Polyacrylamide Gel Research Articles

Escherichia coli alcohol dehydrogenase YahK is a protein that binds both iron and zinc

Background Previous studies have highlighted the catalytic activity of Escherichia coli alcohol dehydrogenase YahK in the presence of coenzyme nicotinamide adenine dinucleotide (NAD) and metal zinc. Notably, competitive interaction between iron and zinc ligands has been shown to influence the catalytic efficiency of several key proteases. This study aims to unravel the intricate mechanisms underlying YahK’s catalytic action, with a particular focus on the pivotal roles played by metal ions zinc and iron. Methods The purified YahK protein from E. coli cells cultivated in LB medium was utilized to investigate its metal-binding properties through UV-visible absorption measurements and determination of metal content. Subsequently, the effects of excess zinc and iron on the metal-binding ability and alcohol dehydrogenase activity of the YahK protein were explored using M9 minimal medium. Furthermore, site-directed mutagenesis technology was employed to determine the iron-binding site location within the YahK protein. Polyacrylamide gel electrophoresis was conducted to examine the relationship between iron and zinc with respect to the YahK protein. Results The study confirmed the presence of iron and zinc in the YahK protein, with the zinc-bound form exhibiting enhanced catalytic activity in alcohol dehydrogenation reactions. Conversely, the presence of iron appears to play a pivotal role in maintaining overall stability of the YahK protein. Furthermore, experimental findings indicate that excessive zinc within M9 minimal medium can competitively bind to iron-binding sites on YahK, thereby augmenting its alcohol dehydrogenase activity. Conclusion The dynamic binding of YahK to iron and zinc unveils its intricate regulatory mechanism as an alcohol dehydrogenase, thereby highlighting the possible physiological role of YahK in E. coli and its significance in governing cellular metabolic processes. This discovery provides a novel perspective for further investigating the specific impact of metal ion binding on YahK and E. coli cell metabolism.

Capillary-Assisted Assembly of Polymer Gel-Supported Lipid Bilayers.

The polymer-supported lipid bilayer represents an attractive supramolecular assembly in numerous biophysical and bioanalytical applications. The assembly of polymer-supported membranes with a polymer layer thickness of just a few nanometers is now well-established, but bilayer properties in such a membrane architecture are still influenced by the nearby solid substrate. Polymer-supported lipid bilayer systems with a several micrometers thick polymer layer will overcome this shortcoming. However, formation of a fluid lipid bilayer on a fully hydrated, micrometer thick polymer film using traditional methods (e.g., vesicle fusion and lipid monolayer deposition techniques) remains a challenging task due to the rather unfavorable interfacial conditions for bilayer formation in such a system. Here, we report for the first time on the facile capillary-assisted formation of a lipid bilayer on the surface of a fully hydrated, several micrometers thick polyacrylamide (PAA) gel, in which forced molecular crowding of lipids at the air-water interface of the capillary results in monolayer instability and collapse, thereby forming a lipid bilayer on the top of the polymer gel inside the capillary. Stable bilayer attachment on the surface of the polymer gel can be achieved via physisorption or specific chemical linkages (tethering) on both cross-linked and non-cross-linked PAA films. Unlike the traditional solid-supported lipid bilayer (SLB), the lipid lateral diffusion in the polymer gel-supported lipid bilayer is not anymore perturbed by a solid substrate. Instead, more like a plasma membrane, it is mainly influenced by the properties of the underlying polymer and the nature/distribution of polymer-bilayer attachments. Polymer gel-supported lipid bilayers built using the capillary-assisted assembly approach show attractive self-healing properties, resulting in superior long-term stability relative to the SLB. We hypothesize that the described capillary-assisted assembly method can be applied to a wide range of polymeric materials and lipid compositions, opening exciting opportunities as an advanced model membrane system.

Comparative venom analysis between melanistic and normally coloured phenotypes of the common adder (Vipera berus).

Snake venom is an ecologically relevant functional trait directly linked with a snake's fitness and survival, facilitating predation and defence. Snake venom variation occurs at all taxonomic levels, but the study at the intraspecific level is still in its early stages. The common adder (Vipera berus) exhibits considerable variation in colour phenotypes across its distribution range. Melanistic (fully black) individuals are the subject of myths and fairytales, and in German folklore such 'hell adders' are considered more toxic than their normally coloured conspecifics despite any formal investigation. Here, we provide the first comparative analysis of venoms from melanistic and normally coloured common adders. Specifically, we compared the venom profiles by sodium dodecylsulfate polyacrylamide gel electrophoresis and reversed-phase high-performance liquid chromatography and tested the venoms' protease, phospholipase A2 and cytotoxic activities. Phospholipase A2 activity was similar in both phenotypes, whereas general protease activity was higher in the melanistic venom, which was also more cytotoxic at two concentrations (6.25 and 12.5 µg ml-1). These minor differences between the venoms of melanistic and normally coloured adders are unlikely to be of clinical relevance in the context of human envenomation. In light of our results, the claim that melanistic adders produce more toxic venom than their normally coloured conspecifics appears rooted entirely in folklore.

Multi-omics approach for understanding the response of Bacteroides fragilis to carbapenems

BackgroundThe prevalence of Bacteroides fragilis isolates resistant to first-line beta-lactam drugs is increasing, resulting in reduced treatment efficacy. Investigating the bacterial transcriptome and proteome can uncover links between bacterial genes and resistance mechanisms. In this study, we experimentally assessed in vitro the transcriptional and proteomic profiles of B. fragilis exposed to SICs of meropenem, an effective antimicrobial agent, collected from patients with intra-abdominal diseases at Astana City Hospital, Kazakhstan. MethodsB. fragilis was cultured in brain heart infusion broth and sub-cultured every 48 h for 8 days in media with and without meropenem. Total RNA was extracted from the liquid cultures using a commercial RNeasy mini kit, and strand-specific RNA sequencing (RNA-seq) was performed on the DNBSEQ platform. Raw RNA-seq data were retrieved from BioProject No. PRJNA531645 and uploaded to the NCBI Sequence Read Archive (accession no. SRX22081155). Proteins of B. fragilis were extracted and separated using sodium dodecyl sulphate–polyacrylamide gel electrophoresis, followed by analysis of the eluted peptides using liquid chromatography–tandem mass spectrometry. Cluster analysis utilised the Database for Annotation, Visualization, and Integrated Discovery. ResultsThe subinhibitory concentration (SIC) of meropenem was determined to be 0.5 μg/L (minimum inhibitory concentration: 1). Mapping of reads to the reference genome identified 2,477 expressed genes in all B. fragilis BFR KZ01 samples. Ten differentially expressed genes (DEGs) were common across comparison groups during and post-antibiotic exposure (wMEM vs. MEM2 and MEM2 vs. rMEM8); however, no substantially enriched Gene Ontology terms were identified. The cluster analysis highlighted a significant enrichment cluster (W-0560 oxidoreductase) of DEGs following antibiotic withdrawal. In total, 859 B. fragilis proteins were identified, with the expressions of three proteins, 3-oxoacyl-[acyl carrier protein] reductase, acetyl-CoA carboxylase biotin carboxylase subunit, and beta-ketoacyl-ACP synthase III, being upregulated in the enriched protein folding category. Notably, chaperone proteins such as FKBP-type peptidyl-prolyl cis-trans isomerases (involved in the cis-trans isomerisation of prolyl peptide bonds) and GroES (a co-chaperone functioning with GroEL) were also identified. ConclusionsUnder the influence of low doses of antibiotics defense mechanisms are activated which contribute to the emergence of resistance. These results provide insight into the response of B. fragilis to meropenem exposure, mainly at the SIC, contributing to the understanding bacterial survival strategies under stress conditions.

Analysis of the genus Bothrops snake venom: An inter and intraspecific comparative study

The genus Bothrops are considered Category 1 of medical importance by the World Health Organization, responsible for approximately 85 % of snakebites occurring throughout Brazil. Main factors determining snake venom variations can be genetics, diet, gender, geographic distribution, age, or even seasonality. In this study, we compared the composition of protein profile, biochemical activities, and immunorecognition of toxins present in the venom of eight adults of Bothrops species (B. alternatus, B. atrox, B. jararaca, B. jararacussu, B. leucurus, B. moojeni, B. neuwiedi and B. pauloensis). The following methods were used to analyze the venoms: protein dosage; electrophoresis in polyacrylamide gel containing SDS; High Performance Liquid Chromatography – Reverse Phase; enzymatic activities, western blotting and Enzyme Linked Immuno Sorbent Assay. The results show inter and intraspecific differences in the electrophoretic profile. LAAO and PLA2 activities, in general, were higher in males than females and proteolytic activity was higher in females than males. The bothropic antivenom produced by Instituto Butantan recognized most of the protein bands in all Bothrops species analyzed, with only the regions between 37 and 25 kDa presenting lower intensity. A notable variability in the chromatograms was observed. Bothrops venom demonstrated inter-intraspecific disparities in protein composition and biochemical activity.

Photo‐induced polymerization of styrene‐maleic acid copolymers for the extraction of membrane proteins

AbstractWe report herein the photoinduced electron/energy transfer–reversible addition‐fragmentation chain transfer (PET‐RAFT) polymerization of styrene‐maleic anhydride (SMAnh) using the photocatalyst, zinc tetraphenylporphirine, under a white cool LED light. PET‐RAFT is an easy and convenient polymerization method that does not require deoxygenation unlike other radical polymerizations. Various parameters, for example, the amount of styrene in the feed or the solvent, and their influence on the polymerization were studied. After hydrolysis of the anhydride moieties, the resulting styrene‐maleic acid (SMA) copolymers obtained by PET‐RAFT copolymerization were evaluated for their solubilization efficiency of three different membrane proteins, BmrA and AcrB, overexpressed in Escherichia coli, and A2AR, expressed in insect cells (Sf9). The different copolymers provided similar solubilization rates to the commercially available SMA; however, a highly improved migration behavior on sodium dodecyl‐sulfate polyacrylamide gel electrophoresis was observed which could, facilitate downstream analyses. Overall, we demonstrated that PET‐RAFT is a versatile oxygen tolerant polymerization technique to yield SMA, a suitable polymer for biochemical applications.

A bioswitchable delivery system for microRNA therapeutics based on a tetrahedral DNA nanostructure.

As microRNAs (miRNA) regulate almost all physiopathological activities in the human body, miRNA therapeutics that deliver miRNA regulators have attracted considerable attention in the field of nucleic acid drug development. The use of tetrahedral DNA nanostructures to deliver miRNA regulators is promising because of their simple fabrication, enhanced cell entry, effective tissue penetration, biocompatibility and functional editability. This protocol extension builds on our previous protocol for the use of tetrahedral DNA nanostructures and was designed to establish an updated bioswitchable delivery system (BDS) for achieving controlled cargo loading and release. A ribonuclease H-sensitive sequence is designed as a bioswitchable apparatus for the targeted release of the miRNA regulator. The functional sequence of the miRNA regulator and minimal secondary structure formation tendency during annealing are two key points in cargo design. We provide two BDS design strategies; BDS-A comprises an intact DNA tetrahedron with the RNA cargo hanging outside, offering the merits of lower cost, simplicity, and more direct structural design. In the BDS-B design, the RNA regulators are embedded into the DNA tetrahedron, which is beneficial for dermal tissue permeation applications. Following sequence design in Oligo 7 and Tiamat, the BDS assembly is completed and then ribonuclease H achieves controlled release of the miRNA regulator by triggering the bioswitchable apparatus. This is verified via polyacrylamide and agarose gel electrophoresis or fluorophore modifications. Both BDSs show promising cellular membrane permeability, tissue permeability and target inhibition in vitro and in vivo. The assembly and characterization of the BDS can be completed in 4 d, and the validation time for biostability and biological applications will depend on the specific use.

Fractionation by Spatially Heterogeneous Diffusion: Experiments and the Two-Component Random Walk Model.

The fundamental question regarding the fractionation phenomenon is whether diffusion alone is responsible for it or whether an additional advection dynamic is involved. We studied the fractionation by diffusion of particles in spatially heterogeneous environments. By experimentally observing the time-sequential fractionation patterns of dye particles diffusing across a solid-solid interface of varying polyacrylamide gel densities, we found that the two-component diffusion model accurately captures the observed fractionation dynamics. In contrast, single-component diffusion models by Fick, Wereide, and Chapman do not. Our results indicate that diffusion alone can explain the fractionation phenomenon and that additional advection dynamics are not involved. The underlying physics in the fractionation phenomenon is discussed by using a two-component random walk model.

Purification and characterization of α-amylase from Acanthoscelides obtectus (Say) (Coleoptera: Chrysomelidae)

Acanthoscelides obtectus is one of the most notorious pests of stored kidney beans (Phaseolus vulgaris) worldwide. Kidney beans are an important source of food for these insects. α-Amylase is the main carbohydrate-digesting enzyme in animals including insects. In the current study, the biochemical analysis revealed higher α-amylase activity (U/ml) in 3rd and 4th larval instars but decreased gradually in subsequent developmental stages. However, the specific activity (U/mg) interestingly was highest in 1st instar and decreased in further developmental stages. During qualitative analysis of α-amylase using starch-agar and native PAGE, the maximum zone of starch lysis and a prominent band on the gel was observed in 3rd and 4th larval stages. The molecular mass of the native enzyme was also estimated and found to be 30.34 kDa. The crude α-amylase was further purified by ammonium sulfate precipitation, gel filtration on a Sephadex G-75, and ion exchange chromatography on the DEAE cellulose column. The purified amylase was found to be a monomer with a molecular mass of 15 kDa. The specific activity of the purified enzyme increased from 1.74 U/mg in the crude sample to 166.35 U/mg in the final purification step resulting in 95-fold purification with a yield of 11.14%. Further characterization of purified α-amylase revealed a pH optimum of 7.0 and a temperature optimum of 35 °C. Lineweaver-Burk plot analysis revealed Km and Vmax to be 0.09% and 3.3 U/mL, respectively. Oxalic acid, tannic acid, and HgCl2 significantly inhibited the enzyme, while the Na+, Ca++, and Mg++ ions acted as activators. In conclusion, the study revealed, the highest α-amylase activity in 3rd and 4th larval stages of Acanthoscelides obtectus followed by native and SDS PAGE resulting in molecular mass of 30.34 and 15 kDa respectively.

Enhancing structural strength and water retention of crosslinked polyacrylamide gel with the T-ZnOw

Crosslinked polyacrylamide gel serves as an effective plugging material, crucial for preventing water/gas channeling in reservoirs and enhancing the swept volume and oil recovery. However, its practical application encounters a significant challenge in maintaining long-term stability of strength and volume, particularly under high temperatures. This study investigates the reinforcement of polyacrylamide gel with three-dimensional (3D) nanomaterials, specifically tetra-needle-like zinc oxide whiskers (T-ZnOw), to enhance its structural stability and bound water content. The stress sweep tests showed that the addition of 0.15 wt% T-ZnOw increased the maximum elastic modulus of the gel to 28.5 Pa, representing a 51.2 % improvement compared to before the addition. After 60 days of high-temperature aging at 130 °C, the T-ZnOw strengthened gel still maintained an elastic modulus of 21.8 Pa. With increasing T-ZnOw concentration, the yield point of the gel increased from 4.74 Pa to 9.57 Pa, the anti-deformation of the gel has significantly increased. The stability test results in high salinity water indicate that at a salinity of 150,000 mg/L, the dehydration rate of the strengthened gel is less than 5 %, with an elastic modulus retention rate exceeding 88 %. Furthermore, thermodynamic testing confirms the enhanced thermal degradation resistance and water retention capacity of the gel. Under reservoir temperature conditions, the T-ZnOw strengthened gel exhibits a weight loss of less than 3 %. Additionally, the inclusion of T-ZnOw results in an increase in the bound water capacity index from 4.65 to 12.28. In conclusion, the integration of T-ZnOw into the polyacrylamide gel network not only enhances its mechanical performance and water retention under high-temperature conditions but also demonstrates superior stability in high-salinity environments. This innovative material shows significant potential for improving oil recovery rates in high-temperature reservoirs and can be applied to other high-temperature underground scenarios such as geothermal development and CO2 utilization and storage.

A Novel Application of Virus Like Particles in the Hemagglutination Inhibition Assay.

The hemagglutination inhibition (HI) assay is a traditional laboratory procedure for detection and quantitation of serum antibodies of hemagglutinating viruses containing the hemagglutinin (HA) gene. The current study aimed to investigate the novel use of virus like particles (VLP) as an antigen for the HI assay. VLPs were prepared from a strain of H5N1 using a baculovirus expression system. The VLPs were characterized using the hemagglutination test, Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and transmission electron microscopy. The comparative HI assay was performed using three different seed antigens: A/chicken/Mexico/232/94 (H5N2), A/chicken/Egypt/18-H/09(H5N1) and A/goose/Guangdong/1/1996(H5N1). The HI assay of serum antibody titrations using homologous antigens to these vaccinal seeds were compared to the VLP's antigens for the same serum. The HI titers were logically relevant to the similarity between VLP antigens and vaccinal seeds, indicating the VLPs behave similarly to the standard HI assay which uses inactivated whole virus as an antigen. VLPs could be considered as an alternative to the HI assay antigen as they show a relatedness between the similarity with vaccinal seed and serum antibodies. Compared to typical entire H5N1 viral antigen prepared in SPF eggs that require proper inactivation to avoid any public health risk, VLPs prepared in tissue culture, plants or insect cells are a safe, inexpensive and scalable alternative to inactivated whole virus antigen.

Upstream alternative polyadenylation in SCN5A produces a short transcript isoform encoding a mitochondria-localized NaV1.5 N-terminal fragment that influences cardiomyocyte respiration.

SCN5A encodes the cardiac voltage-gated Na+ channel, NaV1.5, that initiates action potentials. SCN5A gene variants cause arrhythmias and increased heart failure risk. Mechanisms controlling NaV1.5 expression and activity are not fully understood. We recently found a well-conserved alternative polyadenylation (APA) signal downstream of the first SCN5A coding exon. This yields a SCN5A-short transcript isoform expressed in several species (e.g. human, pig, and cat), though rodents lack this upstream APA. Reanalysis of transcriptome-wide cardiac APA-seq and mRNA-seq data shows reductions in both upstream APA usage and short/full-length SCN5A mRNA ratios in failing hearts. Knock-in of the human SCN5A APA sequence into mice is sufficient to enable expression of SCN5A -short transcript, while significantly decreasing expression of full-length SCN5A mRNA. Notably, SCN5A -short transcript encodes a novel protein (NaV1.5-NT), composed of an N-terminus identical to NaV1.5 and a unique C-terminus derived from intronic sequence. AAV9 constructs were able to achieve stable NaV1.5-NT expression in mouse hearts, and western blot of human heart tissues showed bands co-migrating with NaV1.5-NT transgene-derived bands. NaV1.5-NT is predicted to contain a mitochondrial targeting sequence and localizes to mitochondria in cultured cardiomyocytes and in mouse hearts. NaV1.5-NT expression in cardiomyocytes led to elevations in basal oxygen consumption rate, ATP production, and mitochondrial ROS, while depleting NADH supply. Native PAGE analyses of mitochondria lysates revealed that NaV1.5-NT expression resulted in increased levels of disassembled complex V subunits and accumulation of complex I-containing supercomplexes. Overall, we discovered that APA-mediated regulation of SCN5A produces a short transcript encoding NaV1.5-NT. Our data support that NaV1.5-NT plays a multifaceted role in influencing mitochondrial physiology: 1) by increasing basal respiration likely through promoting complex V conformations that enhance proton leak, and 2) by increasing overall respiratory efficiency and NADH consumption by enhancing formation and/or stability of complex I-containing respiratory supercomplexes, though the specific molecular mechanisms underlying each of these remain unresolved.

Robust peroxidase from Bacillus mojavensis TH309: Immobilization on walnut shell hydrochar and evaluation of its potential in dye decolorization

Peroxidases have received considerable attention as a cost-effective and environmentally friendly catalyst for bioremediation. Their rapid activity loss under harsh environmental conditions and inability to be used repetitively limit their exploitation in real-world wastewater treatment. First, a peroxidase was produced extracellularly by Bacillus mojavensis TH309 and purified 8.12-fold with a final yield of 47.10 % using Sephadex G-100 superfine resin. The pure peroxidase (BmPer) possessed a relatively low molecular weight of ∼21 kDa and was active against L-DOPA on acrylamide gel after electrophoresis. BmPer was immobilized by adsorption functionalized walnut shell hydrochar (WsH) with 61.99 ± 1.34 % efficiency and 37.07 ± 4.16 % activity loss. BmPer and its immobilized form (WsH-BmPer) exhibited maximum activity at 50 °C and pH 9. WsH-BmPer exhibited 3.23-, 2.37-, 1.65-, and 2.25-fold longer half-life than BmPer at 50, 60, 70, and 80 °C, respectively. Immobilization significantly enhanced the stability of the enzyme under acidic conditions. BmPer and WsH-BmPer showed maximal activity in the presence of 1 % salt and retained more than 85 % of their activity even after pre-incubation with 2.5 M salt for 60 min at 50 °C. Their catalytic efficiency was significantly stimulated by pre-incubation with Triton X-100 (1 mM), Tween20 (1 mM), and Mg2+ (1 and 10 mM). Immobilization strongly reduced the loss of activity caused by inhibitors including Ba2+, Hg2+, and Cu2+. Moreover, both forms of the enzyme were compatible with solvents. The Michaelis constant (Km) values of BmPer and WsH-BmPer were 0.88 and 2.66 mM for 2,4 DCP, respectively. WsH-BmPer peroxidase maintained about 82 % and 85 % of its activity when stored at 4 °C for 30 days and reused for up to 10 cycles, respectively. Furthermore, it decolorized Cibacron red (CR), Poly R-478 (PR), Remazol Brilliant Blue R (RBBR), and Methyl red (MR) dyes by 60.13 %, 91.34 %, 86.41 %, and 50.51 % within 60 min, respectively.

DELINEATING GENETIC DIVERSITY IN SOYBEAN (GLYCINE MAX L.) GENOTYPES: INSIGHTS FROM A-PAGE ANALYSIS OF GLOBULIN RESERVE PROTEINS

This study explores the genetic diversity of soybean (Glycine max) genotypes using polyacrylamide gel electrophoresis (A-PAGE) to analyze globulin reserve proteins. The research utilized 51 soybean genotypes from various origins, including Canada, Turkey, Uzbekistan and others. The A-PAGE method was employed to extract and analyze the globulin storage proteins in the grains of soybean samples. The proteins were categorized into ω, γ, β and α zones, revealing distinct electrophoretic patterns among the genotypes. A total of 22 spectra and 107 patterns were identified, demonstrating polymorphism based on the frequency of occurrence. The genetic diversity index, calculated using Nei’s formula, indicated varying levels of genetic diversity across the different zones.

Synthesis and color-tunable emission of ZnGaOx with different cation ratios by a polyacrylamide gel method

ZnGaOx nanoparticles with varied ratios of Zn2+ to Ga3+ were synthesized by a polyacrylamide gel route. The phase purity and structure are confirmed by X-ray diffraction (XRD), Fourier transform infrared spectrometer (FTIR) and X-ray photoelectron spectrometer (XPS). High crystal quality and elemental distribution are determined by transmission electron microscopy (TEM) measurements. The band gaps of the samples ranging from 3.22 to 4.47 eV are estimated by ultraviolet–visible (UV–Vis) diffuse refectance spectroscopy (DRS). Under the excitation of 325 nm laser light, ZnGaOx nanoparticles with the Zn2+/Ga3+ molar ratios of 0.8:2 and 0.7:2 show two emission peaks at 415 and 510 nm. A marked emission band at 695 nm is observed from the samples with Zn2+/Ga3+ molar ratios of 0.6:2, 0.5:2 and 0.4:2. The luminescence mechanism is discussed based on the experimental results. The luminescence color of as-synthesized samples gradually shifts from blue-green to green-yellow region with decreasing Zn2+/Ga3+ ratio. Interestingly, the sample with Zn2+/Ga3+ ratio of 0.7:2 has the CIE chromaticity coordinates of (x = 0.26, y = 0.33) close to the coordinates (x = 0.33, y = 0.33) of pure white light. Together, ZnGaOx phosphors with different emission colors without doping with rare earths have considerable potential applications in tunable light emitting diodes (LEDs) and color display.

Oligomerization of protein arginine methyltransferase 1 and its effect on methyltransferase activity and substrate specificity.

Proper protein arginine methylation by protein arginine methyltransferase 1 (PRMT1) is critical for maintaining cellular health, while dysregulation is often associated with disease. How the activity of PRMT1 is regulated is therefore paramount, but is not clearly understood. Several studies have observed higher order oligomeric species of PRMT1, but it is unclear if these exist at physiological concentrations and there is confusion in the literature about how oligomerization affects activity. We therefore sought to determine which oligomeric species of PRMT1 are physiologically relevant, and quantitatively correlate activity with specific oligomer forms. Through quantitative western blotting, we determined that concentrations of PRMT1 available in a variety of human cell lines are in the sub-micromolar to low micromolar range. Isothermal spectral shift binding data were modeled to a monomer/dimer/tetramer equilibrium with an EC50 for tetramer dissociation of ~20 nM. A combination of sedimentation velocity and Native polyacrylamide gel electrophoresis experiments directly confirmed that the major oligomeric species of PRMT1 at physiological concentrations would be dimers and tetramers. Surprisingly, the methyltransferase activity of a dimeric PRMT1 variant is similar to wild type, tetrameric PRMT1 with some purified substrates, but dimer and tetramer forms of PRMT1 show differences in catalytic efficiencies and substrate specificity for other substrates. Our results define an oligomerization paradigm for PRMT1, show that the biophysical characteristics of PRMT1 are poised to support a monomer/dimer/tetramer equilibrium in vivo, and suggest that the oligomeric state of PRMT1 could be used to regulate substrate specificity.

Unveiling the transformative influence of sonochemistry on formation of whey protein isolate and green tea extract (WPI-GTE) conjugates

This study investigated the formation of conjugates between whey protein isolate (WPI) and green tea extract (GTE) using three methods: redox-pair (R), ultrasound-assisted redox-pair (RU), and ultrasonication (UL). Ultrasonication significantly reduced the reaction time for synthesizing WPI-GTE conjugates compared to the standard R method (p < 0.05). The UL methods had the highest conjugate yield determined by polyphenol binding (p < 0.05). Fourier-transform infrared spectroscopy (FTIR) and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the conjugate formation, indicating an increased molecular weight due to protein binding with polyphenols through covalent and non-covalent bonds. Conjugates produced via ultrasonication exhibited enhanced solubility, smaller particle size, better emulsifying capacity, and improved foaming ability compared to those formed using the traditional R method (p < 0.05). However, conjugates from the R method showed higher antioxidant activity, as evidenced by DPPH•and ABTS•+ scavenging activities (p < 0.05). In conclusion, WPI-GTE conjugates created through ultrasonic treatment demonstrate potential as dual-functional ingredients, serving as both antioxidant and emulsifier.

Mild osmotic stress offers photoprotection in Chlamydomonas reinhardtii under high light

The exposure of autotrophs to high light intensities significantly impacts their photosynthetic performance. When combined with unpredictable climate changes, the lethality of these effects is exacerbated and , often surpassing the organisms' threshold for tolerance. In this regard, our study centres on examining the mitigating effects of mild osmotic stress induced by 2% Polyethylene Glycol (PEG) in conjunction with high-light conditions, using Chlamydomonas reinhardtii as a model system. Cells were cultivated under low PEG-induced osmotic stress at various light intensities, and their responses were analyzed through biochemical and biophysical approaches. Remarkably, cells grown under lower PEG concentrations exhibited superior growth, increased biomass, and enhanced photosynthetic efficiency under high light compared to non-PEG-treated cells. Surprisingly, their non-photochemical quenching (NPQ) levels were lower, indicating the operation of a distinct photoprotective mechanism in PEG-grown samples. The PEG-grown cells demonstrated higher chlorophyll content but lower carotenoid content, supporting the NPQ data. Circular dichroism analysis suggested that the macro-organization of super-complexes was minimally disrupted in PEG-grown samples, even under high light. This was further supported by Blue native PAGE, which showed greater stability of the super-complexes in PEG-grown cells, implying heightened stability in pigment-protein interactions. Immunoblot analysis revealed minimal differences in core reaction center proteins between PEG-grown and non-PEG cells. Notably, this protective mechanism was absent in the cell wall-deficient mutant CC503. We propose that the partial photoprotection observed is attributed to the PEG shielding the cell wall. This result holds promise for enhancing algal biomass production under natural environmental conditions influenced by fluctuating light intensity.

Regulation of mitochondrial metabolism by hibernating bear serum: Insights into seasonal metabolic adaptations

Hibernating animals undergo a unique and reversible decrease in their whole-body metabolism, which is often accompanied by a suppression of mitochondrial respiration. However, the precise mechanisms underlying these seasonal shifts in mitochondrial metabolism remain unclear. In this study, the effect of the serum from active and hibernating Japanese black bears on mitochondrial respiration was assessed. Stromal–vascular cells were obtained from bear white adipose tissue and cultured with or without an adipocyte differentiation cocktail. When the oxygen consumption was measured in the presence of bear serum, the hibernating bear serum reduced maximal respiration by 15.5 % (p < 0.05) and spare respiratory capacity by 46.0 % (p < 0.01) in the differentiated adipocytes in comparison to the active bear serum. Similar reductions of 23.4 % (p = 0.06) and 40.6 % (p < 0.05) respectively were observed in undifferentiated cells, indicating the effect is cell type-independent. Blue native PAGE analysis revealed that hibernating bear serum suppressed cellular metabolism independently of the assembly of mitochondrial respiratory chain complexes. RNA-seq analysis identified 1094 differentially expressed genes (fold change>1.5, FDR<0.05) related to insulin signaling and glucose metabolism pathways. These findings suggest that the rapid alterations in mitochondrial metabolism during hibernation are likely induced by a combination of reduced insulin signaling and suppressed mitochondrial function, rather than changes in respiratory complex assembly.

Recyclable NaCl template assisted preparation of N/O co-doped porous carbon for zinc-ion hybrid capacitor

The traditional strong alkaline activation method for preparing porous carbon faces with complex preparation processes, equipment corrosion, excessive disordered microporous structures, and environmental pollution. Hence, a green, facile and sustainable method is proposed for synthesizing N/O co-doped porous carbon derived from polyacrylamide gel with the assistance of recyclable sodium chloride (NaCl) template. NaCl template is dissolved in gel with water and can give full play to the template effect after recrystallization. Due to a suitable specific surface area (499.8 m2 g−1) and massive N (11.38 at. %) and O (8.71 at. %) functional groups, the optimized NPC-5 electrode possesses a high specific capacitance of 283.3 F g−1 at 0.5 A g−1, superior rate characteristics and excellent cycle stability in 6 M KOH aqueous electrolyte. In addition, the constructed NPC-5//NPC-5 symmetric supercapacitor shows a high energy density of 34.0 Wh kg−1 in 1 M ZnSO4 aqueous electrolyte. More importantly, the assembled Zn//ZnSO4//NPC-5 hybrid capacitor shows a high capacity of 115.44 mAh g−1 and an energy density of 85.3 Wh kg−1.