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Personalized dosages of monoclonal antibodies are being used more regularly to treat various diseases, rendering their quantitation more essential than ever for the right dose administration to the patients. A promising alternative, which overcomes the obstacles of the well-established chromatographic techniques regarding the quantification of biopharmaceuticals, is Raman spectroscopy. This study aimed to develop and validate a novel analytical method for the quantitation of bevacizumab in solutions via Raman spectroscopy. For this purpose, a droplet of the solution was left to dry on a highly reflective carrier and a home-made apparatus was employed for rotation of the sample. Hence, each recorded Raman spectrum was the average of the signal acquired simultaneously from multiple points on a circular circumference. The method was validated, and the detection limit of the antibody was found to be 1.06 mg/mL. Bevacizumab was found to be highly distributed at the formed coffee ring of the dried droplet, though this was a function of solution concentration. Finally, Raman spectra at different distances on the coffee ring were obtained from the four quarters. The lowest bevacizumab detection limit was found at a distance of 75 μm from the external side of the coffee ring and it was determined to be equal to 0.53 mg/mL., Competing Interests: The authors declare no conflicts of interest.

Raman microspectroscopy and X-ray microcomputed tomography (micro-CT) were used for assessment of the quality of the femur and tibia bones in apolipoprotein-deficient mice compared to control littermates. The cortical and trabecular bone was investigated separately. Raman spectra revealed no differences in the bioapatite-to-collagenous matrix ratio of the cortical bone. The quantities of calcium and collagen, which were measured using atomic absorption spectrometry and thermogravimetric analysis, respectively, were also found to be equal in the two groups. Density and morphometric parameters, which were measured using micro-CT, verified the cortical mineral stability. Bone quality indices were measured using Raman spectra. A decreased collagen crosslink (trivalent-to-divalent) ratio revealed delayed maturation of the collagen network. Such a decrease has been reported in the literature to be connected to decreased bone strength. For the trabecular bone, micro-CT revealed severe osteoporosis in the knock-out group, which was evident from a decreased mineral density, trabecular thickness and increased bone surface/volume ratio. The trabecular bone was not accessible for Raman spectroscopy. According to these results, the cortical and trabecular femur bone is expected to exhibit proneness to fracturing, each for a different reason. A combination of the two techniques was regarded as necessary for an overall assessment of bone quality.

The measurement of ethanol and toxic alcohol (methanol and isopropanol) strengths in beverages and spirits is crucial for health reasons but also for the identification of adulterated products. Many methodologies have been reported in the literature, based mainly on chromatographic and on spectroscopic techniques. Chromatographic techniques are laborious and time-consuming, while spectroscopic techniques are rapid and need no special sample pretreatment. All techniques were only applied to off-line or at-line manner. In the present work, Raman spectroscopy was used for fast and non-destructive measurements. A "through the container" method was developed for a non-invasive analysis, i.e., analysis without unsealing the bottles. This method, coupled with a miniature portable Raman, can serve for in-line measurements in a production line. The optimum laser focus for maximum spirit signal and minimum glass-wall signal was investigated. Calibration curves for the alcohols of interest were constructed and validated. The limits of detections were calculated and proved to be lower than the legitimate values. The influences of the liquor color and the bottle color, shape, and thickness were checked. Twenty-eight alcoholic products were studied. The concentrations found were compared against the nominal values (from the bottle labels).

The functional role(s) of plant calcium oxalate (CaOx) crystals are still poorly understood. Recently, it was shown that crystals function as dynamic carbon pools whose decomposition could provide CO 2 to photosynthesis when stomata are closed (e.g. under drought conditions) and CO 2 starvation conditions may be created within the mesophyll. This biochemical process, named as 'alarm photosynthesis', can become crucial for plant survival under adverse conditions. Here, we study crystal decomposition under controlled CO 2 starvation conditions (either in the shoot or in the root) to obtain a better insight into the process of crystal formation and function. Hydroponically grown pigweed plants were kept in CO 2 -free air and/or CO 2 -free nutrient medium for 9 days. Crystal volume was monitored daily, and carbon stable isotope composition (δ 13 C) and Fourier transformation Raman spectra were obtained at the end of the experiment. A considerable reduction in the leaf crystal volume was observed in shoot-CO 2 -starved plants at the end of the experiment. The smallest crystals were isolated from the plants in which carbon was excluded from both the shoot and the root and contained potassium nitrate. Crystal δ 13 C of CO 2 -starved plants was altered in a predicted way. Specifically, it depended on the average calculated isotope fractionation of all carbon fixation processes considered to be contributing in each experimental treatment. The results of the present study confirmed the correlation between CO 2 starvation conditions and the CaOx crystal decomposition. Inorganic carbon fixed in the root may represent a major carbon source for CaOx formation., (© 2018 Scandinavian Plant Physiology Society.)

Electronic cigarettes are considered healthier alternatives to conventional cigarettes containing tobacco. They produce vapor through heating of the refill liquids (e-liquids) which consist of propylene glycol, vegetable glycerin, nicotine (in various concentrations), water and flavoring agents. Heavy metals may enter the refill liquid during the production, posing a risk for consumer's health due to their toxicity. The objective of the present study was the development of a methodology for the detection and quantitative analysis of cadmium (Cd), lead (Pb), nickel (Ni), copper (Cu), arsenic (As) and chromium (Cr), employing Total Reflection X-Ray Fluorescence Spectroscopy (TXRF) as an alternative technique to ICP-MS or ICP-OES commonly used for this type of analysis. TXRF was chosen due to its advantages, which include short analysis time, promptness, simultaneous multi-element analysis capability and minimum sample preparation, low purchase and operational cost. The proposed methodology was applied to a large number of electronic cigarette liquids commercially available, as well as their constituents, in order to evaluate their safety. TXRF may be a valuable tool for probing heavy metals in electronic cigarette refill liquids to serve for the protection of human health., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Land plants face the perpetual dilemma of using atmospheric carbon dioxide for photosynthesis and losing water vapors, or saving water and reducing photosynthesis and thus growth. The reason behind this dilemma is that this simultaneous exchange of gases is accomplished through the same minute pores on leaf surfaces, called stomata. In a recent study we provided evidence that pigweed, an aggressive weed, attenuates this problem exploiting large crystals of calcium oxalate as dynamic carbon pools. This plant is able to photosynthesize even under drought conditions, when stomata are closed and water losses are limited, using carbon dioxide from crystal decomposition instead from the atmosphere. Abscisic acid, an alarm signal that causes stomatal closure seems to be implicated in this function and for this reason we named this path "alarm photosynthesis." The so-far "enigmatic," but highly conserved and widespread among plant species calcium oxalate crystals seem to play a crucial role in the survival of plants.

Calcium oxalate crystals are widespread among animals and plants. In land plants, crystals often reach high amounts, up to 80% of dry biomass. They are formed within specific cells, and their accumulation constitutes a normal activity rather than a pathological symptom, as occurs in animals. Despite their ubiquity, our knowledge on the formation and the possible role(s) of these crystals remains limited. We show that the mesophyll crystals of pigweed (Amaranthus hybridus) exhibit diurnal volume changes with a gradual decrease during daytime and a total recovery during the night. Moreover, stable carbon isotope composition indicated that crystals are of nonatmospheric origin. Stomatal closure (under drought conditions or exogenous application of abscisic acid) was accompanied by crystal decomposition and by increased activity of oxalate oxidase that converts oxalate into CO2 Similar results were also observed under drought stress in Dianthus chinensis, Pelargonium peltatum, and Portulacaria afra Moreover, in A. hybridus, despite closed stomata, the leaf metabolic profiles combined with chlorophyll fluorescence measurements indicated active photosynthetic metabolism. In combination, calcium oxalate crystals in leaves can act as a biochemical reservoir that collects nonatmospheric carbon, mainly during the night. During the day, crystal degradation provides subsidiary carbon for photosynthetic assimilation, especially under drought conditions. This new photosynthetic path, with the suggested name "alarm photosynthesis," seems to provide a number of adaptive advantages, such as water economy, limitation of carbon losses to the atmosphere, and a lower risk of photoinhibition, roles that justify its vast presence in plants., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Imbalances in lipid metabolism affect bone homeostasis, altering bone mass and quality. A link between bone mass and high-density lipoprotein (HDL) has been proposed. Indeed, it has been recently shown that absence of the HDL receptor scavenger receptor class B type I (SR-B1) causes dense bone mediated by increased adrenocorticotropic hormone (ACTH). In the present study we aimed at further expanding the current knowledge as regards the fascinating bone-HDL connection studying bone turnover in apoA-1-deficient mice. Interestingly, we found that bone mass was greatly reduced in the apoA-1-deficient mice compared with their wild-type counterparts. More specifically, static and dynamic histomorphometry showed that the reduced bone mass in apoA-1(-/-) mice reflect decreased bone formation. Biochemical composition and biomechanical properties of ApoA-1(-/-) femora were significantly impaired. Mesenchymal stem cell (MSC) differentiation from the apoA-1(-/-) mice showed reduced osteoblasts, and increased adipocytes, relative to wild type, in identical differentiation conditions. This suggests a shift in MSC subtypes toward adipocyte precursors, a result that is in line with our finding of increased bone marrow adiposity in apoA-1(-/-) mouse femora. Notably, osteoclast differentiation in vitro and osteoclast surface in vivo were unaffected in the knock-out mice. In whole bone marrow, PPARγ was greatly increased, consistent with increased adipocytes and committed precursors. Further, in the apoA-1(-/-) mice marrow, CXCL12 and ANXA2 levels were significantly decreased, whereas CXCR4 were increased, consistent with reduced signaling in a pathway that supports MSC homing and osteoblast generation. In keeping, in the apoA-1(-/-) animals the osteoblast-related factors Runx2, osterix, and Col1a1 were also decreased. The apoA-1(-/-) phenotype also included augmented CEPBa levels, suggesting complex changes in growth and differentiation that deserve further investigation. We conclude that the apoA-1 deficiency generates changes in the bone cell precursor population that increase adipoblast, and decrease osteoblast production resulting in reduced bone mass and impaired bone quality in mice.

Numerous calibration models were developed and tested for the quantitative analysis of collagen and bioapatite in bone using Raman spectroscopy. The ν1 phosphate vibration at 960 cm(-1) was used as indicator of the content of bioapatite while for collagen three markers were used: the C-H2 band at 2940 cm(-1) , the amide I band at 1667 cm(-1) and the vibrations of proline and hydroxyproline at 855 and 878 cm(-1) , respectively. Also a calibration model based on the PLS algorithm was developed, too. Validation of the derived calibration models indicated that the model that makes use of the height ratio of the peaks 960/(855+878) exhibits the best accuracy., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the present study, the possible reversal effects of saffron against established aluminum (Al)-toxicity in adult mice, were investigated. Control, Al-treated (50 mg AlCl(3)/kg/day diluted in the drinking water for 5 weeks) and Al+saffron (Al-treatment as previously plus 60 mg saffron extract/kg/day intraperitoneally for the last 6 days), groups of male Balb-c mice were used. We assessed learning/memory, the activity of acetylcholinesterase [AChE, salt-(SS)/detergent-soluble(DS) isoforms], butyrylcholinesterase (BuChE, SS/DS isoforms), monoamine oxidase (MAO-A, MAO-B), the levels of lipid peroxidation (MDA) and reduced glutathione (GSH), in whole brain and cerebellum. Brain Al was determined by atomic absorption spectrometry, while, for the first time, crocetin, the main active metabolite of saffron, was determined in brain after intraperitoneal saffron administration by HPLC. Al intake caused memory impairment, significant decrease of AChE and BuChE activity, activation of brain MAO isoforms but inhibition of cerebellar MAO-B, significant elevation of brain MDA and significant reduction of GSH content. Although saffron extract co-administration had no effect on cognitive performance of mice, it reversed significantly the Al-induced changes in MAO activity and the levels of MDA and GSH. AChE activity was further significantly decreased in cerebral tissues of Al+saffron group. The biochemical changes support the neuroprotective potential of saffron under toxicity., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In several bone deproteination protocols the chemical agent used for protein cleavage is hydrazine. The effect of hydrazine deproteination method on the crystal size and crystallinity of the bone mineral was studied. Bovine bones were subjected to this protocol and the crystal size and crystallinity of the remaining bone mineral were determined by X-ray Diffraction (XRD), by measuring the width at the half of the maximum intensity of the (002) reflection. It was found that hydrazine deproteination induces noteworthy increase of crystal size and crystallinity. The effect was enhanced by increasing hydrazine temperature from 25 to 55°C. Furthermore, infrared spectroscopy revealed that hydrazine facilitates the removal of carbonate and acid phosphate ions from bone mineral. It is proposed that the mechanism of modification of crystal size and crystallinity lies on the removal of these ions thus, resulting in crystal re-organization.

Human Arkadia is a nuclear protein consisted of 989 amino acid residues, with a characteristic RING domain in its C-terminus. The RING domain harbours the E3 ubiquitin ligase activity needed by Arkadia to ubiquitinate its substrates such as negative regulators of TGF-beta signaling. The RING finger domain of Arkadia is a RING-H2 type and its structure and stability is strongly dependent on the presence of two bound Zn(II) ions attached to the protein frame through a defined Cys3-His2-Cys3 motif. In the present paper we transform the RING-H2 type of Arkadia finger domain to nonnative RING sequence, substituting the zinc-binding residues Cys(955) or His(960) to Arginine, through site-directed mutagenesis. The recombinant expression, in Escherichia coli, of the mutants C955R and H960R reveal significant lower yield in respect with the native polypeptide of Arkadia RING-H2 finger domain. In particular, only the C955R mutant exhibits expression yield sufficient for recombinant protein isolation and preliminary studies. Atomic absorption measurements and preliminary NMR data analysis reveal that the C955R point mutation in the RING Finger domain of Arkadia diminishes dramatically the zinc binding affinity, leading to the breakdown of the global structural integrity of the RING construct.

A simple, non-destructive, methodology based on FT-Raman spectroscopy was developed for the quantitative analysis of risperidone in commercially available film-coated tablets. A simple linear regression model was constructed based on standard tablets, prepared using the same manufacturing process as the commercially available. The tablets contained 0.27, 0.54, 1.08, 1.62, 2.16, 3.24 and 4.32 wt% risperidone. The most prominent Raman vibration of the active pharmaceutical ingredient at 1533 cm(-1), recorded using a home-made rotating system, was plotted against concentration. The model was tested on commercial film-coated tablets. The results were compared against those obtained by application of HPLC on the same samples.

Two different nondestructive spectroscopy methods based on near-infrared (NIR) and Fourier transform (FT) Raman spectroscopy were developed for the determination of ticlopidine-hydrochloride (TCL) in pharmaceutical formulations and the results were compared to those obtained by high-performance liquid chromatography (HPLC). An NIR assay was performed by reflectance over the 850-1700 nm region using a partial least squares (PLS) prediction model, while the absolute FT-Raman intensity of TCL's most intense vibration was used for constructing the calibration curve. For both methodologies the spectra were obtained from the as-received film-coated tablets of TCL. The two quantitative techniques were built using five "manual compressed" tablets containing different concentrations and validated by evaluating the calibration model as well as the accuracy and precision. The models were applied to commercial preparations (Ticlid). The results were compared to those obtained from the application of HPLC using the methodology described by "Sanofi Research Department" and were found to be in excellent agreement, proving that NIR, using fiber-optic probes, and FT-Raman spectroscopy can be used for the fast and reliable determination of the major component in pharmaceutical analysis.

Raman spectroscopy was applied for the direct non-destructive analysis of amiodarone hydrochloride (ADH), the active ingredient of the liquid formulation Angoron((R)). The FT-Raman spectra were obtained through the un-broken as-received ampoules of Angoron((R)). Using the most intense vibration of the active pharmaceutical ingredient (API) at 1568cm(-1), a calibration model, based on solutions with known concentrations, was developed. The model was applied to the Raman spectra recorded from three as-purchased commercial formulations of Angoron((R)) having nominal strength of 50mgml(-1) ADH. The average value of the API in these samples was found to be 48.56+/-0.64mgml(-1) while the detection limit of the proposed technique was found to be 2.11mgml(-1). The results were compared to those obtained from the application of HPLC using the methodology described in the European Pharmacopoeia and found to be in excellent agreement. The proposed analytical methodology was also validated by evaluating the linearity of the calibration line as well as its accuracy and precision. The main advantage of Raman spectroscopy over HPLC method during routine analysis is that it is considerably faster and no solvent consuming. Furthermore, Raman spectroscopy is non-destructive for the sample. However, the detection limit for Raman spectroscopy is much higher than the corresponding for the HPLC methodology.

Identification of the crystal phase of an active pharmaceutical ingredient (API) in a pharmaceutical tablet is of outmost importance since different polymorphs exhibit different physicochemical properties. Furthermore, some of the crystal phases are protected by patents. Identification of Risperidone polymorph A in film coated commercial tablets was attempted using IR spectroscopy, Raman spectroscopy and X-ray powder diffraction (XRPD). The stability of this polymorph through time and during the manufacturing process was also examined. The inability of IR and Raman techniques to identify the presence of polymorph A in the tablets, despite their lower detection limits for Risperidone, left the XRPD as the only technique that could be used for identifying the presence of Risperidone A against the other crystal phases in the presence of the excipients. Polymorph A was proved to be stable during the manufacturing process and after a storage period of 2 years.

The capability of FT-Raman spectroscopy for the fast and non-destructive quantitative analysis of liquid formulations was tested and the results were compared to those obtained by HPLC. Diphenhydramine hydrochloride (DPH), the active ingredient of Benadryl, was determined in the presence of the numerous excipients of the elixir. A Raman calibration model was developed by measuring the peak intensities of different standard solutions of DPH vibration at 1003 cm(-1). Application of the calibration model on the peak intensity recorded from the as-received commercially available sample with 2.5 mg ml(-1) DPH nominal value yielded a value of 2.49+/-0.05 mg ml(-1) DPH. The reliability of this method was verified by testing it against the conventionally used HPLC. The results from both methods were in excellent agreement. The main advantage of Raman over HPLC method during routine analysis is that is considerably faster and less solvent consuming. Furthermore, Raman spectroscopy is non-destructive for the sample. On the other hand, the detection limit for Raman spectroscopy is much higher than the corresponding for the HPLC methodology.

Methodologies based on FT-Raman spectroscopy and HPLC were developed for monitoring the stability of lovastatin in the solid state in the presence of gallic acid, a natural antioxidant. A Raman calibration curve was constructed using the area of the strong but overlapping vibration mode of lovastatin at 1645 cm(-1) and of the gallic acid at 1595 cm(-1). Mixtures of the active ingredient with the antioxidant were heated in the presence of atmospheric air up to 120 degrees C. The molar ratios of lovastatin and gallic acid in the artificially oxidized mixtures were determined from their Raman spectra using the calibration curve. The results were compared to those obtained from the application of the HPLC methodology and found to match satisfactorily. The HPLC analysis was based on a reserved-phase Zorbax C(g), 10 microm (4.6mm x 25 cm i.d.) column, using a gradient elution program by varying the proportion of solvent A acetonitrile 100% to solvent B 0.1% v/v phosphoric acid, and a programmable diode array detection at 225 nm. The Raman methodology was simpler and non-destructive for the sample but yielded only molar ratios as opposed to the HPLC technique where the moles of the both ingredients were determined.

In the present study, the effect of mesopore and particle size distributions on the kinetics of dissolution of powdered Pentelic marble was investigated. Powders obtained by grinding marble slabs in an agate mortar with a pestle (1-3.5 min) were found to be non-porous by nitrogen absorption measurements. These powders, upon dissolution under conditions of constant under-saturation, 25 degrees C, pH 8.25, showed that the kinetics in the absence of mesopores depended on the number of active sites on the exposed surface. Thus, powders consisting of smaller particles, having higher specific surface areas, yielded higher rates of dissolution. Powders, however, which were prepared by grinding marble slabs in a cylinder mill for time periods between 2.0 and 30.0 min, and which exhibited considerable mesoporosity, showed the opposite trend. The rates of dissolution measured for these powders in under-saturated solutions increased with increasing mean particle size and decreased with increasing specific surface area. This finding suggested that the presence of mesopores resulted in lower dissolution rates even though the exposed total surface area was larger. Furthermore, the larger the number of mesopores in a powder sample the slower the corresponding dissolution rates in under-saturated solutions.

Dissolution processes play an important role in marble weathering. Investigation of the dissolution kinetics of powdered Pentelic marble, a calcitic natural stone, showed that the rates measured at constant undersaturation decreased with time. It was found that the rate of dissolution decreased sharply to about 50% of the initial value before 5% of the initially suspended powder was dissolved. Past a dissolution extent corresponding to 25% with respect to the suspended solid, the rates of dissolution at the same constant undersaturation reached a stable value corresponding to 20-25% of the initial rate measured. The reduction in the rates of dissolution up to 25% of mass loss was in agreement with the reduction in the total surface area exposed to the solution, suggesting a dependence of the rate on the number of active sites on the surface of the dissolving material. The decrease in the dissolution rates did not depend on the undersaturation of the solution or on the physical characteristics of the particles such as specific surface area, porosity, and particle size distribution. Equal mass losses, with respect to the initially suspended solid, of different samples exhibited the same relative decrease in rate regardless of the conditions of the aqueous solutions.

Dissolution of natural stone such as marble is not limited to its surface. The porous structure, known to play an important role in stone decay, is also affected by the conditions of dissolution. In the present work, the changes in pore size distribution of Pentelic marble particles accompanying chemical dissolution in undersaturated solutions and at alkaline pH 8.25 were investigated. The specific surface area and the mesopore distribution of the Pentelic marble tested showed a pronounced decrease to very low values. On the other hand, the sizes of macropores exhibited a tendency to increase with the extent of dissolution due either to dissolution in the interior of the pores or to fusion of small pores into larger. Furthermore, the number of small particles decreased significantly, reaching complete disappearance, depending on the extent of dissolution. At the same time, the relative number of particles of intermediate size increased. Copyright 2001 Academic Press.