Heavy Metals Content in Selected Medicinal Plants Produced and Consumed in Serbia and Their Daily Intake in Herbal Infusions.

The heavy metals content (HMs) was investigated in 14 different medicinal plants collected from the three regions in Central Serbia, Zlatar, Sokobanja, and Kopaonik. The concentrations of Cd, Cr, Ni, Hg and Pb were determined: Cd (<0.03-2.72 mg/kg); Cr (<0.08-12.1 mg/kg); Ni (<0.08-12.2 mg/kg); Pb (0.6-49.0 mg/kg); the Hg concentration was below the detection limit of 0.06 mg/kg in all samples. The daily intake of HMs due to ingestion of 200 mL of herbal infusion was in all cases below the recommended limit prescribed by the World Health Organization. The estimated daily intake values were below the values for the oral reference dose regulated by the U.S. Environmental Protection Agency (USA EPA). The target hazard quotient and hazard index for Cd, Cr Ni, and Pb were below 1. Nevertheless, due to the tendency of heavy metals to accumulate in the organism, attention should be paid to the daily intake of herbal infusion during long-term usage. Specifically, it is recommended to consume not more than one cup (200 mL) of infusion per day made from thyme (Mt. Zlatar) and blueberry (Mt. Kopaonik), and not more than two cups per day for other herbs.

An Insight into an Olive Scab on the "Istrska Belica" Variety: Host-Pathogen Interactions and Phyllosphere Mycobiome.

The olive tree is one of the most important agricultural plants, affected by several pests and diseases that cause a severe decline in health status leading to crop losses. Olive leaf spot disease caused by the fungus Venturia oleaginea can result in complete tree defoliation and consequently lower yield. The aim of the study was to obtain new knowledge related to plant-pathogen interaction, reveal mechanisms of plant defense against the pathogen, and characterize fungal phyllosphere communities on infected and symptomless leaves that could contribute to the development of new plant breeding strategies and identification of novel biocontrol agents. The highly susceptible olive variety "Istrska Belica"' was selected for a detailed evaluation. Microscopy analyses led to the observation of raphides in the mesophyll and parenchyma cells of infected leaves and gave new insight into the complex V. oleaginea pathogenesis. Culturable and total phyllosphere mycobiota, obtained via metabarcoding approach, highlighted Didymella, Aureobasidium, Cladosporium, and Alternaria species as overlapping between infected and symptomless leaves. Only Venturia and Erythrobasidium in infected and Cladosporium in symptomless samples with higher abundance showed statistically significant differences. Based on the ecological role of identified taxa, it can be suggested that Cladosporium species might have potential antagonistic effects on V. oleaginea.

Small regions as key sources of traditional knowledge: a quantitative ethnobotanical survey in the central Balkans.

BACKGROUND: Starting from the idea that unexplored areas may yield new and different ethnobotanical information, we performed a survey of traditional uses of plants in two neighboring districts situated in east Serbia (Bor and Aleksinac), both lacking in previous ethnobotanical reports, but characterized by an interesting history and culture, together with some specific features. In this study, we hypothesized that such small and specific areas could be of high ethnobotanical importance. METHODS: Semi-structured interviews were used with 155 informants. Relative cultural importance (RCI) indices, such as the frequency of citation (FC), relative frequency of citation (RFC), relative importance index (RI), informant consensus factor (ICF-FIC), use value (UV), fidelity level (FL) and Jaccard index (JI), were calculated, and principal coordinate analysis (PCoA) was performed. RESULTS: In this study, 2333 use-reports and 114 plants were recorded. Of the 101 medical herbs, 33 are included in the European Pharmacopoeia Edition 8.0. The most frequently used mode of preparation was as an infusion (50.0%), while leaf (44.7%) was the most used plant part. The highest FC and RFC values were recorded for Hypericum perforatum L. (13.1 and 0.2, respectively), while the highest RI was documented for Urtica dioica L. (1.0). ICF and FL indices showed important differences among selected groups of informants. The PCoA showed three homogeneous plant groups. Plants were mostly used for the treatment of digestive (49.1%), circulatory (41.2%) and respiratory system disorders (35.1%). Thirty-seven (32.5%) herbs were used for human nutrition, 14 (12.3%) in veterinary medicine, 17 (14.9%) in rituals and ethnoculture, while 24 (21.0%) for miscellaneous purposes. The highest degree of similarity was determined with studies conducted in close proximity. Four species are new to Balkan ethnobotany. New uses for some well-known plants are highlighted. CONCLUSION: The study indicated that small and specific areas in the Balkans may be an important reservoir of ethnobotanical knowledge.

New perspectives of purple starthistle (Centaurea calcitrapa) leaf extracts: phytochemical analysis, cytotoxicity and antimicrobial activity.

Ethnobotanical and ethnopharmacological studies of many Centaurea species indicated their potential in folk medicine so far. However, investigations of different Centaurea calcitrapa L. extracts in terms of cytotoxicity and antimicrobial activity against phytopathogens are generally scarce. The phenolic profile and broad antimicrobial activity (especially towards bacterial phytopathogens) of methanol (MeOH), 70% ethanol (EtOH), ethyl-acetate (EtOAc), 50% acetone (Me2CO) and dichloromethane: methanol (DCM: MeOH, 1: 1) extracts of C. calcitrapa leaves and their potential toxicity on MRC-5 cell line were investigated for the first time. A total of 55 phenolic compounds were identified: 30 phenolic acids and their derivatives, 25 flavonoid glycosides and aglycones. This is also the first report of the presence of centaureidin, jaceidin, kaempferide, nepetin, flavonoid glycosides, phenolic acids and their esters in C. calcitrapa extracts. The best results were obtained with EtOAc extract with lowest MIC values expressed in µg/mL ranging from 13 to 25, while methicillin resistant Staphylococcus aureus was the most susceptible strain. The most susceptible phytopathogens were Pseudomonas syringae pv. syringae, Xanthomonas campestris pv. campestris and Agrobacterium tumefaciens. The highest cytotoxicity was recorded for EtOAc and Me2CO extracts with the lowest relative and absolute IC50 values between 88 and 102 µg/mL, while EtOH extract was the least toxic with predicted relative IC50 value of 1578 µg/mL. Our results indicate that all tested extracts at concentration considered as non-toxic can be one of great importance in combat towards phytopathogenic and human pathogenic strains, as well as natural sources of antimicrobials.

Activity concentrations of 137Cs, 40K, and 210Pb radionuclides in selected medicinal herbs from Central Serbia and their effective dose due to ingestion.

Specific activity of 137Cs, 40K, and 210Pb radionuclides in fifteen selected medicinal herbs from three locations in Central Serbia (two mountains, Kopaonik and Zlatar, and a valley, Sokobanja) was measured using two semiconductor HPGe spectrometer systems. The obtained values are in intervals (<0.3 ÷ 9.7) Bq/kg, (<0.2 ÷ 24.7) Bq/kg, and (<0.2 ÷ 5.7) Bq/kg for 137Cs; in intervals (125 ÷ 1100) Bq/kg, (104 ÷ 872) Bq/kg, and (103 ÷ 954) Bq/kg for 40K, and in intervals (3.6 ÷ 49.0) Bq/kg, (3.9 ÷ 57.9) Bq/kg, and (2.8 ÷ 103) Bq/kg for 210Pb, for herbs from Kopaonik, Sokobanja and Zlatar, respectively. The highest activity measured in individual herbs was: 24.7 Bq/kg for 137Cs (Sokobanja valley), 1100 Bq/kg for 40K (Mt. Kopaonik) and 103 Bq/kg for 210Pb (Mt. Zlatar). The corresponding individual annual effective doses due to ingestion calculated from the measured activity concentrations, of the radionuclides are: in intervals (<1.7 ÷ 82.9) nSv, (<1.4 ÷ 211) nSv, and (<1.1 ÷ 48.7) nSv for 137Cs; in intervals (0.76 ÷ 4.5) µSv, (0.64 ÷ 4.4) µSv, and (0.63 ÷ 4.9) µSv for 40K, and in intervals (1.1 ÷ 18.2) µSv, (1.3 ÷ 21.6) µSv, (0.9 ÷ 38.3) µSv for 210Pb, respectively. The specific activity concentration values of 137Cs and 40K obtained for the investigated herbs are similar to the literature data, while no information was found in literature about specific activity concentration of 210Pb. The all values obtained for individual annual effective dose due to ingestion are less than 100 µSv, which means that the daily use of 200 mL of herbal infusion during a year made from the investigated herbs does not represent a radiological risk for health. However, a high individual annual effective dose of ingestion of 210Pb obtained for some herbs indicate that their consumption in larger amounts 2-3 cups of infusion daily over a year could make the dose to exceed the recommended level, and points to necessity for extensive investigation of 210Pb activity concentration worldwide.

Frankincense and myrrh essential oils and burn incense fume against micro-inhabitants of sacral ambients. Wisdom of the ancients?

ETHNOPHARMACOLOGICAL RELEVANCE: Essential oils obtained from resins of Boswellia carteri Birdw. and Commiphora myrrha (Nees) Engl., commonly known as frankincense and true myrrh respectively, have been used extensively since 2800 BCE for the treatment of skin sores, wounds, teeth, inflammation, and urinary tract diseases in traditional medicine; for preparation of mummification balms and unguents; and also as incense and perfumes. Since ancient times, burning of frankincense and myrrh in places of worship for spiritual purposes and contemplation (a ubiquitous practice across various religions) had hygienic functions, to refine the smell and reduce contagion by purifying the indoor air. AIM OF THE STUDY: The general purpose of the study was to assess the in vitro antimicrobial potential of the liquid and vapour phases of B. carteri and C. myrrha essential oils and burn incense, as well as to test the effectiveness of their in situ application to cleanse microbially-contaminated air within the ambient of an investigated 17th-century church. MATERIALS AND METHODS: The chemical composition of B. carteri and C. myrrha essential oils, obtained by hydrodistillation of frankincense and true myrrh oleo gum resins was determined using GC/MS, and antimicrobial properties of their liquid and vapour phases were assessed by the broth microdilution and microatmosphere diffusion methods. Chemical analysis of burn incense fume obtained using bottle gas washing with dichloromethane as a solvent was performed by GC/MS, while its antimicrobial activity was evaluated using a modified microatmosphere diffusion method to evaluate germination inhibition for fungi and CFU count reduction for bacteria. The in situ antimicrobial activity of B. carteri burn incense and essential oil vapour phase was assessed in the sealed nave and diaconicon of the church, respectively. RESULTS: The dominant compounds of B. carteri EO were α-pinene (38.41%) and myrcene (15.21%), while C. myrrha EO was characterized by high content of furanoeudesma-1,3-diene (17.65%), followed by curzerene (12.97%), ß-elemene (12.70%), and germacrene B (12.15%). Burn incense fume and soot had α-pinene (68.6%) and incensole (28.6%) as the most dominant compounds, respectively. In vitro antimicrobial assays demonstrated high bacterial and fungal sensitivity to the liquid and vapour phases of EOs, and burn incense fume. In situ application of B. carteri EO vapour and incense fume resulted in reduction of air-borne viable microbial counts by up to 45.39 ±â€¯2.83% for fungi and 67.56 ±â€¯3.12% for bacteria (EO); and by up to 80.43 ±â€¯2.07% for fungi and 91.43 ±â€¯1.26% for bacteria (incense fume). CONCLUSIONS: The antimicrobial properties of essential oil derived from frankincense, a compound with well-known traditional use, showed that it possesses a clear potential as a natural antimicrobial agent. Moreover, the results suggest possible application of B. carteri EO vapour and incense fume as occasional air purifiers in sacral ambients, apart from daily church rituals.

Scanning Electron Microscopy Coupled with Energy Dispersive Spectrometric Analysis Reveals for the First Time Weddellite and Sylvite Crystals on the Surface of Involucral Bracts and Petals of two Xeranthemum L. (Compositae) Species.

In this work, weddellite and sylvite crystals are identified for the first time on the involucral bracts and petals of Xeranthemum annuum and Xeranthemum cylindraceum using scanning electron microscopy coupled with energy dispersive spectrometric (SEM-EDS) analysis. Well-developed crystals of weddellite (CaC2O4·2H2O) occur in the form of a tetragonal bipyramid (hhl), rarely in combination of a bipyramid and tetragonal prism (h00). Indumentum of involucral bracts of X. cylindraceum consists of nonglandular and glandular trichomes. Sylvite (KCl) crystals are observed only on the petal surface of X. cylindraceum. The crystals of sylvite occur in the form of perfect cubes (hexahedrons), but some crystals are deformed, i.e., partially elongated. Taxonomic significance of investigated microcharacters as well as the use of SEM-EDS analysis in taxonomic studies of plants are discussed.

Blind color decomposition of histological images.

Cancer diagnosis is based on visual examination under a microscope of tissue sections from biopsies. But whereas pathologists rely on tissue stains to identify morphological features, automated tissue recognition using color is fraught with problems that stem from image intensity variations due to variations in tissue preparation, variations in spectral signatures of the stained tissue, spectral overlap and spatial aliasing in acquisition, and noise at image acquisition. We present a blind method for color decomposition of histological images. The method decouples intensity from color information and bases the decomposition only on the tissue absorption characteristics of each stain. By modeling the charge-coupled device sensor noise, we improve the method accuracy. We extend current linear decomposition methods to include stained tissues where one spectral signature cannot be separated from all combinations of the other tissues' spectral signatures. We demonstrate both qualitatively and quantitatively that our method results in more accurate decompositions than methods based on non-negative matrix factorization and independent component analysis. The result is one density map for each stained tissue type that classifies portions of pixels into the correct stained tissue allowing accurate identification of morphological features that may be linked to cancer.

Visualising individual sequence-specific protein-DNA interactions in situ.

Gene expression - a key feature for modulating cell fate-is regulated in part by histone modifications, which modulate accessibility of the chromatin to transcription factors. Until now, protein-DNA interactions (PDIs) have mostly been studied in bulk without retrieving spatial information from the sample or with poor sequence resolution. New tools are needed to reveal proteins interacting with specific DNA sequences in situ for further understanding of the orchestration of transcriptional control within the nucleus. We present herein an approach to visualise individual PDIs within cells, based on the in situ proximity ligation assay (PLA). This assay, previously used for the detection of protein-protein interactions in situ, was adapted for analysis of target PDIs, using padlock probes to identify unique DNA sequences in complex genomes. As a proof-of-principle we detected histone H3 interacting with a 26 bp consensus sequence of the Alu-repeat abundantly expressed in the human genome, but absent in mice. However, the mouse genome contains a highly similar sequence, providing a model system to analyse the selectivity of the developed methods. Although efficiency of detection currently is limiting, we conclude that in situ PLA can be used to achieve a highly selective analysis of PDIs in single cells.

Automated classification of multicolored rolling circle products in dual-channel wide-field fluorescence microscopy.

Specific single-molecule detection opens new possibilities in genomics and proteomics, and automated image analysis is needed for accurate quantification. This work presents image analysis methods for the detection and classification of single molecules and single-molecule interactions detected using padlock probes or proximity ligation. We use simple, widespread, and cost-efficient wide-field microscopy and increase detection multiplexity by labeling detection events with combinations of fluorescence dyes. The mathematical model presented herein can classify the resulting point-like signals in dual-channel images by spectral angles without discriminating between low and high intensity. We evaluate the methods on experiments with known signal classes and compare to classical classification algorithms based on intensity thresholding. We also demonstrate how the methods can be used as tools to evaluate biochemical protocols by measuring detection probe quality and accuracy. Finally, the method is used to evaluate single-molecule detection events in situ.

[Surgical techniques and outcomes in the treatment of malignant tongue base tumors].

INTRODUCTION: Prognosis of patients with malignant tongue base tumors is poor. Survival is low, in spite of different treatment modalities. Most patients seek treatment too late, when their disease has already progressed to stage III or IV. The aim of this investigation was to compare different treatment modalities in patients with malignant tongue base tumors. MATERIAL AND METHODS: We have analyzed a total of 82 patients (72 men and 6 women) treated at the ENT Clinic, Clinical Center Novi Sad, between 1992 and 2004. The average age of our patients was 59 years. In regard to lifestyle habits, out of 82 patients, 67 were smokers and 57 were alcohol users (16 of the latter were treated alcoholics). The majority (54/82) of patients were both smokers and alcohol users. RESULTS: There were 6% of patients (5/82) with stage I disease, 15% ofJ patients (12/82) with stage 11, 24% of patients (20/82) with stage III and 55% of patient (45/82) with stage IV disease. Planocellular cancer was diagnosed in 79/82 patients, and the remaining (3182) had transitional cell carcinoma. Surgical treatment alone was performed in 17 patients, seven were treated with radiation only, and nine only with chemotherapy. Combined surgical and radiation therapy was performed in 28 patients, and 5 were treated with all three. Ten patients were not treated with any therapy. Tongue base resection only was performed in 12 patients, tongue base resection with epiglottectomy in 20, tongue base resection with supraglottic laryngectomy in 13 and tongue base resection with total laryngectomy in 5 patients. In radiation only cases, 25% of patients survived 20 months, whereas in surger) only cases, 25% of patients survived 27 months. Five-year survival after combined surgical and radiation therapy was 35% CONCLUSION: Development of tongue base carcinoma is strongly associated with alcohol and tobacco consumption. Survival is low, despite various treatment modalities. However, combined therapy is the therapy of choice in cases with tongue base carcinoma.