FoxO3a, a forkhead family member of transcription factors, is involved in the regulation of cell metabolism, proliferation, differentiation and apoptosis. However, whether FoxO3a participates in the regulation of glucocorticoids induced-hypothalamic-pituitary-adrenal (HPA) dysfunction is still unknown. Our present results indicate that dexamethasone(DEX) increased FoxO3a expression in PC12 and hypothalamic neuronal cultures in correlation to reduced expression of NPW, a process that could be blocked by GR2 antagonist. DEX restrained the phosphorylation of Akt and FoxO3a, but not ERK1/2 phosphorylation, resulting with FoxO3a nuclear localization. Overexpression of FoxO3a inhibited NPW expression, while FoxO3a knockdown by siRNA had the opposite effect. The regulatory region of NPW promoter contains multiple FoxO3a binding sites, and FoxO3a bonding to these sites inhibited its transcriptional activity. In a rat model, chronic administration of corticosterone reduced animals' body weight and sucrose consumption and caused stress-depression like behavior. Corticosterone treatment induced a marked increase in FoxO3a levels, while decreased the expression of NPW protein in the hypothalamus. Immunofluorescent double labeling demonstrated that FoxO3a and NPW were collocated in the hypothalamus. Taken together, these data indicate that NPW is a new direct downstream target gene of FoxO3a. FoxO3a suppressed the transcription of NPW and modulated glucocorticoids-induced HPA dysfunction by directly regulating the expression of NPW. Thus, present findings suggest that FoxO3a and NPW may be potential therapeutic targets for endocrine and psychiatric disorders.

Peripheral nerve injuries can be extremely debilitating, resulting in sensory and motor loss-of-function. Endogenous repair is limited to non-severe injuries in which transection of nerves necessitates surgical intervention. Traditional treatment approaches include the use of biological grafts and alternative engineering approaches have made progress. The current article serves as a comprehensive, in-depth perspective on peripheral nerve regeneration, particularly nerve guidance conduits and drug delivery strategies. A detailed background of peripheral nerve injury and repair pathology, and an in-depth look into augmented nerve regeneration, nerve guidance conduits, and drug delivery strategies provide a state-of-the-art perspective on the field.

The average respiration rate for an adult is 12-20 breaths per minute, which constantly exposes the lungs to allergens and harmful particles. As a result, respiratory diseases, which includes asthma, chronic obstructive pulmonary disease (COPD) and acute lower respiratory tract infections (LTRI), are a major cause of death worldwide. Although asthma, COPD and LTRI are distinctly different diseases with separate mechanisms of disease progression, they do share a common feature - airway inflammation with intense recruitment and activation of granulocytes and mast cells. Neutrophils, eosinophils, basophils, and mast cells are crucial players in host defense against pathogens and maintenance of lung homeostasis. Upon contact with harmful particles, part of the pulmonary defense mechanism is to recruit these cells into the airways. Despite their protective nature, overactivation or accumulation of granulocytes and mast cells in the lungs results in unwanted chronic airway inflammation and damage. As such, understanding the bright and the dark side of these leukocytes in lung physiology paves the way for the development of therapies targeting this important mechanism of disease. Here we discuss the role of granulocytes in respiratory diseases and summarize therapeutic strategies focused on granulocyte recruitment and activation in the lungs.

Many toxins from a variety of venomous animals and plants have evolved to target neuronal ion channels and receptors. However, a significant obstacle in the study of these toxins is the finding and characterization of their specific molecular target. Here, we describe a method for fast and efficient screening of venom and toxin activity using live-cell calcium imaging. We describe the use of Fura-2, a calcium indictor that changes its fluorescence properties in response to intracellular calcium elevations, to measure the activity of neurons from the dorsal root and trigeminal ganglia. Calcium imaging is an efficient technique for testing many of the venom's components on large numbers of neurons simultaneously. This technique offers a novel tool for low-cost and rapid characterization of functionally active toxins and their target receptors.

Biodegradable poly(l-lactide-co-$ε$-caprolactone) (PLCL) are used to prepare inflatable balloon implants in treating rotator-cuff injuries and tissue separation. These balloon implants act as a temporary spacer for tissues, while reducing pain and allowing rehabilitation after surgery. It is essential to ensure that each balloon fulfill two requirements after implantation: (1) display a well-defined degradation profile, and (2) remain unaffected by premature rapture or leakage. Storage also affects the stability of a polymer-based implant. Since the balloons are implanted into humans, it is essential to understand their in vitro and in vivo degradation along with their physicochemical properties. It is unpredictable if balloon storage on their performance. Therefore, the in vitro and in vivo degradation behavior of PLCL balloons was examined during one year, and the information obtained was used to correlate reliability under prolonged storage conditions. We investigated changes in weight, melting temperature (T(m)), molecular weight distribution (M(w), M(n) and PDI), crystallinity (Χ), optical activity [$\alpha$], and inherent viscosity ($η$) of the balloons during the entire degradation time. We also examined the molecular properties of the balloons under annealing and extreme temperature conditions, such as the combined effect of temperature and humidity that simulate various storage conditions. We have concluded that degradation of the PLCL balloons is slow, and they remain stable during the test period. Results reveal that the balloons retain their molecular properties under long-term storage, annealing, and extreme temperature conditions. The balloons did not show any variation from reference samples, and they exhibited a constant stability profile even after shelf-storage of more than 3 years. These findings can serve as a case study for evaluating various other biodegradable materials.

This study investigated the effects of drought stress on Chinese cabbage (Chcab) by measuring plant growth responses, total antioxidant enzyme activities, the contents of bioactive compounds including glucosinolates (GLS, aliphatic and indolic), and binding with human serum albumin (HSA). Forty-day-old Chinese cabbage (Brassica rapa L. ssp. pekinensis) seedlings were transplanted into pots and maintained for three weeks at 10% (drought-treated, D-T) and 30% (control, C) soil water. The total leaf number, leaf area, and fresh and dry weights were significantly lower in D-T Chcab than in controls. Total GLSs and catalase activities were found to be significantly higher in D-T Chcab than in controls. Indolic GLSs were significantly higher than aliphatic GLSs in D-T Chcab. These results show that D-T Chcab reduced growth parameters and binding properties with HSA and influenced total contents of GLSs, polyphenols, flavonoids, total antioxidant enzyme activities, catalase and peroxidase.

The B7 family member, B7H6, is a ligand for the natural killer cell receptor NKp30. B7H6 is hardly expressed on normal tissues, but undergoes upregulation on different types of tumors, implicating it as an attractive target for cancer immunotherapy. The molecular mechanisms that control B7H6 expression are poorly understood. We report that in contrast to other NK cell ligands, endoplasmic reticulum (ER) stress upregulates B7H6 mRNA levels and surface expression. B7H6 induction by ER stress requires protein kinase R-like ER kinase (PERK), one of the three canonical sensors of the unfolded protein response. PERK phosphorylates eIF2$\alpha$, which regulates protein synthesis and gene expression. Because eIF2$\alpha$ is phosphorylated by several kinases following different stress conditions, the program downstream to eIF2$\alpha$ phosphorylation is called the integrated stress response (ISR). Several drugs were reported to promote the ISR. Nelfinavir and lopinavir, two clinically approved HIV protease inhibitors, promote eIF2$\alpha$ phosphorylation by different mechanisms. We show that nelfinavir and lopinavir sustainably instigate B7H6 expression at their pharmacologically relevant concentrations. As such, ER stress and ISR conditions sensitize melanoma targets to CAR-T cells directed against B7H6. Our study highlights a novel mechanism to induce B7H6 expression and suggests a pharmacological approach to improve B7H6-directed immunotherapy. KEY MESSAGES: B7H6 is induced by ER stress in a PERK-dependent mechanism. Induction of B7H6 is obtained pharmacologically by HIV protease inhibitors. Exposure of tumor cells to the HIV protease inhibitor nelfinavir improves the recognition by B7H6-directed CAR-T.

Recent developments in near-infrared (NIR) dyes and imaging modalities enable tumor fluorescent images in preclinical and clinical settings. However, NIR dyes have several drawbacks, and therefore, there is an unmet diagnostic need for NIR dye encapsulation in appropriate pharmaceutical nanocarriers with targeting abilities for the purpose of achieving effective diagnosis and image-guided surgeries. Because integrin receptors are established diagnostic targets, the cyclic Arg-Gly-Asp (RGD) peptides, recognizing the $\alpha$(V)$\beta$(3) integrin, have been extensively investigated for radiology and bioimaging of tumors. However, the Lys(Arg)-Thr-Ser [K(R)TS] cyclic peptides, selective for collagen receptors $\alpha$(1)$\beta$(1)/$\alpha$(2)$\beta$(1) integrins, which are overexpressed in many tumors, were not yet investigated and therefore used here for tumor bioimaging with a unique $\alpha$(2)$\beta$(1)-integrin-targeted nanocarrier, encapsulating the indocyanine green NIR dye. We synthesized three kinds of peptides: two cyclic RTS peptides functional only in the cyclic conformation and a linear peptide lacking the cyclic cysteine constrained RTS loop. We used them for the preparation of integrin-targeted self-assembled nanocarriers (ITNCs), referred to as OF5 and OF27, and a nontargeted control nanocarrier, referred to as OF70. Their selective association was demonstrated with $\alpha$(2)$\beta$(1) integrin expressing cell cultures and three-dimensional tumor spheroids and by competition with a $\alpha$(2)$\beta$(1) selective disintegrin. Cytotoxicity experiments in vitro demonstrated the safety of the ITNCs. The targeting potential and the biodistribution of the ITNCs, applied intravenously in A431 tumor-bearing nude mice, were evaluated in vivo using NIR bioimaging. Time-dependent biodistributions indicated that the ITNC OF27 showed higher fluorescent signals in main tissues, with no cytotoxic effects to major organs, and presented higher accumulation in tumors. Cumulatively, these results highlight the potential of the ITNC OF27 as an optical and innovative pharmaceutical bioimaging system, suitable for integrin $\alpha$(2)$\beta$(1) receptor in vivo tumor targeting and visualization in the NIR region.

For highly lipophilic drugs, passage into the intestinal lymphatic system rather than the portal vein following oral administration may represent a major alternative route of delivery into the general circulation. Increasing intestinal lymphatic transport provides an effective strategy to improve oral bioavailability when hepatic first-pass metabolism is a major rate-limiting step hampering access to the systemic circulation after oral dosing. The transfer of orally administered, highly lipid-soluble drugs to the lymphatic system is mediated by their association with chylomicrons, large intestinal lipoproteins that are assembled in the enterocytes in the presence of long-chain triglycerides or long-chain fatty acids. Due to its very high lipophilicity, cannabidiol (CBD) has physicochemical features (e.g. logP = 6.3) consistent with an oral absorption mediated at least in part by transport via the intestinal lymphatic system. CBD also undergoes extensive first-pass hepatic metabolism. Formulation changes favoring diversion of orally absorbed CBD from the portal to the lymphatic circulation pathway can result in reduced first-pass liver metabolism, enhanced oral bioavailability, and reduced intra- and intersubject variability in systemic exposure. In this manuscript, we discuss (1) evidence for CBD undergoing hepatic first-pass liver metabolism and lymphatic absorption to a clinically important extent; (2) the potential interplay between improved oral absorption, diversion of orally absorbed drug to the lymphatic system, and magnitude of presystemic elimination in the liver; and (3) strategies by which innovative chemical and/or pharmaceutical delivery systems of CBD with improved bioavailability could be developed.

The cannabis plant has been widely researched for many therapeutic indications and found to be effective in many chronic conditions such as epilepsy, neuropathic or chronic pain and more. However, biased opinion against compounds of the plant, regulatory as well as compounding challenges have led to very few approved cannabinoid medicinal products. Those formulations which are approved are dosed several times a day, creating an unmet need for controlled release (CR) formulations of cannabinoids. Conventional CR formulations rely on prolonged absorption of the drug, including absorption from the colon. The purpose of this work is to investigate regional absorption of major cannabinoids THC and CBD from the colon and develop a suitable CR formulation. As hypothesized by researchers, THC and CBD have poor absorption from the colon compared to small intestine, suggesting that these compounds have a narrow absorption window. The suggested CR formulation examined in-vitro was a floating gastro retentive tablet based on egg albumin matrix, gas generating agents and surfactants. In-vivo investigation of CBD containing formulation in the freely moving rat model proved a prolonged absorption phase with a substantial increase in bioavailability compared to CBD solution. The findings of this paper answer a crucial question regarding potential application of CR dosage forms for cannabinoids and shed light on the regional intestinal absorption of these compounds. Ultimately, these results cement the way for future development of cannabinoid gastro retentive dosage forms.

We report here on ion-exchange polymeric nanoparticles from a linear copolymer of maleic anhydride methyl vinyl ether esterified with 30% octadecanol. The side chains for the polymer structure were optimized through metadynamics simulations, which revealed the use of octadecanol esters generates ideal free energy surfaces for drug encapsulation and release. Nanoparticles were synthesized using a solvent evaporation-precipitation method by mixing the polymer solution in acetone into water; upon acetone evaporation, a nanodispersion with an average particle size of ∼150 nm was obtained. Gentamicin sulfate, possessing five amino groups, was spontaneously entrapped in the nanocarrier by ionic interactions. Encapsulation efficiency increases significantly with the increase in pH and ionic strength. In vivo results demonstrate high gentamicin (GM) content in the enteric chamber (AUC 8207 ± 1334 ($μ$g min)/mL) compared to 3% GM solution (AUC 2024 ± 438 ($μ$g min)/mL). The formulation was also able to significantly extend the release of gentamicin when applied to rabbit cornea. These anionic nanoparticles can be used for extended-release of other cationic drugs.

Amiodarone is an anti-arrhythmic drug that was approved by the US Food and Drug Administration (FDA) in 1985. Pre-clinical studies suggest that Amiodarone induces cytotoxicity in several types of cancer cells, thus making it a potential candidate for use as an anti-cancer treatment. However, it is also known to cause a variety of severe side effects. We hypothesized that in addition to the cytotoxic effects observed in cancer cells Amiodarone also has an indirect effect on angiogensis, a key factor in the tumor microenvironment. In this study, we examined Amiodarone's effects on a murine tumor model comprised of U-87 MG glioblastoma multiforme (GBM) cells, known to form highly vascularized tumors. We performed several in vitro assays using tumor and endothelial cells, along with in vivo assays utilizing three murine models. Low dose Amiodarone markedly reduced the size of GBM xenograft tumors and displayed a strong anti-angiogenic effect, suggesting dual cancer fighting properties. Our findings lay the ground for further research of Amiodarone as a possible clinical agent that, used in safe doses, maintains its dual properties while averting the drug's harmful side effects.

BACKGROUND: Accumulating public health and epidemiological literature support the hypothesis that arsenic in drinking water or food affects the brain adversely. METHODS: Experiments on the consequences of nitric oxide (NO) formation in neuronal cell culture and mouse brain were conducted to probe the mechanistic pathways of nitrosative damage following arsenic exposure. RESULTS: After exposure of mouse embryonic neuronal cells to low doses of sodium arsenite (SA), we found that Ca(2+) was released leading to the formation of large amounts of NO and apoptosis. Inhibition of NO synthase prevented neuronal apoptosis. Further, SA led to concerted S-nitrosylation of proteins significantly associated with synaptic vesicle recycling and acetyl-CoA homeostasis. Our findings show that low-dose chronic exposure (0.1-1 ppm) to SA in the drinking water of mice led to S-nitrosylation of proteomic cysteines. Subsequent removal of arsenic from the drinking water reversed the biochemical alterations. CONCLUSIONS: This work develops a mechanistic understanding of the role of NO in arsenic-mediated toxicity in the brain, incorporating Ca(2+) release and S-nitrosylation as important modifiers of neuronal protein function.

Snake and spider envenomation have a considerable impact on public health. Their pathology is induced by a variety of toxins composing the venom which induce cytotoxicity to cells of different organs by several cell death pathways. Described in this chapter are methods in vitro used to assess venoms and toxin-induced cell death using mammalian cell cultures. The chapter is divided into five sections: (1) a brief overview of in vitro cytotoxicity and categories of cell death induced by venoms and toxins; (2) a common method to measure necrotic cell death using lactate dehydrogenase (LDH) release; (3) a flow cytometry method that simultaneously measures necrosis and apoptosis; (4) measurements of nuclear morphology; and (5) measurements of the autophagy following microtubule-associated protein light chain 3 (LC3) expression, by immunoblotting and by fluorescence microscopy of LC3-positive vesicles, to assess the levels of autophagosomes.

Pt(II) complexes are commonly used to treat cancer. To reduce their side effects and improve their pharmacological properties, Pt(IV) complexes are being developed as prodrug candidates that are activated by reduction in cancer cells. Concomitantly, Ru(II) polypyridine complexes have gained much attention as photosensitizers for use in photodynamic therapy due to their attractive characteristics. In this article, a novel Pt(IV) -Ru(II) conjugate, which combines cancer activated chemotherapy with PDT, is presented. Upon entering the cancer cell, the Pt(IV) centre is reduced to Pt(II) and the axial ligands including the Ru(II) complex and phenylbutyrate are released. As each component has its individual targets, the conjugate exerts a multi-target and multi-action effect with (photo-)cytotoxicity values upon irradiation up to 595 nm in the low nanomolar range in various (drug resistant) 2D monolayer cancer cells and 3D multicellular tumour spheroids.

Multiaction Pt(IV) prodrugs can overcome resistance associated with the FDA approved Pt(II) drugs like cisplatin. Intracellular reduction of the octahedral Pt(IV) derivatives of cisplatin releases cisplatin and the two axial ligands. When the released axial ligands act synergistically with cisplatin to kill the cancer cells, we have multiaction prodrugs. Most Pt(IV) multiaction prodrugs have bioactive ligands possessing a carboxylate that is conjugated to the Pt(IV) because breaking the Pt(IV)-ligand bond releases the active moiety. As many drugs that act synergistically with cisplatin do not have carboxylates, a major challenge is to prepare multiaction Pt(IV) complexes with drugs that have amino groups or hydroxyl groups such that following reduction, the drugs are released in their active form. Our objective was to prepare multiaction Pt(IV) prodrugs that release bioactive molecules having amino groups. Because we cannot conjugate amino groups to the axial position of Pt(IV), we developed a novel and efficient approach for the synthesis of Pt(IV)-carbamato complexes and demonstrated that following reduction of the Pt(IV), the released carbamates undergo rapid decarboxylation, releasing the free amine, as in the case of the PARP-1 inhibitor 3-aminobenzamide and the amino derivative of the HDAC inhibitor SAHA. Pt(IV)-carbamato complexes are stable in cell culture medium and are reduced by ascorbate. They are reduced slower than their carboxylato and carbonato analogues. We believe that this approach paves the way for preparing novel classes of multiaction Pt(IV) prodrugs with amino containing bioactive molecules that up to now were not accessible.

Dysregulated myeloid cell activity underlies a variety of pathologies, including immunosuppression in malignant cancers. Current treatments to alter myeloid cell behavior also alter other immune cell subpopulations and nonimmune cell types with deleterious side effects. Therefore, improved selectivity of myeloid treatment is an urgent need. To meet this need, we demonstrate a novel, targeted nanoparticle system that achieves superior myeloid selectivity both in vitro and in vivo. This system comprises: (1) granulocyte-colony stimulating factor (G-CSF) as a targeting ligand to promote accumulation in myeloid cells, including immunosuppressive myeloid-derived suppressor cells (MDSCs); (2) albumin nanoparticles 100-120 nm in diameter that maintain morphology and drug payload in simulated physiological conditions; and (3) a fluorophore that enables nanoparticle tracking and models a therapeutic molecule. Here, we show that this strategy achieves high myeloid uptake in mixed primary immune cells and that nanoparticles successfully infiltrate the 4T1 triple-negative breast tumor murine microenvironment, where they preferentially accumulate in myeloid cells in a mouse model. Further development will realize diagnostic myeloid cell tracking applications and therapeutic delivery of myeloid-reprogramming drugs.

Cancer recurrence is one of the most imminent problems in the current world of medicine, and it is responsible for most of the cancer-related death rates worldwide. Long-term administration of anticancer cytotoxic drugs may act as a double-edged sword, as necrosis may lead to renewed cancer progression and treatment resistance. The lack of nutrients, coupled with the induced hypoxia, triggers cell death and secretion of signals that affect the tumor niche. Many efforts have been made to better understand the contribution of hypoxia and metabolic stress to cancer progression and resistance, but mostly with respect to inflammation. Here we provide an overview of the direct anticancer effects of necrotic signals, which are not necessarily mediated by inflammation and the role of DAMPs (damage-associated molecular patterns) on the formation of a pro-cancerous environment.

Polyneuropathy is a disease involving multiple peripheral nerves injuries. Axon regrowth remains the major prerequisite for plasticity, regeneration, circuit formation, and eventually functional recovery and therefore, regulation of neurite outgrowth might be a candidate for treating polyneuropathies. In a recent study, we synthesized and established the methylene-cycloalkylacetate (MCAs) pharmacophore as a lead for the development of a neurotropic drug (inducing neurite/axonal outgrowth) using the PC12 neuronal model. In the present study we extended the characterizations of the in vitro neurotropic effect of the derivative 3-(3-allyl-2-methylenecyclohexyl) propanoic acid (MCA-13) on dorsal root ganglia and spinal cord neuronal cultures and analyzed its safety properties using blood biochemistry and cell counting, acute toxicity evaluation in mice and different in vitro "off-target" pharmacological evaluations. This MCA derivative deserves further preclinical mechanistic pharmacological characterizations including therapeutic efficacy in in vivo animal models of polyneuropathies, toward development of a clinically relevant neurotropic drug.

The goal of this work was to study the characteristics of a new phospholipid nanovesicular carrier for nasal administration of drugs. Multilamellar vesicles were visualized by electron microscopy, and their mean distribution size of 200 nm was evaluated by DLS. Measured pH and viscosity values were found adequate for a nasal delivery carrier. CLS micrographs of the nasal mucosa of rats following administration of the carrier incorporating probes with various properties show delivery into the nasal mucosa layers. Tramadol containing systems were characterized and tested for their analgesic effect in two pain animal models. In mice, a significantly higher antinociceptive effect and a rapid onset of action were obtained as compared to other nasal delivery carriers and to oral treatment. This enhanced analgesic effect was further confirmed in rat pain model and sustained by drug plasma and brain levels. To test the systems behavior in a larger animal, a pharmacokinetic crossover study was carried out in sheep after administrating Tramadol nasally in the nanocarrier and IV. The plasma and CSF absolute bioavailability values were 1.09 and 0.87, respectively. HPLC and LC-MS/MS methods for quantification of Tramadol in plasma, brain and CSF were developed and are presented here. It is noteworthy that no pathological alterations or inflammation signs were observed in rat nasal mucosa following sub-chronic treatment. The results obtained in this work encourage further investigation of using the new carrier for nasal delivery of drugs in humans.