Search Results (61)

Celiac disease (CD) is an autoimmune disorder triggered by pepsin-resistant, gluten-derived immunogenic peptides (GIPs) in genetically predisposed individuals. Enzyme therapy targeting GIPs has been suggested as a complementary practice to a gluten-free diet to help reduce the symptoms of CD. Here, we present the crystal structure of RmuAP1, a pepsin-like aspartic protease from Rhodotorula mucilaginosa, which effectively degrades the toxic 33-mer and 26-mer GIPs under postprandial gastric conditions (pH 3.0–6.0). RmuAP1 has a canonical fold characteristic of the aspartic protease subfamily A1; however, it features a distinct flap and a flexible loop structure. Compared to pepsin, RmuAP1 accommodates the tetrapeptides PQQP and PQPQ, motifs frequently repeated on GIPs, via an adaptable binding cleft. Molecular dynamics (MD) simulations have shown that RmuAP1 stably engages these ligands, maintaining both the catalytic water in position and a closed flap conformation, primarily through ligand-induced remodeling of the S1′ pocket. In contrast, pepsin neither binds these ligands effectively nor achieves a catalytically competent conformation. Structural comparisons and dihedral analysis further support an induced-fit mechanism underlying RmuAP1’s pocket remodeling. Together, this study clarifies the structural basis for RmuAP1 to hydrolyze GIPs, emphasizing the potential of RmuAP1 as a platform for developing enhanced oral peptidase for CD patients through protein engineering approaches. Full article

Background and Objectives: Human Papillomavirus (HPV) is a leading cause of cervical and other anogenital cancers, with over 200 known genotypes classified into high-risk, probable high-risk, and low-risk groups. While conventional diagnostic and classification approaches often rely on sequence alignment, phylogenetic relationships, or protein structure analyses, these methods are limited in scalability, cost efficiency, and generalizability to emerging HPV types. This study aims to develop a novel, machine learning-based framework for classifying HPV genotypes by oncogenic risk using genome-derived numerical features. A key objective is to introduce TATA-box, CAAT-box, and CpG-island-based features to HPV risk prediction for the first time. Methods: We constructed a comprehensive feature set that integrates regulatory sequence motifs (TATA-box, CAAT-box, CpG islands) with dinucleotide and trinucleotide (k-mer) composition derived from full HPV genomes. Multiple machine learning algorithms were implemented to evaluate classification performance across all risk categories. Model accuracy, precision, recall, and F1-score were calculated to assess the effectiveness and robustness of the proposed feature set. Results: The proposed method achieves an average precision of 0.95, a recall of 0.95, an F1-score of 0.95, and an accuracy of 97.47%. The experimental findings indicate that the proposed method not only attains high classification accuracy across all HPV risk groups but also surpasses existing models in generalizability by utilizing genomic data and novel biologically informed features. Conclusions: This study introduces regulatory motif-based numerical features to HPV classification for the first time and demonstrates that integrating these with k-mer descriptors yields a highly accurate and scalable machine learning model. Unlike previous studies, which often focus on specific HPV genes or a limited subset of types, our method is scalable, robust, and capable of classifying known and emerging HPV types with high reliability. This highlights its potential for real-world deployment in large-scale epidemiological screening and vaccine development programs. Full article

The common carp Cyprinus carpio L. is a cornerstone aquaculture species, yet transcriptome interpretation is complicated by its paleotetraploid genome and extensive alternative splicing. A species-tailored oligonucleotide microarray was developed to deliver reproducible, gene-level expression profiling. Probe design was anchored to the SPL01 reference and implemented on an Agilent platform using a gene-level strategy that collapsed transcripts to genes, selected the longest isoform, and placed 3′-anchored 60-mer probes. The workflow incorporated embedded technical controls and a standardized two-color pipeline to ensure stable measurements across arrays. Baseline functional organization of the head kidney was defined using 614 C. carpio L. -Danio rerio orthologs and complementary enrichment tools. Coherent signatures emerged for hemoglobin-mediated oxygen transport, heme and porphyrin metabolism, antioxidant defense driven by peroxiredoxin and thioredoxin systems, including hydrogen peroxide detoxification, protease regulation through SERPIN, SPINK, and WFDC families, and elements of innate and humoral immunity. Targets bearing c-Myc motifs showed a modest positive bias consistent with ongoing hematopoiesis. These resolved baseline modules provide a reference against which infection- or exposure-induced programs such as interferon-stimulated genes, chemokines and chemotaxis, complement activation, and degranulation can be detected and quantified. The platform complements RNA-seq by offering cost-efficient, rapid, and comparable measurements suited to large cohorts and longitudinal designs. Anticipated applications include host–pathogen studies for viral and bacterial agents and the assessment of chemical contaminants in aquaculture surveillance, supporting standardized, cross-study decision-making in research and health monitoring. Full article

Molecular simulation is central to modern drug discovery but is often limited by high computational cost and the complexity of molecular interactions. Deep-learning drug–target interaction (DTI) prediction can accelerate screening; however, many models underuse the local functional structure features—binding motifs, reactive groups, and residue-level fragments—that drive recognition. We present LoF-DTI, a framework that explicitly represents and couples such local features. Drugs are converted from SMILES into molecular graphs and targets from sequences into feature representations. On the drug side, a Jumping Knowledge (JK) enhanced Graph Isomorphism Network (GIN) extracts atom- and neighborhood-level patterns; on the target side, residual CNN blocks with progressively enlarged receptive fields, augmented by N-mer substructural statistics, capture multi-scale local motifs. A Gated Cross-Attention (GCA) module then performs atom-to-residue interaction learning, highlighting decisive local pairs and providing token-level interpretability through attention scores. By prioritizing locality during both encoding and interaction, LoF-DTI delivers competitive results across multiple benchmarks and improves early retrieval relevant to virtual screening. Case analyses show that the model recovers known functional binding sites, suggesting strong potential to provide mechanism-aware guidance for molecular simulation and to streamline the drug design pipeline. Full article

Background/Objectives: Sepiella japonica is a highly adaptable cephalopod with an advanced nervous system and complex reproductive behavior, capable of reproducing two to three generations annually depending on water temperature. However, the absence of a complete genome assembly has limited molecular investigations of its unique biological characteristics. This study aimed to perform a genome survey of female and male S. japonica, systematically characterize and compare key genomic characteristics. Methods: Quality-filtered short reads enabled K-mer-based estimation of genome size, heterozygosity, repeat content, and GC content; generation of draft genome assemblies, SSR identification from the draft assemblies, complete mitogenome assemblies and annotations with ML phylogeny based on 13 concatenated PCGs, and PSMC-based demographic inference. Results: The estimated genome sizes were 4317 Mb (female) and 4222 Mb (male), with revised estimates of 4310 Mb and 4215 Mb, respectively. K-mer analysis revealed heterozygosity rates of 0.85% (female) and 0.77% (male) and repeat content of 76.05% (female) and 75.91% (male). The assembled genome sizes were 4197 Mb for females (N50: 508 bp) and 4206 Mb for males (N50: 511 bp); the GC content was 34.15% for both genomes. Deduplicated data showed GC content of 35.16% (female) and 35.27% (male). Microsatellite analysis revealed that mononucleotide repeats were the most abundant simple sequence repeat motif. The mitochondrial genome sequences measured 16,729 bp for the female genome and 16,725 bp for the male genome. Conclusions: This study provides fundamental data for subsequent high-quality whole-genome assembly and comparative analysis of female and male genomes. Full article

K-mer analysis is a powerful tool for understanding genome structure and evolution. A “k-mer” refers to a short DNA sequence made up of k nucleotides (where k is a specific integer), while an “m-mer” is a similar concept but with a shorter sequence length. The functional mechanisms of CG-containing k-mers, as well as their potential role in evolutionary processes, remain unclear. To explore this issue, we analyzed 8-mers in several species with varying genomic complexities and evolutionary divergences: Homo sapiens, Saccharomyces cerevisiae, Bombyx mori, Ciona intestinalis, Danio rerio, and Caenorhabditis elegans, which were grouped by CG dinucleotide content (0CG, 1CG, and 2CG). We examined the relative frequencies of shorter m-mers (with m = 3 and 4) within each CG-defined group, using information-theoretic, distance-based, and angular metrics. Our results show that 0CG motifs follow random patterns, while 1CG and 2CG motifs display significant deviations, likely due to functional constraints such as nucleosome-binding and CpG island association. The observed unimodal distribution of 8-mers arises from the convergence of the three CG-defined groups. Among them, the 2CG group shows the highest divergence in m-mer composition, followed by 1CG, reflecting varying degrees of selective pressure. Furthermore, species-specific differences in CG-classified 8-mer patterns could provide valuable insights into phylogenetic relationships. Through extensive comparison, we explore how CG content and sequence composition influence genomic organization and contribute to evolutionary divergence across different taxa. These findings deepen our understanding of short motif functions, genome organization, and sequence evolution. Full article

Biological-sequence representation methods are pivotal for advancing machine learning in computational biology, transforming nucleotide and protein sequences into formats that enhance predictive modeling and downstream task performance. This review categorizes these methods into three developmental stages: computational-based, word embedding-based, and large language model (LLM)-based, detailing their principles, applications, and limitations. Computational-based methods, such as k-mer counting and position-specific scoring matrices (PSSM), extract statistical and evolutionary patterns to support tasks like motif discovery and protein–protein interaction prediction. Word embedding-based approaches, including Word2Vec and GloVe, capture contextual relationships, enabling robust sequence classification and regulatory element identification. Advanced LLM-based methods, leveraging Transformer architectures like ESM3 and RNAErnie, model long-range dependencies for RNA structure prediction and cross-modal analysis, achieving superior accuracy. However, challenges persist, including computational complexity, sensitivity to data quality, and limited interpretability of high-dimensional embeddings. Future directions prioritize integrating multimodal data (e.g., sequences, structures, and functional annotations), employing sparse attention mechanisms to enhance efficiency, and leveraging explainable AI to bridge embeddings with biological insights. These advancements promise transformative applications in drug discovery, disease prediction, and genomics, empowering computational biology with robust, interpretable tools. Full article

A novel and general approach to the practical ROMP polymerization of cinchona alkaloid derivatives providing novel hybrid materials having quinine attached on a poly(norbornene-5,6-dicarboxyimide) matrix is presented. The concept involves an easy modification of quinine (in general, any cinchona alkaloid) toward clickable 9-azide that reacts with N-propargyl-cis-5-norbornene-exo-2,3-dicarboxylic imide in Cu(I)-catalyzed Huisgen cycloaddition (click chemistry). The resulting monomers undergo a controllable ROMP reaction that leads to novel polymers of a desired length and solubility. This sequence allows for the facile preparation of a regularly decorated polymeric material having one quinine moiety per single mer of the polymer chain inaccessible using typical immobilization methods. A poly(norbornene-5,6-dicarboxyimide) type of polymeric matrix was selected due to the high reactivity of the exo-norbornene motif in Ru(II)-catalyzed ROMP and its chemical and thermal stability as well as convenient, scalable access from inexpensive cis-5-norbornene-exo-2,3-dicarboxylic anhydride (‘one-pot’ Diels–Alder reaction of dicyclopentadiene and maleic anhydride). An appropriate combination of a Grubbs catalyst, Ru(II) (G1, G2), and ROMP conditions allowed for the efficient synthesis of well-defined soluble polymers with mass parameters in the range Mn = 2.24 × 10⁴ – 2.26 × 10⁴ g/mol and Mw = 2.90 × 10⁴–3.05 × 10⁴ g/mol with good polydispersity, Đ_M = 1.32–1.35, and excellent thermal stability (up to 309°C T_d10). Spectroscopic studies (NMR and electronic circular dichroism (ECD)) of these products revealed a linear structure with the slight advantage of a trans-configuration of an olefinic double bond. The resulting short-chain polymer discriminates mandelic acid enantiomers with a preference for the (R)-stereoisomer in spectrofluorimetric assays. This concept seems to be rather general with respect to other molecules dedicated to incorporation into the poly(norbornene-5,6-dicarboxyimide) chain. Full article

Background/Objectives: The use of virus-like particles (VLPs) in vaccinology has expanded significantly in recent years. VLPs have the advantage of being non-infectious while effectively stimulating B cell responses through the repetitive presentation of epitope motifs on their surface. Since VLPs are often derived from human-infecting viruses, preexisting immunity may influence the immune response they elicit, warranting further investigation. Methods: We have developed a 60-mer VLP derived from human adenovirus type 3, a common pathogen. We investigated the impact of pre-existing adenovirus immunity on the immunization outcome against the linear S14P5 epitope of SARS-CoV-2, which was engineered into the particle (Ad-VLP-S14P5). To this end, antibody responses to S14P5 were evaluated following immunization with Ad-VLP-S14P5 in either naive or vector-primed mice. Results: Mice with pre-existing anti-vector immunity exhibited significantly greater anti-S14P5 antibody responses compared to vector-naive animals, demonstrating a beneficial impact of prior anti-adenovirus responses. However, the addition of an oil-in-water adjuvant for the immunizations abolished this positive impact, even leading to a deleterious effect of the pre-existing anti-vector immunity. Conclusions: The data suggest that the immune status against immunizing VLPs must be taken into consideration when designing immunization protocols. Importantly, the effects of prior immunity may vary depending on the nature of the protocol, including factors such as adjuvant use. Full article

Mycobacteriophages are viruses that specifically infect bacterial species within the genera Mycobacterium and Mycolicibacterium. Over 2400 mycobacteriophages have been isolated on the host Mycolicibacterium smegmatis and sequenced. This wealth of genomic data indicates that mycobacteriophage genomes are diverse, mosaic, and contain numerous (35–60%) genes for which there is no predicted function based on sequence similarity to characterized orthologs, many of which are essential to lytic growth. To fully understand the molecular aspects of mycobacteriophage–host interactions, it is paramount to investigate the function of these genes and gene products. Here we show that the temperate mycobacteriophage, Alexphander, makes stable lysogens with a frequency of 2.8%. Alexphander gene 94 is essential for lytic infection and encodes a protein predicted to contain a C-terminal MerR family helix–turn–helix DNA-binding motif (HTH) and an N-terminal DinB/YfiT motif, a putative metal-binding motif found in stress-inducible gene products. Full-length and C-terminal gp94 constructs form high-order nucleoprotein complexes on 100–500 base pair double-stranded DNA fragments and full-length phage genomic DNA with little sequence discrimination for the DNA fragments tested. Maximum gene 94 mRNA levels are observed late in the lytic growth cycle, and gene 94 is transcribed in a message with neighboring genes 92 through 96. We hypothesize that gp94 is an essential DNA-binding protein for Alexphander during lytic growth. We proposed that gp94 forms multiprotein complexes on DNA through cooperative interactions involving its HTH DNA-binding motif at sites throughout the phage chromosome, facilitating essential DNA transactions required for lytic propagation. Full article

We report on a highly significant, positive association between anthrax vaccination and occurrence of Gulf War Illness (GWI) in 111 Gulf War veterans (42 with GWI and 69 controls). GWI was diagnosed in 47.1% of vaccinated veterans but only in 17.2% of non-vaccinated veterans (Pearson χ² = 7.08, p = 0.008; odds ratio = 3.947; relative risk = 2.617), with 1.6x higher GWI symptom severity in vaccinated veterans (p = 0.007, F-test in analysis of covariance). Next, we tested the hypothesis that the susceptibility to GWI following anthrax vaccination could be due to inability to make antibodies against the anthrax protective antigen (PA), the key protein contained in the vaccine. Since the first step in initiating antibody production would be the binding of PA peptide fragments (typically 15-amino acid long [15-mer]) to peptide-binding motifs of human leukocyte antigen (HLA) Class II molecules, we assessed the binding-motif affinities of such HLA specific molecules to all linear 15-mer peptide fragments of the anthrax PA. We identified a total of 58 HLA Class II alleles carried by the veterans in our sample and found that, of those, 18 (31%) were present in the vaccinated group that did not develop GWI but were absent from the vaccinated group who developed GWI. Remarkably, in silico analyses revealed very high binding affinities of peptide-binding motifs of those 18 HLA alleles with fragments of anthrax vaccine PA, leading to the successful production of anti-PA antibodies. Conversely, the absence of these protective HLA alleles points to a reduced ability to develop antibodies against PA, thus resulting in harmful PA persistence and development of GWI. Full article

Nucleotide substitutions have played an important role in molecular evolution, and understanding their dynamics would contribute to genetic studies. Related research with defined DNA sequences lasted for decades until whole-genome sequencing arose. UV radiation (UVR) can generate base changes and other genetic variations in a short period of time, so it would be more meaningful to explore mutations caused by UVR from a genomic perspective. The monokaryon enoki strain WT583 was selected as the experimental material in this study because it can spontaneously produce large amounts of oidia on PDA plates, and the monokaryons originating from oidia have the same genotype as their mother monokaryon. After exposure to UV radiation, 100 randomly selected mutants, with WT583 as the reference genome, were sent for genome sequencing. BWA, samtools, and GATK software were employed for SNP calling, and the R package CMplot was used to visualize the distribution of the SNPs on the contigs of the reference genome. Furthermore, a k-mer-based method was used to detect DNA fragment deletion. Moreover, the non-synonymous genes were functionally annotated. A total of 3707 single-base substitutions and 228 tandem mutations were analyzed. The immediate adjacent bases showed different effects on the mutation frequencies of adenine and cytosine. For adenine, the overall effects of the immediate 5′-side and 3′-side bases were T > A > C > G and A > T > G > C, respectively; for cytosine, the overall effects of the immediate 5′-side and 3′-side bases were T > C > A > G and C > T > A > G, respectively. Regarding tandem mutations, the mutation frequencies of double-transition, double-transversion, 3′-side transition, and 5′-side transition were 131, 8, 72, and 17, respectively. Transitions at the 3′-side with a high mutation frequency shared a common feature, where they held transversions at the 5′-side of A→T or T→A without covalent bond changes, suggesting that the sequence context of tandem motifs might be related to their mutation frequency. In total, 3707 mutation sites were non-randomly distributed on the contigs of the reference genome. In addition, pyrimidines at the 3′-side of adenine promoted its transversion frequency, and UVR generated DNA fragment deletions over 200 bp with a low frequency in the enoki genome. The functional annotation of the genes with non-synonymous mutation indicated that UVR could produce abundant mutations in a short period of time. Full article

The overexpression of one or more somatostatin receptors (SST_1–5R) in human tumors has provided an opportunity for diagnosis and therapy with somatostatin-like radionuclide carriers. The application of “pansomatostatin” analogs is expected to broaden the clinical indications and upgrade the diagnostic/therapeutic efficacy of currently applied SST₂R-prefering radioligands. In pursuit of this goal, we now introduce two bicyclic somatostatin-14 (SS14) analogs, AT5S (DOTA-Ala¹-Gly²-c[Cys³-Lys⁴-Asn⁵-c[Cys⁶-Phe⁷-DTrp⁸-Lys⁹-Thr¹⁰-Cys¹¹]-Thr¹²-Ser¹³-Cys¹⁴]) and AT6S (DOTA-Ala¹-Gly²-c[Cys³-Lys⁴-c[Cys⁵-Phe⁶-Phe⁷-DTrp⁸-Lys⁹-Thr¹⁰-Phe¹¹-Cys¹²]-Ser¹³-Cys¹⁴]), suitable for labeling with trivalent radiometals and designed to sustain in vivo degradation. Both AT5S and AT6S and the respective [¹¹¹In]In-AT5S and [¹¹¹In]In-AT6S were evaluated in a series of in vitro assays, while radioligand stability and biodistribution were studied in mice. The 8/12-mer bicyclic AT6S showed expanded affinity for all SST_1–5R and agonistic properties at the SST₂R, whereas AT5S lost all affinity to SST_1–5R. Both [¹¹¹In]In-AT5S and [¹¹¹In]In-AT6S remained stable in the peripheral blood of mice, while [¹¹¹In]In-AT6S displayed low, but specific uptake in AR4-2J tumors and higher uptake in HEK293-SST₃R tumors in mice. In summary, high radioligand stability was acquired by the two disulfide bridges introduced into the SS14 motif, but only the 8/12-mer ring AT6S retained a pansomatostatin profile. In consequence, [¹¹¹In]In-AT6S targeted SST₂R-/SST₃R-positive xenografts in mice. These results call for further research on pansomatostatin-like radioligands for cancer theranostics. Full article

The specific characteristics of k-mer words (2 ≤ k ≤ 11) regarding genomic distribution and evolutionary conservation were recently found. Among them are, in high abundance, words with a tandem repeat structure (repeat unit length of 1 bp to 3 bp). Furthermore, there seems to be a class of extremely short tandem repeats (≤12 bp), so far overlooked, that are non-random-distributed and, therefore, may play a crucial role in the functioning of the genome. In the following article, the positional distributions of these motifs we call super-short tandem repeats (SSTRs) were compared to other functional elements, like genes and retrotransposons. We found length- and sequence-dependent correlations between the local SSTR density and G+C content, and also between the density of SSTRs and genes, as well as correlations with retrotransposon density. In addition to many general interesting relations, we found that SINE Alu has a strong influence on the local SSTR density. Moreover, the observed connection of SSTR patterns to pseudogenes and -exons might imply a special role of SSTRs in gene expression. In summary, our findings support the idea of a special role and the functional relevance of SSTRs in the genome. Full article

Medicinal chemistry is constantly searching for new approaches to develop more effective and targeted therapeutic molecules. The design of peptidomimetics is a promising emerging strategy that is aimed at developing peptides that mimic or modulate the biological activity of proteins. Among these, stapled peptides stand out for their unique ability to stabilize highly frequent helical motifs, but they have failed to be systematically reported. Here, we exploit chemically diverse helix-inducing i, i + 4 constraints—lactam, hydrocarbon, triazole, double triazole and thioether—on two distinct short sequences derived from the N-terminal peptidase domain of hACE2 upon structural characterization and in silico alanine scan. Our overall objective was to provide a sequence-independent comparison of α-helix-inducing staples using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. We identified a 9-mer lactam stapled peptide derived from the hACE2 sequence (His34-Gln42) capable of reaching its maximal helicity of 55% with antiviral activity in bioreporter- and pseudovirus-based inhibition assays. To the best of our knowledge, this study is the first comprehensive investigation comparing several cyclization methods with the goal of generating stapled peptides and correlating their secondary structures with PPI inhibitions using a highly topical model system (i.e., the interaction of SARS-CoV-2 Spike RBD with hACE2). Full article