Proteomic-miRNA Biomics Profile Reveals 2D Cultures of Human iPSC-Derived Neural Progenitor Cells More Sensitive than 3D Spheroid System Against the Experimental Exposure to Arsenic.

The iPSC-derived 3D models are considered to be a connective link between 2D culture and in vivo studies. However, the sensitivity of such 3D models is yet to be established. We assessed the sensitivity of the hiPSC-derived 3D spheroids against 2D cultures of neural progenitor cells. The sub-toxic dose of Sodium Arsenite (SA) was used to investigate the alterations in miRNA-proteins in both systems. Though SA exposure induced significant alterations in the proteins in both 2D and 3D systems, these proteins were uncommon except for 20 proteins. The number and magnitude of altered proteins were higher in the 2D system compared to 3D. The association of dysregulated miRNAs with the target proteins showed their involvement primarily in mitochondrial bioenergetics, oxidative and ER stress, transcription and translation mechanism, cytostructure, etc., in both culture systems. Further, the impact of dysregulated miRNAs and associated proteins on these functions and ultrastructural changes was compared in both culture systems. The ultrastructural studies revealed a similar pattern of mitochondrial damage, while the cellular bioenergetics studies confirm a significantly higher energy failure in the 2D system than to 3D. Such a higher magnitude of changes could be correlated with a higher amount of internalization of SA in 2D cultures than in 3D spheroids. Our findings demonstrate that a 2D culture system seems better responsive than a 3D spheroid system against SA exposure.

Proteome architecture of human-induced pluripotent stem cell-derived three-dimensional organoids as a tool for early diagnosis of neuronal disorders.

BACKGROUND AND OBJECTIVES: Induced pluripotent stem cells (iPSCs) derived three-dimensional (3D) model for rare neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) is emerging as a novel alternative to human diseased tissue to explore the disease etiology and potential drug discovery. In the interest of the same, we have generated a TDP-43-mutated human iPSCs (hiPSCs) derived 3D organoid model of ALS disease. The high-resolution mass spectrometry (MS)-based proteomic approach is used to explore the differential mechanism under disease conditions and the suitability of a 3D model to study the disease. MATERIALS AND METHODS: The hiPSCs cell line was procured from a commercial source, grown, and characterized following standard protocols. The mutation in hiPSCs was accomplished using CRISPR/Cas-9 technology and predesigned gRNA. The two groups of organoids were produced by normal and mutated hiPSCs and subjected to the whole proteomic profiling by high-resolution MS in two biological replicates with three technical replicas of each. RESULTS: The proteomic analysis of normal and mutated organoids revealed the proteins associated with pathways of neurodegenerative disorders, proteasomes, autophagy, and hypoxia-inducible factor-1 signaling. Differential proteomic analysis revealed that the mutation in TDP-43 gene caused proteomic deregulation, which impaired protein quality mechanisms. Furthermore, this impairment may contribute to the generation of stress conditions that may ultimately lead to the development of ALS pathology. CONCLUSION: The developed 3D model represents the majority of candidate proteins and associated biological mechanisms altered in ALS disease. The study also offers novel protein targets that may uncloud the precise disease pathological mechanism and be considered for future diagnostic and therapeutic purposes for various neurodegenerative disorders.

Crosstalk Between miRNA and Protein Expression Profiles in Nitrate-Exposed Brain Cells.

Growing evidence reported a strong association between the nitrate ingestion and adverse health consequences in humans, including its detrimental impact on the developing brain. The present study identified miRNAs and proteins in SH-SY5Y human neuroblastoma cells and HMC3 human microglial cells using high-throughput techniques in response to nitrate level most prevalent in the environment (India) as X dose and an exceptionally high nitrate level as 5X dose that can be reached in the near future. Cells were exposed to mixtures of nitrates for 72 h at doses of X and 5X, 320 mg/L and 1600 mg/L, respectively. OpenArray and LCMS analysis revealed maximum deregulation in miRNAs and proteins in cells exposed to 5X dose. Top deregulated miRNAs include miR-34b, miR-34c, miR-155, miR-143, and miR-145. The proteomic profiles of both cell types include proteins that are potential targets of deregulated miRNAs. These miRNAs and their targeted proteins involve in multiple functions, including metabolic processes, mitochondrial functions, autophagy, necroptosis, apoptosis, neuronal disorders, brain development, and homeostasis. Furthermore, measuring mitochondrial bioenergetics in cells exposed to nitrate revealed that a 5X dose causes a significant reduction in oxygen consumption rate (OCR) and other bioenergetic parameters in both cell types. In summary, our studies have demonstrated that a 5X dose of nitrate significantly alters cellular physiology and functions by deregulating several miRNAs and proteins. However, X dose of nitrate has not caused any adverse effects on any cell type.

Insights of Extracellular Vesicles of Mesenchymal Stem Cells: a Prospective Cell-Free Regenerative Medicine for Neurodegenerative Disorders.

Mesenchymal stem cells (MSCs) are multipotent, adult stem cells which are found in numerous tissues like the umbilical cord, Wharton's jelly, bone marrow, and adipose tissue. They possess the capacity of self-renewal by dividing and differentiating into various cellular lineages. Their characteristic therapeutic potential exploited so far has made them a desirable candidate in regenerative medicine. Neurodegenerative diseases (NDs) like Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), and ischemic stroke have been treated with MSCs and MSC-derived products. Over the past few decades, we have witnessed significant contributions in discovering the etiology of various NDs and their possible therapeutic solutions. One of the MSC-based therapeutics is extracellular vesicles (EVs), which contain multiple biologically active molecules like nucleic acids and proteins. The contents of EVs are ferried between cells for intercellular communication which then leads to regulation of the homeostasis of recipient cells. EVs serve as a considerable means of cell-free therapies like for tissue repair or regeneration as EVs can maintain therapeutically effective cargo of parent cells and are free of various ethical issues in cell-based therapies. Due to paucity of standard protocols in extraction procedures of EVs and their pharmacological properties and mechanisms, the development of new EV dependent therapies is challenging. With this review, an attempt has been made to annotate these mechanisms, which can help advance the novel therapeutic approaches towards the treat and define a more narrowed down approach for each ND to devise effective MSC-based therapies to cure and avert these diseases.

Efficient antileishmanial activity of amphotericin B and piperine entrapped in enteric coated guar gum nanoparticles.

Amphotericin B (AmB) exhibits potential antileishmanial activity, with only a little rate of recurrence. However, low bioavailability and severe nephrotoxicity are among the major shortcomings of AmB-based therapy. Various AmB nanoformulations have been developed, which to an extent, have reduced its toxicity and increased the drug efficacy. To further reduce the nonspecific tissue distribution and the cost of the treatment, the current AmB-based formulations require additional improvements. Combination of natural bioenhancers with AmB is expected to further increase its bioavailability. Therefore, we developed a nanoformulation of AmB and piperine (Pip), a plant alkaloid, known to enhance the bioavailability of various drugs, by entrapping them in guar gum, a macrophage targeting polymer. Owing to the ease of oral delivery, these nanoparticles (NPs) were coated with eudragit to make them suitable for oral administration. The formulated eudragit-coated AmB and Pip-loaded NPs (Eu-HDGG-AmB-Pip-NPs) exhibited controlled release of the loaded therapeutic agents and protected the drug from acidic pH. These NPs exhibited effective suppression of growth of both promastigotes and amastigotes of Leishmania donovani parasite under in vitro. In vivo evaluation of these NPs for therapeutic efficacy in golden hamster-L. donovani model demonstrated enhanced drug bioavailability, non-nephrotoxic nature, and potential antileishmanial activity with up to 96% inhibition of the parasite. Graphical abstract.

Secretome of Differentiated PC12 Cells Restores the Monocrotophos-Induced Damages in Human Mesenchymal Stem Cells and SHSY-5Y Cells: Role of Autophagy and Mitochondrial Dynamics.

A perturbed cellular homeostasis is a key factor associated with xenobiotic exposure resulting in various ailments. The local cellular microenvironment enriched with secretory components aids in cell-cell communication that restores this homeostasis. Deciphering the underlying mechanism behind this restorative potential of secretome could serve as a possible solution to many health hazards. We, therefore, explored the protective efficacy of the secretome of differentiated PC12 cells with emphasis on induction of autophagy and mitochondrial biogenesis. Monocrotophos (MCP), a widely used neurotoxic organophosphate, was used as the test compound at sublethal concentration. The conditioned medium (CM) of differentiated PC12 cells comprising of their secretome restored the cell viability, oxidative stress and apoptotic cell death in MCP-challenged human mesenchymal stem cells and SHSY-5Y, a human neuroblastoma cell line. Delving further to identify the underlying mechanism of this restorative effect we observed a marked increase in the expression of autophagy markers LC3, Beclin-1, Atg5 and Atg7. Exposure to autophagy inhibitor, 3-methyladenine, led to a reduced expression of these markers with a concomitant increase in the expression of pro-apoptotic caspase-3. Besides that, the increased mitochondrial fission in MCP-exposed cells was balanced with increased fusion in the presence of CM facilitated by AMPK/SIRT1/PGC-1α signaling cascade. Mitochondrial dysfunctions are strongly associated with autophagy activation and as per our findings, cellular secretome too induces autophagy. Therefore, connecting these three potential apices can be a major breakthrough in repair and rescue of xenobiotic-damaged tissues and cells.

Secretome of Differentiated PC12 Cells Enhances Neuronal Differentiation in Human Mesenchymal Stem Cells Via NGF-Like Mechanism.

The secretome-mediated responses over cellular physiology are well documented. Stem cells have been ruling the field of secretomics and its role in regenerative medicine since the past few years. However, the mechanistic aspects of secretome-mediated responses and the role of other cells in this area remain somewhat elusive. Here, we investigate the effects of secretome-enriched conditioned medium (CM) of neuronally differentiated PC12 cells on the neuronal differentiation of human mesenchymal stem cells (hMSCs). The exposure to CM at a ratio of 1:1 (CM: conditioned medium of PC12 cells) led to neuronal induction in hMSCs. This neuronal induction was compared with a parallel group of cells exposed to nerve growth factor (NGF). There was a marked increase in neurite length and expression of neuronal markers (ß-III tubulin, neurofilament-M (NF-M), synaptophysin, NeuN in exposed hMSCs). Experimental group co-exposed to NGF and CM showed an additive response via MAPK signaling and directed the cells particularly towards cholinergic lineage. The ability of CM to enhance the neuronal properties of stem cells could aid in their rapid differentiation into neuronal subtypes in case of stem cell transplantation for neuronal injuries, thus broadening the scope of non-stem cell-based applications in the area of secretomics.

Resveratrol Prevents the Cellular Damages Induced by Monocrotophos via PI3K Signaling Pathway in Human Cord Blood Mesenchymal Stem Cells.

The role of resveratrol (RV) as a neuroprotectant is well recognized, and cellular molecules involved in imparting the physiological effect have been well illustrated. However, some ambiguity still prevails as the specific receptor, and downstream signaling molecules are not yet clearly stated. So, we investigated the signaling pathway(s) involved in its cellular protection in the human umbilical cord blood mesenchymal stem cell (hUCB-MSC) derived neuronal cells. The mesenchymal stem cells were exposed to various concentrations (10, 100, 1000 µM) of monocrotophos (MCP), a known developmental neurotoxic organophosphate pesticide, for a period of 24 h. The MAPK signaling pathways (JNK, p38, and ERK) known to be associated with MCP-induced damages were also taken into consideration to identify the potential connection. The biological safe dose of RV (10 µM) shows a significant restoration in the MCP-induced alterations. Under the specific growth conditions, RV exposure was found to promote neuronal differentiation in the hUCB-MSCs. The exposure of cells to a specific pharmacological inhibitor (LY294002) of PI3K confirms the significant involvement of PI3K-mediated pathway in the ameliorative responses of RV against MCP exposure. Our data identifies the substantial role of RV in the restoration of MCP-induced cellular damages, thus proving to have a therapeutic potential against organophosphate pesticide-induced neurodegeneration.

Hyaluronic acid-grafted PLGA nanoparticles for the sustained delivery of berberine chloride for an efficient suppression of Ehrlich ascites tumors.

To promote the specific targeting and elimination of CD44-positive cancer cells, berberine chloride (BRB)-encapsulated hyaluronic acid-grafted poly(lactic-co-glycolic acid) copolymer (BRB-d(HA)-g-PLGA) nanoparticles (NPs) were prepared. The targeted action of these NPs was compared to non-targeted BRB-loaded PLGA NPs and bulk BRB. The in vitro studies demonstrated faster release of BRB and increased cytotoxicity of BRB-d(HA)-g-PLGA NPs in Hela and MCF-7 cells in comparison to BRB-PLGA NPs and bulk BRB. The uptake of BRB-d(HA)-g-PLGA NPs was increased in case of MCF-7 cells as compared to HeLa cells owing to the higher expression of CD44 receptors on MCF-7 cells. The CD44 receptor-mediated uptake of these NPs was confirmed through competitive inhibition experiments. The in vitro results were further validated in vivo in Ehrlich Ascites Carcinoma (EAC)-bearing mice. EAC-bearing mice were injected intravenously with these NPs and the results obtained were compared with that of BRB-PLGA NPs and bulk BRB. BRB-d(HA)-g-PLGA NPs were found to significantly enhance apoptosis, sub-G1 content, life span, mean survival time, and ROS levels in EAC cells with subsequent decrease in mitochondrial membrane potential and tumor burden ion tumor-bearing mice. Taking into account the findings of in vitro and in vivo studies, the enhanced and targeted anti-tumor activity of HA-grafted PLGA copolymer-encapsulated NPs of BRB cannot be negated. Therefore, HA-grafted nanoparticle-based delivery of BRB may offer a promising and improved alternative for anti-tumor therapy.

PKA-GSK3ß and ß-Catenin Signaling Play a Critical Role in Trans-Resveratrol Mediated Neuronal Differentiation in Human Cord Blood Stem Cells.

The role of resveratrol (RV), a natural polyphenol, is well documented, although its role on neurogenesis is still controversial and poorly understood. Therefore, to decipher the cellular insights of RV on neurogenesis, we investigated the potential effects of the compound on the survival, proliferation, and neuronal differentiation of human cord blood-derived mesenchymal stem cells (hCBMSCs). For neuronal differentiation, purified and characterized hCBMSCs were exposed to biological safe doses of RV (10 µM) alone and in combination with nerve growth factor (NGF-50 ng). The cells exposed only to NGF (50 ng/mL) served as positive control for neuronal differentiation. The genes showing significant involvement in the process of neuronal differentiation were further funneled down at transcriptional and translational level. It was observed that RV promotes PKA-mediated neuronal differentiation in hCBMSCs by inducing canonical pathway. The studies with pharmacological inhibitors also confirmed that PKA significantly induces ß-catenin expression via GSK3ß induction and stimulates CREB phosphorylation and pERK1/2 induction. Besides that, the studies also revealed that RV additionally possesses the binding sites for molecules other than PKA and GSK3ß, with which it interacts. The present study therefore highlights the positive impact of RV over the survival, proliferation, and neuronal differentiation in hCBMSCs via PKA-mediated induction of GSK3ß, ß catenin, CREB, and ERK1/2.

Curcumin loading potentiates the chemotherapeutic efficacy of selenium nanoparticles in HCT116 cells and Ehrlich's ascites carcinoma bearing mice.

The anticancer properties of selenium (Se) and curcumin nanoparticles in solo formulations as well as in combination with other therapeutic agents have been proved time and again. Exploiting this facet of the two, we clubbed their tumoricidal characteristics and designed curcumin loaded Se nanoparticles (Se-CurNPs) to achieve an enhanced therapeutic effect. We evaluated their therapeutic effects on different cancer cell lines and Ehrlich's ascites carcinoma mouse model. In vitro results showed that Se-CurNPs were most effective on colorectal carcinoma cells (HCT116) compared to the other cancer cell lines used and possessed pleiotropic anticancer effects. The therapeutic effect on HCT116 was primarily attributed to an elevated level of autophagy and apoptosis as evident from significant up-regulation of autophagy associated (LC3B-II) and pro-apoptotic (Bax) proteins, down-regulation of anti-apoptotic (Bcl-2) protein and Cytochrome c (cyt c) release from mitochondria along with reduced NFκB signaling and EMT based machineries marked by downregulation of inflammation (NFκB, phospho-NFκB) and epithelial-mesenchymal transition (CD44, N-cadherin) associated proteins. In vivo studies on Ehrlich's ascites carcinoma (EAC) mice model indicated that Se-CurNPs significantly reduced the tumor load and enhanced the mean survival time (days) of tumor-bearing EAC mice.

Adoptive Autophagy Activation: a Much-Needed Remedy Against Chemical Induced Neurotoxicity/Developmental Neurotoxicity.

The profound significance of autophagy as a cell survival mechanism under conditions of metabolic stress is a well-proven fact. Nearly a decade-long research in this area has led scientists to unearth various roles played by autophagy other than just being an auto cell death mechanism. It is implicated as a vital cell survival pathway for clearance of all the aberrant cellular materials in case of cellular injury, metastasis, disease states, cellular stress, neurodegeneration and so on. In this review, we emphasise the critical role of autophagy in the environmental stressors-induced neurotoxicity and its therapeutic implications for the same. We also attempt to shed some light on the possible protective role of autophagy in developmental neurotoxicity (DNT) which is a rapidly growing health issue of the human population at large and hence a point of rising concern amongst researchers. The intimate association between DNT and neurodegenerative disorders strongly indicates towards adopting autophagy activation as a much-needed remedy for DNT.

Neurotrophic factor mediated neuronal differentiation of human cord blood mesenchymal stem cells and their applicability to assess the developmental neurotoxicity.

Plasticity and developmental capacity of stem cells have now been established as a promising tool to restore the degenerative disorders. The linearity differentiation of human mesenchymal stem cells (hMSCs) into adipogenic, chondrogenic, osteogenic and even in neuronal subtypes has been demonstrated. The number of xenobiotics such as dexamethasone, insulin, isobutyl 1-methyle xanthine and retinoic acid has been reported for the potential to differentiate hMSCs into neuronal subtypes. But, the applicability of indigenous neurotrophic factor-nerve growth factor (NGF) has not been explored for the purpose. Thus, the present investigations were carried out to study the NGF induced neuronal differentiation of hMSCs. Following the isolation, purification and characterization of hMSCs were allowed to differentiate into neuronal subtypes under the influence of NGF (50 ng/mL). At various concentrations of NGF, the neuronal makers were analysed at both mRNA and protein levels. Cells, exposed with NGF were showing the significant and gradual increase in the neuronal markers in differentiating cells. The magnitude of expression of markers was maximum at day 4 of differentiation. NGF at 50 ng/mL concentration was found to induce neuronal differentiation of hMSCs into neuronal subtypes.

Stem Cells in Neurotoxicology/Developmental Neurotoxicology: Current Scenario and Future Prospects.

Stem cell biology has played a pivotal role in the field of disease modeling, regenerative medicine, and tissue engineering. The scope of stem cell research has been further extended to address the issues associated with toxicity and biosafety. However, its role in the field of neurotoxicity (NT) and the emerging field of developmental neurotoxicity (DNT) is somewhat underrepresented and needs thorough investigation. Several challenges have hindered the progress of NT and DNT studies, and there is a dire need for human-specific high-throughput in vitro system(s) as a tool with better predictivity, reliability, and reproducibility. The unique proliferation and pluripotency of stem cells makes them a tremendous resource for human material, allowing the prediction of drug toxicity and metabolic effects of chemicals. Recognizing the growing importance of NT and DNT and the application of stem cell biology, in this review article, we provide the diversified approaches of stem cell research which can be effectively applied to the NT and DNT studies and provide an update of the recent progress made so far. We further provide a futuristic approach towards novel stem cell-based strategies for NT and DNT testing. We have further discussed the current technologies, role of induced pluripotent stem cells, the application of three-dimensional (3D) cultures and role of stem cell-derived organs in the NT and DNT studies.

Lead Intoxication Synergies of the Ethanol-Induced Toxic Responses in Neuronal Cells--PC12.

Lead (Pb)-induced neurodegeneration and its link with widespread neurobehavioral changes are well documented. Experimental evidences suggest that ethanol could enhance the absorption of metals in the body, and alcohol consumption may increase the susceptibility to metal intoxication in the brain. However, the underlying mechanism of ethanol action in affecting metal toxicity in brain cells is poorly understood. Thus, an attempt was made to investigate the modulatory effect of ethanol on Pb intoxication in PC12 cells, a rat pheochromocytoma. Cells were co-exposed to biological safe doses of Pb (10 µM) and ethanol (200 mM), and data were compared to the response of cells which received independent exposure to these chemicals at similar doses. Ethanol (200 mM) exposure significantly aggravated the Pb-induced alterations in the end points associated with oxidative stress and apoptosis. The finding confirms the involvement of reactive oxygen species (ROS)-mediated oxidative stress, and impairment of mitochondrial membrane potential, which subsequently facilitate the translocation of triggering proteins between cytoplasm and mitochondria. We further confirmed the apoptotic changes due to induction of mitochondria-mediated caspase cascade. These cellular changes were found to recover significantly, if the cells are exposed to N-acetyl cysteine (NAC), a known antioxidant. Our data suggest that ethanol may potentiate Pb-induced cellular damage in brain cells, but such damaging effects could be recovered by inhibition of ROS generation. These results open up further possibilities for the design of new therapeutics based on antioxidants to prevent neurodegeneration and associated health problems.

Galactomannan-PEI based non-viral vectors for targeted delivery of plasmid to macrophages and hepatocytes.

Intracellular nature and diversified locations of infectious and parasitic diseases such as leishmaniasis, trypanosomiasis, tuberculosis and hepatitis B and C pose a significant global burden and challenge to the scientists working in the area of drug discovery and drug delivery. The macrophages and hepatocytes are considered as potential target sites as they together play an important role in various infectious diseases. The present study scrutinizes the applicability of a natural biopolymer-based chemical vectors, capable of targeting both macrophages and hepatocytes, that can form a complex with plasmid and administer it into cells to produce a desired protein. The investigations were made to develop a novel series of gene carriers by conjugating depolymerized galactomannan (guar gum), a biocompatible polysaccharide with low molecular weight branched PEI (LMWP). A series of conjugates were developed and characterized using physicochemical techniques. All the GP/pDNA complexes showed significantly higher transfection efficiency with GP-3/pDNA, one of the best formulations, showed ~2.0-7.7-folds higher transfection efficacy when compared with the standard transfection reagents. Further, GP-3/pDNA displayed significantly higher target specific transfection efficiency under both in vitro and in vivo conditions. The data demonstrate the potential of GP vectors to deliver nucleic acids simultaneously to macrophages and hepatocytes in gene delivery applications.

Virtual Screening of Natural and Synthetic Ligands Against Diabetic Retinopathy by Molecular Interaction With Angiopoietin-2.

PURPOSE: Diabetic retinopathy (DR) is the most common diabetic eye disease and a leading cause of blindness. The role of angiopoietin-2 a tyrosine kinase receptor is well-reported in angiogenesis during the onset of the disease. The purpose of this study is to screen out more potential herbal molecules which can evidently be used as a better, natural and safe herbal drug against this disease. DESIGN: In silico virtual screening and molecular interaction studies were performed. METHODS: The current course of work focused on molecular interactions on angiopoietin-2 protein with selected natural ligands, namely allicin, ajoene, D-pinitol and salacinol, along with synthetic ones like nateglinide, biguanide, tolbutamide and tolazamide. There was an attempt to carry out the virtual comparative study between natural and synthetic ligands. Proceeding toward this approach, docking of all molecules was performed using the Autodock 4.2 program. RESULTS: Inference of this interaction study is that D-pinitol, which is the herbal extract of Glycine max, shows a very reliable docking pattern as compared with the synthetic ligand tolazamide. Although the binding energy of a synthetic ligand is lower compared to that of the natural ones, the binding energy of synthetic and natural ligands are at an approximate level. The lower the binding energy, the better the ligand molecular interaction. CONCLUSIONS: Our findings suggest that D-pinitol, the natural, safe ligand, can be used in the treatment of diabetic retinopathy with few or no side effects after estimating and calculating proper doses using in vitro approaches.