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Lactococcus lactis is a Gram-positive bacterium used to produce fermented foods and heterologous proteins. Its Nisin-controlled gene expression system stands out for its versatility and safety. However, the lower GC content in its genome may lead to some limitations in protein production. In this study, we explored the importance and effect of codon optimization on fluorescent reporter protein expression in L. lactis. Three non-optimized fluorescent reporter genes (gfp, rfp, and mcherry) were compared to the codon-optimized variant (mcherry-O). Parameters such as Codon Adaptation Index (CAI), Effective Number of Codons (Enc) and Guanine-Cytosine percentage (% GC) were determined to assess their influence on gene expression and protein synthesis. The production of non-optimized fluorescent proteins does not correlate with their gene expression levels, except for the codon-optimized mCherry-O protein, which was detected in the SDS-PAGE gel and the extracted lysate (visually detected). Expression of the mcherry gene was similar to the mcherry-O gene, but protein was only detected with the optimized gene. The gfp gene showed the highest expression levels, but the quantity of protein was undetectable by SDS-PAGE. The rfp gene was revealed to be an optimized gene but not tailored for L. lactis. These findings underscore the necessity of comprehensive codon optimization for foreign genes in L. lactis and reveal intriguing complexities between expression levels, RNA stability and protein synthesis.

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Tomato brown rugose fruit virus (ToBRFV) poses a significant threat to tomato production worldwide, prompting extensive research into its genetic diversity, evolutionary dynamics, and adaptive strategies. In this study, we conducted a comprehensive analysis of ToBRFV at the codon level, focusing on codon usage bias, selection pressures, and evolutionary patterns across multiple genes. Our analysis revealed distinct patterns of codon usage bias and selection pressures within the ToBRFV genome, with varying levels of genetic diversity and evolutionary constraints among different genes. We observed a transition/transversion bias of 2.07 across the entire ToBRFV genome, with the movement protein (MP) gene exhibiting the highest transition/transversion bias and SNP density, suggesting potential evolutionary pressures or a higher mutation rate in this gene. Furthermore, our study identified episodic positive selection primarily in the MP gene, highlighting specific codons subject to adaptive changes in response to host immune pressures or environmental factors. Comparative analysis of codon usage bias in the coat protein (CP) and RNA-dependent RNA polymerase (RdRp) genes revealed gene-specific patterns reflecting functional constraints and adaptation to the host's translational machinery. Our findings provide valuable insights into the molecular mechanisms driving ToBRFV evolution and adaptation, with implications for understanding viral pathogenesis, host-virus interactions, and the development of control strategies. Future research directions include further elucidating the functional significance of codon usage biases, exploring the role of episodic positive selection in viral adaptation, and leveraging these insights to inform the development of effective antiviral strategies and crop protection measures.

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Alphaviruses are enveloped, single-stranded, positive-sense RNA viruses that often require transmission between arthropod and vertebrate hosts for their sustained propagation. Most alphaviruses encode an opal (UGA) termination codon in nonstructural protein 3 (nsP3) upstream of the viral polymerase, nsP4. The selective constraints underlying the conservation of the opal codon are poorly understood. Using primate and mosquito cells, we explored the role and selective pressure on the nsP3 opal codon through extensive mutational analysis in the prototype alphavirus, Sindbis virus (SINV). We found that the opal codon is highly favored over all other codons in primate cells under native 37°C growth conditions. However, this preference is diminished in mosquito and primate cells grown at a lower temperature. Thus, the primary determinant driving the selection of the opal stop codon is not host genetics but the passaging temperature. We show that the opal codon is preferred over amber and ochre termination codons because it results in the highest translational readthrough and polymerase production. However, substituting the opal codon with sense codons leads to excessive full-length polyprotein (P1234) production, which disrupts optimal nsP polyprotein processing, delays the switch from minus-strand to positive-strand RNA production, and significantly reduces SINV fitness at 37°C; this fitness defect is relieved at lower temperatures. A naturally occurring suppressor mutation unexpectedly compensates for a delayed transition from minus to genomic RNA production by also delaying the subsequent transition between genomic and sub-genomic RNA production. Our study reveals that the opal stop codon is the best solution for alphavirus replication at 37°C, producing enough nsP4 protein to maximize replication without disrupting nsP processing and RNA replication transitions needed for optimal fitness. Our study uncovers the intricate strategy dual-host alphaviruses use at a single codon to optimize fitness.

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More and more attention has been paid to the role of synonymous substitution in evolution, in which codon usage preference can affect gene expression distribution and protein structure and function. Vesicular glutamate transporter (VGLUT) consists of three isoforms, among which VGLUT3 is significantly different from other VGLUTs in functional importance, expression level, and distribution range, whose reason is still unclear. This study sought to analyze the role of codon preference in VGLUT differentiation. To conduct an evolutionary analysis of the three VGLUTs, this paper uses bioinformatics research methods to analyze the coding sequences of the three VGLUTs in different species and compare the codon usage patterns. Furthermore, the differences among the three VGLUTs were analyzed by combining functional importance, expression level, distribution range, gene structure, protein relationship network, expression at specific developmental stages, and phylogenetic tree, and the influence of codon usage pattern was explored. The results showed that the VGLUT with greater codon preference had less functional importance, lower expression levels, more peripheral distribution away from the CNS, smaller exon density of gene, less conserved and farther away from the CDS region miRNA regulatory sites, simpler and less tight protein interaction networks, delayed developmental expression, and more distant evolutionary relationships. Codon usage preference is a potential factor affecting VGLUT developmental expression and protein evolution.

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BACKGROUND: Viral diseases of sweet potatoes are causing severe crop losses worldwide. More than 30 viruses have been identified to infect sweet potatoes among which the sweet potato latent virus (SPLV), sweet potato mild speckling virus (SPMSV), sweet potato virus G (SPVG) and sweet potato virus 2 (SPV2) have been recognized as distinct species of the genus Potyvirus in the family Potyviridae. The sweet potato virus 2 (SPV2) is a primary pathogen affecting sweet potato crops. METHODS: In this study, we detected an SPV2 isolate (named SPV2-LN) in Ipomoea nil in China. The complete genomic sequence of SPV2-LN was obtained using sequencing of small RNAs, RT-PCR, and RACE amplification. The codon usage, phylogeny, recombination analysis and selective pressure analysis were assessed on the SPV2-LN genome. RESULTS: The complete genome of SPV2-LN consisted of 10,606 nt (GenBank No. OR842902), encoding 3425 amino acids. There were 28 codons in the SPV2-LN genome with a relative synonymous codon usage (RSCU) value greater than 1, of which 21 end in A/U. Among the 12 proteins of SPV2, P3 and P3N-PIPO exhibited the highest variability in their amino acid sequences, while P1 was the most conserved, with an amino acid sequence identity of 87-95.3%. The phylogenetic analysis showed that 21 SPV2 isolates were clustered into four groups, and SPV2-LN was clustered together with isolate yu-17-47 (MK778808) in group IV. Recombination analysis indicated no major recombination sites in SPV2-LN. Selective pressure analysis showed dN/dS of the 12 proteins of SPV2 were less than 1, indicating that all were undergoing negative selection, except for P1N-PISPO. CONCLUSION: This study identified a sweet potato virus, SPV2-LN, in Ipomoea nil. Sequence identities and genome analysis showed high similarity between our isolate and a Chinese isolate, yu-17-47, isolated from sweet potato. These results will provide a theoretical basis for understanding the genetic evolution and viral spread of SPV2.

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While bioactivity and a favorable safety profile for biotherapeutics is of utmost importance, manufacturability is also worth of consideration to ease the manufacturing process. Manufacturability in the scientific literature is mostly related to stability of formulated drug substances, with limited focus on downstream process-related manufacturability, that is, how easily can a protein be purified. Process-related impurities or biological impurities like viruses and host cell proteins (HCP) are present in the harvest which have mostly acid isoelectric points and need to be removed to ensure patient safety. Therefore, during molecule design, the surface charge of the target molecule should preferably differ sufficiently from the surface charge of the impurities to enable an efficient purification strategy. In this feasibility study, we evaluated the possibility of improving manufacturability by adapting the surface charge of the target protein. We generated several variants of a GLP1-receptor-agonist-Fc-domain-FGF21-fusion protein and demonstrated proof of concept exemplarily for an anion exchange chromatography step which then can be operated at high pH values with maximal product recovery allowing removal of HCP and viruses. Altering the surface charge distribution of biotherapeutic proteins can thus be useful allowing for an efficient manufacturing process for removing HCP and viruses, thereby reducing manufacturing costs.

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Translation elongation inhibitors are commonly used to study different cellular processes. Yet, their specific impact on transcription and mRNA decay has not been thoroughly assessed. Here we use TimeLapse sequencing to investigate how translational stress impacts mRNA dynamics in human cells. Our results reveal that a distinct group of transcripts is stabilized in response to the translation elongation inhibitor emetine. These stabilized mRNAs are short-lived at steady state and many of them encode C2H2 zinc finger proteins. The codon usage of these stabilized transcripts is suboptimal compared to other expressed transcripts, including other short-lived mRNAs that are not stabilized after emetine treatment. Finally, we show that stabilization of these transcripts is independent of ribosome quality control factors and signaling pathways activated by ribosome collisions. Our data describe a group of short-lived transcripts whose degradation is particularly sensitive to the inhibition of translation elongation.

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White spot syndrome virus (WSSV) causes highly destructive infection in crustacean aquaculture, often resulting in 100% mortality within a week. However, there is lack of studies addressing the safety issues of WSSV vaccines in shrimps. In this study, WSSV VP28 mRNA vaccines were developed using codon deoptimization approach. These vaccines were administered to Litopenaeus vannamei shrimps at various dosages to access their safety and the shrimps' immune responses using quantification PCR (qPCR). The findings of this study indicate that the expression level of codon deoptimized VP28 mRNA vaccines are lower compared to the wild type VP28 vaccines, as observed through a comparison of bioinformatic predictions and experimental results. Additionally, the total haemocyte count (THC) in shrimps injected with codon deoptimized VP28 vaccine was higher than those injected with wild type VP28 vaccines. Furthermore, the expression of immune-related genes differed between codon deoptimized and wild type VP28 vaccines. In summary, the results suggest that 0.01µg codon deoptimized VP28-D1 mRNA vaccine is the most promising WSSV mRNA vaccine, displaying low pathogenicity and expression in shrimps. To the best of our knowledge, this research represents the first attempt to attenuate WSSV using codon deoptimization method and development of a potential mRNA vaccine for shrimp purpose. The study addresses an important gap in shrimp vaccine research, offering potential solutions for WSSV control in shrimps.

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To predict potential epidemic outbreaks, we tested our strategy, Epi-Clock, which applies the novel ZHU algorithm to different SARS-CoV-2 datasets before outbreaks to search for significant mutational accumulation patterns correlated with outbreak events. Surprisingly, some inter-species genetic distances in Coronaviridae may represent intermediate states of different species or subspecies in the evolutionary history of Coronaviridae. The insertions and deletions in whole-genome sequences between different hosts were separately associated with important roles in host transmission and shifts in Coronaviridae. Furthermore, we believe that non-nucleosomal DNA may play a dominant role in the divergence of different lineages of SARS-CoV-2 in different regions of the world owing to the lack of nucleosome protection. We suggest that strong selective variation among different lineages of SARS-CoV-2 is required to produce strong codon usage bias, which appears in B.1.640.2 and B.1.617.2 (Delta). Notably, we found that an increasing number of other types of substitutions, such as those resulting from the hitchhiking effect, accumulated, especially in the pre-breakout phase, although some of the previous substitutions were replaced by other dominant genotypes. From most validations, we could accurately predict the potential pre-phase of outbreaks with a median interval of 5 days.

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Mouse (Mus musculus) models have been heavily utilized in developmental biology research to understand mammalian embryonic development, as mice share many genetic, physiological, and developmental characteristics with humans. New explorations into the integration of temporal (stage-specific) and transcriptional (tissue-specific) data have expanded our knowledge of mouse embryo tissue-specific gene functions. To better understand the substantial impact of synonymous mutational variations in the cell-state-specific transcriptome on a tissue's codon and codon pair usage landscape, we have established a novel resource-Mouse Embryo Codon and Codon Pair Usage Tables (Mouse Embryo CoCoPUTs). This webpage not only offers codon and codon pair usage, but also GC, dinucleotide, and junction dinucleotide usage, encompassing four strains, 15 murine embryonic tissue groups, 18 Theiler stages, and 26 embryonic days. Here, we leverage Mouse Embryo CoCoPUTs and employ the use of heatmaps to depict usage changes over time and a comparison to human usage for each strain and embryonic time point, highlighting unique differences and similarities. The usage similarities found between mouse and human central nervous system data highlight the translation for projects leveraging mouse models. Data for this analysis can be directly retrieved from Mouse Embryo CoCoPUTs. This cutting-edge resource plays a crucial role in deciphering the complex interplay between usage patterns and embryonic development, offering valuable insights into variation across diverse tissues, strains, and stages. Its applications extend across multiple domains, with notable advantages for biotherapeutic development, where optimizing codon usage can enhance protein expression; one can compare strains, tissues, and mouse embryonic stages in one query. Additionally, Mouse Embryo CoCoPUTs holds great potential in the field of tissue-specific genetic engineering, providing insights for tailoring gene expression to specific tissues for targeted interventions. Furthermore, this resource may enhance our understanding of the nuanced connections between usage biases and tissue-specific gene function, contributing to the development of more accurate predictive models for genetic disorders.

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Macropsini is a tribe of Eurymelinae in the family Cicadellidae that is widely distributed worldwide. Still, its taxonomic status has been unstable, and the classification of certain clades at the genus level has been controversial. The aim of this study is to address the patterns and processes that explain the structure and the evolution of the mitogenomes of Macropsini, while contributing to the resolution of systematic issues involving five of their genera. To this task, the mitogenomes of 26 species of the tribe were sequenced and characterized, and their phylogenetic relationships were reconstructed. The results revealed that the nucleotide composition of mitochondrial genes in these 26 species was significantly skewed toward A and T. Codons ending with T or A in relative synonymous codon usage were significantly more prevalent than those ending with C or G. The parity plot, neutrality plot, and correspondence analysis revealed that mutation and selective pressure affect codon usage patterns. In the phylogenetic relationships of the Macropsini, the monophyly of Pedionis and Macropsis was well-supported. Meanwhile, Oncopsis revealed paraphyletic regarding Pediopsoides. In conclusion, this research not only contributes the valuable data to the understanding of the mitogenome of the Macropsini but also provides a reference for future investigations on codon usage patterns, potential adaptive evolution, and the phylogeny of the mitogenome within the subfamily Eurymelinae.

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BACKGROUND: The global spread of the plasmid-mediated mcr (mobilized colistin resistance) gene family presents a significant threat to the efficacy of colistin, a last-line defense against numerous Gram-negative pathogens. The mcr-9 is the second most prevalent variant after mcr-1. METHODS: A dataset of 698 mcr-9-positive isolates from 44 countries is compiled. The historical trajectory of the mcr-9 gene is reconstructed using Bayesian analysis. The effective reproduction number is used innovatively to study the transmission dynamics of this mobile-drug-resistant gene. FINDINGS: Our investigation traces the origins of mcr-9 back to the 1960s, revealing a subsequent expansion from Western Europe to the America and East Asia in the late 20th century. Currently, its transmissibility remains high in Western Europe. Intriguingly, mcr-9 likely emerged from human-associated Salmonella and exhibits a unique propensity for transmission within the Enterobacter. Our research provides a new perspective that this host preference may be driven by codon usage biases in plasmids. Specifically, mcr-9-carrying plasmids prefer the nucleotide C over T compared to mcr-1-carrying plasmids among synonymous codons. The same bias is seen in Enterobacter compared to Escherichia (respectively as their most dominant genus). Furthermore, we uncovered fascinating patterns of coexistence between different mcr-9 subtypes and other resistance genes. Characterized by its low colistin resistance, mcr-9 has used this seemingly benign feature to silently circumnavigate the globe, evading conventional detection methods. However, colistin-resistant Enterobacter strains with high mcr-9 expression have emerged clinically, implying a strong risk of mcr-9 evolving into a global "true-resistance-gene". INTERPRETATION: This study explores the mcr-9 gene, emphasizing its origin, adaptability, and dissemination potential. Given the high mcr-9 expression colistin-resistant strains was observed in clinically the prevalence of mcr-9 poses a significant challenge to drug resistance prevention and control within the One Health framework. FUNDING: This work was partially supported by the National Natural Science Foundation of China (Grant No. 32141001 and 81991533).

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Plant non-specific lipid transfer protein (nsLTP) is able to bind and transport lipids and essential oils, as well as engage in various physiological processes, including defense against phytopathogens. Kalanchoe fedtschenkoi (Lavender Scallops) is an attractive and versatile succulent. To investigate the functional mechanism of Kalanchoe fedtschenkoi nsLTP (Ka-nsLTP), we expressed, purified and successfully obtained monomeric Ka-nsLTP. Mutational experiments revealed that the C6A variant retained the same activity as the wild-type (WT) Ka-nsLTP. Ka-nsLTP showed weak antiphytopathogenic bacterial activity, but inhibited fungal growth. Ka-nsLTP possessed a hydrophobic cavity effectively binding lauric acid. Our results offer novel molecular insights into the functional mechanism of nsLTP, which broadens our knowledge of the biological function of nsLTP in crops and provides a useful locus for genetic improvement of plants.

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Oxygenases catalyze crucial reactions throughout all domains of life, cleaving molecular oxygen (O2) and inserting one or two of its atoms into organic substrates. Many oxygenases, including those in the cytochrome P450 (P450) and Rieske oxygenase enzyme families, function as multicomponent systems, which require one or more redox partners to transfer electrons to the catalytic center. As the identity of the reductase can change the reactivity of the oxygenase, characterization of the latter with its cognate redox partners is critical. However, the isolation of the native redox partner or partners is often challenging. Here, we report the preparation and characterization of PbdB, the native reductase partner of PbdA, a bacterial P450 enzyme that catalyzes the O-demethylation of para-methoxylated benzoates. Through production in a rhodoccocal host, codon optimization, and anaerobic purification, this procedure overcomes conventional challenges in redox partner production and allows for robust oxygenase characterization with its native redox partner. Key lessons learned here, including the value of production in a related host and rare codon effects are applicable to a broad range of Fe-dependent oxygenases and their components.

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Introduction: Among cultivated tea plants (Camellia sinensis), only four mitogenomes for C. sinensis var. assamica (CSA) have been reported so far but none for C. sinensis var. sinensis (CSS). Here, two mitogenomes of CSS (CSSDHP and CSSRG) have been sequenced and assembled. Methods: Using a combination of Illumina and Nanopore data for the first time. Comparison between CSS and CSA mitogenomes revealed a huge heterogeneity. Results: The number of the repetitive sequences was proportional to the mitogenome size and the repetitive sequences dominated the intracellular gene transfer segments (accounting for 88.7%- 92.8% of the total length). Predictive RNA editing analysis revealed that there might be significant editing in NADH dehydrogenase subunit transcripts. Codon preference analysis showed a tendency to favor A/T bases and T was used more frequently at the third base of the codon. ENc plots analysis showed that the natural selection play an important role in shaping the codon usage bias, and Ka/Ks ratios analysis indicated Nad1 and Sdh3 genes may have undergone positive selection. Further, phylogenetic analysis shows that six C. sinensis clustered together, with the CSA and CSS forming two distinct branches, suggesting two different evolutionary pathway. Discussion: Altogether, this investigation provided an insight into evolution and phylogeny relationship of C. sinensis mitogenome, thereby enhancing comprehension of the evolutionary patterns within C. sinensis species.

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The nearly neutral theory of molecular evolution posits variation among species in the effectiveness of selection. In an idealized model, the census population size determines both this minimum magnitude of the selection coefficient required for deleterious variants to be reliably purged, and the amount of neutral diversity. Empirically, an 'effective population size' is often estimated from the amount of putatively neutral genetic diversity and is assumed to also capture a species' effectiveness of selection. A potentially more direct measure of the effectiveness of selection is the degree to which selection maintains preferred codons. However, past metrics that compare codon bias across species are confounded by among-species variation in %GC content and/or amino acid composition. Here, we propose a new Codon Adaptation Index of Species (CAIS), based on Kullback-Leibler divergence, that corrects for both confounders. We demonstrate the use of CAIS correlations, as well as the Effective Number of Codons, to show that the protein domains of more highly adapted vertebrate species evolve higher intrinsic structural disorder.

Evolution is the process through which populations change over time, starting with mutations in the genetic sequence of an organism. Many of these mutations harm the survival and reproduction of an organism, but only by a very small amount. Some species, especially those with large populations, can purge these slightly harmful mutations more effectively than other species. This fact has been used by the 'drift barrier theory' to explain various profound differences amongst species, including differences in biological complexity. In this theory, the effectiveness of eliminating slightly harmful mutations is specified by an 'effective' population size, which depends on factors beyond just the number of individuals in the population. Effective population size is normally calculated from the amount of time a 'neutral' mutation (one with no effect at all) stays in the population before becoming lost or taking over. Estimating this time requires both representative data for genetic diversity and knowledge of the mutation rate. A major limitation is that these data are unavailable for most species. A second limitation is that a brief, temporary reduction in the number of individuals has an oversized impact on the metric, relative to its impact on the number of slighly harmful mutations accumulated. Weibel, Wheeler et al. developed a new metric to more directly determine how effectively a species purges slightly harmful mutations. Their approach is based on the fact that the genetic code has 'synonymous' sequences. These sequences code for the same amino acid building block, with one of these sequences being only slightly preferred over others. The metric by Weibel, Wheeler et al. quantifies the proportion of the genome from which less preferred synonymous sequences have been effectively purged. It judges a population to have a higher effective population size when the usage of synonymous sequences departs further from the usage predicted from mutational processes. The researchers expected that natural selection would favour 'ordered' proteins with robust three-dimensional structures, i.e., that species with a higher effective population size would tend to have more ordered versions of a protein. Instead, they found the opposite: species with a higher effective population size tend to have more disordered versions of the same protein. This changes our view of how natural selection acts on proteins. Why species are so different remains a fundamental question in biology. Weibel, Wheeler et al. provide a useful tool for future applications of drift barrier theory to a broad range of ways that species differ.

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Monogeneans of the genus Dactylogyrus Diesing, 1850, the largest genus in the family Dactylogyridae, mostly parasitize the gills of cyprinoid hosts; however, only 3 Dactylogyrus' mitochondrial genomes (mitogenomes) are studied so far. The aim of this research is to extend our understanding of the mitogenomes of Dactylogyrus. We sequenced the mitogenomes of D. crucifer and D. zandti isolated from Rutilus rutilus and Abramis brama orientalis in northwest China, and then we compared these mitogenomes with other monogeneans. We used Illumina NovaSeq to sequence the entire mitochondrial genomes of D. crucifer and D. zandti and characterized the mitogenomes to understand the gene structure, gene identity, the secondary structures of the 22 tRNA genes, and relative synonymous codon usage. We used the analytic Bayesian Information and Maximum Likelihood methods to determine their associated phylogenetic trees. The mitogenomes of D. crucifer and D. zandti were 14,403 and 18,584 bp, respectively. Organization and positioning of these genes were in accordance with Dactylogyrus lamellatus and Dactylogyrus tuba. The nucleotide composition of Dactylogyridae was different from other families of Monogenea, and the A+T count of genus Dactylogyrus (54 - 58.4 %) was lower than other genus species of the family Dactylogyridea (63.9 - 78.4 %) in protein-coding genes. Dactylogyrus members displayed a codon usage bias. The relative synonymous codon used by Dactylogyrus was not conserved and was lower than other monogeneans. The codon use patterns of closely-related species isolated from closely-related hosts were identical. Phylogenetic analyses using mitogenomic dataset produced Dactylogyrus isolated from host subfamily Leuciscinae formed a sister-group. Our results contributed significantly to an increased database of mitogenomes, more than 50 %, for Dactylogyrus that may help future studies of mitochondrial genes and codon uses for the analysis of monogenean phylogenetics.

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BACKGROUND: The tribe Ampelopsideae plants are important garden plants with both medicinal and ornamental values. The study of codon usage bias (CUB) facilitates a deeper comprehension of the molecular genetic evolution of species and their adaptive strategies. The joint analysis of CUB in chloroplast genomes (cpDNA) offers valuable insights for in-depth research on molecular genetic evolution, biological resource conservation, and elite breeding within this plant family. RESULTS: The base composition and codon usage preferences of the eighteen chloroplast genomes were highly similar, with the GC content of bases at all positions of their codons being less than 50%. This indicates that they preferred A/T bases. Their effective codon numbers were all in the range of 35-61, which indicates that the codon preferences of the chloroplast genomes of the 18 Ampelopsideae plants were relatively weak. A series of analyses indicated that the codon preference of the chloroplast genomes of the 18 Ampelopsideae plants was influenced by a combination of multiple factors, with natural selection being the primary influence. The clustering tree generated based on the relative usage of synonymous codons is consistent with some of the results obtained from the phylogenetic tree of chloroplast genomes, which indicates that the clustering tree based on the relative usage of synonymous codons can be an important supplement to the results of the sequence-based phylogenetic analysis. Eventually, 10 shared best codons were screened on the basis of the chloroplast genomes of 18 species. CONCLUSION: The codon preferences of the chloroplast genome in Ampelopsideae plants are relatively weak and are primarily influenced by natural selection. The codon composition of the chloroplast genomes of the eighteen Ampelopsideae plants and their usage preferences were sufficiently similar to demonstrate that the chloroplast genomes of Ampelopsideae plants are highly conserved. This study provides a scientific basis for the genetic evolution of chloroplast genes in Ampelopsideae species and their suitable strategies.

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FEM1B is a substrate-recognition component of the CRL2 E3 ubiquitin-protein ligase. This multi-protein complex targets specific proteins for ubiquitylation, which leads to their degradation. Here, we demonstrate the regulation of FEM1B expression by stop codon readthrough (SCR). In this process, translating ribosomes readthrough the stop codon of FEM1B to generate a C-terminally extended isoform that is highly unstable. A total of 81 nucleotides in the proximal 3'UTR of FEM1B constitute the necessary and sufficient cis-signal for SCR. Also, they encode the amino acid sequence responsible for the degradation of the SCR product. CRISPR-edited cells lacking this region, and therefore SCR of FEM1B, showed increased FEM1B expression. This in turn resulted in reduced expression of SLBP (a target of FEM1B-mediated degradation) and replication-dependent histones (target of SLBP for mRNA stability), causing cell cycle delay. Evolutionary analysis revealed that this phenomenon is specific to the genus Pan and Homo (Hominini). Overall, we show a relatively recently evolved SCR process that relieves the cell cycle from the negative regulation by FEM1B.

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Research has shown that voraxin α derived from male ticks stimulates blood feeding to engorge in female ticks. Whereas, the oviposition rate, egg weight, and body weight of female ticks were reduced in animals vaccinated with recombinant (r-) voraxin α. These data suggest a potential role of r-voraxin α as a functional anti-tick antigen in Rhipicephalus appendiculatus and Amblyomma hebraeum tick infestation. This study investigated the immunogenicity of r-voraxin α protein from Hyalomma anatolicum (H. anatolicum) tick as an anti-tick vaccine in rabbits. The H. anatolicum voraxin α sequence was optimized according to the codon usage in E. coli before being sub-cloned into pQE30. The gene sequence of the voraxin α was synthesized, verified by DNA sequencing, cloned in a pQE30 vector, and transformed into E. coli. Then, the expression of the r-voraxin α protein was confirmed by SDS-PAGE and Western blot analysis. Subsequently, three rabbits were immunized with the r-voraxin α as the vaccinated group, whereas three rabbits without injection were considered the control group. The result indicated the success of cloning of codon-optimized H. anatolicum voraxin α gene. Moreover, the expression of the r-voraxin α protein (approximately 18 kDa) in the bacterial expression system was confirmed by SDS-PAGE and Western blot analysis. The results of this study showed that the mortality rate in vaccine recipients increased compared to the control group (P < 0.01). Also, the egg weight, oviposition rate, and engorgement weight of female ticks fed from vaccinated animals were significantly reduced compared to the control group (P < 0.01). The results confirmed that the codon-optimized H. anatolicum voraxin α gene expressed in the bacterial expression system could be a suitable anti-tick vaccine against H. anatolicum tick infestation.

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