Automated image analysis and hyperspectral imagery with enhanced dark field microscopy applied to biochars produced at different temperatures.

Biochar from agricultural biomasses and solid wastes represents a win-win solution for a rational waste management. Its sustainable usage requires the identification and standardization of biochar characteristics. The aim of this work was to identify the physical-chemical and spatial characteristics of biochars from pruning residues (PR), poultry litter (PL), and anaerobic cattle digestate (CD) at two pyrolysis temperatures (350 °C and 550 °C). The biochar characterization was carried out by applying emerging imaging techniques, 2D automated optical image analysis and hyperspectral enhanced dark-field microscopy (EDFM), and by SEM analysis. As predictable, the feedstock composition and the pyrolysis temperature strongly influence the physical structures of the biochar samples. Irrespective of charring temperature, PR biochar was mainly characterized by a broken and fragmented structure with an irregular and rough particle surface, completely different from the original PR wood cell. The EDFM imaging analysis evidenced the thermal degradation of PR vegetal products, composed primarily of hemicellulose, cellulose and lignin. On the contrary, small and regular particles with a smooth surface were produced by the PL pyrolysis, especially at 550 °C, due to the lower PL morphological homogeneity in comparison with the other biomasses. Finally, CD charring at both temperatures was characterized by changes in chemical composition, suggested by a lower pixel intensity. In conclusion, the emerging imaging techniques used in this study proved to be very effective in analyzing some properties of biochars, and can, therefore be considered as promising experimental strategies for detecting the feedstock and pyrolysis temperature of biochar.

In Vitro Effects of Tea Tree Oil (Melaleuca Alternifolia Essential Oil) and its Principal Component Terpinen-4-ol on Swine Spermatozoa.

The growing interest towards essential oils stems from their biological capabilities that include antibacterial and antioxidant effects. Such properties may be extremely useful in the reproductive field; nonetheless essential oils show toxic effects that can lead to cell disruption. The present study aimed to evaluate and compare the effects of tea tree oil (TTO) and its principal component terpinen-4-ol (TER) on the morpho-functional parameters of swine spermatozoa. Experimental samples were prepared by suspending 15 × 107 spermatozoa in 5 mL of medium with different concentrations of the above-mentioned compounds: from 0.2 to 2 mg/mL at an interval of 0.2 for TTO, while TER concentrations were adjusted according to its presence in TTO (41.5%). After 3 h incubation at 16 °C, samples were analyzed for pH, viability, acrosome status, and objective motility. The results highlighted a concentration-dependent effect of TTO with total motility as the most sensitive parameter. TER was better tolerated, and the most sensitive parameters were related to membrane integrity, suggesting a different pattern of interaction. The study confirms the importance of evaluating the effects of natural compounds on spermatozoa before exploiting their beneficial effects. Spermatozoa seem to be good candidates for preliminary toxicological screenings in the light of their peculiar properties.

Thymbra capitata (L.) Cav. and Rosmarinus officinalis (L.) Essential Oils: In Vitro Effects and Toxicity on Swine Spermatozoa.

Essential oils possess a variety of biological properties (i.e., antioxidant, antibacterial, and cytotoxic) that could possibly be applied in reproductive medicine, but their effects on spermatozoa are still partially unknown. The aim of the study was to describe the effects of Thymbra capitata (L.) Cav. and Rosmarinus officinalis (L.) essential oils on the main morpho-functional parameters of swine spermatozoa. Essential oils were preliminary characterized by gas chromatography and added with emulsifiers to facilitate diffusion. Experimental samples were prepared by suspending a fixed number of spermatozoa in 5 mL of medium with 10 different concentrations of essential oil (0.2-2 mg/mL, at intervals of 0.2). After 3 h of incubation, samples were analyzed for pH, viability, objective motility, and acrosome status. Results showed that the effects of the essential oils are concentration-dependent and that R. officinalis is well tolerated up to 0.6 mg/mL. T. capitata impaired the spermatozoa starting from the lowest concentration, with complete spermicidal effect from 0.4 mg/mL. The patterns of damage, confirmed by SEM, were different and quite distinct. As expected, spermatozoa proved to be sensitive to external stimuli and capable of showing different functional patterns, providing interesting insights to the action/toxicity mechanisms. The results of the present work represent the first step towards the systematic characterization of the effects of these compounds on spermatozoa. This kind of studies are necessary to strengthen the idea of future applications of essential oils in the reproductive field due to their antioxidant, antibacterial, or spermicidal properties.

Detection of Fumonisins in Fresh and Dehydrated Commercial Garlic.

An epidemic fungal disease caused by Fusarium proliferatum, responsible for fumonisin production (FB1, FB2, and FB3), has been reported in the main garlic-producing countries in recent years. Fumonisins are a group of structurally related toxic metabolites produced by this pathogen. The aim of this work was to establish an enzyme-linked immunosorbent assay (ELISA) procedure, mostly applied to cereals, that is suitable for fumonisin detection in garlic and compare these results to those obtained by high-performance liquid chromatography (HPLC) and screening of fresh and dehydrated garlic for toxicological risk. The results show good correlation between the two analytical methods. In fresh symptomatic garlic, fumonisin levels were higher in the basal plates than those in the portions with necrotic spots. Among the 56 commercially dehydrated garlic samples screened, three were positive by ELISA test and only one was above the limit of quantitation. The same samples analyzed by HPLC showed the presence of FB1 in trace amounts that was below the limit of quantitation; FB2 and FB3 were absent. The results are reassuring, because no substantial contamination by fumonisins was found in commercial garlic.

Bifidobacterium aerophilum sp. nov., Bifidobacterium avesanii sp. nov. and Bifidobacterium ramosum sp. nov.: Three novel taxa from the faeces of cotton-top tamarin (Saguinus oedipus L.).

Forty-five microorganisms were isolated on bifidobacteria selective medium from one faecal sample of an adult subject of the cotton-top tamarin (Saguinus oedipus L.). All isolates were Gram-positive, catalase-negative, anaerobic, fructose-6-phosphate phosphoketolase positive, and asporogenous rod-shaped bacteria. In this study, only eight out of the forty-five strains were characterized more deeply, whereas the others are still currently under investigation. They were grouped by BOX-PCR into three clusters: Cluster I (TRE 17(T), TRE 7, TRE 26, TRE 32, TRE 33, TRE I), Cluster II (TRE C(T)), and Cluster III (TRE M(T)). Comparative analysis of 16S rRNA gene sequences confirmed the results from the cluster analysis and revealed relatively low level similarities to each other (mean value 95%) and to members of the genus Bifidobacterium. All eight isolates showed the highest level of 16S rRNA gene sequence similarities with Bifidobacterium scardovii DSM 13734(T) (mean value 96.6%). Multilocus sequence analysis (MLSA) of five housekeeping genes (hsp60, rpoB, clpC, dnaJ and dnaG) supported their independent phylogenetic position to each other and to related species of Bifidobacterium. The G+C contents were 63.2%, 65.9% and 63.0% for Cluster I, Cluster II and Cluster III, respectively. Peptidoglycan types were A3α l-Lys-l-Thr-l-Ala, A4ß l-Orn (Lys)-d-Ser-d-Glu and A3ß l-Orn-l-Ser-l-Ala in Clusters I, II and III, respectively. Based on the data provided, each cluster represented a novel taxon for which the names Bifidobacterium aerophilum sp. nov. (TRE 17(T)=DSM 100689=JCM 30941; TRE 26=DSM 100690=JCM 30942), Bifidobacterium avesanii sp. nov. (TRE C(T)=DSM 100685=JCM 30943) and Bifidobacterium ramosum sp. nov. (TRE M=DSM 100688=JCM 30944) are proposed.

Microbial inoculants for the biocontrol of Fusarium spp. in durum wheat.

BACKGROUND: Fusarium head blight (FHB) is a severe disease caused by different Fusarium species, which affects a wide range of cereal crops, including wheat. It determines from 10 to 30% of yield loss in Europe. Chemical fungicides are mainly used to reduce the incidence of FHB, but low environmental impact solutions are looked forward. Applications of soil/rhizobacteria as biocontrol agents against FHB in wheat are described in literature, whereas the potential use of lactobacilli in agriculture has scarcely been explored. RESULTS: The aim of this work was to study the inhibitory effect of two bacterial strains, Lactobacillus plantarum SLG17 and Bacillus amyloliquefaciens FLN13, against Fusarium spp. in vitro and to assess their efficacy in field, coupled to the study of the microbial community profile of wheat seeds. Antimicrobial assays were performed on agar plates and showed that the two antagonistic strains possessed antimicrobial activity against Fusarium spp. In the field study, a mixture of the two strains was applied to durum wheat i) weekly from heading until anthesis and ii) at flowering, compared to untreated and fungicide treated plots. The FHB index, combining both disease incidence and disease severity, was used to evaluate the extent of the disease on wheat. A mixture of the two microorganisms, when applied in field from heading until anthesis, was capable of reducing the FHB index. Microbial community profile of seeds was studied via PCR-DGGE, showing the presence of L. plantarum SLG17 in wheat seeds and thus underlining an endophytic behavior of the strain. CONCLUSIONS: L. plantarum SLG17 and B. amyloliquefaciens FLN13, applied as biocontrol agents starting from the heading period until anthesis of wheat plants, are promising agents for the reduction of FHB index.

Physiological responses in roots of the grapevine rootstock 140 Ruggeri subjected to Fe deficiency and Fe-heme nutrition.

Iron (Fe)-heme containing fertilizers can effectively prevent Fe deficiency. This paper aims to investigate root physiological responses after a short period of Fe-heme nutrition and Fe deficiency under two pH conditions (with or without HEPES) in the Fe chlorosis-tolerant grapevine rootstock 140 Ruggeri. Organic acids in root exudates, Fe reduction capacity, both roots and root exudates contributions, together with other physiological parameters associated to plant Fe status were evaluated in plants grown in hydroponics. Analyses of root tips by SEM, and Raman and IR spectra of the precipitates of Fe-heme fertilizers were performed. The physiological responses adopted by the tolerant 140 Ruggeri to the application of Fe-heme indicated an increased Fe reduction capacity of the roots. This is the first report showing oxalic, tartaric, malic and ascorbic as major organic acids in Vitis spp. root exudates. Plants reacted to Fe deficiency condition exuding a higher amount of ascorbic acid in the rhizosphere. The presence of HEPES in the medium favoured the malic acid exudation. The lowest concentration of oxalic acid was found in exudates of plants subjected to Fe-heme and could be associated to a higher accumulation in their root tips visualized by SEM analysis.

Bifidobacterium lemurum sp. nov., from faeces of the ring-tailed lemur (Lemur catta).

Four Gram-positive-staining, microaerophilic, non-spore-forming, fructose-6-phosphate phosphoketolase-positive bacterial strains were isolated from a faecal sample of a 5-year-old ring-tailed lemur (Lemur catta). The strains showed a peculiar morphology, resembling a small coiled snake, a ring shape, or forming a little 'Y' shape. The isolated strains appeared identical, and LMC 13T was chosen as a representative strain and characterized further. Strain LMC 13T showed an A3ß peptidoglycan type, similar to that found in Bifidobacterium longum. The DNA base composition was 57.2 mol% G+C. Almost-complete 16S rRNA, hsp60, rpoB, dnaJ, dnaG, purF, clpC and rpoC gene sequences were obtained, and phylogenetic relationships were determined. Comparative analysis of 16S rRNA gene sequences showed that strain LMC 13T showed the highest similarity to B. longum subsp. suis ATCC 27533T (96.65 %) and Bifidobacterium saguini DSM 23967T (96.64 %). Strain LMC 13T was located in an actinobacterial cluster and was more closely related to the genus Bifidobacteriumthan to other genera in the Bifidobacteriaceae. On the basis of these results, strain LMC 13T represents a novel species within the genus Bifidobacterium, for which the name Bifidobacterium lemurum sp. nov. is proposed; the type strain is LMC 13T ( = DSM 28807T = JCM 30168T).

Study of fungal colonization of wheat kernels in syria with a focus on fusarium species.

Wheat is one of the main crops in Mediterranean countries, and its cultivation has an important role in the Syrian economy. In Syria, Fusarium head blight (FHB) has not been reported so far. Mycological analysis of 48 samples of wheat kernels collected from cultivation areas with different climatic conditions were performed in 2009 and 2010. Fungal isolates were identified at the genus level morphologically; Fusarium species were characterized morphologically and by species-specific PCR. The most frequent fungal genera found were Alternaria spp. and Cladosporium spp., with frequencies of 24.7% and 8.1%, respectively, while the frequency of Fusarium spp. was 1.5% of kernels. Most frequent Fusarium species were F. tricinctum (30% of all Fusarium isolates), F. culmorum (18%), F. equiseti (14%) and F. graminearum (13%). The mycotoxin production potential of selected Fusarium isolates was assessed by HPLC-MS analysis of rice cultures; chemotyping by PCR was carried out for comparison. All six F. graminearum strains tested produced small amounts (<3 mg/kg) of nivalenol (NIV). All ten F. culmorum strains tested produced large amounts of trichothecenes (>100 mg/kg); four strains produced NIV and six strains produced deoxynivalenol (DON) and 3-acetyl-deoxynivalenol (3Ac-DON). PCR chemotyping lead to an oversimplified picture, because all 3Ac-DON chemotype strains produced more DON than 3Ac-DON; furthermore, the strongest NIV producers produced significant amounts of DON. All tested strains of F. culmorum, F. graminearum, F. pseudograminearum (two strains) and most F. equiseti strains (five of six strains) produced zearalenone. Grains of durum wheat were more frequently colonized by Fusarium spp. than grains of soft wheat. Incidence of Fusarium spp. in irrigated fields was higher than in rainfed fields. The incidence of Fusarium strains producing mycotoxins raises concerns about the risk of Fusarium head blight to Syria and its consequences for public health.

The key role of segmented filamentous bacteria in the coordinated maturation of gut helper T cell responses.

Microbiota-induced cytokine responses participate in gut homeostasis, but the cytokine balance at steady-state and the role of individual bacterial species in setting the balance remain elusive. Herein, systematic analysis of gnotobiotic mice indicated that colonization by a whole mouse microbiota orchestrated a broad spectrum of proinflammatory T helper 1 (Th1), Th17, and regulatory T cell responses whereas most tested complex microbiota and individual bacteria failed to efficiently stimulate intestinal T cell responses. This function appeared the prerogative of a restricted number of bacteria, the prototype of which is the segmented filamentous bacterium, a nonculturable Clostridia-related species, which could largely recapitulate the coordinated maturation of T cell responses induced by the whole mouse microbiota. This bacterium, already known as a potent inducer of mucosal IgA, likely plays a unique role in the postnatal maturation of gut immune functions. Changes in the infant flora may thus influence the development of host immune responses.

Discrimination of healthy and neoplastic human colon tissues by ex vivo HR-MAS NMR spectroscopy and chemometric analyses.

The metabolic profile of human healthy and neoplastic colorectal tissues was obtained using ex vivo High-Resolution Magic Angle Spinning (HR-MAS) NMR spectroscopy. Principal Components Analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA) were applied to NMR data in order to highlight the biochemical differences between healthy and neoplastic colorectal tissues. The synergic combination of ex vivo HR-MAS NMR spectroscopy with Multivariate Data Analysis enables discrimination between healthy and tumoral colorectal tissues and identification of the increase of taurine, acetate, lactate, and lipids, and the decrease of polyols and sugars as tumoral characteristics. Moreover, it was found that macroscopically/histologically normal colorectal tissues, collected at least 15 cm from the adenocarcinoma, are characterized by a metabolic pattern quite similar to that typical of tumoral lesions. It was shown that ex vivo HR-MAS NMR spectroscopy, performed on intact specimens, may be of great potentiality in the clinical evaluation of human neoplastic colorectal tissues and that the biochemical data represent the molecular basis for an accurate and noninvasive clinical applications of in vivo NMR spectroscopy.

Structural characteristics of 'Hayward' kiwifruits from elephantiasis-affected plants studied by DRIFT, FT-Raman, NMR, and SEM techniques.

This is the first study on structural and ultrastructural changes taking place in Actinidia deliciosa kiwifruits affected by "elephantiasis syndrome", by means of DRIFT, FT-Raman, NMR, and SEM techniques. The fruits arising from elephantiasis-affected plants assume a round and smaller shape, limiting their marketing. Despite etiological studies on this disease, so far no information is available on the structural and ultrastructural characteristics of the fruits. The SEM and spectroscopic data showed significant modifications regarding the polysaccharide fraction in kiwifruits from diseased plants. The pectins seem to be the polysaccharide fraction more involved in the structural variations of the fruits. These structural and ultrastructural variations are related to the elephantiasis syndrome, and they could be adopted as markers for early diagnosis of the disease.

Biochemical alterations from normal mucosa to gastric cancer by ex vivo magnetic resonance spectroscopy.

BACKGROUND AND AIMS: The metabolic profile and morphologic aspects of normal and pathologic human gastric mucosa were studied. The aim of the present research was the application of ex vivo high-resolution magic angle spinning magnetic resonance spectroscopy (HR-MAS MRS) to the human gastric tissue to get information on the molecular steps involved in gastric carcinogenesis and the identification of biochemical markers useful for the development of in vivo MRS methodologies to diagnose gastric pathologies in clinical situations. METHODS: Twelve normal subjects, five with autoimmune atrophic gastritis, five with Helicobacter pylori infection, and five with adenocarcinoma were examined. Ten biopsies were taken during endoscopy from each patient. Specimens from carcinoma were also obtained during gastrectomy. Of the 10 biopsies, 4 were used for histologic evaluation, 4 were fixed in glutaraldehyde and processed for transmission and scanning electron microscopy, and 2 were immersed in liquid nitrogen and stored at -85 degrees C for monodimensional and bidimensional ex vivo HR-MAS MRS analysis. RESULTS: Ex vivo HR-MAS MRS identified glycine, alanine, free choline, and triglycerides as possible molecular markers related to the human gastric mucosa differentiation toward preneoplastic and neoplastic conditions. Ultrastructural studies of autoimmune atrophic gastritis and gastric adenocarcinoma revealed lipid accumulations intracellularly and extracellularly associated with a severe prenecrotic hypoxia and mitochondria degeneration. CONCLUSIONS: This is the first report of synergic applications of ex vivo HR-MAS MRS and electron microscopy in studying the human gastric mucosa differentiation. This research provides useful information about some molecular steps involved in gastric carcinogenesis. The biochemical data obtained on gastric pathologic tissue could represent the basis for clinical applications of in vivo MRS.