Methods for imaging DNA in liquid with lateral molecular-force microscopy

Shear force microscopy is not normally associated with the imaging of biomolecules in a liquid environment. Here we show that the recently developed scattered evanescent wave (SEW) detection system, combined with custom-designed vertically oriented cantilevers (VOCs), can reliably produce true non-contact images in liquid of DNA molecules. The range of cantilever spring constants for successful shear force imaging was experimentally identified between 0.05 and 0.09 N?m(-1). Images of λ-DNA adsorbed on mica in distilled water were obtained at scan rates of 8000?pixels?s(-1). A new constant-height force mapping mode for VOCs is also presented. This method is shown to control the vertical position of the tip in the sample plane with better than 1?nm accuracy. The force mode is demonstrated by mapping the shear force above λ-DNA molecules adsorbed on mica in a liquid environment at different tip-sample separations.     

Transferable Antibiotic Resistances in Marketed Edible Grasshoppers (Locusta migratoria migratorioides)

Grasshoppers are the most commonly eaten insects by humans worldwide, as they are rich in proteins and micronutrients. This study aimed to assess the occurrence of transferable antibiotic resistance genes in commercialized edible grasshoppers. To this end, the prevalence of 12 selected genes [aac(6’)‐Ie aph(2″)‐Ia, blaZ, erm(A), erm(B), erm(C), mecA, tet(M), tet(O), tet(S), tet(K), vanA, vanB] coding for resistance to antibiotics conventionally used in clinical practice was determined. The majority of samples were positive for tet(M) (70.0%), tet(K) (83.3%) and blaZ (83.3%). A low percentage of samples were positive for erm(B) (16.7%), erm(C) (26.7%), and aac(6’)‐Ie aph(2″)‐Ia (13.3%), whereas no samples were positive for erm(A), vanA, vanB, tet(O), and mecA. Cluster analysis identified 4 main clusters, allowing a separation of samples on the basis of their country of origin.     

The Occurrence of Beer Spoilage Lactic Acid Bacteria in Craft Beer Production

Beer is one of the world's most ancient and widely consumed fermented alcoholic beverages produced with water, malted cereal grains (generally barley and wheat), hops, and yeast. Beer is considered an unfavorable substrate of growth for many microorganisms, however, there are a limited number of bacteria and yeasts, which are capable of growth and may spoil beer especially if it is not pasteurized or sterile‐filtered as craft beer. The aim of this research study was to track beer spoilage lactic acid bacteria (LAB) inside a brewery and during the craft beer production process. To that end, indoor air and work surface samples, collected in the brewery under study, together with commercial active dry yeasts, exhausted yeasts, yeast pellet (obtained after mature beer centrifugation), and spoiled beers were analyzed through culture‐dependent methods and PCR‐DGGE in order to identify the contaminant LAB species and the source of contamination. Lactobacillus brevis was detected in a spoiled beer and in a commercial active dry yeast. Other LAB species and bacteria ascribed to Staphylococcus sp., Enterobaceriaceae, and Acetobacter sp. were found in the brewery. In conclusion, the PCR‐DGGE technique coupled with the culture‐dependent method was found to be a useful tool for identifying the beer spoilage bacteria and the source of contamination. The analyses carried out on raw materials, by‐products, final products, and the brewery were useful for implementing a sanitization plan to be adopted in the production plant.     

The effect of starch isolation method on physical and functional properties of Portuguese nut starches. II. Q. rotundifolia Lam. and Q. suber Lam. acorns starches

A new starch was isolated from fruits of two acorn species, Quercus rotundifolia and Quercus suber by alkaline (A3S) and enzymatic (ENZ) methods and physical and functional properties were studied. The isolation method induced changes in most of those properties in the isolated starches, mainly in resistant starch content, syneresis, pasting, thermal and rheological properties. Isolated acorn starches presented high amylose content (53–59%) and resistant starch content (30.8–41.4%). Acorn starches showed limited and similar solubility values and swelling power values, showing a gradual increase from 60 °C to 90 °C. The pasting temperatures ranged from 67.5 to 72.0 °C and pastes did not present breakdown, which is suggestive of a high paste stability of acorn starches during heating. At ambient temperature the turbidity and syneresis values were low, but when held at freezing temperatures the syneresis significantly increased. Thermal analysis revealed that the acorn starches easily undergo transition phenomena as shown by the low T_o and enthalpy values (4.1–4.3 J/g), these effects were more evident in starches isolated by ENZ method. Pastes are more elastic than viscous and form strong gels after cooling. Q. suber starch was shown to be more sensitive to the effect of isolation method. Generally, starch isolated by enzymatic method presented less interesting functional properties, since this isolation procedure greater affected the raw structure of starches.     

Deep cryogenic treatment of AISI 302 stainless steel: Part II – Fatigue and corrosion

During the last decade, the interest about deep cryogenic treatment (DCT) is grown beyond its successful application on tool steels. The use of such cold treatment has shown positive effects on carburized steel fatigue life and some promising results were also noticed on stainless steels and on other materials. In this article, the DCT effects on fatigue and corrosion resistance of the AISI 302 austenitic stainless steel are analysed starting from the results of an extensive experimental campaign that was performed on both hardened and solubilized material conditions. The analysis includes an evaluation of the influence that the most important treatment parameters have on the final result. Considering their statistical significance at different reliability levels, the results show that the DCT can improve the fatigue behaviour of the solubilized AISI 302, while no important changes are noticed on the hardened material. Moreover, no difference was detected on the corrosion resistance of the cryotreated material, ensuring that such key-property for this class of steel is not compromised by the cryogenic treatment.     

In-House Validation and Comparison of Two Wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>) Taxon-Specific Real-Time PCR Methods for GMO Quantification Supported by Droplet Digital PCR

Common wheat is one of the most important staple food crops worldwide. However, unlike other important staple crops such as maize or soybean, genetically modified (GM) wheat is not yet present in the global food market. Nonetheless, in the recent past, the adventitious presence of GM glyphosate-tolerant volunteers was reported in open wheat fields in the USA. The European Union Reference Laboratory for GM Food and Feed (EURL-GMFF) was therefore called to develop a strategy to detect such unauthorised GM wheat in wheat samples by using both taxon-specific and screening tests. Two candidate common wheat taxon-specific real-time PCR methods were suggested, one targeting ssII-D gene coding for starch synthase and the other targeting waxy-D1 gene, coding for granule-bound starch synthase. In the present study, the two above-mentioned real-time PCR taxon-specific methods were in-house verified and compared, proposing droplet digital PCR (ddPCR) as a new tool for supporting the application of the European Network of GMO Laboratories (ENGL) established method performance criteria. Preliminary performance data of waxy-D1 and ssII-D methods in ddPCR format are shown too to give a contribution to the bridging process from the consolidated to the emerging quantitative PCR methodology.     

Chitosan biobased and intelligent films: Monitoring pH variations

Many factors can influence the shelf-life product, such as pH, water activity, nutrient levels available oxygen, etc. Variations in food pH could occur due to microorganism growth and chemical reactions that may impact flavour, consistency and shelf-life. The objective of this paper was to obtain and characterize the fast pH-colourimetric indicator device, applying a simple manufacturing technique, using food grade and biodegradable materials. The intelligent film was based on natural compounds as chitosan and anthocyanin (pH-colourimetric indicator). Chitosan intelligent films (C-ATH, 2.0 g/100 g) were obtained incorporating anthocyanin (1.0 g/100 g) in matrix films. Initially, a dark violet colour was observed in chitosan films dried. When immersed in different pH buffers, the colour range of the films varied from pink (in acid pH) to bluish-green (in neutral pH) and to violet (in basic pH). The water solubility and water vapour transmission rate of C-ATH were decreased to 60% and 48%, respectively, as compared to control film (CF, without indicator). The mechanical properties such as tensile strength and rigidity were maintained, and the elongation at break was reduced to 47% compared to CF. The advantages of this system were the simple manufacturing process, biodegradability and usage of natural and safe compounds.     

Chitinous polymers: extraction from fungal sources,characterization and processing towards value-added applications

Chitin, chitosan and their complexes with β-glucan (chitin–glucan complex, CGC, and chitosan–glucan complex, ChGC) are value-added polysaccharides extracted from the cell-walls of many fungi. Commercial chitin and its deacetylated form, chitosan, are currently obtained from marine waste material, mostly animal sources (crustaceans and marine invertebrates), through harsh chemical procedures that have low reproducibility due to the variability of the composition of the sources and their seasonal character. These disadvantages are overcome by using fungi as sources of chitinous polymers. The extraction of chitin/chitosan from fungi cell-walls has the great advantage of yielding products with stable composition and properties, using simpler procedures, with the added benefit of also generating CGC and ChGC, two copolymers that combine the proven properties of chitin/chitosan with those of β-glucans. Over the last decades, fungal chitinous polymers have been the focus of extensive research that included optimization of the cultivation conditions of a wide range of species and the development of optimized extraction, purification and characterization techniques, as well as the demonstration of the biopolymers' biological properties, which include immunomodulatory, anticancer, antioxidant and antimicrobial activity. Given these properties, several attempts were made to develop applications for them in areas ranging from biomedicine and pharmaceuticals to food and agriculture. Despite their wide range of proven functional properties that include the ability to form different polymeric structures, as well as biological activity, fungal chitinous biopolymers are still underexplored. Nevertheless, these biopolymers hold great potential for development into valuable products or applications that are surely worth further investigation. © 2019 Society of Chemical Industry     

Immunohistochemical Analysis of DNA Repair- and Drug-Efflux-Associated Molecules in Tumor and Peritumor Areas of Glioblastoma

Glioblastoma (GBM), the most commonly occurring primary tumor arising within the central nervous system, is characterized by high invasiveness and poor prognosis. In spite of the improvement in surgical techniques, along with the administration of chemo- and radiation therapy and the incessant investigation in search of prospective therapeutic targets, the local recurrence that frequently occurs within the peritumoral brain tissue makes GBM the most malignant and terminal type of astrocytoma. In the current study, we investigated both GBM and peritumoral tissues obtained from 55 hospitalized patients and the expression of three molecules involved in the onset of resistance/unresponsiveness to chemotherapy: O6-methylguanine methyltransferase (MGMT), breast cancer resistance protein (BCRP1), and A2B5. We propose that the expression of these molecules in the peritumoral tissue might be crucial to promoting the development of early tumorigenic events in the tissue surrounding GBM as well as responsible for the recurrence originating in this apparently normal area and, accordingly, for the resistance to treatment with the standard chemotherapeutic regimen. Notably, the inverse correlation found between MGMT expression in peritumoral tissue and patients’ survival suggests a prognostic role for this protein.     

Glass-ceramic composites for high-power white-light-emitting diodes

In this paper we report on the study of sintered ceramic composites based on a low melting glass in which ZnO nanocrystals and commercial YAG:Ce³⁺ phosphor are embedded. A low T_g is necessary to avoid high temperature sintering which can damage the optical properties of the embedded phosphor, while ZnO is introduced to increase the thermal conductivity of the system. The compositions have been optimized in terms of stability, sintering efficiency and thermal conductivity. Selected samples were optically characterized using a GaN high power multimode 450 nm Laser Diode, with a maximum output power of 1.6 W at 1.5 A.