The development and growth of tissues derived from cranial neural crest and primitive mesoderm is dependent on the ligation status of retinoic acid receptor γ:evidence that retinoic acid receptor γ functions to maintain stem/progenitor cells in the absence of retinoic acid

Retinoic acid (RA) signaling is important to normal development. However, the function of the different RA receptors (RARs)-RARα, RARβ, and RARγ-is as yet unclear. We have used wild-type and transgenic zebrafish to examine the role of RARγ. Treatment of zebrafish embryos with an RARγ-specific agonist reduced somite formation and axial length, which was associated with a loss of hoxb13a expression and less-clear alterations in hoxc11a or myoD expression. Treatment with the RARγ agonist also disrupted formation of tissues arising from cranial neural crest, including cranial bones and anterior neural ganglia. There was a loss of Sox 9-immunopositive neural crest stem/progenitor cells in the same anterior regions. Pectoral fin outgrowth was blocked by RARγ agonist treatment. However, there was no loss of Tbx-5-immunopositive lateral plate mesodermal stem/progenitor cells and the block was reversed by agonist washout or by cotreatment with an RARγ antagonist. Regeneration of the caudal fin was also blocked by RARγ agonist treatment, which was associated with a loss of canonical Wnt signaling. This regenerative response was restored by agonist washout or cotreatment with the RARγ antagonist. These findings suggest that RARγ plays an essential role in maintaining stem/progenitor cells during embryonic development and tissue regeneration when the receptor is in its nonligated state.

Pyroligneous acid:a farmer’s flexible friend?

This article is a review of the findings of key studies into the potential benefits of pyroligneous acid, arising from charcoal production, to the agricultural industry. Through a review of bioassay studies conducted on known plant and human pathogens (e.g., Agrobacterium tumefacien and Xanthomonas campestris) and arthropods, and germination studies on selected crops, the article highlights a number of potential benefits of smoke recovery in the production of charcoal. In addition, the article calls for further research into the impact, if any, of its long-term use on environmental receptors/humans and for the development of a methodology to guarantee consistency in product composition, quality, and efficacy. In doing so, it is hoped that its widespread use as part of sustainable management practices adopted in the agricultural industry will be secured.

GPCR-styrene maleic acid lipid particles (GPCR-SMALPs):their nature and potential

G-protein-coupled receptors (GPCRs) form the largest class of membrane proteins and are an important target for therapeutic drugs. These receptors are highly dynamic proteins sampling a range of conformational states in order to fulfil their complex signalling roles. In order to fully understand GPCR signalling mechanisms it is necessary to extract the receptor protein out of the plasma membrane. Historically this has universally required detergents which inadvertently strip away the annulus of lipid in close association with the receptor and disrupt lateral pressure exerted by the bilayer. Detergent-solubilized GPCRs are very unstable which presents a serious hurdle to characterization by biophysical methods. A range of strategies have been developed to ameliorate the detrimental effect of removing the receptor from the membrane including amphipols and reconstitution into nanodics stabilized by membrane scaffolding proteins (MSPs) but they all require exposure to detergent. Poly(styrene-co-maleic acid) (SMA) incorporates into membranes and spontaneously forms nanoscale poly(styrene-co-maleic acid) lipid particles (SMALPs), effectively acting like a 'molecular pastry cutter' to 'solubilize' GPCRs in the complete absence of detergent at any stage and with preservation of the native annular lipid throughout the process. GPCR-SMALPs have similar pharmacological properties to membrane-bound receptor, exhibit enhanced stability compared with detergent-solubilized receptors and being non-proteinaceous in nature, are fully compatible with downstream biophysical analysis of the encapsulated GPCR.

Syntheses of aza analogues of kainic acid

The kainate receptors are one of the three major groups of ionotropic glutamate receptors in the mammalian central nervous system. They are so named after their most potent agonist, kainic acid (KA), a natural product isolated from the seaweed Diginea simplex. This compound shows both neuroexcitatory and excitotoxic activities, and is an important pharmacological tool for neurophysiological studies. We predict that the more synthetically accessible aza analogues of kainic acid, could act as functional mimics of KA. These could be produced by the 1,3-dipolar cycloaddition of diazoalkanes with trans glutaconate esters. ^ 1,3-Dipolar cycloadditions have been shown to produce 1-pyrazolines that isomerize into 2-pyrazolines. The 1- and 2-pyrazolines can be precursors to aza analogs of kainoids. The regioselectivity, relative stereochemistry and isomerization of the 1-pyrazolines into 2-pyrazolines have been evaluated. Reductions of the 1- and 2-pyrazolines produced aza analogs of kainoids. TMS diazomethane was used as the dipole in 1,3-dipolar cycloaddition reactions leading to aza KA analogs via 2-pyrazolines. A systematic study of cycloaddition-isomerization processes involving TMS-diazomethane and various α, β-unsaturated dipolarophiles has been undertaken. 1H-NMR monitoring of the reaction mixture compositions during the cycloaddition reaction revealed evidence of retro-dipolar cycloaddition processes. Faster formation of 4,5- trans-1-pyrazoline at the beginning of the reaction and subsequent isomerization of this product into 4,5-cis-1-pyrazoline via a retro-dipolar cycloaddition has been observed. Increased reaction time and/or reaction temperature preferentially caused the irreversible isomerization of 4,5-cis-1-pyrazoline into 4,5-cis-2-pyrazoline, which led to high yields of 4,5-cis-2-pyrazolines in the overall process. ^ Two syntheses of the 5-unsubstituted aza-kainic acid have been performed; first, via the reduction of the TMS-eliminated 2-pyrazoline from TMS diazomethane; second by the direct reduction of 1-pyrazoline with Hg/Al-amalgam. 5-Phenyl aza-kainic acid has been produced by direct reduction of 1-pyrazoline, obtained in the reaction of phenyldiazomethane and dibenzyl glutaconate, with Hg/Al-amalgam. ^ Current responses to aza kainate analogs in Aplysia whole cell buccal ganglia indicate potent neuroexcitatory activity. The repetitive exposure of neuronal cells to the 5-unsubstituted aza-kainic acid led to non-desensitizing current responses, showing both binding affinity and neuronal ion-channel activation by the synthesized agonist compound. ^

Asymmetric syntheses of analogs of kainic acid

Kainic acid has been used for nearly 50 years as a tool in neuroscience due to its pronounced neuroexcitatory properties. However, the significant price increase of kainic acid resulting from the disruption in the supply from its natural source, the alga Digenea Simplex, as well as inefficient synthesis of kainic acid, call for the exploration of functional mimics of kainic acid that can be synthesized in a simpler way. Aza kainoids analog could be one of them. The unsubstituted aza analog of kainoids has demonstrates its ability as an ionotropic glutamate receptor agonist and showed affinity in the chloride dependent glutamate (GluCl) binding site. This opened a question of the importance of the presence of one nitrogen or both nitrogens in the aza kainoid analogs for binding to glutamate receptors. Therefore, two different pyrrolidine analogs of kainic acid, trans -4-(carboxymethyl)pyrrolidine-3-carboxylic acid and trans -2-carboxy-3-pyrrolidineacetic acid, were synthesized through multi-step sequences. The lack of the affinity of both pyrrolidine analogs in GluCl binding site indicated that both nitrogens in aza kainoid analogs are involved in hydrogen bonding with receptors, significantly enhancing their affinity in GluCl binding site. Another potential functional mimic of kainic acid is isoxazolidine analogs of kainoids whose skeleton can be constituted directly via a 1, 3 dipolar cycloaddition as the key step. The difficulty in synthesizing N-unsubstituted isoxazolidines when applying such common protecting groups as alkyl, phenyl and benzyl groups, and the requirement of a desired enantioselectivity due to the three chiral ceneters in kainic acid, pose great challenges. Hence, several different protected nitrones were studied to establish that diphenylmethine nitrone may be a good candidate as the dipole in that the generated isoxazolidines can be deprotected in mild conditions with high yields. Our investigations also indicated that the exo/endo selectivity of the 1, 3 dipolar cycloaddition can be controlled by Lewis acids, and that the application of a directing group in dipolarophiles can accomplish a satisfied enantioselectivity. Those results demonstrated the synthesis of isoxazoldines analogs of kainic acid is very promising.

Asymmetric Syntheses of Analogs of Kainic Acid

Kainic acid has been used for nearly 50 years as a tool in neuroscience due to its pronounced neuroexcitatory properties. However, the significant price increase of kainic acid resulting from the disruption in the supply from its natural source, the alga Digenea Simplex, as well as inefficient synthesis of kainic acid, call for the exploration of functional mimics of kainic acid that can be synthesized in a simpler way. Aza kainoids analog could be one of them. The unsubstituted aza analog of kainoids has demonstrates its ability as an ionotropic glutamate receptor agonist and showed affinity in the chloride dependent glutamate (GluCl) binding site. This opened a question of the importance of the presence of one nitrogen or both nitrogens in the aza kainoid analogs for binding to glutamate receptors. Therefore, two different pyrrolidine analogs of kainic acid, trans-4-(carboxymethyl)pyrrolidine-3-carboxylic acid and trans-2-carboxy-3-pyrrolidineacetic acid, were synthesized through multi-step sequences. The lack of the affinity of both pyrrolidine analogs in GluCl binding site indicated that both nitrogens in aza kainoid analogs are involved in hydrogen bonding with receptors, significantly enhancing their affinity in GluCl binding site. Another potential functional mimic of kainic acid is isoxazolidine analogs of kainoids whose skeleton can be constituted directly via a 1, 3 dipolar cycloaddition as the key step. The difficulty in synthesizing N-unsubstituted isoxazolidines when applying such common protecting groups as alkyl, phenyl and benzyl groups, and the requirement of a desired enantioselectivity due to the three chiral ceneters in kainic acid, pose great challenges. Hence, several different protected nitrones were studied to establish that diphenylmethine nitrone may be a good candidate as the dipole in that the generated isoxazolidines can be deprotected in mild conditions with high yields. Our investigations also indicated that the exo/endo selectivity of the 1, 3 dipolar cycloaddition can be controlled by Lewis acids, and that the application of a directing group in dipolarophiles can accomplish a satisfied enantioselectivity. Those results demonstrated the synthesis of isoxazoldines analogs of kainic acid is very promising.

The Photooxidation of Domoic Acid

Domoic acid (DA) is a naturally occurring cyanotoxin, which upon ingestion, is responsible for amnesic shellfish poisoning (ASP) in both humans and animals. Produced by the marine diatom, Pseudonitzschia, DA is accumulated by a number of marine organisms including shellfish, clams and mussels which upon consumption can lead to headaches, nausea and seizures. Possessing a variety of functional groups the structure of DA contains three carboxyl groups, a pyrrole ring and a potent conjugated diene region allowing for binding to glutamate receptors in the dorsal hippocampus of the brain causing the described detrimental effects. Although limitations have been placed regarding the amount of DA that may be contained in seafood no limitations have been placed on the amount present in drinking water. Natural degradation of the toxin may occur through reactive oxygen species such as the hydroxyl radical and singlet oxygen at the conjugated diene region. In this work the photooxidation of DA via singlet oxygen has been studied using sorbic acid as a model compound. The three major reaction pathways observed during the photooxdiation process for both acids include 2 + 4 cycloaddition to produce endoperoxides , 2 + 2 reaction to afford aldehydes and ketones or an ene reaction to generate hydroperoxides. Under similar reaction conditions for SA and DA, the endoperoxide has been seen to be the major product for photoxidation of SA while the hydroperoxide has been seen to be the dominant product during photooxidation of DA.

The Crosslinking Degree Controls The Mechanical, Rheological, And Swelling Properties Of Hyaluronic Acid Microparticles.

Viscosupplements, used for treating joint and cartilage diseases, restore the rheological properties of synovial fluid, regulate joint homeostasis and act as scaffolds for cell growth and tissue regeneration. Most viscosupplements are hydrogels composed of hyaluronic acid (HA) microparticles suspended in fluid HA. These microparticles are crosslinked with chemicals to assure their stability against enzyme degradation and to prolong the action of the viscosupplement. However, the crosslinking also modifies the mechanical, swelling and rheological properties of the HA microparticle hydrogels, with consequences on the effectiveness of the application. The aim of this study is to correlate the crosslinking degree (CD) with these properties to achieve modulation of HA/DVS microparticles through CD control. Because divinyl sulfone (DVS) is the usual crosslinker of HA in viscosupplements, we examined the effects of CD by preparing HA microparticles at 1:1, 2:1, 3:1, and 5:1 HA/DVS mass ratios. The CD was calculated from inductively coupled plasma spectrometry data. HA microparticles were previously sized to a mean diameter of 87.5 µm. Higher CD increased the viscoelasticity and the extrusion force and reduced the swelling of the HA microparticle hydrogels, which also showed Newtonian pseudoplastic behavior and were classified as covalent weak. The hydrogels were not cytotoxic to fibroblasts according to an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2014.

Geochemistry Of Acid Mine Drainage From A Coal Mining Area And Processes Controlling Metal Attenuation In Stream Waters, Southern Brazil.

Acid drainage influence on the water and sediment quality was investigated in a coal mining area (southern Brazil). Mine drainage showed pH between 3.2 and 4.6 and elevated concentrations of sulfate, As and metals, of which, Fe, Mn and Zn exceeded the limits for the emission of effluents stated in the Brazilian legislation. Arsenic also exceeded the limit, but only slightly. Groundwater monitoring wells from active mines and tailings piles showed pH interval and chemical concentrations similar to those of mine drainage. However, the river and ground water samples of municipal public water supplies revealed a pH range from 7.2 to 7.5 and low chemical concentrations, although Cd concentration slightly exceeded the limit adopted by Brazilian legislation for groundwater. In general, surface waters showed large pH range (6 to 10.8), and changes caused by acid drainage in the chemical composition of these waters were not very significant. Locally, acid drainage seemed to have dissolved carbonate rocks present in the local stratigraphic sequence, attenuating the dispersion of metals and As. Stream sediments presented anomalies of these elements, which were strongly dependent on the proximity of tailings piles and abandoned mines. We found that precipitation processes in sediments and the dilution of dissolved phases were responsible for the attenuation of the concentrations of the metals and As in the acid drainage and river water mixing zone. In general, a larger influence of mining activities on the chemical composition of the surface waters and sediments was observed when enrichment factors in relation to regional background levels were used.

Changes In Chromatin Structure In Nih 3t3 Cells Induced By Valproic Acid And Trichostatin A.

Valproic acid (VPA) and trichostatin A (TSA) are known histone deacetylase inhibitors (HDACIs) with epigenetic activity that affect chromatin supra-organization, nuclear architecture, and cellular proliferation, particularly in tumor cells. In this study, chromatin remodeling with effects extending to heterochromatic areas was investigated by image analysis in non-transformed NIH 3T3 cells treated for different periods with different doses of VPA and TSA under conditions that indicated no loss of cell viability. Image analysis revealed chromatin decondensation that affected not only euchromatin but also heterochromatin, concomitant with a decreased activity of histone deacetylases and a general increase in histone H3 acetylation. Heterochromatin protein 1-α (HP1-α), identified immunocytochemically, was depleted from the pericentromeric heterochromatin following exposure to both HDACIs. Drastic changes affecting cell proliferation and micronucleation but not alteration in CCND2 expression and in ratios of Bcl-2/Bax expression and cell death occurred following a 48-h exposure of the NIH 3T3 cells particularly in response to higher doses of VPA. Our results demonstrated that even low doses of VPA (0.05 mM) and TSA (10 ng/ml) treatments for 1 h can affect chromatin structure, including that of the heterochromatin areas, in non-transformed cells. HP1-α depletion, probably related to histone demethylation at H3K9me3, in addition to the effect of VPA and TSA on histone H3 acetylation, is induced on NIH 3T3 cells. Despite these facts, alterations in cell proliferation and micronucleation, possibly depending on mitotic spindle defects, require a longer exposure to higher doses of VPA and TSA.

A Highly Improved Method For Sensitive Determination Of Amitriptyline In Pharmaceutical Formulations Using An Unmodified Carbon Nanotube Electrode In The Presence Of Sulfuric Acid.

The present paper describes a novel, simple and reliable differential pulse voltammetric method for determining amitriptyline (AMT) in pharmaceutical formulations. It has been described for many authors that this antidepressant is electrochemically inactive at carbon electrodes. However, the procedure proposed herein consisted in electrochemically oxidizing AMT at an unmodified carbon nanotube paste electrode in the presence of 0.1 mol L(-1) sulfuric acid used as electrolyte. At such concentration, the acid facilitated the AMT electroxidation through one-electron transfer at 1.33 V vs. Ag/AgCl, as observed by the augmentation of peak current. Concerning optimized conditions (modulation time 5 ms, scan rate 90 mV s(-1), and pulse amplitude 120 mV) a linear calibration curve was constructed in the range of 0.0-30.0 μmol L(-1), with a correlation coefficient of 0.9991 and a limit of detection of 1.61 μmol L(-1). The procedure was successfully validated for intra- and inter-day precision and accuracy. Moreover, its feasibility was assessed through analysis of commercial pharmaceutical formulations and it has been compared to the UV-vis spectrophotometric method used as standard analytical technique recommended by the Brazilian Pharmacopoeia.

Efficacy Of Ethylene-diamine-tetra-acetic Acid Associated With Chlorhexidine On Intracanal Medication Removal: A Scanning Electron Microscopy Study.

The aim of this study was to evaluate the effectiveness of 17% ethylene-diamine-tetra-acetic acid (EDTA) used alone or associated with 2% chlorhexidine gel (CHX) on intracanal medications (ICM) removal. Sixty single-rooted human teeth with fully formed apex were selected. The cervical and middle thirds of each canal were prepared with Gates Glidden drills and rotary files. The apical third was shaped with hand files. The specimens were randomly divided into two groups depending on the ICM used after instrumentation: calcium hydroxide Ca(OH)(2) +CHX or Ca(OH)(2) +sterile saline (SS). After seven days, each group was divided into subgroups according to the protocol used for ICM removal: instrumentation and irrigation either with EDTA, CHX+EDTA, or SS (control groups). All specimens were sectioned and processed for observation of the apical thirds by using scanning electron microscopy. Two calibrated evaluators attributed scores to each specimen. The differences between the protocols for ICM removal were analyzed with Kruskal-Wallis and Mann-Whitney U tests. Friedman and Wilcoxon signed rank tests were used for comparison between the score of debris obtained in each root canal third. Remains of Ca(OH)(2) were found in all specimens independently of the protocol and ICM used (P > 0.05). Seventeen percent EDTA showed the best results in removing ICM when used alone (P < 0.05), particularly in those associated with CHX. It was concluded that the chelating agent 17% EDTA significantly improved the removal of ICM when used alone. Furthermore, the type of the vehicle associated with Ca(OH)(2) also plays a role in the ICM removal.

Fatty Acid Synthase Inhibitors Induce Apoptosis In Non-tumorigenic Melan-a Cells Associated With Inhibition Of Mitochondrial Respiration.

The metabolic enzyme fatty acid synthase (FASN) is responsible for the endogenous synthesis of palmitate, a saturated long-chain fatty acid. In contrast to most normal tissues, a variety of human cancers overexpress FASN. One such cancer is cutaneous melanoma, in which the level of FASN expression is associated with tumor invasion and poor prognosis. We previously reported that two FASN inhibitors, cerulenin and orlistat, induce apoptosis in B16-F10 mouse melanoma cells via the intrinsic apoptosis pathway. Here, we investigated the effects of these inhibitors on non-tumorigenic melan-a cells. Cerulenin and orlistat treatments were found to induce apoptosis and decrease cell proliferation, in addition to inducing the release of mitochondrial cytochrome c and activating caspases-9 and -3. Transfection with FASN siRNA did not result in apoptosis. Mass spectrometry analysis demonstrated that treatment with the FASN inhibitors did not alter either the mitochondrial free fatty acid content or composition. This result suggests that cerulenin- and orlistat-induced apoptosis events are independent of FASN inhibition. Analysis of the energy-linked functions of melan-a mitochondria demonstrated the inhibition of respiration, followed by a significant decrease in mitochondrial membrane potential (ΔΨm) and the stimulation of superoxide anion generation. The inhibition of NADH-linked substrate oxidation was approximately 40% and 61% for cerulenin and orlistat treatments, respectively, and the inhibition of succinate oxidation was approximately 46% and 52%, respectively. In contrast, no significant inhibition occurred when respiration was supported by the complex IV substrate N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). The protection conferred by the free radical scavenger N-acetyl-cysteine indicates that the FASN inhibitors induced apoptosis through an oxidative stress-associated mechanism. In combination, the present results demonstrate that cerulenin and orlistat induce apoptosis in non-tumorigenic cells via mitochondrial dysfunction, independent of FASN inhibition.

Petroleomics By Electrospray Ionization Ft-icr Mass Spectrometry Coupled To Partial Least Squares With Variable Selection Methods: Prediction Of The Total Acid Number Of Crude Oils.

Negative-ion mode electrospray ionization, ESI(-), with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) was coupled to a Partial Least Squares (PLS) regression and variable selection methods to estimate the total acid number (TAN) of Brazilian crude oil samples. Generally, ESI(-)-FT-ICR mass spectra present a power of resolution of ca. 500,000 and a mass accuracy less than 1 ppm, producing a data matrix containing over 5700 variables per sample. These variables correspond to heteroatom-containing species detected as deprotonated molecules, [M - H](-) ions, which are identified primarily as naphthenic acids, phenols and carbazole analog species. The TAN values for all samples ranged from 0.06 to 3.61 mg of KOH g(-1). To facilitate the spectral interpretation, three methods of variable selection were studied: variable importance in the projection (VIP), interval partial least squares (iPLS) and elimination of uninformative variables (UVE). The UVE method seems to be more appropriate for selecting important variables, reducing the dimension of the variables to 183 and producing a root mean square error of prediction of 0.32 mg of KOH g(-1). By reducing the size of the data, it was possible to relate the selected variables with their corresponding molecular formulas, thus identifying the main chemical species responsible for the TAN values.

Analysis Of The Contribution Of Immunologically-detectable Her2, Steroid Receptors And Of The Triple-negative" Tumor Status To Disease-free And Overall Survival Of Women With Epithelial Ovarian Cancer."

We assessed associations between steroid receptors including: estrogen-alpha, estrogen-beta, androgen receptor, progesterone receptor, the HER2 status and triple-negative epithelial ovarian cancer (ERα-/PR-/HER2-; TNEOC) status and survival in women with epithelial ovarian cancer. The study included 152 women with primary epithelial ovarian cancer. The status of steroid receptor and HER2 was determined by immunohistochemistry. Disease-free and overall survival were calculated and compared with steroid receptor and HER2 status as well as clinicopathological features using the Cox Proportional Hazards model. A mean follow-up period of 43.6 months (interquartile range=41.4 months) was achieved where 44% of patients had serous tumor, followed by mucinous (23%), endometrioid (9%), mixed (9%), undifferentiated (8.5%) and clear cell tumors (5.3%). ER-alpha staining was associated with grade II-III tumors. Progesterone receptor staining was positively associated with a Body Mass Index≥25. Androgen receptor positivity was higher in serous tumors. In stand-alone analysis of receptor contribution to survival, estrogen-alpha positivity was associated with greater disease-free survival. However, there was no significant association between steroid receptor expression, HER2 status, or TNEOC status, and overall survival. Although estrogen-alpha, androgen receptor, progesterone receptor and the HER2 status were associated with key clinical features of the women and pathological characteristics of the tumors, these associations were not implicated in survival. Interestingly, women with TNEOC seem to fare the same way as their counterparts with non-TNEOC.