Codon Usage Research Articles

A statistical-physics approach for codon usage optimisation

The concept of “codon optimisation” involves adjusting the coding sequence of a target protein to account for the inherent codon preferences of a host species and maximise protein expression in that species. However, there is still a lack of consensus on the most effective approach to achieve optimal results. Existing methods typically depend on heuristic combinations of different variables, leaving the user with the final choice of the sequence hit. In this study, we propose a new statistical-physics model for codon optimisation. This model, called the Nearest-Neighbour interaction (NN) model, links the probability of any given codon sequence to the “interactions” between neighbouring codons. We used the model to design codon sequences for different proteins of interest, and we compared our sequences with the predictions of some commercial tools. In order to assess the importance of the pair interactions, we additionally compared the NN model with a simpler method (Ind) that disregards interactions. It was observed that the NN method yielded similar Codon Adaptation Index (CAI) values to those obtained by other commercial algorithms, despite the fact that CAI was not explicitly considered in the algorithm. By utilising both the NN and Ind methods to optimise the reporter protein luciferase, and then analysing the translation performance in human cell lines and in a mouse model, we found that the NN approach yielded the highest protein expression in vivo. Consequently, we propose that the NN model may prove advantageous in biotechnological applications, such as heterologous protein expression or mRNA-based therapies.

Decoding the Chloroplast Genome of Tetrastigma (Vitaceae): Variations and Phylogenetic Selection Insights.

Tetrastigma (Vitaceae) is known for its ornamental, medicinal, and ecological significance. However, the structural and variational characteristics of the Tetrastigma chloroplast genome and their impact on phylogenetic relationships remain underexplored. This study utilized bioinformatics methods to assemble and annotate the chloroplast genomes of 10 Tetrastigma species and compare them with five previously sequenced species. This study analyzed gene composition, simple sequence repeats, and codon usage patterns, revealing a high A/T content, uniquely identified pentanucleotide repeats in five species and several preferred codons. In addition, comparative analyses were conducted of the chloroplast genomes of 15 Tetrastigma species, examining their structural differences and identifying polymorphic hotspots (rps16, rps16-trnQ, trnS, trnD, psbC-trnS-psbZ, accD-psaI, psbE-petL-petG, etc.) suitable for DNA marker development. Furthermore, phylogenetic and selective pressure analyses were performed based on the chloroplast genomes of these 15 Tetrastigma species, validating and elucidating intra-genus relationships within Tetrastigma. Futhermore, several genes under positive selection, such as atpF and accD, were identified, shedding light on the adaptive evolution of Tetrastigma. Utilizing 40 Vitaceae species, the divergence time of Tetrastigma was estimated, clarifying the evolutionary relationships within Tetrastigma relative to other genera. The analysis revealed diverse divergences of Tetrastigma in the Miocene and Pliocene, with possible ancient divergence events before the Eocene. Furthermore, family-level selective pressure analysis identified key features distinguishing Tetrastigma from other genera, showing a higher degree of purifying selection. This research enriches the chloroplast genome data for Tetrastigma and offers new insights into species identification, phylogenetic analysis, and adaptive evolution, enhancing our understanding of the genetic diversity and evolutionary history of these species.

Differentially used codons among essential genes in bacteria identified by machine learning-based analysis.

Codon usage bias (CUB), the uneven usage of synonymous codons encoding the same amino acid, differs among genes within and across bacteria genomes. CUB is known to be influenced by gene expression and accordingly, CUB differs between the high-expression and low-expression genes in several bacteria. In this article, we have extended codon usage study considering gene essentiality as a feature. Using machine learning (ML) based approaches, we have analysed Relative Synonymous Codon Usage (RSCU) values between essential and non-essential genes in Escherichia coli and thirty-four other bacterial genomes whose gene essentiality features were available in public databases. We observed significant differences in codon usage patterns between essential and non-essential genes for majority of the bacterial genomes and accordingly, ML based classifiers achieved high area under curve (AUC) scores, with a minimum score of 70.0 across twenty-eight organisms. Further, importance of the codons towards classifying genes found to differ among the codons in each genome. Arg codon CGT and Gly codon GGT were observed to be the most preferred codons among essential genes in Escherichia coli. Interestingly, some of the codons like CGT, ATA, GGT and GGG observed to be contributing consistently towards classifying essential genes across thirty-five bacteria genomes studied. In other hand, codons TGY and CAY encoding amino acids Cys and His respectively were among the least contributing codons towards classification among all these bacteria. This study demonstrates the gene essentiality based differences in synonymous codon usage in bacteria genomes and presents a common codon usage pattern across bacteria.

The Complete Mitogenome of Apostasia fujianica Y.Li & S.Lan and Comparative Analysis of Mitogenomes across Orchidaceae.

Apostasia fujianica belongs to the genus Apostasia and is part of the basal lineage in the phylogenetic tree of the Orchidaceae. Currently, there are only ten reported complete mitochondrial genomes in orchids, which greatly hinders the understanding of mitochondrial evolution in Orchidaceae. Therefore, we assembled and annotated the mitochondrial genome of A. fujianica, which has a length of 573,612 bp and a GC content of 44.5%. We annotated a total of 44 genes, including 30 protein-coding genes, 12 tRNA genes, and two rRNA genes. We also performed relative synonymous codon usage (RSCU) analysis, repeat sequence analysis, intergenomic transfer (IGT) analysis, and Ka/Ks analysis for A. fujianica and conducted RNA editing site analysis on the mitochondrial genomes of eight orchid species. We found that most protein-coding genes are under purifying selection, but nad6 is under positive selection, with a Ka/Ks value of 1.35. During the IGT event in A. fujianica's mitogenome, the trnN-GUU, trnD-GUC, trnW-CCA, trnP-UGG, and psaJ genes were identified as having transferred from the plastid to the mitochondrion. Compared to other monocots, the family Orchidaceae appears to have lost the rpl10, rpl14, sdh3, and sdh4 genes. Additionally, to further elucidate the evolutionary relationships among monocots, we constructed a phylogenetic tree based on the complete mitogenomes of monocots. Our study results provide valuable data on the mitogenome of A. fujianica and lay the groundwork for future research on genetic variation, evolutionary relationships, and breeding of Orchidaceae.

De novo assembly and comprehensive analysis of the mitochondrial genome of Taxus wallichiana reveals different repeats mediate recombination to generate multiple conformations

Taxus plants are the exclusive source of paclitaxel, an anticancer drug with significant medicinal and economic value. Interspecies hybridization and gene introgression during evolution have obscured distinctions among Taxus species, complicating their phylogenetic classification. While the chloroplast genome of Taxus wallichiana, a widely distributed species in China, has been sequenced, its mitochondrial genome (mitogenome) remains uncharacterized.We sequenced and assembled the T. wallichiana mitogenome using BGI short reads and Nanopore long reads, facilitating comparisons with other gymnosperm mitogenomes. The T. wallichiana mitogenome spanning 469,949 bp, predominantly forms a circular configuration with a GC content of 50.51%, supplemented by 3 minor configurations mediated by one pair of LRs and two pairs of IntRs. It includes 32 protein-coding genes, 7 tRNA genes, and 3 rRNA genes, several of which exist in multiple copies.We detailed the mitogenome's structure, codon usage, RNA editing, and sequence migration between organelles, constructing a phylogenetic tree to elucidate evolutionary relationships. Unlike typical gymnosperm mitochondria, T. wallichiana shows no evidence of mitochondrial-plastid DNA transfer (MTPT), highlighting its unique genomic architecture. Synteny analysis indicated extensive genomic rearrangements in T. wallichiana, likely driven by recombination among abundant repetitive sequences. This study offers a high-quality T. wallichiana mitogenome, enhancing our understanding of gymnosperm mitochondrial evolution and supporting further cultivation and utilization of Taxus species.

A computational approach to developing a multi-epitope vaccine for combating Pseudomonas aeruginosa-induced pneumonia and sepsis.

Pseudomonas aeruginosa is a complex nosocomial infectious agent responsible for numerous illnesses, with its growing resistance variations complicating treatment development. Studies have emphasized the importance of virulence factors OprE and OprF in pathogenesis, highlighting their potential as vaccine candidates. In this study, B-cell, MHC-I, and MHC-II epitopes were identified, and molecular linkers were active to join these epitopes with an appropriate adjuvant to construct a vaccine. Computational tools were employed to forecast the tertiary framework, characteristics, and also to confirm the vaccine's composition. The potency was weighed through population coverage analysis and immune simulation. This project aims to create a multi-epitope vaccine to reduce P. aeruginosa-related illness and mortality using immunoinformatics resources. The ultimate complex has been determined to be stable, soluble, antigenic, and non-allergenic upon inspection of its physicochemical and immunological properties. Additionally, the protein exhibited acidic and hydrophilic characteristics. The Ramachandran plot, ProSA-web, ERRAT, and Verify3D were employed to ensure the final model's authenticity once the protein's three-dimensional structure had been established and refined. The vaccine model showed a significant binding score and stability when interacting with MHC receptors. Population coverage analysis indicated a global coverage rate of 83.40%, with the USA having the highest coverage rate, exceeding 90%. Moreover, the vaccine sequence underwent codon optimization before being cloned into the Escherichia coli plasmid vector pET-28a (+) at the EcoRI and EcoRV restriction sites. Our research has developed a vaccine against P. aeruginosa that has strong binding affinity and worldwide coverage, offering an acceptable way to mitigate nosocomial infections.

Identification and Analysis of PPO Gene Family Members in Paulownia fortunei.

Polyphenol oxidase (PPO) is a common metalloproteinase in plants with important roles in plant responses to abiotic and biotic stresses. There is evidence that PPOs contribute to stress responses in Paulownia fortunei. In this study, PPO gene family members in P. fortunei were comprehensively identified and characterized using bioinformatics methods as well as analyses of phylogenetic relationships, gene and protein structure, codon usage bias, and gene expression in response to stress. The genome contained 10 PPO gene family members encoding 406-593 amino acids, with a G/C bias. Most were localized in chloroplasts. The motif structure was conserved among family members, and α-helices and random coils were the dominant elements in the secondary structure. The promoters contained many cis-acting elements, such as auxin, gibberellin, salicylic acid, abscisic acid, and photoresponsive elements. The formation of genes in this family was linked to evolutionary events, such as fragment replication. Real-time quantitative PCR results showed that PfPPO7, PfPPO10, PfPPO1, PfPPO2, PfPPO3, PfPPO4, PfPPO5, and PfPPO8 may be key genes in drought stress resistance. PfPPO1, PfPPO3, PfPPO4, and PfPPO10 were resistant stress-sensitive genes. These results provide a reliable basis for fully understanding the potential functions of these genes and the selection of resistance breeding.

Dengue virus preferentially uses human and mosquito non-optimal codons

Codon optimality refers to the effect that codon composition has on messenger RNA (mRNA) stability and translation level and implies that synonymous codons are not silent from a regulatory point of view. Here, we investigated the adaptation of virus genomes to the host optimality code using mosquito-borne dengue virus (DENV) as a model. We demonstrated that codon optimality exists in mosquito cells and showed that DENV preferentially uses nonoptimal (destabilizing) codons and avoids codons that are defined as optimal (stabilizing) in either human or mosquito cells. Human genes enriched in the codons preferentially and frequently used by DENV are upregulated during infection, and so is the tRNA decoding the nonoptimal and DENV preferentially used codon for arginine. We found that adaptation during single-host passaging in human or mosquito cells results in the selection of synonymous mutations towards DENV’s preferred nonoptimal codons that increase virus fitness. Finally, our analyses revealed that hundreds of viruses preferentially use nonoptimal codons, with those infecting a single host displaying an even stronger bias, suggesting that host–pathogen interaction shapes virus-synonymous codon choice.

Phylogenetic and Comparative Genomics Study of Papilionidae Based on Mitochondrial Genomes.

Most species of Papilionidae are large and beautiful ornamental butterflies. They are recognized as model organisms in ecology, evolutionary biology, genetics, and conservation biology but present numerous unresolved phylogenetic problems. Complete mitochondrial genomes (mitogenomes) have been widely used in phylogenetic studies of butterflies, but mitogenome knowledge within the family Papilionidae is limited, and its phylogeny is far from resolved. In this study, we first report the mitogenome of Byasa confusa from the subfamily Papilioninae of Papilionidae. The mitogenome of B. confusa is 15,135 bp in length and contains 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and an AT-rich control region (CR), closely mirroring the genomic structure observed in related butterfly species. Comparative analysis of 77 Papilionidae mitogenomes shows gene composition and order to be identical to that of an ancestral insect, and the AT bias, Ka/Ks, and relative synonymous codon usage (RSCU) are all consistent with that of other reported butterfly mitogenomes. We conducted phylogenetic analyses using maximum-likelihood (ML) and Bayesian-inference (BI) methods, with 77 Papilionidae species as ingroups and two species of Nymphalidae and Lycaenidae as outgroups. The phylogenetic analysis indicated that B. confusa were clustered within Byasa. The phylogenetic trees show the monophyly of the subfamily Papilioninae and the tribes Leptocircini, Papilionini, and Troidini. The data supported the following relationships in tribe level on Papilioninae: (((Troidini + Papilionini) + Teinopalpini) + Leptocircini). The divergence time analysis suggests that Papilionidae originated in the late Creataceous. Overall, utilizing the largest number of Papilionidae mitogenomes sequenced to date, with the current first exploration in a phylogenetic analysis on Papilionidae (including four subfamilies), this study comprehensively reveals the mitogenome characteristics and mitogenome-based phylogeny, providing information for further studies on the mitogenome, phylogeny, evolution, and taxonomic revision of the Papilionidae family.

Integration of Illumina and PacBio HiFi Sequencing Reveals a Three-Linear-Molecule Mitogenome with RNA-Editing Sites and Phylogeny in Arrow Bamboo (Fargesia qinlingensis)

Arrow bamboo (Fargesia qinlingensis) is endemic to the Qinling Mountains and has remarkable adaptive resilience to changing climates. However, its complete mitogenome remains unknown. Using the Illumina and PacBio HiFi sequencing platforms, we found that the mitogenome assembly of the F. qinlingensis has a multi-branched skeleton comprising three linear molecules (M1, M2, and M3), with a length of 442,368 bp and a GC content of 44.05%. Thirty-five unique PCGs were identified in the complete mitogenome, including twenty-four core structural genes, eleven noncore structural genes, three rRNAs, and sixteen tRNAs. The GCU for alanine and CAA for glutamine represented the most significant frequency (RSCU = 1.55) in the codon usage preference. A total of 51, 28, and 14 SSRs were determined on M1, M2, and M3, respectively. The mitogenome contained 149 pairs of dispersed repeats with lengths greater than 30 bp, the most abundant of which were 82 forward and 67 palindromic repeats. A long repeat sequence (14,342 bp) was characterized in mediating mitogenome recombination. DNA transfer analyses suggested that 44 MTPTs (30,943 bp, 6.99%) originated from the plastome. Among the 482 potential C-U/T RNA-editing sites predicted in 35 PCGs, ccmFn (38 times) and ccmC (36 times) shoed the highest frequency. Collinearity and phylogenetic trees revealed the close relationship between F. qinlingensis and Bambusa oldhamii. The primary features of the mitogenome of F. qinlingensis will help decipher the functional mitochondrial traits related to growth performance and climate resilience. Moreover, our findings provide insights into the evolution, environmental adaptation, and sustainable use of subalpine bamboo resources in the Qinling Mountains.

Stop Codon Usage of Three Gene Families in Selected Medical Insects, and their Phylogenetic Relationships

Medical insects pose significant health risks, transmitting disease causing agents with significant morbidity and mortality globally. Understanding their genetic composition and evolutionary relationship is crucial to developing control techniques to combat them. This study aims to compare the stop codon usage of gustatory, odorant and kinase genes, and assess their use as markers for evaluating the phylogenetic relationship between selected medical insects. Fifteen genes from three gene families including gustatory receptors, odorant receptors, and kinases were studied in thirty-four insect species. Gene and transcript sequences were retrieved from Ensembl Metazoa database. The stop codon usage was obtained by identifying the stop codon present at the end of the transcript sequences. Multiple sequence alignment of the gene sequences was performed and phylogenetic trees were computed. There were variations in the stop codon usage across the different insect species. The odorant receptor and kinase genes grouped the insects into two major clusters. The stop codon usage highlighted the variation within and among the species. The phylogenetic analysis results supported existing insect classification. These approaches could be considered for phylogenetic analyses of other arthropod groups. Accurate measurement of stop codon usage will be important for understanding natural selection, the genetic variation of coding sequences of genes, and useful in design of more efficient expression constructs in gene-editing techniques for vector control.

Unveiling a Comprehensive Multi-epitope Subunit Vaccine Strategy Against Salmonella subsp. enterica: Bridging Core, Subtractive Proteomics, and Immunoinformatics.

Salmonella subsp. enterica (SE) presents a significant global health challenge in both developed and developing countries. Current SE vaccines have limitations, targeting specific strains and demonstrating moderate efficacy in adults, while also being unsuitable for young children and often unaffordable in regions with lower income levels where the disease is prevalent. To address these challenges, this study employed a computational approach integrating core proteomics, subtractive proteomics, and immunoinformatics to develop a universal SE vaccine and identify potential drug targets. Analysis of the core proteome of 185 SE strains revealed 1964 conserved proteins. Subtractive proteomics identified 9 proteins as potential vaccine candidates and 41 as novel drug targets. Using reverse vaccinology-based immunoinformatics, four multi-epitope-based subunit vaccine constructs (MESVCs) were designed, aiming to stimulate cytotoxic T lymphocyte, helper T lymphocyte, and linear B lymphocyte responses. These constructs underwent comprehensive evaluations for antigenicity, immunogenicity, toxicity, hydropathicity, and physicochemical properties. Predictive modeling, refinement, and validation were conducted to determine the secondary and tertiary structures of the SE-MESVCs, followed by docking studies with MHC-I, MHC-II, and TLR4 receptors. Molecular docking assessments showed favorable binding with all three receptors, with SE-MESVC-4 exhibiting the most promising binding energy. Molecular dynamics simulations confirmed the binding affinity and stability of SE-MESVC-4 with the TLR4/MD2 complex. Additionally, codon optimization and in silico cloning verified the efficient translation and successful expression of SE-MESVC-4 in Escherichia coli (E. coli) str. K12. Subsequent in silico immune simulation evaluated the efficacy of SE-MESVC-4 in triggering an effective immune response. These results suggest that SE-MESVC-4 may induce both humoral and cellular immune responses, making it a potential candidate for an effective SE vaccine. However, further experimental investigations are necessary to validate the immunogenicity and efficacy of SE-MESVC-4, bringing us closer to effectively combating SE infections.

The first complete mitochondrial genome of the genus Laelaps with novel gene arrangement reveals extensive rearrangement and phylogenetics in the superfamily Dermanyssoidea.

We collected 56 specimens ofLaelaps chini from the endemic Hengduan Mountain rat species (Eothenomys miletus) and obtained the first complete mitochondrial genome of L. chini by next-generation sequencing (NGS). The L. chini mitogenome is 16,507bp in size and contains 37 genes and a control region of 2380bp in length. The L. chini mitogenome has a high AT content and a compact arrangement with four overlapping regions ranging from 1 to 2bp and 16 spacer regions ranging from 1 to 48bp. We analyzed 13 protein-coding genes of L. chini mitogenome andfound that protein-coding genes in the L. chini mitogenome preferred codons ending in A/U and codon usage pattern was mainly influenced by natural selection. Cox1 has the slowest evolution rate and cox3 has the fastest evolution rate. We combined the mitochondrial genome of eight species ofgamasid mites in the superfamily Dermanyssoidea from Genbank and theL. chini mitochondrial genometo analyze its rearrangement patterns and breakpoint numbers. We found that the L. chini mitogenome showed a novel arrangement pattern and nine species ofgamasid mites in the superfamily Dermanyssoidea, which have been sequenced complete mitochondrial genomes to date, all showed different degrees of rearrangement. Laelaps chini, Echinolaelaps echidninus and Echinolaelaps fukinenensis were closely related species based on genetic distance and phylogenetic analyses. Notably they are clustered with Varroa destructor of the family Varroidae, suggesting that the family Varroidae is more closely related to the family Laelapidae, but more data are needed to test whether Varroa can be classified under the family Laelapidae. The L. chini mitogenome is the first complete mitochondrial genome for the genus Laelaps, and contributes to further exploration of the mitochondrial gene rearrangements and phylogeny for the superfamily Dermanyssoidea.

RNA editing sites and triplet usage in exomes of bat RNA virus genomes of the family Paramyxoviridae

Bats contain a diverse spectrum of viral species in their bodies. The RNA virus family Paramyxoviridae tends to infect several vertebrate species, which are accountable for a variety of devastating infections in both humans and animals. Viruses of this kind include measles, mumps, and Hendra. Some synonymous codons are favoured over others in mRNAs during gene-to-protein synthesis process. Such phenomenon is termed as codon usage bias (CUB). Our research emphasized many aspects that shape the CUB of genes in the Paramyxoviridae family found in bats. Here, the nitrogenous base A occurred the most. AT was found to be abundant in the coding sequences of the Paramyxoviridae family. RSCU data revealed that A or T ending codons occurred more frequently than predicted. Furthermore, 3 overrepresented codons (CAT, AGA, and GCA) and 7 underrepresented codons (CCG, TCG, CGC, CGG, CGT, GCG and ACG) were detected in the viral genomes. Correspondence analysis, neutrality plot, and parity plots highlight the combined impact of mutational pressure and natural selection on CUB. The neutrality plot of GC12 against GC3 yielded a regression coefficient value of 0.366, indicating that natural selection had a significant (63.4 %) impact. Moreover, RNA editing analysis was done, which revealed the highest frequency of C to T mutations. The results of our research revealed the pattern of codon usage and RNA editing sites in Paramyxoviridae genomes.

Comparative Analysis of the Mitochondrial Genomes of Three Species of Yangiella (Hemiptera: Aradidae) and the Phylogenetic Implications of Aradidae.

The mitochondrial genomes of three species of Yangiella were sequenced, annotated, and analyzed. The genome length of the three species of the genus is 15,070-15,202 bp, with a typical gene number, including a control region, 2 ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), and 13 protein-coding genes (PCGs). It was found that the mitochondrial genome of Yangiella had AT bias. Except for the lack of a DHU arm of the trnS1 gene, the other tRNAs had a typical cloverleaf structure, and the codon usage preferences of the three species exhibited high similarity. In addition, tRNA gene rearrangements were observed among the three subfamilies of Aradidae (Mezirinae, Calisiinae, Aradinae), and it was found that codon usage preferences appeared to be less affected by base mutation and more by natural selection. The Pi and Ka/Ks values indicated that cox1 was the most conserved gene in the mitochondrial genome of Aradidae, while atp8 and nad6 were rapidly evolved genes. Substitution saturation level analysis showed that the nucleic acid sequence of mitochondrial protein-coding genes in Aradidae did not reach saturation, suggesting the rationality of the phylogenetic analysis data. Bayesian and maximum likelihood methods were used to analyze the phylogeny of 16 species of Hemiptera insects, which supported the monophyly of Aneurinae, Carventinae, and Mezirinae, as well as the monophyly of Yangiella. Based on fossils and previous studies, the differentiation time was inferred, indicating that Yangiella diverged about 57 million years ago.

Structural proteomics guided annotation of vaccine targets and designing of multi-epitopes vaccine to instigate adaptive immune response against Francisella tularensis

Francisella tularensis can cause severe disease in humans via the respiratory or cutaneous routes and a case fatality ratio of up to 10 % is reported due to lack of proper antibiotic treatment, while F. novicida causes disease in severely immunocompromised individuals. Efforts are needed to develop effective vaccine candidates against Francisella species. Thus, in this study, a systematic computational work frame was used to deeply investigate the whole proteome of Francisella novicida containing 1728 proteins to develop vaccine against F. tularensis and related species. Whole-proteome analysis revealed that four proteins including (A0Q492) (A0Q7Y4), (A0Q4N4), and (A0Q5D9) are the suitable vaccine targets after the removal of homologous, paralogous and prediction of subcellular localization. These proteins were used to predict the T cell, B cell, and HTL epitopes which were joined together through suitable linkers to construct a multi-epitopes vaccine (MEVC). The MEVC was found to be highly immunogenic and non-allergenic while the physiochemical properties revealed the feasible expression and purification. Moreover, the molecular interaction of MEVC with TLR2, molecular simulation, and binding free energy analyses further validated the immune potential of the construct. According to Jcat analysis, the refined sequence demonstrates GC contents of 41.48 % and a CAI value of 1. The in-silico cloning and optimization process ensured compatibility with host codon usage, thereby facilitating efficient expression. Computational immune simulation studies underscored the capacity of MEVC to induce both primary and secondary immune responses. The conservation analysis further revealed that the selected epitopes exhibit 100 % conservation across different species and thus provides wider protection against Francisella.

Designing multi-epitope vaccine against human cytomegalovirus integrating pan-genome and reverse vaccinology pipelines

Human cytomegalovirus (HCMV) is accountable for high morbidity in neonates and immunosuppressed individuals. Due to the high genetic variability of HCMV, current prophylactic measures are insufficient. In this study, we employed a pan-genome and reverse vaccinology approach to screen the target for efficient vaccine candidates. Four proteins, envelope glycoprotein M, UL41A, US23, and US28, were shortlisted based on cellular localization, high solubility, antigenicity, and immunogenicity. A total of 29 B-cell and 44 T-cell highly immunogenic and antigenic epitopes with high global population coverage were finalized using immunoinformatics tools and algorithms. Further, the epitopes that were overlapping among the finalized B-cell and T-cell epitopes were linked with suitable linkers to form various combinations of multi-epitopic vaccine constructs. Among 16 vaccine constructs, Vc12 was selected based on physicochemical and structural properties. The docking and molecular simulations of VC12 were performed, which showed its high binding affinity (−23.35 kcal/mol) towards TLR4 due to intermolecular hydrogen bonds, salt bridges, and hydrophobic interactions, and there were only minimal fluctuations. Furthermore, Vc12 eliciting a good response was checked for its expression in Escherichia coli through in silico cloning and codon optimization, suggesting it to be a potent vaccine candidate.

The Complete Chloroplast Genome Sequence of the Medicinal Moss Rhodobryum giganteum (Bryaceae, Bryophyta): Comparative Genomics and Phylogenetic Analyses.

Rhodobryum giganteum (Bryaceae, Bryophyta), a rare medicinal bryophyte, is valued for its cardiovascular therapeutic properties in traditional Chinese medicine. This study presents the first complete chloroplast genome sequence of R. giganteum, including its assembly and annotation. The circular chloroplast genome of R. giganteum is 124,315 bp in length, displaying a typical quadripartite structure with 128 genes: 83 protein-coding genes, 37 tRNAs, and 8 rRNAs. Analyses of codon usage bias, repetitive sequences, and simple sequence repeats (SSRs) revealed an A/U-ending codon preference, 96 repetitive sequences, and 385 SSRs in the R. giganteum chloroplast genome. Nucleotide diversity analysis identified 10 high mutational hotspots. Ka/Ks ratio analysis suggested potential positive selection in rpl20, rps18, petG, and psbM genes. Phylogenetic analysis of whole chloroplast genomes from 38 moss species positioned R. giganteum within Bryales, closely related to Rhodobryum laxelimbatum. This study augments the chloroplast genomic data for Bryales and provides a foundation for molecular marker development and genetic diversity analyses in medicinal bryophytes.

Analysis of Codon Usage Bias in Chloroplast Genomes of Dryas octopetala var. asiatica (Rosaceae).

Dryas octopetala var. asiatica, a dwarf shrub belonging to the Rosaceae family and native to Asia, exhibits notable plasticity in photosynthesis in response to temperature variations. However, the codon usage patterns and factors influencing them in the chloroplast genome of this species have not yet been documented. This study sequenced and assembled the complete genome of D. octopetala var. asiatica. The annotated genes in the chloroplast genome were analyzed for codon composition through multivariate statistical methods including a neutrality plot, a parity rule 2 (PR2) bias plot, and an effective number of codons (ENC) plot using CodonW 1.4.2 software. The results indicated that the mean GC content of 53 CDSs was 38.08%, with the average GC content at the third codon base position being 27.80%, suggesting a preference for A/U(T) at the third codon position in chloroplast genes. Additionally, the chloroplast genes exhibited a weak overall codon usage bias (CUB) based on ENC values and other indicators. Correlation analysis showed a significant negative correlation between ENC value and GC2, an extremely positive correlation with GC3, but no correlation with GC1 content. These findings highlight the importance of the codon composition at the third position in influencing codon usage bias. Furthermore, our analysis indicated that the CUB of the chloroplast genome of D. octopetala var. asiatica was primarily influenced by natural selection and other factors. Finally, this study identified UCA, CCU, GCU, AAU, GAU, and GGU as the optimal codons. These results offer a foundational understanding for genetic modification and evolutionary dynamics of the chloroplast genome of D. octopetala var. asiatica.

The complete chloroplast genome sequences of nine melon varieties (Cucumis melo L.): lights into comparative analysis and phylogenetic relationships.

Melon (Cucumis melo L.) is one of the most extensively grown horticulture crops of the world. Based on the morphological characters, melon was formerly divided into two subspecies, Cucumis melo ssp. melo and C. melo ssp. agrestis. However, the present methods are still inadequate to distinguish between them. The phylogenetic analysis based on chloroplast genome sequences could provide essential evidence for the classification of melon varieties. We sequenced the chloroplast genomes of nine different melon varieties by the Illumina Hiseq and performed bioinformatic analyses including repeat element analysis, genome comparison and phylogenetic analysis. The results showed that the melon chloroplast genome has a typical quadripartite structure that was conserved across the analyzed sequences. Its length ranges between 155, 558 and 156, 569bp, with a total GC content varying from 36.7% to 37%. We found 127-132 genes in melon chloroplast genomes, including 85-87 protein-coding regions, 34-37 tRNA and 6-8 rRNA genes. The molecular structure, gene order, content, codon usage, long repeats, and simple sequence repeats (SSRs) were mostly conserved among the nine sequenced genomes. Phylogenetic analysis showed that the chloroplast genome could clearly distinguish between C. melo ssp. melo and C. melo ssp. agrestis. This study not only provides valuable knowledge on melon chloroplasts, but also offers a theoretical basis and technical support for the genetic breeding of melons.