Identification Drug Targets for Oxaliplatin-Induced Cardiotoxicity without Affecting Cancer Treatment through Inter Variability Cross-Correlation Analysis (IVCCA).

The successful treatment of side effects of chemotherapy faces two major limitations: the need to avoid interfering with pathways essential for the cancer-destroying effects of the chemotherapy drug, and the need to avoid helping tumor progression through cancer promoting cellular pathways. To address these questions and identify new pathways and targets that satisfy these limitations, we have developed the bioinformatics tool Inter Variability Cross-Correlation Analysis (IVCCA). This tool calculates the cross-correlation of differentially expressed genes, analyzes their clusters, and compares them across a vast number of known pathways to identify the most relevant target(s). To demonstrate the utility of IVCCA, we applied this platform to RNA-seq data obtained from the hearts of the animal models with oxaliplatin-induced CTX. RNA-seq of the heart tissue from oxaliplatin treated mice identified 1744 differentially expressed genes with False Discovery Rate (FDR) less than 0.05 and fold change above 1.5 across nine samples. We compared the results against traditional gene enrichment analysis methods, revealing that IVCCA identified additional pathways potentially involved in CTX beyond those detected by conventional approaches. The newly identified pathways such as energy metabolism and several others represent promising target for therapeutic intervention against CTX, while preserving the efficacy of the chemotherapy treatment and avoiding tumor proliferation. Targeting these pathways is expected to mitigate the damaging effects of chemotherapy on cardiac tissues and improve patient outcomes by reducing the incidence of heart failure and other cardiovascular complications, ultimately enabling patients to complete their full course of chemotherapy with improved quality of life and survival rates.

DRG Explant Model: Elucidating Mechanisms of Oxaliplatin-Induced Peripheral Neuropathy and Identifying Potential Therapeutic Targets.

Oxaliplatin triggered chemotherapy induced peripheral neuropathy (CIPN) is a common and debilitating side effect of cancer treatment which limits the efficacy of chemotherapy and negatively impacts patients quality of life dramatically. For better understanding the mechanisms of CIPN and screen for potential therapeutic targets, it is critical to have reliable in vitro assays that effectively mirror the neuropathy in vivo . In this study, we established a dorsal root ganglia (DRG) explant model. This model displayed dose-dependent inhibition of neurite outgrowth in response to oxaliplatin, while oxalic acid exhibited no significant impact on the regrowth of DRG. The robustness of this assay was further demonstrated by the inhibition of OCT2 transporter, which facilitates oxaliplatin accumulation in neurons, fully restoring the neurite regrowth capacity. Using this model, we revealed that oxaliplatin triggered a substantial increase of oxidative stress in DRG. Notably, inhibition of TXNIP with verapamil significantly reduced oxidative stress level. Our results demonstrated the use of DRG explants as an efficient model to study the mechanisms of CIPN and screen for potential treatments.

Oxaliplatin-induced cardiotoxicity in mice is connected to the changes in energy metabolism in the heart tissue.

Oxaliplatin is a platinum-based alkylating chemotherapeutic agent used for cancer treatment. At high cumulative dosage, the negative effect of oxaliplatin on the heart becomes evident and is linked to a growing number of clinical reports. The aim of this study was to determine how chronic oxaliplatin treatment causes the changes in energy-related metabolic activity in the heart that leads to cardiotoxicity and heart damage in mice. C57BL/6 male mice were treated with a human equivalent dosage of intraperitoneal oxaliplatin (0 and 10 mg/kg) once a week for eight weeks. During the treatment, mice were followed for physiological parameters, ECG, histology and RNA sequencing of the heart. We identified that oxaliplatin induces strong changes in the heart and affects the heart's energy-related metabolic profile. Histological post-mortem evaluation identified focal myocardial necrosis infiltrated with a small number of associated neutrophils. Accumulated doses of oxaliplatin led to significant changes in gene expression related to energy related metabolic pathways including fatty acid (FA) oxidation, amino acid metabolism, glycolysis, electron transport chain, and NAD synthesis pathway. At high accumulative doses of oxaliplatin, the heart shifts its metabolism from FAs to glycolysis and increases lactate production. It also leads to strong overexpression of genes in NAD synthesis pathways such as Nmrk2. Changes in gene expression associated with energy metabolic pathways can be used to develop diagnostic methods to detect oxaliplatin-induced cardiotoxicity early on as well as therapy to compensate for the energy deficit in the heart to prevent heart damage.

3D Media Stabilizes Membrane and Prolongs Lifespan of Defolliculated Xenopus laevis Oocytes.

Xenopus laevis oocytes are commonly used in many fundamental biological studies. One of the major limitations of X. laevis oocytes is their short storage lifespan with most defolliculated oocytes physically deteriorating in 10 days or less. Herein, we identified a 3D Cultrex-based storage media that incorporates extracellular membrane-based hydrogels to maintain oocyte integrity. Under these treatments, the lifespan of the oocytes increased to more than 20 days compared to standard conditions. The treatment preserved the oocytes membrane integrity and did not interfere with mRNA- or cDNA-derived protein expression.

New in vitro highly cytotoxic platinum and palladium cyanoximates with minimal side effects in vivo.

Several biologically active bivalent Pd and Pt complexes with two structurally similar cyanoxime ligands abbreviated as H(DECO): 2-oximino-2-cyano-N,N'-diethylacetamide, and H(PyrCO): 2-oximino-2-cyan-N-pyrrolidine acetamide were synthesized and characterized using spectroscopic methods, thermal analysis and X-ray crystallography. Structures revealed planar cis-geometry of studied complexes. Freshly obtained Pt(DECO)2, Pd(DECO)2, Pt(PyrCO)2 and Pd(PyrCO)2 complexes were used in for in vitro cytotoxicity assays using two different etiology human cancer cell lines HeLa and WiDr cells. Investigated compounds showed cytotoxicity levels at or above cisplatin. Pt(DECO)2 was also tested in vivo in healthy C57BL/6 mice. The complex was administered at three different dosage (0, 7.5, 15 mg/kg, i.p. once/week), over a total period of 8 weeks. No changes were observed in the animal weight in the treated mice compared to the control dextrose-treated group. The levels of erythrocytes, leukocytes, and hemoglobin were within the normal level suggesting low myelotoxicity. Negligible cardiotoxicity was observed from the histological evaluation of the hearts from the treated animals. Results from the tail nerve conduction velocity (NCV) and nerve histomorphometry suggested no impact of Pt(DECO)2 on peripheral nerves. The complex, however, induced certain hepatotoxicity and lead to the elevation of IL-6, a pro-inflammatory cytokine. Overall, Pt(DECO)2 showed minimal in vivo toxicity, thus presenting a promising candidate for future testing in animal models of cancer.

Using Xenopus oocytes in neurological disease drug discovery.

Introduction: Neurological diseases present a difficult challenge in drug discovery. Many of the current treatments have limited efficiency or result in a variety of debilitating side effects. The search of new therapies is of a paramount importance, since the number of patients that require a better treatment is growing rapidly. As an in vitro model, Xenopus oocytes provide the drug developer with many distinct advantages, including size, durability, and efficiency in exogenous protein expression. However, there is an increasing need to refine the recent breakthroughs.Areas covered: This review covers the usage and recent advancements of Xenopus oocytes for drug discovery in neurological diseases from expression and functional measurement techniques to current applications in Alzheimer's disease, painful neuropathies, and amyotrophic lateral sclerosis (ALS). The existing limitations of Xenopus oocytes in drug discovery are also discussed.Expert opinion: With the rise of aging population and neurological disorders, Xenopus oocytes, will continue to play an important role in understanding the mechanism of the disease, identification and validation of novel molecular targets, and drug screening, providing high-quality data despite the technical limitations. With further advances in oocytes-related techniques toward an accurate modeling of the disease, the diagnostics and treatment of neuropathologies will be becoming increasing personalized.

Fluorescence lifetime imaging reveals heterogeneous functional distribution of eGFP expressed in Xenopus oocytes.

The oocytes from Xenopus laevis are well known for their polarity, presenting a distinct animal and vegetal pole. Other heterogeneities are less known. To study the heterogeneity of the Xenopus oocyte, we expressed eGFP and analyzed the protein distribution with fluorescence lifetime microscopy. The vegetal pole exhibited higher levels of fluorescence, than the animal pole. However, the fluorescence lifetimes between the two areas were indistinguishable, suggesting similar environments. In contrast, we observed a substantial and gradual decrease in the fluorescence lifetime from 2.9 ns to 2.6 ns as slices approached the periphery. This has an important implication for future oocyte studies as it demonstrates the environment inside the oocyte is not uniform and might affect the fluorescence intensity. As a result, it cannot be assumed that the observed fluorescence intensity reflects the expression of the proteins but might reflect the environment within the oocyte.

Imaging in the repair of peripheral nerve injury.

Surgical intervention followed by physical therapy remains the major way to repair damaged nerves and restore function. Imaging constitutes promising, yet underutilized, approaches to improve surgical and postoperative techniques. Dedicated methods for imaging nerve regeneration will potentially provide surgical guidance, enable recovery monitoring and postrepair intervention, elucidate failure mechanisms and optimize preclinical procedures. Herein, we present an outline of promising innovations in imaging-based tracking of in vivo peripheral nerve regeneration. We emphasize optical imaging because of its cost, versatility, relatively low toxicity and sensitivity. We discuss the use of targeted probes and contrast agents (small molecules and nanoparticles) to facilitate nerve regeneration imaging and the engineering of grafts that could be used to track nerve repair. We also discuss how new imaging methods might overcome the most significant challenges in nerve injury treatment.

cAMP, Ca2+, pHi, and NO Regulate H-like Cation Channels That Underlie Feeding and Locomotion in the Predatory Sea Slug Pleurobranchaea californica.

A systems approach to regulation of neuronal excitation in the mollusc Pleurobranchaea has described novel interactions of cyclic AMP-gated cation current (INa,cAMP), Ca2+, pHi, and NO. INa,cAMP appears in many neurons of feeding and locomotor neuronal networks. It is likely one of the family of hyperpolarization-activated, cyclic-nucleotide-gated currents (h-current) of vertebrate and invertebrate pacemaker networks. There are two isoforms. Ca2+ regulates both voltage dependence and depolarization-sensitive inactivation in both isoforms. The Type 1 INa,cAMP of the feeding network is enhanced by intracellular acidification. A direct dependence of INa,cAMP on cAMP allows the current to be used as a reporter on cAMP concentrations in the cell, and from there to the intrinsic activities of the synthetic adenyl cyclase and the degradative phosphodiesterase. Type 2 INa,cAMP of the locomotor system is activated by serotonergic inputs, while Type 1 of the feeding network is thought to be regulated peptidergically. NO synthase activity is high in the CNS, where it differs from standard neuronal NO synthase in not being Ca2+ sensitive. NO acidifies pHi, potentiating Type 1, and may act to open proton channels. A cGMP pathway does not mediate NO effects as in other systems. Rather, nitrosylation likely mediates its actions. An integrated model of the action of cAMP, Ca2+, pHi, and NO in the feeding network postulates that NO regulates proton conductance to cause neuronal excitation in the cell body on the one hand, and relief of activity-induced hyperacidification in fine dendritic processes on the other.

Nitric oxide potentiates cAMP-gated cation current in feeding neurons of Pleurobranchaea californica independent of cAMP and cGMP signaling pathways.

Critical roles for nitric oxide (NO) in regulating cell and tissue physiology are broadly appreciated, but aspects remain to be explored. In the mollusk Pleurobranchaea, NO synthase activity is high in CNS ganglia containing motor networks for feeding and locomotion, where a cAMP-gated cation current (I(Na,cAMP)) is also prominent in many neurons. We examined effects of NO on I(Na,cAMP) using voltage-clamp methods developed to analyze cAMP signaling in the live neuron, focusing on the identified metacerebral giant neuron of the feeding network. NO donors enhanced the I(Na,cAMP) response to injected cAMP by an averaged 85%. In dose-response measures, NO increased the current stimulated by cAMP injection without altering either apparent cAMP binding affinity or cooperativity of current activation. NO did not detectably alter levels of native cAMP or synthesis or degradation rates as observable in both current saturation and decay rate of I(Na,cAMP) responses to cAMP injection. NO actions were not exerted by cGMP signaling, as they were not mimicked by cGMP analogue nor blocked by inhibitors of guanylate cyclase and protein kinase G. NO potentiation of I(Na,cAMP) was broadly distributed among many other neurons of the feeding motor network in the buccal ganglion. However, NO did not affect a second type of I(Na,cAMP) found in locomotor neurons of the pedal ganglia. These results suggest that NO acts through a novel mechanism to regulate the gain of cAMP-dependent neuromodulatory pathways that activate I(Na,cAMP) and may thereby affect the set points of feeding network excitability and reactivity to exogenous input.