Search Results (1,833)

Carnitine plays an essential role in fatty acid metabolism, and its biosynthesis is tightly regulated by γ-butyrobetaine hydroxylase (BBOX), an Fe(II)/α-ketoglutarate-dependent dioxygenase. BBOX is the target of mildronate (THP), a clinically used drug for treating ischemic heart diseases. However, the detailed mechanisms of BBOX-catalyzed hydroxylation and the atypical oxidative rearrangement underlying THP inhibition remain elusive. In this study, we employed combined quantum mechanics/molecular mechanics (QM/MM) methods to systematically elucidate these mechanisms at the atomic level. Our calculations reveal that the hydroxylation of γBB proceeds via a classical three-step mechanism in the quintet state, with hydrogen atom abstraction as the rate-determining step. Remarkably, substitution of the C4 methylene group in γBB with an amino group in THP redirects the reaction pathway, as the lone pair electrons on the adjacent nitrogen atom render N-N bond cleavage kinetically favored over hydroxyl rebound, thereby blocking carnitine synthesis. Through systematic evaluation of possible rearrangement pathways, we rule out the previously proposed direct 1,2-H migration and suggest a revised mechanism featuring imine-mediated hydrogen transfer, hydroxyl rebound preceding C-C bond formation, and final radical coupling. This work provides a detailed atomic-level understanding of both the catalytic and inhibitory mechanisms of BBOX, revealing how substrate electronic effects dictate reaction outcomes. The elucidated mechanistic insights offer a theoretical foundation for understanding the catalytic versatility of the αKG-dependent dioxygenase family and provide valuable guidance for the rational design of novel BBOX inhibitors. Full article

PPV5 NS1 is a nonstructural and multifunctional protein comprising helicase and nuclease domains. The helicase domain contains conserved motifs from superfamily 3 helicases, including Walker A, Walker B, Motif B’, Motif C, and Box VII, whereas the nuclease domain consists of glutamate, a HUH motif, lysine, and tyrosine. In Mexico, the reported prevalence of PPV5 is higher than in other countries, with notable amino acid differences compared with pathogenic PPVs. This study compares the helicase and nuclease domains from a full-length PPV5 NS1 sequence with porcine parvovirus 1 (PPV1) and canine parvovirus (CPV) to characterize the protein further and perform three-dimensional (3D) modeling using bioinformatic tools, including solvent-accessible surface area (SASA) and electrostatic potential assessments. The main findings highlight the ATP-binding pocket, showing electrostatic values in PPV5 that contrast with PPV1 and CPV. The electrostatic potential 3D models suggest those differences involve non-conserved regions. In particular, the PPV5 Box VII surface is predominantly negative due to a glutamate substitution at position 7. In the nuclease domain, the interaction with Mg²⁺ differs between PPV5 and pathogenic PPV. The electrostatic findings suggest that these differences may have functional implications for both domains, but confirmation must be completed with functional assays. Full article

Type 1 diabetes (T1D) is driven by autoreactive CD4⁺ T-cell responses to pancreatic beta cell antigens presented by disease-associated human leucocyte antigen (HLA) class II molecules. However, the molecular mechanisms by which subtle antigenic modifications promote pathogenic immunity remain incompletely defined. Recent immunopeptidomic studies have identified a cysteine-to-serine substitution at position 19 of the insulin B chain, referred to as InsC19S, as a microenvironment-driven neoepitope that can be presented by HLA class II molecules, including HLA-DQ8, and is recognized by diabetogenic CD4⁺ T cells. In this study we explore potential structural and thermodynamic mechanisms that may contribute to the enhanced immunogenicity associated with this single-amino-acid modification. Using molecular dynamics simulations combined with coarse-grained free-energy-perturbation analyses, we compared HLA DQ8 complexes bound to wild-type (WT) insulin and InsC19S peptides. The InsC19S variant is predicted in simulations to exhibit enhanced binding stability, characterized by increased hydrogen bond occupancy, reduced peptide conformational mobility, and a more favorable binding free energy. In addition, the modified peptide is predicted to induce peptide-dependent conformational adjustments within the HLA-DQ8 peptide-binding groove, resulting in expansion of the conformational landscape and stabilization of distinct low-energy states that are not accessed by the WT complex. Principal component analysis and free-energy landscape mapping suggest that this mutation may promote altered collective motions within HLA DQ8 that are consistent with enhanced peptide major histocompatibility complex (MHC) persistence and optimized antigen presentation geometry. Together, these computational observations suggest a structural framework that may help explain the preferential presentation and pathogenic recognition of InsC19S reported in experimental studies. These findings provide a molecular-level framework that may help link microenvironment-driven insulin neoepitope formation to altered peptide–MHC stability and conformational dynamics in HLA-DQ8. Full article

A 7-week study was conducted to investigate the effects of replacing fish meal (FM) with Spirulina residue meal (SPRM) on the growth, feed utilization, carcass composition, antioxidant ability, liver and intestinal histology of juvenile rainbow trout (Oncorhynchus mykiss) (initial body weight 5.36 ± 0.04 g). Four isonitrogenous (42%) and isolipidic (16%) diets were formulated to replace FM protein with SPRM at 0 (SPRM0), 10% (SPRM10), 20% (SPRM20) and 30% (SPRM30), respectively. Results showed that growth, feed utilization, carcass amino acid profile, serum biochemical indices, antioxidant ability, intestinal and liver histology were not significantly affected by dietary SPRM levels. Whole-body lipid content decreased as dietary SPRM replacement levels increased, and fish fed diet SPRM30 had lower lipid content than that fish fed diet SPRM0 (p < 0.05). Fish fed diet SPRM30 had higher C16:1n-7, C20:3n-6, total saturated fatty acid (SFA) and total fatty acid (TFA) contents in muscle than those fed other diets (p < 0.05), while these fatty acids had no change when FM was substituted with 10% and 20% SPRM (p > 0.05). The muscle C22:6n-3 (DHA) content decreased, but C18:3n-6 and n-6/n-3 polyunsaturated fatty acid (PUFA) ratio increased with increasing SPRM levels, and fish fed diet SPRM30 had significantly lower DHA content and higher n-6/n-3 ratio than the group fed SPRM0 (p < 0.05). The C22:1n-9, C18:2n-6c, C20:4n-6, total n-6 PUFA, and monounsaturated fatty acid (MUFA) content in muscle observed in SPRM30 were similar to the SPRM0 group (p > 0.05), but higher than the SPRM10 and SPRM20 groups (p < 0.05). In conclusion, 30% of FM protein could be replaced by SPRM in diets of juvenile rainbow trout without having a significant negative effect on growth, feed efficiency, antioxidant ability, and structure of liver and intestine, but could reduce DHA content, increase n-6 PUFA and n-6/n-3 PUFA ratio in muscle. Full article

Ricinus communis is the primary commercial source of ricinoleic acid (RA), a hydroxy fatty acid (HFA) synthesized by the fatty acid hydroxylase FAH12, which evolved from the Δ12-oleate desaturase FAD2. However, the evolutionary origins and diversification mechanisms of FAH12 across HFA-producing plants remain poorly understood. Here, we performed a comprehensive cross-species analysis of FAH12 and FAD2 homologs by integrating sequence analysis, structural modeling, phylogenetic reconstruction, and transcriptomic profiling. Across all currently available HFA-producing plant lineages, we found that amino acid substitutions associated with hydroxylase activity exhibit strong lineage-specific patterns rather than universal conservation, indicating multiple evolutionary solutions to catalytic divergence. Phylogenetic and synteny analyses further suggest that FAH12 arose independently from ancestral FAD2 duplications in distinct plant lineages, supporting a model of recurrent independent neofunctionalization. Transcriptomic and qRT-PCR analyses reveal that FAH12 exhibits a highly specialized endosperm-preferential expression pattern and is embedded within a regulatory network that is partially decoupled from that of FAD2. Together, these findings demonstrate that FAH12 evolution is driven by recurrent independent origins coupled with transcriptional specialization, providing a framework linking structural variation, evolutionary history, and regulatory divergence for understanding and engineering hydroxy fatty acid biosynthesis in plants. Full article

Phosphorus Starvation Tolerance 1 in rice (OsPSTOL1, known as Phosphorus uptake 1, Pup1) is a receptor-like cytoplasmic protein kinase that confers tolerance to phosphorus deficiency. The OsPSTOL1 gene possesses a Ser/Thr kinase and shows high amino-acid sequence similarity with the leaf rust receptor-like kinase (OsLrK10). We hypothesise that the putative wheat genes TaPSTOL1 and TaLrK10 have a common ancestral origin and that putative TaPSTOL1 diverged recently, acquiring new structural modifications and biological functions in the process. In this study, we identified all putative TaPSTOL1 homeologs and examined the evolutionary relationship between TaPSTOL1 and TaLrK10 in Triticum species. Our results indicate that the putative TaPSTOL1 diverged recently without possessing the amino-terminal domain, which is a typical characteristic of TaLrK10. We observed numerous conversion tracts between these two genes, and the substitution pattern of randomly selected amino acids indicates that dynamic selection pressures acted on both genes. The putative TaPSTOL1 shows high nucleotide diversity compared to TaLrK10 within Triticum species. Further, a multiple-sequence analysis reveals that the third exon of TaLrK10 appears to have been duplicated and diverged as a putative single-exon-based TaPSTOL1 in bread wheat. Overall, our comparative analysis indicates that both TaPSTOL1 and TaLrK10 appear to have diverged from a common ancestor, acquiring distinct structural organisations and biological functions. Full article

Feline panleukopenia virus (FPLV) is the primary causative agent of a highly contagious and often fatal disease affecting domestic cats and other felids. The increasing isolation of species-specific FPLV variants from multiple host species has garnered considerable attention, highlighting the need to investigate their genetic diversity. In this study, three FPLV isolates were obtained and phylogenetically classified into two distinct FPLV-China groups within separate clusters. Compared to the prototype FPLV (M38246.1), these isolates exhibited seven amino acid substitutions in the NS1 (n = 6) and VP2 (n = 1) proteins. Further analysis of 157 NS1 sequences and 947 VP2 sequences retrieved from the NCBI database revealed 113 and 479 synonymous substitutions and 71 and 279 non-synonymous substitutions, respectively. Notably, the majority of these substitutions occurred as single events (57% in NS1, 40/71; 55% in VP2, 153/279) or were present in no more than five FPLV sequences (23% in NS1, 16/71; 32% in VP2, 89/279). However, three non-synonymous substitutions in the NS1 protein (Ile443Val, His595Gln, and Val596Leu) were detected in more than half of the 157 sequences analyzed. In the VP2 protein, six non-synonymous substitutions (Ala91Ser, Thr101Ile, Val232Ile, Lys93Asn, Asp323Asn, and Val562Leu) were each found in 20 to 40 FPLV sequences. Furthermore, ten sites in the NS protein and 224 sites in the VP2 protein exhibited both synonymous and non-synonymous substitutions simultaneously. Additionally, 75 sites in VP2 harbored multiple non-synonymous substitutions. These findings provide valuable insights for future research on the genetic determinants and vaccine development of FPLV. Full article

The orphan insulin receptor-related receptor (IRR), in contrast to its homologs from the insulin receptor family, is activated by a mildly alkaline extracellular medium. We have previously demonstrated that IRR activation is defined by two synergistic sites located in the dimeric extracellular domain. Here, we describe artificial mutations in the IRR transmembrane domain that promote receptor activation. First, using molecular modeling based on the NMR-derived structure, we proposed amino acid substitutions that could enhance non-covalent interactions between the transmembrane segments of the IRR dimer. These mutations were subsequently tested for effects on pH sensing by IRR. We showed that double-mutant A938E-A939R was highly phosphorylated at neutral pH and still sensitive to alkaline pH. Remarkably, the double substitution of V929E-G930R resulted in strong basal phosphorylation of the receptor over the pH titration range. Through site-directed mutagenesis, we demonstrated that the transmembrane domain plays a critical role in IRR activation, allowing for targeted control of functioning of the receptor, including its pH sensitivity. Full article

Background/Objectives: The envelope (E) protein of orthoflaviviruses contains antigenic sites that are composed of one or more epitopes, which can vary in antigenic specificity, including between viral species, strains, and even substrains. Monoclonal antibodies (mAbs) that bind these epitopes vary in functionality based on their specificity. This makes mAbs useful to study the differences in phenotypes between strains of viruses, such as the wild type (WT) and live attenuated vaccine strains of yellow fever virus (YFV). mAb 2D12 was raised against the 17D-204 YFV vaccine substrain virus (YF VAX^®) by Schlesinger et al. in 1983. However, it only neutralizes Asibi WT virus, not the 17D-204 vaccine substrain virus. Results: We confirmed these results and demonstrated that mAb 2D12 fails to neutralize all 17D vaccine substrains (17D-204, 17DD, and 17D-213), indicating that the minor differences between these virus substrains do not affect the epitope or functionality of mAb 2D12. In addition, mAb 2D12 was found to neutralize WT strain of French viscerotropic virus (FVV), with statistically indistinguishable neutralization from the WT strain Asibi. All but one of the live attenuated French neurotropic vaccine (FNV) derivative viruses had significantly lower neutralization than WT strains Asibi and FVV. FVV, Asibi, 17D, and FNV have many amino acid differences in the membrane (M) and E proteins. It is unclear which of them contributes to this differential neutralization. However, FNV and 17D have common amino acid substitutions from WT FVV and Asibi at positions M-36 and E-331, suggesting that one or both of these residues may contribute to the 2D12 epitope. Conclusions: Overall, mAb 2D12 is a valuable tool to distinguish WT virulent strains of YFV from live attenuated vaccine strains. Full article

The ubiquitously transcribed tetratricopeptide repeat Y-linked gene (UTY/KDM6C), a catalytically impaired histone demethylase encoded on the Y chromosome, has garnered increasing attention for its emerging roles in tumorigenesis and cancer progression. Despite high sequence homology with its X-linked paralog UTX/KDM6A, UTY exhibits markedly reduced or absent H3K27me3 demethylase activity due to critical amino acid substitutions in its Jumonji C domain. Consequently, UTY primarily functions through non-enzymatic mechanisms, acting as a scaffold in chromatin-remodelling complexes like COMPASS and SWI/SNF, or mediating protein–protein interactions that regulate transcriptional programs independent of demethylation. This aligns with epigenetic dysregulation in cancers, where imbalances in repressive H3K27me3 and active H3K4me either drive tumour suppressor silencing or oncogene activation. Unlike frequently mutated UTX in cancers such as breast, renal cell carcinoma, and acute myeloid leukaemia, UTY’s contributions in cancer are less defined, constrained by male-specific expression. Emerging evidence suggests UTY as a context-dependent tumour suppressor in AML and squamous-like pancreatic ductal adenocarcinoma. While direct functional validation remains limited in several cancer types, UTY is increasingly implicated as a potential tumour suppressor in haematological malignancies and prostate cancer. Therapeutically targeting UTY’s scaffold functions shows promise for male-specific cancers and merits future investigation. Full article

Aquaculture is expanding rapidly, creating a greater need for sustainable and cost-effective feed ingredients to reduce reliance on traditional protein sources such as fishmeal (FM) and soybean meal (SBM). Insect-derived frass, which consists of insect excrement, molted exoskeletons, uneaten substrate, plus associated microbial biomass, has shown potential as a viable and sustainable ingredient in aquafeed. Although traditionally used as an organic fertilizer, its richness in essential nutrients and bioactive compounds highlights its potential as a partial substitute for conventional feedstuffs. This study synthesizes current research on insect-derived frass, focusing on its nutritional composition and effects on growth performance, immunity, health, and gut microbiota in aquaculture species, alongside environmental, economic, safety, and regulatory considerations. Although a wide range of insect species have been evaluated for use in aquafeeds, research on insect frass has primarily focused on black soldier fly and yellow mealworm, with most studies examining its application in omnivorous fish species. Despite its promise as a circular economy-aligned aquafeed ingredient, challenges remain due to nutritional and amino acid variability, largely influenced by the quality of the original insect rearing substrate, as well as species-specific responses and potential contamination risks. To promote widespread adoption of insect-derived frass in aquafeed, there is a need to optimize insect rearing substrate selection and processing, define inclusion levels by insect and target aquatic species, establish safety protocols, and develop harmonized international standards. Full article

The appressorium is a specialized infection structure formed by Metarhizium anisopliae during host invasion. To investigate the correlation between appressorium formation and fungal pathogenicity, as well as its impact on insect cuticular metabolism, different concentrations of sulforaphane were used to inhibit the appressorium formation rate of M. anisopliae. The relationship between appressorium formation rate and pathogenicity against Opisina arenosella larvae was evaluated, and LC-MS-based metabolomics was employed to characterize changes in cuticular compounds during the appressorium formation stage, thereby elucidating the chemical responses of the insect cuticle to appressorium formation. The appressorium formation rate of M. anisopliae was significantly and positively correlated with its pathogenicity (p ≤ 0.05). As the appressorium formation rate increased, pathogenicity against O. arenosella larvae increased and the killing speed accelerated. LC-MS metabolomics revealed that after appressorium formation, 102 differential cuticular compounds unique to O. arenosella larvae were identified, mainly including benzenes and substituted derivatives, amino acids and derivatives, and heterocyclic compounds. In addition, metabolic pathways associated with immune defense (tyrosine metabolism), antifungal defense (histidine metabolism), and toxin degradation (flavonoid degradation) in the larval cuticle were activated. These results demonstrate that the appressorium plays an important role in host infection by M. anisopliae, markedly alters cuticular metabolism, and activates defense- and detoxification-related metabolic pathways in the host. This study provides a theoretical basis for further investigations into fungus–insect cuticle interactions. Full article

Background/Objectives: Longer oligoarginines are very effective cell-penetrating peptides. It has been shown that a minimal number of positively charged side chains is necessary for efficient cellular uptake. But a highly positively charged peptide may interact with its cargo molecule, thereby reducing its efficiency. Several chemical modifications were tested to improve the internalization of short tetraarginine derivatives. Aromatic groups, such as Dabcyl at the N-terminus, Trp in the sequence, and AMBA or PABA in the backbone, were used to improve internalization. The other useful modification was the aza-glycine substitution in the case of penetratin. Methods: In this study, the effect of aza-glycine insertion into the peptide Dabcyl-RRRRK(Cf) on internalization was studied and compared with that of the Trp-modified peptide Dabcyl-RRWRRK(Cf). To explain the noticed difference in the biological activity of peptides, DFT calculations and the prediction of membrane-binding free energy (ΔΔF) from a peptide sequence were performed. Results: It turned out that the position of the aza-glycine moiety does not have an influence on the cellular uptake. The aza-glycine-containing peptide showed higher internalization than the Dabcyl-RRRRK(Cf) peptide. Besides this, these peptides have similar or higher cellular uptake than that of octaarginine at lower concentrations (c < 2 µM). The aza-glycine affected not only cellular uptake but also the entry mechanism. The structure of peptides depended on the amino acids (Trp, Gly, or azaGly) in their sequences and their positions. Conclusions: These may result in the different amphiphilicity of peptides, and thus changes in the hydrophobic moment and in the binding affinity of peptides to the negatively charged membrane surface. Full article

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major cause of morbidity and mortality worldwide, particularly due to the emergence of drug-resistant strains. Membrane-active antimicrobial peptides (AMPs) represent attractive therapeutic candidates because they target bacterial envelope integrity and disrupt essential cellular processes. We evaluated two rationally designed LL-37-derived peptides: a truncated C-terminally amidated analog (LL37-1) and a modified variant incorporating N-terminal acetylation and a single D-amino acid substitution (D-LL37). Dose–response analysis demonstrated that D-LL37 exhibited greater antimycobacterial potency, with lower inhibitory concentrations of 90% (IC₉₀) and 50% (IC₅₀) values (18.40 ± 0.39 μM and 10.11 ± 0.60 μM, respectively) compared with LL37-1 (25.44 ± 0.36 μM and 15.45 ± 1.40 μM). Fluorescence-based permeability assays revealed partial membrane disruption (36% and 44% at IC₉₀ for LL37-1 and D-LL37, respectively), which was supported by ultrastructural alterations observed by scanning electron microscopy, including bacillary shortening, rough surface formation, cell clusters, and the presence of cellular debris, all of which are consistent with membrane damage. RT-qPCR analysis demonstrated significant upregulation of the P-type ATPase genes ctpF, ctpA, and ctpH following D-LL37 exposure. Collectively, these findings indicate that optimized LL-37-derived peptides exert antitubercular activity associated with envelope perturbation and coordinated activation of ion transport-related stress responses. Full article

Background/Objectives: Investigating the modified derivatives of known cell-penetrating peptides can highlight the important residues in the peptide sequence and help understand the cellular uptake mechanism better. Moreover, comparing peptides with different fluorescent-dye positions can highlight the importance of the conjugation site. Earlier, it was demonstrated that the fluorescence quencher 4-((4-(dimethylamino)phenyl)azo)benzoyl (Dabcyl) group can enhance the internalization efficiency of highly cationic oligoarginine peptides. However, its effect in the case of arginine-rich penetratin, a secondary amphipathic cationic CPP, remains undiscovered. Methods: Here, several penetratin derivatives were studied in which the aromatic residues were substituted and the effect of Dabcyl modification was also studied on the cellular uptake of peptides by flow cytometry. Results: The triple Nal-substituted penetratin and dodeca-penetratin with N-terminally positioned carboxyfluoresein (Cf) dye demonstrated remarkable internalization efficiency compared to penetratin. Moreover, almost all the Dabcyl-modified peptides were superior to penetratin except two peptides with C-terminal Cf-labelling. This result highlights the importance of the structure of the conjugate. The position of the cargo molecule may have a high impact on internalization ability. The relatively low cellular uptake of the Trp48 residue-substituted Dabcyl-Pen12 points to the importance of this residue in the cellular uptake of dodeca-penetratin. The confocal microscopic studies revealed that, besides the greater penetration efficiency of Dabcyl penetratin derivatives, these peptides enter the cytoplasm of cells in an increased manner. Conclusions: We identified several intriguing derivatives and expanded the applicability of Dabcyl, while also highlighting its limitations. Additionally, the critical role of Trp48 in the penetratin sequence was reaffirmed, along with the importance of the fluorescent molecule’s position. Full article