Paclitaxel-Induced Epidermal Alterations: An In Vitro Preclinical Assessment in Primary Keratinocytes and in a 3D Epidermis Model.

Paclitaxel is a microtubule-stabilizing chemotherapeutic agent approved for the treatment of ovarian, non-small cell lung, head, neck, and breast cancers. Despite its beneficial effects on cancer and widespread use, paclitaxel also damages healthy tissues, including the skin. However, the mechanisms that drive these skin adverse events are not clearly understood. In the present study, we demonstrated, by using both primary epidermal keratinocytes (NHEK) and a 3D epidermis model, that paclitaxel impairs different cellular processes: paclitaxel increased the release of IL-1α, IL-6, and IL-8 inflammatory cytokines, produced reactive oxygen species (ROS) release and apoptosis, and reduced the endothelial tube formation in the dermal microvascular endothelial cells (HDMEC). Some of the mechanisms driving these adverse skin events in vitro are mediated by the activation of toll-like receptor 4 (TLR-4), which phosphorylate transcription of nuclear factor kappa B (NF-κb). This is the first study analyzing paclitaxel effects on healthy human epidermal cells with an epidermis 3D model, and will help in understanding paclitaxel's effects on the skin.

Functional In Vitro Assessment of VEGFA/NOTCH2 Signaling Pathway and pRB Proteasomal Degradation and the Clinical Relevance of Mucolipin TRPML2 Overexpression in Glioblastoma Patients.

Glioblastoma (GBM) is the most malignant glioma with an extremely poor prognosis. It is characterized by high vascularization and its growth depends on the formation of new blood vessels. We have previously demonstrated that TRPML2 mucolipin channel expression increases with the glioma pathological grade. Herein by ddPCR and Western blot we found that the silencing of TRPML2 inhibits expression of the VEGFA/Notch2 angiogenic pathway. Moreover, the VEGFA/Notch2 expression increased in T98 and U251 cells stimulated with the TRPML2 agonist, ML2-SA1, or by enforced-TRPML2 levels. In addition, changes in TRPML2 expression or ML2-SA1-induced stimulation, affected Notch2 activation and VEGFA release. An increased invasion capability, associated with a reduced VEGF/VEGFR2 expression and increased vimentin and CD44 epithelial-mesenchymal transition markers in siTRPML2, but not in enforced-TRPML2 or ML2-SA1-stimulated glioma cells, was demonstrated. Furthermore, an increased sensitivity to Doxorubicin cytotoxicity was demonstrated in siTRPML2, whereas ML2-SA1-treated GBM cells were more resistant. The role of proteasome in Cathepsin B-dependent and -independent pRB degradation in siTRPML2 compared with siGLO cells was studied. Finally, through Kaplan-Meier analysis, we found that high TRPML2 mRNA expression strongly correlates with short survival in GBM patients, supporting TRPML2 as a negative prognostic factor in GBM patients.

Assessment and Imaging of Intracellular Magnesium in SaOS-2 Osteosarcoma Cells and Its Role in Proliferation.

Magnesium is an essential nutrient involved in many important processes in living organisms, including protein synthesis, cellular energy production and storage, cell growth and nucleic acid synthesis. In this study, we analysed the effect of magnesium deficiency on the proliferation of SaOS-2 osteosarcoma cells. When quiescent magnesium-starved cells were induced to proliferate by serum addition, the magnesium content was 2-3 times lower in cells maintained in a medium without magnesium compared with cells growing in the presence of the ion. Magnesium depletion inhibited cell cycle progression and caused the inhibition of cell proliferation, which was associated with mTOR hypophosphorylation at Serine 2448. In order to map the intracellular magnesium distribution, an analytical approach using synchrotron-based X-ray techniques was applied. When cell growth was stimulated, magnesium was mainly localized near the plasma membrane in cells maintained in a medium without magnesium. In non-proliferating cells growing in the presence of the ion, high concentration areas inside the cell were observed. These results support the role of magnesium in the control of cell proliferation, suggesting that mTOR may represent an important target for the antiproliferative effect of magnesium. Selective control of magnesium availability could be a useful strategy for inhibiting osteosarcoma cell growth.

Direct Inhibition of the Allergic Effector Response by Raw Cow's Milk-An Extensive In Vitro Assessment.

The mechanisms underlying the allergy-protective effects of raw cow's milk are poorly understood. The current focus is mainly on the modulation of T cell responses. In the present study, we investigated whether raw cow's milk can also directly inhibit mast cells, the key effector cells in IgE-mediated allergic responses. Primary murine bone marrow-derived mast cells (BMMC) and peritoneal mast cells (PMC), were incubated with raw milk, heated raw milk, or shop milk, prior to IgE-mediated activation. The effects on mast cell activation and underlying signaling events were assessed. Raw milk was furthermore fractionated based on molecular size and obtained fractions were tested for their capacity to reduce IgE-mediated mast cell activation. Coincubation of BMMC and PMC with raw milk prior to activation reduced ß-hexosaminidase release and IL-6 and IL-13 production, while heated raw milk or shop milk had no effect. The reduced mast cell activation coincided with a reduced intracellular calcium influx. In addition, SYK and ERK phosphorylation levels, both downstream signaling events of the FcεRI, were lower in raw milk-treated BMMC compared to control BMMC, although differences did not reach full significance. Raw milk-treated BMMC furthermore retained membrane-bound IgE expression after allergen stimulation. Raw milk fractionation showed that the heat-sensitive raw milk components responsible for the reduced mast cell activation are likely to have a molecular weight of > 37 kDa. The present study demonstrates that raw cow's milk can also directly affect mast cell activation. These results extend the current knowledge on mechanisms via which raw cow's milk prevents allergic diseases, which is crucial for the development of new, microbiologically safe, nutritional strategies to reduce allergic diseases.

Shikonin Attenuates Hepatic Steatosis by Enhancing Beta Oxidation and Energy Expenditure via AMPK Activation.

Shikonin, a natural plant pigment, is known to have anti-obesity activity and to improve insulin sensitivity. This study aimed to examine the effect of shikonin on hepatic steatosis, focusing on the AMP-activated protein kinase (AMPK) and energy expenditure in Hepa 1-6 cells and in high-fat fed mice. Shikonin increased AMPK phosphorylation in a dose- and time-dependent manner, and inhibition of AMPK with compound C inhibited this activation. In an oleic acid-induced steatosis model in hepatocytes, shikonin suppressed oleic acid-induced lipid accumulation, increased AMPK phosphorylation, suppressed the expression of lipogenic genes, and stimulated fatty acid oxidation-related genes. Shikonin administration for four weeks decreased body weight gain and the accumulation of lipid droplets in the liver of high-fat fed mice. Furthermore, shikonin promoted energy expenditure by activating fatty acid oxidation. In addition, shikonin increased the expression of PPARγ coactivator-1α (PGC-1α), carnitine palmitoyltransferase-1 (CPT1) and other mitochondrial function-related genes. These results suggest that shikonin attenuated a high fat diet-induced nonalcoholic fatty liver disease by stimulating fatty acid oxidation and energy expenditure via AMPK activation.

Beta2 -adrenergic agonist clenbuterol increases energy expenditure and fat oxidation, and induces mTOR phosphorylation in skeletal muscle of young healthy men.

Clenbuterol is a beta2 -adrenoceptor agonist marketed as an asthma reliever but is not approved for human use in most countries due to concerns of adverse cardiac effects. Given its demonstrated hypertrophic and lipolytic actions in rodents, clenbuterol is one of the most widely abused doping substances amongt athletes and recreational body-builders seeking leanness. Herein, we examined the effect of clenbuterol ingestion on metabolic rate as well as skeletal muscle mammalian target of rapamycin (mTOR) phosphorylation and protein kinase A (PKA)-signaling in six young men. Before and 140 min after ingestion of 80 µg clenbuterol, resting metabolic rate and contractile function of the quadriceps muscle were measured, and blood samples as well as vastus lateralis muscle biopsies were collected. Clenbuterol increased resting energy expenditure by 21% (P < 0.001), and fat oxidation by 39% (P = 0.006), whereas carbohydrate oxidation was unchanged. Phosphorylation of mTORSer2448 and PKA substrates increased by 121% (P = 0.004) and 35% (P = 0.006), respectively, with clenbuterol. Maximal voluntary contraction torque decreased by 4% (P = 0.026) and the half-relaxation time shortened by 9% (P = 0.046), while voluntary activation, time to peak twitch, and peak twitch torque did not change significantly with clenbuterol. Glycogen content of the vastus lateralis muscle did not change with clenbuterol. Clenbuterol increased circulating levels of glucose (+30%; P < 0.001), lactate (+90%; P = 0.004), insulin (+130%; P = 0.009), and fatty acids (+180%; P = 0.001). Collectively, these findings indicate that clenbuterol is an efficient thermogenic substance that possibly also exerts muscle hypertrophic actions in humans. For these reasons, the restrictions imposed against clenbuterol in competitive sports seem warranted.

Role of non-PTS dependent glucose permease (GlcU) in maintaining the fitness cost during acquisition of nisin resistance by Enterococcus faecalis.

Nisin is used for food preservation due to its antibacterial activity. However, some bacteria survive under the prevailing conditions owing to the acquisition of resistance. This study aimed to characterize nisin-resistant Enterococcus faecalis isolated from raw buffalo milk and investigate their fitness cost. FE-SEM, biofilm and cytochrome c assay were used for characterization. Growth kinetics, HPLC, qPCR and western blotting were performed to confer their fitness cost. Results revealed that nisin-resistant E. faecalis were morphologically different from sensitive strain and internalize more glucose. However, no significant difference was observed in the growth pattern of the resistant strain compared to that of the sensitive strain. A non-phosphotransferase glucose permease (GlcU) was found to be associated with enhanced glucose uptake. Conversely, Mpt, a major phosphotransferase system responsible for glucose uptake, did not play any role, as confirmed by gene expression studies and western blot analysis of HPr protein. The phosphorylation of His-15 residue of HPr phosphoprotein was reduced, while that of the Ser-46 residue increased with progression in nisin resistance, indicating that it may be involved in the regulation of pathogenicity. In conclusion, resistance imposes a significant fitness cost and GlcU plays a key role in maintaining the fitness cost in nisin-resistant variants.

Amelioration of diabetic nephropathy using pomegranate peel extract-stabilized gold nanoparticles: assessment of NF-κB and Nrf2 signaling system.

BACKGROUND: Diabetic nephropathy (DN), an end-stage renal disorder, has posed a menace to humankind globally, because of its complex nature and poorly understandable intricate mechanism. In recent times, functional foods as potential health benefits have been gaining attention of consumers and researchers alike. Rich in antioxidants, the peel and seed of pomegranate have previously demonstrated protection against oxidative-stress-related diseases, including cardiovascular disorders, diabetes, and cancer. PURPOSE: This study was designed to investigate the ameliorative role of pomegranate peel extract-stabilized gold nanoparticle (PPE-AuNP) on streptozotocin (STZ)-induced DN in an experimental murine model. METHODS: Following the reduction methods, AuNP was prepared using the pomegranate peel ellagitannins and characterized by particle size, physical appearance, and morphological architecture. Modulatory potential of PPE-AuNP was examined through the plethora of biochemical and high throughput techniques, flow cytometry, immunoblotting, and immunofluorescence. RESULTS: The animals treated with PPE-AuNP markedly reduced the fasting blood glucose, renal toxicity indices, and serum TC and TG in a hyperglycemic condition. As evident from an increased level of plasma insulin level, PPE-AuNP normalized the STZ-induced pancreatic ß-cell dysfunction. The STZ-mediated suppression of endogenous antioxidant response was restored by the PPE-AuNP treatment, which reduced the generation of LPO as well as iROS. Furthermore, the hyperglycemia-mediated augmentation of protein glycation, followed by the NOX4/p-47phox activation, diminished with the application of PPE-AuNP. The histological and immunohistochemical findings showed the protective efficacy of PPE-AuNP in reducing STZ-induced glomerular sclerosis and renal fibrosis. In addition, it reduced proinflammatory burden through the modulation of the MAPK/NF-κB/STAT3/cytokine axis. Simultaneously, PI3K/AKT-guided Nrf2 activation was evident upon the PPE-AuNP application, which enhanced the antioxidant response and maintained hyperglycemic homeostasis. CONCLUSION: The findings indicate that the use of PPE-AuNPs might act as an economic therapeutic remedy for alleviating DN.

Atomic force microscopy technique used for assessment of the anti-arthritic effect of licochalcone A via suppressing NF-κB activation.

Atomic force microscopy (AFM) is appropriately applied to the examination of hard surfaces and soft samples with extremely high resolution and ultrasensitive force, which cannot be obtained by other imaging techniques, including optical and electron microscopy. In the current study, AFM was employed to evaluate the anti-arthritic effect of licochalcone A (LCA), a flavonoid isolated from the root of Chinese medicinal herb Glycyrrhiza inflate, on rheumatoid arthritis synovial fibroblasts (RASFs) at the nanoscale for the first time. The morphology, ultrastructure and stiffness of RASFs was modified by LCA as determined by AFM, suggesting that LCA most likely exerts an anti-arthritic effect based on the key role of RASFs in the progression of RA. Further studies showed that the inhibitory effect of LCA on IκBα phosphorylation and degradation as well as on p65 nuclear translocation and phosphorylation contributed to altering the morphology, ultrastructure and stiffness of the RASF membrane. Interestingly, IKKß phosphorylation was not detectable in RASFs, indicating that LCA altered the morphology, ultrastructure and stiffness of the RASF membrane by inhibiting NF-κB activation independent of IKKß phosphorylation. Antigen-induced arthritis (AIA) was established in Sprague Dawley (SD) rats to validate the anti-arthritic effect of LCA, and LCA significantly decreased both the arthritis scores and paw swelling in the AIA rats, suggesting that LCA inhibits the progression and development of arthritis in vivo. Collectively, AFM provides evidence at the nanoscale to predict the anti-arthritic effect of drugs on RASFs, and LCA should be further investigated as a candidate agent for the treatment of arthritis.

Phosphorylation of intracellular signalling molecules in peripheral blood cells from patients with psoriasis on originator or biosimilar infliximab.

BACKGROUND: Psoriasis vulgaris is a chronic, inflammatory skin disease characterized by a dysregulated immune response and it is associated with substantial systemic comorbidities. Biological drugs such as tumour necrosis factor (TNF)-α inhibitors can ameliorate the disease but are expensive. Biosimilar drugs have the same amino-acid sequence as the originator, but differences in manufacturing can affect biological activity, efficacy and tolerability. OBJECTIVES: To explore potential differences in intracellular phosphorylation of signalling molecules in peripheral blood cells from patients with psoriasis treated with the TNF-α inhibitor infliximab compared with healthy controls, and to investigate if the phosphorylation pattern was influenced by switching from the originator infliximab to the biosimilar CT-P13. METHODS: By flow cytometry, we measured phosphorylation of nuclear factor kappa B, extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase and signal transducer and activator of transcription 3, before and after TNF-α stimulation in monocytes and T, B, natural killer and CD3+  CD56+ cells from 25 patients with psoriasis treated with infliximab and 19 healthy controls. RESULTS: At inclusion, phosphorylation levels of peripheral blood mononuclear cells (PBMCs) were increased in patients with psoriasis compared with healthy controls, even though clinical remission had already been achieved. Phosphorylation levels declined in patients on both originator infliximab and biosimilar during continued treatment. No significant differences were detected between the two medications after 12 months. CONCLUSIONS: Patients with psoriasis on infliximab have higher activation levels of PBMCs than do healthy controls, possibly reflecting systemic inflammation. Switching from the originator infliximab to biosimilar CT-P13 did not affect phosphorylation levels or clinical parameters, suggesting that CT-P13 is a noninferior treatment alternative to the originator infliximab.

The V2 receptor antagonist tolvaptan raises cytosolic calcium and prevents AQP2 trafficking and function: an in vitro and in vivo assessment.

Tolvaptan, a selective vasopressin V2 receptor antagonist, is a new generation diuretic. Its clinical efficacy is in principle due to impaired vasopressin-regulated water reabsorption via aquaporin-2 (AQP2). Nevertheless, no direct in vitro evidence that tolvaptan prevents AQP2-mediated water transport, nor that this pathway is targeted in vivo in patients with syndrome of inappropriate antidiuresis (SIAD) has been provided. The effects of tolvaptan on the vasopressin-cAMP/PKA signalling cascade were investigated in MDCK cells expressing endogenous V2R and in mouse kidney. In MDCK, tolvaptan prevented dDAVP-induced increase in ser256-AQP2 and osmotic water permeability. A similar effect on ser256-AQP2 was found in V1aR -/- mice, thus confirming the V2R selectively. Of note, calcium calibration in MDCK showed that tolvaptan per se caused calcium mobilization from the endoplasmic reticulum resulting in a significant increase in basal intracellular calcium. This effect was only observed in cells expressing the V2R, indicating that it requires the tolvaptan-V2R interaction. Consistent with this finding, tolvaptan partially reduced the increase in ser256-AQP2 and the water permeability in response to forskolin, a direct activator of adenylyl cyclase (AC), suggesting that the increase in intracellular calcium is associated with an inhibition of the calcium-inhibitable AC type VI. Furthermore, tolvaptan treatment reduced AQP2 excretion in two SIAD patients and normalized plasma sodium concentration. These data represent the first detailed demonstration of the central role of AQP2 blockade in the aquaretic effect of tolvaptan and underscore a novel effect in raising intracellular calcium that can be of significant clinical relevance.

In Vivo Analysis of Hypertension: Induction of Hypertension, In Vivo Kinase Manipulation, and Assessment of Physiologic Outputs.

Using an in vivo model system to study signal transduction will include several steps: (1) induce hypertension in the animal, (2) manipulate kinase activation and signal transduction pathways as desired, and (3) observe physiologic outputs. This chapter provides the reader with overviews of the techniques our lab uses to manipulate signal transduction pathways and determine the effects on hypertension.

A Single Oral Administration of Theaflavins Increases Energy Expenditure and the Expression of Metabolic Genes.

Theaflavins are polyphenols found in black tea, whose physiological activities are not well understood. This study on mice evaluated the influence of a single oral administration of theaflavins on energy metabolism by monitoring the initial metabolic changess in skeletal muscle and brown adipose tissue (BAT). Oxygen consumption (VO2) and energy expenditure (EE) were increased significantly in mice treated with theaflavin rich fraction (TF) compared with the group administered vehicle alone. There was no difference in locomotor activity. Fasting mice were euthanized under anesthesia before and 2 and 5, 20-hr after treatment with TF or vehicle. The mRNA levels of uncoupling protein-1 (UCP-1) and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) in BAT were increased significantly 2-hr after administration ofTF. The levels of UCP-3 and PGC-1α in the gastrocnemius muscle were increased significantly 2 and 5-hr after administration of TF. The concentration of phosphorylated AMP-activated protein kinase (AMPK) 1α was also increased significantly in the gastrocnemius 2 and 5-hr after treatment with TF. These results indicate that TF significantly enhances systemic energy expenditure, as evidenced by an increase in expression of metabolic genes.

Assessment of mTOR-Dependent Translational Regulation of Interferon Stimulated Genes.

Type-I interferon (IFN)-induced activation of the mammalian target of rapamycin (mTOR) signaling pathway has been implicated in translational control of mRNAs encoding interferon-stimulated genes (ISGs). However, mTOR-sensitive translatomes commonly include mRNAs with a 5' terminal oligopyrimidine tract (TOP), such as those encoding ribosomal proteins, but not ISGs. Because these translatomes were obtained under conditions when ISG expression is not induced, we examined the mTOR-sensitive translatome in human WISH cells stimulated with IFN ß. The mTOR inhibitor Torin1 resulted in a repression of global protein synthesis, including that of ISG products, and translation of all but 3 ISG mRNAs (TLR3, NT5C3A, and RNF19B) was not selectively more sensitive to mTOR inhibition. Detailed studies of NT5C3A revealed an IFN-induced change in transcription start site resulting in a switch from a non-TOP to a TOP-like transcript variant and mTOR sensitive translation. Thus, we show that, in the cell model used, translation of the vast majority of ISG mRNAs is not selectively sensitive to mTOR activity and describe an uncharacterized mechanism wherein the 5'-UTR of an mRNA is altered in response to a cytokine, resulting in a shift from mTOR-insensitive to mTOR-sensitive translation.

Enhancement of energy expenditure following a single oral dose of flavan-3-ols associated with an increase in catecholamine secretion.

Numerous clinical studies have reported that ingestion of chocolate reduces the risk of metabolic syndrome. However, the mechanisms by which this occurs remain unclear. In this murine study, the metabolic-enhancing activity of a 10 mg/kg mixture of flavan-3-ol fraction derived from cocoa (FL) was compared with the same single dose of (-)-epicatechin (EC). Resting energy expenditure (REE) was significantly increased in mice treated with the FL versus the group administered the distilled water vehicle (Cont) during periods of ad libitum feeding and fasting. Mice were euthanized under the effect of anesthesia 2, 5, and 20 hr after treatment with FL or Cont while subsequently fasting. The mRNA levels of the uncoupling protein-1 (UCP-1) and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) in brown adipose tissue (BAT) were significantly increased 2 hr after administration of FL. UCP-3 and PGC-1α in the gastrocnemius were significantly increased 2 and 5 hr after administration of the FL. The concentrations of phosphorylated AMP-activated protein kinase (AMPK) 1α were found to be significant in the gastrocnemius of mice 2 and 5 hr after ingesting FL. However, these changes were not observed following treatment with EC. Plasma was collected for measurement of catecholamine levels in other animals euthanized by decapitation 2 and 4 hr after their respective group treatment. Plasma adrenaline level was significantly elevated 2 hr after treatment with FL; however, this change was not observed following the administration of EC alone. The present results indicated that FL significantly enhanced systemic energy expenditure, as evidenced by an accompanying increase in the type of gene expression responsible for thermogenesis and lipolysis, whereas EC exhibited this less robustly or effectively. It was suggested the possible interaction between thermogenic and lipolytic effects and the increase in plasma catecholamine concentrations after administration of a single oral dose of FL.

Hamiltonian Monte Carlo methods for efficient parameter estimation in steady state dynamical systems.

BACKGROUND: Parameter estimation for differential equation models of intracellular processes is a highly relevant bu challenging task. The available experimental data do not usually contain enough information to identify all parameters uniquely, resulting in ill-posed estimation problems with often highly correlated parameters. Sampling-based Bayesian statistical approaches are appropriate for tackling this problem. The samples are typically generated via Markov chain Monte Carlo, however such methods are computationally expensive and their convergence may be slow, especially if there are strong correlations between parameters. Monte Carlo methods based on Euclidean or Riemannian Hamiltonian dynamics have been shown to outperform other samplers by making proposal moves that take the local sensitivities of the system's states into account and accepting these moves with high probability. However, the high computational cost involved with calculating the Hamiltonian trajectories prevents their widespread use for all but the smallest differential equation models. The further development of efficient sampling algorithms is therefore an important step towards improving the statistical analysis of predictive models of intracellular processes. RESULTS: We show how state of the art Hamiltonian Monte Carlo methods may be significantly improved for steady state dynamical models. We present a novel approach for efficiently calculating the required geometric quantities by tracking steady states across the Hamiltonian trajectories using a Newton-Raphson method and employing local sensitivity information. Using our approach, we compare both Euclidean and Riemannian versions of Hamiltonian Monte Carlo on three models for intracellular processes with real data and demonstrate at least an order of magnitude improvement in the effective sampling speed. We further demonstrate the wider applicability of our approach to other gradient based MCMC methods, such as those based on Langevin diffusions. CONCLUSION: Our approach is strictly benefitial in all test cases. The Matlab sources implementing our MCMC methodology is available from https://github.com/a-kramer/ode_rmhmc.

Assessment of mitochondrial biogenesis and mTORC1 signaling during chronic rapamycin feeding in male and female mice.

Chronic inhibition of the protein synthesis regulator mTORC1 through rapamycin extends life span in mice, with longer extension in females than in males. Whether rapamycin treatment inhibits protein synthesis or whether it does so differently between sexes has not been examined. UM-HET3 mice were fed a control or rapamycin-supplemented (Rap) diet for 12 weeks. Protein synthesis in mixed, cytosolic (cyto), and mitochondrial (mito) fractions and DNA synthesis and mTORC1 signaling were determined in skeletal muscle, heart, and liver. In both sexes, mito protein synthesis was maintained in skeletal muscle from Rap despite decreases in mixed and cyto fractions, DNA synthesis, and rpS6 phosphorylation. In the heart, no change in protein synthesis occurred despite the decreased DNA synthesis. In the heart and liver, Rap males were more sensitive to mTORC1 inhibition than Rap females. In conclusion, we show changes in protein synthesis and mTORC1 signaling that differ by sex and tissue.

Cytofluorometric assessment of cell cycle progression.

One of the most prominent features of cellular senescence, a stress response that prevents the propagation of cells that have accumulated potentially oncogenic alterations, is a permanent loss of proliferative potential. Thus, at odds with quiescent cells, which resume proliferation when stimulated to do so, senescent cells cannot proceed through the cell cycle even in the presence of mitogenic factors. Here, we describe a set of cytofluorometric techniques for studying how chemical and/or physical stimuli alter the cell cycle in vitro, in both qualitative and quantitative terms. Taken together, these methods allow for the identification of bona fide cytostatic effects as well as for a refined characterization of cell cycle distributions, providing information on proliferation, DNA content as well as on the presence of cell cycle phase-specific markers. At the end of the chapter, a set of guidelines is offered to assist researchers that approach the study of the cell cycle with the interpretation of results.

Proteomics of Drosophila compound eyes: early studies, now, and the future--light-induced protein phosphorylation as an example.

In the past three decades, efforts to understand the molecular mechanisms underlying photoreceptor transduction of the fruit fly Drosophila melanogaster experienced drastic waves of technological development that involve multiple areas of scientific disciplines; the multidisciplinary approach includes a classical genetic manipulation in which random mutations are created and phenotypes are screened, a modern genetics maneuver in which a specific gene relevant to a hypothesis is molecularly cloned and manipulated, and, more recently, direct studies of proteins by proteomics technologies in combination with modern molecular biology and electrophysiology. This paper will review efforts that originated three decades ago in Professor William L. Pak's laboratory at Purdue University to study proteins involved in the Drosophila photoreceptor transduction process and show the power of such multidisciplinary approach that involves collaboration between molecular genetics, electrophysiology, and proteomics.