Growth and quality attributes were quantified in Cichorium intybus L. and Cichorium endivia L. in response to the nitrate supply. Chicory was grown in Italy, in a cold greenhouse from the 11 of February 2020, in a pot with commercial soil and sand with and without 12 mM of Ca(NO3)(2). Seventy-six days after sowing, the growth variables, contents of leaf and root carbohydrates (glucose, fructose sucrose starch and fructans), carbon, nitrate sulphate and phosphate were measured. Fertilization significantly increased the yield and specific leaf dry weight (SLDW) of the C. endivia. The shoot/root ratio was increased in C. intybus with high N; this also increased the carbohydrate content in leaves and roots of C. endivia compared to the value measured in C. intybus. The interaction between fertilization and genotype significantly affected fructans and nitrate accumulation in taproots. Fertilization decreased the sulphate and phosphate contents in the leaves and roots of both chicory species. The yield and quality of young chicory plants can be modulated by species selection and nitrate fertilization. The genotype and nitrogen supply interact in modulating the yield and the inulin and nitrate accumulation in the taproot, thereby affecting its nutritional value and representing a powerful tool to cultivate this new produce for healthy human nutrition.

Background: Infant milk formula (IMF) was produced at pilot scale by cascade membrane filtration (IMF-CMF) as an alternative to high temperature (IMF-HT) processing. Methods: At weaning, 20 piglets were randomly assigned to two treatment groups (1) IMFHT OR (2) IMF-CMF. Piglets were fed twice daily and water was available ad libitum for 28 days. Piglets were slaughtered 3 hours after their final feeding. Results: Piglets fed IMF-CMF had significantly higher average daily feed intake from day 0 - 7 in comparison with IMF-HT fed piglets (269.49 V's 200.87 ± 17.85 g/day; P <0.001). Piglets fed IMF-HT had significantly lower average daily gain in contrast with piglets fed IMF-CMF (140.20 V's 200.82 ± 22.63 g/day; P = 0.013). The degree of protein hydrolysis was significantly higher (P = 0.027) in the duodenum of piglets who received IMF-CMF (1530 ± 136 µmol of NH2/mg of protein) versus IMF-HT (1174 ± 124 µmol of NH2/mg of protein). The IMF-CMF fed piglets had a significantly higher number of goblet cells in the jejunum versus the IMF-HT fed piglets (17.4 V's 11.7 ± 1.9 goblet cells: P = 0.028). Conclusions: IMF-CMF increases feed intake, milk protein hydrolysis during duodenal digestion, and goblet cells in jejunum.

The present study addresses the effects of dietary Tenebrio molitor (TM) larvae meal inclusion on cytoprotective, cell death pathways, antioxidant defence, and intermediate metabolism in the heart, muscle, and digestive tract of gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax). Three experimental diets were formulated to contain 0%, 25%, or 50% inclusion TM levels. Heat Shock Proteins (HSPs) induction was apparent in both species' muscle at 50% inclusion. Conversely, p44/42 Mitogen-Activated Protein Kinase (MAPK) activation was increased (p < 0:05) in both species' muscle and digestive tract at 25% inclusion. Regarding the apoptotic machinery, TM inclusion exerted no influence on gilthead seabream, while suppression through autophagy may have occurred in the muscle. However, significant apoptosis (p < 0:05) was evident in European sea bass muscle and digestive tract. Both fish species' heart seemed to additionally rely on lipids compared to muscle and digestive tract. In contrast to gilthead seabream, European sea bass exhibited increased (p < 0:05) antioxidant activity at 50% TM inclusion. The present findings highlight the dietary derived induction of cellular responses in a species and tissue-specific manner, whereas European sea bass appears to be more susceptible to TM inclusion.

The use of micro- and nanoparticles in biological applications has dramatically grown during the last few decades due to the ease of protocols development and compatibility with microfluidics devices. Particles can be composed by different materials, i.e., polymers, inorganic dielectrics, and metals. Among them, silica is a suitable material for the development of biosensing applications. Depending on their final application, the surface properties of particles, including silica, are tailored by means of chemical modification or polymeric coating. The latter strategy represents a powerful tool to create a hydrophilic environment that enables the functionalization of particles with biomolecules and the further interaction with analytes. Here, the use of MCP-6, a dimethylacrylamide (DMA)-based ter-copolymer, to coat silica microspheres is presented. MCP-6 offers unprecedented ease of coating, imparting silica particles a hydrophilic coating with antifouling properties that is able to provide high-density immobilization of biological probes.

A fundamental requirement to predict the native conformation, address questions of sequence design and optimization, and gain insights into the folding mechanisms of proteins lies in the definition of an unbiased reaction coordinate that reports on the folding state without the need to compare it to reference values, which might be unavailable for new (designed) sequences. Here, we introduce such a reaction coordinate, which does not depend on previous structural knowledge of the native state but relies solely on the energy partition within the protein: the spectral gap of the pair nonbonded energy matrix (ENergy Gap, ENG). This quantity can be simply calculated along unbiased MD trajectories. We show that upon folding the gap increases significantly, while its fluctuations are reduced to a minimum. This is consistently observed for a diverse set of systems and trajectories. Our approach allows one to promptly identify residues that belong to the folding core as well as residues involved in non-native contacts that need to be disrupted to guide polypeptides to the folded state. The energy gap and fluctuations criteria are then used to develop an automatic detection system which allows us to extract and analyze folding transitions from a generic MD trajectory. We speculate that our method can be used to detect conformational ensembles in dynamic and intrinsically disordered proteins, revealing potential preorganization for binding.

Perfluorinated organic compounds (PFCs) are nontoxic, biocompatible, bioavailable, and bioorthogonal species which possess the unique ability to segregate away from both polar and nonpolar solvents producing a compact fluorophilic phase. Traditional techniques of fluorous chemical proteomics are generally applied to enrich biological samples in target protein(s) exploiting this property of PFCs to build fluorinated probes able to covalently bind to protein ensembles and being selectively extracted by fluorophilic solvents. Aiming at building a strategy able to avoid irreversible modification of the analyzed biosystem, a novel fully noncovalent probe is presented as an enabling tool for the recognition and isolation of biological protein(s). In our strategy, both the fluorophilic extraction and the biorecognition of a selected protein successfully occur via the establishment of reversible but selective interactions.

This review outlines the current state of knowledge on the nutritive value of black cumin (Nigella sativa L.). The popularity of this plant, which is an annual herbaceous plant belonging to the Ranunculaceae family and is native to Iran, Pakistan and Turkey, is due to its beneficial actions. Black cumin has many therapeutic effects and is considered one of the most important medicinal plants in the world because of its antioxidant, anticoccidial, anthelminthic and antimicrobial activities. The nutritive value of black cumin is result of its carbohydrate, fatty acid, protein contents as well as its several bioactive compounds. The seeds or their byproducts can be used in feeds for farm animals, with positive effects on the compositional characteristics of eggs, milk and meat.

Microalgae can produce other metabolites that can be used in the prevention and management of diseases including cancer, microbial infection, oxidative stress, diabetes, and hyperlipidaemia. In addition, microalgae can be associated with negative health effects. This chapter will focus only on bioactive peptides derived from microalgal proteins and will present the current state of the art regarding microalgal biorefinery.

Using a surface forces apparatus and an atomic force microscope, we characterized the adhesive properties of adsorbed layers of two recombinant variants of Perna viridis foot protein 5 (PVFP-5), the main surface-binding protein in the adhesive plaque of the Asian green mussel. In one variant, all tyrosine residues were modified into 3,4-dihydroxy-L-phenylalanine (DOPA) during expression using a residue-specific incorporation strategy. DOPA is a key molecular moiety underlying underwater mussel adhesion. In the other variant, all tyrosine residues were preserved. The layer was adsorbed on a mica substrate and pressed against an uncoated surface. While DOPA produced a stronger adhesion than tyrosine in contact with the nanoscopic Si3N4 probe of the atomic force microscope, the two variants produced comparable adhesion on the curved macroscopic mica surfaces of the surface forces apparatus. These findings show that the presence of DOPA is not a sufficient condition to generate strong underwater adhesion. Surface chemistry and contact geometry affect the strength and abundance of protein-surface bonds created during adsorption and surface contact. Importantly, the adsorbed protein layer has a random and dynamic polymer-network structure that should be optimized to transmit the tensile stress generated during surface separation to DOPA surface bonds rather than other weaker bonds.

Non-specific lipid transfer proteins (nsLTPs) are characterized by an eight-cysteine motif backbone that is stabilized by four disulphide bonds. The strong interest towards this protein family is mainly due to the fact that nsLTPs are involved in many biological processes and have been identified as major human allergens. Since tomato (Solanum lycopersicum L.) is one of the most consumed and allergenic vegetables, a full characterization of this family is needed. In this study, hidden Markov model profiles were used to identify nsLTPs within the tomato protein complement. Following manual curation, 64 nsLTP genes were classified into six sub-families. Furthermore, nsLTP gene structure, distribution and arrangement along tomato chromosomes were investigated. Available RNA-seq expression profile data and Real-Time PCR analyses were used to derive expression patterns of tomato nsLTPs in different tissues/ organs. Non-specific LTP genes with high level of expression in tomato fruits were filtered out since they could play a key role in tomato allergenicity. Among these genes was Solyc10g075090 that encodes the allergen Sola l 3. Finally, cloning, heterologous expression, purification and biochemical characterization of the recombinant protein Sola l 3 was performed.

Amyloidosis is a group of diseases occurring in humans and animals, due to the deposition of misfolding proteins in different organs. In humans, deposits were characterized using proteomics and more recently the role of miRNAs in the pathogenesis was proposed. In Abyssinian cat the main target is the kidney. Little is known on the mechanisms underlying the disease. The aim of this study is to profile proteins and miRNAs in healthy and affected Abyssinian cats, to evaluate their differential expression and clarify the pathogenesis mechanisms. Formalin-fixed paraffin-embedded kidney slices were collected from 7 affected and 5 healthy Abyssinians and used for proteomic and miRNAs analyzes. Peptides were analyzed with an LTQ-Orbitrap Velos mass spectrometer (MS). MS spectra were searched against the F.catus NCBI sequence database (release31.01.2017) by MaxQuant. Bioinformatic analysis was performed with DAVID and Panther softwares. MiRNAs were sequenced on the Illumina NextSeq500 platform. MiRDeep2 was used to map reads on the genome vs9.0, identify putative miRNAs, quantify their expression and identify homologous. Filtered proteins and miRNs statistically different were identified using a student t-test and a moderate t-test respectively (p- value<=.05). Part of the proteins was exclusively detected in the affected (n.175), part in the healthy (n.47) and part were common to the two groups (n.160). A fraction of the latter resulted upregulated (n.16) or down regulated (n.18) in affected compared to the healthy cats (p-value<=.05). Annotation and functional grouping suggested an effect on extracellular matrix and macromolecular complex subunit organization. MiRNA analysis detected 341 representatives, 22 differentially expressed between affected and healthy (p<.05). Six miRNAs out of 22 (four with a P-value<.009) are known to be involved in Alzheimer Disease. Interestingly, miR-26a-5p (P-value 0.120) is involved in the human immunoglobulin light chain amyloidosis onset. This study identified different miRNA and protein renal compositions in Abyssinian affected and healthy cats. The pathways involving these molecules are under investigation, providing new insights for the pathogenesis understanding.

The endoplasmic reticulum (ER) is a continuous cell-wide membrane network. Network formation has been associated with proteins producing membrane curvature and fusion, such as reticulons and atlastin. Regulated network fragmentation, occurring in different physiological contexts, is less understood. Here we find that the ER has an embedded fragmentation mechanism based upon the ability of reticulon to produce fission of elongating network branches. In Drosophila, Rtnl1-facilitated fission is counterbalanced by atlastin-driven fusion, with the prevalence of Rtnl1 leading to ER fragmentation. Ectopic expression of Drosophila reticulon in COS-7 cells reveals individual fission events in dynamic ER tubules. Consistently, in vitro analyses show that reticulon produces velocity-dependent constriction of lipid nanotubes leading to stochastic fission via a hemifission mechanism. Fission occurs at elongation rates and pulling force ranges intrinsic to the ER, thus suggesting a principle whereby the dynamic balance between fusion and fission controlling organelle morphology depends on membrane motility.

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In an attempt to produce a SERS-active and low cost sensor for label free detection of trace amounts and small volumes of biomolecule samples, silver nanowires with high aspect ratio were implemented in a flow-through self assembly process on hydrophobic PTFE membrane. This bottom up assembly was followed by spot arrays design and fabrication through laser patterning of the silver nanostructured surface, leading to the formation of circular spots of defined size aimed at both concentrating microliter volumes of aqueous solution droplets and enhancing the Raman signal of micromolar amounts of biomolecules. The spot arrays so produced were efficiently tested as SERS sensors via direct detection, i.e. by direct measurement of the analyte molecular Raman fingerprint, with several proteins and biomarkers of neurodegenerative diseases and they showed excellent reproducibility and sensitivity. The specificity of the detection system is intrinsic in the Raman spectroscopic signal, which enables a structural characterization of the biomolecules while a rapid and effective detection is perfomed. The silver spot arrays can also represent a versatile label free SERS platform for indirect biomolecular detection, thanks to the simple and easy functionalization of the silver spots by thiolated small molecules and receptors.

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Despite the large variety of dyes reported so far, the combination of large Stokes shift, high molar extinction coefficient and high quantum yield (QY) to give high brightness values in aqueous solution,1 as well as low cytotoxicity in the same molecule is still challenging. Among polyaromatic compounds, spirobifluorene-based emitters attract growing interest due to chemical stability and versatility allowing their use in several technological applications.2 Nonetheless, the lack of water solubility precluded the application of spirobifluorene compounds in biological field to date. Here, we disclose the synthetic approach to obtain the first water soluble spirobifluorene-based fluorescent dye and its interaction with proteins as model system for biological purposes.3 The dye is characterized by high blue-greenish photoluminescence QY (50-70%) and very large Stokes shift (> 6000 cm-1) in both organic and aqueous solution. Noteworthy, in presence of bovine serum albumin (BSA) the emission peak shows a hypsochromic shift with an impressive increased QY in water from 50% to 95%. The outstanding detection limit of BSA and the effect of BSA:dye interactions and local microenvironment on the emission will be also presented. Investigations on cellular uptake and cytotoxicity revealed that this chromophore is biocompatible and taken up by living cells, indicating the potential application for live cell imaging. To conclude, we show the earliest example of a new class of spirobifluorene-based emitters with outstanding fluorescent properties in water, opening novel scenario for bio-applications.

Fluorescent markers suitable for cellular organelles or biomolecules staining require a series of features, like large Stokes shift, high molar extinction coefficient (e) and high quantum yield (QY) to give high brightness values in aqueous solution,[1] as well as low cytotoxicity. Despite the large variety of dyes reported so far, the combination of the whole set of requirements in the same molecule is still challenging. Among polyaromatic compounds, spirobifluorene-based emitters attract growing interest due to chemical stability and versatility allowing their use in several technological applications.[2] Nonetheless, the lack of water solubility precluded the application of spirobifluorene compounds in biological field to date. Here, we disclose the synthesis and photophysical properties of the first water soluble spirobifluorene-based fluorescent dye and its interaction with proteins as model system for biological purposes.[3] The dye is characterized by high blue-greenish photoluminescence QY (50-70%) and very large Stokes shift (> 6000 cm-1) in both organic and aqueous solution. Noteworthy, in presence of bovine serum albumin (BSA) the emission peak shows a hypsochromic shift with increased QY in water from 50% to 95%. By using this dye as non covalent probe for BSA, we were able to reach the outstanding detection limit of 7.3 nM in a wide range with small concentration of dye. The effect of the BSA:dye interactions and the local microenvironment on the emission will be also presented. Investigations on cellular uptake and cytotoxicity revealed that this chromophore is biocompatible and taken up by living cells, indicating the potential application for live cell imaging. To conclude, we show the earliest example of a new class of spirobifluorene-based emitters with outstanding fluorescent properties in water, opening novel scenario for bio applications.

An advanced optofluidic system for protein detection based on Raman signal amplification via dewetting and molecular gathering within temporary mesoscale assemblies is presented. The evaporation of a microliter volume of protein solution deposited in a circular microwell precisely follows an outward-receding geometry. Herein the combination of liquid withdrawal with intermolecular interactions induces the formation of self-assembled molecular domains at the solid-liquid interface. Through proper control of the evaporation rate, amplitude of the assemblies and time for spectral collection at the liquid edge are extensively raised, resulting in a local enhancement and refinement of the Raman response, respectively. Further signal amplification is obtained by taking advantage of the intense local electromagnetic fields generated upon adding a plasmonic coating to the microwell. Major advantages of this optofluidic method lie in the obtainment of high-quality, high-sensitivity Raman spectra with detection limit down to sub-micromolar values. Peculiarly, the assembled proteins in the liquid edge region maintain their native-like state without displaying spectral changes usually occurring when dried drop deposits are considered.

The term "Shankopathies" identifies neurodevelopmental diseases, such as autism Spectrum Disorders (ASD), Intellectual Disability (ID), and schizophrenia (SCZ) caused by deletion or mutations of SHANK/ProSAP genes. The three SHANK genes code for a postsynaptic scaffold protein which has a main function of regulating synaptic formation, development and plasticity. The review summarizes the major genetic, molecular and electrophysiological studies that provide new information about the function of Shanks proteins and are prodromic in identifying therapeutic approaches, pharmacological targets for treating patients with SHANK deletions and mutations and eventually for other patients affected by neuropsychiatric and neurodevelopmental disorders.

The Mediterranean fruitfly Ceratitis capitata (medfly) is an invasive agricultural pest of high economic impact and has become an emerging model for developing new genetic control strategies as an alternative to insecticides. Here, we report the successful adaptation of CRISPR-Cas9-based gene disruption in the medfly by injecting in vitro pre-assembled, solubilized Cas9 ribonucleoprotein complexes (RNPs) loaded with gene-specific single guide RNAs (sgRNA) into early embryos. When targeting the eye pigmentation gene white eye (we), a high rate of somatic mosaicism in surviving G0 adults was observed. Germline transmission rate of mutated we alleles by G0 animals was on average above 52%, with individual cases achieving nearly 100%. We further recovered large deletions in the we gene when two sites were simultaneously targeted by two sgRNAs. CRISPR-Cas9 targeting of the Ceratitis ortholog of the Drosophila segmentation paired gene (Ccprd) caused segmental malformations in late embryos and in hatched larvae. Mutant phenotypes correlate with repair by non-homologous end-joining (NHEJ) lesions in the two targeted genes. This simple and highly effective Cas9 RNP-based gene editing to introduce mutations in C. capitata will significantly advance the design and development of new effective strategies for pest control management.