Nanoformulation of EPZ011989 Attenuates EZH2-c-Myb Epigenetic Interaction by Proteasomal Degradation in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a malignant disorder of hematopoietic progenitor cells with a poor prognosis of 26% of patients surviving 5 years after diagnosis. Poor bioavailability and solubility are significant factors limiting the efficacy of chemopreventive agents. In AML, the epigenetic regulator polycomb group of protein member EZH2 is highly expressed and is essential for the survival of leukemic cells. An EZH2-specific inhibitor, EPZ011989, encapsulated in human serum albumin nanoparticles (HSANPs) was synthesized for the first time via the desolvation method. The noncovalent interactions between EPZ011989 and HSANPs in nanocomposites facilitating the efficient loading and sustainable release of the drug showed enhanced cellular uptake and nuclear localization of EPZ011989-loaded HSANPs in human AML cell lines. The reduction of cell viability, colony formation inhibition, cell cycle arrest at the G2/M phase, and cell proliferation assay promoting apoptosis through the loss of mitochondrial homeostasis exerting antileukemic activity were evident. The real-time polymerase chain reaction (PCR) and western blot-based studies showed that the present nanoformulation reduces the level of PcG proteins, including EZH2, BMI-1, etc. This downregulation is associated with reduced H3K27me3 and H2AK119ub modifications conferring chromatin compaction. The immunoprecipitation study showed the physical interaction of EZH2 and c-Myb can be linked to the regulation of leukemogenesis. Further investigation revealed the mechanism of EZH2 and c-Myb downregulation via ubiquitination and proteasomal degradation pathway, confirmed by using proteasome inhibitor, suggesting the key role of proteasomal degradation machinery. Moreover, c-Myb interacted with the EZH2 promoter, which is evident by the chromatin immunoprecipitation assay and siRNA silencing. Furthermore, the formulation of EPZ011989 in HSANPs improved its biodistribution in vivo and showed excellent aqueous dispersibility and biocompatibility. In vivo studies further showed that EPZ011989-loaded HSANPs reduce the expression of CD11b+ and CD45+ markers in immunophenotyping from peripheral blood and bone marrow in engrafted nude mice. Targeted depletion of EZH2 alleviated the disease progression in nude mice and prolonged their survival. The findings provide valuable experimental evidence for the targeted epigenetic therapy of AML. The present results demonstrate an epigenetic regulation-based superior antileukemic therapy.

Recuperative effect of metformin loaded polydopamine nanoformulation promoting EZH2 mediated proteasomal degradation of phospho-α-synuclein in Parkinson's disease model.

Posttranslational modification and agglomeration of α-synuclein (α-Syn), mitochondrial dysfunction, oxidative stress and loss of dopaminergic neurons are hallmark of Parkinson's disease (PD). This paper evaluates neuroprotection efficacy of nature inspired biocompatible polydopamine nanocarrier for metformin delivery (Met encapsulated PDANPs) by crossing blood brain barrier in in vitro, 3D and in vivo experimental PD models. The neuroprotective potential was arbitrated by downregulation of phospho-serine 129 (pSer129) α-Syn, with reduction in oxidative stress, prevention of apoptosis and anti-inflammatory activities. The neuroprotective mechanism proved novel interaction of epigenetic regulator EZH2 mediated ubiquitination and proteasomal degradation of aggregated pSer129 α-Syn. In summary, this study divulges the neuroprotective role of Met loaded PDANPs by reversing the neurochemical deficits by confirming an epigenetic mediated nanotherapeutic approach for the PD prevention.

Mycobacterium tuberculosis WhiB4 regulates oxidative stress response to modulate survival and dissemination in vivo.

Host-generated oxidative stress is considered one of the main mechanisms constraining Mycobacterium tuberculosis (Mtb) growth. The redox-sensing mechanisms in Mtb are not completely understood. Here we show that WhiB4 responds to oxygen (O2) and nitric oxide (NO) via its 4Fe-4S cluster and controls the oxidative stress response in Mtb. The WhiB4 mutant (MtbΔwhiB4) displayed an altered redox balance and a reduced membrane potential. Microarray analysis demonstrated that MtbΔwhiB4 overexpresses the antioxidant systems including alkyl hydroperoxidase (ahpC-ahpD) and rubredoxins (rubA-rubB). DNA binding assays showed that WhiB4 [4Fe-4S] cluster is dispensable for DNA binding. However, oxidation of the apo-WhiB4 Cys thiols induced disulphide-linked oligomerization, DNA binding and transcriptional repression, whereas reduction reversed the effect. Furthermore, WhiB4 binds DNA with a preference for GC-rich sequences. Expression analysis showed that oxidative stress repressed whiB4 and induced antioxidants in Mtb, while their hyper-induction was observed in MtbΔwhiB4. MtbΔwhiB4 showed increased resistance to oxidative stress in vitro and enhanced survival inside the macrophages. Lastly, MtbΔwhiB4 displayed hypervirulence in the lungs of guinea pigs, but showed a defect in dissemination to their spleen. These findings suggest that WhiB4 systematically calibrates the activation of oxidative stress response in Mtb to maintain redox balance, and to modulate virulence.

Ultrafine particles in urban ambient air and their health perspectives.

Ultrafine particles (UfPs, PM<0.1) are constituents of urban ambient air aerosol. We have reviewed literature on UfPs in urban ambient air and their health perspectives. Generally traffic-linked and of anthropogenic origin, these are toxicants and a health risk factor for urban subjects. UfPs occur in single and agglomerate forms. Studies on the number concentrations of UfPs show tens of thousand times greater levels in urban aerosol than in nonurban aerosol. These nanosize pollutants seem to have more aggressive implications than other respirable fractions of urban aerosol. In literature, it is hypothesized that a chronic exposure to their high number concentrations and their vast surface area, transporting various toxicants, injure tissues or cells and induce inflammation or, eventually, adverse health effects. UfPs are deposited deep in the tissues, translocate, and skip the innate clearance mechanisms. After retention for a long time, these can infiltrate into the interstitium and permeate cells. Traffic-linked UfPs have been found to be toxic to the respiratory, cardiovascular, and nervous systems. At the molecular level, UfPs influence signaling cascade, actin-cytoskeleton pathway, immunoregulation, reactive oxygen species generation to trigger histaminic response, mast cell activation, and pro-inflammatory changes; their mutagenic and carcinogenic effects are also tacit in view of the carcinogenic potential of diesel exhaust in humans. The molecular changes are proposed to be the subclinical effects that manifest disease exacerbations or the predisposition of subjects to pathologies after exposure to UfP. A legislatively regulated monitoring of UfP-contaminated urban ambient air environment is also endorsed to reduce the disease load or its exacerbation that is growing in diesel exhaust (a human carcinogen)-polluted urban areas.

Coupled catalytic dephosphorylation and complex phosphate ion-exchange in networked hierarchical lanthanum carbonate grafted asymmetric bio-composite membrane.

The remediation of non-reactive phosphate pollutants in the aquatic system is essential for protecting the ecological niche. In this work, a highly robust protein nanoparticles networked rare-earth metal carbonate-grafted bio-composite membrane (abbreviated as REMC) was fabricated via chemical crosslinking of three-dimensional (3D) hierarchical lanthanum carbonate (mREM) and casein nanoparticles (CsNPs) for selective rejection of non-reactive phosphates. The main components of the REMC membrane are mREM and CsNPs, which were prepared via SDS/CTAB templated homogeneous precipitation and the coacervation/desolvation hybrid method, respectively. The active lanthanum ion (La3+) on the 3D spherulitic surface of mREM exhibited excellent phosphate adsorption capacity (maximum adsorption capacity was 358 mg.g-1) across a wide pH range and in a multi-ionic environment. A series of batch testing and characterizations revealed that the active La3+ and dominating phosphate centers in the REMC membrane framework enable non-enzymatic phosphatase-like activity, cleaving the phosphate ester bond of organic phosphates and releasing free phosphate anions. These released phosphate ions are retained in the REMC membrane via an ion exchange mechanism, where they contribute to improved phosphate removal capacities. Furthermore, CsNPs have a dual function in the membrane, acting as a matrix in the REMC membrane framework and contributing to phosphate ion sequestrations in a synergistic manner. The catalysis of para-nitrophenyl phosphates (pNPP) to paranitrophenol (pNP) in a sequential dephosphorylation by REMC offers an estimate of reaction kinetics and elucidates the underlying mechanism of improved phosphate selectivity in a multi-ionic environment. Furthermore, phosphate specificity, homogeneous binding capacity, reusability, and visual observation of REMC membrane saturation binding direct it's useful economic, industrial applications in aqueous phosphate contaminant removal, which could be beneficial for the active recovery of the aquatic ecosystem.

Examining the role of nickel and NiTi nanoparticles promoting inflammation and angiogenesis.

Nickel titanium (NiTi, or Nitinol) alloy is used in several biomedical applications, including cardiac, peripheral vascular, and fallopian tube stents. There are significant biocompatibility issues of metallic implants to nickel ions and nano-/micro-sized alloy particles. Our laboratories have recently shown that microscale CoCr wear particles from metal-on-metal hips crosslink with the innate immune signaling Toll-like receptor 4 (TLR4), prompting downstream signaling that results in interleukin (IL)-1ß and IL-8 gene expression. In vivo, NiTi alloy can also generate wear particles on the nanoscale (NP) that have thus far not been studied for their potential to induce inflammation and angiogenesis that can, in turn, contribute to implant (e.g. stent) failure. Earlier studies by others demonstrated that nickel could induce contact hypersensitivity by crosslinking the human, but not the mouse, TLR4. In the present work, it is demonstrated that NiCl2 ions and NiTi nanoparticles induce pro-inflammatory and pro-angiogenic cytokine/chemokine expression in human endothelial and monocyte cell lines in vitro. These observations prompt concerns about potential mechanisms for stent failure. The data here showed a direct correlation between intracellular uptake of Ni2+ and generation of reactive oxygen species. To determine a role for nickel and NiTi nanoparticles in inducing angiogenesis in vivo, 1-cm silicone angioreactors were implanted subcutaneously into athymic (T-cell-deficient) nude mice. The angioreactors contained Matrigel (a gelatinous protein mixture that resembles extracellular matrix) in addition to one of the following: PBS (negative control), VEGF/FGF-2 (positive control), NiCl2, or NiTi NP. The implantation of angioreactors represents a potential tool for quantification of angiogenic potentials of medical device-derived particles and ions in vivo. By this approach, NiTi NP were found to be markedly angiogenic, while Ni2+ was less-so. The angioreactors may provide a powerful tool to examine if debris shed from medical devices may promote untoward biological effects.

Neuronal Bmi-1 is critical for melatonin induced ubiquitination and proteasomal degradation of α-synuclein in experimental Parkinson's disease models.

Epigenetic polycomb repressor complex-1 subunit BMI-1 plays a pivotal role in the process of gene repression to maintain the self-renewal and differentiation state of neurogenic tissues. Accumulating reports links lower expression of BMI-1 fails to regulate the repression of anti-oxidant response genes disrupt mitochondrial homeostasis underlying neurodegeneration. Interestingly, this negative relation between BMI-1 function and neurodegeneration is distinct but has not been generalized as a potential biomarker particularly in Parkinson's disease (PD). Hyperphosphorylated BMI-1 undergoes canonical polycomb E3 ligase function loss, thereby leads to reduce monoubiquitylation of histone 2A at lysine 119 (H2AK119ub) corroborates cellular accumulation of α-synuclein protein phosphorylated at serine 129 (pα-SYN (S129). In general, neuroprotectant suppressing pα-SYN (S129) level turns ineffective upon depletion of neuronal BMI-1. However, it has been observed that our neuroprotectant exposure suppresses the cellular pα-SYN (S129) and restore the the BMI-1 expression level in neuronal tissues. The pharmacological inhibition and activation of proteasomal machinery promote the cellular accumulation and degradation of neuronal pα-SYN (S129), respectively. Furthermore, our investigation reveals that accumulated pα-SYN (S129) are priorly complexed with BMI-1 undergoes ubiquitin-dependent proteasomal degradation and established as key pathway for therpeutic effect in PD. These findings linked the unestablished non-canonical role of BMI-1 in the clearance of pathological α-SYN and suspected to be a novel therapeutic target in PD.

Melatonin/polydopamine nanostructures for collective neuroprotection-based Parkinson's disease therapy.

Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra and localized deposition of cytoplasmic fibrillary inclusions as Lewy bodies in the brain. The aberrant phosphorylation of α-synuclein at serine 129 is the key process on its early onset, which alters the cellular conformation to oligomers and insoluble aggregates, underpinning cellular oxidative stress and mitochondrial dysfunction, leading to devastating PD synucleinopathy. The multiple neuroprotective roles of dopamine and melatonin are often demonstrated separately; however, this approach suffers from low and short bioavailability and is associated with side-effects upon overdosing. Herein, highly pleiotropic melatonin-enriched polydopamine nanostructures were fabricated, which showed efficient brain tissue retention, sustainable and prolonged melatonin release, and prevented neuroblastoma cell death elicited by Parkinson's disease-associated and mitochondrial damaging stimuli. The synergistic neuroprotection re-established the mitochondrial membrane potential, reduced the generation of cellular reactive oxygen species (ROS), inhibited the activation of both the caspase-dependent and independent apoptotic pathways, and exhibited an anti-inflammatory effect. At the molecular level, it suppressed α-synuclein phosphorylation at Ser 129 and reduced the cellular deposition of high molecular weight oligomers. The therapeutic assessment on ex vivo organotypic brain slice culture, and in vivo experimental PD model confirmed the superior brain targeting, collective neuroprotection on dopaminergic neurons with reduced alpha-synuclein phosphorylation and deposition in the hippocampal and substantia nigra region of the brain. Thus, nature-inspired melatonin-enriched polydopamine nanostructures conferring collective neuroprotective effects attributes activation of anti-oxidative, anti-inflammatory, and anti-apoptotic pathways may be superior for application in a nanomedicine-based PD therapy.

Near-Infrared Responsive Dopamine/Melatonin-Derived Nanocomposites Abrogating in Situ Amyloid ß Nucleation, Propagation, and Ameliorate Neuronal Functions.

Alzheimer's disease (AD) is one of the common causes of dementia and mild cognitive impairments, which is progressively expanding among the elderly population worldwide. A short Amyloid-ß (Aß) peptide generated after amyloidogenic processing of amyloid precursor protein exist as intermolecular ß-sheet rich oligomeric, protofibriler, and fibrillar structures and believe to be toxic species which instigate neuronal pathobiology in the brain and deposits as senile plaque. Enormous efforts are being made to develop an effective anti-AD therapy that can target Aß processing, aggregation, and propagation and provide a synergistic neuroprotective effect. However, a nanodrug prepared from natural origin can confer a multimodal synergistic chemo/photothermal inhibition of Aß pathobiology is not yet demonstrated. In the present work, we report a dopamine-melatonin nanocomposite (DM-NC), which possesses a synergistic near-infrared (NIR) responsive photothermal and pharmacological modality. The noncovalent interaction-mediated self-assembly of melatonin and dopamine oxidative intermediates leads to the evolution of DM-NCs that can withstand variable pH and peroxide environment. NIR-activated melatonin release and photothermal effect collectively inhibit Aß nucleation, self-seeding, and propagation and can also disrupt the preformed Aß fibers examined using in vitro Aß aggregation and Aß-misfolding cyclic amplification assays. The DM-NCs display a higher biocompatibility to neuroblastoma cells, suppress the AD-associated generation of intracellular reactive oxygen species, and are devoid of any negative impact on the axonal growth process. In okadaic acid-induced neuroblastoma and ex vivo midbrain slice culture-based AD model, DM-NCs exposure suppresses the intracellular Aß production, aggregation, and accumulation. Therefore, this nature-derived nanocomposite demonstrates a multimodal NIR-responsive synergistic photothermal and pharmacological modality for effective AD therapy.

A NIR-responsive indocyanine green-genistein nanoformulation to control the polycomb epigenetic machinery for the efficient combinatorial photo/chemotherapy of glioblastoma.

Combinatorial photodynamics and chemotherapy have drawn enormous attention as therapeutic modalities via precise stimuli-responsive drug delivery for glioblastoma, which can overcome the limitations associated with conventional therapies. Herein, we have prepared an indocyanine green tagged, genistein encapsulated casein nanoformulation (ICG-Gen@CasNPs) that exhibits the near infra-red region responsive controlled release of genistein and enhanced cellular uptake in the human glioblastoma monolayer and a three-dimensional raft culture model via the enhanced retention effect. ICG-Gen@CasNPs, with the integrated photosensitizer indocyanine green within the nanoformulation, triggered oxidative stress, activating the apoptosis cascade, promoting cell cycle arrest and damaging the mitochondrial membrane potential, collectively directing glioblastoma cell death. The suppression of the polycomb group of proteins in the glioblastoma upon ICG-Gen@CasNPs/NIR exposure revealed the involvement of the epigenetic repression complex machinery in the regulation. Furthermore, ICG-Gen@CasNPs/PDT/PTT directed ubiquitination and proteasomal degradation of EZH2 and BMI1 indicates the implication of the polycomb in conferring glioblastoma survival. The increased activation of the apoptotic pathways and the generation of cellular reactive oxygen species upon inhibiting the expression of EZH2 and BMI1 strengthen our observations. It is worth noting that ICG-Gen@CasNPs robustly accumulated in the brain after crossing the blood-brain barrier, which represents the eminent biocompatibility and means that the system is devoid of any nonspecific toxicity in vivo. Moreover, a superior anti-tumor effect was demonstrated on a three-dimensional glioma spheroid model. Thus, this combinatorial chemo/photodynamic therapy revealed that ICG-Gen@CasNPs mediated epigenetic regulation, which is a crucial molecular mechanism of GBM suppression.

Nanostructure Endows Neurotherapeutic Potential in Optogenetics: Current Development and Future Prospects.

Optogenetics have evolved as a promising tool to control the processes at a cellular level via photons. Specially, it confers a specific control over cellular function through real-time cytomodulation even in freely moving animals. Neuronal stimulation is prerequisite for deep tissue light penetration or insertion of optrode for light illumination to the neurons that have been proven to be compromised due to poor light penetration and invasiveness of the procedure, respectively. In this review, the application of nanotechnology is being elaborated by the use of metal nanoparticles (AuNPs), upconversion nanocrystals (UCNPs), and quantum dots (CdSe) for targeting particular organs or tissues, and their potential to emit a specific light on excitation to overcome the limitations associated with earlier methods has been elucidated. The optothermal and magnetothermal properties, photoluminescence, and higher photostability of nanomaterials are explored in context of therapeutic applicability of optogenetics. The nanostructure characteristics and specific ion channel targeting have shown promising therapeutic potential against neurodegenerative disorders (Alzheimer's, Parkinson's, Huntington's), epilepsy, and blindness. This review compiles mechanical and optical characteristics of nanomaterials that endow superior optogenetic therapeutic potentials to cure immedicable infirmities.

1, 3ß-Glucan anchored, paclitaxel loaded chitosan nanocarrier endows enhanced hemocompatibility with efficient anti-glioblastoma stem cells therapy.

Recurrence of glioblastoma is one of the major concerns due to its heterogeneous nature and association of Glioma Initiating stem-like Cells (GICs). Nanoparticles mediated delivery of chemotherapeutic agent targeting both cancer and glioma stem cells could provide a solution to recurrent malignancies of the glioblastoma tumor. The approach described here provides enhanced chemotherapeutic potency utilizing 1,3ß-Glucan as an outer shell to the chitosan nanoparticles (Cs-NPs) loaded with paclitaxel to prevent hemolysis with, the core-shell nano-structure (Cs-PTX-NP) enabling effective chemotherapy against malignant glioblastoma. The prepared nanoparticles (1,3ß-Cs-PTX-NPs) with sustained release of the paclitaxel provide a targeted therapeutic approach that overcome systemic toxicities with the 1,3ß-Glucan shell and improve drug bioavailability. Hemolysis investigation indicated that 1,3ß-Cs-PTX-NP was significantly less hemolytic than paclitaxel enabling intravenous delivery. Also, 1,3ß-Cs-PTX-NPs were considerably more cytotoxic (IC50) against glioma cancer LN18 cells and C6 stem-like cells compared with the PTX. In conclusion, this study found that 1,3ß-Cs-PTX-NP addressed serious limitation with systemic delivery of paclitaxel by preventing hemolysis and providing chemotherapeutic delivery with significant anti-cancer efficacy against recurrent glioblastoma.

Role of the bradykinin B2 receptor in a rat model of local heart irradiation.

PURPOSE: Radiation-induced heart disease (RIHD) is a delayed effect of radiotherapy for cancers of the chest, such as breast, esophageal, and lung. Kinins are small peptides with cardioprotective properties. We previously used a rat model that lacks the precursor kininogen to demonstrate that kinins are involved in RIHD. Here, we examined the role of the kinin B2 receptor (B2R) in early radiation-induced signaling in the heart. MATERIALS AND METHODS: Male Brown Norway rats received the B2R-selective antagonist HOE-140 (icatibant) via osmotic minipump from 5 days before until 4 weeks after 21 Gy local heart irradiation. At 4 weeks, signaling events were measured in left ventricular homogenates and nuclear extracts using western blotting and real-time polymerase chain reaction. Numbers of CD68-positive (monocytes/macrophages), CD2-positive (T-lymphocytes), and mast cells were measured using immunohistochemistry. RESULTS: Radiation-induced c-Jun phosphorylation and nuclear translocation were enhanced by HOE-140. HOE-140 did not modify endothelial nitric oxide synthase (eNOS) phosphorylation or alter numbers of CD2-positive or mast cells, but enhanced CD68-positive cell counts in irradiated hearts. CONCLUSIONS: B2R signaling may regulate monocyte/macrophage infiltration and c-Jun signals in the irradiated heart. Although eNOS is a main target for kinins, the B2R may not regulate eNOS phosphorylation in response to radiation.

The ß3 subunit contributes to vascular calcium channel upregulation and hypertension in angiotensin II-infused C57BL/6 mice.

Voltage-gated L-type Ca(2+) (Ca(v)1.2) channels in vascular smooth muscle cells are a predominant Ca(2+) influx pathway that mediates arterial tone. Channel biogenesis is accomplished when the pore-forming α(1C) subunit coassembles with regulatory Ca(v)ß subunits intracellularly, and the multiprotein Ca(v)1.2 channel complex translocates to the plasma membrane to form functional Ca(2+) channels. We hypothesized that the main Ca(v)ß isoform in vascular smooth muscle cells, Ca(v)ß3, is required for the upregulation of arterial Ca(v)1.2 channels during the development of hypertension, an event associated with abnormal Ca(2+)-dependent tone. Ca(v)1.2 channel expression and function were compared between second-order mesenteric arteries of C57BL/6 wild-type (WT) and Ca(v)ß3(-/-) mice infused with saline (control) or angiotensin II (Ang II) for 2 weeks to induce hypertension. The mesenteric arteries of Ang II-infused WT mice showed increased Ca(v)1.2 channel expression and accentuated Ca(2+)-mediated contractions compared with saline-infused WT mice. In contrast, Ca(v)1.2 channels failed to upregulate in mesenteric arteries of Ang II-infused Ca(v)ß3(-/-) mice, and Ca(2+)-dependent reactivity was normal in these arteries. Basal systolic blood pressure was not significantly different between WT and Ca(v)ß3(-/-) mice (98 ± 2 and 102 ± 3 mm Hg, respectively), but the Ca(v)ß3(-/-) mice showed a blunted pressor response to Ang II infusion. Two weeks after the start of Ang II administration, the systolic blood pressure of Ca(v)ß3(-/-) mice averaged 149 ± 4 mm Hg compared with 180 ± 5 mm Hg in WT mice. Thus, the Ca(v)ß3 subunit is a critical regulatory protein required to upregulate arterial Ca(v)1.2 channels and fully develop Ang II-dependent hypertension in C57BL/6 mice.