Search Results (57)

(1) Background: The ongoing panzootic of highly pathogenic avian influenza virus (HPAIV) of subtype H5N1, clade 2.3.4.4b, has decimated wild/domestic birds and mammals’ populations worldwide with reports of sporadic cases in humans. (2) Methods: This study aimed to compare the mutational profile of H5N1 avian Influenza virus isolated from a Peruvian natural reserve, with recent data from other related international studies made in human and different species of domestic and wild birds and mammals. Briefly, the near complete protein sequences of the Influenza virus coming from a Calidris alba were analyzed at a multisegmented level, together with 55 samples collected between 2022 and 2024 in different countries. Moreover, the glycosylation patterns were also predicted in silico. (3) Results: A total of 603 amino acid changes were found among H5N1 viruses analyzed, underscoring the detection of critical mutations HA:11I, HA:211I, HA:336T, HA:492D, HA:527I, NA:10T, NA:269L, NA:405T, NP:377N, PA:57R, PA:68S, PA:322V/L, PA:432I, PB2:539V, PB1:207R, PB1:375N, PB1:264D, PB1:429R, PA-X:250Q, PB1-F2:65R, and PB1-F2:42Y, as well as PA:13V, PA-X:13V, PA20T, PA-X:20T, PA:36T PA-X:36T, PA:45S, PA-X:45S, PA:57Q, PA-X:57Q, PA:61I, PA-X:61I, PA:68S, PA-X:68S, PA:70V, PA-X:70V, PA:75Q, PA-X:75Q, PA:85T, PA-X:85T, PA:86I, PA-X:86I, PA:100I, PA-X:100I, PA:142E, PA-X:142E, PA:160E, PA-X:160E, PA:211I, PA-X:211Y, among others, considered of importance under the One Health perspective. Similarly, changes in the N-linked glycosylation sites (NLGs) predicted in both HA and NA proteins were found, highlighting the loss/acquisition or changes in some NLGs, such as 209NNTN, 100 NPTT, 302NSSM (HA) and 70NNTN, 68NISS, and 50NGSV (NA). (4) Conclusions: This study provides our understanding about the evolution of current Influenza A viruses H5N1 HPAIV circulating globally. These findings outline the importance of surveillance updating mutational profiles and glycosylation patterns of these highly evolved viruses. Full article

Due to their catalytic performance, laccases constitute one of the most promising groups of enzymes for potential applications in modern biotechnology. In this study, we aimed to chemically modify Ensifer meliloti (Sinorhizobium meliloti) and Cerrena unicolor laccase and comparatively characterize the structures of both enzymes. The most characteristic feature was the spatial localization of lysine residues, predominantly positioned distal to the active site region for both compared enzymes. The solvent-accessible surface area (SASA) analysis showed that bacterial laccase was characterized by a larger hydrophobic SASA than the fungal enzyme. The pK_a prediction identified only one Lys in the E. meliloti laccase structure susceptible to modification. Modifications were achieved by using mono- and bifunctional crosslinking agents, and glycosylations were also performed. The degree of protein modification ranged from 0% for glucose- and galactose-modified E. meliloti laccase and citraconic anhydride-modified (CA) C. unicolor laccase to 62.94% for the palmitic acid N-hydroxysuccinimide ester-modified E. meliloti enzyme. The stability of covalently modified laccases over a wide pH and temperature ranges and in the presence of inhibitors was investigated. Protein modifications with polymeric sucrose (PS) and ethylene glycol bis-(succinimidyl succinate) (EGNHS) significantly increased the activity of the bacterial and fungal laccases by 15 and 19%, respectively. Although pH optima remained relatively unchanged by modifications, certain variants, especially CA-modified bacterial protein and EGNHS-modified C. unicolor enzyme, exhibited improved stability at near-neutral pH (6–7). Modification of the bacterial enzyme with glutaraldehyde-carbodiimide (GA-CDI-ver) and of the fungal enzyme with CA was the most effective in improving its thermal stability. Chemical modifications using GA, CDI, GA-CDI, and PS allowed E. meliloti L 3.8 laccase to retain full activity in the presence of 5 mM NaI, whereas CA-, PS-, and EGNHS-modified C. unicolor variants retained their activity even at elevated NaCl concentrations. The results clearly demonstrate that the outcome of chemical modifications is closely linked to enzyme-specific structural features and that selecting an appropriate modification strategy is critical to achieving the desired effect. Full article

Background/Objectives: Obesity has reached pandemic proportions, with predictions suggesting that, by 2030, over 1.5 billion people will be affected. Pancreatic lipase (PL), the enzyme primarily responsible for the absorption of dietary lipids, presents a potential target for obesity management. However, while porcine pancreatic lipase (PPL) is commonly used as the enzyme source for screening potential inhibitors, its effect on human pancreatic lipase (HPL) is rarely reported. This work aimed to screen the inhibitory effects of a library of flavonoids with different functional groups on the activity of PL from the human pancreas (triacylglycerol acyl hydrolase, EC 3.1.1.3) and compare it to the effects of the porcine pancreas (type II, EC 3.1.1.3), establishing, whenever possible, a structure–activity relationship. Methods: The inhibitory effects of a library of 48 flavonoids with different hydroxy, glycosyl, rutinosyl, galloyl, and extended alkyl groups were evaluated against PPL and HPL. The kinetic parameters and inhibitory mechanisms of the most active flavonoids were determined, and in silico docking studies of the more potent flavonoids were also performed, using the active site of HPL. Results/Conclusions: Variations in enzyme catalytic activity were observed depending on the source of the enzyme. The inhibitory effect was particularly influenced by the presence of extended alkyl groups at the C-3 of the C-ring and the C2=C3 double bond of the C-ring and the presence of a pyrogallol group at the C-2′, C-3′ and C-4′ of the B-ring. Docking results showed a strong correlation between docking scores and observed inhibitory activities, highlighting the critical role of specific substituents on the flavonoid backbone in enhancing detailed interaction dynamics with key amino acids. Compounds 28, 29, and 30, with alkyl groups, showed the highest docking scores, interacting with residues HIS151, PHE215, ARG256, and HIS263. Further analysis also revealed that specific substituents improved pocket occupancy and formed additional interactions with residues TYR114, PRO180, ILE209, and PHE215, which are crucial for inhibition. These binding characteristics closely mimic those observed with orlistat, reinforcing their mechanistic similarities in inhibiting HPL and validating their inhibitory activities. Full article

Despite extensive experience with influenza surveillance in humans in Senegal, there is limited knowledge about the actual situation and genetic diversity of avian influenza viruses (AIVs) circulating in the country, hindering control measures and pandemic risk assessment. Therefore, as part of the “One Health” approach to influenza surveillance, we conducted active AIV surveillance in two live bird markets (LBMs) in Dakar to better understand the dynamics and diversity of influenza viruses in Senegal, obtain genetic profiles of circulating AIVs, and assess the risk of emergence of novel strains and their transmission to humans. Cloacal swabs from poultry and environmental samples collected weekly from the two LBMs were screened by RT-qPCR for H5, H7, and H9 AIVs. Subsequently, a subset of H9-positive samples was selected for whole sequencing. From December 2023 to October 2024, 499 samples were tested, and AIV was detected in 58.3% of them. Among these, A/H9N2 was the only subtype detected in both markets, with a detection rate of 47.7% (82/172) in Thiaroye and 35.3% (42/119) in Tilene, resulting in an overall positivity rate of 42.6% (124/291). Genome sequencing of 22 A/H9N2 isolates, including 11 poultry drinking water samples, 7 carcass wash water samples, 3 fecal samples, and 1 cloacal swab, yielded 7 complete and 15 partial genomic sequences. Phylogenetic analyses of the resulting sequences showed that the A/H9N2 isolates obtained in this study formed a monophyletic cluster and were closely related to the Senegalese human strain (A/Senegal/0243/2019) identified through the national influenza sentinel surveillance program. These strains were also closely related to the A/H9N2 viruses of the G1 lineage circulating in neighboring countries, suggesting cross-border transmission. The A/H9N2 strains carried the low pathogenicity RSSR/GLF motif at the HA cleavage site and possessed several key amino acid mutations, including HA-I155T and HA-Q226L, which are associated with human host adaptation, PB2-T105V, PB2-A661T, and PB2-A588V, which are linked to the human-to-human transmission and increased polymerase activity, NS2-T14M, NS2-M100I, NS1-I106M, NS1-V222M, NS1-E223A, NS1-I226V, NS1-E227G, and NS1-P228S, which are known to alter virulence (increased or reduced) in humans or mice, and M2-S31N, which promotes drug resistance. Seven potential N-glycosylation sites were predicted in the HA protein and six in the NA protein. The selection pressure analysis revealed that the A/H9N2 isolates were primarily under neutral evolution or purifying selection pressure. Overall, our findings highlight the potential for cross-species transmission of Senegalese A/H9N2 viruses, emphasizing the need for sustained monitoring of these viruses in both animal and human populations. Full article

Glycosylation is a critical post-translational modification that influences protein folding, stability and function. While extensively studied in extracellular and intracellular regions, glycosylation within transmembrane (TM) regions and at membrane interfaces remains poorly understood. This study aimed to map O- and N-glycosylation sites in these regions using a comprehensive database search and structural validation where possible. Extensive database searches revealed glycosylation sites in a range of membrane proteins. Only the sites falling in the TM regions and at the membrane interface (according to Uniprot annotations) were retained. The location of these sites was confirmed based on available 3D structures. We identified 32 O-glycosylation sites and 7 N-glycosylation sites in the TM domains of 29 proteins. O-GlcNAc sites validated as located within TM regions presented side chains either oriented toward the lipid bilayer or buried within the protein. N-glycosylation sites predicted in protein TM regions were largely confined to interface or extracellular domains. The results obtained here highlight the occurrence of glycosylation in TM regions of proteins and at membrane interfaces. This dataset provides a valuable foundation for the further exploration of structural and functional roles of glycosylation in membrane-associated regions. Full article

Background/Objectives: Indolo[2,3-a]pyrrolo[3,4-c]carbazole scaffold is successfully used as an efficient structural motif for the design and development of different antitumor agents. In this study, we investigated the anti-glioblastoma therapeutic potential of glycosylated indolocarbazole analog LCS1269 utilizing in vitro, in vivo, and in silico approaches. Methods: Cell viability was estimated by an MTT assay. The distribution of cell cycle phases was monitored using flow cytometry. Mitotic figures were visualized by fluorescence microscopy. Quantitative RT-PCR was used to evaluate the gene expression. The protein expression was assessed by Western blotting. Molecular docking and computational ADMET were approved for the probable protein target simulations and predicted pharmacological assessments, respectively. Results: Our findings clearly suggest that LCS1269 displayed a significant cytotoxic effect against diverse glioblastoma cell lines and patient-derived glioblastoma cultures as well as strongly suppressed xenograft growth in nude mice. LCS1269 exhibited more potent anti-proliferative activity toward glioblastoma cell lines and patient-derived glioblastoma cultures compared to conventional drug temozolomide. We further demonstrated that LCS1269 treatment caused the severe G2 phase arrest of cell cycle in a dose-dependent manner. Mechanistically, we proposed that LCS1269 could affect the CDK1 activity both by targeting active site of this enzyme and indirectly, in particular through the modulation of the Wee1/Myt1 and FOXM1/Plk1 signaling pathways, and via p21 up-regulation. LCS1269 also showed favorable pharmacological characteristics in in silico ADME prediction in comparison with staurosporine, rebeccamycin, and becatecarin as reference drugs. Conclusions: Further investigations of LCS1269 as an anti-glioblastoma medicinal agent could be very promising. Full article

Lactopontin (LPN) is a highly phosphorylated O-glycosylated acidic protein closely associated with infant gut, brain, and immune development, and its recognition is urgent due to its rising application in fortified dairy products and infant formula. In this study, an ssDNA aptamer against LPN was obtained, among which two kinds of matrix-background-assisted systematic evolution of ligands via exponential enrichment (SELEX) approaches were performed and compared. The direct approach was to utilize the sample matrix as the mixing-incubation background between the ssDNA library and LPN that can theoretically increase screening pressure and simulate practical application scenarios. The indirect approach was to utilize a PBS buffer as a screening background and to include counter-screening steps that adopt the “sample matrix” as a whole as the counter-screening target. Their screening evolutions were monitored through qPCR assays from sequence diversity convergences of each sub-library based on the change in the proportion of hetero- and homo-duplexes from the dissociation curve and melting temperature, which were also verified from the sequence statistics of high-throughput sequencing. The common sequence of Seq.I1II3 from the two approaches was finally fished out as the aptamer through multiple analyses of combining the sequence frequency, secondary structures, homology, and binding assessments, which was demonstrated good specificity and low-nanomolar affinity by qPCR assay (K_D, 5.9 nM). In addition, molecular docking and a dynamics simulation were performed for their binding site prediction and affinity confirmation. This study provides a potential identifying element and a basis for accelerating the development of methods for LPN detection in dairy products. Full article

Laccase is an enzyme that plays an important role in fungi, including lignin degradation, stress defense, and formation of fruiting bodies. Auricularia auricula-judae is a white-rot fungus in the Basidiomycota phylum, capable of delignifying wood. In this study, seven genes belonging to the laccase family were identified through de novo sequencing, containing Cu-Oxidase, Cu-Oxidase_2, and Cu-Oxidase_3 domains. Subsequently, the physical characteristics, phylogenetic relationships, protein secondary structure, and tertiary structure of the laccase family (AaLac1–AaLac7) were analyzed. Prediction of N-glycosylation sites identified 2 to 10 sites in the laccase family, with AaLac7 having the highest number of sites at 10. Sequence alignment and analysis of the laccase family showed high consistency in signature sequences. Phylogenetic analysis confirmed the relationship among laccases within the family, with AaLac3–AaLac4 and AaLac5–AaLac6 being closely positioned on the tree, exhibiting high similarity in tertiary structure predictions. This study identified and analyzed laccase family genes in Auricularia auricula-judae using de novo sequencing, offering a simple method for identifying and analyzing the laccase family in organisms with unknown genetic information. Full article

Seasonal influenza viruses continuously evolve via antigenic drift. This leads to recurring epidemics, globally significant mortality rates, and the need for annually updated vaccines. Co-occurring mutations in hemagglutinin (HA) and neuraminidase (NA) are suggested to have synergistic interactions where mutations can increase the chances of immune escape and viral fitness. Association rule mining was used to identify temporal relationships of co-occurring HA–NA mutations of influenza virus A/H3N2 and its role in antigenic evolution. A total of 64 clusters were found. These included well-known mutations responsible for antigenic drift, as well as previously undiscovered groups. A majority (41/64) were associated with known antigenic sites, and 38/64 involved mutations across both HA and NA. The emergence and disappearance of N-glycosylation sites in the pattern of N-X-[S/T] were also identified, which are crucial post-translational processes to maintain protein stability and functional balance (e.g., emergence of NA:339ASP and disappearance of HA:187ASP). Our study offers an alternative approach to the existing mutual-information and phylogenetic methods used to identify co-occurring mutations, enabling faster processing of large amounts of data. Our approach can facilitate the prediction of critical mutations given their occurrence in a previous season, facilitating vaccine development for the next flu season and leading to better preparation for future pandemics. Full article

Chalcones, secondary plant metabolites, exhibit various biological properties. The introduction of a chlorine and a glucosyl substituent to the chalcone could enhance its bioactivity and bioavailability. Such compounds can be obtained through a combination of chemical and biotechnological methods. Therefore, 4-chloro-2′-hydroxychalcone and 5′-chloro-2′-hydroxychalcone were obtained by synthesis and then glycosylated in two filamentous fungi strains cultures, i.e., Isaria fumosorosea KCH J2 and Beauveria bassiana KCH J1.5. The main site of the glycosylation of both compounds by I. fumosorosea KCH J2 was C-2′ and C-3 when the second strain was utilized. The pharmacokinetics of these compounds were predicted using chemoinformatics tools. Furthermore, antimicrobial activity tests were performed. Compounds significantly inhibited the growth of the bacteria strains Escherichia coli 10536, Staphylococcus aureus DSM 799, and yeast Candida albicans DSM 1386. Nevertheless, the bacterial strain Pseudomonas aeruginosa DSM 939 exhibited significant resistance to their effects. The growth of lactic acid bacteria strain Lactococcus acidophilus KBiMZ 01 bacteria was moderately inhibited, but strains Lactococcus rhamnosus GG and Streptococcus thermophilus KBM-1 were completely inhibited. In summary, chalcones substituted with a chlorine demonstrated greater efficacy in inhibiting the microbial strains under examination compared to 2′-hydroxychalcone, while aglycones and their glycosides exhibited similar effectiveness. Full article

Vascular Ehlers–Danlos syndrome or Ehlers–Danlos syndrome type IV (vEDS) is a connective tissue disorder characterised by skin hyperextensibility, joint hypermobility and fatal vascular rupture caused by COL3A1 mutations that affect collagen III expression, homo-trimer assembly and secretion. Along with collagens I, II, V and XI, collagen III plays an important role in the extracellular matrix, particularly in the inner organs. To date, only symptomatic treatment for vEDS patients is available. Fibroblasts derived from vEDS patients carrying dominant negative and/or haploinsufficiency mutations in COL3A1 deposit reduced collagen III in the extracellular matrix. This study explored the potential of an antisense oligonucleotide (ASO)-mediated splice modulating strategy to bypass disease-causing COL3A1 mutations reported in the in-frame exons 10 and 15. Antisense oligonucleotides designed to redirect COL3A1 pre-mRNA processing and excise exons 10 or 15 were transfected into dermal fibroblasts derived from vEDS patients and a healthy control subject. Efficient exon 10 or 15 excision from the mature COL3A1 mRNA was achieved and intracellular collagen III expression was increased after treatment with ASOs; however, collagen III deposition into the extracellular matrix was reduced in patient cells. The region encoded by exon 10 includes a glycosylation site, and exon 15 encodes hydroxyproline and hydroxylysine-containing triplet repeats, predicted to be crucial for collagen III assembly. These results emphasize the importance of post-translational modification for collagen III homo-trimer assembly. In conclusion, while efficient skipping of target COL3A1 exons was achieved, the induced collagen III isoforms generated showed defects in extracellular matrix formation. While therapeutic ASO-mediated exon skipping is not indicated for the patients in this study, the observations are restricted to exons 10 and 15 and may not be applicable to other collagen III in-frame exons. Full article

Lectins are proteins of a non-immune nature with activity against microorganisms, insects, and tumor cells. The aim of this work was to predict the physicochemical characteristics, structure, and functional properties of a Bauhinia holophylla lectin (BhL), sequenced from genomic material obtained from calli cultures, through bioinformatics tools. The results showed a high similarity between the Bhl gene and nucleotide sequences that encode lectins expressed by Bauhinia species and a high identity between the protein sequence of BhL and lectins from B. forficata (90%), B. variegata (79.04%), B. purpurea (78.01%), and B. ungulata (85.27%). BhL has 289 amino acids, of which 30, 85, and 174 residues are related to α-helix, β-sheet, and disordered regions, respectively. Their estimated molecular weight is 31.9 kDa and the theoretical isoelectric point is 5.79. Bauhinia holophylla lectin possibly undergoes phosphorylation and glycosylation at specific sites. Conserved protein domains, catalytic sites, and conserved amino acids were observed in BhL, bringing it closer to lectin families from other legume species. The prediction signaled the presence of a sequence of 28 amino acids at the N-terminal end of BhL, with a high hydropathicity index and conceptualized as a signal peptide. The molecular function predicted for BhL was associated with carbohydrate recognition activity. BhL could be an extracellular protein, and its three-dimensional structure showed 78.82% identity with the B. purpurea lectin. Full article

Worldwide, human respiratory syncytial virus (HRSV) is a major cause of severe infections of the lower respiratory system, affecting individuals of all ages. This study investigated the genetic variability of HRSV during the COVID-19 outbreak in Yaoundé; nasopharyngeal samples positive for HRSV were collected from different age groups between July 2020 and October 2021. A semi-nested RT-PCR was performed on the second hypervariable region of the G gene of detected HRSV, followed by sequencing and phylogenetic assessment. Throughout the study, 40 (37.7%) of the 106 HRSV-positive samples successfully underwent G-gene amplification. HRSV A and HRSV B co-circulated at rates of 47.5% and 52.5%, respectively. HRSV A clustered in the GA2.3.5 genetic lineage (ON1) and HRSV B clustered in the GB5.0.5a genetic lineage (BA9). Differences in circulating genotypes were observed between pre- and post-pandemic years for HRSV A. Predictions revealed potential N-glycosylation sites at positions 237-318 of HRSV A and positions 228-232-294 of HRSV B. This study reports the molecular epidemiology of HRSV in Cameroon during the COVID-19 pandemic. It describes the exclusive co-circulation of two genetic lineages. These findings highlight the importance of implementing comprehensive molecular surveillance to prevent the unexpected emergence of other diseases. Full article

Glycosylation, a crucial and the most common post-translational modification, coordinates a multitude of biological functions through the attachment of glycans to proteins and lipids. This process, predominantly governed by glycosyltransferases (GTs) and glycoside hydrolases (GHs), decides not only biomolecular functionality but also protein stability and solubility. Mutations in these enzymes have been implicated in a spectrum of diseases, prompting critical research into the structural and functional consequences of such genetic variations. This study compiles an extensive dataset from ClinVar and UniProt, providing a nuanced analysis of 2603 variants within 343 GT and GH genes. We conduct thorough MTR score analyses for the proteins with the most documented variants using MTR3D-AF2 via AlphaFold2 (AlphaFold v2.2.4) predicted protein structure, with the analyses indicating that pathogenic mutations frequently correlate with Beta Bridge secondary structures. Further, the calculation of the solvent accessibility score and variant visualisation show that pathogenic mutations exhibit reduced solvent accessibility, suggesting the mutated residues are likely buried and their localisation is within protein cores. We also find that pathogenic variants are often found proximal to active and binding sites, which may interfere with substrate interactions. We also incorporate computational predictions to assess the impact of these mutations on protein function, utilising tools such as mCSM to predict the destabilisation effect of variants. By identifying these critical regions that are prone to disease-associated mutations, our study opens avenues for designing small molecules or biologics that can modulate enzyme function or compensate for the loss of stability due to these mutations. Full article

The influenza A(H1N1) pdm09 virus, which emerged in 2009, has been circulating seasonally since then. In this study, we conducted a comprehensive genome-based investigation to gain a detailed understanding of the genetic and evolutionary characteristics of the hemagglutinin (HA) and neuraminidase (NA) surface proteins of A/H1N1pdm09 strains circulating in Italy over a fourteen-year period from 2009 to 2023 in relation to global strains. Phylogenetic analysis revealed rapid transmission and diversification of viral variants during the early pandemic that clustered in clade 6B.1. In contrast, limited genetic diversity was observed during the 2023 season, probably due to the genetic drift, which provides the virus with a constant adaptability to the host; furthermore, all isolates were split into two main groups representing two clades, i.e., 6B.1A.5a.2a and its descendant 6B.1A.5a.2a.1. The HA gene showed a faster rate of evolution compared to the NA gene. Using FUBAR, we identified positively selected sites 41 and 177 for HA and 248, 286, and 455 for NA in 2009, as well as sites 22, 123, and 513 for HA and 339 for NA in 2023, all of which may be important sites related to the host immune response. Changes in glycosylation acquisition/loss at prominent sites, i.e., 177 in HA and 248 in NA, should be considered as a predictive tool for early warning signs of emerging pandemics, and for vaccine and drug development. Full article