Native Molecule Research Articles

Prion-like Spreading of Disease in TDP-43 Proteinopathies

TDP-43 is a ubiquitous nuclear protein that plays a central role in neurodegenerative disorders collectively known as TDP-43 proteinopathies. Under physiological conditions, TDP-43 is primarily localized to the nucleus, but in its pathological form it aggregates in the cytoplasm, contributing to neuronal death. Given its association with numerous diseases, particularly ALS and FTLD, the mechanisms underlying TDP-43 aggregation and its impact on neuronal function have been extensively investigated. However, little is still known about the spreading of this pathology from cell to cell. Recent research has unveiled the possibility that TDP-43 may possess prion-like properties. Specifically, misfolded TDP-43 aggregates can act as templates inducing conformational changes in native TDP-43 molecules and propagating the misfolded state across neural networks. This review summarizes the mounting and most recent evidence from in vitro and in vivo studies supporting the prion-like hypothesis and its underlying mechanisms. The prion-like behavior of TDP-43 has significant implications for diagnostics and therapeutics. Importantly, emerging strategies such as small molecule inhibitors, immunotherapies, and gene therapies targeting TDP-43 propagation offer promising avenues for developing effective treatments. By elucidating the mechanisms of TDP-43 spreading, we therefore aim to pave the way for novel therapies for TDP-43-related neurodegenerative diseases.

Variability in individual native fibrin fiber mechanics

Fibrin fibers are important structural elements in blood coagulation. They form a mesh network that acts as a scaffold and imparts mechanical strength to the clot. A review of published work measuring the mechanics of fibrin fibers reveals a range of values for fiber extensibility. This study investigates fibrinogen concentration as a variable responsible for variability in fibrin mechanics. It expands previous work to describe the modulus, strain hardening, extensibility, and the force required for fiber failure when fibers are formed with different fibrinogen concentrations using lateral force atomic force microscopy. Analysis of the mechanical properties showed fibers formed from 1 mg ml-1and 2 mg ml-1fibrinogen had significantly different mechanical properties. To help clarify our findings we developed two behavior profiles to describe individual fiber mechanics. The first describes a fiber with low initial modulus and high extensible, that undergoes significant strain hardening, and has moderate strength. Most fibers formed with 1 mg ml-1fibrinogen had this behavior profile. The second profile describes a fiber with a high initial modulus, minimal strain hardening, high strength, and low extensibility. Most fibrin fibers formed with 2 mg ml-1fibrinogen were described by this second profile. In conclusion, we see a range of behaviors from fibers formed from native fibrinogen molecules but various fibrinogen concentrations. Potential differences in fiber formation are investigated with SEM. It is likely this range of behaviors also occursin vivo. Understanding the variability in mechanical properties could contribute to a deeper understanding of pathophysiology of coagulative disorders.

Lactoferrin: a secret weapon in the war against pathogenic bacteria

The excessive use of antibiotics to treat bacterial infectious diseases in all living beings has caused a global epidemic of bacterial resistance to antibiotics, leading to the emergence of multidrug-resistant and pandrug-resistant strains. In 2019, the World Health Organization (WHO) reported that antimicrobial resistance causes at least 700,000 deaths per year worldwide. Therefore, in this global war against microorganisms, a therapeutic alternative is necessary to help us win this battle. A key in this race against the clock could be lactoferrin (Lf), a cationic glycoprotein of the mammalian innate immune system that is highly conserved among mammals. Lf is a multifunctional glycoprotein with immunomodulatory, anticarcinogenic, wound-healing, antioxidant, antimicrobial, and bone regeneration properties, in addition to improving the gut microbiota. Lf limits the growth of microorganisms through the sequestration of iron but can also interact directly with some components of the outer membrane of Gram-negative bacteria or bind to teichoic acids in Gram-positive bacteria, destabilizing the membrane and resulting in lysis. Moreover, cleavage of the Lf molecule could promote the production of lactoferricins (Lfcins) and lactoferrampin (Lfampin) from the N-terminal end, which are known to often have stronger antimicrobial effects than the native molecule, as well as analogous peptides, such as HLopt2, which have also shown enhanced antimicrobial activity. Bovine Lf (bLf) has been approved by the US Food and Drug Administration (FDA), and the European Food Safety Authority for its use as a dietary supplement in food products. Because of its effectiveness, accessibility, low cost, and nontoxicity, Lf could be a promising alternative for preventing or treating infections in animals and humans.

Precision DNA methylation typing via hierarchical clustering of Nanopore current signals and attention-based neural network.

Decoding DNA methylation sites through nanopore sequencing has emerged as a cutting-edge technology in the field of DNA methylation research, as it enables direct sequencing of native DNA molecules without the need for prior enzymatic or chemical treatments. During nanopore sequencing, methylation modifications on DNA bases cause changes in electrical current intensity. Therefore, constructing deep neural network models to decode the electrical signals of nanopore sequencing has become a crucial step in methylation site identification. In this study, we utilized nanopore sequencing data containing diverse DNA methylation types and motif sequence diversity. We proposed a feature encoding method based on current signal clustering and leveraged the powerful attention mechanism in the Transformer framework to construct the PoreFormer model for identifying DNA methylation sites in nanopore sequencing. The model demonstrated excellent performance under conditions of multi-class methylation and motif sequence diversity, offering new insights into related research fields.

Biochemical and Epigenetic Regulation of Glutamate Metabolism in Maize (Zea mays L.) Leaves under Salt Stress.

The effect of salt stress (150 mM NaCl) on the expression of genes, methylation of their promoters, and enzymatic activity of glutamate dehydrogenase (GDH), glutamate decarboxylase (GAD), and the 2-oxoglutarate (2-OG)-dehydrogenase (2-OGDH) complex was studied in maize (Zea mays L.). GDH activity increased continuously under salt stress, being 3-fold higher after 24 h. This was accompanied by the appearance of a second isoform with lower electrophoretic mobility. The expression of the Gdh1 gene strongly increased after 6-12 h of incubation, which corresponded to the demethylation of its promoter, while Gdh2 gene expression slightly increased after 2-6 h and then decreased. GAD activity gradually increased in the first 12 h, and then returned to the control level. This corresponded to the increase of Gad expression and its demethylation. Salt stress led to a 2-fold increase in the activity of 2-OGDH during the first 6 h of NaCl treatment, then the activity returned to the control level. Expression of the genes Ogdh1 and Ogdh3 peaked after 1-2 h of incubation. After 6-8 h with NaCl, the expression of these genes declined below the control levels, which correlated with the higher methylation of their promoters. We conclude that salt stress causes a redirection of the 2-OG flux to the γ-aminobutyric acid shunt via its amination to glutamate, by altering the expression of the Gdh1 and Gdh2 genes, which likely promotes the assembly of the native GDH molecule having a different subunit composition and greater affinity for 2-OG.

Enhanced detection of RNA modifications and read mapping with high-accuracy nanopore RNA basecalling models.

In recent years, nanopore direct RNA sequencing (DRS) became a valuable tool for studying the epitranscriptome, due to its ability to detect multiple modifications within the same full-length native RNA molecules. While RNA modifications can be identified in the form of systematic basecalling 'errors' in DRS datasets, N6-methyladenosine (m6A) modifications produce relatively low 'errors' compared to other RNA modifications, limiting the applicability of this approach to m6A sites that are modified at high stoichiometries. Here, we demonstrate that the use of alternative RNA basecalling models, trained with fully unmodified sequences, increases the 'error'signal of m6A, leading to enhanced detection and improved sensitivity even at low stoichiometries. Moreover, we find that high-accuracy alternative RNA basecalling models can show up to 97% median basecalling accuracy, outperforming currently available RNA basecalling models, which show 91% median basecalling accuracy. Notably, the use of high-accuracy basecalling models is accompanied by a significant increase in the number of mapped reads -especially in shorter RNA fractions- and increased basecalling error signatures at pseudouridine (Ψ) and N1-methylpseudouridine (m1Ψ) modified sites. Overall, our work demonstrates that alternative RNA basecalling models can be used to improve the detection of RNA modifications, read mappability, and basecalling accuracy in nanopore DRS datasets.

The separation between mRNA-ends is more variable than expected.

Effective circularization of mRNA molecules is a key step for the efficient initiation of translation. Research has shown that the intrinsic separation of the ends of mRNA molecules is rather small, suggesting that intramolecular arrangements could provide this effective circularization. Considering that the innate proximity of RNA ends might have important unknown biological implications, we aimed to determine whether the close proximity of the ends of mRNA molecules is a conserved feature across organisms and gain further insights into the functional effects of the proximity of RNA ends. To do so, we studied the secondary structure of 274 full native mRNA molecules from 17 different organisms to calculate the contour length (CL) of the external loop as an index of their end-to-end separation. Our computational predictions show bigger variations (from 0.59 to 31.8 nm) than previously reported and also than those observed in random sequences. Our results suggest that separations larger than 18.5 nm are not favored, whereas short separations could be related to phenotypical stability. Overall, our work implies the existence of a biological mechanism responsible for the increase in the observed variability, suggesting that the CL features of the exterior loop could be relevant for the initiation of translation and that a short CL could contribute to the stability of phenotypes.

Human NGF "Painless" Ocular Delivery for Retinitis Pigmentosa: An In Vivo Study.

Retinitis pigmentosa (RP) is a family of genetically heterogeneous diseases still without a cure. Despite the causative genetic mutation typically not expressed in cone photoreceptors, these cells inevitably degenerate following the primary death of rods, causing blindness. The reasons for the "bystander" degeneration of cones are presently unknown but decrement of survival factors, oxidative stress, and inflammation all play a role. Targeting these generalized biological processes represents a strategy to develop mutation-agnostic therapies for saving vision in large populations of RP individuals. A classical method to support neuronal survival is by employing neurotrophic factors, such as NGF. This study uses painless human NGF (hNGFp), a TrkA receptor-biased variant of the native molecule with lower affinity for nociceptors and limited activity as a pain inducer; the molecule has identical neurotrophic power of the native form but a reduced affinity for the p75NTR receptors, known to trigger apoptosis. hNGFp has a recognized activity on brain microglial cells, which are induced to a phenotype switch from a highly activated to a more homeostatic configuration. hNGFp was administered to RP-like mice in vivo with the aim of decreasing retinal inflammation and also providing retinal neuroprotection. However, the ability of this treatment to counteract the bystander degeneration of cones remained limited.

Biomimetic Hyaluronan Binding Biomaterials to Capture the Complex Regulation of Hyaluronan in Tissue Development and Function.

Within native ECM, Hyaluronan (HA) undergoes remarkable structural remodeling through its binding receptors and proteins called hyaladherins. Hyaladherins contain a group of tandem repeat sequences, such as LINK domains, BxB7 homologous sequences, or 20-50 amino acid long short peptide sequences that have high affinity towards side chains of HA. The HA binding sequences are critical players in HA distribution and regulation within tissues and potentially attractive therapeutic targets to regulate HA synthesis and organization. While HA is a versatile and successful biopolymer, most HA-based therapeutics have major differences from a native HA molecule, such as molecular weight discrepancies, crosslinking state, and remodeling with other HA binding proteins. Recent studies showed the promise of HA binding domains being used as therapeutic biomaterials for osteoarthritic, ocular, or cardiovascular therapeutic products. However, we propose that there is a significant potential for HA binding materials to reveal the physiological functions of HA in a more realistic setting. This review is focused on giving a comprehensive overview of the connections between HA's role in the body and the potential of HA binding material applications in therapeutics and regenerative medicine. We begin with an introduction to HA then discuss HA binding molecules and the process of HA binding. Finally, we discuss HA binding materials anf the future prospects of potential HA binding biomaterials systems in the field of biomaterials and tissue engineering.

Total Biosynthesis of Circular Bacteriocins by Merging the Genetic Engineering and Enzymatic Catalysis.

Circular bacteriocins are known for their structural stability and effective antimicrobial properties, positioning them as potential natural food preservatives. However, their widespread application is impeded by restricted availability. This research developed a total biosynthesis platform for circular bacteriocins, with a focus on AS-48 by involving recombinant production of the linear precursor in Escherichia coli, followed by enzymatic cyclization of the precursor into cyclic AS-48 using the ligase butelase-1 in vitro. An important discovery is that, aside from fusion tags, the C-terminal motif LE and LEKKK also could affect the expression yield of the precursor. This biosynthesis platform is both versatile and high-yielding, achieving yields of 10-20 mg/L of AS-48. Importantly, the biosynthetic AS-48 exhibited a secondary structure and antimicrobial activities comparable to those of the native molecules. As such, this work proposes an effective synthetic approach for circular bacteriocins, facilitating their advancement and application in the food industry.

Impact of nanosilver surface electronic distributions on serum protein interaction and hemocompatibility.

Translation of silver-based nanotechnology "from bench to bedside" requires a deep understanding of the molecular aspects of its biological action, which remains controversial at low concentrations and non-spherical morphologies. Here, we present a hemocompatibility approach based on the effect of the distinctive electronic charge distribution in silver nanoparticles (nanosilver) on blood components. On basis of spectroscopic, volumetric, microscopic, dynamic light scattering measurements, pro-coagulant activity tests and cellular inspection we determine that, at extremely low nanosilver concentrations (0.125 - 2.5 μg mL-1) there is a relevant interaction effect on serum albumin and on red blood cells. The explanation has its origin in the surface charge distribution of nanosilver and their electron-mediated energy transfer mechanism. Prism-shaped nanoparticles, with anisotropic charge distributions, act at the surface level generating a compaction of the native protein molecule, while the spherical nanosilver, by exhibiting isotropic surface charge, generates a polar environment comparable to the solvent. Both morphologies induce aggregation at NPs / BSA ≅ 0.044 molar ratio values without altering the coagulation cascade tests, although the spherical-shaped nanosilver has a negative impact on red blood cells. Overall, our results suggest that the electron distributions of nanosilver, even at extremely low concentrations, are a critical factor influencing the molecular structure of blood proteins and red blood cells' membranes. Isotropic forms of nanosilver should be considered with caution, as they are not always the least harmful.

Determination of the molecular weight of native molecules of γ-hydroxybutyrate dehydrogenase isoforms obtained from maize (Zea mays L.) seedlings by gel chromatography

It is known that under oxygen deficiency the functioning of the tricarboxylic acid cycle is impaired, resulting in the activation of an alternative pathway, the GABA shunt. It maintains the functioning of the citric acid cycle by providing succinic acid. The key phase of this bypass is a reaction catalysed by succinate semialdehyde dehydrogenase (SSADH, EC 1.1.1.16). Under oxygen deficiency, SSADH ceases to function efficiently. This leads to the accumulation of succinic acid semialdehyde in the mitochondrial matrix and its high level adversely affects the plant cell metabolism. γ–hydroxybutyrate dehydrogenase (HBDH, EC 1.1.1.61) is an enzyme belonging to the group of oxidoreductases. It transforms γ–hydroxybutyrate into succinic acid semialdehyde, participating in the process of its detoxification, which is important in the maintenance of plant metabolism under oxygen deficiency. To date, unfortunately, there are no data on the biochemical and kinetic features of γ–hydroxybutyrate dehydrogenase. In this regard, our laboratory developed a method for purification of HBDH from green maize leaves, which makes it possible to study the physicochemical properties of this enzyme. During the study, we obtained γ-hydroxybutyrate dehydrogenase from 7-day-old seedlings of Zea mays L. by a five-stage purification. Homogenised plant material with extracted proteins was subjected to two-stage ammonium sulphate fractionation. The catalytic activity was determined spectrophotometrically at λ=340 nm by the amount of reduced NAD+. To remove ammonium salts, we used gel filtration through Sephadex G-25. Proteins were separated according to their charge by DEAE-Sephacel ion exchange chromatography. The enzyme was desorbed using a linear sodium chloride gradient (100-300 mM). The use of gel chromatography through Sephadex G-200 made it possible to determine the molecular mass of the purified enzyme isoforms. The homogeneity of enzyme preparations was confirmed by electrophoresis in polyacrylamide gel with universal AgNO3 staining. We used the tetrazolium method to confirm that the obtained protein preparations were γ-hydroxybutyrate dehydrogenase. As a result, homogeneous preparations of two isoforms of the enzyme (HBDH1 and HBDH2) were obtained. The first isoform of γ-hydroxybutyrate dehydrogenase was 185.7 times purified with a yield of 10% and had a specific activity of 343.6 U/mg of protein. The purification rate of the second isoform was 209 times with a yield of 7.74%. The specific activity of the obtained preparation was 386.7 U/mg of protein. Using gel chromatography through Sephadex G-200, we determined the molecular mass of native molecules of hydroxybutyrate dehydrogenase. We found that in 7-day-old maize seedlings the investigated enzyme was presented in low-molecular and high-molecular forms: for HBDH1 the Mr value was ⁓60.3 kDa, while for HBDH2 the molecular mass of the enzyme was 286 kDa.

Enhanced stabilisation and reduced fibril forming potential of an amyloidogenic light chain using a variable heavy domain to mimic the homodimer complex.

Light chain amyloidosis (AL), is classified as a plasma cell dyscrasia, whereby a mutant plasma cell multiplies uncontrollably and secretes enormous amounts of immunoglobulin-free light chain (FLC) fragments. These FLCs undergo a process of misfolding and aggregation into amyloid fibrils, that can cause irreversible system-wide damage. Current treatments that focus on depleting the underlying plasma cell clone are often poorly tolerated, particularly in patients with severe cardiac involvement, meaning patient prognosis is poor. An alternative treatment approach currently being explored is the inhibition of FLC aggregation by stabilisation of the native conformer. Here, we aimed to identify and characterise antibody fragments that target FLC domains and promote their stabilisation. Using phage-display screening methods, we identified a variable heavy (VH) domain, termed VH1, targeted towards the FLC. Using differential scanning fluorimetry and surface plasmon resonance, VH1 was characterised to bind and kinetically stabilise an amyloidogenic FLC, whereby a > 5.5 °C increase in thermal stability was noted. This improved stability corresponded to the inhibition of fibril formation, where 10 : 1 LC : VH1 concentration reduced aggregation to baseline levels. X-ray crystallographic structures of the LC : VH1 complex at atomic resolution revealed binding in a 1 : 1 ratio, mimicking the dimeric antigen binding sites of the native immunoglobulin molecule and the native LC homodimer.

Starch-based biodegradable films amended with nano-starch and tannic acid-coated nano-starch exhibit enhanced mechanical and functional attributes with antimicrobial activity

Starch-based biofilms are biodegradable, but their application is limited by lower mechanical strength and absence of antimicrobial properties. In this context, the present study attempted to unleash the potential of nanotechnology for synthesizing nano-starch (NS) and tannic acid-coated nano-starch (T-NS) for augmenting the tensile strength and antimicrobial properties of starch-based biofilms. Moreover, this study reports one of the first such attempts to improve the commercial viability of starch extracted from the corms of Amorphophallus paeoniifolius. In this study, NS and T-NS samples were first synthesized by the physical and chemical modification of the native starch (S) molecules. The NS and T-NS samples showed significantly smaller granule size, lower moisture content, and swelling power. Further, amendments with NS and T-NS samples (25 % and 50 %) to the native starch molecules were performed to obtain biofilm samples. The NSB (NS amended) and T-NSB (T-NS amended) biofilms showed comparatively higher tensile strength than SB films (100 % starch-based). The T-NSB showed greater antimicrobial activity against gram-positive and gram-negative bacteria. All the biofilms showed almost complete biodegradation in soil (in 10 days). Therefore, it can be concluded that additives like NS and T-NS can improve starch-based biofilms' mechanical strength and antimicrobial properties with considerable biodegradability.

Controlling CAR-T cell activity and specificity with synthetic SparX adapters

While conventional chimeric antigen-receptor (CAR)-T therapies have shown remarkable clinical activity in some settings, they can induce severe toxicities and are rarely curative. To address these challenges, we developed a controllable cell therapy where synthetic D-domain-containing proteins (soluble protein antigen-receptor X-linker [SparX]) bind one or more tumor antigens and mark those cells for elimination by genetically modified Tcells (antigen-receptor complex [ARC]-T). The chimeric antigen receptor was engineered with a D-domain that specifically binds to the SparX protein via a unique TAG, derived from human alpha-fetoprotein. The interaction is mediated through an epitope on the TAG that is occluded in the native alpha-fetoprotein molecule. Invitro and invivo data demonstrate that the activation and cytolytic activity of ARC-T cells is dependent on the dose of SparX protein and only occurs when ARC-T cells are engaged with SparX proteins bound to antigen-positive cells. ARC-T cell specificity was also redirected invivo by changing SparX proteins that recognized different tumor antigens to combat inherent or acquired tumor heterogeneity. The ARC-SparX platform is designed to expand patient and physician access to cell therapy by controlling potential toxicities through SparX dosing regimens and enhancing tumor elimination through sequential or simultaneous administration of SparX proteins engineered to bind different tumor antigens.

Third Generation Solid Dispersion-Based Formulation of Novel Anti-Tubercular Agent Exhibited Improvementin Solubility, Dissolution and Biological Activity.

The long treatment period and development of drug resistance in tuberculosis (TB) necessitates the discovery of new anti-tubercular agents. The drug discovery program of the institute leads to the development of an anti-tubercular lead (IIIM-019), which is an analogue of nitrodihydroimidazooxazole and exhibited promising anti-tubercular action. However, IIIM-019 displays poor aqueous solubility (1.2µg/mL), which demands suitable dosage form for its efficient oral administration. In the present study, third generation solid dispersion-based formulation was developed to increase the solubility and dissolution of IIIM-019. The solubility profile of IIIM-019 using various polymeric carriers was determined and subsequently, PVP K-30 and P-407 were selected for preparation of binary and ternary solid dispersion. The third-generation ternary solid dispersion comprising PVP K-30 and P-407 revealed a remarkable enhancement in the aqueous solubility of IIIM-019. Physicochemical characterization of the developed formulations was done by employing FTIR spectroscopy, scanning electron microscopy, X-ray diffraction analysis, differential scanning calorimetry, and dynamic light scattering analysis. The dissolution study indicated an impressive release profile with the optimized formulation. The optimized formulation was further examined for cytotoxicity, cellular uptake, and hemolytic activity. The results indicated that the formulation had no apparent cytotoxicity on Caco-2 cells and was non-hemolytic in nature. Moreover, the optimized formulation showed significantly improved anti-tubercular activity compared to the native molecule. These findings showed that the developed third generation ternary solid dispersion could be a promising option for the oral delivery of investigated anti-tubercular molecule.

Thrombopoietin, the Primary Regulator of Platelet Production: From Mythos to Logos, a Thirty-Year Journey.

Thrombopoietin, the primary regulator of blood platelet production, was postulated to exist in 1958, but was only proven to exist when the cDNA for the hormone was cloned in 1994. Since its initial cloning and characterization, the hormone has revealed many surprises. For example, instead of acting as the postulated differentiation factor for platelet precursors, megakaryocytes, it is the most potent stimulator of megakaryocyte progenitor expansion known. Moreover, it also stimulates the survival, and in combination with stem cell factor leads to the expansion of hematopoietic stem cells. All of these growth-promoting activities have resulted in its clinical use in patients with thrombocytopenia and aplastic anemia, although the clinical development of the native molecule illustrated that "it's not wise to mess with mother nature", as a highly engineered version of the native hormone led to autoantibody formation and severe thrombocytopenia. Finally, another unexpected finding was the role of the thrombopoietin receptor in stem cell biology, including the development of myeloproliferative neoplasms, an important disorder of hematopoietic stem cells. Overall, the past 30 years of clinical and basic research has yielded many important insights, which are reviewed in this paper.

Homo- and hetero-oligomeric protein–protein associations explain autocrine and heterologous pheromone-cell interactions in Euplotes

In Euplotes, protein pheromones regulate cell reproduction and mating by binding cells in autocrine or heterologous fashion, respectively. Pheromone binding sites (receptors) are identified with membrane-bound pheromone isoforms determined by the same genes specifying the soluble forms, establishing a structural equivalence in each cell type between the two twin proteins. Based on this equivalence, autocrine and heterologous pheromone/receptor interactions were investigated analyzing how native molecules of pheromones Er-1 and Er-13, distinctive of mating compatible E. raikovi cell types, associate into crystals. Er-1 and Er-13 crystals are equally formed by molecules that associate cooperatively into oligomeric chains rigorously taking a mutually opposite orientation, and each burying two interfaces. A minor interface is pheromone-specific, while a major one is common in Er-1 and Er-13 crystals. A close structural inspection of this interface suggests that it may be used by Er-1 and Er-13 to associate into heterodimers, yet inapt to further associate into higher complexes. Pheromone-molecule homo-oligomerization into chains accounts for clustering and internalization of autocrine pheromone/receptor complexes in growing cells, while the heterodimer unsuitability to oligomerize may explain why heterologous pheromone/receptor complexes fail clustering and internalization. Remaining on the cell surface, they are credited with a key role in cell–cell mating adhesion.

Co-encapsulation of curcumin and piperine in whey protein isolate fibrils improves their water dispersibility and antioxidant activity

In this study, whey protein isolate (WPI) fibrils prepared by acid heat treatment for different times (5, 10, 15, or 20 h) were used for the co-encapsulation and co-delivery of curcumin (Cur) and piperine (Pip). Co-encapsulation of both nutraceuticals in the fibrils significantly improved their water dispersibility, chemical stability, and antioxidant activity. For instance, the water dispersibility of Cur and Pip in native WPI solutions was around 47.0% and 47.5%, respectively. In contrast, the water dispersibility of the same two nutraceuticals in WPI fibrils (15 h treatment) was 94.3% and 66.6%, respectively. The nutraceutical-loaded WPI fibrils did not undergo significant flocculation or precipitation after 14 d storage at 25 °C. After encapsulation, the DPPH free radical scavenging ability of the Cur-Pip combination experienced a surge, rising from approximately 35.4% to 72.0%. The Stern-Volmer quenching constants (Ksv) of Cur-WPI (3.03 × 104 M−1) and Pip-WPI (7.08 × 104 M−1) were much smaller than those of Cur-WPI fibrils (3.88 × 104–4.69 × 104 M−1) and Pip-WPI fibrils (8.26 × 104–9.41 × 104 M−1), indicating that the nutraceuticals bind stronger to the protein fibrils than to the native protein molecules. In summary, WPI fibrils have the capability to encapsulate, protect, and deliver hydrophobic nutraceuticals, suggesting their suitability for application in functional foods and beverages.

A simple and rapid preparation of smooth muscle myosin 2 for the electron microscopic analysis.

There has been an increase in the demand for purified protein as a result of recent developments in the structural biology of myosin 2. Although promising, current practices in myosin purification are usually time-consuming and cumbersome. The reported increased actin to myosin ratio in smooth muscles adds to the complexity of the purification process. Present study outlines a streamlined approach to isolate smooth muscle myosin 2 molecules from actomyosin suspension of chicken gizzard tissues. The procedure entails treating actomyosin for a brief period with actin-binding peptide phalloidin, followed by co-sedimentation and short column size exclusion chromatography. Typical myosin molecule with heavy and light chains and approximately 95% purity was examined using gel electrophoresis. Negative staining electron microscopy and image processing showed intact 10S myosin 2 molecules, proving that phalloidin is effective at eliminating majority of actin in the form of F-actin without dramatic alteration in the structure of myosin. The entire purification discussed here can be completed in a few hours, and further analysis can be done the same day. Thus, by offering quick and fresh supplies of native myosin molecules suited for structural research, specially cryo-electron microscopy, this innovative approach can be adapted to get around the drawbacks of time-intensive myosin purifying processes.