Comparative evaluation of electrospraying and lyophilization techniques on solid state properties of Erlotinib nanocrystals: Assessment of In-vitro cytotoxicity

Publication date: 1 January 2018
Source:European Journal of Pharmaceutical Sciences, Volume 111
Author(s): Shreya Thakkar, Dilip Sharma, Manju Misra
IntroductionErlotinib is a well known FDA approved drug from category of tyrosine kinase inhibitors; used for the treatment of lung cancer. However its use is limited because of its poor water solubility.ObjectiveThe aim of present work was to improve solubility by developing a stable nanocrystal based drug delivery system of ERL with the aid of sodium lauryl sulfate as potential stabilizer and to carry out comparative evaluation of electrospraying and lyophilization as solidification techniques on its solid state properties.ExperimentalNanocrystal formulation was developed with antisolvent precipitation method having particle size, polydispersity index and zetapotential of 232.4±4.3nm, 0.162 and −9.82mV respectively. Further comparative evaluation of lyophilization and electrospraying was commenced as potential solidification techniques and solid powder matrix obtained from both the solidification techniques were compared in terms of size after re-dispersion (260±4.8 and 329±5.2nm respectively), particle morphology, surface area (0.984±0.11 and 0.341±0.05m²/g respectively), pore volume (0.0014 and 0.0009cc/g respectively), solid state of drug present and % drug release (~100% and ~78% respectively in 600min). In vitro cytotoxicity studies shared that obtained formulation was having reduced IC50 values in comparison to drug. Further intracellular reactive oxygen species production was found to be higher for formulation treated cells when compared to free drug. Overall developed formulation was found to be potential drug delivery system for lung cancer therapy.

Graphical abstract

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Statistical investigation of the full concentration range of fasted and fed simulated intestinal fluid on the equilibrium solubility of oral drugs

Publication date: 1 January 2018
Source:European Journal of Pharmaceutical Sciences, Volume 111
Author(s): Jeremy Perrier, Zhou Zhou, Claire Dunn, Ibrahim Khadra, Clive G. Wilson, Gavin Halbert
Upon oral administration the solubility of a drug in intestinal fluid is a key property influencing bioavailability. It is also recognised that simple aqueous solubility does not reflect intestinal solubility and to optimise in vitro investigations simulated intestinal media systems have been developed. Simulated intestinal media which can mimic either the fasted or fed state consists of multiple components each of which either singly or in combination may influence drug solubility, a property that can be investigated by a statistical design of experiment technique. In this study a design of experiment covering the full range from the lower limit of fasted to the upper limit of fed parameters and using a small number of experiments has been performed. The measured equilibrium solubility values are comparable with literature values for simulated fasted and fed intestinal fluids as well as human fasted and fed intestinal fluids. The equilibrium solubility data range is statistically equivalent to a combination of published fasted and fed design of experiment data in six (indomethacin, phenytoin, zafirlukast, carvedilol, fenofibrate and probucol) drugs with three (aprepitant, tadalafil and felodipine) drugs not equivalent. In addition the measured equilibrium solubility data sets were not normally distributed. Further studies will be required to determine the reasons for these results however it implies that a single solubility measurement without knowledge of the solubility distribution will be of limited value. The statistically significant media factors which promote equilibrium solubility (pH, sodium oleate and bile salt) were in agreement with published results but the number of determined significant factors and factor interactions was fewer in this study, lecithin for example did not influence solubility. This may be due to the reduction in statistical sensitivity from the lower number of experimental data points or the fact that using the full range will examine media parameters ratios that are not biorelevant. Overall the approach will provide an estimate of the solubility range and the most important media factors but will not be equivalent to larger scale focussed studies. Further investigations will be required to determine why some drugs do not produce equivalent DoE solubility distributions, for example combined fasted and fed DoE, but this simply may be due to the complexity and individuality of the interactions between a drug and the media components.

Graphical abstract

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Studying of drug solubility in water and alcohols using drug-ammonium ionic liquid-compounds

Publication date: 1 January 2018
Source:European Journal of Pharmaceutical Sciences, Volume 111
Author(s): Mohammad Halayqa, Aneta Pobudkowska, Urszula Domańska, Maciej Zawadzki
Synthesis of three mefenamic acid (MEF) derivatives - ionic liquid compounds composed of MEF in an anionic form and ammonium cation (choline, MEF1), or {di(2-hydroxyethyl)dimethyl ammonium (MEF2)}, or {tri(2-hydroxyethyl)methyl ammonium compound (MEF3)} is presented. The basic thermal properties of pure compounds i.e. fusion temperatures, and the enthalpy of fusion of these compounds have been measured with differential scanning microcalorimetry technique (DSC). Molar volumes have been calculated with the Barton group contribution method. The solubilities of MEF1, MEF2 and MEF3 using the dynamic method were measured at constant pH in a range of temperature from (290 to 370) K in three solvents: water, ethanol and 1-octanol. The experimental solubility data have been correlated by means of three commonly known G^E equations: the Wilson, NRTL and UNIQUAC with the assumption that the systems studied here present simple eutectic behaviour. The activity coefficients of pharmaceuticals at saturated solutions in each binary mixture were calculated from the experimental data. The formation of MEF-ionic liquid compounds greatly increases the solubility in water in comparison with pure MEF or complexes with 2-hydroxypropyl-β-cyclodextrin. The development of these compounds formulations will assist in medication taking into account oral solid or gel medicines.

Graphical abstract

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Binding affinity toward human prion protein of some anti-prion compounds — Assessment based on QSAR modeling, molecular docking and non-parametric ranking

Publication date: 1 January 2018
Source:European Journal of Pharmaceutical Sciences, Volume 111
Author(s): Strahinja Kovačević, Milica Karadžić, Sanja Podunavac-Kuzmanović, Lidija Jevrić
The present study is based on the quantitative structure-activity relationship (QSAR) analysis of binding affinity toward human prion protein (huPrP^C) of quinacrine, pyridine dicarbonitrile, diphenylthiazole and diphenyloxazole analogs applying different linear and non-linear chemometric regression techniques, including univariate linear regression, multiple linear regression, partial least squares regression and artificial neural networks. The QSAR analysis distinguished molecular lipophilicity as an important factor that contributes to the binding affinity. Principal component analysis was used in order to reveal similarities or dissimilarities among the studied compounds. The analysis of in silico absorption, distribution, metabolism, excretion and toxicity (ADMET) parameters was conducted. The ranking of the studied analogs on the basis of their ADMET parameters was done applying the sum of ranking differences, as a relatively new chemometric method. The main aim of the study was to reveal the most important molecular features whose changes lead to the changes in the binding affinities of the studied compounds. Another point of view on the binding affinity of the most promising analogs was established by application of molecular docking analysis. The results of the molecular docking were proven to be in agreement with the experimental outcome.

Graphical abstract

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Insight into lipophilicity of deoxyribonucleoside‑boron cluster conjugates

Publication date: 1 January 2018
Source:European Journal of Pharmaceutical Sciences, Volume 111
Author(s): Aneta Dąbrowska, Michał Matuszewski, Krzysztof Zwoliński, Anna Ignaczak, Agnieszka B. Olejniczak
Lipophilicity was investigated for 20 2′-deoxyribonucleoside derivatives modified with electron-neutral 1,2-dicarba-closo-dodecaborane, 1,12-dicarba-closo-dodecaborane, 7,8-dicarba-nido-undecaborate anion, and metallacarborane containing Co, Fe, or Cr. The partition coefficient (P) for neutral conjugates and the distribution coefficient (D7.4) for ionic compounds were determined as a lipophilicity descriptor using a shake-flask method. All modified nucleosides had P/D7.4 values higher than those of an appropriate unmodified 2′-closo-dodecaborane and metallacarborane was found to be three orders of magnitude higher than that of its unmodified counterpart. The lowest impact on the P/D7.4 values of the conjugates was observed for the 7,8-dicarba-nido-undecaborate anion. A preliminary molecular modeling study of a thymidine-carborane conjugate with β-cyclodextrin confirmed the ability of the components to form an inclusion complex.

Graphical abstract

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Tuning the rheological properties of an ammonium methacrylate copolymer for the design of adhesives suitable for transdermal patches

Publication date: 1 January 2018
Source:European Journal of Pharmaceutical Sciences, Volume 111
Author(s): Gaia M.G. Quaroni, Chiara G.M. Gennari, Francesco Cilurzo, Guylaine Ducouret, Costantino Creton, Paola Minghetti
Eudragit® RL (EuRL) matrices have been proposed to release a drug to the skin. However, no information is available on both viscoelastic and adhesive properties of such compositions. This work focuses on the evaluation of both rheological and texture properties of EuRL differently plasticized with tributyl citrate (TBC) or triacetin (TRI) in order to design a pressure sensitive adhesive suitable for transdermal patch preparation. The patch adhesive properties (i.e. tack, peel adhesion and shear adhesion) as well as its in vitro biopharmaceutical performances were determined after loading ibuprofen, ketoprofen or flurbiprofen. The addition of 40–60% w/w TBC or 40–50% w/w TRI to EuRL permitted to obtain matrices with the desired adhesive properties. Moreover, the increase of plasticizer content and loading of the drug reduced the relaxation time (τR). Consequently, the shear adhesion values decreased and the in vitro drug release constants (k) increased. Indeed, the k values from patches containing TBC were lower than the corresponding with TRI because of the lower fluidity of such matrices. In conclusion, the 60/40 EuRL/TBC binary blend is suitable for the design of transdermal patches since the in vitro permeability of the three selected drugs appeared comparable to those described in literature for marketed products.

Graphical abstract

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Effect of a high-cholesterol diet on lipoprotein metabolism and xanthoma formation in rabbits

Summary

Background

Xanthelasma is the most common type of cutaneous xanthoma and often occurs on the eyelids. Xanthoma has been reported to be highly correlated with abnormal lipoprotein metabolism.

Aims

In this study, we wanted to investigate the effects of a high-cholesterol diet on xanthoma formation and lipoprotein metabolism in rabbits.

Methods

In animals fed with high-cholesterol diet, deteced plasma lipid [ie, total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (L-DLC)] levels and pathology of xanthoma.

Results

Plasma lipid levels were dramatically elevated within 8 weeks. In addition, high dietary cholesterol promoted xanthoma formation on the napex. Microscopic examination showed that foam cells laden with cholesterol deposits accumulated around the dermal capillaries and cutaneous appendages within the skin of the napex.

Conclusion

These findings indicate a critical role for a high-cholesterol diet in the dysregulation of lipoprotein metabolism and the development of xanthoma in rabbits. The results suggest that abnormal lipid metabolism may increase the occurrence of xanthoma.

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Basophils are recruited and localized at the site of tick bites in humans

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Reconsidering the H&E stain as the gold standard in assessing the depth of burn wounds

While histological examination is considered by most as the gold standard for burn depth assessment, it has no practical use in the clinical setting. It has, however, been used in the research setting, as a mean for evaluating emerging techniques of depth measurement. Due to the limitations of the H&E stain, other stains have also been explored, such as lactate dehydrogenase (LDH), as presented in this issue, in "Improving the Histologic Characterization of Burn Depth." As the determination of burn depth is not a typical subject in dermatopathology, a summary of selected techniques and the possible role for the LDH stain in future research, is described herein.

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Differential Processing of Isolated Object and Multi-item Pop-Out Displays in LIP and PFC

Abstract

Objects that are highly distinct from their surroundings appear to visually "pop-out." This effect is present for displays in which: (1) a single cue object is shown on a blank background, and (2) a single cue object is highly distinct from surrounding objects; it is generally assumed that these 2 display types are processed in the same way. To directly examine this, we applied a decoding analysis to neural activity recorded from the lateral intraparietal (LIP) area and the dorsolateral prefrontal cortex (dlPFC). Our analyses showed that for the single-object displays, cue location information appeared earlier in LIP than in dlPFC. However, for the display with distractors, location information was substantially delayed in both brain regions, and information first appeared in dlPFC. Additionally, we see that pattern of neural activity is similar for both types of displays and across different color transformations of the stimuli, indicating that location information is being coded in the same way regardless of display type. These results lead us to hypothesize that 2 different pathways are involved processing these 2 types of pop-out displays.

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Missing ozone-induced potential aerosol formation in a suburban deciduous forest

Publication date: Available online 10 October 2017
Source:Atmospheric Environment
Author(s): T. Nakayama, Y. Kuruma, Y. Matsumi, Y. Morino, K. Sato, H. Tsurumaru, S. Ramasamy, Y. Sakamoto, S. Kato, Y. Miyazaki, T. Mochizuki, K. Kawamura, Y. Sadanaga, Y. Nakashima, K. Matsuda, Y. Kajii
As a new approach to investigating formation processes of secondary organic aerosol (SOA) in the atmosphere, ozone-induced potential aerosol formation was measured in summer at a suburban forest site surrounded by deciduous trees, near Tokyo, Japan. After passage through a reactor containing high concentrations of ozone, increases in total particle volume (average of 1.4 × 10⁹ nm³/cm³, which corresponds to 17% that of pre-existing particles) were observed, especially during daytime. The observed aerosol formations were compared with the results of box model simulations using simultaneously measured concentrations of gaseous and particulate species. According to the model, the relative contributions of isoprene, monoterpene, and aromatic hydrocarbon oxidation to SOA formation in the reactor were 24, 21, and 55%, respectively. However, the model could explain, on average, only ∼40% of the observed particle formation, and large discrepancies between the observations and model were found, especially around noon and in the afternoon when the concentrations of isoprene and oxygenated volatile organic compounds were high. The results suggest a significant contribution of missing (unaccounted-for) SOA formation processes from identified and/or unidentified volatile organic compounds, especially those emitted during daytime. Further efforts should be made to explore and parameterize this missing SOA formation to assist in the improvement of atmospheric chemistry and climate models.

Graphical abstract

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MTA promotes chemotaxis and chemokinesis of immune cells through distinct calcium-sensing receptor signaling pathways

Publication date: January 2018
Source:Biomaterials, Volume 150
Author(s): Fengjiao Chang, Jin Man Kim, Youngnim Choi, Kyungpyo Park
Mineral trioxide aggregate (MTA) has been introduced as a choice material for regenerative dentistry. To date, the diverse biological activities of MTA, including its anti-inflammatory effects, have been extensively discussed. However, there is limited insight into the link between MTA and immune cell migration. In this study, we report the role of MTA in enhancing both chemotactic and chemokinetic immune cell migration through distinct signaling pathways. By using versatile live imaging techniques, we demonstrated that MTA-mediated CaSR activation induced diverse downstream pathways to govern cell migratory capacity. In this context, Cdc42 generates cytoskeleton-driven cellular protrusions to steer directional cell migration (chemotaxis) whereas Ca²⁺-calmodulin dependent myosin light chain kinase induces cell contractility that plays an important role in speeding up the average migration speed (chemokinesis). Our findings illuminate an unrecognized role for MTA and the related CaSR signaling network in immune cell migration, providing evidence that can drive development of novel approaches to immunological therapy.



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Enhanced antitumor efficacy of arginine modified amphiphilic nanoparticles co-delivering doxorubicin and iSur-pDNA via the multiple synergistic effect

Publication date: January 2018
Source:Biomaterials, Volume 150
Author(s): Yudong Song, Cui Tang, Chunhua Yin
Arginine and α-tocopherol succinate (α-TOS) double grafted N-trimethyl chitosan chloride (TMC) nanoparticles (TAS NPs) were designed and developed for effective co-delivery of doxorubicin (DOX) and Survivin shRNA-expressing pDNA (iSur-pDNA). With DOX loading into the hydrophobic core and iSur-pDNA combining to the hydrophilic shell, TAS/DOX/pDNA NPs demonstrated favorable structural stability and sustained release properties in vitro. With the special non-clathrin-dependent endocytosis, TAS/DOX/pDNA NPs presented higher cellular uptake and mainly distributed in ER and Golgi rather than lysosomes following internalization. The in vitro nuclear localization, gene silencing efficiency, cell apoptosis, and growth inhibition of tumor cells were significantly promoted by arginine modification. In the tumor-bearing mice model, TAS/DOX/pDNA NPs possessed the maximum antitumor efficiency as compared with single delivery of DOX or iSur-pDNA. Particularly, blank TAS NPs were selectively be toxic to tumor cells as evidenced by their capabilities to inhibit proliferation and induce apoptosis of tumor cells. The promising tumor treatment of TAS/DOX/pDNA NPs via a multiple synergistic manner arising from DOX and pDNA as well as the vectors would provide a potential strategy for a dual-delivery system to improve their therapeutic efficacies.



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Editorial board

Publication date: December 2017
Source:Biomaterials, Volume 148





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Down-regulation of miR-26a-5p in hepatocellular carcinoma: a qRT-PCR and bioinformatics study

Publication date: Available online 10 October 2017
Source:Pathology - Research and Practice
Author(s): Liang Liang, Jiang-hui Zeng, Jie-yu Wang, Rong-quan He, Jie Ma, Gang Chen, Xiao-yong Cai, Xiao-hua Hu
BackgroundTo practically verify the clinical value of miR-26a-5p and thoroughly explore its target genes as well as its potential functions in hepatocellular carcinoma (HCC).MethodsHCC and adjacent non-cancerous hepatic tissues of 95 HCC patients were collected for analysis using reverse transcription quantitative real-time PCR (qRT-PCR). For the bioinformatics analysis, we identified potential target genes for miR-26a-5p from differentially expressed genes (DEGs) in Gene Expression Omnibus (GEO) data sets and miRWalk predicted database. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and protein-protein interaction (PPI) analyses were applied to analyze the prospective mechanisms of the predicted target genes.ResultsMiR-26a-5p showed a significantly lower expression level in HCC tissues (1.56±1.07) than adjacent benign liver tissues (2.28±1.06, P <0.001). The area under the curve (AUC) of the receiver operating characteristic (ROC) was 0.665 (95% CI: 0.588-0.743, P <0.001). Significant correlations between miR-26a-5p expression and clinicopathological features such as gender (r=0.275, P <0.01), clinical TNM stage (r=−0.306, P <0.01), and metastasis (r=−0.321, P <0.01) were observed. To examine potential target genes, we obtained 175 genes for further function analysis, by attaining the intersection of 2062 up-regulated DEGs and 1390 online-predicted target genes. The GO and KEGG pathway annotation indicated focal adhesion, regulation of actin cytoskeleton and the PI3K-Akt signaling pathway as significant prospective mechanisms. The PPI network indicated that NRAS was the most essential hub gene in the whole network.ConclusionDown-regulated miR-26a-5p was closely correlated with the status of metastasis and the progression of HCC. MiR-26a-5p might play protective roles by targeting diverse genes and pathways.



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Aberrant promoter methylation of the PAQR3 gene is associated with prostate cancer

Publication date: Available online 10 October 2017
Source:Pathology - Research and Practice
Author(s): Kowit Lounglaithong, Andrey Bychkov, Pichet Sampatanukul
Methylation markers are promising tools for diagnosis, prognosis and targeted treatment of cancer. In prostate carcinoma, aberrant promoter hypermethylation occurs earlier in the disease course and more consistently than recurrent somatic mutations. PAQR3, a tumor suppressor gene, was recently found to be downregulated in prostate cancer cell lines. We hypothesized that promoter methylation could be responsible for PAQR3 silencing in prostate cancer tissues. We aimed to investigate PAQR3 promoter methylation in prostate cancer by comparing it to benign prostatic hyperplasia (BPH). A total of 154 human prostate tissue samples, including 92 cases with prostate cancer and 62 cases with BPH, were examined by methylation-specific PCR. Clinicopathological correlation between PAQR3 promoter methylation and prognostically relevant variables was studied by statistical analysis. Promoter methylation of PAQR3 was significantly more frequent in prostate carcinoma compared to BPH (73.9% vs. 25.8%, p <0.01). The high prevalence of PAQR3 methylation in cancer foci was also confirmed with microdissection technique in 12 samples of prostate adenocarcinoma. PAQR3 hypermethylation was associated with perineural invasion (p=0.03), an adverse clinicopathological feature of prostate cancer. We concluded that PAQR3 can be a promising methylation marker candidate for the detection and monitoring of prostate cancer.



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Over expression of HIF1α is associated with inactivation of both LimD1 and VHL in renal cell carcinoma: clinical importance

Publication date: Available online 10 October 2017
Source:Pathology - Research and Practice
Author(s): Subhayan Sur, Arun Kumar Maurya, Anup Roy, Tyson V. Sharp, Dilip Kumar Pal, Chinmay Kumar Panda
It is evident that the association of LimD1 and VHL could regulate stabilization of HIF1α protein. In this study, the expression of LimD1, VHL and HIF1α was analyzed in primary renal cell carcinoma (RCC) samples to understand their association with development of the disease. For this purpose, immunohistochemical expression analysis of LimD1, VHL and HIF1α was performed along with proliferation marker PCNA in 32 primary RCC samples in different subtypes at different clinical stages of Indian patients (year: 2014–2016). Significant decrease (P<0.05) in LimD1 and VHL expression was observed in different subtypes of RCC and in early invasive lesions. High nuclear expression of HIF1α was seen in different subtypes as well as in early invasive lesions. The LimD1 and VHL expression strongly correlated with each other, while inversely correlated with HIF1α. On the other hand, increased expression of PCNA was seen in different sub types and in early invasive lesions. The PCNA expression was negatively correlated with LimD1 and VHL expression and positively correlated with HIF1α. Thus, our data indicates that the reduced expression of LimD1 and VHL might have synergistic effect on induction of HIF1α resulting increased cellular proliferation and progression of the disease.



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Oceanic dispersion of Fukushima-derived Cs-137 simulated by multiple oceanic general circulation models

Publication date: December 2017
Source:Journal of Environmental Radioactivity, Volume 180
Author(s): Hideyuki Kawamura, Akiko Furuno, Takuya Kobayashi, Teiji In, Tomoharu Nakayama, Yoichi Ishikawa, Yasumasa Miyazawa, Norihisa Usui
To understand the concentration and amount of Fukushima-derived Cs-137 in the ocean, this study simulated the oceanic dispersion of Cs-137 by atmospheric and oceanic dispersion simulations. The oceanic dispersion simulations were carried out with an oceanic dispersion model and multiple oceanic general circulation models. The Cs-137 concentrations were sensitive to ocean currents in the coastal, offshore, and open oceans. The mean Cs-137 concentrations of the multiple models relatively well agreed with the observed concentrations in the coastal and offshore oceans during the first few months after the Fukushima disaster, and in the open ocean during the first year after the disaster. The Cs-137 amounts were quantified in the coastal, offshore, and open oceans during the first year after the disaster. It was suggested that Cs-137 actively dispersed from the coastal and offshore oceans to the open ocean, and from the surface layer to the deeper layers in the North Pacific.



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Application of ISOCS system in the laboratory efficiency calibration

Publication date: Available online 10 October 2017
Source:Journal of Environmental Radioactivity
Author(s): Dominik Grządziel, Krzysztof Kozak, Jadwiga Mazur, Mariusz Mroczek
ISOCS (In Situ Counting Object System) from Canberra is applied in laboratory for creating efficiency calibrations of good quality without using radioactive standards. Besides of typical sample containers used in laboratory, ISOSC system also allows modelling containers and objects of almost any shape and elemental composition.The study was based on gamma spectrometry with HPGe semiconductor detector with electronics and software spectrum analysis GENIE 2000 + ISOCS. Measuring set is equipped with portable shield system with set of collimators ISOCS Shield Systems Model ISOXSHLD from Canberra. This shielding system provides attenuation of gamma background radiation with average value 33 (for gamma energies from 186 keV to 2615.5 keV).The portable shield system can be used for low-background laboratory measurements. For this purpose a measuring vessel of new geometry was constructed: the polystyrene cylinder with a height of 40 mm and a diameter of 70 mm. The efficiency calibration for this container was performed using both ISOCS system and classical calibration standard in the same geometry. In order to verify the correctness of performed calibration procedures, the measurements of radioactive standard CBSS 2 were made. The results of both calibrations were compared with the data from the standard certificate. Satisfactory agreement was achieved. Mean percentage difference between results from ISOCS calibration compared to reference values is 6% for all isotopes activities in CBSS 2 standard.The set of collimators was used to develop efficiency calibration for in situ measurements of the soil surface. Test measurements were carried out at the area of the Institute of Nuclear Physics Polish Academy of Sciences in Kraków, Poland (IFJ PAN). Two measurement methods were compared: in situ and laboratory gamma spectroscopy. The obtained average results (from all 10 measuring points) are consistent within the range of measurement uncertainty.



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Simultaneous profiles of sulfonated androgens, sulfonated estrogens and sulfonated progestogens in postpubertal boars (sus scrofa domestica) measured by LC-MS/MS

Publication date: Available online 10 October 2017
Source:The Journal of Steroid Biochemistry and Molecular Biology
Author(s): G. Schuler, A. Sánchez-Guijo, M.F. Hartmann, S.A. Wudy
1 Sulfonated steroids (s-St) have been usually regarded as inactive metabolites but are progressively considered as precursors for the intra-tissue formation of bioactive steroids. Moreover, independent effects without preceding removal of the sulfate group have been observed. We use the porcine testicular-epididymal compartment as a model to investigate the still largely unknown s-St physiology as the boar exhibits an intriguingly broad s-St spectrum predominantly originating from the testis. The application of LC-MS/MS in steroidomics enables the determination of unconjugated and intact sulfonated steroids with currently highest specificity and good sensitivity, allowing the concurrent measuring of numerous analytes in larger quantities of samples. Profiles (6hours, 20min intervals) were generated for sulfonated 5-androstene-3ß,17ß-diol (Adiol-S), androsterone (A-S), dehydroepiandrosterone (DHEA-S), epiandrosterone (EA-S), epitestosterone (ET-S), estrone (E1-S), estradiol-17β (E2-S), pregnenolone (P5-S), 17αOH-pregnenolone (OHP5-S) and unconjugated testosterone (T) in four unstimulated and four hCG-stimulated boars. Moreover, concentrations were measured in individual samples collected from testicular afferent and efferent blood to differentiate between testicular vs. extratesticular origin. Highest concentrations were found for EA-S, followed by ET-S, Adiol-S and DHEA-S, which mostly exceeded the levels of E1-S and A-S. Lowest concentrations were obtained for E2-S, P5-S and OHP5-S. The analytical profile also included sulfonated T, 5α-dihydrotestosterone and cholesterol. However, their concentrations were below the limit of quantification. Profiles of quantifiable s-St were consistent with a wave-like pattern associated with T pulses. In postpartal females (n=5) concentrations of all analytes assessed were undetectable, suggesting that in pigs the adrenals are not a quantitatively significant source of s-St.



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Fanconi-Anemia-Associated Mutations Destabilize RAD51 Filaments and Impair Replication Fork Protection

Publication date: 10 October 2017
Source:Cell Reports, Volume 21, Issue 2
Author(s): Karina Zadorozhny, Vincenzo Sannino, Ondrej Beláň, Jarmila Mlčoušková, Mário Špírek, Vincenzo Costanzo, Lumír Krejčí
Fanconi anemia (FA) is a genetic disorder characterized by a defect in DNA interstrand crosslink (ICL) repair, chromosomal instability, and a predisposition to cancer. Recently, two RAD51 mutations were reported to cause an FA-like phenotype. Despite the tight association of FA/HR proteins with replication fork (RF) stabilization during normal replication, it remains unknown how FA-associated RAD51 mutations affect replication beyond ICL lesions. Here, we report that these mutations fail to protect nascent DNA from MRE11-mediated degradation during RF stalling in Xenopus laevis egg extracts. Reconstitution of DNA protection in vitro revealed that the defect arises directly due to altered RAD51 properties. Both mutations induce pronounced structural changes and RAD51 filament destabilization that is not rescued by prevention of ATP hydrolysis due to aberrant ATP binding. Our results further interconnect the FA pathway with DNA replication and provide mechanistic insight into the role of RAD51 in recombination-independent mechanisms of genome maintenance.

Graphical abstract

Teaser

Zadorozhny et al. find that RAD51 mutations associated with Fanconi anemia not only impair DNA crosslink repair but also affect DNA replication during fork stalling. The mutant proteins cause RAD51 filament destabilization and induce pronounced structural changes resulting from aberrant ATP binding and hydrolysis.


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Live Imaging Reveals that the First Division of Differentiating Human Embryonic Stem Cells Often Yields Asymmetric Fates

Publication date: 10 October 2017
Source:Cell Reports, Volume 21, Issue 2
Author(s): Katharine Brown, Kyle M. Loh, Roel Nusse
How do stem cells respond to signals to initiate differentiation? Here, we show that, despite uniform exposure to differentiation-inducing extracellular signals, individual human embryonic stem cells (hESCs) respond heterogeneously. To track how hESCs incipiently exit pluripotency, we established a system to differentiate hESCs as single cells and conducted live imaging to track their very first cell division. We followed the fate of their earliest daughters as they remained undifferentiated or differentiated toward the primitive streak (the earliest descendants of pluripotent cells). About 30%–50% of the time, hESCs divided to yield one primitive streak and one undifferentiated daughter. The undifferentiated daughter cell was innately resistant to WNT signaling and could not respond to this primitive-streak-specifying differentiation signal. Hence, the first division of differentiating hESCs sometimes yields daughters with diverging fates, with implications for the efficiency of directed differentiation protocols and the underlying rules of lineage commitment.

Graphical abstract

Teaser

Brown et al. show that 30%–50% of the time, the first division of differentiating human embryonic stem cells generates one differentiated and one undifferentiated daughter cell. These asymmetric fate outcomes have implications for the efficiency of stem cell differentiation protocols and the mechanisms underlying lineage commitment.


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Functional Plasticity of Odor Representations during Motherhood

Publication date: 10 October 2017
Source:Cell Reports, Volume 21, Issue 2
Author(s): Amit Vinograd, Yael Fuchs-Shlomai, Merav Stern, Diptendu Mukherjee, Yuan Gao, Ami Citri, Ian Davison, Adi Mizrahi
Motherhood is accompanied by new behaviors aimed at ensuring the wellbeing of the offspring. Olfaction plays a key role in guiding maternal behaviors during this transition. We studied functional changes in the main olfactory bulb (OB) of mothers in mice. Using in vivo two-photon calcium imaging, we studied the sensory representation of odors by mitral cells (MCs). We show that MC responses to monomolecular odors become sparser and weaker in mothers. In contrast, responses to biologically relevant odors are spared from sparsening or strengthen. MC responses to mixtures and to a range of concentrations suggest that these differences between odor responses cannot be accounted for by mixture suppressive effects or gain control mechanisms. In vitro whole-cell recordings show an increase in inhibitory synaptic drive onto MCs. The increase of inhibitory tone may contribute to the general decrease in responsiveness and concomitant enhanced representation of specific odors.

Graphical abstract

Teaser

Motherhood is associated with changes in neural circuits that affect how the mother senses her surroundings. Vinograd et al. show that the olfactory bulb is a locus of plasticity. Output neurons of the bulb have elevated inhibition, and odor coding of natural odors is improved.


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Overproduction of Neurons Is Correlated with Enhanced Cortical Ensembles and Increased Perceptual Discrimination

Publication date: 10 October 2017
Source:Cell Reports, Volume 21, Issue 2
Author(s): Wei-Qun Fang, Rafael Yuste
Brains vary greatly in neuronal number and density, even across individuals within the same species, yet it remains unclear whether such variation leads to differences in brain function or behavior. By imaging cortical activity of a mouse model in which neuronal production is moderately enhanced in utero, we find that animals with more cortical neurons also develop enhanced functional correlations and more distinct neuronal ensembles in primary visual cortex. These mice also have sharper orientation discrimination in their visual behavior. These results unveil a correlation between neuronal ensembles and behavior and suggest that neuronal number is linked to functional modularity and perceptual discrimination of visual cortex. By experimentally linking differences in neuronal number and behavior, our findings could help explain how evolutionary and developmental variability of individual and species brain size may lead to perceptual and cognitive differences.

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Fang et al. report that mice engineered to have more cortical neurons also have more neuronal ensembles and improved visual discrimination compared to wild-type mice. These findings suggest that neuronal production is linked to functional modularity and perceptual discrimination in the visual cortex.


http://ift.tt/2ybqYvk

Uncoupling of Metabolic Health from Longevity through Genetic Alteration of Adipose Tissue Lipid-Binding Proteins

Publication date: 10 October 2017
Source:Cell Reports, Volume 21, Issue 2
Author(s): Khanichi N. Charles, Min-Dian Li, Feyza Engin, Ana Paula Arruda, Karen Inouye, Gökhan S. Hotamisligil
Deterioration of metabolic health is a hallmark of aging and generally assumed to be detrimental to longevity. Exposure to a high-calorie diet impairs metabolism and accelerates aging; conversely, calorie restriction (CR) prevents age-related metabolic diseases and extends lifespan. However, it is unclear whether preservation of metabolic health is sufficient to extend lifespan. We utilized a genetic mouse model lacking Fabp4/5 that confers protection against metabolic diseases and shares molecular and lipidomic features with CR to address this question. Fabp-deficient mice exhibit extended metabolic healthspan, with protection against insulin resistance and glucose intolerance, inflammation, deterioration of adipose tissue integrity, and fatty liver disease. Surprisingly, however, Fabp-deficient mice did not exhibit any extension of lifespan. These data indicate that extension of metabolic healthspan in the absence of CR can be uncoupled from lifespan, indicating the potential for independent drivers of these pathways, at least in laboratory mice.

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Deterioration of metabolic health is a hallmark of aging and generally thought to be detrimental to longevity. Charles et al. utilize FABP-deficient mice as a model to demonstrate that the preservation of metabolic health in this model persists throughout life, even under metabolic stress, but does not increase longevity.


http://ift.tt/2ybPa0y

A Specific ChREBP and PPARα Cross-Talk Is Required for the Glucose-Mediated FGF21 Response

Publication date: 10 October 2017
Source:Cell Reports, Volume 21, Issue 2
Author(s): Alison Iroz, Alexandra Montagner, Fadila Benhamed, Françoise Levavasseur, Arnaud Polizzi, Elodie Anthony, Marion Régnier, Edwin Fouché, Céline Lukowicz, Michèle Cauzac, Emilie Tournier, Marcio Do-Cruzeiro, Martine Daujat-Chavanieu, Sabine Gerbal-Chalouin, Véronique Fauveau, Solenne Marmier, Anne-Françoise Burnol, Sandra Guilmeau, Yannick Lippi, Jean Girard, Walter Wahli, Renaud Dentin, Hervé Guillou, Catherine Postic
While the physiological benefits of the fibroblast growth factor 21 (FGF21) hepatokine are documented in response to fasting, little information is available on Fgf21 regulation in a glucose-overload context. We report that peroxisome-proliferator-activated receptor α (PPARα), a nuclear receptor of the fasting response, is required with the carbohydrate-sensitive transcription factor carbohydrate-responsive element-binding protein (ChREBP) to balance FGF21 glucose response. Microarray analysis indicated that only a few hepatic genes respond to fasting and glucose similarly to Fgf21. Glucose-challenged Chrebp^−/− mice exhibit a marked reduction in FGF21 production, a decrease that was rescued by re-expression of an active ChREBP isoform in the liver of Chrebp^−/− mice. Unexpectedly, carbohydrate challenge of hepatic Pparα knockout mice also demonstrated a PPARα-dependent glucose response for Fgf21 that was associated with an increased sucrose preference. This blunted response was due to decreased Fgf21 promoter accessibility and diminished ChREBP binding onto Fgf21 carbohydrate-responsive element (ChoRE) in hepatocytes lacking PPARα. Our study reports that PPARα is required for the ChREBP-induced glucose response of FGF21.

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FGF21 is a hepatokine with beneficial metabolic effects, including control of sucrose preference. Iroz et al. demonstrate that Fgf21 is a unique hepatic gene inducible by both fasting and glucose signals and that the transcription factors PPARα and ChREBP both regulate the endocrine control of sugar intake by hepatic FGF21.


http://ift.tt/2yal7pY

Molecular Mechanism by which Prominent Human Gut Bacteroidetes Utilize Mixed-Linkage Beta-Glucans, Major Health-Promoting Cereal Polysaccharides

Publication date: 10 October 2017
Source:Cell Reports, Volume 21, Issue 2
Author(s): Kazune Tamura, Glyn R. Hemsworth, Guillaume Déjean, Theresa E. Rogers, Nicholas A. Pudlo, Karthik Urs, Namrata Jain, Gideon J. Davies, Eric C. Martens, Harry Brumer
Microbial utilization of complex polysaccharides is a major driving force in shaping the composition of the human gut microbiota. There is a growing appreciation that finely tuned polysaccharide utilization loci enable ubiquitous gut Bacteroidetes to thrive on the plethora of complex polysaccharides that constitute "dietary fiber." Mixed-linkage β(1,3)/β(1,4)-glucans (MLGs) are a key family of plant cell wall polysaccharides with recognized health benefits but whose mechanism of utilization has remained unclear. Here, we provide molecular insight into the function of an archetypal MLG utilization locus (MLGUL) through a combination of biochemistry, enzymology, structural biology, and microbiology. Comparative genomics coupled with growth studies demonstrated further that syntenic MLGULs serve as genetic markers for MLG catabolism across commensal gut bacteria. In turn, we surveyed human gut metagenomes to reveal that MLGULs are ubiquitous in human populations globally, which underscores the importance of gut microbial metabolism of MLG as a common cereal polysaccharide.

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Mixed-linkage β(1,3)/β(1,4)-glucan (MLG) is an important complex dietary polysaccharide (dietary fiber), the degradation of which in the human gut depends on the resident microbiota. Tamura et al. outline the molecular mechanism of MLG utilization by Bacteroides ovatus and reveal that the majority of surveyed humans possess MLG-utilizing Bacteroidetes.


http://ift.tt/2yc3PsI

Differential Regulation of Lipoprotein and Hepatitis C Virus Secretion by Rab1b

Publication date: 10 October 2017
Source:Cell Reports, Volume 21, Issue 2
Author(s): Constantin N. Takacs, Ursula Andreo, Viet Loan Dao Thi, Xianfang Wu, Caroline E. Gleason, Michelle S. Itano, Gabriella S. Spitz-Becker, Rachel L. Belote, Brenna R. Hedin, Margaret A. Scull, Charles M. Rice, Sanford M. Simon
Secretory cells produce diverse cargoes, yet how they regulate concomitant secretory traffic remains insufficiently explored. Rab GTPases control intracellular vesicular transport. To map secretion pathways, we generated a library of lentivirus-expressed dominant-negative Rab mutants and used it in a large-scale screen to identify regulators of hepatic lipoprotein secretion. We identified several candidate pathways, including those mediated by Rab11 and Rab8. Surprisingly, inhibition of Rab1b, the major regulator of transport from the endoplasmic reticulum to the Golgi, differently affected the secretion of the very-low-density lipoprotein components ApoE and ApoB100, despite their final association on mature secreted lipoprotein particles. Since hepatitis C virus (HCV) incorporates ApoE and ApoB100 into its virus particle, we also investigated infectious HCV secretion and show that its regulation by Rab1b mirrors that of ApoB100. These observations reveal differential regulation of hepatocyte secretion by Rab1b and advance our understanding of lipoprotein assembly and lipoprotein and HCV secretion.

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Takacs et al. show that Rab1b, a major regulator of transport from the endoplasmic reticulum to the Golgi, differentially controls the secretion of lipoprotein components ApoE and ApoB100 and infectious hepatitis C virus particles.


http://ift.tt/2yaZEgR

Degradation of Bcl-2 by XIAP and ARTS Promotes Apoptosis

Publication date: 10 October 2017
Source:Cell Reports, Volume 21, Issue 2
Author(s): Natalia Edison, Yael Curtz, Nicole Paland, Dana Mamriev, Nicolas Chorubczyk, Tali Haviv-Reingewertz, Nir Kfir, David Morgenstern, Meital Kupervaser, Juliana Kagan, Hyoung Tae Kim, Sarit Larisch
We describe a mechanism by which the anti-apoptotic B cell lymphoma 2 (Bcl-2) protein is downregulated to induce apoptosis. ARTS (Sept4_i2) is a tumor suppressor protein that promotes cell death through specifically antagonizing XIAP (X-linked inhibitor of apoptosis). ARTS and Bcl-2 reside at the outer mitochondrial membrane in living cells. Upon apoptotic induction, ARTS brings XIAP and Bcl-2 into a ternary complex, allowing XIAP to promote ubiquitylation and degradation of Bcl-2. ARTS binding to Bcl-2 involves the BH3 domain of Bcl-2. Lysine 17 in Bcl-2 serves as the main acceptor for ubiquitylation, and a Bcl-2 K17A mutant has increased stability and is more potent in protection against apoptosis. Bcl-2 ubiquitylation is reduced in both XIAP- and Sept4/ARTS-deficient MEFs, demonstrating that XIAP serves as an E3 ligase for Bcl-2 and that ARTS is essential for this process. Collectively, these results suggest a distinct model for the regulation of Bcl-2 by ARTS-mediated degradation.

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Many cancers avoid cell death (apoptosis) by expressing high levels of apoptosis inhibitors, such as Bcl-2. Thus, Bcl-2 is a major target for cancer therapy. Edison et al. describe a mechanism by which the ARTS protein promotes proteasome-mediated degradation of Bcl-2 and thereby stimulates cell death.


http://ift.tt/2yb10bi

Activation of the p53 Transcriptional Program Sensitizes Cancer Cells to Cdk7 Inhibitors

Publication date: 10 October 2017
Source:Cell Reports, Volume 21, Issue 2
Author(s): Sampada Kalan, Ramon Amat, Miriam Merzel Schachter, Nicholas Kwiatkowski, Brian J. Abraham, Yanke Liang, Tinghu Zhang, Calla M. Olson, Stéphane Larochelle, Richard A. Young, Nathanael S. Gray, Robert P. Fisher
Cdk7, the CDK-activating kinase and transcription factor IIH component, is a target of inhibitors that kill cancer cells by exploiting tumor-specific transcriptional dependencies. However, whereas selective inhibition of analog-sensitive (AS) Cdk7 in colon cancer-derived cells arrests division and disrupts transcription, it does not by itself trigger apoptosis efficiently. Here, we show that p53 activation by 5-fluorouracil or nutlin-3 synergizes with a reversible Cdk7^as inhibitor to induce cell death. Synthetic lethality was recapitulated with covalent inhibitors of wild-type Cdk7, THZ1, or the more selective YKL-1-116. The effects were allele specific; a CDK7^as mutation conferred both sensitivity to bulky adenine analogs and resistance to covalent inhibitors. Non-transformed colon epithelial cells were resistant to these combinations, as were cancer-derived cells with p53-inactivating mutations. Apoptosis was dependent on death receptor DR5, a p53 transcriptional target whose expression was refractory to Cdk7 inhibition. Therefore, p53 activation induces transcriptional dependency to sensitize cancer cells to Cdk7 inhibition.

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Kalan et al. find that activation of the p53 tumor suppressor protein in human colon cancer-derived cells can induce transcriptional dependency on Cdk7, analogous to constitutive dependencies described in other tumors driven by oncogenic transcription factors. This work provides a proof of concept for combining p53-activating agents with Cdk7 inhibitors to elicit synthetic lethality.


http://ift.tt/2ycck71

Erasure of Tet-Oxidized 5-Methylcytosine by a SRAP Nuclease

Publication date: 10 October 2017
Source:Cell Reports, Volume 21, Issue 2
Author(s): Soo-Mi Kweon, Bing Zhu, Yibu Chen, L. Aravind, Shuang-Yong Xu, Douglas E. Feldman
Enzymatic oxidation of 5-methylcytosine (5mC) in DNA by the Tet dioxygenases reprograms genome function in embryogenesis and postnatal development. Tet-oxidized derivatives of 5mC such as 5-hydroxymethylcytosine (5hmC) act as transient intermediates in DNA demethylation or persist as durable marks, yet how these alternative fates are specified at individual CpGs is not understood. Here, we report that the SOS response-associated peptidase (SRAP) domain protein Srap1, the mammalian ortholog of an ancient protein superfamily associated with DNA damage response operons in bacteria, binds to Tet-oxidized forms of 5mC in DNA and catalyzes turnover of these bases to unmodified cytosine by an autopeptidase-coupled nuclease. Biallelic inactivation of murine Srap1 causes embryonic sublethality associated with widespread accumulation of ectopic 5hmC. These findings establish a function for a class of DNA base modification-selective nucleases and position Srap1 as a determinant of 5mC demethylation trajectories during mammalian embryonic development.

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Kweon et al. uncover a function for the SRAP domain, which couples autoproteolytic cleavage to activation of a nuclease selective for DNA containing Tet-oxidized derivatives of 5-methylcytosine. These findings reveal a mechanism for targeted erasure of DNA methylation via the stepwise enzymatic actions of Tet and SRAP.


http://ift.tt/2yccmf9

Brachyury-YAP Regulatory Axis Drives Stemness and Growth in Cancer

Publication date: 10 October 2017
Source:Cell Reports, Volume 21, Issue 2
Author(s): Sagar R. Shah, Justin M. David, Nathaniel D. Tippens, Ahmed Mohyeldin, Juan C. Martinez-Gutierrez, Sara Ganaha, Paula Schiapparelli, Duane H. Hamilton, Claudia Palena, Andre Levchenko, Alfredo Quiñones-Hinojosa
Molecular factors that define stem cell identity have recently emerged as oncogenic drivers. For instance, brachyury, a key developmental transcriptional factor, is also implicated in carcinogenesis, most notably of chordoma, through mechanisms that remain elusive. Here, we show that brachyury is a crucial regulator of stemness in chordoma and in more common aggressive cancers. Furthermore, this effect of brachyury is mediated by control of synthesis and stability of Yes-associated protein (YAP), a key regulator of tissue growth and homeostasis, providing an unexpected mechanism of control of YAP expression. We further demonstrate that the brachyury-YAP regulatory pathway is associated with tumor aggressiveness. These results elucidate a mechanism of controlling both tumor stemness and aggressiveness through regulatory coupling of two developmental factors.

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Malignant neoplasms exhibit uninhibited and dysregulated growth coupled with acquisition of stem-like properties that are integral to the development and progression of disease. Shah et al. demonstrate a critical role of brachyury in regulating stemness and growth by activating YAP through direct transcriptional and post-transcriptional mechanisms in various cancers.


http://ift.tt/2yc1BJE

Human Organ Chip Models Recapitulate Orthotopic Lung Cancer Growth, Therapeutic Responses, and Tumor Dormancy In Vitro

Publication date: 10 October 2017
Source:Cell Reports, Volume 21, Issue 2
Author(s): Bryan A. Hassell, Girija Goyal, Esak Lee, Alexandra Sontheimer-Phelps, Oren Levy, Christopher S. Chen, Donald E. Ingber
Here, we show that microfluidic organ-on-a-chip (organ chip) cell culture technology can be used to create in vitro human orthotopic models of non-small-cell lung cancer (NSCLC) that recapitulate organ microenvironment-specific cancer growth, tumor dormancy, and responses to tyrosine kinase inhibitor (TKI) therapy observed in human patients in vivo. Use of the mechanical actuation functionalities of this technology revealed a previously unknown sensitivity of lung cancer cell growth, invasion, and TKI therapeutic responses to physical cues associated with breathing motions, which appear to be mediated by changes in signaling through epidermal growth factor receptor (EGFR) and MET protein kinase. These findings might help to explain the high level of resistance to therapy in cancer patients with minimal residual disease in regions of the lung that remain functionally aerated and mobile, in addition to providing an experimental model to study cancer persister cells and mechanisms of tumor dormancy in vitro.

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Hassell et al. demonstrate that, when human lung cancer cells are grown within organ-on-a-chip culture devices that mimic lung structure and function, tumor cells recapitulate tumor growth and invasion patterns, as well as responses to therapy, observed in human patients. They also discover that breathing motions influence these responses.


http://ift.tt/2yaBGSP

Self-Organized Cerebral Organoids with Human-Specific Features Predict Effective Drugs to Combat Zika Virus Infection

Publication date: 10 October 2017
Source:Cell Reports, Volume 21, Issue 2
Author(s): Momoko Watanabe, Jessie E. Buth, Neda Vishlaghi, Luis de la Torre-Ubieta, Jiannis Taxidis, Baljit S. Khakh, Giovanni Coppola, Caroline A. Pearson, Ken Yamauchi, Danyang Gong, Xinghong Dai, Robert Damoiseaux, Roghiyh Aliyari, Simone Liebscher, Katja Schenke-Layland, Christine Caneda, Eric J. Huang, Ye Zhang, Genhong Cheng, Daniel H. Geschwind, Peyman Golshani, Ren Sun, Bennett G. Novitch
The human cerebral cortex possesses distinct structural and functional features that are not found in the lower species traditionally used to model brain development and disease. Accordingly, considerable attention has been placed on the development of methods to direct pluripotent stem cells to form human brain-like structures termed organoids. However, many organoid differentiation protocols are inefficient and display marked variability in their ability to recapitulate the three-dimensional architecture and course of neurogenesis in the developing human brain. Here, we describe optimized organoid culture methods that efficiently and reliably produce cortical and basal ganglia structures similar to those in the human fetal brain in vivo. Neurons within the organoids are functional and exhibit network-like activities. We further demonstrate the utility of this organoid system for modeling the teratogenic effects of Zika virus on the developing brain and identifying more susceptibility receptors and therapeutic compounds that can mitigate its destructive actions.

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Cerebral organoids recapitulate many aspects of human corticogenesis and are a useful platform for modeling neurodevelopmental mechanisms and diseases. Watanabe et al. describe enhanced organoid methods and model ZIKV pathology. More susceptibility receptors for ZIKV are identified, and differential effects of various compounds to mitigate ZIKV-induced cytopathy are demonstrated.


http://ift.tt/2ycb5oy

Biodegradable zwitterionic sulfobetaine polymer and its conjugate with paclitaxel for sustained drug delivery

Publication date: Available online 10 October 2017
Source:Acta Biomaterialia
Author(s): Haotian Sun, Michael Yu Zarng Chang, Wei-I Cheng, Qing Wang, Alex Commisso, Meghan Capeling, Yun Wu, Chong Cheng
A fully biodegradable zwitterionic polymer and the corresponding conjugate with paclitaxel (PTX) were synthesized as promising biomaterials. Allyl-functionalized polylactide (PLA) was employed as the precursor of polymer backbones. UV-induced thiol-ene reaction was conducted to conjugate thiol-functionalized sulfobetaine (SB) with the PLA-based backbone. The resulting zwitterionic polymer did not exhibit considerable cytotoxicity. A polymer-drug conjugate was also obtained by thiol-ene reaction of both thiol-functionalized SB and PTX with allyl-functionalized PLA. The conjugate could readily form narrowly-dispersed nanoparticles in aqueous solutions with a volume-average hydrodynamic diameter (Dh,V) of 19.3 ± 0.2 nm. Such a polymer-drug conjugate-based drug delivery system showed full degradability, well-suppressed non-specific interaction with biomolecules, and sustained drug release. In vitro assessments also confirmed the significant anti-cancer efficacy of the conjugate. After 72 h incubation with PLA-SB/PTX containing 10 µg/mL of PTX, the cell viabilities of A549, MCF7, and PaCa-2 cells were as low as 20.0 ± 2.5%, 1.7 ± 1.7%, and 14.8 ± 0.9%, respectively. Both flow cytometry and confocal microscopy suggested that the conjugates could be easily uptaken by A549 cells before the major release of PTX moieties. Overall, this work elucidates promising potentials of biodegradable zwitterionic polymer-based materials in biomedical applications.Statement of SignificanceThe applicability of FDA-approved biodegradable aliphatic polyesters has been significantly restricted because they are hydrophobic and lack functionalities. Recently zwitterionic polymers have emerged as promising hydrophilic biomaterials, but most of the reported zwitterionic polymers are non-biodegradable. This study reports a novel aliphatic polyester-based zwitterionic polymer and the corresponding polymer-drug conjugate. Their aliphatic polyester and zwitterionic components provide them with high enzymatic degradability and low nonspecific interactions with biomolecules, respectively. While the zwitterionic polymer did not show noticeable cytotoxicity, the corresponding polymer-anticancer drug conjugate exhibited acid-sensitive sustained drug release, remarkable effectiveness in killing cancer cells, as well as the ready cellular internalization. This work lays a foundation for the further development of synthetic biodegradable zwitterionic polymer-based materials which potentially may have broad and significant biomedical applications.

Graphical abstract

http://ift.tt/2wN6Oq8

EGFR-targeted multifunctional polymersomal doxorubicin induces selective and potent suppression of orthotopic human liver cancer in vivo

Publication date: Available online 10 October 2017
Source:Acta Biomaterialia
Author(s): Yuan Fang, Weijing Yang, Liang Cheng, Fenghua Meng, Jian Zhang, Zhiyuan Zhong
Liver cancer is a globally leading malignancy that has a poor five-year survival rate of less than 20%. The systemic chemotherapeutics are generally ineffective for liver cancers partly due to fast clearance and low tumor uptake. Here, we report that GE11 peptide functionalized polymersomal doxorubicin (GE11-PS-DOX) effectively targets and inhibits epidermal growth factor receptor (EGFR)-positive SMMC7721 orthotopic human liver tumor xenografts in mice. GE11-PS-DOX with a GE11 surface density of 10% displayed a high drug loading of 15.4 wt.%, a small size of 78 nm, and glutathione-triggered release of DOX. MTT assays, flow cytometry and confocal microscopy studies revealed that GE11-PS-DOX mediated obviously more efficient DOX delivery into SMMC7721 cells than the non-targeting PS-DOX and clinically used liposomal doxorubicin (Lipo-DOX) controls. The in vivo studies showed that GE11-PS-DOX had a long circulation time and an extraordinary accumulation in the tumors (13.3%ID/g). Interestingly, GE11-PS-DOX caused much better treatment of SMMC7721 orthotopic liver tumor-bearing mice as compared to PS-DOX and Lipo-DOX. The mice treated with GE11-PS-DOX (12 mg DOX equiv./kg) exhibited a significantly improved survival rate (median survival time: 130 days versus 70 and 38 days for PS-DOX at 12 mg DOX equiv./kg and Lipo-DOX at 6 mg DOX equiv./kg, respectively) and achieved 50% complete regression. Notably, GE11-PS-DOX induced obviously lower systemic toxicity than Lipo-DOX. EGFR-targeted multifunctional polymersomal doxorubicin with improved efficacy and safety has a high potential for treating human liver cancers.Statement of SignificanceLiver cancer is one of the top five leading causes of cancer death worldwide. The systemic chemotherapeutics and biotherapeutics generally have a low treatment efficacy for hepatocellular carcinoma partly due to fast clearance and/or low tumor uptake. Nanomedicines based on biodegradable micelle and polymersomes offer a most promising treatment for malignant liver cancers. Their clinical effectiveness remains, however, suboptimal owing to issues like inadequate systemic stability, low tumor accumulation and selectivity, and poor control over drug release. Here we report that GE11 peptide-functionalized, disulfide-crosslinked multifunctional polymersomal doxorubicin (GE11-PS-DOX) can effectively suppress the growth of orthotopic SMMC7721 human liver tumors in nude mice. They showed significantly decreased systemic toxicity and improved mouse survival rate with 3.4-fold longer median survival time as compared to clinically used pegylated liposomal doxorubicin (Lipo-DOX) and achieving 50% complete regression. GE11-PS-DOX, based on PEG-PTMC is biodegradable, nontoxic, and easy to prepare, appears as a safe, robust, versatile and all-function-in-one nanoplatform that has a high potential in targeted chemotherapy of EGFR expressed hepatocellular carcinoma.

Graphical abstract

http://ift.tt/2wMdC7n

Engineering fibrin hydrogels to promote the wound healing potential of mesenchymal stem cell spheroids

Publication date: Available online 5 October 2017
Source:Acta Biomaterialia
Author(s): Kaitlin C. Murphy, Jacklyn Whitehead, Dejie Zhou, Steve S. Ho, J. Kent Leach
Mesenchymal stem cells (MSCs) secrete endogenous factors such as vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2) that promote angiogenesis, modulate the inflammatory microenvironment, and stimulate wound repair, and MSC spheroids secrete more trophic factors than dissociated, individual MSCs. Compared to injection of cells alone, transplantation of MSCs in a biomaterial can enhance their wound healing potential by localizing cells at the defect site and upregulating trophic factor secretion. To capitalize on the therapeutic potential of spheroids, we engineered a fibrin gel delivery vehicle to simultaneously enhance the proangiogenic and anti-inflammatory potential of entrapped human MSC spheroids. We used multifactorial statistical analysis to determine the interaction between four input variables derived from fibrin gel synthesis on four output variables (gel stiffness, gel contraction, and secretion of VEGF and PGE2). Manipulation of the four input variables tuned fibrin gel biophysical properties to promote the simultaneous secretion of VEGF and PGE2 by entrapped MSC spheroids while maintaining overall gel integrity. MSC spheroids in stiffer gels secreted the most VEGF, while PGE2 secretion was highest in more compliant gels. Simultaneous VEGF and PGE2 secretion was greatest using hydrogels with intermediate mechanical properties, as small increases in stiffness increased VEGF secretion while maintaining PGE2 secretion by entrapped spheroids. The fibrin gel formulation predicted to simultaneously increase VEGF and PGE2 secretion stimulated endothelial cell proliferation, enhanced macrophage polarization, and promoted angiogenesis when used to treat a wounded three-dimensional human skin equivalent. These data demonstrate that a statistical approach is an effective strategy to formulate fibrin gel formulations that enhance the wound healing potential of human MSCs.Statement of SignificanceMesenchymal stem cells (MSCs) are under investigation for wound healing applications due to their secretion of bioactive factors that enhance granulation tissue formation, blood vessel ingrowth, and reduce inflammation. However, the effectiveness of cell-based therapies is reduced due to poor engraftment and high rates of cell death when transplanted into harsh environments characteristic of large wounds. Compared to dissociated cells, MSCs exhibit increased overall function when aggregated into three-dimensional spheroids, and transplantation of cells using biomaterials is one strategy for guiding cell function in the defect site. The present study demonstrates that the biophysical properties of fibrin hydrogels, designed for use as a cell carrier, can be engineered to dictate the secretion of bioactive factors by entrapped MSC spheroids. This strategy enables MSCs to contribute to wound healing by synergistically promoting neovascularization and modulating the inflammatory milieu.

Graphical abstract

http://ift.tt/2wMduop

Entirely S-protected chitosan: A promising mucoadhesive excipient for metronidazole vaginal tablets

Publication date: Available online 10 October 2017
Source:Acta Biomaterialia
Author(s): Noemi Lupo, Benjamin Fodor, Ijaz Muhammad, Muhammad Yaqoob, Barbara Matuszczak, Andreas Bernkop-Schnürch
AimSynthesis and evaluation of an entirely S-protected chitosan as mucoadhesive excipient for vaginal drug delivery.MethodsN-acetyl-cysteine was linked to 6-mercaptonicotinamide via disulphide exchange reaction. The obtained ligand, NAC-6-MNA, was subsequently attached to chitosan by carbodiimide mediated amide bond formation in two concentrations. The synthesized S-protected chitosan was chemically characterized and mucoadhesive properties and stability against oxidation were investigated. Moreover, metronidazole tablets comprising the S-protected chitosan were evaluated regarding water uptake capacity, disintegration behaviour, residence time on vaginal mucosa, release of the encapsulated drug and antimicrobial activity.ResultsS-protected chitosan displayed 160±19 (CS-MNA-160) and 320±38 (CS-MNA-320) µmol of ligand per gram of polymer. At pH 4.2, CS-MNA-160 and CS-MNA-320 showed 5.2-fold and 6.2-fold increase in mucus viscosity in comparison to unmodified chitosan (One-way ANOVA, p<0.001), whereas, 9.9-fold (CS-MNA-160) and 15.6-fold (CS-MNA-320) (One-way ANOVA, p<0.001) increase in viscosity was measured at pH 6. The S-protected chitosan remained stable against oxidation in presence of 0.5% v/v hydrogen peroxide. Metronidazole tablets consisting in S-protected chitosan showed prolonged residence time on vaginal mucosa and improved water uptake capacity and disintegration time in comparison to tablets consisting of unmodified chitosan. Moreover, CS-MNA-320 metronidazole tablets displayed prolonged drug release and antimicrobial activity.ConclusionsOn the basis of the achieved results, entirely S-protected chitosan represents a promising excipient for the development of metronidazole vaginal tablets.Statement of significanceS-protected thiomers are polymers modified with thiol groups protected by aromatic ligands and characterized by strong mucoadhesive properties and high stability against oxidation. Up to date, the entirely S-protection of thiol groups was achieved via the synthesis of the ligand 2-((2-amino-2-carboxyethyl)disulfanyl)nicotinic acid) which can be directly bound to the backbone of polymers bearing carboxylic moieties as pectin. However, this ligand is not suitable for positively charged polymers due to the negative charge. In this paper, the synthesis of a suitable ligand for the entirely S-protection of positively charged polymers is presented. The first entirely S-protected chitosan was synthetized, characterized and its mucoadhesive properties were assessed. Moreover, metronidazole tablets comprising the entirely S-protected chitosan were developed and evaluated as vaginal drug delivery system.

Graphical abstract

http://ift.tt/2wMsl2a

Electrospun formulations of bevacizumab for sustained release in the eye

Publication date: Available online 10 October 2017
Source:Acta Biomaterialia
Author(s): Ukrit Angkawinitwong, Sahar Awwad, Peng T. Khaw, Steve Brocchini, Gareth R. Williams
Medicines based on vascular endothelial growth factor (VEGF) neutralising antibodies such as bevacizumab have revolutionized the treatment of age related macular degeneration (AMD), a common blinding disease, and have great potential in preventing scarring after surgery or accelerating the healing of corneal injuries. However, at present, frequent invasive injections are required to deliver these antibodies. Such administration is uncomfortable for patients and expensive for health service providers. Much effort is thus focused on developing dosage forms that can be administered less frequently. Here we use electrospinning to prepare a solid form of bevacizumab designed for prolonged release while maintaining antibody stability. Electrospun fibers were prepared with bevacizumab encapsulated in the core, surrounded by a poly-ε-caprolactone sheath. The fibers were generated using aqueous bevacizumab solutions buffered at two different pH values: 6.2 (the pH of the commercial product; Fbeva) and 8.3 (the isoelectric point of bevacizumab; FbevaP). The fibers had smooth and cylindrical morphologies, with diameters of ca. 500 nm. Both sets of bevacizumab loaded fibers gave sustained release profiles in an aqueous outflow model of the eye. Fbeva displayed first order kinetics with t1/2 of 11.4 ± 4.4 days, while FbevaP comprises a zero-order reservoir type release system with t1/2 of 52.9 ± 14.8 days. Both SDS-PAGE and surface plasmon resonance demonstrate that the bevacizumab in FbevaP did not undergo degradation during fiber fabrication or release. In contrast, the antibody released from Fbeva had degraded, and failed to bind to VEGF. Our results demonstrate that pH control is crucial to maintain antibody stability during the fabrication of core/shell fibers and ensure release of functional protein.Statement of significanceBevacizumab is a potent protein drug which is highly effective in the treatment of degenerative conditions in the eye. To be effective, frequent injections into the eye are required, which is deeply unpleasant for patients and expensive for healthcare providers. Alternative methods of administration are thus greatly sought after to produce more effective medicines. In our work, we use the electrospinning technique to prepare fiber-based formulations loaded with bevacizumab. By careful control of the experimental parameters we are able to stabilize the protein during processing and ensure a constant rate of release of the protein over two months. These fibers could thus be used to reduce the frequency of dosing required, reducing cost and improving patient outcomes.

Graphical abstract

http://ift.tt/2wMdlRT

Electrospraying of microfluidic encapsulated cells for the fabrication of cell-laden electrospun hybrid tissue constructs

Publication date: Available online 10 October 2017
Source:Acta Biomaterialia
Author(s): L. Weidenbacher, A. Abrishamkar, M. Rottmar, A.G. Guex, K. Maniura-Weber, A.J. deMello, S.J. Ferguson, R.M. Rossi, G. Fortunato
The fabrication of functional 3D tissues is a major goal in tissue engineering. While electrospinning is a promising technique to manufacture a structure mimicking the extracellular matrix, cell infiltration into electrospun scaffolds remains challenging. The robust and in situ delivery of cells into such biomimetic scaffolds would potentially enable the design of tissue engineered constructs with spatial control over cellular distribution but often solvents employed in the spinning process are problematic due to their high cytotoxicity. Herein, microfluidic cell encapsulation is used to establish a temporary protection vehicle for the in situ delivery of cells for the development of a fibrous, cell-laden hybrid biograft. Therefore a layer-by-layer process is used by alternating fiber electrospinning and cell spraying procedures.Both encapsulation and subsequent electrospraying of capsules has no negative effect on the viability and myogenic differentiation of murine myoblast cells. Propidium iodide positive stained cells were analyzed to quantify the amount of dead cells and the presence of myosin heavy chain positive cells after the processes was shown. Furthermore, encapsulation successfully protects cells from cytotoxic solvents (such as dimethylformamide) during in situ delivery of the cells into electrospun poly(vinylidene fluoride-co-hexafluoropropylene) scaffolds. The resulting cell-populated biografts demonstrate the clear potential of this approach in the creation of viable tissue engineering constructs.Statement of SignificanceInfiltration of cells and their controlled spatial distribution within fibrous electrospun membranes is a challenging task but allows for the development of functional highly organized 3D hybrid tissues. Combining polymer electrospinning and cell electrospraying in a layer-by-layer approach is expected to overcome current limitations of reduced cell infiltration after traditional static seeding. However, organic solvents, used during the electrospinning process, impede often major issues due to their high cytotoxicity. Utilizing microfluidic encapsulation as a mean to embed cells within a protective polymer casing enables the controlled deposition of viable cells without interfering with the cellular phenotype. The presented techniques allow for novel cell manipulation approaches being significant for enhanced 3D tissue engineering based on its versatility in terms of material and cell selection.

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Interaction of cruciferin-based nanoparticles with Caco-2 cells and Caco-2 /HT29-MTX co-cultures

Publication date: Available online 10 October 2017
Source:Acta Biomaterialia
Author(s): Ali Akbari, Afsaneh Lavasanifar, Jianping Wu
The objective of this work was to assess the potential of Cruciferin/Calcium (Cru/Ca) and Cruciferin/Chitosan (Cru/Cs) nanoparticles for oral drug delivery. For this purpose, Cru/Ca and Cru/Cs nanoparticles were developed through cold gelation of Cruciferin, a major canola protein, and in interaction with calcium and chitosan, respectively. The extent and rate of particle uptake in Caco-2 cells and Caco-2/HT29 co-culture was then evaluated by fluorescence spectroscopy as well as flow cytometry. Through pre-incubation of Caco-2 cell monolayer with specific endocytosis inhibitors, the mechanism of cell uptake was investigated. Our results showed that the uptake of negatively-charged Cru/Ca particles to be ∼3 times higher than positively-charged Cru/Cs ones by Caco-2 cells. Presence of mucus secreted by HT29 cells in their co-culture with Caco-2 had negligible influence on the uptake and transport of both particles. In contrast to Cru/Ca particles which were dissociated in the simulated gastrointestinal conditions, digestion of Cru/Cs particles resulted in 6- and 2-fold increase in the cellular uptake and transport of encapsulated coumarin in the latter particles, respectively. While the presence of mucus in Caco-2/HT29 co-culture caused 40-50% decrease of cellular uptake and transport for coumarin encapsulated in digested Cru/Cs particles, it had no significant effect on the cell uptake and transport of coumarin associated with Cru/Ca particles after digestion. Energy-dependent mechanisms were the dominant mechanism for uptake of both undigested and digested particles. Therefore, in Caco-2/HT29 co-culture which closely simulated intestinal epithelial cells, undigested Cru/Ca and Cru/Cs particles had the ability to penetrate mucus layers, while digested Cru/Cs particles showed mucoadhesive property, and digested Cru/Ca particles were dissociated. Our results points to a potential for cruciferin based nanoparticles for oral drug delivery.Statement of significanceThe long-term objective of this research is to investigate the potential of edible and safe biopolymer in enhanced oral delivery of drugs and/or vaccines. Here, we investigated the potential application of nanoparticles based on a protein extracted from Canola seeds, i.e., cruciferin, for oral delivery of a model small molecule, i.e., coumarin, through cells representing gastrointestinal epithelium, Caco-2 and Caco-2/HT29 cell monolayer. This study was completed for intact cruciferin nanoparticles and cruciferin coated chitosan nanoparticles, before and after digestion with gastric or intestine simulating fluids. This comparison was useful to understand the fate the cruciferin based particles in digestive mucosal tissues and their potential mucoadhesive and/or mucus-penetrating property.

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Erythrokeratoderma - a manifestation associated with multiple types of ichthyoses with different gene defects

Erythrokeratoderma (OMIM #133200) refers to a group of closely related disorders of cornification manifesting with hyperkeratotic, often transient and migratory, erythematous figurate plaques with sharply demarcated borders that typically develop in early childhood with or without palmoplantar involvement.¹ Erythrokeratodermas have been historically classified into the two main categories: (a) erythrokeratodermia variabilis et progressiva (EKVP, also known as erythrokeratoderma figurata variabilis and Mendes da Costa disease), and (b) progressive symmetric erythrokeratoderma (PSEK, also known as Gottron syndrome).

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The optimal regimen of brentuximab vedotin for CD30+ cutaneous lymphoma: Are we there yet?

Brentuximab vedotin (BV) is an anti-CD30 antibody–drug conjugate that is approved for refractory Hodgkin lymphoma and systemic anaplastic large-cell lymphoma. In the ALCANZA trial, the proportion of CD30+ cutaneous T-cell lymphoma (CTCL) patients achieving an objective response lasting at least 4 months was 56.3% with BV versus 12.5% with conventional therapy (oral methotrexate or bexarotene), and progression-free survival, complete response rate and improvement in symptom burden were significantly improved.¹ As in previous studies, 1.8 mg/kg dose was administered every 3 weeks in this study.^1,2 We routinely use this regimen that has been well studied in clinical trials, starting with 1.8 mg/kg and decrease to 1.2 mg/kg if needed. The safety profile for BV is similar to conventional therapies and although peripheral neuropathy occurs in 67%, it usually resolves or improves after cessation or completion of treatment.¹

This article is protected by copyright. All rights reserved.

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Second-line therapy in patients with metastatic castration-resistant prostate cancer with progression after or under docetaxel: A systematic review of nine randomized controlled trials

Publication date: Available online 10 October 2017
Source:Seminars in Oncology
Author(s): M.H.F. Poorthuis, R.W.M. Vernooij, R.J.A. van Moorselaar, Th.M. de Reijke
Treatment decision in patients with metastatic castration-resistant prostate cancer with progression after or under docetaxel are challenging. We systematically searched the published literature on all treatment options and assessed the risk of bias and quality of evidence. We found the best available evidence for effective prolongation of overall survival and progression-free survival for abiraterone acetate plus prednisone versus placebo plus prednisone and enzalutamide versus placebo. Other treatment modalities could be beneficial for individual patients by taking the selection criteria of the randomized clinical trials, the risk of bias, the subgroup analyses, and the quality of life and adverse events into consideration. Further research is needed to determine the sequence, timing and combination of different treatments.



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Metabolic Symbiosis in Cancer and Its Therapeutic Implication

Publication date: Available online 10 October 2017
Source:Seminars in Oncology
Author(s): Mark R. Lipstein, Ipsita Pal, Susan E. Bates, Changchun Deng




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Pilot study demonstrating metabolic and anti-proliferative effects of in vivo anti-oxidant supplementation with N-Acetylcysteine in Breast Cancer

Publication date: Available online 10 October 2017
Source:Seminars in Oncology
Author(s): Daniel Monti, Federica Sotgia, Diana Whitaker Menezes, Madalina Tuluc, Ruth Birbe, Adam Berger, Melissa Lazar, Paolo Cotzia, Rossitza Draganova-Tacheva, Zhao Lin, Marina Domingo-Vidal, Andrew Newberg, Michael P. Lisanti, Ubaldo Martinez-Outschoorn
BackgroundHigh oxidative stress as defined by hydroxyl and peroxyl activity is often found in the stroma of human breast cancers. Oxidative stress induces stromal catabolism, which promotes cancer aggressiveness. Stromal cells exposed to oxidative stress release catabolites such as lactate, which are up-taken by cancer cells to support mitochondrial oxidative phosphorylation. The transfer of catabolites between stromal and cancer cells leads to metabolic heterogeneity between these cells and increased cancer cell proliferation and reduced apoptosis in preclinical models. N-Acetylcysteine (NAC) is an antioxidant, which reduces oxidative stress, reverses stromal catabolism, and stromal-carcinoma cell metabolic heterogeneity resulting in reduced proliferation and increased apoptosis of cancer cells in experimental models of breast cancer. The purpose of this clinical trial was to determine if NAC could reduce markers of stromal-cancer metabolic heterogeneity and markers of cancer cell aggressiveness in human breast cancer.MethodsSubjects with newly diagnosed stage 0 and I breast cancer who were not going to receive neoadjuvant therapy prior to surgical resection were treated with NAC before definitive surgery to assess intra-tumoral metabolic markers. NAC was administered once a week intravenously (IV) at a dose of 150mg/kg and 600mg twice daily orally on the days not receiving NAC IV. Histochemistry (HC) for the stromal metabolic markers monocarboxylate transporter 4 (MCT4) and caveolin-1 (CAV1) and the Ki67 proliferation assay and TUNEL apoptosis assay in carcinoma cells were performed in pre- and post-NAC specimens.ResultsThe range of days on NAC was 14-27 and the mean was 19 days. Post-treatment biopsies showed significant decrease in stromal MCT4 and reduced Ki67 in carcinoma cells. NAC did not significantly change stromal CAV1 and carcinoma TUNEL staining. NAC was well tolerated.ConclusionsNAC as a single agent reduces MCT4 stromal expression, which is a marker of glycolysis in breast cancer with reduced carcinoma cell proliferation. This study suggests that modulating metabolism in the tumor microenvironment has the potential to impact breast cancer proliferation.



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Metabolic Coupling and the Reverse Warburg Effect in Cancer, implications for novel biomarker and anticancer agent development

Publication date: Available online 10 October 2017
Source:Seminars in Oncology
Author(s): Lindsay Wilde, Megan Roche, Marina Domingo-Vidal, Katherina Tanson, Nancy Philp, Joseph Curry, Ubaldo Martinez-Outschoorn
Glucose is a key metabolite used by cancer cells to generate ATP, maintain redox state and create biomass. Glucose can be catabolized to lactate in the cytoplasm, which is termed glycolysis or alternatively can be catabolized to carbon dioxide and water in the mitochondria via oxidative phosphorylation (OXPHOS). Metabolic heterogeneity exists in a subset of human tumors, with some cells maintaining a glycolytic phenotype while others predominantly utilize OXPHOS. Cells within tumors interact metabolically with transfer of catabolites from supporting stromal cells to adjacent cancer cells. The Reverse Warburg Effect describes when glycolysis in the cancer-associated stroma metabolically supports adjacent cancer cells. This catabolite transfer, which induces stromal-cancer metabolic coupling, allows cancer cells to generate ATP, increase proliferation and reduce cell death. Catabolites implicated in metabolic coupling include the monocarboxylates lactate, pyruvate and ketone bodies. Monocarboxylate transporters (MCT) are critically necessary for release and uptake of these catabolites. MCT4 is involved in the release of monocarboxylates from cells, is regulated by catabolic transcription factors such as hypoxia inducible factor 1 alpha (HIF1A) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and is highly expressed in cancer-associated fibroblasts. Conversely, MCT1 is predominantly involved in the uptake of these catabolites and is highly expressed in a subgroup of cancer cells. MYC and TIGAR, which are genes involved in cellular proliferation and anabolism are inducers of MCT1. Profiling human tumors on the basis of an altered redox balance and intra-tumoral metabolic interactions may have important biomarker and therapeutic implications. Alterations in the redox state and mitochondrial function of cells can induce metabolic coupling. Hence, there is interest in redox and metabolic modulators as anticancer agents. Also, markers of metabolic coupling have been associated with poor outcomes in numerous human malignancies and may be useful prognostic and predictive biomarkers.



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Hodgkin Lymphoma: A Complex Metabolic Ecosystem with Glycolytic Reprogramming of the Tumor Microenvironment

Publication date: Available online 10 October 2017
Source:Seminars in Oncology
Author(s): Lekha Mikkilineni, Diana Whitaker-Menezes, Marina Domingo-Vidal, John Sprandio, Paola Avena, Paolo Cotzia, Alina Dulau-Florea, Jerald Gong, Guldeep Uppal, Tingting Zhan, Benjamin Leiby, Zhao Lin, Barbara Pro, Federica Sotgia, Michael P. Lisanti, Ubaldo Martinez-Outschoorn
BackgroundTwenty percent of patients with classical Hodgkin Lymphoma (cHL) have aggressive disease defined as relapsed or refractory disease to initial therapy. At present we cannot identify these patients pre-treatment. The microenvironment is very important in cHL since non-cancer cells constitute the majority of the cells in these tumors. Non-cancer intra-tumoral cells such as tumor-associated macrophages (TAMs) have been shown to promote tumor growth in cHL via crosstalk with the cancer cells. Metabolic heterogeneity is defined as high mitochondrial metabolism in some tumor cells and glycolysis in others. We hypothesized that there are metabolic differences between cancer cells and non-cancer tumor cells such as TAMs and tumor-infiltrating lymphocytes in cHL and that greater metabolic differences between cancer cells and TAMs are associated with poor outcomes.MethodsA case-control study was conducted with 22 tissue samples of cHL at diagnosis from a single institution. The case samples were from 11 patients with aggressive cHL who had relapsed after standard treatment with adriamycin bleomycin vinblastine and dacarbazine (ABVD) or were refractory to this treatment. The control samples were from 11 patients with cHL who achieved a remission and never relapsed after ABVD. Reactive non-cancerous lymph nodes from 4 subjects served as additional controls. Samples were stained by immunohistochemistry for three metabolic markers: translocase of the outer mitochondrial membrane 20 (TOMM20), monocarboxylate transporter 1 (MCT1) and monocarboxylate transporter 4 (MCT4). TOMM20 is a marker of mitochondrial oxidative phosphorylation (OXPHOS) metabolism. Monocarboxylate transporter 1 (MCT1) is the main importer of lactate into cells and is a marker of OXPHOS. Monocarboxylate transporter 4 (MCT4) is the main lactate exporter out of cells and is a marker of glycolysis. The immunoreactivity for TOMM20, MCT1 and MCT4 was scored based on staining intensity and percentage of positive cells, as follows: 0 for no detectable staining in > 50% of cells; 1+ for faint to moderate staining in > 50% of cells, and 2+ for high or strong staining in >50% of cells.ResultsTOMM20, MCT1 and MCT4 expression was significantly different in Hodgkin and Reed Sternberg (HRS) cells, which are the cancerous cells in cHL compared to tumor associated macrophages (TAMs) and tumor-associated lymphocytes. HRS have high expression of TOMM20 and MCT1 while TAMs have absent expression of TOMM20 and MCT1 in all but 2 cases. Tumor-infiltrating lymphocytes have low TOMM20 expression and absent MCT1 expression. Conversely, high MCT4 expression was found in TAMs, but absent in HRS cells in all but 1 case. Tumor-infiltrating lymphocytes had absent MCT4 expression. Reactive lymph nodes in contrast to cHL tumors had low TOMM20, MCT1and MCT4 expression in lymphocytes and macrophages. High TOMM20 and MCT1 expression in cancer cells with high MCT4 expression in TAMs is a signature of high metabolic heterogeneity between cancer cells and the tumor microenvironment (TME). A high metabolic heterogeneity signature was associated with relapsed or refractory cHL with a hazard ratio of 5.87 [1.16-29.71] (two sided p< 0.05) compared to the low metabolic heterogeneity signature.ConclusionAggressive cHL exhibits features of metabolic heterogeneity with high mitochondrial metabolism in cancer cells and high glycolysis in TAMs, which is not seen in reactive lymph nodes. Future studies will need to confirm the value of these markers as prognostic and predictive biomarkers in clinical practice. Treatment intensity may be tailored in the future to the metabolic profile of the tumor microenvironment and drugs that target metabolic heterogeneity may be valuable in this disease.



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Mitochondrial and glycolytic metabolic compartmentalization in Diffuse Large B Cell Lymphoma

Publication date: Available online 10 October 2017
Source:Seminars in Oncology
Author(s): Mahasweta Gooptu, Diana Whitaker-Menezes, John Sprandio, Marina Domingo-Vidal, Zhao Lin, Guldeep Uppal, Jerald Gong, Roberto Fratamico, Benjamin Leiby, Alina Dulau-Florea, Jaime Caro, Ubaldo Martinez-Outschoorn
Metabolic heterogeneity between neoplastic cells and surrounding stroma has been described in several epithelial malignancies; however, the metabolic phenotypes of neoplastic lymphocytes and neighboring stroma in diffuse large B-cell lymphoma (DLBCL) is unknown. We investigated the metabolic phenotypes of human DLBCL tumors by using immunohistochemical markers of glycolytic and mitochondrial oxidative phosphorylation (OXPHOS) metabolism. The lactate importer MCT4 is a marker of glycolysis, whereas the lactate importer MCT1 and TOMM20 are markers of OXPHOS metabolism. Staining patterns were assessed in 33 DLBCL samples as well as 18 control samples (non-neoplastic lymph nodes). TOMM20 and MCT1 were highly expressed in neoplastic lymphocytes, indicating an OXPHOS phenotype, whereas non-neoplastic lymphocytes in the control samples did not express these markers. Stromal cells in DLBCL samples strongly expressed MCT4, displaying a glycolytic phenotype, a feature not seen in stromal elements of non-neoplastic lymphatic tissue. Furthermore, the differential expression of lactate exporters (MCT4) on tumor associated stroma and lactate importers (MCT1) on neoplastic lymphocytes support the hypothesis that neoplastic cells are metabolically linked to the stroma likely via mutually beneficial reprogramming. MCT4 is a marker of tumor-associated stroma in neoplastic tissue. Our findings suggest that disruption of neoplastic-stromal cell metabolic heterogeneity including MCT1 and MCT4 blockade should b studied to determine if it could represent a novel treatment target in DLBCL.



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Corrigendum to “Avoidable cancer cases in the Nordic countries – The impact of overweight and obesity” [Eur J Cancer 79 (2017) 106–118]

Publication date: Available online 10 October 2017
Source:European Journal of Cancer
Author(s): Therese M.-L. Andersson, Elisabete Weiderpass, Gerda Engholm, Anne-Sofie Q. Lund, Elinborg Olafsdottir, Eero Pukkala, Magnus Stenbeck, Hans Storm




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Thromboprophylaxis in autologous breast reconstruction

Dear Sir,

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Medical Safety Huddles in Rehabilitation – A Novel Patient Safety Strategy

Publication date: Available online 10 October 2017
Source:Archives of Physical Medicine and Rehabilitation
Author(s): Meiqi Guo, Gaetan Tardif, Mark Bayley
ObjectiveTo describe the implementation process, outcomes, and lessons learned in the implementation of medical safety huddles, a novel patient safety monitoring strategy that promotes physician engagement with patient safety.DesignSingle-centre observational study.SettingThe Brain and Spinal Cord Injury Rehabilitation Program (BSCIRP) at an urban, academic adult rehabilitation hospital.Participants18 physicians associated with the program.InterventionsWeekly physicians' safety huddles were implemented to review, anticipate, and address patient safety issues.Main Outcome MeasuresThe main outcome measures were the number and nature of identified and anticipated patient safety incidents, actions taken and physician attendance during huddles. The number of adverse events in the program before and after huddle implementation were secondary measures.ResultsOver a 7-month period, average physician attendance at medical huddles was 76.0%. 1.0±0.8 patient safety incidents and 3.2±2.1 anticipated patient safety issues were identified in each weekly huddle. The majority of patient safety incidents identified were clinical administrative and clinical process-related, which differed from information gathered from the organization's pre-existing patient safety monitoring strategies. A total of 79 actions, or 3.3±1.8 actions/huddle were taken in response to improve patient safety for the program. Adverse events decreased from 31.2/month (95% confidence interval[CI] 27.0-35.3) to 22.9/month (CI 19.3-26.5) after implementation.Conclusion(s)Medical safety huddles are a novel strategy to engage physicians in patient safety and organizational quality improvement. They have the potential to enhance organizational anticipation of safety risks by supplementing existing methods. Other rehabilitation settings may wish to consider implementing and evaluating similar huddles into their existing patient safety and quality improvement frameworks.



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Best practice in research: Consensus Statement on Ethnopharmacological Field Studies – ConSEFS

Publication date: 30 January 2018
Source:Journal of Ethnopharmacology, Volume 211
Author(s): Michael Heinrich, Andreas Lardos, Marco Leonti, Caroline Weckerle, Merlin Willcox, Wendy Applequist, Ana Ladio, Chun Lin Long, Pulok Mukherjee, Gary Stafford
BackgroundEthnopharmacological research aims at gathering information on local and traditional uses of plants and other natural substances. However, the approaches used and the methods employed vary, and while such a variability is desirable in terms of scientific diversity, research must adhere to well defined quality standards and reproducible methodsObjectivesWith ConSEFS (the Consensus Statement on Ethnopharmacological Field Studies) we want to define best-practice in developing, conducting and reporting field studies focusing on local and traditional uses of medicinal and food plants, including studies using a historical approach.MethodsAfter first developing an initial draft the core group invited community-wide feedback from researchers both through a web-based consultation and a series of workshops at conferences during 2017.OutcomesThe consultation resulted in a large number of responses. Feedback was received via a weblink on the Journal of Ethnopharmacology's website (ca. 100 responses), other oral and written responses (ca. 50) and discussions with stakeholders at four conferences. The main outcome is a checklist, covering best practice for designing, implementing and recording ethnopharmacological field studies and historical studies.ConclusionsPrior to starting ethnopharmacological field research, it is essential that the authors are fully aware of the best practice in the field. For the first time in the field of ethnopharmacology a community-wide document defines guidelines for best practice on how to conduct and report such studies. It will need to be updated and further developed. While the feedback has been based on responses by many experienced researchers, there is a need to test it in practice by using it both in implementing and reporting field studies (or historical studies), and peer-review.

Graphical abstract

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The role of endemic plants in Mauritian folkloric medicine - Therapeutic efficacy or placebo effect?

Publication date: Available online 10 October 2017
Source:Journal of Ethnopharmacology
Author(s): Nawraj Rummun, Vidushi S. Neergheen-Bhujun, Kersley B. Pynee, Cláudia Baider, Theeshan Bahorun
Ethnopharmacological relevanceThe Mauritian endemic flora has been recorded to be used as medicinal for nearly 300 years. Despite acceptance of these endemic plants among the local population, proper documentation of their therapeutic uses is scarce. This review aims at summarizing documented traditional uses of Mauritian endemic plants with existing scientific data of their alleged bioactivities, showing there is a need for more stringent evidence-based validations for clinical efficacy.Material and MethodsWe made a comprehensive bibliographic search of Mauritian endemic plants on ethnobotanical textbooks, regional and local herbal pharmacopeia and dissertations and theses, concurrently with screening of peer-reviewed journals. Available data on the usage of Mauritian endemic plants in the folklore medicine and scientific investigation were correlated.Results and DiscussionAnalysis of the available literature shows that a small percentage of the Mauritian endemic plants are used for their medicinal value. Endemic plants are either used as part of complex herbal formulations or singly, and are prescribed by herbalists to mitigate a myriad of diseases from metabolic disorders, dermatological pathologies, arthritis to sexually transmissible diseases. However, these species have undergone a limited consistent evaluation to validate their purported ethnomedicinal claims. As the World Health Organization Traditional Medicine Strategy 2014–2023 emphasizes on moving traditional medicine into mainstream medicine on an equally trusted footage, the re-evaluation and modernization of Mauritius cultural heritage become necessary.ConclusionsWith a consumer-driven 'return to nature', scientific validation and valorization of the herbal remedies, including evidence-based efficacy and safety are, therefore, important. The use of optimized methodologies to investigate the claims of these traditional medicines is mandatory for effective treatments to emanate from unfounded superstitions.

Graphical abstract

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Research on the antioxidant, wound healing, and anti-inflammatory activities and the phytochemical composition of maritime pine (Pinus pinaster Ait)

Publication date: 30 January 2018
Source:Journal of Ethnopharmacology, Volume 211
Author(s): İbrahim Tümen, Esra Küpeli Akkol, Hakkı Taştan, Ipek Süntar, Mehmet Kurtca
Ethnopharmacological relevanceEthnobotanical investigations have shown that the Pinus species have been used against rheumatic pain and for wound healing in Turkish folk medicine.Material and methodsIn this study, phytochemical composition, antioxidant, anti-inflammatory, and wound healing activities of Maritime Pine (Pinus pinaster Ait.) that is collected in Turkey are investigated. Essential oil composition and the amount of extracts (lipophilic and hydrophilic) of maritime pine wood and fresh cone samples had been tested.ResultsThe essential oil from cones of P. pinaster revealed the highest activities, whereas other parts of the plant did not display any appreciable wound healing, anti-inflammatory, or antioxidant effects. α-Pinene was the main constituent of the essential oil obtained from the cones of P. pinaster.ConclusionExperimental studies shown that P. pinaster's remarkable anti-inflammatory and wound healing activities support the traditional use of the plant, and suggest it could have a place in modern medicine.

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