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Polymer coatings on cellulosic fibres are widely used to enhance the natural fibre properties by improving, for example, the hydrophobicity and wet strength. Here, we investigate the effects of a terpolymer P(S-co-MABP-co-PyMA) coating on cotton linters and eucalyptus fibres to improve the resistance of cellulose fibres against wetness. Coated and uncoated fibres were characterised by using scanning electron microscopy, contact angle measurements, Raman spectroscopy and atomic force microscopy with the objective of correlating macroscopic properties such as the hydrophobicity of the fleece with microscopic properties such as the coating distribution and local nanomechanics. The scanning electron and fluorescence microscopy results revealed the distribution of the coating on the paper fleeces and fibres. Contact angle measurements proved the hydrophobic character of the coated fleece, which was also confirmed by Raman spectroscopy measurements that investigated the water uptake in single fibres. The water uptake also induced a change in the local mechanical properties, as measured by atomic force microscopy. These results verify the basic functionality of the hydrophobic coating on fibres and paper fleeces but call into question the homogeneity of the coating., Comment: 24 pages, 5 figures, submitted

The productive potential, apparent nitrogen conversion efficiency, nitrogen contained in the plant tissue, and apparent nitrogen recovery of Mombasa grass subjected to nitrogen fertilization (0, 100, 300, and 500 kg ha -1 ) and cutting heights were evaluated (0.20 and 0.40 m). The experimental design was a complete Randomized block with a 4 x 2 factorial arrangement, in plots subdivided over time with four replications. The dry mass production (DMP) was determined for the whole plant (PI) and its fractions, and only for the whole plant the apparent nitrogen conversion efficiency (ANCE), contained nitrogen (NC) and apparent nitrogen recovery (ANR). The residual heights evaluated did not interfere (P> 0.05) in the production results and the nitrogen dynamics. An effect (P

Summary Internalized and ubiquitinated signaling receptors are silenced by their intraluminal budding into multivesicular bodies aided by the endosomal sorting complexes required for transport (ESCRT) machinery. HD-PTP, an ESCRT protein, forms complexes with ESCRT-0, -I and -III proteins, and binds to Endofin, a FYVE-domain protein confined to endosomes with poorly understood roles. Using proximity biotinylation, we showed that Endofin forms a complex with ESCRT constituents and Endofin depletion increased integrin α5-and EGF-receptor plasma membrane density and stability by hampering their lysosomal delivery. This coincided with sustained receptor signaling and increased cell migration. Complementation of Endofin- or HD-PTP-depleted cells with wild-type Endofin or HD-PTP, but not with mutants harboring impaired Endofin/HD-PTP association or cytosolic Endofin, restored EGFR lysosomal delivery. Endofin also promoted Hrs indirect interaction with HD-PTP. Jointly, our results indicate that Endofin is required for HD-PTP and ESCRT-0 interdependent sorting of ubiquitinated transmembrane cargoes to ensure efficient receptor desensitization and lysosomal delivery., Graphical abstract, Highlights • Endofin forms a complex with ESCRT proteins and EGFR on early endosomes • Endofin is required for activated EGFR and integrin α5 lysosomal targeting • Endofin promotes HD-PTP colocalization with ESCRT-0 and -III on early endosomes • Endofin depletion increases cell migration and sustains receptor signaling, Biological sciences; Molecular biology; Cell biology

Enzyme additives have been added to forage at ensiling to improve nutritive value. The aim of this study was to evaluate effects of adding exogenous enzymes to silage on fermentative losses and profile, aerobic stability, chemical composition, in vitro degradation, microbial quality, and nutrients intake and digestibility. Treatments were control (CON); addition of exo-1,4-α-glycosidase glucoamylase (GLU; Kerazyme 3035, Kera Nutrição Animal, Bento Gonçalves, Brazil); addition of β-glucan 4-glucanhydrolase (CEL); and GLU + CEL. Data from the silo experiment were analyzed using PROC MIXED of SAS, with fixed effects of glucoamylase and cellulase, and interaction effect between them. In vivo experiment analyses also included fixed effect of Latin Square and period, and random effect of animal within Latin Square. CEL increased (P ≤ 0.038) gas losses and effluents production, CEL and GLU decreased (P = 0.039) DM recovery compared to control but not differ from GLU+CEL. CEL silage had higher (P ≤ 0.021) starch and crude protein and in vitro digestibility of DM and NDF (P ≤ 0.032), while GLU had higher (P = 0.001) ADF. CEL showed lower (P = 0.012) ethanol content and higher (P = 0.02) anaerobic bacteria counts, while GLU showed higher (P = 0.012) lactate concentration and lower (P ≤ 0.002) counts of bacteria and fungi. Lambs fed with CEL presented higher (P ≤ 0.012) digestibility coefficients for DM, OM, CP and NDF. Decrease on DM recovery indicates no improvements on the nutritive value of silage. On the other hand, cellulolytic enzyme positively affected animal digestion.

Cell surface receptors trigger the activation of signaling pathways to regulate key cellular processes, including cell survival and proliferation. Internalization, sorting, and trafficking of activated receptors, therefore, play a major role in the regulation and attenuation of cell signaling. Efficient sorting of endocytosed receptors is performed by the ESCRT machinery, which targets receptors for degradation by the sequential establishment of protein complexes. These events are tightly regulated and malfunction of ESCRT components can lead to abnormal trafficking and sustained signaling and promote tumor formation or progression. In this review, we analyze the modular domain organization of the alternative ESCRT protein HD-PTP and its role in receptor trafficking and tumorigenesis.

Folliculin (FLCN) is a tumor suppressor gene responsible for the inherited Birt-Hogg-Dubé (BHD) syndrome, which affects kidneys, skin and lungs. FLCN is a highly conserved protein that forms a complex with folliculin interacting proteins 1 and 2 (FNIP1/2). Although its sequence does not show homology to known functional domains, structural studies have determined a role of FLCN as a GTPase activating protein (GAP) for small GTPases such as Rag GTPases. FLCN GAP activity on the Rags is required for the recruitment of mTORC1 and the transcriptional factors TFEB and TFE3 on the lysosome, where mTORC1 phosphorylates and inactivates these factors. TFEB/TFE3 are master regulators of lysosomal biogenesis and function, and autophagy. By this mechanism, FLCN/FNIP complex participates in the control of metabolic processes. AMPK, a key regulator of catabolism, interacts with FLCN/FNIP complex. FLCN loss results in constitutive activation of AMPK, which suggests an additional mechanism by which FLCN/FNIP may control metabolism. AMPK regulates the expression and activity of the transcriptional cofactors PGC1α/β, implicated in the control of mitochondrial biogenesis and oxidative metabolism. In this review, we summarize our current knowledge of the interplay between mTORC1, FLCN/FNIP, and AMPK and their implications in the control of cellular homeostasis through the transcriptional activity of TFEB/TFE3 and PGC1α/β. Other pathways and cellular processes regulated by FLCN will be briefly discussed.

Increased autophagy and lysosomal activity promote tumor growth, survival and chemo-resistance. During acute starvation, autophagy is rapidly engaged by AMPK activation and mTORC1 inhibition to maintain energy homeostasis and cell survival. TFEB and TFE3 are master transcriptional regulators of autophagy and lysosomal activity and their cytoplasm/nuclear shuttling is controlled by mTORC1-dependent multisite phosphorylation. However, it is not known whether and how the transcriptional activity of TFEB or TFE3 is regulated. We show that AMPK mediates phosphorylation of TFEB and TFE3 on three serine residues, leading to TFEB/TFE3 transcriptional activity upon nutrient starvation, FLCN depletion and pharmacological manipulation of mTORC1 or AMPK. AMPK loss does not affect TFEB/TFE3 nuclear localization nor protein levels but reduces their transcriptional activity. Collectively, we show that mTORC1 specifically controls TFEB/TFE3 cytosolic retention whereas AMPK is essential for TFEB/TFE3 transcriptional activity. This dual and opposing regulation of TFEB/TFE3 by mTORC1 and AMPK is reminiscent of the regulation of another critical regulator of autophagy, ULK1. Surprisingly, we show that chemoresistance is mediated by AMPK-dependent activation of TFEB, which is abolished by pharmacological inhibition of AMPK or mutation of serine 466/467/469 to alanine residues within TFEB. Altogether, we show that AMPK is a key regulator of TFEB/TFE3 transcriptional activity, and we validate AMPK as a promising target in cancer therapy to evade chemotherapeutic resistance.

Microfluidic systems made out of polydimethylsiloxane (PDMS) offer a platform to mimic vascular flow conditions in model systems at well-defined shear stresses. However, extracellular matrix (ECM) proteins that are physisorbed on the PDMS are not reliably attached under high shear stress conditions, which makes long-term experiments difficult. To overcome this limitation, we functionalized PDMS surfaces with 3-aminopropyltriethoxysilane (APTES) by using different surface activation methods to develop a stable linkage between the PDMS surface and collagen, which served as a model ECM protein. The stability of the protein coating inside the microfluidic devices was evaluated in perfusion experiments with phosphate-buffered saline (PBS) at 10−40 dynes/cm2 wall shear stress. To assess the stability of cell adhesion, endothelial cells were grown in a multi-shear device over a shear stress range of 20−150 dynes/cm2. Cells on the APTES-mediated collagen coating were stable over the entire shear stress range in PBS (pH 9) for 48 h. The results suggest that at high pH values, the electrostatic interaction between APTES-coated surfaces and collagen molecules offer a very promising tool to modify PDMS-based microfluidic devices for long-term endothelialization under high shear stress conditions.

The signalling output of many transmembrane receptors that mediate cell-cell communication is restricted by the endosomal sorting complex required for transport (ESCRT), but the impact of this machinery on Eph tyrosine kinase receptor function is unknown. We identified the ESCRT-associated adaptor protein HD-PTP as part of an EphB2 proximity-dependent biotin identification (BioID) interactome, and confirmed this association using co-immunoprecipitation. HD-PTP loss attenuates the ephrin-B2:EphB2 signalling-induced collapse of cultured cells and axonal growth cones, and results in aberrant guidance of chick spinal motor neuron axons in vivo. HD-PTP depletion abrogates ephrin-B2-induced EphB2 clustering, and EphB2 and Src family kinase activation. HD-PTP loss also accelerates ligand-induced EphB2 degradation, contrasting the effects of HD-PTP loss on the relay of signals from other cell surface receptors. Our results link Eph function to the ESCRT machinery and demonstrate a role for HD-PTP in the earliest steps of ephrin-B:EphB signalling, as well as in obstructing premature receptor depletion.

Background Neuroblastoma is the most common extracranial solid tumor in children. This cancer has a low frequency of TP53 mutations and its downstream pathway is usually intact. This study assessed the efficacy of the p53 activator, PRIMA-1MET, in inducing neuroblastoma cell death. Methods CellTiter 2.0 was used to study susceptibility and specificity of NB cell lines to PRIMA-1MET. Real-time PCR and western blot were used to assess the most common p53 transactivation targets. Induction of p53 and Noxa, and inhibition of Cas3/7, were used to assess impact on cell death after PRIMA-1MET treatment. Flow cytometry was used to analyze cell cycle phase and induction of apoptosis, reactive oxygen species, and the collapse of mitochondrial membrane potential. Results Neuroblastoma cell lines were at least four times more susceptible to PRIMA-1MET than were primary fibroblasts and keratinocyte cell lines. PRIMA-1MET induced cell death rapidly and in all cell cycle phases. Although PRIMA-1MET activated p53 transactivation activity, p53’s role is likely limited because its main targets remained unaffected, whereas pan-caspase inhibitor demonstrated no ability to prevent cell death. PRIMA-1MET induced oxidative stress and modulated the methionine/cysteine/glutathione axis. Variations of MYCN and p53 modulated intracellular levels of GSH and resulted in increased/decreased sensitivity of PRIMA-1MET. PRIMA-1MET inhibited thioredoxin reductase, but the effect of PRIMA-1MET was not altered by thioredoxin inhibition. Conclusions PRIMA-1MET could be a promising new agent to treat neuroblastoma because it demonstrated good anti-tumor action. Although p53 is involved in PRIMA-1MET-mediated cell death, our results suggest that direct interaction with p53 has a limited role in neuroblastoma but rather acts through modulation of GSH levels. Electronic supplementary material The online version of this article (10.1186/s13046-019-1066-6) contains supplementary material, which is available to authorized users.

The endosomal sorting complexes required for transport (ESCRT) comprise a major trafficking pathway for plasma membrane proteins and are fundamental for ubiquitin-dependent cargo endocytosis. Here, we describe a method for studying the effect of ESCRT complexes on endo-lysosomal membrane trafficking and their role in receptor integrin α5β1 downregulation. Single cell fluorescence ratio image analysis (FRIA), using appropriate fluorescence probes, enables the measurement of dynamics of integrin α5β1 containing vesicles and represents a live cell-based method for studying the role of ESCRTs.

Summary: TFEB and TFE3 are transcriptional regulators of the innate immune response, but the mechanisms regulating their activation upon pathogen infection are poorly elucidated. Using C. elegans and mammalian models, we report that the master metabolic modulator 5′-AMP-activated protein kinase (AMPK) and its negative regulator Folliculin (FLCN) act upstream of TFEB/TFE3 in the innate immune response, independently of the mTORC1 signaling pathway. In nematodes, loss of FLCN or overexpression of AMPK confers pathogen resistance via activation of TFEB/TFE3-dependent antimicrobial genes, whereas ablation of total AMPK activity abolishes this phenotype. Similarly, in mammalian cells, loss of FLCN or pharmacological activation of AMPK induces TFEB/TFE3-dependent pro-inflammatory cytokine expression. Importantly, a rapid reduction in cellular ATP levels in murine macrophages is observed upon lipopolysaccharide (LPS) treatment accompanied by an acute AMPK activation and TFEB nuclear localization. These results uncover an ancient, highly conserved, and pharmacologically actionable mechanism coupling energy status with innate immunity. : El-Houjeiri et al. show that loss of FLCN or pharmacological activation of AMPK induces TFEB/TFE3-dependent pro-inflammatory cytokine expression and phagocytosis in macrophages and confers pathogen resistance in C. elegans. These results uncover an ancient, highly conserved, and pharmacologically actionable mechanism coupling energy status to innate immunity. Keywords: TFEB, TFE3, FLCN, AMPK, innate immune response, pathogen resistance, autophagy, lysosomal biogenesis, phagocytosis

The method of electroless plating offers a facile synthesis route to high-aspect ratio metal nanotubes. Despite the simplicity of the method, the traditional approach involves hazardous and toxic chemicals; to change this Green Chemistry has come into play. Since both, nanotechnology and Green Chemistry, have become important research topics, the combination of these offers new opportunities. According to the concept of Green Chemistry, Pd nanotubes were produced by using nonhazardous chemicals. The challenge was to rebuild the sensitisation and activation process of electroless plating and to find green reaction parameters for controlling the auto-catalysed heterogeneous nucleation of Pd. The produced Pd nanotubes were characterised by SEM and EDX. Furthermore TEM characterisation was done for determining the structural properties of Pd NTs. The efficiency of the reaction was quantified by ICP-OES and XPS measurements. To illustrate that green synthesized nanomaterials can be compared with conventionally prepared catalysts, the Pd nanotubes were tested in a model reaction to determine their catalytic activity. For this purpose the reduction of 4-nitrophenol to 4-aminophenol with NaBH4 was chosen, which also finds application in the synthesis of acetaminophen.

Activation of AMPK has been associated with pro‐atrophic signaling in muscle. However, AMPK also has anti‐inflammatory effects, suggesting that in cachexia, a syndrome of inflammatory‐driven muscle wasting, AMPK activation could be beneficial. Here we show that the AMPK agonist AICAR suppresses IFNγ/TNFα‐induced atrophy, while the mitochondrial inhibitor metformin does not. IFNγ/TNFα impair mitochondrial oxidative respiration in myotubes and promote a metabolic shift to aerobic glycolysis, similarly to metformin. In contrast, AICAR partially restored metabolic function. The effects of AICAR were prevented by the AMPK inhibitor Compound C and were reproduced with A‐769662, a specific AMPK activator. AICAR and A‐769662 co‐treatment was found to be synergistic, suggesting that the anti‐cachectic effects of these drugs are mediated through AMPK activation. AICAR spared muscle mass in mouse models of cancer and LPS induced atrophy. Together, our findings suggest a dual function for AMPK during inflammation‐driven atrophy, wherein it can play a protective role when activated exogenously early in disease progression, but may contribute to anabolic suppression and atrophy when activated later through mitochondrial dysfunction and subsequent metabolic stress.

Cachexia is a deadly muscle wasting syndrome that arises under conditions linked to chronic inflammation, such as cancer. Cytokines, including interferon γ (IFNγ), tumor necrosis factor α (TNFα) and interleukin-6 (IL-6), and their downstream effectors such as Signal Transducer and Activator of Transcription 3 (STAT3), have been shown to play a prominent role in muscle wasting. Previously, we demonstrated that Pateamine A (PatA), a compound that targets eukaryotic initiation factor 4A (eIF4A), could prevent muscle wasting by modulating the translation of the inducible Nitric Oxide Synthase (iNOS) mRNA. Here we show that hippuristanol, a compound that impedes eIF4A in a manner distinct from PatA, similarly inhibits the iNOS/NO pathway and cytokine-induced muscle wasting. Furthermore, we show that hippuristanol perturbs the activation of the STAT3 pathway and expression of STAT3-gene targets such as IL-6. The decreased activation of STAT3, which resulted from a decrease in STAT3 protein expression, was due to the inhibition of STAT3 translation as there were no changes in STAT3 mRNA levels. These effects are likely dependent on the inhibition of eIF4A activity since we observed similar results using PatA. Our results identify the inhibition of eIF4A-responsive transcripts, such as STAT3, as a viable approach to alleviate cachexia.

Glycogen is a main carbohydrate energy storage primarily found in fungi and animals. It is a glucose polymer that comprises α(1-4) glycosidic linkages attaching UDP-glucose molecules linearly and α(1-6) linkages branching glucose chains every 8-10 molecules to the main backbone chain. Glycogen synthase, branching enzyme, and glycogen phosphorylase are key enzymes involved in glycogen synthesis and degradation. These enzymes are tightly regulated by upstream kinases and phosphatases that respond to hormonal cues in order to coordinate storage and degradation and meet the cellular and organismal metabolic needs. The 5'AMP-activated protein kinase (AMPK) is one of the main regulators of glycogen metabolism. Despite extensive research, the role of AMPK in glycogen synthesis and degradation remains controversial. Specifically, the level and duration of AMPK activity highly influence the outcome on glycogen reserves. Here, we describe a rapid and robust protocol to efficiently measure the levels of glycogen in vitro. We use the commercially available glycogen determination kit to hydrolyze glycogen into glucose, which is oxidized to form D-gluconic acid and hydrogen peroxide that react with the OxiRed/Amplex Red probe generating a product that could be detected either in a colorimetric or fluorimetric plate format. This method is quantitative and could be used to address the role of AMPK in glycogen metabolism in cells and tissues. Summary This chapter provides a quick and reliable biochemical quantitative method to measure glycogen in cells and tissues. Briefly, this method is based on the degradation of glycogen to glucose, which is then specifically oxidized to generate a product that reacts with the OxiRed probe with maximum absorbance at 570 nm. This method is very accurate and highly sensitive. In the notes of this chapter, we shed the light on important actions that should be followed to get reliable results. We also state advantages and disadvantages of this method in comparison to other glycogen measurement techniques.

Tunable diode laser absorption spectroscopy combined with extractive sampling is used to quantify fuel and acetylene concentrations in a 32% ethylene-diluted, nitrogen diffusion flame. Additional major species concentrations are determined using quartz microprobe, direct-sampling mass spectrometry. Quantification of these mass spectra used a multilinear regression technique anchored by computed nitrogen concentration profiles in a simulation from Yale University. Because of the overlap in mass spectra features for ethylene, nitrogen, and carbon monoxide, both optical and mass spectrometric techniques are required to determine the suite of major species in this flame system. Good agreement is observed between these experimental measurements and numerical simulation of this flame system. Probe clogging in these sooting flames remains a challenge for all probe techniques and additional, non-intrusive optical diagnostics must be applied to characterize flame regions with substantial soot formation.

We describe a study using the ASIA-Eagle hyperspectral sensor to measure the spectral response of spring barley over an entire climate-controlled growing season and correlate those results with the results of 25 biophysical and biochemical parameters. The spectrum of each hyperspectral image was used to calculate a range of vegetation indices (VIs) that have been recorded in the literature. Furthermore, all combinations of the 252 spectral bands were tested to calculate a range of difference vegetation indices (VIs(xy)) and reflectance value indices (R(x)) at the central wavelength (x nm) of each band (R(x)). For all three index types we examined the relationship with the vegetation variables measured on the ground by using a cross-validation procedure. The relationship between the estimated and the measured canopy chlorophyll content (CCC) was (CV, covariance of variation). An was obtained when modelling leaf area index (LAI), chlorophyll content (Chl-SPAD) as well as leaf gravimetric water content (GWC)...

libRadtran is a widely used software package for radiative transfer calculations. It allows one to compute (polarized) radiances, irradiance, and actinic fluxes in the solar and thermal spectral regions. libRadtran has been used for various applications, including remote sensing of clouds, aerosols and trace gases in the Earth's atmosphere, climate studies, e.g., for the calculation of radiative forcing due to different atmospheric components, for UV forecasting, the calculation of photolysis frequencies, and for remote sensing of other planets in our solar system. The package has been described in Mayer and Kylling (2005). Since then several new features have been included, for example polarization, Raman scattering, a new molecular gas absorption parameterization, and several new parameterizations of cloud and aerosol optical properties. Furthermore, a graphical user interface is now available, which greatly simplifies the usage of the model, especially for new users. This paper gives an overview of libRadtran version 2.0.1 with a focus on new features. Applications including these new features are provided as examples of use. A complete description of libRadtran and all its input options is given in the user manual included in the libRadtran software package, which is freely available at http://www.libradtran.org.

Busulfan (Bu) is an important component of the myeloablative conditioning regimen prior to hematopoietic stem cell transplantation (HSCT) especially in children. Intravenously administered Bu exhibits a therapeutic window phenomenon requiring therapeutic drug monitoring. Analytical methods developed for Bu routine monitoring were aimed at using low volumes of biological fluids and development of simple procedures to facilitate the dosage adjustment. In this report, we describe a simple, rapid method for Bu measurement using dried blood spots (DBS) from only 5 μL of whole blood.

Mechanisms of adaptation to environmental changes in osmolarity are fundamental for cellular and organismal survival. Here we identify a novel osmotic stress resistance pathway in Caenorhabditis elegans (C. elegans), which is dependent on the metabolic master regulator 5’-AMP-activated protein kinase (AMPK) and its negative regulator Folliculin (FLCN). FLCN-1 is the nematode ortholog of the tumor suppressor FLCN, responsible for the Birt-Hogg-Dubé (BHD) tumor syndrome. We show that flcn-1 mutants exhibit increased resistance to hyperosmotic stress via constitutive AMPK-dependent accumulation of glycogen reserves. Upon hyperosmotic stress exposure, glycogen stores are rapidly degraded, leading to a significant accumulation of the organic osmolyte glycerol through transcriptional upregulation of glycerol-3-phosphate dehydrogenase enzymes (gpdh-1 and gpdh-2). Importantly, the hyperosmotic stress resistance in flcn-1 mutant and wild-type animals is strongly suppressed by loss of AMPK, glycogen synthase, glycogen phosphorylase, or simultaneous loss of gpdh-1 and gpdh-2 enzymes. Our studies show for the first time that animals normally exhibit AMPK-dependent glycogen stores, which can be utilized for rapid adaptation to either energy stress or hyperosmotic stress. Importantly, we show that glycogen accumulates in kidneys from mice lacking FLCN and in renal tumors from a BHD patient. Our findings suggest a dual role for glycogen, acting as a reservoir for energy supply and osmolyte production, and both processes might be supporting tumorigenesis., Author Summary The ability of an organism to adapt to sudden changes in environmental osmolarity is critical to ensure growth, propagation, and survival. The synthesis of organic osmolytes is a common adaptive strategy to survive hyperosmotic stress. However, it was not well understood, which biosynthetic pathways and storage strategies were used by organisms to rapidly generate osmolytes upon acute hyperosmotic stress. Here, we demonstrate that glycogen is an essential reservoir that is used upon acute hyperosmotic stress to generate the organic osmolyte glycerol promoting fast and efficient protection. Importantly, we show that this pathway is regulated by FLCN-1, an ortholog of the human tumor suppressor Folliculin responsible for the Birt-Hogg-Dubé cancer syndrome, and by AMPK, the master regulator of energy homeostasis.

Under conditions of reduced tissue oxygenation, hypoxia-inducible factor (HIF) controls many processes, including angiogenesis and cellular metabolism, and also influences cell proliferation and survival decisions. HIF is centrally involved in tumour growth in inherited diseases that give rise to renal cell carcinoma (RCC), such as Von Hippel-Lindau syndrome and tuberous sclerosis complex. In this study, we examined whether HIF is involved in tumour formation of RCC in Birt-Hogg-Dube syndrome. For this, we analysed a Birt-Hogg-Dube patient-derived renal tumour cell line (UOK257) that is devoid of the Birt-Hogg-Dube protein (BHD) and observed high levels of HIF activity. Knockdown of BHD expression also caused a threefold activation of HIF, which was not as a consequence of more HIF1 alpha or HIF2 alpha protein. Transcription of HIF target genes VEGF, BNIP3 and CCND1 was also increased. We found nuclear localization of HIF1 alpha and increased expression of VEGF, BNIP3 and GLUT1 in a chromophobe carcinoma from a Birt-Hogg-Dube patient. Our data also reveal that UOK257 cells have high lactate dehydrogenase, pyruvate kinase and 3-hydroxyacyl-CoA dehydrogenase activity. We observed increased expression of pyruvate dehydrogenase kinase 1 (a HIF gene target), which in turn leads to increased phosphorylation and inhibition of pyruvate dehydrogenase. Together with increased protein levels of GLUT1, our data reveal that UOK257 cells favour glycolytic rather than lipid metabolism (a cancer phenomenon termed the 'Warburg effect'). UOK257 cells also possessed a higher expression level of the L-lactate influx monocarboxylate transporter 1 and consequently utilized L-lactate as a metabolic fuel. As a result of their higher dependency on glycolysis, we were able to selectively inhibit the growth of these UOK257 cells by treatment with 2-deoxyglucose. This work suggests that targeting glycolytic metabolism may be used therapeutically to treat Birt-Hogg-Dube-associated renal lesions. Oncogene (2011) 30, 1159-1173; doi:10.1038/onc.2010.497; published online 8 November 2010

A structure-based approach was pursued in designing novel bisphosphonate inhibitors of the human farnesyl pyrophosphate synthase (hFPPS). Preliminary SAR and structural evidence for the simultaneous binding of these inhibitors into the isopentenyl pyrophosphate (IPP) and the geranyl pyrophosphate (GPP) substrate sub-pockets of the enzyme are presented.

Androstenone has often been discussed as a human social chemosignal, as it is one of the major contributing substances to the human body odor for which humans possess specific receptors. Here, we investigated whether male sexual orientation influences the perception of androstenone. Sensitivity to androstenone, subjective ratings of intensity, pleasantness, unpleasantness, and familiarity of the odor, as well as self-reported emotional responses (valence, arousal, dominance) to the odor were assessed in 13 homosexual and 14 heterosexual men. Isovaleric acid served as a control substance. Homosexual men displayed significantly higher olfactory sensitivity to the odor of androstenone than heterosexual men (p 0.25). Moreover, both groups did not differ in their judgments of or in their emotional response to androstenone or isovaleric acid. The current results indicate that men's sexual orientation significantly impacts the perception of androstenone but that this effect does not necessarily extend to subjective judgments.

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved. Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

Continuous intrathecal administration of baclofen with implanted programmable pump systems is recommended in the treatment of severe spasticity of cerebral origin. Prior to pump implantation, a baclofen bolus test (BBT) is used to assess the effectiveness of intrathecal baclofen using clinical scales such as the Modified Ashworth Scale (MAS). In the literature, the time and period of maximum effect of a bolus dose of intrathecally administered baclofen in patients with cerebral spasticity is variously reported. The aim of the study was, therefore, to reveal the time course of the effect of a BBT on severe cerebral spasticity by the use of a recently described spasticity measurement method. Spasticity in knee joints of 13 patients with severe cerebral spasticity was repeatedly assessed using the MAS and also continuously recorded by the measurement of force under circular fibreglass casts. Force was recorded as nettorque by multiplying the force by the distance between sensor and joint axis, thus allowing inter-individual comparison. Half-hour time integrals (TI) of net-torque were determined 9 hours before and 22 hours after intrathecal baclofen administration. Post-BBT half-hour time integrals (TI+0, TI+0.5, to TI+22) were compared with the mean of 17 pre-BBT half-hour time integrals % MathType!Translator!2!1!AMS LaTeX.tdl!TeX -- AMS-LaTeX! % MathType!MTEF!2!1!+- % feaafeart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn % hiov2DGi1BTfMBaeXafv3ySLgzGmvETj2BSbqefm0B1jxALjhiov2D % aebbnrfifHhDYfgasaacH8srps0lbbf9q8WrFfeuY-Hhbbf9v8qqaq % Fr0xc9pk0xbba9q8WqFfea0-yr0RYxir-Jbba9q8aq0-yq-He9q8qq % Q8frFve9Fve9Ff0dmeaabaqaciGacaGaaeqabaWaaeaaeaaakeaada % qadaqaamaanaaabaGaaeivaiaabMeaaaWaaSbaaSqaaiabgkHiTiaa % iMdacaaMe8UaaeiDaiaab+gacaaMe8UaeyOeI0IaaGymaaqabaaaki % aawIcacaGLPaaaaaa!4430! $$ {\left( {\overline{{{\text{TI}}}} _{{ - 9\;{\text{to}}\; - 1}} } \right)} $$ . Significantly lower post-BBT half-hour time integrals compared with % MathType!Translator!2!1!AMS LaTeX.tdl!TeX -- AMS-LaTeX! % MathType!MTEF!2!1!+- % feaafeart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn % hiov2DGi1BTfMBaeXafv3ySLgzGmvETj2BSbqefm0B1jxALjhiov2D % aebbnrfifHhDYfgasaacH8srps0lbbf9q8WrFfeuY-Hhbbf9v8qqaq % Fr0xc9pk0xbba9q8WqFfea0-yr0RYxir-Jbba9q8aq0-yq-He9q8qq % Q8frFve9Fve9Ff0dmeaabaqaciGacaGaaeqabaWaaeaaeaaakeaada % qadaqaamaanaaabaGaaeivaiaabMeaaaWaaSbaaSqaaiabgkHiTiaa % iMdacaaMe8UaaeiDaiaab+gacaaMe8UaeyOeI0IaaGymaaqabaaaki % aawIcacaGLPaaaaaa!4430! $$ {\left( {\overline{{{\text{TI}}}} _{{ - 9\;{\text{to}}\; - 1}} } \right)} $$ were found between TI+2 and TI+8 (Dunnett adjusted p < 0.05). The median lowest TI after BBT of the 13 patients was TI+4. The lowest mean MAS scores were found 4 hours after BBT. The findings suggest that the greatest effect of BBT on cerebral spasticity occurs between 2 and 8.5 hours, with a maximal effect at 4 hours after intrathecal baclofen injection. Clinical scales used to determine the effect of BBT should thus be carried out during this period—ideally at 4 hours after baclofen injection.

Nonwoven protective garments possess a poor thermo-physiological wearing comfort due to their insufficient breathability. The problem can be solved by applying phase change material (PCM) to the fabrics that the garment consists of PCM is a highly-productive thermal storage medium, which can be used to absorb excessive heat released by the human body under strenuous activities. The heat absorption by the PCM results in a delay in the microclimate temperature increase and hence a substantial decrease of the skin's moisture release. Both lead to an overall enhancement of the thermo-physiological wearing comfort and a prevention of heat stress situations as a serious health risk. Furthermore, the wearing times of nonwoven protective garments can be extended, resulting in an increased productivity.

Continuous intrathecal administration of baclofen with implanted programmable pump systems is recommended in the treatment of severe spasticity of cerebral origin. Prior to pump implantation, a baclofen bolus test (BBT) is used to assess the effectiveness of intrathecal baclofen using the modified Ashworth Scale (MAS) and Penn Spasm Frequency Scales (SFS). The result of a BBT may be difficult to interpret in patients with reduced joint mobility caused by contractures. The aim of this study was to apply a new spasticity measurement which would quantify and visualise the effect of a BBT in 10 patients with severe cerebral spasticity and contractures. Spasticity was recorded continuously by the measurement of force under circular fibreglass casts in 10 knee joint contractures. Force was recorded as net-torque by multiplying the force and distance between sensor and joint axis, thus allowing inter-individual comparison. MAS, SFS, and two three-hour time integrals of net-torque were determined before and after intrathecally administered baclofen. No significant changes in MAS (p = 0.1) and SFS (p = 0.07) were observed; however, a significant reduction of time integrals of net-torque after baclofen administration (p = 0.005) was found. The present study shows that the antispastic effect of intrathecally administered BBT can be quantitatively assessed and visualised using the described method. It also suggests that this method can be helpful in the assessment of the effectiveness of the BBT in patients with severe spasticity of cerebral origin and contractures.

In adult therapy, arsenic trioxide (ATO) and all-trans-retinoic acid (ATRA) are recognized as active treatment of relapsed acute promyelocytic leukemia (APL). The efficacy of this combination in pediatric APL has not yet been well established. We report the case of a 6-year-old girl with relapsed APL, with a PML-RARα mutation, treated with a combination of ATO and ATRA. Over a period of 5 months, she received in total, 75 doses of intravenous ATO and 40 doses of oral ATRA. Currently, 22 months after relapse, she is still in complete remission. Here, we describe treatment of a relapsed APL in a child with limited treatment of ATO and ATRA and review the literature.

The idea that significant ion/radical interactions should vary with solvent if they do exist in the liquid phase was pursued by an investigation of the dissociative electron-transfer reactivity of carbon tetrachloride and 4-cyanobenzyl chloride in four different solvents, 1,2-dichloroethane, N,N-dimethylformamide, ethanol, and formamide, by means of their cyclic voltammetric responses. Modification of the conventional dissociative electron transfer theory to take account of an interaction between fragments in the ion/radical pair resulting from the dissociative electron reaction allows a satisfactory fitting of the experimental data leading to the determination of the interaction energy. There is an approximate correlation between the interaction energies in the ion/radical pair and the solvation free energies of the leaving anion, Cl(-). The interaction is maximal in 1,2-dichloroethane, which is both the least polar and the least able to solvate Cl(-). The interaction is smaller in the polar solvents, albeit distinctly measurable. The two protic solvents, ethanol and formamide, which are the most able to solvate Cl(-), give rise to similar interaction energies. The interaction is definitely stronger in N,N-dimethylformamide, which has a lesser ability to solvate Cl(-) than the two other polar solvents. The existence of significant ion/radical interactions in polar media is thus confirmed and a route to their determination opened.

The electrochemical (cyclic voltammetry) and photoinduced (fluorescence quenching, quantum yields) reductive cleavages of four compounds, 4-cyano-alpha-trifluorotoluene (1), dimethylphenyl sulfonium (2), 4-cyanobenzylmethylphenyl sulfonium (3), and 4-cyanobenzyl chloride (4), are investigated and compared in terms of concerted vs stepwise mechanisms. Bearing in mind that an increase of the thermodynamic driving force shifts the mechanism from concerted to stepwise and that the driving force is larger under photochemical than under electrochemical conditions, 1 and 2 are typical examples where a stepwise mechanism is followed with compatible kinetic characteristics under both regimes. 4 undergoes a concerted electrochemical reductive cleavage, and the same mechanism is followed in the photoinduced reaction with consistent kinetic characteristics. The case of 3 is of particular interest, since a trend of passing from a concerted to a stepwise mechanism when going from the electrochemical to the photochemical conditions indeed appears upon analysis of the experimental results. The change of mechanism is, however, not complete since, in the photoinduced reaction, there is a balanced competition between the two pathways. In the same families of compounds, the unsubstituted benzylmethylphenyl sulfonium cations shows such a borderline behavior during the electrochemical reaction. In the photoinduced reaction, it is the 4-cyano derivative which behaves in a borderline manner, in line with the fact that it gives rise more readily to a concerted mechanism than the unsubstituted compound.

Mutations of the VHL tumor suppressor gene occur in patients with VHL disease and in the majority of sporadic clear cell renal carcinomas (VHL(-/-) RCC). Loss of VHL protein function is associated with constitutive expression of mRNAs encoding hypoxia-inducible proteins, such as vascular endothelial growth factor. Overproduction of angiogenic factors might explain why VHL(-/-) RCC tumors are so highly vascularized, but whether this overproduction is sufficient for oncogenesis still remains unknown. In this report, we examined the activity of transforming growth factor-alpha (TGF-alpha), another VHL-regulated growth factor. We show that TGF-alpha mRNA and protein are hypoxia-inducible in VHL(-/-) RCC cells expressing reintroduced VHL. In addition to its overexpression by VHL(-/-) RCC cells, TGF-alpha can also act as a specific growth-stimulatory factor for VHL(-/-) RCC cells expressing reintroduced wild-type VHL, as well as primary renal proximal tubule epithelial cells, the likely site of origin of RCC. This role is in contrast to those of other growth factors overexpressed by VHL(-/-) RCC cells, such as vascular endothelial growth factor and TGF-beta1, which do not stimulate RCC cell proliferation. A TGF-alpha-specific antisense oligodeoxynucleotide blocked TGF-alpha production in VHL(-/-) RCC cells, which led to the dependence of those cells on exogenous growth factors to sustain growth in culture. Growth of VHL(-/-) RCC cells was also significantly reduced by a drug that specifically inhibits the epidermal growth factor receptor, the receptor through which TGF-alpha stimulates proliferation. These results suggest that the generation of a TGF-alpha autocrine loop as a consequence of VHL inactivation in renal proximal tubule epithelial cells may provide the uncontrolled growth stimulus necessary for the initiation of tumorigenesis.

Environmental cocontamination by polycyclic aromatic hydrocarbons (PAHs) and metals could affect the carcinogenic consequences of PAH exposure by modifying PAH induction of PAH-bioactivating CYP1A. The effect of As, Pb, Hg, or Cd (ranked as the most hazardous environmental metals by EPA and ATSDR) on CYP1A1 and 1A2 induction by benzo[a]pyrene (BaP), benzo[b]fluoranthene (BbF), dibenzo[a,h]anthracene (DBahA), benzo[a]anthracene (BaA), and benzo[k]fluoranthene (BkF) has thus been investigated in fresh human hepatocyte cultures. Induction was probed by ethoxyresorufin-O-deethylase activity, by immunoblots, and by RT-PCR. Uptake of PAHs into the hepatocytes varied according to PAH and liver donor: 84% of 5 microM BaA and 25-40% of 5 microM DBahA was taken up in 24 h. Hepatocytes retained viability up to 1 microM Cd and 5 microM Pb, Hg, or As and 5 microM PAHs. PAH induction of CYP1A in hepatocytes was variable, some cultures expressed CYP1A1 and others CYP1A1 and 1A2, and to variable extents. Induction efficiency (relative to DMSO controls) at 2.5 microM PAH concentration was in the order BkF (7.6-fold) > DBahA (6.1 fold) > BaP (5.7 fold) > BbF (3.9-fold) > BaA (2.5-fold). All four metals (1-5 microM) decreased CYP1A1/1A2 induction by some of the PAHs with dose-, metal-, and PAH-dependency. Arsenic (5 microM) decreased induction by 47% for BaP, 68% for BaA, 45% for BbF, 79% for BkF, and 53% for DBahA. Induced CYP1A2 protein was much more extensively decreased than 1A1 protein, and CYP1A2 mRNA and, to variable extents, CYP1A1 mRNA were decreased by As. Thus the metals in PAH/metal mixtures could diminish PAH carcinogenicity by decreasing induction of their bioactivation by CYP1A1/1A2.

In the course of exploring the electronic properties and chemistry of stable non-annelated and annelated heterocyclic carbenes and carbene analogues (silylenes, germylenes and nitrenium ions), we investigated ground state configurations of such structures as well as their one-electron reduction by means of ab initio calculations. We used a DFT approach, which proved to be reliable for obtaining the geometries of the ground state molecules and was thus employed for all calculations. The compounds investigated are presented in Scheme 2. Injection of one electron into these molecules leads to the formation of radical anions, except for with cation 4a, which gives a neutral radical. For all molecules, ground states are singlet states and comparison has been made in terms of geometrical parameters with known experimental structures and results obtained at the MP2 level of calculation. These results, along with experimental data on the reduction of carbenes, nitrenium ions and germylenes obtained by cyclic voltammetry, give some clues about their stability, properties and potential redox activity.

It has been shown recently that the electrochemical reduction of carbon tetrachloride in N,N'-dimethylformamide follows a mechanism in which electron transfer and bond cleavage are concerted. We report here results concerning photoinduced electron transfer from the singlet excited state of two aromatic molecules, 2-ethyl-9,10-dimethoxyanthracene and perylene, to CCl4 , which is characterised by a quantum yield of complete quenching fragmentation ranging from 0.7 to 0.8. It is shown that a quantum yield below unity is compatible with a dissociative mechanism and arises from partitioning of the system at the intersection of the product- and ground-state potential energy surfaces. This phenomenon predominates over back electron transfer from the clustered fragments state. The photoinduced reductive cleavage of CCl4 thus provides a clear illustration of the recent theoretical prediction, that photoinduced dissociative electron transfers are not necessarily endowed with a unity quantum yield. This offers an opportunity to estimate the magnitude of the electronic matrix element that couples the fragmented product state and the ground reactant state.

The electrochemical reduction of carbon tetrachloride in N,N‘-dimethylformamide follows a mechanism in which electron transfer and bond cleavage are concerted, at least at low and moderate driving forces. A detailed analysis of the kinetics of the reductive cleavage reveals that a small but significant interaction between the Cl- and Cl3C• fragments exists in the product state and is responsible for a strong acceleration of the reaction. An extension of the theory of dissociative electron transfer is proposed to rationalize the kinetic results and estimate the magnitude of the interaction energy. The model explains how a relatively small interaction energy results in a substantial acceleration of the reaction, caused by both an increase of the driving force and a decrease of the intrinsic barrier. Due to the strong polarization of the CCl3 radical, the reaction is a particularly clear example of the possibility that attractive interactions between fragments survive in a polar solvent. Another attractive f...

Androstenone is a boar pheromone. and has also been found within different human body fluids. However, it is still unclear whether it carries pheromonal information in humans and whether it contributes significantly to the complex human body odor at all. Some humans fail to perceive the odor of androstenone, but most of these anosmics can achieve sensitivity by daily sniffing. The following study was designed to investigate whether sensitivity to androstenone influences the perception of body odors. Four females osmic to and four females anosmic to androstenone attended two EEG sessions. Anosmics were successfully sensitized to androstenone between sessions. CSERPs (chemosensory event-related potentials) were obtained while subjects perceived their own body odor and a male body odor within an olfactory oddball paradigm. The CSERPs showed a general decrease in amplitude from the first to the second session except for the sensitized anosmics in response to male body odor. The results indicate that the sensitivity to androstenone in females is associated with a stronger brain response to male body odor.

Zusammenfassung. Bereits mehrfach sind in der Fachliteratur spezifische Anosmien - im Volksmund auch als Geruchsblindheit bezeichnet - für unterschiedliche Duftstoffe bei Menschen und Tieren beschrieben worden. Die durchgeführte Studie befaßt sich in der Hauptsache mit der Frage, ob bei Vorliegen einer spezifischen Anosmie olfaktorische Sensitivität induzierbar ist. Zu diesem Zweck wurden Frauen mit spezifischer Anosmie für den Duftstoff Androstenon auf Induzierbarkeit der Wahrnehmung geprüft, indem die absoluten Wahrnehmungsschwellen für diesen Duftstoff vor und nach wiederholter Duftexposition bestimmt wurde. Androstenon ist eine Komponente des menschlichen Körpergeruchs und findet sich im Achselschweiß von Männern in stärkerer Konzentration als bei Frauen. Die Resultate der vorliegenden Untersuchung zeigen, daß bei über 80 % der Duft-exponierten Teilnehmerinnen nach anfänglicher Anosmie Wahrnehmung induziert werden konnte.

Protein tyrosine phosphatases (PTPs) are key enzymes in the regulation of cellular homeostasis and signaling pathways. Strikingly, not all PTPs bear enzymatic activity. A considerable fraction of PTPs are enzymatically inactive and are known as pseudophosphatases. Despite the lack of activity they execute pivotal roles in development, cell biology and human disease. The present review is focused on the methods used to identify pseudophosphatases, their targets, and physiological roles. We present a strategy for detailed enzymatic analysis of inactive PTPs, regulation of inactive PTP domains and identification of binding partners. Furthermore, we provide a detailed overview of human pseudophosphatases and discuss their regulation of cellular processes and functions in human pathologies.

Androstenone, a compound of human male body odor, might act as a chemosensory signal communicating dominance or aggressiveness. In order to clarify its communicative significance, the relationship between androstenone perception and the level of circulating steroid hormones was investigated in both men and women. Androstenone perception was assessed within n=26 men and n=25 women. Female participants were not currently using hormonal contraception and were in their follicular menstrual cycle phase. Androstenone perception was assessed in terms of olfactory sensitivity, quality judgments, and emotional self-ratings. The perception of isovaleric acid served as a control. Over the course of 2h five saliva samples were collected, aliquots were mixed and levels of estradiol and testosterone were analyzed via enzyme-linked immunosorbent assays. In men, higher testosterone levels were associated with lower olfactory sensitivity to androstenone (p=0.014) and negative feelings when exposed to it (p=0.047). In women, higher estradiol levels were related to judging androstenone as less pleasant (p=0.009) and more unpleasant (p=.0036). The perception of isovaleric acid was unrelated to sex-hormone levels. The current results support the notion of androstenone communicating dominance, aggression or competition. Men with higher testosterone levels are more sensitive to androstenone and dislike its odor, possibly indicating that androstenone signals the readiness for competition in men. Similarly, the fact that women with higher estradiol levels dislike androstenone may be due to androstenone being a signal of reduced willingness for social cooperation and an increased likelihood to engage in extramarital sex.

Infection of cells with picornaviruses, such as poliovirus and encephalomyocarditis virus (EMCV), causes a shutoff of host protein synthesis. The molecular mechanism of the shutoff has been partly elucidated for poliovirus but not for EMCV. Translation initiation in eukaryotes is facilitated by the mRNA 5' cap structure to which the multisubunit translation initiation factor eIF4F binds to promote ribosome binding. Picornaviruses use a mechanism for the translation of their RNA that is independent of the cap structure. Poliovirus infection engenders the cleavage of the eIF4G (formerly p220) component of eIF4F and renders this complex inactive for cap-dependent translation. In contrast, EMCV infection does not result in eIF4G cleavage. Here, we report that both EMCV and poliovirus activate a translational repressor, 4E-BP1, that inhibits cap-dependent translation by binding to the cap-binding subunit eIF4E. Binding of eIF4E occurs only to the underphosphorylated form of 4E-BP1, and this interaction is highly regulated in cells. We show that 4E-BP1 becomes dephosphorylated upon infection with both EMCV and poliovirus. Dephosphorylation of 4E-BP1 temporally coincides with the shutoff of protein synthesis by EMCV but lags behind the shutoff and eIF4G cleavage in poliovirus-infected cells. Dephosphorylation of 4E-BP1 by specifically inhibiting cap-dependent translation may be the major cause of the shutoff phenomenon in EMCV-infected cells.

Mechanisms of adaptation to acute changes in osmolarity are fundamental for life. When exposed to hyperosmotic stress, cells and organisms utilize conserved strategies to prevent water loss and maintain cellular integrity and viability. The production of glycerol is a common strategy utilized by the nematode Caenorhabditis elegans (C. elegans) and many other organisms to survive hyperosmotic stress. Specifically, the transcriptional upregulation of glycerol-3-phosphate dehydrogenase, a rate-limiting enzyme in the production of glycerol, has been previously implicated in many model organisms. However, what fuels this massive and rapid production of glycerol upon hyperosmotic stress has not been clearly elucidated. We have recently discovered an AMPK-dependent pathway that mediates hyperosmotic stress resistance in C. elegans. Specifically, we demonstrated that the chronic activation of AMPK leads to glycogen accumulation, which under hyperosmotic stress exposure, is rapidly degraded to mediate glycerol production. Importantly, we demonstrate that this strategy is utilized by flcn-1 mutant C. elegans nematodes in an AMPK-dependent manner. FLCN-1 is the worm homolog of the human renal tumor suppressor Folliculin (FLCN) responsible for the Birt-Hogg-Dube neoplastic syndrome. Here, we comment on the dual role for glycogen in stress resistance: it serves as an energy store and a fuel for osmolyte production. We further discuss the potential utilization of this mechanism by organisms in general and by human cancer cells in order to survive harsh environmental conditions and notably hyperosmotic stress.

The role of busulfan (Bu) metabolites in the adverse events seen during hematopoietic stem cell transplantation and in drug interactions is not explored. Lack of availability of established analytical methods limits our understanding in this area. The present work describes a novel gas chromatography-tandem mass spectrometric assay for the analysis of sulfolane (Su) in plasma of patients receiving high-dose Bu. Su and Bu were extracted from a single 100 μL plasma sample by liquid-liquid extraction. Bu was separately derivatized with 2,3,5,6-tetrafluorothiophenolfluorinated agent. Mass spectrometric detection of the analytes was performed in the selected reaction monitoring mode on a triple quadrupole instrument after electronic impact ionization. Bu and Su were analyzed with separate chromatographic programs, lasting 5min each. The assay for Su was found to be linear in the concentration range of 20-400ng/mL. The method has satisfactory sensitivity (lower limit of quantification, 20ng/mL) and precision (relative standard deviation less than 15%) for all the concentrations tested with a good trueness (100 ± 5%). This method was applied to measure Su from pediatric patients with samples collected 4h after dose 1 (n = 46), before dose 7 (n = 56), and after dose 9 (n = 54) infusions of Bu. Su (mean ± SD) was detectable in plasma of patients 4h after dose 1, and higher levels were observed after dose 9 (249.9 ± 123.4ng/mL). This method may be used in clinical studies investigating the role of Su on adverse events and drug interactions associated with Bu therapy. Figure Overall sample preparation procedure for quantification of sulfolane and busulfan in plasma from patients receiving higher doses of busulfan

The cloning is described of two related human complementary DNAs encoding polypeptides that interact specifically with the translation initiation factor eIF-4E, which binds to the messenger RNA 5'-cap structure. Interaction of these proteins with eIF-4E inhibits translation but treatment of cells with insulin causes one of them to become hyperphosphorylated and dissociate from eIF-4E, thereby relieving the translational inhibition. The action of this new regulator of protein synthesis is therefore modulated by insulin, which acts to stimulate the overall rate of translation and promote cell growth.

Saccharomyces cerevisiae translation initiation factor eIF-4A, an RNA helicase of the Asp-Glu-Ala-Asp (DEAD) box protein family, was mutated in the putative ATP binding site and expressed in Escherichia coli. Mutant proteins with alanine at position 66 replaced by glycine [eIF-4A(A66G)] or valine [eIF-4A(A66V)] were purified from Escherichia coli extracts and analyzed in vitro for activity in ATP crosslinking, ATP hydrolysis, RNA helicase, and translation assays. The results show that in vitro ATP hydrolysis activity, RNA helicase activity, and translation activity of eIF-4A correlate with in vivo activity of the factor. Whereas eIF-4A(A66G) showed wild-type activity in all assays, eIF-4A(A66V) was active in ATP crosslinking but inactive in ATP hydrolysis and RNA helicase assays. In vitro translation was supported by wild-type eIF-4A and eIF-4A(A66G) but not by eIF-4A(A66V). The results show that, for their translation, the majority of mRNAs from Saccharomyces cerevisiae including an mRNA with the initiator AUG positioned 8 nucleotides downstream of the cap structure require eIF-4A that is able to hydrolyze ATP.