Isoelectric Point Research Articles

Characterization, expression and functional analysis of Hsp40 during LPS challenge in blood parrot Amphilophus citrinellus ×Vieja melanura

Heat shock protein 40 belonging to heat shock protein family plays an important role in the immune responses of organisms. In this study, the full length cDNA of Hsp40 was 2426 bp including a 1368 bp open reading frame (ORF) encoding 455 amino acids with a molecular weight of 49.16 kDa and a theoretical isoelectric point of 9.34 in blood parrot Vieja synspila ♀ × Amphilophus citrinellus ♂, an important ornamental fish in China. It had three conserved domains DnaJ, CRR and DnaJ_C. Phylogenetic analysis showed that the sequence of Hsp40 among species was conserved, and the blood parrot Hsp40 was closely related to Neolamprologus brichardi. Blood parrot Hsp40 mRNA could be detected in all of the tissues examined and mainly distributed in the cytoplasm. The expression of Hsp40 was upregulated during lipopolysaccharide (LPS) challenge. Upregulated Hsp40 inhibited the activity of nuclear factor κB (NF-κB) and activated protein 1 (AP-1) and reduced the ratio of Bax/Bcl-2 mRNA expression. This study provides a theoretical basis for further exploring the role of Hsp40 gene in the anti-bacterial immunity of blood parrot.

Characterization of α-hairpinin in the genome of lima bean (Phaseolus lunatus)

Antimicrobial α-Hairpinin peptides are sources of bioactive molecules with significant structural diversity and various biological activities. Phaseolus lunatus, known as lima bean, is a plant from the Fabaceae family, native to Central and South America. It is the second most important legume agronomically and economically in tropical regions, after the common bean. Due to its importance, several omics studies have been recently conducted on this species. The research aimed to characterize the antimicrobial peptides of the α-hairpinin family in the lima bean genome by analyzing their physicochemical properties, in silico biological activities, and three-dimensional structure. To this end, α-hairpinin sequences were identified according to the cysteine pattern characteristic of this protein family. After target identification, a search for conserved domains, signal peptides, and subcellular localization was conducted. Molecular mass, isoelectric point, grand average of hydropathicity (GRAVY), and biological activities were also analyzed, and a three-dimensional model of the candidate was generated. Following the mining process, an α-hairpinin sequence from P. lunatus (PlHrp1) was identified. PlHrp1 consisted of 41 amino acids, was predicted to be in the extracellular region, had an isoelectric point of 6.28, a mass of 4.81 kDa, and a gravy of -1.078. The alignment revealed that PlHrp1 contained the 4 conserved cysteine residues. In silico activity analyses demonstrated that PlHrp1 exhibited antifungal and antibacterial activities, with no antiviral activity. The secondary structure displayed two antiparallel α-helices connected by a loop. The model described 94.4% of amino acid residues in favorable positions. This study provides a comprehensive analysis of α-hairpinin peptides in the Phaseolus lunatus genome, highlighting the presence of this peptide family in the lima bean species and offering insights into their potential.

Correlation between zeta potential and electron paramagnetic resonance of thulium, europium co-doped yttria based suspensions

The formation of advanced ceramic components with homogeneous microstructure and functional characteristics demands a suitable control of particle dispersion. Thus, the characterization of particle stability as immersed in a liquid medium is important. The present paper reports an approach to evaluate the stability of europium, thulium co-doped yttria (YET) nanoparticles by a correlation between zeta potential and Electron Paramagnetic Resonance (EPR) techniques. Based on results, YET suspensions exhibited high stability apart from pH 10, while their isoelectric point presented a slight variation from pHIEP 8.5 to 9.2 according to thulium content 0 and 2 at.%, respectively. The peak-to-peak amplitude of EPR spectra of the YET suspensions increased as pH shifted toward alkaline condition, following zeta potential curves features. The present achievements are very useful parameters to form stable suspensions based on rare-earth oxides and to advance toward new materials for radiation dosimetry.

Comparison of Colorimetric Methods for Measuring the Solubility of Legume Proteins

Increasing the use of plant proteins in foods requires improving their physical and chemical properties, such as emulsification, gelation capacity, and thermal stability. These properties determine the acceptability and functionality of food products. Higher protein solubility significantly impacts these properties by affecting denaturation and the stability of emulsifiers or gels. Therefore, developing plant-based protein ingredients requires accurately and conveniently measuring their solubility. Colorimetric solubility methods overcome many issues of more robust combustion and titration methods, but complicated chemical mechanisms limit their applicability for certain proteins. This study aims to compare the effectiveness of four common colorimetric solubility measurement methods for pulse and non-pulse legume proteins and hydrolysates. Pea, chickpea, lentil, and soy protein isolates were made from defatted flour and their solubility at a range of pHs was measured using the Bradford, Lowry, bicinchoninic acid (BCA), and biuret methods. Solubility was also measured for chickpea and soy protein hydrolysates made using Alcalase and Flavourzyme. A comparison of the methods for solubility quantification revealed that the Bradford and Lowry methods most closely match the expected results for the unhydrolyzed protein, with the BCA and biuret methods underestimating solubility by 30%. The Lowry method was the preferred method for hydrolysate solubility measurement, with the Bradford method measuring 0% solubility at the isoelectric point due to an inability to interact with peptides that are soluble at this pH. This study identifies reliable methods for measuring plant protein solubility that establish uniform outcomes and enable a better comparison across studies, giving a consensus for key functional properties in food applications.

Influence of life-history traits on mitochondrial DNA substitution rates exceeds that of metabolic rates in teleost fishes

Abstract Understanding the molecular relevance of metabolic rate (MR) is crucial for unveiling the mechanisms driving the evolution of animals. In this study, we investigated the association between mitochondrial DNA characteristics and both resting and maximal MRs in conjunction with life-history traits among 139 species of teleost fish. We gathered fish MR data from various sources and procured sequences of 13 mitochondrial protein-encoding genes. We calculated the absolute substitution rate for entire nucleotide sequences and 4-fold degenerate sites of each gene, along with encoding amino acid sequences. Using the phylogenetic comparative method, we then explored the associations between MR and mitochondrial DNA absolute substitution rate. Additionally, we screened MR-associated single nucleotide variants in mitochondrial DNA. The findings indicate no positive correlation between MRs and any substitution rate values of both combined sequences and individual mitochondrial protein-coding genes, refuting the MR hypothesis. Instead, both maximum body size and longevity correlated negatively with molecular substitution rates, suggesting their influences on both mutation and fixation within mitochondrial genes in fish. Results also revealed significant correlations between base variation at ATP6_169 and both resting MR and maximum MR, identifying the unique ATP6_169G in Scombridae fish, which results in an extremely low isoelectric point (pI) value of the ATP6 protein. Considering its functional significance, the ATP6_169G in Scombridae fish might link to their lifestyle characterized by fast locomotion and high metabolic demands alongside a slower molecular evolutionary rate.

Selective Inhibition Mechanisms of Fe(III) in the Flotation of Lepidolite

Lepidolite, crucial for lithium extraction, is primarily processed through flotation. However, conventional flotation inhibitors pose environmental challenges. This study introduces Fe(III) as a selective and eco-friendly inhibitor in lepidolite flotation. We investigated its impact on flotation performance and interaction mechanisms with feldspar, quartz, and lepidolite. Adsorption studies revealed that dodecylamine (DDA) selectively adsorbs onto lepidolite when Fe(III) is present. Consistent contact angle and flotation results showed reduced hydrophobicity and recovery rates for feldspar and quartz, with minimal impact on lepidolite. Zeta potential measurements indicated lower potentials for feldspar and quartz compared to lepidolite. Notably, the addition of Fe(III) altered the isoelectric points of quartz and feldspar, suggesting stronger Fe(III) adsorption on these minerals. Infrared spectroscopy and X-ray photoelectron spectroscopy confirmed reduced DDA adsorption on feldspar and quartz surfaces due to Fe(III) adsorption, while DDA adsorption on lepidolite remained largely unaffected. The mechanism underlying Fe(III)’s selective inhibition on feldspar and quartz involves their more negative surfaces compared to lepidolite, which facilitates Fe(III) adsorption and inhibits DDA adsorption. This study offers insights into mechanisms relevant to systems using metal ions as depressants, providing valuable references for similar research.

Nanogel particles formed from ethanol-treated whey proteins: Effect of heating, pH and NaCl on their stability.

Whey protein particles have gained significant attention for their ability to enhance the functional properties of foods as they can be used for emulsions and foams stabilization or fat substitution. This study explores the ability of whey protein nanogel particles formed in the presence of ethanol (30–70 % w/w) by controlling pH and ionic strength, to retain their morphological properties after ethanol removal. The stability of the produced nanogel particles after the removal of ethanol was investigated under various conditions, including heat treatment (90 °C, 20 min), pH modification (4−7), and the addition of NaCl (50–300 mM). Morphological analysis was conducted using confocal laser scanning microscopy, laser diffraction analysis, and scanning electron microscopy. The results showed that the exclusion of ethanol resulted in the formation of spherical-like nanogel particles with different sizes ranging from 100 to 200 nm in diameter. The pH of the solution affected the stability of the ethanol-free particles, leading to aggregation when the net charge of the protein approached zero. However, no aggregation phenomena were observed away from the isoelectric point. High concentrations of NaCl led to extensive aggregation, but no substantial changes were found upon heating. The production of nanogel particles is a promising advance with potential for a wide range of food applications.

LEP Gene Promotes Milk Fat Synthesis via the JAK2-STAT3 and mTOR Signaling Pathways in Buffalo Mammary Epithelial Cells.

Leptin (LEP), a protein hormone well-known for its role in metabolic regulation, has recently been linked to lipid metabolism in cattle. However, its function in buffalo mammary glands remains unclear. To address this issue, we isolated and identified the LEP gene and conducted experiments to investigate its function in buffalo mammary epithelial cells (BuMECs). In this study, two transcript variants of LEP, designated as LEP_X1 and LEP_X2, were identified. The coding sequences (CDS) of LEP_X1 and LEP_X2 are 504 bp and 579 bp in length, encoding 167 and 192 amino acid residues, respectively. Bioinformatics analysis revealed that LEP_X2 is a hydrophobic protein with an isoelectric point below 7 and contains a signal peptide, while LEP_X1 is hydrophilic and lacks a signal peptide. Our study found that LEP gene expression in lactating BuMECs was significantly higher than in non-lactating cells, with LEP_X2 expression remarkably higher than LEP_X1 in lactating BuMECs. Overexpression of both LEP_X1 and LEP_X2 significantly promoted the expression of genes related to milk fat synthesis in lactating BuMECs, including STAT3, PI3K, mTOR, SCD, and SREBF1, accompanied by an increase in cellular triglycerides (TG). Interestingly, LEP_X2 overexpression significantly suppressed LEP_X1 expression while increasing intracellular TG concentration by 12.10-fold compared to LEP_X1 overexpression, suggesting an antagonistic relationship between the two variants and supposing LEP_X2 plays a dominant role in milk fat synthesis in lactating BuMECs. Additionally, four nucleotide substitutions were identified in the buffalo LEP CDS, including a nonsynonymous substitution c.148C>T (p.Arg50Cys), which was predicted to decrease the stability of the LEP protein without affecting its function. These results collectively underscore the significant role of LEP in milk fat synthesis and can provide a basis for molecular breeding strategies of buffalo.

Toll-like receptor 21 in Labeo rohita recognizes double-stranded RNA and lipopolysaccharides by engaging the critical motifs in the LRR domain and gets activated against bacterial assaults

TLR (Toll-like receptor)-21 is a non-mammalian TLR and exhibits a unique function within the innate immune systems of fishes, birds, and amphibians. Despite its important role as PRR (pattern recognition receptor), research on TLR21 in many fish species, as well as in rohu (Labeo rohita), remains relatively limited. This article describes the molecular cloning of LrTLR21 (TLR21 in L. rohita), 3D (3-dimensional) modelling of its LRR (leucine-rich repeat)-regions, prediction of LRR18 to LRR20 as the LPS (lipopolysaccharide)-binding site, and LRR1 to LRR5 and LRR21 to LRR23 as the poly I:C (polyinosinic-polycytidylic acid) binding sites. It also describes the response of LrTLR21 in response to Aeromonas hydrophila and Edwardsiella tarda infections and PAMPs (pathogen-associated molecular patterns) (LPS and poly I:C)-stimulations. The ORF (open reading frame) of the LrTLR21 comprises 2955 nucleotides, encoding 984 aa (amino acid) residues with molecular weight and isoelectric point of 113.791 kDa and ∼8.79, respectively. Domain analysis of the deduced LrTLR21 displayed the existence of a signal peptide (residues 1–24), 26 LRR regions (residues 61–685), a TM (transmembrane) domain (residues 736–758), and a TIR (Toll/interleukin-1 receptor) domain (residues 790–937). The 3D model of LrTLR21-LRR regions has parallel β-sheets and few α-helices. Phylogenetically, LrTLR21 is closely related to the Onychostoma macrolepis and Carassius gibelio TLR21, and during ontogenesis, it is expressed in most of the developmental stages. In rohu fingerlings, it is consistently expressed in all examined tissues viz., skin, liver, heart, blood, eye, muscle, kidney, intestine, brain, gill, and spleen. Upon exposure to E. tarda and A. hydrophila infections, as well as LPS and poly I:C stimulations, the expression of LrTLR21 is significantly (p < 0.05) increased in the blood, kidney, liver, and gill. In the RBCs (red blood cells), PBLs (peripheral blood leukocytes), and kidney macrophages, LrTLR21 is also significantly induced following in-vivo and in-vitro LPS and poly I: C-stimulations. These findings on LrTLR21 are the first ones showing its structural insights and PAMPs binding motifs and its key role in recognizing pathogens and their PAMPs in RBCs, PBLs, and macrophages.

Insight into the self-assembly process, microrheological properties and microstructure of acid-soluble collagen from lamb skin under different environmental conditions

The self-assembly behavior of collagen under the regulation of the external environment is important for the construction of collagen-based materials. In this study, the effects of temperature, pH, and ion concentration on the self-assembly property, microrheology, and microstructure of acid-soluble collagen (ASC) extracted from lamb skin were investigated. The kinetics of ASC self-assembly under different environmental conditions was captured by monitoring the turbidity over time. The results showed that the ASC assembly process was accelerated by temperature increase in the range of 25–37 °C, and the self-assembly was unfavorable when the temperature exceeded 37 °C. The pH closed to the isoelectric point of ASC was conducive to assembly, and appropriate ion concentration increased the assembly rate of ASC. The microrheology reflected the influence of environmental conditions on the assembly process through the changes in the elastic and solid-liquid transition points of the solution system, and the results were consistent with the conventional turbidimetry method. Scanning electron microscopy showed that ASC formed fibril structures with different diameters in different environments. In addition, infrared spectroscopy showed the changes of secondary structure of ASC during the assembly process. The amino acid composition, UV spectrum, secondary structure, and morphology of ASC were characterized. The above results provide a foundation for the construction of controllable ASC self-assemblies from lambskins.

Investigation into the impact of Mn2+ and its interaction with calcite in the flotation of rhodochrosite

The unavoidable Mn2+ ions in pulp were considered in the flotation of rhodochrosite, and its effect and relative interaction mechanism on the floatability of calcite served as a typical gangue mineral were investigated through microflotation tests, zeta potential measurements, SEM-EDS detection, solution chemistry calculation, adsorption experiments, FTIR and XPS analyses. There exhibited a good separation performance of rhodochrosite against calcite with OHA collector, but in the presence of Mn2+ ions, the floatability of calcite was closed to rhodochrosite within a pH of 8.0–10.0, which resulted in the separation difficulty between them. As evidenced, the surface of calcite could be modified by Mn2+ ions, its isoelectric point shifted positively from 8.91 to 10.06, and Mn2+ species detected on calcite surfaces were in close proximity to rhodochrosite. It indicated that MnCO3 precipitates were generated on the surface of calcite. After modified by Mn2+, the calcite surface became more active and obviously increased the adsorption amount of OHA. It was verified from zeta potential and XPS analyses that Mn species on the surface of calcite were active sites for OHA adsorption, likely the hydroxamate group of OHA collector chelated with Mn species to form hydrophobic compounds. As a result, Mn2+ ions in pulp would reduce the flotation selectivity of rhodochrosite from calcite, thus it is necessary to eliminate the disadvantage of Mn2+ for strengthening flotation.

Up-conversion luminescence and Bi SPR effect promoted Z-scheme BiVO4(040)-Bi-(NaYF4:Er,Yb,Tm@BiOBr)/Bi photocatalyst for efficient photocatalytic hydrogen production with simultaneous degradation of malachite green

Photocatalytic hydrogen production with simultaneous degradation of organic pollutants by using solar energy provide a promising strategy to solve the energy and environmental issue in the future. However, for onefold wide band-gap semiconductor, the poor photocatalytic efficiency limits its application on a large scale due to its easy recombination of photo-generated electron-hole pairs and low solar energy utilization. Herein, a novel Z-scheme BiVO4(040)-Bi-(NaYF4:Er,Yb,Tm@BiOBr)/Bi photocatalyst is prepared via photo-assisted isoelectric point method. In this composite photocatalyst, up-convention luminescence material, NaYF4:Er,Yb,Tm, can offer more ultraviolet light and visible light to intensify the activations of BiOBr and metal Bi nanoparticles, respectively. Furthermore, the formation of Z-scheme BiVO4(040)-BiOBr photocatalystic sytem, the spatial separation of the BiVO4(040) and BiVO4(110) facets and the presence of metal Bi surface plasmon resonance effect are contributed to the high-efficiency separation of the photo-generated electron-hole pairs. The experimental results show that as-prepared Z-scheme BiVO4(040)-Bi-(NaYF4:Er,Yb,Tm@BiOBr)/Bi photocatalyst exhibits advanced photocatalytic hydrogen production amount (192.0 μm) and excellent degradation ratio of malachite green (88.51 %) under simulated sunlight irradiation for 5.0 h. The apparent quantum yield of the Z-scheme BiVO4(040)-Bi-(NaYF4:Er,Yb,Tm@BiOBr)/Bi photocatalyst at 420 nm reaches a specific value of 5.8 %. In addition, Z-scheme BiVO4(040)-Bi-(NaYF4:Er,Yb,Tm@BiOBr)/Bi photocatalyst also remains a high stability in the photocatalytic hydrogen production with simultaneous degradation of malachite green within six reuse. At last, the possible reaction mechanism and degradation pathways of malachite green caused by Z-scheme BiVO4(040)-Bi-(NaYF4:Er,Yb,Tm@BiOBr)/Bi photocatalyst under simulated sunlight irradiation are put forward.

Quantitative study on the selective depressing effect of polysaccharides on some polar minerals

This study presents a systematic summary on the depressing effect of polysaccharides on some polar minerals, revealing that the order of their depressing strength on some polar minerals was primarily associated with the surface properties of the minerals, showing a limited correlation with the structure of polysaccharides. Subsequently, the selective depressing mechanism of polysaccharides on polar minerals was quantitatively explained from the perspective of acid–base interaction mechanism, and a criterion system for determining the depressing effect of polysaccharide on minerals was proposed. Specifically, it was found that the hydroxylation reaction of metal sites on mineral surface was a prerequisite for polysaccharide adsorption. Under the above–mentioned conditions, the selective depressing effect of polysaccharides on these minerals with different metal sites could be assessed through a parameter system in which the ion potential φ of surface metal sites served as the primary determinant, and the solubility product constant of metal hydroxides played a secondary role. In cases where minerals shared identical surface metal sites, the selective depressing effect of polysaccharides was analyzed by comparing the coordination numbers of these sites. Specifically, minerals with a higher number of coordinated surface metal sites tend to show a weaker response to depressing effect of polysaccharide. Consequently, this study theoretically elucidated the selective depressing effect of polysaccharides on polar minerals, and the criterion system proposed in this study outperforms the prediction results based on the isoelectric point (pHiep). These research findings have important guiding significance for a deeper understanding of the selective depressing mechanism of polysaccharides and the regulation of flotation.

Partition coefficient screening – An effective approach for finding the best conditions for byproduct removal as demonstrated by a bispecific antibody purification case

In recombinant protein purification, differences in isoelectric point (pI)/surface charge and hydrophobicity between the product and byproducts generally form the basis for separation. For bispecific antibodies (bsAbs), in many cases the physicochemical difference between product and byproducts is subtle, making byproduct removal considerably challenging. In a previous report, with a bsAb case study, we showed that partition coefficient (Kp) screening for the product and byproducts under various conditions facilitated finding conditions under which effective separation of two difficult-to-remove byproducts was achieved by anion exchange (AEX) chromatography. In the current work, as a follow-up study, we demonstrated that the same approach enabled identification of conditions allowing equally good byproduct removal by mixed-mode chromatography with remarkably improved yield. Results from the current and previous studies proved that separation factor determination based on Kp screening for product and byproduct is an effective approach for finding conditions enabling efficient and maximum byproduct removal, especially in challenging cases.

Large-scale analysis of the PAC domain structure of arogenate dehydratases reveals their evolutionary patterns in angiosperms

Arogenate dehydratase (ADT) is the key limiting enzyme of plant phenylalanine biosynthesis, but some ADTs display a prephenate decarboxylase/dehydratase activity-conferring (PAC) domain. The genome resources of 70 species were employed to identify genes and outline their characteristics, especially the number and type of PAC domain structures. We obtained 522 ADTs, and their size, exon number, amino acid number and putative protein isoelectric point greatly varied from 306 to 2520 bp, 1 to 15, 101 to 839 and 4.37 to 11.18, respectively. We classified the ADTs into Class α (without a PAC domain) (115, 22.0 %), β (with a type I PAC domain) (244, 46.7 %) and γ (with a type II PAC domain) (163, 31.2 %), and their distribution frequencies exhibited large differences among various branches of angiosperms. We found that Class γ members are more conserved than Class β members, although they commonly experienced multiple duplication events and strong purifying selection, which resulted in a small number, and the putative origin order was from Class α to β and then to γ. In addition, the co-occurrence of both Class β and γ members could ensure the survival of angiosperms, while their optimized composition and strategically intertwined regulation may facilitate core eudicot success.

Cloning and expression profiling of voltage-gated sodium channel gene (VGSC) from Spodoptera frugiperda

Spodoptera frugiperda is a long-distance migratory pest with strong dispersal ability, fast reproduction speed and destructive feeding, so it is difficult to prevent and control. Pyrethroid insecticides are commonly used in pest insects control, And since the voltage-gated sodium channel (VGSC) serves as a major target of pyrethroids, it is important to study this gene for pest control. VGSC is an integral transmembrane protein consisting of approximately 2,000 amino acid residues found in neurons, myocytes, endocrine cells, and ovarian cells and involved in the initiation and propagation of excitable cellular action potentials. In this study, the cDNA sequence of the VGSC was identified from S. frugiperda by rapid amplification of cDNA ends (RACE) which contained an open reading frame of 6,261 bp encoding a protein of 2,086 amino acids. The molecular weight of this protein was predicted to be 236 kDa, and the theoretical isoelectric point was 5.21. A phylogenetic tree constructed based on lepidopteran insects showed that the VGSC of S. frugiperda was most closely relative to that of Spodoptera litura. VGSC is a highly conserved protein with Ion channel conserved structural domains of transmembrane proteins. qPCR showed that the VGSC gene was highly expressed in the epidermis of 2nd instar larvae, and its expression level was low in other tissues, such as the foregut and Malpighian tubules. In addition, VGSC was also detected in the prepupal stage, then gradually increased in abundance after entering the adult stage, peaked at the adult males on the 4th day of pupal stage, and decreased afterwards. The recombinant plasmid of pSumo-mut-VGSC was constructed and induced to express a His tag fused VGSC protein. Polyclonal antibodies were prepared from purified recombinant VGSC protein. The antibody was ELISA-titered, and the western blotting results showed that it specifically recognized VGSC, whether it was recombinant or endogenous protein. These results have laid the foundation for future studies on the physiological function of this gene in the growth and development of S. frugiperda.

Spiro[2.3]hexane‐ and Spiro[3.3]heptane‐derived α‐Amino Acids: Synthesis and Isoelectric Point Evaluation

AbstractAn efficient synthesis of spiro[2.3]hexane‐ and spiro[3.3]heptane‐derived α‐amino acids is reported. The used methods are divided on two different approaches. First of them involves modification of commercially available spirocyclic diesters via monohydrolysis, Curtius rearrangement and functional groups deprotection sequence. Alternative approach was based on Bucherer ‐ Bergs hydantoine synthesis with its subsequent cleavage to the target amino acid fragment. The proposed synthetic schemes offer the desired product in 31–52 % overall yield on up to 22.5 g scale. Acid‐base titration of the obtained spirocyclic amino acids revealed lowered pI values compared to the parent 1‐amino‐cyclohexane‐ and ‐cycloheptanecarboxylic acids.

A comprehensive study on the multifunctional properties of galectin-4 in red-lip mullet (Planiliza haematocheilus): Insights into molecular interactions, antimicrobial defense, and cell proliferation

Galectin-4 belongs to the galactoside-binding protein family and is a type of tandem repeat galectin. Despite previous studies indicating its importance in fish immunology, a comprehensive investigation is necessary to fully understand its role in immunomodulatory functions and cellular dynamics. Therefore, this study aimed to explore the immunomodulatory functions of galectin-4 with a particular focus on its antimicrobial and cellular proliferative properties. The open reading frame of PhGal4 spans 1092 base pairs and encodes a soluble protein of 363 amino acids with a theoretical isoelectric point (IEP) of 6.39 and a molecular weight of 39.411 kDa. Spatial expression analysis under normal physiological conditions revealed ubiquitous expression of PhGal4 across all examined tissues, with the highest level observed in intestinal tissue. Upon stimulation with poly I:C, LPS, and L. garvieae, a significant increase (p < 0.05) in PhGal4 expression was observed in both blood and spleen tissues. Subsequent subcellular localization assay demonstrated that PhGal4 was predominantly localized in the cytoplasm. The recombinant PhGal4 (rPhGal4) exhibited specific binding capabilities to pathogen-associated molecular patterns (PAMPs), including LPS and peptidoglycan, but not poly I:C. The rPhGal4 negatively affected the bacterial growth kinetics. Additionally, rPhGal4 demonstrated complete hemagglutination of fish erythrocytes, which could be inhibited by the presence of D-galactose and α-lactose. The overexpression of PhGal4 in FHM epithelial cells demonstrated a significant suppression of viral replication during VHSV infection. Furthermore, the in vitro scratch assay and WST-1 assay demonstrated a wound healing effect of PhGal4 overexpression in FHM cells, potentially achieved through the promotion of cell proliferation by activating genes involved in cell cycle regulation. In conclusion, the responsive expression to immune stimuli, antimicrobial properties, and cell proliferation promotion of PhGal4 suggest that it plays a crucial role in immunomodulation and cellular dynamics of red-lip mullet. The findings in this study shed light on the multifunctional nature of galectin-4 in teleost fish.

Lysine methylation: A strategy to improve in-cell NMR spectroscopy of proteins

BackgroundIn-cell NMR is a valuable technique for investigating protein structure and function in cellular environments. However, challenges arise due to highly crowded cellular environment, where nonspecific interactions between the target protein and other cellular components can lead to signals broadening or disappearance in NMR spectra. ResultsWe implemented chemical reduction methylation to selectively modify lysine residues on protein surfaces aiming to weaken charge interactions and recover obscured NMR signals. This method was tested on six proteins varying in molecular size and lysine content. While methylation did not disrupt the protein's native conformation, it successful restored some previously obscured in-cell NMR signals, particularly for proteins with high isoelectric points that decreased post-methylation. SignificanceThis study affirms lysine methylation as a feasible approach to enhance the sensitivity of in-cell NMR spectra for protein studies. By mitigating signal loss due to nonspecific interactions, this method expands the utility of in-cell NMR for investigating proteins in their natural cellular environment, potentially leading to more accurate structural and functional insights.

Extremely acidic proteomes and metabolic flexibility in bacteria and highly diversified archaea thriving in geothermal chaotropic brines.

Few described archaeal, and fewer bacterial, lineages thrive under salt-saturating conditions, such as solar saltern crystallizers (salinity above 30% w/v). They accumulate molar K+ cytoplasmic concentrations to maintain osmotic balance ('salt-in' strategy) and have proteins adaptively enriched in negatively charged acidic amino acids. Here we analysed metagenomes and metagenome-assembled genomes from geothermally influenced hypersaline ecosystems with increasing chaotropicity in the Danakil Depression. Normalized abundances of universal single-copy genes confirmed that haloarchaea and Nanohaloarchaeota encompass 99% of microbial communities in the near-life-limiting conditions of the Western-Canyon Lakes. Danakil metagenome- and metagenome-assembled-genome-inferred proteomes, compared with those of freshwater, seawater and solar saltern ponds up to saturation (6-14-32% salinity), showed that Western-Canyon Lake archaea encode the most acidic proteomes ever observed (median protein isoelectric points ≤4.4). We identified previously undescribed haloarchaeal families as well as an Aenigmatarchaeota family and a bacterial phylum independently adapted to extreme halophily. Despite phylum-level diversity decreasing with increasing salinity-chaotropicity, and unlike in solar salterns, adapted archaea exceedingly diversified in Danakil ecosystems, challenging the notion of decreasing diversity under extreme conditions. Metabolic flexibility to utilize multiple energy and carbon resources generated by local hydrothermalism along feast-and-famine strategies seemingly shapes microbial diversity in these ecosystems near life limits.