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A-kinase-anchoring proteins (AKAPs) act as scaffold proteins that anchor the regulatory subunits of the cAMP-dependent protein kinase A (PKA) to coordinate and compartmentalize signaling elements and signals downstream of Gs-coupled G protein-coupled receptors (GPCRs). The beta-2-adrenoceptor (β 2 AR), as well as the Gs-coupled EP2 and EP4 receptor subtypes of the E-prostanoid (EP) receptor subfamily, are effective regulators of multiple airway smooth muscle (ASM) cell functions whose dysregulation contributes of asthma pathobiology. Here, we identify specific roles of the AKAPs Ezrin and Gravin, in differentially regulating PKA substrates downstream of the β 2 AR, EP2 receptor (EP2R) and EP4 receptor (EP4R). Knockdown of Ezrin, Gravin, or both in primary human ASM cells caused differential phosphorylation of the PKA substrates vasodilator-stimulated phosphoprotein (VASP) and heat shock protein 20 (HSP20). Ezrin knockdown, as well as combined Ezrin + Gravin knockdown significantly reduced the induction of phospho-VASP and phospho-HSP20 by β 2 AR, EP2R, and EP4R agonists. Gravin knockdown inhibited the induction of phospho-HSP20 by β 2 AR, EP2R, and EP4R agonists. Knockdown of Ezrin, Gravin, or both also attenuated histamine-induced phosphorylation of MLC20. Moreover, knockdown of Ezrin, Gravin or both suppressed the inhibitory effects of Gs-coupled receptor agonists on cell migration in ASM cells. These findings demonstrate the role of AKAPs in regulating Gs-coupled GPCR signaling and function in ASM, and suggest the therapeutic utility of targeting specific AKAP family members in the management of asthma.

Croton bonplandianus , a natural source traditionally used for treating various illnesses, including rheumatoid arthritis, was evaluated in this study. The effects of ethanolic extracts (CBEE) and aqueous fractions (CBAF) of C. bonplandianus leaves on arthritis-induced inflammation were studied using an albino rat model of inflammation induced by Freund's complete adjuvant. Eight test groups (n = 5 per group) and one vehicle control were used to evaluate the antiarthritic effects of different doses of CBEE and CBAF (125 mg.kg -1 , 250 mg.kg -1 , and 500 mg.kg -1 ) on days 5, 10, 15, and 20 compared to arthritic and vehicle controls. Arthritis severity was assessed using macroscopic arthritis grading, histological analysis, body weights, and paw thickness. CBEE and CBAF were found to reduce the prevalence of arthritis, increase body weight, and decrease paw inflammation compared to the vehicle control group by the 23rd day. In addition, they showed no effect on biochemical parameters, but a significant difference (p < 0.05) in hematological parameters compared to the arthritic control group. The study identified Hentriacontane compound as a potential contributor to the anti-inflammatory effect of C. bonplandianus , as it showed the lowest dock score for IL-1β and IL-6. Palmitoylethanol amide was identified as a potential contributor to the anti-inflammatory effect of TNF-α. Gene expression of IL-6, IL-1β, and TNF-α was down-regulated significantly (p < 0.05) in a dose-dependent manner in all treatment groups compared to the arthritic control group. In conclusion, this study validated the anti-arthritic and anti-inflammatory properties of CBEE and CBAF in a time and dose-dependent manner., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

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 (<0.05) to ONO-259 in relaxing MCh-contracted PCLSs (log half maximal effective concentration [logEC 50 ]: 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.

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.

Exaggerated airway smooth muscle (ASM) contraction regulated by the Gq family of G protein-coupled receptors causes airway hyperresponsiveness in asthma. Activation of Gq-coupled G protein-coupled receptors leads to phospholipase C (PLC)-mediated generation of inositol triphosphate (IP 3 ) and diacylglycerol (DAG). DAG signaling is terminated by the action of DAG kinase (DGK) that converts DAG into phosphatidic acid (PA). Our previous study demonstrated that DGKζ and α isoform knockout mice are protected from the development of allergen-induced airway hyperresponsiveness. Here we aimed to determine the mechanism by which DGK regulates ASM contraction. Activity of DGK isoforms was inhibited in human ASM cells by siRNA-mediated knockdown of DGKα and ζ, whereas pharmacological inhibition was achieved by pan DGK inhibitor I (R59022). Effects of DGK inhibition on contractile agonist-induced activation of PLC and myosin light chain (MLC) kinase, elevation of IP 3, and calcium levels were assessed. Furthermore, we used precision-cut human lung slices and assessed the role of DGK in agonist-induced bronchoconstriction. DGK inhibitor I attenuated histamine- and methacholine-induced bronchoconstriction. DGKα and ζ knockdown or pretreatment with DGK inhibitor I resulted in attenuated agonist-induced phosphorylation of MLC and MLC phosphatase in ASM cells. Furthermore, DGK inhibition decreased Gq agonist-induced calcium elevation and generation of IP 3 and increased histamine-induced production of PA. Finally, DGK inhibition or treatment with DAG analog resulted in attenuation of activation of PLC in human ASM cells. Our findings suggest that DGK inhibition perturbed the DAG:PA ratio, resulting in inhibition of Gq-PLC activation in a negative feedback manner, resulting in protection against ASM contraction.

The power industry is in the process of grid modernization with the introduction of phasor measurement units (PMUs), advanced metering infrastructure (AMI), and other technologies. Although these technologies enable more reliable and efficient operation, the risk of cyber threats has increased, as evidenced by the recent blackouts in Ukraine and New York. One of these threats is false data injection attacks (FDIAs). Most of the FDIA literature focuses on the vulnerability of DC estimators and AC estimators to such attacks. This paper investigates FDIAs for PMU-based state estimation, where the PMUs are comparable. Several states can be manipulated by compromising one PMU through the channels of that PMU. A Phase Locking Value (PLV) technique was developed to detect FDIAs. The proposed approach is tested on the IEEE 14-bus and the IEEE 30-bus test systems under different scenarios using a Monte Carlo simulation where the PLV demonstrated an efficient performance.

Gas sensors are critical components when adhering to health safety and environmental policies in various manufacturing industries, such as the petroleum and oil industry; scent and makeup production; food and beverage manufacturing; chemical engineering; pollution monitoring. In recent times, gas sensors have been introduced to medical diagnostics, bioprocesses, and plant disease diagnosis processes. There could be an adverse impact on human health due to the mixture of various gases (e.g., acetone (A), ethanol (E), propane (P)) that vent out from industrial areas. Therefore, it is important to accurately detect and differentiate such gases. Towards this goal, this paper presents a novel electronic nose (e-nose) detection method to classify various explosive gases. To detect explosive gases, metal oxide semiconductor (MOS) sensors are used as reliable tools to detect such volatile gases. The data received from MOS sensors are processed through a multivariate analysis technique to classify different categories of gases. Multivariate analysis was done using three variants-differential, relative, and fractional analyses-in principal components analysis (PCA). The MOS sensors also have three different designs: loading design, notch design, and Bi design. The proposed MOS sensor-based e-nose accurately detects and classifies three different gases, which indicates the reliability and practicality of the developed system. The developed system enables discrimination of these gases from the mixture. Based on the results from the proposed system, authorities can take preventive measures to deal with these gases to avoid their potential adverse impacts on employee health.

Francisella tularensis, a highly infectious, intracellular bacterium possesses an atypical type VI secretion system (T6SS), which is essential for its virulence. The chaperone ClpB, a member of the Hsp100/Clp family, is involved in Francisella T6SS disassembly and type VI secretion (T6S) is impaired in its absence. We asked if the role of ClpB for T6S was related to its prototypical role for the disaggregation activity. The latter is dependent on its interaction with the DnaK/Hsp70 chaperone system. Key residues of the ClpB-DnaK interaction were identified by molecular dynamic simulation and verified by targeted mutagenesis. Using such targeted mutants, it was found that the F. novicida ClpB-DnaK interaction was dispensable for T6S, intracellular replication, and virulence in a mouse model, although essential for handling of heat shock. Moreover, by mutagenesis of key amino acids of the Walker A, Walker B, and Arginine finger motifs of each of the two Nucleotide-Binding Domains, their critical roles for heat shock, T6S, intracellular replication, and virulence were identified. In contrast, the N-terminus was dispensable for heat shock, but required for T6S, intracellular replication, and virulence. Complementation of the ΔclpB mutant with a chimeric F. novicida ClpB expressing the N-terminal of Escherichia coli, led to reconstitution of the wild-type phenotype. Collectively, the data demonstrate that the ClpB-DnaK interaction does not contribute to T6S, whereas the N-terminal and NBD domains displayed critical roles for T6S and virulence., Competing Interests: The authors have declared that no competing interests exist.

Platinum-catalyzed selective C-H acylation of 2-aryloxypyridines with ethyl chlorooxoacetate provides an efficient way of introducing an α-keto ester functional group. The reaction is oxidant-free, additive-free, and, more significantly, free of any decarbonylative side reactions. The reaction tolerates a variety of substituents from strongly electron-donating to strongly electron-withdrawing groups. Double acylation is feasible for 2-phenoxypyridine and its derivatives with only one substituent at the para position. Although the reaction of 2-(2-methylphenoxy)pyridine with ethyl malonyl chloride did not produce the desired β-keto ester, the reaction with ethyl succinyl chloride proceeded smoothly to give the γ-keto ester. Ethyl chlorooxoacetate is much more reactive than ethyl succinyl chloride in this Pt-catalyzed C-H acylation reaction., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

Bladder dysfunction is associated with the overexpression of the intermediate filament (IF) proteins desmin and vimentin in obstructed bladder smooth muscle (BSM). However, the mechanisms by which these proteins contribute to BSM dysfunction are not known. Previous studies have shown that desmin and vimentin directly participate in signal transduction. In this study, we hypothesized that BSM dysfunction associated with overexpression of desmin or vimentin is mediated via c-Jun N-terminal kinase (JNK). We employed a model of murine BSM tissue in which increased expression of desmin or vimentin was induced by adenoviral transduction to examine the sufficiency of increased IF protein expression to reduce BSM contraction. Murine BSM strips overexpressing desmin or vimentin generated less force in response to KCl and carbachol relative to the levels in control murine BSM strips, an effect associated with increased JNK2 phosphorylation and reduced myosin light chain (MLC 20 ) phosphorylation. Furthermore, desmin and vimentin overexpressions did not alter BSM contractility and MLC 20 phosphorylation in strips isolated from JNK2 knockout mice. Pharmacological JNK2 inhibition produced results qualitatively similar to those caused by JNK2 knockout. These findings suggest that inhibition of JNK2 may improve diminished BSM contractility associated with obstructive bladder disease., (© 2019 Federation of American Societies for Experimental Biology.)

Background: This study addresses an ongoing debate, i.e. whether microstates have a relation to specific oscillations or frequency bands. The previous literature on this has been inconclusive. Due to stochastic calculation of microstates it is important to address this issue because instead of providing further insights, it might lead us to ambiguous interpretations., New Method: Here we propose a new method that allows to remove the time-frequency trade-off, which hampered previous works, using Empirical Mode Decomposition (EMD) and the AM-FM model. The method is applied to two resting-state EEG datasets., Results: First, our analysis confirmed that, indeed, when overlooking time-dependence in frequency domain, the results are inconclusive and consequently, highlighted the importance of preserving time-information in the spectral domain. Second, it is confirmed using synthetic data that the local peaks in global field potential (GFP) waveform are influenced by spectral powers present in composite signals. Based on synthetic results, it is inferred that in our dataset, an average frequency range of 10-15 Hz dominates the formation and the temporal dynamics of microstates. Third, it is shown that multiple overlapping patterns of synchronized activities described by a single meta-process in full band microstate studies can be identified using the proposed frequency-band subdivision. The results are consistent across both datasets., Conclusion: This study opens several new ventures to be explored in the future: e.g. analysis of temporally overlapping patterns described so far by single topographic patterns, which we show to be spectrally differentiable via band-wise topographic segmentation proposed in the present study., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Caveolins (CAVs) are structural proteins of caveolae that function as signaling platforms to regulate smooth muscle contraction. Loss of CAV protein expression is associated with impaired contraction in obstruction-induced bladder smooth muscle (BSM) hypertrophy. In this study, microarray analysis of bladder RNA revealed down-regulation of CAV1, CAV2, and CAV3 gene transcription in BSM from models of obstructive bladder disease in mice and humans. We identified and characterized regulatory regions responsible for CAV1, CAV2, and CAV3 gene expression in mice with obstruction-induced BSM hypertrophy, and in men with benign prostatic hyperplasia. DNA affinity chromatography and chromatin immunoprecipitation assays revealed a greater increase in binding of GATA-binding factor 6 (GATA-6) and NF-κB to their cognate binding motifs on CAV1, CAV2, and CAV3 promoters in obstructed BSM relative to that observed in control BSM. Knockout of NF-κB subunits, shRNA-mediated knockdown of GATA-6, or pharmacologic inhibition of GATA-6 and NF-κB in BSM increased CAV1, CAV2, and CAV3 transcription and promoter activity. Conversely, overexpression of GATA-6 decreased CAV2 and CAV3 transcription and promoter activity. Collectively, these data provide new insight into the mechanisms by which CAV gene expression is repressed in hypertrophied BSM in obstructive bladder disease., (Copyright © 2019 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Francisella tularensis, a highly infectious, intracellular bacterium possesses an atypical type VI secretion system (T6SS), which is essential for the virulence of the bacterium. Recent data suggest that the HSP100 family member, ClpB, is involved in T6SS disassembly in the subspecies Francisella novicida. Here, we investigated the role of ClpB for the function of the T6SS and for phenotypic characteristics of the human pathogenic subspecies holarctica and tularensis. The ∆clpB mutants of the human live vaccine strain, LVS, belonging to subspecies holarctica, and the highly virulent SCHU S4 strain, belonging to subspecies tularensis, both showed extreme susceptibility to heat shock and low pH, severely impaired type VI secretion (T6S), and significant, but impaired intracellular replication compared to the wild-type strains. Moreover, they showed essentially intact phagosomal escape. Infection of mice demonstrated that both ΔclpB mutants were highly attenuated, but the SCHU S4 mutant showed more effective replication than the LVS strain. Collectively, our data demonstrate that ClpB performs multiple functions in the F. tularensis subspecies holarctica and tularensis and its function is important for T6S, intracellular replication, and virulence.

Background: Simultaneous electroencephalography (EEG) and functional magnetic resonance image (fMRI) acquisitions provide better insight into brain dynamics. Some artefacts due to simultaneous acquisition pose a threat to the quality of the data. One such problematic artefact is the ballistocardiogram (BCG) artefact., Methods: We developed a hybrid algorithm that combines features of empirical mode decomposition (EMD) with principal component analysis (PCA) to reduce the BCG artefact. The algorithm does not require extra electrocardiogram (ECG) or electrooculogram (EOG) recordings to extract the BCG artefact., Results: The method was tested with both simulated and real EEG data of 11 participants. From the simulated data, the similarity index between the extracted BCG and the simulated BCG showed the effectiveness of the proposed method in BCG removal. On the other hand, real data were recorded with two conditions, i.e. resting state (eyes closed dataset) and task influenced (event-related potentials (ERPs) dataset). Using qualitative (visual inspection) and quantitative (similarity index, improved normalized power spectrum (INPS) ratio, power spectrum, sample entropy (SE)) evaluation parameters, the assessment results showed that the proposed method can efficiently reduce the BCG artefact while preserving the neuronal signals., Comparison With Existing Methods: Compared with conventional methods, namely, average artefact subtraction (AAS), optimal basis set (OBS) and combined independent component analysis and principal component analysis (ICA-PCA), the statistical analyses of the results showed that the proposed method has better performance, and the differences were significant for all quantitative parameters except for the power and sample entropy., Conclusions: The proposed method does not require any reference signal, prior information or assumption to extract the BCG artefact. It will be very useful in circumstances where the reference signal is not available., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A unique, platinum-catalyzed, direct C-H acylation of 2-(aryloxy)pyridines with acyl chlorides is discovered. The reaction requires neither an oxidant nor other additives. When both ortho positions of the aryl group are accessible, the double acylation occurs readily to produce the diacylated products. Aliphatic, aromatic, and α,β-unsaturated acyl groups can all be introduced. The acylation reaction may proceed through an analogous aromatic electrophilic substitution triggered by the nucleophilic attack of the platinum at the acyl chloride.

Splenic injuries after an endoscopic retrograde cholangiopancreatography are a rare but lethal complication. We describe a subcapsular splenic hematoma requiring emergent splenectomy after an endoscopic retrograde cholangiopancreatography in a liver transplant recipient.

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Approximately half of traumatic spinal cord injury (SCI) cases affect cervical regions, resulting in chronic respiratory compromise. The majority of these injuries affect midcervical levels, the location of phrenic motor neurons (PMNs) that innervate the diaphragm. A valuable opportunity exists following SCI for preventing PMN loss that occurs during secondary degeneration. One of the primary causes of secondary injury is excitotoxicity due to dysregulation of extracellular glutamate homeostasis. Astrocytes express glutamate transporter 1 (GLT1), which is responsible for the majority of CNS glutamate clearance. Given our observations of GLT1 dysfunction post-SCI, we evaluated intraspinal transplantation of Glial-Restricted Precursors (GRPs)--a class of lineage-restricted astrocyte progenitors--into ventral horn following cervical hemicontusion as a novel strategy for reconstituting GLT1 function, preventing excitotoxicity and protecting PMNs in the acutely injured spinal cord. We find that unmodified transplants express low levels of GLT1 in the injured spinal cord. To enhance their therapeutic properties, we engineered GRPs with AAV8 to overexpress GLT1 only in astrocytes using the GFA2 promoter, resulting in significantly increased GLT1 protein expression and functional glutamate uptake following astrocyte differentiation in vitro and after transplantation into C4 hemicontusion. Compared to medium-only control and unmodified GRPs, GLT1-overexpressing transplants reduced lesion size, diaphragm denervation and diaphragm dysfunction. Our findings demonstrate transplantation-based replacement of astrocyte GLT1 is a promising approach for SCI.

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., (Copyright © 2014 the authors 0270-6474/14/337622-17$15.00/0.)

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) <1. 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.

Invasion and metastasis are the major causes of cancer-related death. Pharmacological or therapeutic interventions such as chemoprevention of the progression stages of neoplastic development could result in substantial reduction in the incidence of cancer mortality. (-)-Epigallocatechin-3-gallate (EGCG), a promising chemopreventive agent, has attracted extensive interest for cancer therapy utilizing its antioxidant, anti- proliferative and inhibitory effects on angiogenesis and tumor cell invasion. In this study, we assessed the influence of EGCG on the proliferative potential of HeLa cells by cell viability assay and authenticated the results by nuclear morphological examination, DNA laddering assay and cell cycle analysis. Further we analyzed the anti-invasive properties of EGCG by wound migration assay and gene expression of MMP-9 and TIMP-1 in HeLa cells. Our results indicated that EGCG induced growth inhibition of HeLa cells in a dose- and time- dependent manner. It was observed that cell death mediated by EGCG was through apoptosis. Interestingly, EGCG effectively inhibited invasion and migration of HeLa cells and modulated the expression of related genes (MMP-9 and TIMP-1) . These results indicate that EGCG may effectively suppress promotion and progression stages of cervical cancer development.