Sulforaphane inhibits growth of human breast cancer cells and augments the therapeutic index of the chemotherapeutic drug, gemcitabine

Phytochemicals are among the natural chemopreventive agents with most potential for delaying, blocking or reversing the initiation and promotional events of carcinogenesis. They therefore offer cancer treatment strategies to reduce cancer related death. One such promising chemopreventive agent which has attracted considerable attention is sulforaphane (SFN), which exhibits anti-cancer, anti-diabetic, and anti-microbial properties. The present study was undertaken to assess effect of SFN alone and in combination with a chemotherapeutic agent, gemcitabine, on the proliferative potential of MCF-7 cells by cell viability assay and authenticated the results by nuclear morphological examination. Further we analyzed the modulation of expression of Bcl-2 and COX-2 on treatment of these cells with SFN by RT-PCR. SFN showed cytotoxic effects on MCF-7 cells in a dose- and time-dependent manner via an apoptotic mode of cell death. In addition, a combinational treatment of SFN and gemcitabine on MCF-7 cells resulted in growth inhibition in a synergistic manner with a combination index (CI)\u3c1. Notably, SFN was found to significantly downregulate the expression of Bcl-2, an anti-apoptotic gene, and COX-2, a gene involved in inflammation, in a time-dependent manner. These results indicate that SFN induces apoptosis and anti-inflammatory effects on MCF-7 cells via downregulation of Bcl-2 and COX-2 respectively. The combination of SFN and gemcitabine may potentiate the efficacy of gemcitabine and minimize the toxicity to normal cells. Taken together, SFN may be a potent anti-cancer agent for breast cancer treatment

Human iPS cell-derived astrocyte transplants preserve respiratory function after spinal cord injury.

Transplantation-based replacement of lost and/or dysfunctional astrocytes is a promising therapy for spinal cord injury (SCI) that has not been extensively explored, despite the integral roles played by astrocytes in the central nervous system (CNS). Induced pluripotent stem (iPS) cells are a clinically-relevant source of pluripotent cells that both avoid ethical issues of embryonic stem cells and allow for homogeneous derivation of mature cell types in large quantities, potentially in an autologous fashion. Despite their promise, the iPS cell field is in its infancy with respect to evaluating in vivo graft integration and therapeutic efficacy in SCI models. Astrocytes express the major glutamate transporter, GLT1, which is responsible for the vast majority of glutamate uptake in spinal cord. Following SCI, compromised GLT1 expression/function can increase susceptibility to excitotoxicity. We therefore evaluated intraspinal transplantation of human iPS cell-derived astrocytes (hIPSAs) following cervical contusion SCI as a novel strategy for reconstituting GLT1 expression and for protecting diaphragmatic respiratory neural circuitry. Transplant-derived cells showed robust long-term survival post-injection and efficiently differentiated into astrocytes in injured spinal cord of both immunesuppressed mice and rats. However, the majority of transplant-derived astrocytes did not express high levels of GLT1, particularly at early times post-injection. To enhance their ability to modulate extracellular glutamate levels, we engineered hIPSAs with lentivirus to constitutively express GLT1. Overexpression significantly increased GLT1 protein and functional GLT1-mediated glutamate uptake levels in hIPSAs both in vitro and in vivo post-transplantation. Compared to human fibroblast control and unmodified hIPSA transplantation, GLT1-overexpressing hIPSAs reduced (1) lesion size within the injured cervical spinal cord, (2) morphological denervation by respiratory phrenic motor neurons at the diaphragm neuromuscular junction, and (3) functional diaphragm denervation as measured by recording of spontaneous EMGs and evoked compound muscle action potentials. Our findings demonstrate that hiPSA transplantation is a therapeutically-powerful approach for SCI

Epicutaneous Sensitization to the Phytocannabinoid β-Caryophyllene Induces Pruritic Inflammation

In recent years, there has been increased accessibility to cannabis for recreational and medicinal use. Incidentally, there has been an increase in reports describing allergic reactions to cannabis including exacerbation of underlying asthma. Recently, multiple protein allergens were discovered in cannabis, yet these fail to explain allergic sensitization in many patients, particularly urticaria and angioedema. Cannabis has a rich chemical profile including cannabinoids and terpenes that possess immunomodulatory potential. We examined whether major cannabinoids of cannabis such as cannabidiol (CBD) and the bicyclic sesquiterpene beta-caryophyllene (β-CP) act as contact sensitizers. The repeated topical application of mice skin with β-CP at 10 mg/mL (50 µL) induced an itch response and dermatitis at 2 weeks in mice, which were sustained for the period of study. Histopathological analysis of skin tissues revealed significant edema and desquamation for β-CP at 10 mg/mL. For CBD and β-CP, we observed a dose-dependent increase in epidermal thickening with profound thickening observed for β-CP at 10 mg/mL. Significant trafficking of CD11b cells was observed in various compartments of the skin in response to treatment with β-CP in a concentration-dependent manner. Mast cell trafficking was restricted to β-CP (10 mg/mL). Mouse proteome profiler cytokine/chemokine array revealed upregulation of complement C5/5a (anaphylatoxin), soluble intracellular adhesion molecule-1 (sICAM-1) and IL-1 receptor antagonist (IL-1RA) in animals dosed with β-CP (10 mg/mL). Moreover, we observed a dose-dependent increase in serum IgE in animals dosed with β-CP. Treatment with β-CP (10 mg/mL) significantly reduced filaggrin expression, an indicator of barrier disruption. In contrast, treatment with CBD at all concentrations failed to evoke scratching and dermatitis in mice and did not result in increased serum IgE. Further, skin tissues were devoid of any remarkable features, although at 10 mg/mL CBD we did observe the accumulation of dermal CD11b cells in skin tissue sections. We also observed increased filaggrin staining in mice repeatedly dosed with CBD (10 mg/mL). Collectively, our studies indicate that repeated exposure to high concentrations of β-CP can induce dermatitis-like pathological outcomes in mice

Overexpression of the astrocyte glutamate transporter GLT1 exacerbates phrenic motor neuron degeneration, diaphragm compromise, and forelimb motor dysfunction following cervical contusion spinal cord injury.

A major portion of spinal cord injury (SCI) cases affect midcervical levels, the location of the phrenic motor neuron (PhMN) pool that innervates the diaphragm. While initial trauma is uncontrollable, a valuable opportunity exists in the hours to days following SCI for preventing PhMN loss and consequent respiratory dysfunction that occurs during secondary degeneration. One of the primary causes of secondary injury is excitotoxic cell death due to dysregulation of extracellular glutamate homeostasis. GLT1, mainly expressed by astrocytes, is responsible for the vast majority of functional uptake of extracellular glutamate in the CNS, particularly in spinal cord. We found that, in bacterial artificial chromosome-GLT1-enhanced green fluorescent protein reporter mice following unilateral midcervical (C4) contusion SCI, numbers of GLT1-expressing astrocytes in ventral horn and total intraspinal GLT1 protein expression were reduced soon after injury and the decrease persisted for ≥6 weeks. We used intraspinal delivery of adeno-associated virus type 8 (AAV8)-Gfa2 vector to rat cervical spinal cord ventral horn for targeting focal astrocyte GLT1 overexpression in areas of PhMN loss. Intraspinal delivery of AAV8-Gfa2-GLT1 resulted in transduction primarily of GFAP(+) astrocytes that persisted for ≥6 weeks postinjury, as well as increased intraspinal GLT1 protein expression. Surprisingly, we found that astrocyte-targeted GLT1 overexpression increased lesion size, PhMN loss, phrenic nerve axonal degeneration, and diaphragm neuromuscular junction denervation, and resulted in reduced functional diaphragm innervation as assessed by phrenic nerve-diaphragm compound muscle action potential recordings. These results demonstrate that GLT1 overexpression via intraspinal AAV-Gfa2-GLT1 delivery exacerbates neuronal damage and increases respiratory impairment following cervical SCI

Intermediate Filaments in Bladder Smooth Muscle Dysfunction

Lower urinary tract symptoms (LUTS) is associated with bladder smooth muscle (BSM) hypertrophy secondary to benign prostatic hyperplasia (BPH)-induced partial bladder outlet obstruction (PBOO), and its incidence and prevalence are increasing as the population ages. Symptoms of lower urinary tract disorder (LUTD) are costly to diagnose and treat; existing therapeutic interventions are neither highly effective nor durable and have side effects of their own. Establishing the molecular pathogenesis of PBOO is an important step in identifying new therapeutic targets. Physiological and biochemical studies have shown reduced contraction, decreased mitochondrial ATP levels and increased reactive oxygen species (ROS) production in the hypertrophied BSM of obstructed bladders. Bladder hypertrophy is also associated with overexpression of the intermediate filament (IF) proteins, desmin and vimentin which are important for the cytoskeletal structure, contraction and mitochondrial respiratory function. Mitochondrial and muscle pathophysiology, including decreased ATP, increased ROS production and diminished contractility of smooth muscle in LUTD suggests a role for desmin and vimentin overexpression in inducing mitochondrial and muscle dysfunction. However, potential mechanisms by which these IF proteins mediate BSM dysfunction remain unexplored. Our preliminary data demonstrate that desmin and vimentin overexpression in human and murine BSM strips and cells significantly reduces carbachol- and KCl- mediated contraction as also seen in BSM with PBOO. Overexpression of IF proteins in BSM strips and cells enhances mitogen-activated protein kinase (MAPK)/JNK2 phosphorylation. Ultrastructural and biochemical studies of bladder outlet obstruction (BOO)-induced remodeled BSM reveal accumulation of damaged mitochondria with concomitant decrease in ATP and increase in reactive oxygen species (ROS) in BSM. Based on our preliminary data, we hypothesize that the activation of JNK2 is involved in desmin- and vimentin-induced BSM contractile dysfunction. The research work presented in this PhD thesis demonstrates that the increased expression of desmin and vimentin reduces bladder smooth muscle contractility via JNK2 and induces mitochondrial dysfunction. Studies in Aim 1 were designed to address the hypothesis that increased expression of IF proteins desmin and vimentin induce BSM hypocontractility and this phenomenon is mediated via JNK2, a member of MAP kinase family protein. Overexpression of desmin and vimentin in murine BSM strips resulted in decreased contraction in response to carbachol and KCl with an associated decrease in phosphorylation of myosin light chain kinase (MLC20) and an increased accumulation of phospho-JNK2. Furthermore, desmin and vimentin overexpression-induced loss of BSM contractility was abrogated in strips isolated from JNK2 knockout mice or in BSM strips treated with JNK2 inhibitor. These findings demonstrate that IF regulate BSM contractility via JNK2. Studies in Aim 2 were designed to address the hypothesis that IF-induced BSM hypocontractility involves mitochondrial mechanisms. Using our in vitro overexpression model, our studies demonstrate that increased expression desmin or vimentin in human BSM cells reduces mitochondrial membrane potential (MMP), mitochondrial respiration and ATP production and increases ROS production in BSM. Overall, our findings suggest JNK2 as a major effector of IF protein overexpression induced BSM contractile dysfunction. Further, our data implicate an IFs/JNK2 dependent mechanism that contributes to the pathologic bladder contractility that occurs following bladder outlet obstruction, a condition in which desmin and vimentin protein expressions are known to be up regulated. Together, these findings suggest that desmin-JNK2 and vimentin-JNK2 axes are likely to contribute to diminished BSM contractility associated with obstructive bladder disease and that JNK2 inhibition may have therapeutic application. In addition, desmin- and vimentin- overexpression induces mitochondrial dysfunction which possibly contributes to BSM hypocontractility. Future studies are needed to ascertain the role of JNK2 in inducing mitochondrial dysfunction in obstructed bladder

Managing Access to Confidential Documents: A Case Study of an Email Security Tool

User adoption and usage of end-to-end encryption tools is an ongoing research topic. A subset of such tools allows users to encrypt confidential emails, as well as manage their access control using features such as the expiration time, disabling forwarding, persistent protection, and watermarking. Previous studies have suggested that protective attitudes and behaviors could improve the adoption of new security technologies. Therefore, we conducted a user study on 19 participants to understand their perceptions of an email security tool and how they use it to manage access control to confidential information such as medical, tax, and employee information if sent via email. Our results showed that the participants’ first impression upon receiving an end-to-end encrypted email was that it looked suspicious, especially when received from an unknown person. After the participants were informed about the importance of the investigated tool, they were comfortable sharing medical, tax, and employee information via this tool. Regarding access control management of the three types of confidential information, the expiration time and disabling forwarding were most useful for the participants in preventing unauthorized and continued access. While the participants did not understand how the persistent protection feature worked, many still chose to use it, assuming it provided some extra layer of protection to confidential information and prevented unauthorized access. Watermarking was the least useful feature for the participants, as many were unsure of its usage. Our participants were concerned about data leaks from recipients’ devices if they set a longer expiration date, such as a year. We provide the practical implications of our findings

Enhancing Functionalities in Nanocomposites for Effective Dye Removal from Wastewater: Isothermal, Kinetic and Thermodynamic Aspects

The adsorption process combined with electrocoagulation is a highly effective technique for dye removal. Calcinized and non-calcinized composites based on bentonite and sodium zeolite were prepared for adsorptive removal of Foron EBL blue, Terasil brown 2RFL, Torque blue PG, and Orange P3R dyes. Factors affecting the adsorption process, such as contact time, initial dye concentration, and temperature, were also explored in this study. Equilibrium data of natural clay composites was explained by Freundlich, Langmuir, Dubinin Radushkevich isotherm, Harkin Jura, and Temkin isothermal models. Harkin Jura isotherm model best fitted on the adsorption mechanism compared to Langmuir and Temkin isotherm model. Morphology of clay-based adsorbents and functional group arrangement were investigated by scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy (FTIR). The calcinized nano-composite material exhibited better adsorption capacity than non-calcinized nano-composite and could be employed as a low-cost alternative for dye removal

Enhancing functionalities in nanocomposites for effective dye removal from wastewater: isothermal, kinetic and thermodynamic aspects

The adsorption process combined with electrocoagulation is a highly effective technique for dye removal. Calcinized and non-calcinized composites based on bentonite and sodium zeolite were prepared for adsorptive removal of Foron EBL blue, Terasil brown 2RFL, Torque blue PG, and Orange P3R dyes. Factors affecting the adsorption process, such as contact time, initial dye concentration, and temperature, were also explored in this study. Equilibrium data of natural clay composites was explained by Freundlich, Langmuir, Dubinin Radushkevich isotherm, Harkin Jura, and Temkin isothermal models. Harkin Jura isotherm model best fitted on the adsorption mechanism compared to Langmuir and Temkin isotherm model. Morphology of clay-based adsorbents and functional group arrangement were investigated by scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy (FTIR). The calcinized nano-composite material exhibited better adsorption capacity than non-calcinized nano-composite and could be employed as a low-cost alternative for dye removal

Epicutaneous Sensitization to the Phytocannabinoid β-Caryophyllene Induces Pruritic Inflammation

In recent years, there has been increased accessibility to cannabis for recreational and medicinal use. Incidentally, there has been an increase in reports describing allergic reactions to cannabis including exacerbation of underlying asthma. Recently, multiple protein allergens were discovered in cannabis, yet these fail to explain allergic sensitization in many patients, particularly urticaria and angioedema. Cannabis has a rich chemical profile including cannabinoids and terpenes that possess immunomodulatory potential. We examined whether major cannabinoids of cannabis such as cannabidiol (CBD) and the bicyclic sesquiterpene beta-caryophyllene (β-CP) act as contact sensitizers. The repeated topical application of mice skin with β-CP at 10 mg/mL (50 µL) induced an itch response and dermatitis at 2 weeks in mice, which were sustained for the period of study. Histopathological analysis of skin tissues revealed significant edema and desquamation for β-CP at 10 mg/mL. For CBD and β-CP, we observed a dose-dependent increase in epidermal thickening with profound thickening observed for β-CP at 10 mg/mL. Significant trafficking of CD11b cells was observed in various compartments of the skin in response to treatment with β-CP in a concentration-dependent manner. Mast cell trafficking was restricted to β-CP (10 mg/mL). Mouse proteome profiler cytokine/chemokine array revealed upregulation of complement C5/5a (anaphylatoxin), soluble intracellular adhesion molecule-1 (sICAM-1) and IL-1 receptor antagonist (IL-1RA) in animals dosed with β-CP (10 mg/mL). Moreover, we observed a dose-dependent increase in serum IgE in animals dosed with β-CP. Treatment with β-CP (10 mg/mL) significantly reduced filaggrin expression, an indicator of barrier disruption. In contrast, treatment with CBD at all concentrations failed to evoke scratching and dermatitis in mice and did not result in increased serum IgE. Further, skin tissues were devoid of any remarkable features, although at 10 mg/mL CBD we did observe the accumulation of dermal CD11b cells in skin tissue sections. We also observed increased filaggrin staining in mice repeatedly dosed with CBD (10 mg/mL). Collectively, our studies indicate that repeated exposure to high concentrations of β-CP can induce dermatitis-like pathological outcomes in mice

Prorelaxant E-type Prostanoid Receptors Functionally Partition to Different Procontractile Receptors in Airway Smooth Muscle

Prostaglandin E2 imparts diverse physiological effects on multiple airway cells through its actions on four distinct E-type prostanoid (EP) receptor subtypes (EP1–EP4). Gs-coupled EP2 and EP4 receptors are expressed on airway smooth muscle (ASM), yet their capacity to regulate the ASM contractile state remains subject to debate. We used EP2 and EP4 subtype-specific agonists (ONO-259 and ONO-329, respectively) in cell- and tissue-based models of human ASM contraction—magnetic twisting cytometry (MTC), and precision-cut lung slices (PCLSs), respectively—to study the EP2 and EP4 regulation of ASM contraction and signaling under conditions of histamine or methacholine (MCh) stimulation. ONO-329 was superior (\u3c0.05) to ONO-259 in relaxing MCh-contracted PCLSs (log half maximal effective concentration [logEC50]: 4.9 × 10−7 vs. 2.2 × 10−6; maximal bronchodilation ± SE, 35 ± 2% vs. 15 ± 2%). However, ONO-259 and ONO-329 were similarly efficacious in relaxing histamine-contracted PCLSs. Similar differential effects were observed in MTC studies. Signaling analyses revealed only modest differences in ONO-329– and ONO-259–induced phosphorylation of the protein kinase A substrates VASP and HSP20, with concomitant stimulation with MCh or histamine. Conversely, ONO-259 failed to inhibit MCh-induced phosphorylation of the regulatory myosin light chain (pMLC20) and the F-actin/G-actin ratio (F/G-actin ratio) while effectively inhibiting their induction by histamine. ONO-329 was effective in reversing induced pMLC20 and the F/G-actin ratio with both MCh and histamine. Thus, the contractile-agonist–dependent differential effects are not explained by changes in the global levels of phosphorylated protein kinase A substrates but are reflected in the regulation of pMLC20 (cross-bridge cycling) and F/G-actin ratio (actin cytoskeleton integrity, force transmission), implicating a role for compartmentalized signaling involving muscarinic, histamine, and EP receptor subtypes