Label-Free Identification of Exosomes using Raman Spectroscopy and Machine Learning.

Exosomes, nano-sized extracellular vesicles (EVs) secreted from cells, carry various cargo molecules reflecting their cells of origin. As EV content, structure, and size are highly heterogeneous, their classification via cargo molecules by determining their origin is challenging. Here, a method is presented combining surface-enhanced Raman spectroscopy (SERS) with machine learning algorithms to employ the classification of EVs derived from five different cell lines to reveal their cellular origins. Using an artificial neural network algorithm, it is shown that the label-free Raman spectroscopy method's prediction ratio correlates with the ratio of HT-1080 exosomes in the mixture. This machine learning-assisted SERS method enables a new direction through label-free investigation of EV preparations by differentiating cancer cell-derived exosomes from those of healthy. This approach will potentially open up new avenues of research for early detection and monitoring of various diseases, including cancer.

Time-course analysis of salicylic acid effects on ROS regulation and antioxidant defense in roots of hulled and hulless barley under combined stress of drought, heat and salinity.

Greater crop losses can result from simultaneous exposure to a combination of drought, heat and salinity in the field. Salicylic acid (SA), a phenolic phytohormone, can affect a range of physiological and biochemical processes in plants and significantly impacts their resistance to these abiotic stresses. Despite numerous reports involving the positive effects of SA by applying each abiotic stress separately, the mechanism of SA-mediated adaptation to combined stresses remains elusive. This study, via a time-course analysis, investigated the role of SA on the roots of hulled and hulless (naked) barley (Hordeum vulgare L. 'Tarm' and 'Özen', respectively), which differed in salt tolerance, under the combined stress of drought, heat and salt. The combined stress caused marked reductions in root length and increases in proline content in both genotypes; however, Tarm exhibited better adaptation to the triple stress. Under the first 24 h of stress, superoxide dismutase (SOD; EC.1.15.1.1) and peroxidase (POX; EC.1.11.1.7) activity in the Tarm roots increased remarkably, while decreasing in the Özen roots. Furthermore, the Tarm roots showed higher catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.11) and glutathione reductase (GR; EC 1.6.4.2) activity than the Özen during the combined stresses. The sensitivity of hulless barley roots may be related to decreasing SOD, POX, CAT and GR activity under stress. Over 72 h of stress, the SA pretreatment improved the APX and GR activity in Tarm and that of POX and CAT in Özen, demonstrating that exogenously applied SA regulates antioxidant defense enzymes in order to detoxify reactive oxygen species. The results of this study suggest that SA treatment may improve the triple-stress combination tolerance in hulled and hulless barley cultivars by increasing the level of antioxidant enzyme activity and promoting the accumulation of proline. Thus, SA alleviated the damaging effects of the triple stress by improving the antioxidant system, although these effects differed depending on characteristic of the hull of the grain.

Exogenous N-Acetylcysteine alleviates heavy metal stress by promoting phenolic acids to support antioxidant defence systems in wheat roots.

N-acetylcysteine (N-Acetyl L-cysteine, NAC) is a thiol compound derived from the addition of the acetyl group to cysteine amino acid. NAC has been used as an antioxidant, free radical scavenger, and chelating agent for reducing the deleterious effects on plants of biotic and abiotic environmental stresses. It can also relieve heavy metal (HM) toxicity, although its alleviating mechanism remains unknown. In this study, we compared HM-stressed (Cu, Hg, Cd and Pb, 100 µM each) wheat seedlings without NAC treatment and in combination with NAC (1 mM). In comparison to HMs alone, NAC treatment in combination with HMs (Cu, Cd, Hg and Pb, respectively) stimulated root growth (1.1-, 1.5-, 10.5- and 1.9-fold), and significantly increased fresh (1.3-, 1.5-, 4.3- and 1.4-fold) and dry (1.2-, 1.5-, 2.5- and 1.2-fold) mass. Combination treatment also led to significant reductions in HM concentrations (1.3-, 1.4-, 4- and 1.1-fold, respectively). GSH (1.1 - 1.8-fold), TBARS (1.4 - 2.7-fold) and H2O2 (1.6 - 1.8-fold) contents in treatment with HMs alone were significantly mitigated by the NAC combination. Some of the antioxidant enzyme activities increased or reduced by some HM treatments alone were stimulated by a combination of NAC with HMs, or remained unchanged or changed only insignificantly, supported by the phenolic pool of the plant. Ferulic, p-comaric and syringic acids were the major phenolic acids (PAs) in the roots in free, ester, glycoside and ester-bound forms, and their concentrations were increased by HM treatments alone, in comparison to the control seedlings, while PAs concentrations were relatively reduced by NAC in combination with HMs. These results indicate that NAC can alleviate HM toxicity and improve the growth of HM-stressed wheat seedlings by coordinated induction of the phenolic pool and the antioxidant defence system.

Nutrient profiles of the hybrid grape cultivar 'Isabel' during berry maturation and ripening.

BACKGROUND: The hybrid grape cultivar 'Isabel' (Vitis labrusca × V. vinifera) is widely grown in the Black Sea region of Turkey and constitutes an important part of the diet of its inhabitants. Phenological and ampelographic studies of the grape have previously been performed, but there are no data concerning nutrient composition. Its nutrient content needs to be investigated to ascertain its potential economic and nutritional uses in the region. RESULTS: The concentrations (range, g kg-1 fresh weight) of major soluble sugars (fructose 2.08-79.70; glucose 3.71-66.74) increased and those of organic acids (tartaric acid 0.05-6.60; malic acid 2.38-9.92) decreased in the skin and pulp during ripening. The major fatty acid was linoleic acid in the skin (50.13), pulp (18.11) and seed (704), while the main minerals (dry weight) were potassium (2.70-19.70), followed by phosphorus (0.53-3.40) and calcium (0.42-5.90) in the parts. CONCLUSION: Berry maturation and ripening significantly influenced the concentrations of sugars and organic acids the most in the skin and pulp, but did not significantly affect the concentrations of fatty acids and minerals in the 'Isabel' grape. Our findings also suggest an optimal harvest time in terms of levels of the nutrients. © 2016 Society of Chemical Industry.

Salicylic acid alleviates the effects of cadmium and drought stress by regulating water status, ions, and antioxidant defense in Pterocarya fraxinifolia.

Introduction: Pterocarya fraxinifolia (Poiret) Spach (Caucasian wingnut, Juglandaceae) is a relict tree species, and little is known about its tolerance to abiotic stress factors, including drought stress and heavy metal toxicity. In addition, salicylic acid (SA) has been shown to have a pivotal role in plant responses to biotic and abiotic stresses. Methods: The current study is focused on evaluating the impact of foliar application of SA in mediating Caucasian wingnut physiological and biochemical responses, including growth, relative water content (RWC), osmotic potential (Ψs), quantum yield (Fv/Fm), electrolyte leakage, lipid peroxidation, hydrogen peroxide, and antioxidant enzymes, to cadmium (Cd; 100 µM) and drought stress, as well as their interaction. Moreover, the antioxidant activity (e.g., ascorbate peroxidase, catalase, glutathione reductase, peroxidase, and superoxide dismutase activities) of the stressed trees was investigated. The study was conducted on 6-month-old seedlings under controlled environmental conditions in a greenhouse for 3 weeks. Results and discussion: Leaf length, RWC, Ψs, and Fv/Fm were decreased under all treatments, although the effect of drought stress was the most pronounced. An efficient antioxidant defense mechanism was detected in Caucasian wingnut. Moreover, SA-treated Caucasian wingnut plants had lower lipid peroxidation, as one of the indicators of oxidative stress, when compared to non-SA-treated groups, suggesting the tolerance of this plant to Cd stress, drought stress, and their combination. Cadmium and drought stress also changed the ion concentrations in Caucasian wingnut, causing excessive accumulation of Cd in leaves. These results highlight the beneficial function of SA in reducing the negative effects of Cd and drought stress on Caucasian wingnut plants.

The Effects of Exogenous Salicylic Acid on Endogenous Phytohormone Status in Hordeum vulgare L. under Salt Stress.

Acclimation to salt stress in plants is regulated by complex signaling pathways involving endogenous phytohormones. The signaling role of salicylic acid (SA) in regulating crosstalk between endogenous plant growth regulators' levels was investigated in barley (Hordeum vulgare L. 'Ince'; 2n = 14) leaves and roots under salt stress. Salinity (150 and 300 mM NaCl) markedly reduced leaf relative water content (RWC), growth parameters, and leaf water potential (LWP), but increased proline levels in both vegetative organs. Exogenous SA treatment did not significantly affect salt-induced negative effects on RWC, LWP, and growth parameters but increased the leaf proline content of plants under 150 mM salt stress by 23.1%, suggesting that SA enhances the accumulation of proline, which acts as a compatible solute that helps preserve the leaf's water status under salt stress. Changes in endogenous phytohormone levels were also investigated to identify agents that may be involved in responses to increased salinity and exogenous SA. Salt stress strongly affected endogenous cytokinin (CK) levels in both vegetative organs, increasing the concentrations of CK free bases, ribosides, and nucleotides. Indole-3-acetic acid (IAA, auxin) levels were largely unaffected by salinity alone, especially in barley leaves, but SA strongly increased IAA levels in leaves at high salt concentration and suppressed salinity-induced reductions in IAA levels in roots. Salt stress also significantly increased abscisic acid (ABA) and ethylene levels; the magnitude of this increase was reduced by treatment with exogenous SA. Both salinity and SA treatment reduced jasmonic acid (JA) levels at 300 mM NaCl but had little effect at 150 mM NaCl, especially in leaves. These results indicate that under high salinity, SA has antagonistic effects on levels of ABA, JA, ethylene, and most CKs, as well as basic morphological and physiological parameters, but has a synergistic effect on IAA, which was well exhibited by principal component analysis (PCA).

Exogenous melatonin ameliorates ionizing radiation-induced damage by modulating growth, osmotic adjustment and photosynthetic capacity in wheat seedlings.

As a multifunctional signal molecule, melatonin (N-acetyl-5-methoxytryptamine) plays many important roles in the regulation of plant growth and development. The effect of melatonin application on enhancing plant stress tolerance has been widely reported, but the ameliorative effect of exogenous melatonin treatment on plants exposed to ionization stress is still unknown. This study investigated the ameliorative effects of two types of melatonin treatment, pre-sowing priming (prMel) and application during growth (ptMel), in wheat (Triticum aestivum L.) seedlings exposed to different radiation doses (100, 200, 300 and 400 Gy) of radioactive cobalt (60Co) gamma rays as dry seeds. The growth parameters, photosynthetic pigments, chlorophyll fluorescence, osmotic potential with soluble sugars, fructans and proline contents were then examined. The results indicated that high doses of ionizing radiation (IR) led to decreases in plant growth, pigment contents, chlorophyll fluorescence ratios and osmotic potential. However, soluble sugar, fructan and proline contents increased under IR stress conditions. Both melatonin applications, but particularly prMel, enhanced the morphological parameters, preserved the photosynthetic machinery and regulated the osmotic adjustment of IR-stressed wheat seedlings. Taken together, the findings show that exogenously applied melatonin, particularly prMel, play a significant role in alleviating IR stress in wheat seedlings.

Fatty acid and amino acid compositions of selected wild-edible mushrooms consumed in Turkey.

The fatty acid and amino acid compositions of 11 mushroom species commonly consumed were collected from the East Black Sea region of Turkey and analyzed. All species were characterized by a high content of linoleic acid (C18:2n-6) and glutamic acid. The highest content of linoleic acid (78.0%) and glutamic acid (29.4 µg/mg dry weight [d.w.]) was found in Agaricus arvensis and the lowest in Cantharellus tubaeformis, 19.8% and 10.9 µg/mg d.w., respectively. The average content of amino acids for all species was 148 µg/mg d.w. Overall, these results demonstrate that the 11 different kinds of wild edible mushrooms gathered from the region represent substantial sources of fatty acids and amino acids that are essential in the diet of humans. Quality of the mushroom protein compares favorably with the FAO/WHO Standard. The present study demonstrates that macrofungi from the East Black Sea region (Turkey) are a good source of many nutrients essential to human well-being.

Genetic Algorithm-Driven Surface-Enhanced Raman Spectroscopy Substrate Optimization.

Surface-enhanced Raman spectroscopy (SERS) is a highly sensitive and molecule-specific detection technique that uses surface plasmon resonances to enhance Raman scattering from analytes. In SERS system design, the substrates must have minimal or no background at the incident laser wavelength and large Raman signal enhancement via plasmonic confinement and grating modes over large areas (i.e., squared millimeters). These requirements impose many competing design constraints that make exhaustive parametric computational optimization of SERS substrates prohibitively time consuming. Here, we demonstrate a genetic-algorithm (GA)-based optimization method for SERS substrates to achieve strong electric field localization over wide areas for reconfigurable and programmable photonic SERS sensors. We analyzed the GA parameters and tuned them for SERS substrate optimization in detail. We experimentally validated the model results by fabricating the predicted nanostructures using electron beam lithography. The experimental Raman spectrum signal enhancements of the optimized SERS substrates validated the model predictions and enabled the generation of a detailed Raman profile of methylene blue fluorescence dye. The GA and its optimization shown here could pave the way for photonic chips and components with arbitrary design constraints, wavelength bands, and performance targets.

The protective effect of exogenous salicylic and gallic acids ameliorates the adverse effects of ionizing radiation stress in wheat seedlings by modulating the antioxidant defence system.

Plant growth regulatory substances play a significant role in maintaining developmental and physiological processes in plants under abiotic stress. Apart from traditional plant hormones, the phenolic acids, salicylic acid (SA) and gallic acid (GaA), are emerging players with pivotal roles in alleviating various environmental perturbations. The present study compared the stress alleviation effect of these two phenolic acids in wheat (Triticum aestivum L. 'Gönen-98') seedling whose seeds were used in this study pre-treated with increasing doses of gamma irradiation (IR, 100 > 400 Gy). Leaves from seedlings hydroponically grown for 10 days in medium containing 100 µmol/l SA and GaA were used in the measurements and determinations. Accordingly, exogenous treatment with SA and GaA significantly improved plant growth and photosynthetic activity and regulated stress-induced osmolyte accumulation against γ-irradiation. Treatments also led to significant reductions in TBARS and H2O2 contents. Antioxidant enzyme activities were further stimulated by SA and GaA treatment in comparison to IR alone. The phenolic pool including phenolic acids and GSH content in whole seedlings were promoted by IR and further SA and GaA applications. Contents in phenolic acids liberated from soluble free, soluble ester-conjugated and soluble glycoside-conjugated SA and GaA contents in roots and leaves increased following SA and GaA treatments alone in comparison to the control and IR groups. The present results indicate that SA and GaA can alleviate the ameliorative effects of IR, leading to further oxidative stress, and can improve the tolerance of stressed wheat seedlings by stimulating enzymatic and non-enzymatic antioxidant defence system components.

Timing-dependent effects of salicylic acid treatment on phytohormonal changes, ROS regulation, and antioxidant defense in salinized barley (Hordeum vulgare L.).

Cross-talk between exogenous salicylic acid (SA) and endogenous phytohormone pathways affects the antioxidant defense system and its response to salt stress. The study presented here investigated the effects of SA treatment before and during salt stress on the levels of endogenous plant growth regulators in three barley cultivars with different salinity tolerances: Hordeum vulgare L. cvs. Akhisar (sensitive), Erginel (moderate), and Kalayci (tolerant). The cultivars' relative leaf water contents, growth parameters, proline contents, chlorophyll a/b ratios, and lipid peroxidation levels were measured, along with the activities of enzymes involved in detoxifying reactive oxygen species (ROS) including superoxide-dismutase, peroxidase, catalase, ascorbate-peroxidase, and glutathione-reductase. In addition, levels of several endogenous phytohormones (indole-3-acetic-acid, cytokinins, abscisic acid, jasmonic acid, and ethylene) were measured. Barley is known to be more salt tolerant than related plant species. Accordingly, none of the studied cultivars exhibited changes in membrane lipid peroxidation under salt stress. However, they responded differently to salt-stress with respect to their accumulation of phytohormones and antioxidant enzyme activity. The strongest and weakest increases in ABA and proline accumulation were observed in Kalayci and Akhisar, respectively, suggesting that salt-stress was more effectively managed in Kalayci. The effects of exogenous SA treatment depended on both the timing of the treatment and the cultivar to which it was applied. In general, however, where SA helped mitigate salt stress, it appeared to do so by increasing ROS scavenging capacity and antioxidant enzyme activity. SA treatment also induced changes in phytohormone levels, presumably as a consequence of SA-phytohormone salt-stress cross-talk.

Nutrient content of carob pod (Ceratonia siliqua L.) flour prepared commercially and domestically.

Although the fruit of the carob tree (Ceratonia siliqua L. Fabaceae) is nutritious and widely available in Turkey, especially in West and South Anatolia, much remains to be learned about its nutrient composition. The main goal of our study was to determine if there are differences in the content of certain nutrients in commercially-prepared carob flour (CPCP) and domestic or home-prepared carob powder (HPCP). Sucrose was the main sugar in CPCP and HPCP. Total protein was 40% lower in CPCP than HPCP due mainly to decreases in the content of several essential amino acids. However, except for lysine in CPCP, HPCP and CPCP compared favourably to a WHO protein standard. There were large differences in terms of their content of the two essential fatty acids, linoleic and alpha-linolenic acid, and the linoleic acid/alpha-linolenic acid ratio was 3.6 for CPCP, and 6.1 for HPCP. Manganese and iron were 2.5-fold higher in HPCP than CPCP. This study demonstrates that carob flour prepared in either the household or industrially is a good source of many, but not all essential nutrients, and that commercial processing of carob fruit into flour seems to affect its content of several important nutrients.

Bog bilberry phenolics, antioxidant capacity and nutrient profile.

Phenolics and nutrient profiles of bog bilberry (Vaccinium uliginosum L.) collected from high mountain pastures in northeast Anatolia (Turkey) were examined for the first time in this study. The major soluble sugar identified in the berry was fructose, following by glucose, and the main organic acid identified was citric acid, followed by malic acid. Eleven phenolic acids and 17 anthocyanin 3-glycosides were identified and quantified. Caffeic acid in the free and glycoside forms and syringic acid in the ester form were the major phenolic acids, and the major individual anthocyanin present in the berry was malvidin 3-glucoside (24%). The highest total phenolics and anthocyanin contents were obtained from the anthocyanin fraction in conjunction with the highest antioxidant capacity, followed by the polyphenolic and aqueous fractions, FRAP, ORAC and DPPH, in that order. Our findings can be used to compare bog bilberry with other Vaccinium berries and to help clarify the relative potential health benefits of different berries.