s of treatment.

Cold rolling is widely used for screw thread manufacturing in industry but is less common in implant dentistry, where cutting is the preferred manufacturing method.

The purpose of this invitro study was to compare the surface finish and mechanical performance of a specific model of prosthetic screw used for direct restorations manufactured by thread rolling and cutting.

The thread profiles were measured in an optical measuring machine, the residual stresses in an X-ray diffractometer, the surface finish in a scanning electron microscope, and then fatigue and static load tests were carried out in a direct stress test bench according to the International Organization for Standardization (ISO) 14801. Finally, linear regression models and 95% interval confidence bands were calculated and compared through ANCOVA for fatigue tests while the t test was used for statistical comparisons (α=.05).

The surface finish was smoother, and compressive residual stresses were higher for the roll-threaded screws. Linear regression models showed a fatigue life 9 times higher for roll-threaded screws (P=1) without affecting static behavior, which showed statistically similar static strengths (P=.54). However, the thread profile in the roll-threaded screws was not accurately reproduced, but this should be easily corrected in future prototypes.

Rolling was demonstrated to be a better thread-manufacturing process for prosthetic screws, producing improved surface quality and fatigue behavior.

Rolling was demonstrated to be a better thread-manufacturing process for prosthetic screws, producing improved surface quality and fatigue behavior.The contribution of food in promotion of health has become of most importance. The challenges that lie before the global food supply chain, such as climate changes, food contamination, and antimicrobial resistance may compromise food safety at international scale. Compounds with strong antimicrobial and antioxidant activity can be extracted from different natural and sustainable sources and may contribute to extend the shelf life of meat and seafood products, enhance food safety and enrich foods with additional biologically active and functional ingredients. This chapter describes the use of bioprotective cultures, essential oils, plant extracts, seaweed extracts and grape pomace compounds in production of value-added meat and seafood products with improved shelf life and safety, following the requests from the market and consumers.Colocasia esculenta (L.) Schott and Xanthosoma sagittifolium (L.) Schott are the most popular tubers among the Araceas family. Their chemical composition related to their nutritional benefits could make these rhizomes a valid option for the nutritional and technological improvement of food products. This chapter provide a clarification about the correct nomenclature of both tubers giving an insight around the principle components and their health effects. The scientific literature review has primarily highlighted several in vitro and animal studies where the consumption (leaves and whole tuber) of Colocasia esculenta (L.) Schott and Xanthosoma sagittifolium (L.) Schott was related with certain antihyperglycemic, antihypertensive, hypoglycemic and prebiotic effects. Owing to their functional properties, different component from these rhizomes, specially starch, mucilage and powders are being used by the food industry. Their ability to behave as thickener and gelling agent has allowed their incorporation in baked food, food paste and beverages. This chapter suggests the development of more research around these rhizomes since they could potentially play, with other crops, an important role in the future sustainable strategies to feed the planet.Seafood, including fish and shellfish, provides an ideal package of nutrients and is an important part of a healthy diet. Strong evidence has shown that eating fish and other seafoods improve brain, eye, and heart health. The new 2020-2025 Dietary Guidelines for Americans (DGA) recommend that Americans of all ages should eat more seafood-at least twice a week-particularly pregnant women and young children. However, less than one in five Americans heed that advice. About one-third of Americans eat seafood once a week, while nearly half eat fish only occasionally or not at all. This calls for a drastic shift in the American diet to vary protein sources and include more seafood products in order to receive the most health benefits. This chapter covers (1) seafood nutrition and health benefits, (2) seafood’s protective effects against mercury toxicity, (3) selenium health benefit values (HBVs), and (4) challenges and opportunities for seafood production, demand and sustainability. This chapter aims to convey recent advances in science-based information to increase public awareness of seafood safety, nutrition and health benefits of seafood as part of a healthy diet, and to advocate healthy eating with smart food choices by promoting two servings of seafood per week. This will support the healthy eating patterns and promotes a minimum two to three servings of seafood recommended by the current DGA.Environmental variations in early life influence brain development, making individuals more vulnerable to psychiatric and metabolic disorders. Early life stress (ELS) has a strong impact on the development of eating behavior. However, eating is a complex behavior, determined by an interaction between signals of energy homeostasis, neuronal circuits involved in its regulation, and circuits related to rewarding properties of the food. Although mechanisms underlying ELS-induced altered feeding behavior are not completely understood, evidence suggest that the effects of ELS on metabolic, mood, and emotional disorders, as well as reward system dysfunctions can contribute directly or indirectly to altered feeding behavior. The focus of this chapter is to discuss the effects of ELS on eating behavior and metabolism, considering different factors that control appetite such as energy homeostasis, hedonic properties of the food, emotional and cognitive status. After highlighting classic studies on the association between ELS and eating behavior alterations, we discuss how exposure to adversity can interact with genetics characteristics to predict variable outcomes.Novel food preservation methods, along with preservatives have been employed to prevent food products from spoilage. There is an increasing demand to substitute synthetic preservatives with natural bioactive compounds since they are safe and environmentally friendly. Bioactive compounds with functional and therapeutic properties are found in foods and have also beneficial physiological and immunological health effects. However, there are some issues associated with bioactive compounds, such as low stability, solubility, and permeability. Encapsulation techniques, especially nano-encapsulation, are a promising technique to overcome these restrictions. A range of the plants’ constituents can be converted into bio-nanomaterials. Major plant constituents are polysaccharides which have good biocompatibility properties and therapeutic activities, such as antioxidant, antiviral, anti-inflammatory, anti-allergic, and anti-tumor. Among plant and marine-based polysaccharides, cellulose, starch, alginates, chitosan, and carrageenans have been used as carrier materials to preserve core material. Moreover, many studies indicated that favorable sources such as plant and marine based polysaccharides are emerging. This chapter will cover plant and marine-based polysaccharides for nano-encapsulation and their application in the food industry.Meat and meat products constitute an important source of nutrients and play vital roles for growth, maintenance and repair of the body. In addition to the high quality of proteins, meat is also regarded as a major resource to produce bioactive peptides. Meat processing industry also produces by-products such as bones, blood and viscera, which could be further used for the production of bioactive compounds. In the physiological analysis, meat bioactive peptides have been reported to exert antioxidant, anti-hypertensive, anti-inflammatory, anti-microbial and antitumoral activities, which endow nutritional and functional value of meat. With the objective to exert the functional effect, the bioavailability should also be considered due to the degradation by digestion enzymes and the absorption process in intestinal mucosa. In this chapter, the general source, the enzymatic hydrolysis, the physiological effects as well as the bioavailability of bioactive peptides in meat are discussed.Current available methods used to measure or estimate the composition, functionality, and sensory properties of foods and food ingredients are destructive and time consuming. Therefore, new approaches are required by both the food industry and R&D organizations. Recent years have witnessed a steady growth on the applications and utilization of vibrational spectroscopy techniques [near (NIR), mid infrared (MIR), Raman] to analyse or estimate several properties in a wide range of foods and food ingredients. This chapter will provide with an overview of vibrational spectroscopy techniques, the combination of these techniques with multivariate data analysis, and examples on the use of these techniques to measure composition, and functional properties in a wide range of foods.Bacteriophages represent the main microbiological threat for the manufacture of fermented foods. The dairy industry is the most affected by this problem, as phages are naturally present in raw milk, surfaces, vats, tanks, floors, and distributed by air displacements. Cheese whey may also contain high phage concentrations. Prophages harbored by lysogenic strains could be induced, generating new lytic phages. In this context, where phages cannot be eradicated from dairies, methods of phage monitoring are mandatory. These are mainly based in microbiological features, like classical methods, that are the most used, economic and simple to carry out. Phage DNA detection and quantification by PCR and qPCR, more complex and expensive, are faster, although not able to discern between viable and non-viable virions. Electron microscopy allows direct visualization and characterization of phage morphology, but the apparatus is expensive. Alternative methods based in other phage traits also exist, though less studied and not applicable on a daily basis. Recognition of contamination sources and correct phage monitoring in dairy factories allow a correct application of control measures. These include general measures such as proper factory design, efficient programs of sanitization, good treatment of raw materials, especially milk, and careful handling of by-products. Additionally, the use of starts cultures should be adequate, with application of rotation schemes when possible. Finally, the selection of bacteriophage insensitive mutants (BIM) is essential, and can be achieved simply and empirically, though the study of CRISPR-Cas and other newly discovered mechanisms provide a more rational basis to obtain BIMs with optimized features.In this study, yeast microbial fuel cells (MFCs) were established as biosensors for in-situ monitoring of dissolved oxygen (DO) levels in environmental waters, with yeast and glucose substrates acting as biocatalyst and fuel, respectively. Diverse environmental factors, such as temperature, pH and conductivity, were considered. The sensor performance was first tested with distilled water with different DO levels ranging from 0 mg/L to 8 mg/L and an external resistance of 1000 Ω. The relationship between DO and current density was non-linear (exponential). This MFC capability was further explored under different environmental conditions (pH, temperature and conductivity), and the current density produced was within the range of 0.14-34.88 mA/m2, which increased with elevated DO concentration. The resulting regression was y = 1.3051e0.3548x, with a regression coefficient (R2) = 0.71, indicating that the MFC-based DO meter was susceptible to interference. When used in environmental water samples, DO measurements using MFC resulted in errors ranging from 6.25 % to 15.15 % when compared with commercial DO meters. The simple yeast-based MFC sensors demonstrate promising prospects for future monitoring in a variety of areas, including developing countries and remote locations.Fructansucrases (FSs), including inulosucrase (IS) and levansucrase (LS), are the members of the Glycoside Hydrolase family 68 (GH68) enzymes. IS and LS catalyze the polymerization of the fructosyl moiety from sucrose to inulin- and levan-type fructans, respectively. Lactobacillus-derived FSs have relatively extended N- and C-terminal sequences. However, the functional roles of these sequences in their enzymatic properties and fructan biosynthesis remain largely unknown. Limosilactobacillus reuteri (basionym Lactobacillus reuteri) 121 could produce both IS and LS, abbreviated as Lare121-IS and Lare121-LS, respectively. In this study, it was found that the terminal truncation displayed an obvious effect on their activities and the N-terminal truncated variants, Lare121-ISΔ177-701 and Lare121-LSΔ154-686, displayed the highest activities. Melting temperature (Tm) and the thermostability at 50 °C were measured to evaluate the stability of various truncated versions, revealing the different effects of N-terminal on the stability. The average molecular weight and polymerization degree of the fructans produced by different truncated variants did not change considerably, indicating that N-terminal truncation had low influence on fructan biosynthesis. In addition, it was found that N-terminal truncation could also improve the activity of other reported FSs from Lactobacillus species.Plants like almost all living organisms, have developed a biological clock or circadian clock (CC) capable of synchronizing and adjusting various metabolic and physiological processes at certain times of the day and in a period of 24 h. This endogenous timekeeping is able to predict the environmental changes providing adaptive advantages against stressful conditions. Therefore, the aim of this work was to analyze the possible link between metabolism of xenobiotic compounds (MXC) and the CC. Synchronized Nicotiana tabacum hairy roots (HRs) were used as a validated plant model system, and peroxidases (PODs), key enzymes of the phase I in the MCX, were evaluated after phenol treatment. Two POD genes were selected and their temporal expression profiles as well as the total POD activity were analyzed in order to find circadian oscillations either under control conditions or phenol treatment. It was demonstrated that these PODs genes showed oscillatory profiles with an ultradian period (period length shorter than t novel information about the performance of PODs, which would be rhythmically controlled at biochemical level, by phenol exposure.Human papilloma virus (HPV) is the primary causative agent of cervical, vaginal, and vulvar cancers. HPV E6/E7 mRNA detection has been proven to improve the specificity and positive predictive value compared with HPV DNA testing in screening, whereby, it may possess higher diagnostic potential. Herein, to establish the ultrasensitive and specific detection of HPV E6/E7 mRNA, we developed a novel triple signal amplification strategy, combined with gold nanoparticles (AuNPs), reverse transcription loop-mediated isothermal amplification (RT-LAMP) and high affinity biotin-avidin system. This novel proposed signal amplification strategy exhibits the desired detection limit of 0.08 fM (approximately 100 copies) and a wide linear range from 0.1 pmol/mL to 100 nmol/mL for HPV16 E6/E7 mRNA detection. Importantly, the present novel biosensor is 10-100 times more sensitive than conventional RT-PCR in detecting HPV16 E6/E7 mRNA positive clinical samples. Conclusively, this biosensor shows good stability, selectivity, and reproducibility, which demonstrates its potential in future clinical diagnosis with desirable sensitivity and specificity.Xylanases are categorized into different family groups, two of which are glycoside hydrolases 10 (GH10) and 11 (GH11) families. These well-characterized xylanases demonstrate different modes of action in hydrolysis of xylans. Imitating certain types of microorganisms to produce bifunctional enzymes such as engineered xylanases has gained considerable attention among researchers. In this study, a recombinant chimeric enzyme (X11-10) was designed by fusing two thermostable xylanases through a peptide linker. The recombinant parental enzymes, xylanase 10 from fungus Bispora sp. MEY-1 (X10) and xylanase 11 from bacterium Thermobacillus xylanilyticus (X11), and their chimera were successfully expressed in Pichia pastoris (P. pastoris), purified, and characterized. Being active over a wide pH range, X11-10 chimera showed higher thermal stability, possessed a lower Km, and a higher catalytic efficiency (kcat/Km) in comparison to the parental enzymes. Also, molecular dynamics simulation (MDS) of X11-10 revealed that its active site residues were free to interact with substrate. This novel chimeric xylanase may have potential applications in different industrial processes since it can substitute two separate enzymes and therefore minimize the production costs.Polyphosphate kinase (PPK) is important for industrial processes involving ATP regeneration. While a variety of methods have been reported for regenerating ATP from ADP, few have explored enzyme catalyzed ATP regeneration from cheaper and stable AMP. In this work, PPKs from different sources were expressed and their catalytic activity were tested at different reaction temperatures, reaction pH and with different polyphosphate (polyPn) types. The ATP regeneration system for glutathione (GSH) synthesis was established using a single PPK capable of phosphorylating AMP to synthesize ATP from AMP and short chain polyPn. GSH yield was obtained using adenosine mono-, di- and triphosphates, which confirmed the flexibility of our constructed ATP regeneration system coupled with GSH synthesis via bifunctional GSH synthase. Finally, optimization of the GSH synthesis yielded conversion value above 80 %. Overall, these results illustrate that PPK is suitable for a broader range of substrates than previously expected, and has great untapped potential for applications involving ATP regeneration.As evidences showed that UOX(Gene ID 391051), a single pseudogene formed after multiple mutations during human evolution, could be transcribed to mature mRNA and translated to two short peptides, we hypothesized that urate oxidase with higher homology with deduced human urate oxidase (dHU) might have lower immunogenicity. In this work, we constructed a „resurrected” human-source urate oxidase (rHU19) based on dHU. It obtained better uricolytic activity (8.29 U/mg) and catalytic efficiency (3.32 s-1 μM-1) compared with wild porcine urate oxidase (wPU) and FDA-approved porcine-baboon chimera (PBC). Maintaining high homology with dHU (93.75 %), rHU19 could be more suitable for the treatment of gout and hyperuricemia theoretically.The popularity and promise of gene therapy for common genetic diseases are currently increasing. Although effective treatments for genetic disorders are rare, editing of the mutated gene is a possible therapeutic approach for conditions caused by stop codon mutations, including either amber (TAG), opal (TGA) or ochre (TAA) stop codons. Restoration of point-mutated RNAs using artificial RNA editing can be used to modify gene-encoded information and generate functionally distinct proteins from a single gene. By linking the catalytic domain of the RNA editing enzyme, adenosine deaminase acting on RNA (ADAR), to an antisense guide RNA, specific adenosines (A) can be converted to inosine (I), which is recognized as guanosine (G) during translation. In this study, we engineered the deaminase domain of ADAR1 and the MS2 system to target a specific adenosine and restore the G to A mutations. To this end, the ADAR1 deaminase domain was fused with the RNA binding protein, MS2, which binds to MS2 RNA. Guide RNAs of 19 bpercentage of edited codons after 24 h, which increased after 48 h, but decreased again after 72 h. Successful establishment of this system has the potential to represent a new era in the field of gene therapy.The rare sugar d-allulose is an attractive sucrose substitute due to its sweetness and ultra-low caloric value. It can be produced from D-fructose using d-allulose 3-epimerase (DAE) as the biocatalyst. However, most of the reported DAEs show low catalytic efficiency and poor thermostability, which limited their further use in food industrial. Here, a putative d-allulose 3-epimerase from a thermophilic organism of Halanaerobium congolense (HcDAE) was characterized, showing optimal activity at pH 8.0 and 70 °C in the presence of Mg2+. Saturation mutagenesis of Y7, C66, and I108, the putative residues responsible for substrate recognition at the O-4, -5, and -6 atoms of D-fructose was performed, and it yielded the triple mutant Y7H/C66L/I108A with improved activity toward D-fructose (345 % of wild-type enzyme). The combined mutant Y7H/C66L/I108A/R156C/K260C exhibited a half-half (t1/2) of 5.2 h at 70 °C and an increase of the Tm value by 6.5 °C due to the introduction of disulfide bridges between intersubunit with increased interface interactions. The results indicate that mutants could be used as industrial biocatalysts for d-allulose production.A lipase from Malassizia globose, named SMG1, is highly desirable for industrial application due to its substrate specificity towards mono- and diacylglycerol. To improve its thermostability, we constructed a mutant library using an error-prone polymerase chain reaction, which was screened for both initial and residual enzymatic activity. Selected mutants were further studied using purified proteins for their kinetic thermostability at 45 ℃, T50 (the temperature at which the enzyme loses half of its activity), and the optimal reaction temperature. Results showed that the majority of mutations with improved thermostability were on the protein surface. D245N and L270P showed the most significant thermostability enhancement with an approximately 3 ℃ increase in T50 compared to wild-type (WT). In addition, combining these two mutations resulted in an increase of T50 by 5 °C. Also, the optimal reaction temperatures of L270P and this double mutant are 10 ℃ higher than WT. The double mutant showed an approximately 100-fold increase in half-life at 45 ℃ and higher enzymatic activities at 30 ℃ and above compared to WT. High-temperature unfolding molecular dynamics simulation suggested that the double mutant stabilized a flexible loop in the catalytic pocket.The signal peptide sequence is known to increase transport efficiency to organelles in eukaryotic cells. In this study, we focus on the signal peptide of the vacuolar protein for vacuolar targeting. The signal peptide sequence QRPL of carboxypeptidase Y (CPY) was inserted inside the interest protein that does not locate in the vacuole for vacuolar targeting. We constructed recombinant strains MBTL-Q-DJ1 and MBTL-Q-DJ2 containing QRPL and green florescent protein (GFP) or aldehyde dehydrogenase 6 (ALD6), respectively. The protein location was then confirmed by confocal microscopy. Fascinatingly, the green fluorescent protein that contains QRPL inside the sequence could be expressed faster than its natural form (within 1 h after induction). Also, the aldehyde removal activity of ALD6 protein in the recombinant yeast was then analyzed by measuring the luminescent intensity in Vibrio fischeri. We confirmed that MBTL-Q-DJ2 containing ALD6 protein has the aldehydes-reducing ability, and in particular, the highest efficiency showed at 500 μg/μL of vacuolar enzyme. In summary, the signal peptide QRPL could be used not only to transport proteins accurately to vacuole but also to improve the protein activity and shorten the induction time.Maltodextrins (dextrins) are glucose chains normally produced by starch hydrolysis. Maltodextrins are characterized by their degree of polymerization (DP), which indicates the average number of glucose units per chain. Maltoheptaose (DP7), also known as amyloheptaose, is one of the maltodextrin mixtures widely used in foods, cosmetics, and pharmaceutical industries. Recently, the enzymatic synthesis of DP7 has attracted considerable attention, owing to its considerable advantages over chemical methods. In this work, we have designed a one-pot cascade reaction bio-synthesis starting from soluble starch to produce a specific degree of polymerization (DP7). The reaction system was catalyzed by cyclodextrin glucotransferase (GaCGT) from Gracilibacillus alcaliphilus SK51.001CGTase (transglycosylation/cyclization reaction) and cyclomaltodextrinase (BsCD) from Bacillus sphaericus E-244CDase (ring-opening reaction). The one-pot cascade reaction exhibited an optimum temperature of 30 °C and pH 7.0, and the addition of Ca2+ enhanced the maltoheptaose production. The optimum enzyme units for the one-pot cascade reaction were 80 U/g of GaCGT and 1 U/g of BsCD. However, the sequential addition of the enzymes exhibited a 5-fold higher conversion rate over simultaneous addition. The one-pot cascade reaction converted 30 g/L of soluble starch to 5.4 g/L of maltoheptaose in 1 h reaction time with a conversion rate of 16 %.Antibiotic resistance has become a major risk to community health over last few years because of antibiotics overuse around the globe and lack of new antibiotics development. Phages and their lytic enzymes are considered as an effective alternative of antibiotics to control drug resistant bacterial pathogens. Endolysins prove to be a promising class of antibacterials due to their specificity and less chances of resistance development in bacterial pathogens. Though large number of endolysins has been reported against gram positive bacteria, very few reported against gram negative bacteria due to the presence of outer membrane, which acts as physical barrier against endolysin attack to peptidoglycan. In the current study, we have expressed endolysin (RL_Lys) and holin fused at the N terminus of endolysin (RL_Hlys) from RL phage infecting multi drug resistant (MDR) Pseudomonas aeruginosa. Both endolysin variants were found active against wide range of MDR strains P. aeruginosa, Klebsella pneumonia, Salmonella Sp. and Methicillin Resistant Staphylococcus aureus (MRSA). Broth reduction assay showed that RL_Hlys is more active than RL_Lys due to presence of holin, which assist the endolysin access towards cell wall. The protein ligand docking and molecular dynamic simulation results showed that C- terminus region of endolysin play vital role in cell wall binding and even in the absence of holin, hydrolyze a broad range of gram negative bacterial pathogens. The significant activity of RL-Lys and RL_Hlys against a broad range of MDR gram negative and positive bacterial pathogens makes them good candidates for antibiotic alternatives.Enzymatic stereospecific reduction of 17-oxosteroids offers an attractive approach to access 17β-hydroxysteroids of pharmaceutical importance. In this study, by adjusting the flexibility of α6-helix at the substrate entrance of the alcohol dehydrogenase from Ralstonia sp. (RasADH), the catalytic activity toward the stereospecific 17β-reduction of androstenedione was improved without sacrifice of the enantioselectivity. Among the mutants, F205I and F205A exhibited up to 623- and 523-fold improvement in catalytic efficiency, respectively, towards a range of different 17-oxosteroids compared to the wild-type enzyme. The corresponding 17β-hydroxysteroids were prepared in optically pure form with high space-time productivity and isolated yields using F205I as the biocatalyst, indicating that these mutants are promising biocatalysts for this useful transformation. These results suggest that modulating the flexibility of the active site lid offers an effective approach to engineer alcohol dehydrogenase for accommodating bulky steroidal substrates.Complex carbohydrates, proteins, and other food components require a longer digestion process to be absorbed by the lining of the alimentary canal. In addition to the enzymes of the gastrointestinal tract, gut microbiota, comprising a large range of bacteria and fungi, has complementary action on the production of digestive enzymes. Within this universe of „hidden soldiers”, lactobacilli are extensively studied because of their ability to produce lactase, proteases, peptidases, fructanases, amylases, bile salt hydrolases, phytases, and esterases. The administration of living lactobacilli cells has been shown to increase nutrient digestibility. However, it is still little known how these microbial-derived enzymes act in the human body. Enzyme secretion may be affected by variations in temperature, pH, and other extreme conditions faced by the bacterial cells in the human body. Besides, lactobacilli administration cannot itself be considered the only factor interfering with enzyme secretion, human diet (microbial substrate) being determinant in their metabolism. This review highlights the potential of lactobacilli to release functional enzymes associated with the digestive process and how this complex metabolism can be explored to contribute to the human diet. Enzymatic activity of lactobacilli is exerted in a strain-dependent manner, i.e., within the same lactobacilli species, there are different enzyme contents, leading to a large variety of enzymatic activities. Thus, we report current methods to select the most promising lactobacilli strains as sources of bioactive enzymes. Finally, a patent landscape and commercial products are described to provide the state of art of the transfer of knowledge from the scientific sphere to the industrial application.d-Aspartate (d-Asp) is an important intermediate for synthetic penicillin and an endogenous amino acid that plays important roles in the endocrine and nervous systems in animals including humans. Lactic acid bacteria (LABs) have been used as probiotics in humans, and some LAB species produce d-Asp as a component of cell wall peptidoglycan. LAB strains with greater d-Asp production would therefore be valuable for industrial d-Asp production. In this study, we developed an enzymatic screening method for d-Asp-producing LABs and isolated a strain with high d-Asp production. The d-Asp concentration in the culture medium was colorimetrically estimated up to 4 mM using d-aspartate oxidase (ChDDO) from the yeast Cryptococcus humicola strain UJ1 coupled with horseradish peroxidase, although a more accurate determination required correction because of interference by the medium component Mn2+. We isolated 628 LAB strains from various foods and screened them for d-Asp production using the enzymatic d-Asp assay method. The screening identified 13 d-Asp-producing LAB strains, which were suggested to belong to the genera Latilactobacillus, Levilactobacillus, Lactococcus, and Enterococcus. d-Asp production ability was likely to widely differ among the strains in the same genera and species. One strain, named strain WDN19, produced much higher d-Asp levels (1.84 mM), and it was closely related to Latilactobacillus curvatus. These results indicated that the enzymatic screening method was useful for identifying and isolating d-Asp-producing LABs rapidly and easily, and it might provide novel findings regarding d-Asp production by LABs.The goal of this work was the autodisplay of the endo β-1,4-xylanase (XynA) from Clostridium cellulovorans in Escherichia coli using the AIDA system to carry out whole-cell biocatalysis and hydrolysate xylans. For this, pAIDA-xynA vector containing a synthetic xynA gene was fused to the signal peptide of the toxin subunit B Vibro cholere (ctxB) and the auto-transporter of the synthetic aida gene, which encodes for the connector peptide and β-barrel of the auto-transporter (AT-AIDA). E. coli TOP10 cells were transformed and the biocatalyst was characterized using beechwood xylans as substrate. Optimal operational conditions were temperature of 55 °C and pH 6.5, and the Michaelis-Menten catalytic constants Vmax and Km were 149 U/gDCW and 6.01 mg/mL, respectively. Xylanase activity was inhibited by Cu2+, Zn2+ and Hg2+ as well as EDTA, detergents, and organic acids, and improved by Ca2+, Co2+ and Mn2+ ions. Ca2+ ion strongly enhanced the xylanolytic activity up to 2.4-fold when 5 mM CaCl2 were added. Also, Ca2+ improved enzyme stability at 60 and 70 °C. Results suggest that pAIDA-xynA vector has the ability to express functional xylanase to perform whole-cell biocatalysis in order to hydrolysate xylans from hemicellulose feedstock.Endoglucanase and xylanase are critical enzymes for liquefaction and enzyme hydrolysis of high solids lignocellulosic biomass to facilitate its transport and production of desired derived products. Here is reported how combinations of different spore concentrations and pH influence microbial morphology, and how this may be used to direct expression and secretion of enzymes by Aspergillus niger. While xylanase production is not affected by A. niger morphology changes, endoglucanase production is enhanced under conditions of lower stress and by morphology that results in pellets. β-glucosidase production is enhanced under dispersed morphology, which results in up to fourfold increase of this enzyme production under the tested experimental conditions. A morphologic scale (Y) is proposed based on a form factor that considers the size and frequency of each morphology class, and that points to conditions that result in high selectivity for either endoglucanase or β-glucosidase production. An equation proposed to relate enzyme activity to morphology provides a useful tool for tuning enzyme production of A. niger, where morphology is a first indication of relative enzyme activities in a fermentation broth.Haloalkane dehalogenase DhaA catalyzes the hydrolytic cleavage of carbon-halogen bonds and produces alcohol, a proton and a halide. However, DhaA suffers from the poor environmental stability, such as sensitivity to high temperature, low pH, hypersaline and organic solvent. In order to improve the environmental stability of DhaA, DhaA was covalently conjugated with inulin, a hydrophilic polysaccharide in the present study. Each DhaA was averagely conjugated with 7∼8 inulin molecules. The conjugated inulin could form a hydration layer around DhaA, which increased the conformational rigidity and decreased the entropy of the enzyme. Conjugation of inulin maintained 75.5 % of the enzymatic activity of DhaA and slightly altered the structure of DhaA. As compared with DhaA, the conjugate (inu-DhaA) showed slightly different kinetic parameters (Km of 2.9 μmol/L and Kcat of 1.0 s-1). Inulin conjugation could delay the structural unfolding and/or slow the protonation process of DhaA under undesirable environment, including the long-term storage, low pH, hypersaline and organic solvent stability. As a result, the environmental stability of DhaA was markedly increased upon conjugation with inulin. Thus, inulin conjugation was an effective approach to enhance the environmental stability of DhaA.The influence of green light on mycelium biomass growth and extracellular enzyme activities of edible mushrooms from the Pleurotus genus, which is popularly cultivated all over the world, were investigated. The mycelium of seven strains of five species of Pleurotus (P. citrinopileatus, P. djamor, P. eryngii, P. ostreatus, and P. pulmonarius) was grown in liquid medium at 28 °C in the dark or under green light (515-530 nm). The light source was light-emitting diodes (LED) with photon flux density adjusted to 20 μmol m-2 s-1 that was kept on throughout the cultivation period. After 12 days of growth, the mycelium was recovered and used for biomass determination and the cultivation medium was used to total cellulase, endoglucanase, xylanase, and laccase activities determination. Green light reduced the mycelial biomass growth of Pleurotus spp. but increased the cellulolytic and xylanolytic activities. The cellulolytic activity of most strains increased in the presence of green light with increases ranging from 1.5 times (P. ostreatus endoglucanase) to 8 times (P. citrinopileatus total cellulase and endoglucanase). Green light reduced laccase activity for most strains with the greatest reduction for P. eryngii (2.2 times lower). The specific enzymatic activity of cellulase and endoglucanase from P. citrinopileatus, increased by 31 times and 30 times, respectively, compared to the dark. Also, the specific laccase and xylanase activities of P. pulmonarius increased 4.4 times and 6.8 times, respectively, under green light. The use of light at particular wavelengths can be a viable strategy to increase the production of enzymes for different biotechnological applications and species of Pleurotus are particularly interesting for this purpose.The recognition and interaction of FEZ-1 from Legionella (FEZ-1) with penicillin V(PV) and cefoxitin(CFX) were investigated using fluorescence spectra in combination with molecular dynamics simulation (MD). The results revealed that the CFX bind with FEZ-1 in stronger interaction and induced larger conformational change than PV, despite all being forced by the electrostatic interaction and along with the changing in an environment of amino acid residues as well as the polypeptide skeleton inside the FEZ-1. Moreover, only the loop1, loop2, and N-terminal were observed locating near the binding pocket of FEZ-1, consisting of a flexible „gate-like” zone with better adaptability that controlled the entrance of antibiotic into the pocket by allowing the newly introduced antibiotic to match the pocket better through the conformational changes of these three substructures in the binding procedure. The current study may provide some valuable information on the antibiotic hydrolytic process by metallo-beta-lactamase and thus the references for the development of new antibiotics for super bacteria.Robotized high throughput screening allows for the assessment of autophagy in a large number of samples. Here, we describe a drug discovery platform for the phenotypic identification of novel autophagy inducers by means of automated cell biology workflows employing robotized cell culture, sample preparation and data acquisition. In this setting, fluorescent biosensor cells that express microtubule-associated proteins 1A/1B light chain 3B (best known as LC3) conjugated to green fluorescent protein (GFP), are utilized together with automated high content microscopy for the image-based assessment of autophagy. In sum, we detail a drug discovery screening workflow from high throughput sample preparation and processing to data acquisition and analysis.Autophagy is a well-conserved self-degrading mechanism, which involves the elimination of unnecessary or damaged cellular constituents. Although extensively studied, many aspects regarding its tight regulation and its implication in health and disease remain elusive. The nematode Caenorhabditis elegans has been widely used as a simple multicellular model organism for studying the autophagic machinery per se, and uncover its multidimensional roles in the maintenance of cellular and organismal homeostasis. The current protocol describes the in vivo detection and biochemical analysis of the autophagic substrate SQST-1, as an indicator of autophagic flux in C. elegans.Imaging flow cytometry allows for the quantitative assessment of fluorescent signals at the subcellular level. Here, we describe the use of a biosensor cell line, namely, U2OS osteosarcoma cells equipped with a fusion protein comprising monomeric red fluorescent protein (mRFP), green fluorescent protein (GFP) and microtubule-associated proteins 1A/1B light chain 3B (best known as LC3), for the assessment of autophagic flux by imaging flow cytometry. We detail all analysis tools required to distinguish autophagosomes (that emit both a red and a green fluorescence) and autolysosomes (that emit a red fluorescence, yet lose the green fluorescent signal) and to quantitate autophagic flux in a convenient fashion.Autophagy is an important intracellular pathway for the degradation of superfluous or harmful subcellular materials, thereby playing a critical role in the maintenance of cell health under normal and stress-related conditions. Researchers interrogating autophagic activity in mammalian cell lines often leverage complementary assay technologies to confirm observations. The Autophagy LC3 HiBiT Reporter assay system utilizes a tandem reporter module, HiBiT-HaloTag, fused to a key marker of autophagic activity, LC3B protein, to enable multiple, cell-based assay modalities. This novel autophagy reporter expressed in a single cell line supports (a) a bioluminescent, homogeneous, plate-reader assay for rapid and quantitative assessment of changes in the level of the LC3-based reporter, (b) a fluorescence-based imaging approach to monitor reporter subcellular distribution in live cells, and (c) an antibody-free, protein blotting method to detect the relative amounts of the LC3-I and LC-II forms of the reporter associated with modulation of autophagic flux. Here we detail protocols for all three assay modalities applied to a U2OS human osteosarcoma cell line stably expressing the novel autophagy reporter, enabling the identification of modulators of autophagic activity and subsequent confirmation of mechanism of action.Autophagy plays a major role in physiological and pathological processes. The quantitation of the abundance of autophagy-specific substrates constitutes an efficient strategy for assessing autophagic activity. Here, we provide a detailed protocol for quantifying the decay of a fusion protein composed by enhanced green fluorescent protein (EGFP) and glutamine repeats (Q74) using regular or high-throughput fluorescence microscopy. This method provides a direct measurement of autophagic flux in a Huntington’s disease model.The lysosome is the main catabolic organelle in the cell, also serving as a signaling platform. Lysosomes maintain a low intraluminal pH where dozens of hydrolytic enzymes degrade a wide variety of macromolecules. Besides degradation of polymers, the lysosome is involved in various cellular processes, including energy metabolism, plasma membrane repair and antigen presentation. Recent work has shown that the lysosome is an important calcium store, modulating diverse cellular functions such as membrane fusion and fission, autophagy and lysosomal biogenesis. Precise measurement of free lysosomal calcium concentration has been hampered by its low luminal pH, since the affinity of most calcium probes decreases with higher proton concentration. Here we detailed an adapted protocol for the simultaneous measurement of lysosomal pH and calcium using dextran-conjugated ratiometric fluorescent dyes. As compared with indirect measurements of lysosomal calcium release using genetically-encoded calcium indicators (GECIs), the present method offers the possibility of obtaining pH-corrected, intraluminal calcium concentrations at single lysosome resolution. It also enables simultaneous temporal resolution of lysosomal calcium and pH.Exosomes are bi-layered vesicles secreted by the cells in physiological and pathological conditions. They are involved in cell-cell communication facilitating the transfer of functional macromolecules, including DNA, RNA, proteins and lipids. In this chapter, we will focus on specific class of RNA, the microRNAs, that are shuttled from the exosome-producing cells to the recipient cells where they affect biological processes. We will describe the recent methodologies developed to detect and isolate exosomal microRNAs providing a suitable workflow that contributes to quickly expand the field of exosomes-derived microRNAs and their potential use as biomarkers.Autophagy is a process that facilitates the maintenance of intracellular homeostasis by removing unnecessary or dysfunctional cellular components. It plays a role in inhibiting tumorigenesis in the early stage of the disease and might promote progression after tumor formation. Microtubule-associated protein light chain 3 (MAPLC3, better known as LC3), isoform B (LC3B), is one of the most commonly used markers of autophagy. The expression of LC3B has been studied in many cancers and was shown to be closely related to tumor progression. Here, we provide detailed experimental steps for the quantitative detection of LC3B expression in cancer tissue by quantum-dot-based molecular imaging. As compared to the traditional immunohistochemistry (IHC) employing standard fluorochromes, the present method has a higher signal amplitude and improved sensitivity enabling the accurate quantitative detection, which provides a foundation for functional research and the clinical application of LC3B biomarker.Ferroptosis is an iron-dependent form of regulated cell death, driven by the accumulation of lipid peroxidation. Autophagy is a lysosome-dependent degradation process that can be used to remove and recover intracellular components, such as dysfunctional proteins and damaged organelles. By regulating iron storage and oxidative stress, excessive autophagy is involved in the induction and execution of ferroptosis. In particular, several types of selective autophagy (e.g., ferritinophagy, lipophagy, clockophagy, and chaperone-mediated autophagy) increase the susceptibility to ferroptotic cell death by degrading anti-ferroptotic regulators (e.g., ferritin, GPX4, ARNTL, and lipid droplets). These two integrated biological processes play a pathological role in the occurrence and development of human diseases, such as cancer, neurodegenerative disorders, ischemia and reperfusion injury. Therefore, it is important to develop reliable methods to evaluate the kinetics of autophagosome formation, iron accumulation, and lipid peroxidation. Here, we introduce some protocols (such as western blotting, lipid peroxidation assay kits and probes, and iron probes) to monitor the process of autophagy-dependent ferroptosis.Selective elimination of damaged mitochondria via macroautophagy (mitophagy) is a conserved cellular process that plays an important role in organismal health. In recent years mitophagy has been studied in parallel to the more general, non-selective autophagy pathway induced in response to amino acid starvation with important similarities and differences noted between the two. The elaborate sequence of membrane rearrangements that give rise to autophagosomes in the non-selective pathway have their counterpart in mitophagy, but with the addition of other factors, such as a ubiquitin mark and mitophagy receptors, which mediate cargo recognition. In some types of mitophagy such as the one induced by ivermectin, the forming autophagosomal structure contains six different elements the targeted mitochondrial fragment, a section of endoplasmic reticulum that provides a cradle, a ubiquitin layer, the mitophagy receptors and the early and late autophagosomal proteins/membranes. Super-resolution microscopy is ideally suited to investigate the spatial relationships between these elements that converge together but retain some distinctive localization, and we provide here a general protocol that can be used for mammalian cells.Autophagy is a major protein degradation pathway responsible for the removal of primarily long-lived and misfolded proteins, contributing to cellular homeostasis. Autophagy dysfunction has been associated with the onset of various human pathologies. Visualizing key proteins that govern autophagy pathway activity, the molecular machinery and cargo is essential to elucidate roles and mechanisms of autophagy function. Although multiple fluorescence-based microscopy approaches exist to assess autophagy, the limit of resolution associated with light microscopy makes precise intracellular protein localization, interaction and molecular distribution challenging. Here we describe a detailed protocol for both super-resolution structured illumination microscopy (SR-SIM) as well as direct stochastic optical reconstruction microscopy (dSTORM) for the visualization of key proteins associated with the autophagy molecular machinery and cargo. The presented method enables to achieve increased resolving power to assess localization and molecular density profiles, typically not achievable with standard confocal or wide field fluorescence microcopy.Autophagy has been described as a catabolic process in which cytoplasmic material is being recycled under various conditions of cellular stress, preventing cell damage and promoting cell survival. Drosophila has been demonstrated to provide an excellent animal model for the study of autophagy. Here, we provide a detailed experimental procedure for the identification of Atg8a interactors, exploiting the iLIR database, followed by the in vitro confirmation of interactions and in situ detection of the respective proteins.Autophagy is an evolutionarily conserved biological process required for the turnover of the cytoplasm of eukaryotic cell. Beyond its catabolic nature, autophagy has a plethora of pro-survival functions, thus combatting hypoxia, nutrient shortage, and unfolded protein accumulation. Here, we introduce the naturally short-lived turquoise killifish Nothobranchius furzeri as an emerging model to study autophagic function in vivo, in response to environmental challenges. We show that starvation in killifish is sufficient to increase autophagic flux in the liver, thus enhancing the lipidation of microtubule-associated protein light chain 3 (LC3) and reducing the abundance of the autophagic substrate sequestosome-1 (SQSTM1). We describe an immunoblot-based comprehensive protocol to monitor fluctuations in autophagy in this model organism.Acyl-CoA binding protein (ACBP), also called diazepam-binding inhibitor (DBI), is a ubiquitous protein that can be secreted from cells by an unconventional pathway. Depending on its levels and on its subcellular localization, ACBP/DBI can regulate lipid metabolism. Several studies have shown that ACBP/DBI is secreted by an autophagy-dependent mechanism, positioning this catabolic pathway as the mechanism that controls lipid metabolism through the intracellular modulation of the levels of this protein. Autophagy is activated, among other stimuli, when cells have increased energy requirements; this causes a drop in the intracellular ACBP/DBI levels due to its release into the extracellular space and triggers an increase in the lipid catabolism. Conversely, when autophagy is inhibited, during pathological (obesity) or physiological (after-meal) situations, the intracellular levels of ACBP/DBI increase resulting in the activation of lipid anabolism, this effect has been demonstrated to be the link between obesity and autophagy inhibition.