Characterization of Polyphenol Composition and Starch and Protein Structure in Brown Rice Flour, Black Rice Flour and Their Mixtures.

The study investigates the structural and chemical properties of brown rice flour (WRF), black rice flour (BRF) and their mixtures in ratios of 25%, 50% and 75% to provide reference information for the gluten-free bakery industry. BRF contains higher concentrations of proteins, lipids, total minerals, crude fiber, total polyphenols, proanthocyanidins and flavonoids than WRF. A higher amylose content in BRF than in WRF resulted in flour mixtures with slower starch digestion and a lower glycemic response depending on the BRF ratio added. Differences in the chemical composition of WRF and BRF led to improved composition of the flour mixtures depending on the BRF ratio. The presence of anthocyanidins and phenolic acids in higher concentrations in the BRF resulted in a red-blue color shift within the flour mixtures. The deconvoluted FTIR spectra showed a higher proportion of α-helixes in the amide I band of BRF proteins, indicating their tighter folding. An analysis of the FTIR spectra revealed a more compact starch structure in BRF than in WRF. By processing reflection spectra, nine optically active compound groups were distinguished in rice flour, the proportion in BRF being 83.02% higher than in WRF. Due to co-pigmentation, the bathochromic shift to higher wavelengths was expressed by the proanthocyanins and phenolic acids associated with the wavelengths 380 nm to 590 nm and at 695 nm. Anthocyanins, protein-tannin complexes, methylated anthocyanins and acylated anthocyanins, associated with wavelengths 619, 644 and 668 nm, exhibited a hypsochromic effect by shifting the wavelengths to lower values. This research represents a first step in the development of rice-based products with increased nutritional value and a lower glycemic index.

Whole Black Rice Flour Improves the Physicochemical, Glycemic, and Sensory Properties of Cracker Snacks.

The present study describes the enhancement of the nutritional values of gluten-free rice crackers by adding whole black rice grain flour. The crackers were prepared by combining whole brown rice flour (WRF) and whole black rice flour (BRF) in ratios of 0% (WRC), 25% (25-BRC), 50% (50-BRC), 75% (75-BRC), and 100% (BRC). The resulting samples underwent in-vivo effects on postprandial blood glucose levels as well as physicochemical and sensory analysis. In comparison to WRC, the samples containing 100% added black rice flour presented higher nutritional qualities in terms of protein, by 16.61%, 8.64% for lipids, 5.61% for ash, 36.94% for crude fiber, 58.04% for total polyphenols, 95.49% for proanthocyanidins, and 88.07% for flavonoids. The addition of BRF had a suppressing effect on lightness (L*) and yellowness (b*), while redness (a*) increased. The results of the glycemic measurements confirmed that consumption of crackers made from brown or black whole-grain rice grain flour does not generate glycemic peaks above the limit of 30 mg/dL in baseline blood glucose levels. The results of developing rice crackers from black and brown flour blends showed promising physicochemical and nutritional properties and could provide a good alternative to wheat flour as a gluten-free product.

Identification of Triazolopyrimidinyl Scaffold SARS-CoV-2 Papain-Like Protease (PLpro) Inhibitor.

The global impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its companion disease, COVID-19, has reminded us of the importance of basic coronaviral research. In this study, a comprehensive approach using molecular docking, in vitro assays, and molecular dynamics simulations was applied to identify potential inhibitors for SARS-CoV-2 papain-like protease (PLpro), a key and underexplored viral enzyme target. A focused protease inhibitor library was initially created and molecular docking was performed using CmDock software (v0.2.0), resulting in the selection of hit compounds for in vitro testing on the isolated enzyme. Among them, compound 372 exhibited promising inhibitory properties against PLpro, with an IC50 value of 82 ± 34 µM. The compound also displayed a new triazolopyrimidinyl scaffold not yet represented within protease inhibitors. Molecular dynamics simulations demonstrated the favorable binding properties of compound 372. Structural analysis highlighted its key interactions with PLpro, and we stress its potential for further optimization. Moreover, besides compound 372 as a candidate for PLpro inhibitor development, this study elaborates on the PLpro binding site dynamics and provides a valuable contribution for further efforts in pan-coronaviral PLpro inhibitor development.

Synthesis and Cholinesterase Inhibitory Activity of Selected Indole-Based Compounds.

Synthesis and anticholinesterase activity of 18 previously unpublished indole- and tryptophan-derived compounds are disclosed. These sp3-rich compounds containing an indole structural unit exhibit selective submicromolar inhibition of human butyrylcholinesterase (hBChE). The structures of the newly synthesized compounds were confirmed by 1H and 13C NMR, IR spectroscopy, and high-resolution mass spectrometry.

The Release of Grape Pomace Phenolics from Alginate-Based Microbeads during Simulated Digestion In Vitro: The Influence of Coatings and Drying Method.

Grape pomace is a byproduct of wineries and a sustainable source of bioactive phenolic compounds. Encapsulation of phenolics with a well-chosen coating may be a promising means of delivering them to the intestine, where they can then be absorbed and exert their health-promoting properties, including antioxidant, anti-inflammatory, anticancer, cardioprotective, and antimicrobial effects. Ionic gelation of grape pomace extract with natural coatings (sodium alginate and its combination with maltodextrins, gelatin, chitosan, gums Tragacanth and Arabic) was performed, and the resulting hydrogel microbeads were then air-, vacuum-, and freeze-dried to prevent spoilage. Freeze-drying showed advantages in preserving the geometrical parameters and morphology of the microbeads compared to other drying techniques. A good relationship was found between the physicochemical properties of the dried microbeads and the in vitro release of phenolics. Freeze-dried microbeads showed the highest cumulative release of phenols in the intestinal phase (23.65-43.27 mgGAE/gMB), while the most suitable release dynamics in vitro were observed for alginate-based microbeads in combination with gelatin, gum Arabic, and 1.5% (w/v) chitosan. The results highlight the importance of developing encapsulated formulations containing a natural source of bioactive compounds that can be used in various functional foods and pharmaceutical products.

Naive Prediction of Protein Backbone Phi and Psi Dihedral Angles Using Deep Learning.

Protein structure prediction represents a significant challenge in the field of bioinformatics, with the prediction of protein structures using backbone dihedral angles recently achieving significant progress due to the rise of deep neural network research. However, there is a trend in protein structure prediction research to employ increasingly complex neural networks and contributions from multiple models. This study, on the other hand, explores how a single model transparently behaves using sequence data only and what can be expected from the predicted angles. To this end, the current paper presents data acquisition, deep learning model definition, and training toward the final protein backbone angle prediction. The method applies a simple fully connected neural network (FCNN) model that takes only the primary structure of the protein with a sliding window of size 21 as input to predict protein backbone ϕ and ψ dihedral angles. Despite its simplicity, the model shows surprising accuracy for the ϕ angle prediction and somewhat lower accuracy for the ψ angle prediction. Moreover, this study demonstrates that protein secondary structure prediction is also possible with simple neural networks that take in only the protein amino-acid residue sequence, but more complex models are required for higher accuracies.

Design and synthesis of amino-substituted N-arylpiperidinyl-based inhibitors of the (immuno)proteasome.

The constitutive proteasome and the immunoproteasome represent validated targets for pharmacological intervention in the context of various diseases, such as cancer, inflammation, and autoimmune diseases. The development of novel chemical scaffolds of non-peptidic nature, capable of inhibiting different catalytically active subunits of both isoforms, is a viable approach against these diseases. Such compounds are also useful as leads for the development of biochemical probes that enable the studies of the roles of both isoforms in various biological contexts. Here, we present a ligand-based computational design of (immuno)proteasome inhibitors, which resulted in the amino-substituted N-arylpiperidine-based compounds that can inhibit different subunits of the (immuno)proteasome in the low micromolar range. The compounds represent a useful starting point for further structure-activity relationship studies that will, hopefully, lead to non-peptidic compounds that could be used in pharmacological and biochemical studies of both proteasomes.

Design of Tetra-Peptide Ligands of Antibody Fc Regions Using In Silico Combinatorial Library Screening.

Peptides, or short chains of amino-acid residues, are becoming increasingly important as active ingredients of drugs and as crucial probes and/or tools in medical, biotechnological, and pharmaceutical research. Situated at the interface between small molecules and larger macromolecular systems, they pose a difficult challenge for computational methods. We report an in silico peptide library generation and prioritization workflow using CmDock for identifying tetrapeptide ligands that bind to Fc regions of antibodies that is analogous to known in vitro recombinant peptide libraries' display and expression systems. The results of our in silico study are in accordance with existing scientific literature on in vitro peptides that bind to antibody Fc regions. In addition, we postulate an evolving in silico library design workflow that will help circumvent the combinatorial problem of in vitro comprehensive peptide libraries by focusing on peptide subunits that exhibit favorable interaction profiles in initial in silico peptide generation and testing.

Identifying Metal Binding Sites in Proteins Using Homologous Structures, the MADE Approach.

In order to identify the locations of metal ions in the binding sites of proteins, we have developed a method named the MADE (MAcromolecular DEnsity and Structure Analysis) approach. The MADE approach represents an evolution of our previous toolset, the ProBiS H2O (MD) methodology, for the identification of conserved water molecules. Our method uses experimental structures of proteins homologous to a query, which are subsequently superimposed upon it. Areas with a particular species present in a similar location among many homologous protein structures are identified using a clustering algorithm. Dense clusters likely represent positions containing species important to the query protein structure or function. We analyze well-characterized apo protein structures and show that the MADE approach can identify clusters corresponding to the expected positions of metal ions in their binding sites. The greatest advantage of our method lies in its generality. It can in principle be applied to any species found in protein records; it is not only limited to metal ions. We additionally demonstrate that the MADE approach can be successfully applied to predict the location of cofactors in computer-modeled structures, e.g., via AlphaFold. We also conduct a careful protein superposition method comparison and find our methodology robust and the results largely independent of the selected protein superposition algorithm. We postulate that with increasing structural data availability, additional applications of the MADE approach will be possible such as non-protein systems, water network identification, protein binding site elaboration, and analysis of binding events, all in a dynamic manner. We have implemented the MADE approach as a plugin for the PyMOL molecular visualization tool. The MADE plugin is available free of charge at https://gitlab.com/Jukic/made_software.

In Vitro Bioaccessibility Assessment of Phenolic Compounds from Encapsulated Grape Pomace Extract by Ionic Gelation.

Grape pomace is a by-product of winemaking characterized by a rich chemical composition from which phenolics stand out. Phenolics are health-promoting agents, and their beneficial effects depend on their bioaccessibility, which is influenced by gastrointestinal digestion. The effect of encapsulating phenol-rich grape pomace extract (PRE) with sodium alginate (SA), a mixture of SA with gelatin (SA-GEL), and SA with chitosan (SA-CHIT) on the bioaccessibility index (BI) of phenolics during simulated digestion in vitro was studied. A total of 27 individual phenolic compounds (IPCs) were quantified by UHPLC. The addition of a second coating to SA improved the encapsulation efficiency (EE), and the highest EE was obtained for SA-CHIT microbeads (56.25%). Encapsulation affected the physicochemical properties (size, shape and texture, morphology, crystallinity) of the produced microbeads, which influenced the delivery of phenolics to the intestine and their BI. Thus, SA-GEL microbeads had the largest size parameters, as confirmed by scanning electron microscopy (SEM), and the highest BI for total phenolic compounds and IPCs (gallic acid, 3,4-dihydroxybenzoic acid and o-coumaric acid, epicatechin, and gallocatechin gallate) ranged from 96.20 to 1011.3%. The results suggest that encapsulated PRE has great potential to be used as a functional ingredient in products for oral administration.

Long-Term Consequences of War Captivity in Military Veterans.

Numerous studies on the health and functioning of veterans and former prisoners of war have shown that the experience of war captivity is one of the most difficult human experiences. Captivity is often characterized by extremely difficult and inhumane conditions, as well as exposure to various forms of both psychological and physical abuse. Such traumatic experiences can lead to serious psychological consequences that can last for years, even decades after release from captivity. The aim of this paper is to present a brief overview of research that points to the specifics of wartime captivity and the long-term psychological consequences in veterans of former camp detainees, as well as the consequences suffered by their families and factors that, apart from the intensity of the trauma, contribute to the emergence and persistence of psychological disorders. From the presented research, it can be concluded that former prisoners of the camp represent an extremely vulnerable group of the social community and require long-term appropriate treatment, while the needs of veterans' families should not be neglected, with the necessity of including spouses and children in psychological and psychosocial treatments.

Physicochemical Characterization and Evaluation of Gastrointestinal In Vitro Behavior of Alginate-Based Microbeads with Encapsulated Grape Pomace Extracts.

Grape pomace is a byproduct of wineries and a rich source of phenolic compounds that can exert multiple pharmacological effects when consumed and enter the intestine where they can then be absorbed. Phenolic compounds are susceptible to degradation and interaction with other food constituents during digestion, and encapsulation may be a useful technique for protecting phenolic bioactivity and controlling its release. Therefore, the behavior of phenolic-rich grape pomace extracts encapsulated by the ionic gelation method, using a natural coating (sodium alginate, gum arabic, gelatin, and chitosan), was observed during simulated digestion in vitro. The best encapsulation efficiency (69.27%) was obtained with alginate hydrogels. The physicochemical properties of the microbeads were influenced by the coatings used. Scanning electron microscopy showed that drying had the least effect on the surface area of the chitosan-coated microbeads. A structural analysis showed that the structure of the extract changed from crystalline to amorphous after encapsulation. The phenolic compounds were released from the microbeads by Fickian diffusion, which is best described by the Korsmeyer-Peppas model among the four models tested. The obtained results can be used as a predictive tool for the preparation of microbeads containing natural bioactive compounds that could be useful for the development of food supplements.

The Influence of the Addition of Hemp Press Cake Flour on the Properties of Bovine and Ovine Yoghurts.

Hemp press cake flour (HPCF) is a by-product of hemp oil production rich in proteins, carbohydrates, minerals, vitamins, oleochemicals, and phytochemicals. The purpose of this study was to investigate how the addition of HPCF to bovine and ovine plain yoghurts at concentrations of 0%, 2%, 4%, 6%, 8%, and 10% could change the physicochemical, microbiological, and sensory properties of the yoghurts, focusing on the improvement of quality and antioxidant activity, and the issue of food by-products and their utilisation. The results showed that the addition of HPCF to yoghurts significantly affected their properties, including an increase in pH and decrease in titratable acidity, change in colour to darker, reddish or yellowish hue, and a rise in total polyphenols and antioxidant activity during storage. Yoghurts fortified with 4% and 6% HPCF exhibited the best sensory properties, thus maintaining viable starter counts in the yoghurts during the study period. There were no statistically significant differences between the control yoghurts and the samples with 4% added HPCF in terms of overall sensory score while maintaining viable starter counts during the seven-day storage. These results suggest that the addition of HPCF to yoghurts can improve product quality and create functional products and may have potential in sustainable food waste management.

Mechanistic Insights of Polyphenolic Compounds from Rosemary Bound to Their Protein Targets Obtained by Molecular Dynamics Simulations and Free-Energy Calculations.

Rosemary represents an important medicinal plant that has been attributed with various health-promoting properties, especially antioxidative, anti-inflammatory, and anticarcinogenic activities. Carnosic acid, carnosol, and rosmanol, as well as the phenolic acid ester rosmarinic acid, are the main compounds responsible for these actions. In our earlier research, we carried out an inverse molecular docking at the proteome scale to determine possible protein targets of the mentioned compounds. Here, we subjected the previously identified ligand-protein complexes with HIV-1 protease, K-RAS, and factor X to molecular dynamics simulations coupled with free-energy calculations. We observed that carnosic acid and rosmanol act as viable binders of the HIV-1 protease. In addition, carnosol represents a potential binder of the oncogene protein K-RAS. On the other hand, rosmarinic acid was characterized as a weak binder of factor X. We also emphasized the importance of water-mediated hydrogen-bond networks in stabilizing the binding conformation of the studied polyphenols, as well as in mechanistically explaining their promiscuous nature.

Barley in the Production of Cereal-Based Products.

Barley (Hordeum vulgare L.) is unjustly neglected today as a food grain. Interest in the use of barley in the food industry has increased recently. The reason for this is its content of dietary fibre, especially ß-glucan, which has been shown to reduce blood cholesterol and lower blood sugar levels. The main nutritional components of barley and barley products, besides the mentioned ß-glucan, are starch, sugar, proteins, fat and ash. Although not common in the production of bakery products, barley can be very easily involved in the production of the same products, and such products have improved nutritional characteristics and acceptable sensory characteristics, which make them desirable. Barley has great potential for use in a wide range of cereal-based foods as a partial or full replacement for currently used grains (such as wheat, oats, rice and corn). This article provides basic and general information about the use of barley in food and the processing of barley grains for use in the manufacturing of cereal-based products, with particular attention to the use of barley in the manufacturing of bread (flatbread and leavened bread), noodles and pasta, muffins and cakes and cookies and biscuits.

The Effect of the Ala16Val Mutation on the Secondary Structure of the Manganese Superoxide Dismutase Mitochondrial Targeting Sequence.

Manganese Superoxide Dismutase (MnSOD) represents a mitochondrial protein that scavenges reactive oxygen species (ROS) responsible for oxidative stress. A known single nucleotide polymorphism (SNP) rs4880 on the SOD2 gene, causing a mutation from alanine to valine (Ala16Val) in the primary structure of immature MnSOD, has been associated with several types of cancer and other autoimmune diseases. However, no conclusive correlation has been established yet. This study aims to determine the effect of the alanine to valine mutation on the secondary structure of the MnSOD mitochondrial targeting sequence (MTS). A model for each variant of the MTS was prepared and extensively simulated with molecular dynamics simulations using the CHARMM36m force field. The results indicate that the alanine variant of the MTS preserves a uniform α-helical secondary structure favorable for the protein transport into mitochondria, whereas the valine variant quickly breaks down its α-helix. Thus, the alanine MTS represents the more active MnSOD variant, the benefits of which have yet to be determined experimentally.

Conserved Water Networks Identification for Drug Design Using Density Clustering Approaches on Positional and Orientational Data.

This work describes the development and testing of a method for the identification and classification of conserved water molecules and their networks from molecular dynamics (MD) simulations. The conserved waters in the active sites of proteins influence protein-ligand binding. Recently, several groups have argued that a water network formed from conserved waters can be used to interpret the thermodynamic signature of the binding site. We implemented a novel methodology in which we apply the complex approach to categorize water molecules extracted from the MD simulation trajectories using clustering approaches. The main advantage of our methodology as compared to current state of the art approaches is the inclusion of the information on the orientation of hydrogen atoms to further inform the clustering algorithm and to classify the conserved waters into different subtypes depending on how strongly certain orientations are preferred. This information is vital for assessing the stability of water networks. The newly developed approach is described in detail as well as validated against known results from the scientific literature including comparisons with the experimental data on thermolysin, thrombin, and Haemophilus influenzae virulence protein SiaP as well as with the previous computational results on thermolysin. We observed excellent agreement with the literature and were also able to provide additional insights into the orientations of the conserved water molecules, highlighting the key interactions which stabilize them. The source code of our approach, as well as the utility tools used for visualization, are freely available on GitHub.

Effect of Tomato Pomace Addition on Chemical, Technological, Nutritional, and Sensorial Properties of Cream Crackers.

The aim of this research was to determine the influence of tomato pomace (TP) addition on the chemical, nutritional, and technological characteristics of cream crackers made from wheat flour and 4%, 6%, 8%, and 10% TP. The TP-enriched cream crackers showed progressively increasing ash (from 0.69 of the control to 1.22 g/100 g dry matter of the 10% TP sample), fat (from 11.39 to 13.04 g/100 g), protein (from 13.53 to 15.60 g/100 g), total dietary fibre (from 4.08 to 7.80), carotenoids (from 0.55 to 8.56 mg/kg), tocols (from 57.59 to 71.63 mg/kg), free phenolic acids (from 100.08 to 277.37 mg/kg), free flavonoids (from 0.0 to 45.28 mg/kg), bound flavonoids (from 0.0 to 27.71 mg/kg), and fatty acids contents, antioxidant activity and dough viscosity. The colour coordinates increased via augmenting the amounts of TP. Thickness, volume, and specific volume decreased gradually with increasing TP; the enrichment reduced cracker hardness from 65.42 N (control) to 26.28 N (crackers with 10% TP), while the snapping force rose. Cream crackers with 8% TP showed the best sensory quality. Tomato pomace addition improves the nutritional quality of foods; furthermore, its recycling will help to solve the problems linked to the disposal of this industry waste.

A Cross-Sectional Study of Psychiatric Comorbidity in Croatian Homeland War Veterans Who Were Held as Prisoners of War and Are Affected by Posttraumatic Stress Disorder.

BACKGROUND: As a extremely traumatic experience, captivity may cause other mental disorders in addition to posttraumatic stress disorder, which is highly prevalent among ex-prisoners of war, and which often occurs in comorbidity with at least one other mental disorder. This objective of this study is to identify the incidence of comorbid mental disorders in Homeland war veterans ex-prisoners of war affected by posttraumatic stress disorder, as well as to identify the factors that influenced psychiatric comorbidity. SUBJECTS AND METHODS: The study sample comprised 264 subjects, all of whom were Croatian Homeland War veterans with combat experience in the defence of the Republic of Croatia, and all of whom fulfilled clinical criteria for posttraumatic stress disorder at the time of the study. The subjects were divided into two groups: the experimental group was composed of ex-prisoners of war, and the control group of veterans who had never been prisoners of war. The methods of sociodemographic questionnaire, posttraumatic stress disorder self-report checklist and the Harvard Trauma Questionnaire were used in the study. Psychiatric comorbidity data were retrieved from the subjects' anamnesis and medical records. RESULTS: The results showed that ex-prisoners of war were exposed to a statistically much higher number of traumatic events, and had a significantly higher total number of psychiatric comorbidities (p<0.01) than the control group. The incidence of acute and transient psychotic disorders, generalized anxiety disorders and psychological and behavioural factors associated with disorders or diseases classified elsewhere was significantly higher among ex-prisoners of war. There was no statistically significant difference in overall posttraumatic stress disorder intensity between the two groups (p<0.05). CONCLUSIONS: The results of the study confirm our hypothesis that the incidence of psychiatric comorbidity is higher in ex-prisoners of war.

New inhibitors of cathepsin V impair tumor cell proliferation and elastin degradation and increase immune cell cytotoxicity.

Cathepsin V is a human lysosomal cysteine peptidase with specific functions during pathological processes and is as such a promising therapeutic target. Peptidase inhibitors represent powerful pharmacological tools for regulating excessive proteolytic activity in various diseases. Cathepsin V is highly related to cathepsin L but differs in tissue distribution, binding site morphology, substrate specificity, and function. To validate its therapeutic potential and extend the number of potent and selective cathepsin V inhibitors, we used virtual high-throughput screening of commercially available compound libraries followed by an evaluation of kinetic properties to identify novel potent and selective cathepsin V inhibitors. We identified the ureido methylpiperidine carboxylate derivative, compound 7, as a reversible, selective, and potent inhibitor of cathepsin V. It also exhibited the most preferable characteristics for further evaluation with in vitro functional assays that simulate the processes in which cathepsin V is known to play an important role. Compound 7 exerted significant effects on cell proliferation, elastin degradation, and immune cell cytotoxicity. The latter was increased because compound 7 impaired conversion of immunosuppressive factor cystatin F to its active monomeric form. Taken together, our results present novel potent inhibitors of cathepsin V and provide new hit compounds for detailed development and optimization. Further, we demonstrate that cathepsin V is a potential target for new approaches to cancer therapy.