Real-Time Monitoring of Electrically Stimulated Norepinephrine Release in Rat Thalamus: I. Resolution of Transmitter and Metabolite Signal Components

Abstract: Electrical stimulation of an ascending path of the locus ceruleus-norepinephrine system was used to elicit release of norepinephrine at noradrenergic terminal fields of the rat thalamus. Overflow into the extracellular fluid space was measured by fast in vivo chronoamperometry. At pretreated carbon fibers, the electrochemical signal consists of a sharp peak of ∼20–30-s duration followed by a slower, plateau-like decay to baseline. The peak, characterized by a variety of pharmacological manipulations and dialysis perfusion, is primarily due to norepinephrine. The plateau was shown to correspond to metabolite efflux of 3,4-dihydroxyphenylacetic acid. By varying the degree of electrochemical pretreatment, the response time and sensitivity of the fibers can be tuned to follow the entire signal or to select the separate components for detailed evaluation. This approach can be used to provide new information on the spatial and temporal characteristics of stimulated neurotransmitter release.     

Role of JNK translocation to mitochondria leading to inhibition of mitochondria bioenergetics in acetaminophen-induced liver injury

Previously, we demonstrated JNK plays a central role in acetaminophen (APAP)-induced liver injury (Gunawan, B. K., Liu, Z. X., Han, D., Hanawa, N., Gaarde, W. A., and Kaplowitz, N. (2006) Gastroenterology 131, 165-178). In this study, we examine the mechanism involved in activating JNK and explore the downstream targets of JNK important in promoting APAP-induced liver injury in vivo. JNK inhibitor (SP600125) was observed to significantly protect against APAP-induced liver injury. Increased mitochondria-derived reactive oxygen species were implicated in APAP-induced JNK activation based on the following: 1) mitochondrial GSH depletion (maximal at 2 h) caused increased H2O2 release from mitochondria, which preceded JNK activation (maximal at 4 h); 2) treatment of isolated hepatocytes with H2O2 or inhibitors (e.g. antimycin) that cause increased H2O2 release from mitochondria-activated JNK. An important downstream target of JNK following activation was mitochondria based on the following: 1) JNK translocated to mitochondria following activation; 2) JNK inhibitor treatment partially protected against a decline in mitochondria respiration caused by APAP treatment; and 3) addition of purified active JNK to mitochondria isolated from mice treated with APAP plus JNK inhibitor (mitochondria with severe GSH depletion, covalent binding) directly inhibited respiration. Cyclosporin A blocked the inhibitory effect of JNK on mitochondria respiration, suggesting JNK was directly inducing mitochondrial permeability transition in isolated mitochondria from mice treated with APAP plus JNK inhibitor. Addition of JNK to mitochondria isolated from control mice did not affect respiration. Our results suggests that APAP-induced liver injury involves JNK activation, due to increased reactive oxygen species generated by GSH-depleted mitochondria, and translocation of activated JNK to mitochondria where JNK induces mitochondrial permeability transition and inhibits mitochondria bioenergetics.     

Differential pathways govern CD4+ CD28- T cell proinflammatory and effector responses in patients with coronary artery disease

Patients with acute coronary syndromes experience circulatory and intraplaque expansion of an aggressive and unusual CD4(+) lymphocyte subpopulation lacking the CD28 receptor. These CD4(+)CD28(-) cells produce IFN-gamma and perforin, and are thought to play an important role in coronary atheromatous plaque destabilization. Aberrant expression of killer Ig-like receptors (KIRs) in CD4(+)CD28(-) cells is broadly thought to be responsible for their cytotoxicity, but the mechanisms involved remain poorly defined. We therefore sought to investigate the mechanism and regulation of CD4(+)CD28(-) cell functionality using T cell clones (n = 536) established from patients with coronary artery disease (n = 12) and healthy volunteers (n = 3). Our functional studies demonstrated that KIR2DS2 specifically interacted with MHC class I-presenting human heat shock protein 60 (hHSP60) inducing cytotoxicity. Further investigations revealed the novel finding that hHSP60 stimulation of TCR alone could not induce a cytotoxic response, and that this response was specific and KIR dependent. Analysis of CD4(+)CD28(-)2DS2(+) clones (n = 162) showed that not all were hHSP60 cytotoxic; albeit, their prevalence correlated with coronary disease status (p = 0.017). A higher proportion of clones responded to hHSP60 by IFN-gamma compared with perforin (p = 0.008). In this study, for the first time, we define the differential regulatory pathways involved in CD4(+)CD28(-) cell proinflammatory and effector responses. We describe in this study that, contrary to previous reports, CD4(+)CD28(-) cell recognition and killing can be specific and discriminate. These results, in addition to contributing to the understanding of CD4(+)CD28(-) cell functionality, may have implications for the monitoring and management of coronary artery disease progression.     

Regulation of H(2)O(2)-induced necrosis by PKC and AMP-activated kinase signaling in primary cultured hepatocytes

Recent studies have suggested that, in certain cases, necrosis, like apoptosis, may be programmed, involving the activation and inhibition of many signaling pathways. In this study, we examined whether necrosis induced by H(2)O(2) is regulated by signaling pathways in primary hepatocytes. A detailed time course revealed that H(2)O(2) treated to hepatocytes is consumed within minutes, but hepatocytes undergo necrosis several hours later. Thus, H(2)O(2) treatment induces a "lag phase" where signaling changes occur, including PKC activation, Akt (PKB) downregulation, activation of JNK, and downregulation of AMP-activated kinase (AMPK). Investigation of various inhibitors demonstrated that PKC inhibitors were effective in reducing necrosis caused by H(2)O(2) (~80%). PKC inhibitor treatment decreased PKC activity but, surprisingly, also upregulated Akt and AMPK, suggesting that various PKC isoforms negatively regulate Akt and AMPK. Akt did not appear to play a significant role in H(2)O(2)-induced necrosis, since PKC inhibitor treatment protected hepatocytes from H(2)O(2) even when Akt was inhibited. On the other hand, compound C, a selective AMPK inhibitor, abrogated the protective effect of PKC inhibitors against necrosis induced by H(2)O(2). Furthermore, AMPK activators protected against H(2)O(2)-induced necrosis, suggesting that much of the protective effect of PKC inhibition was mediated through the upregulation of AMPK. Work with PKC inhibitors suggested that atypical PKC downregulates AMPK in response to H(2)O(2). Knockdown of PKC-alpha using antisense oligonucleotides also slightly protected (~22%) against H(2)O(2). Taken together, our data demonstrate that the modulation of signaling pathways involving PKC and AMPK can alter H(2)O(2)-induced necrosis, suggesting that a signaling "program" is important in mediating H(2)O(2)-induced necrosis in primary hepatocytes.     

Biomedical applications of carbon nanomaterials: Drug and gene delivery potentials

One of the major components in the development of nanomedicines is the choice of the right biomaterial, which notably determines the subsequent biological responses. The popularity of carbon nanomaterials (CNMs) has been on the rise due to their numerous applications in the fields of drug delivery, bioimaging, tissue engineering, and biosensing. Owing to their considerably high surface area, multifunctional surface chemistry, and excellent optical activity, novel functionalized CNMs possess efficient drug-loading capacity, biocompatibility, and lack of immunogenicity. Over the past few decades, several advances have been made on the functionalization of CNMs to minimize their health concerns and enhance their biosafety. Recent evidence has also implied that CNMs can be functionalized with bioactive peptides, proteins, nucleic acids, and drugs to achieve composites with remarkably low toxicity and high pharmaceutical efficiency. This review focuses on the three main classes of CNMs, including fullerenes, graphenes, and carbon nanotubes, and their recent biomedical applications.     

Computational fluid dynamics modeling,a novel,and effective approach for developing scalable cell therapy manufacturing processes

Induced pluripotent stem cells (iPSCs) hold great potential to generate novel, curative cell therapy products. However, current methods to generate these novel therapies lack scalability, are labor-intensive, require a large footprint, and are not suited to meet clinical and commercial demands. Therefore, it is necessary to develop scalable manufacturing processes to accommodate the generation of high-quality iPSC derivatives under controlled conditions. The current scale-up methods used in cell therapy processes are based on empirical, geometry-dependent methods that do not accurately represent the hydrodynamics of 3D bioreactors. These methods require multiple iterations of scale-up studies, resulting in increased development cost and time. Here we show a novel approach using computational fluid dynamics modeling to effectively scale-up cell therapy manufacturing processes in 3D bioreactors. Using a GMP-compatible iPSC line, we translated and scaled-up a small-scale cardiomyocyte differentiation process to a 3-L computer-controlled bioreactor in an efficient manner, showing comparability in both systems.     

Breast cancer-linked lncRNA u-Eleanor is upregulated in breast of healthy women with lack or short duration of breastfeeding

Recently, it has been revealed that estrogen-related reproductive factors are linked with some early gene expression lesions associated with malignancy in clinically healthy breasts. Accordingly, the aim of the current study was to evaluate the association of expression levels of estrogen-related long noncoding RNAs (lncRNAs) upstream Eleanor (u-Eleanor) and HOX antisense intergenic RNA (HOTAIR) with the different patterns of reproductive factors in breast tissue of healthy women. The subjects of this study were 98 cancer-free women who had undergone cosmetic mammoplasty. The expression levels of u-Eleanor and HOTAIR were measured using quantitative real-time polymerase chain reaction. The results of the current study showed that the women without a history of breastfeeding had a high-level expression of u-Eleanor compared with the women with a breastfeeding duration greater than 6 to 24 months (P = 0.03) as well as the women with a breastfeeding duration of more than 24 months (P = 0.005). Furthermore, a higher expression of u-Eleanor was found in the women with a short breastfeeding duration for 1 to 6 months than that in the women with a breastfeeding duration of greater than 24 months (P = 0.02). In the same way, the results of correlation test (r = −0.258; P = 0.036) and multivariate regression model (β = −0.321; P = 0.023) are indicative of a significant relationship of elevated expression of u-Eleanor with decreasing breastfeeding duration in the women. These findings could be important to identify the molecular mechanisms behind the relationship between a lack or short duration of the breastfeeding and the risk of breast cancer, which has previously been reported by epidemiological studies.     

Resting heart rate,heart rate variability and functional decline in old age

Background:

Heart rate and heart rate variability, markers of cardiac autonomic function, have been linked with cardiovascular disease. We investigated whether heart rate and heart rate variability are associated with functional status in older adults, independent of cardiovascular disease.

Methods:

We obtained data from the Prospective Study of Pravastatin in the Elderly at Risk (PROSPER). A total of 5042 participants were included in the present study, and mean follow-up was 3.2 years. Heart rate and heart rate variability were derived from baseline 10-second electrocardiograms. Heart rate variability was defined as the standard deviation of normal-to-normal RR intervals (SDNN). Functional status in basic (ADL) and instrumental (IADL) activities of daily living was measured using Barthel and Lawton scales, at baseline and during follow-up.

Results:

The mean age of the study population was 75.3 years. At baseline, higher heart rate was associated with worse ADL and IADL, and lower SDNN was related to worse IADL (all p values < 0.05). Participants in the highest tertile of heart rate (range 71–117 beats/min) had a 1.79-fold (95% confidence interval [CI] 1.45–2.22) and 1.35-fold (95% CI 1.12–1.63) higher risk of decline in ADL and IADL, respectively (p for trend < 0.001 and 0.001, respectively). Participants in the lowest tertile of SDNN (range 1.70–13.30 ms) had 1.21-fold (95% CI 1.00–1.46) and 1.25-fold (95% CI 1.05–1.48) higher risk of decline in ADL and IADL, respectively (both p for trends < 0.05). All associations were independent of sex, medications, cardiovascular risk factors and comorbidities.

Interpretation:

Higher resting heart rate and lower heart rate variability were associated with worse functional status and with higher risk of future functional decline in older adults, independent of cardiovascular disease. This study provides insight into the role of cardiac autonomic function in the development of functional decline.Elevated heart rate and reduced heart rate variability — the beat-to-beat variation in heart rate intervals — both reflect an altered balance of the autonomic nervous system tone characterized by increased sympathetic and/or decreased parasympathetic activity.^1–3 Sympathetic overactivity has been linked to a procoagulant state and also to risk factors for atherosclerosis, including metabolic syndrome, obesity and subclinical inflammation.^2–4 Moreover, increased heart rate is related to atherosclerosis, not only as an epiphenomenon of sympathetic overactivity, but also through hemodynamic mechanisms, such as high pulsatile shear stress, which leads to endothelial dysfunction.⁵Atherosclerosis has been linked to increased risk of functional decline in older people via cardiovascular events.⁶ As the world population is aging, the burden of functional disability is expected to increase.⁶ It has been hypothesized that heart rate and heart rate variability are markers of frailty, an increased vulnerability to stressors and functional decline.⁷ However, the direct link between these 2 parameters and risk of functional decline has not been fully established, and it is uncertain whether this association is independent of cardiovascular comorbidities.In this study, we examined whether heart rate and heart rate variability were cross-sectionally and longitudinally associated with functional status in older adults at high risk of cardiovascular disease, independent of cardiovascular risk factors and comorbidities.     

Formulation and In Vitro,In Vivo Evaluation of Effervescent Floating Sustained-Release Imatinib Mesylate Tablet

Introduction

Imatinib mesylate is an antineoplastic agent which has high absorption in the upper part of the gastrointestinal tract (GIT). Conventional imatinib mesylate (Gleevec) tablets produce rapid and relatively high peak blood levels and requires frequent administration to keep the plasma drug level at an effective range. This might cause side effects, reduced effectiveness and poor therapeutic management. Therefore, floating sustained-release Imatinib tablets were developed to allow the tablets to be released in the upper part of the GIT and overcome the inadequacy of conventional tablets.

Methodology

Floating sustained-release Imatinib mesylate tablets were prepared using the wet granulation method. Tablets were formulated using Hydroxypropyl Methylcellulose (HPMC K4M), with Sodium alginate (SA) and Carbomer 934P (CP) as release-retarding polymers, sodium bicarbonate (NaHCO₃) as the effervescent agent and lactose as a filler. Floating behavior, in vitro drug release, and swelling index studies were conducted. Initial and total drug release duration was compared with a commercial tablet (Gleevec) in 0.1 N HCl (pH 1.2) at 37 ± 0.5°C for 24 hours. Tablets were then evaluated for various physical parameters, including weight variation, thickness, hardness, friability, and drug content. Consequently, 6 months of physical stability studies and in vitro gastro-retentive studies were conducted.

Results and Discussion

Statistical data analysis revealed that tablets containing a composition of 14.67% w/w HPMC K4M, 10.67%, w/w Na alginate, 1.33%, w/w Carbomer 934P and 9.33%, w/w NaHCO₃ produced the most favorable formulation to develop 24-hour sustained-release tablets with optimum floating behavior and satisfactory physicochemical characteristics. Furthermore, in vitro release study revealed that the formulated SR tablet had significantly lower C_max and higher T_max compared to the conventional tablet (Gleevec). Thus, formulated SR tablets preserved persistent concentration of plasma up to 24 hours.

Conclusion

In conclusion, in order to suggest a better drug delivery system with constant favorable release, resulting in optimized absorption and less side effects, formulated CP-HPMC-SA based imatinib mesylate floating sustained-release tablets can be a promising candidate for cancer chemotherapy.