozoa might be of clinical relevance for fertility preservation, as this sperm fraction shows no activated apoptosis during the cryopreservation process.