Synonymous Substitutions Research Articles

The novel HLA-A*30:01:23 allele characterised by two different sequencing-based typing techniques.

The novel allele HLA-A*30:01:23 differs from HLA-A*30:01:01:01 by one synonymous nucleotide substitution in exon 2.

The novel HLA-B*44:48:02 allele characterised by two different sequencing-based typing techniques.

The novel allele HLA-B*44:48:02 differs from HLA-B*44:48:01 by one synonymous nucleotide substitution in exon 3.

A new look at the fluorescent protein-based approach for identifying optimal coding sequence for recombinant protein expression in E. coli.

Due to the degeneracy of the genetic code, most amino acids are encoded by several codons. The choice among synonymous codons at the N-terminus of genes has a profound effect on protein expression in Escherichia coli. This is often explained by the different contributions of synonymous codons to mRNA secondary structure formation. Strong secondary structures at the 5'-end of mRNA interfere with ribosome binding and affect the process of translation initiation. In silico optimization of the gene 5'-end can significantly increase the level of protein expression; however, this method is not always effective due to the uncertainty of the exact mechanism by which synonymous substitutions affect expression; thus, it may produce nonoptimal variants as well as miss some of the best producers. In this paper, an alternative approach is proposed based on screening a partially randomized library of expression constructs comprising hundreds of selected synonymous variants. The effect of such substitutions was evaluated using the gene of interest fused to the reporter gene of the fluorescent protein with subsequent screening for the most promising candidates according to the reporter's signal intensity. The power of the approach is demonstrated by a significant increase in the prokaryotic expression of three proteins: canine cystatin C, human BCL2-associated athanogene 3 and human cardiac troponin I. This simple approach was suggested which may provide an efficient, easy, and inexpensive optimization method for poorly expressed proteins in bacteria.

Chromosome-scale genomes of Quercus sichourensis and Quercus rex provide insights into the evolution and adaptation of Fagaceae

The Fagaceae, a plant family with a wide distribution and diverse adaptability, has garnered significant interest as a subject of study in plant speciation and adaptation. Meanwhile, certain Fagaceae species are regarded as highly valuable wood resources due to the exceptional quality of their wood. In this study, we present two high-quality, chromosome-scale genome sequences for Quercus sichourensis (848.75 Mb) and Quercus rex (883.46 Mb). Comparative genomics analysis reveals that the difference in the number of plant disease resistance genes and the nonsynonymous and synonymous substitution ratio (Ka/Ks) of protein-coding genes among Fagaceae species are related to different environmental adaptations. Interestingly, most genes related to starch synthesis in the investigated Quercoideae species are located on a single chromosome, as compared to the outgroup species, Fagus sylvatica. Furthermore, resequencing and population analysis of Q. sichourensis and Q. rex reveal that Q. sichourensis has lower genetic diversity and higher deleterious mutations compared to Q. rex. The high-quality, chromosome-level genomes and the population genomic analysis of the critically endangered Q. sichourensis and Q. rex will provide an invaluable resource as well as insights for future study in these two species, even the genus Quercus, to facilitate their conservation.

Auto-phylo v2 and auto-phylo-pipeliner: building advanced, flexible, and reusable pipelines for phylogenetic inferences, estimation of variability levels and identification of positively selected amino acid sites.

The vast amount of genome sequence data that is available, and that is predicted to drastically increase in the near future, can only be efficiently dealt with by building automated pipelines. Indeed, the Earth Biogenome Project will produce high-quality reference genome sequences for all 1.8 million named living eukaryote species, providing unprecedented insight into the evolution of genes and gene families, and thus on biological issues. Here, new modules for gene annotation, further BLAST search algorithms, further multiple sequence alignment methods, the adding of reference sequences, further tree rooting methods, the estimation of rates of synonymous and nonsynonymous substitutions, and the identification of positively selected amino acid sites, have been added to auto-phylo (version 2), a recently developed software to address biological problems using phylogenetic inferences. Additionally, we present auto-phylo-pipeliner, a graphical user interface application that further facilitates the creation and running of auto-phylo pipelines. Inferences on S-RNase specificity, are critical for both cross-based breeding and for the establishment of pollination requirements. Therefore, as a test case, we develop an auto-phylo pipeline to identify amino acid sites under positive selection, that are, in principle, those determining S-RNase specificity, starting from both non-annotated Prunus genomes and sequences available in public databases.

Influence of Environmental Factors and Genome Diversity on Cumulative COVID-19 Cases in the Highland Region of China: Comparative Correlational Study.

The novel coronavirus SARS-CoV-2 caused the global COVID-19 pandemic. Emerging reports support lower mortality and reduced case numbers in highland areas; however, comparative studies on the cumulative impact of environmental factors and viral genetic diversity on COVID-19 infection rates have not been performed to date. The aims of this study were to determine the difference in COVID-19 infection rates between high and low altitudes, and to explore whether the difference in the pandemic trend in the high-altitude region of China compared to that of the lowlands is influenced by environmental factors, population density, and biological mechanisms. We examined the correlation between population density and COVID-19 cases through linear regression. A zero-shot model was applied to identify possible factors correlated to COVID-19 infection. We further analyzed the correlation of meteorological and air quality factors with infection cases using the Spearman correlation coefficient. Mixed-effects multiple linear regression was applied to evaluate the associations between selected factors and COVID-19 cases adjusting for covariates. Lastly, the relationship between environmental factors and mutation frequency was evaluated using the same correlation techniques mentioned above. Among the 24,826 confirmed COVID-19 cases reported from 40 cities in China from January 23, 2020, to July 7, 2022, 98.4% (n=24,430) were found in the lowlands. Population density was positively correlated with COVID-19 cases in all regions (ρ=0.641, P=.003). In high-altitude areas, the number of COVID-19 cases was negatively associated with temperature, sunlight hours, and UV index (P=.003, P=.001, and P=.009, respectively) and was positively associated with wind speed (ρ=0.388, P<.001), whereas no correlation was found between meteorological factors and COVID-19 cases in the lowlands. After controlling for covariates, the mixed-effects model also showed positive associations of fine particulate matter (PM2.5) and carbon monoxide (CO) with COVID-19 cases (P=.002 and P<.001, respectively). Sequence variant analysis showed lower genetic diversity among nucleotides for each SARS-CoV-2 genome (P<.001) and three open reading frames (P<.001) in high altitudes compared to 300 sequences analyzed from low altitudes. Moreover, the frequencies of 44 nonsynonymous mutations and 32 synonymous mutations were significantly different between the high- and low-altitude groups (P<.001, mutation frequency>0.1). Key nonsynonymous mutations showed positive correlations with altitude, wind speed, and air pressure and showed negative correlations with temperature, UV index, and sunlight hours. By comparison with the lowlands, the number of confirmed COVID-19 cases was substantially lower in high-altitude regions of China, and the population density, temperature, sunlight hours, UV index, wind speed, PM2.5, and CO influenced the cumulative pandemic trend in the highlands. The identified influence of environmental factors on SARS-CoV-2 sequence variants adds knowledge of the impact of altitude on COVID-19 infection, offering novel suggestions for preventive intervention.

BFS2Adv: Black-box adversarial attack towards hard-to-attack short texts

The advent of Machine Learning as a Service (MLaaS) and deep learning applications has increased the susceptibility of models to adversarial textual attacks, particularly in black-box settings. Prior work on black-box adversarial textual attacks generally follows a stable strategy that involves leveraging char-level, world-level, and sentence-level perturbations, as well as using queries to the target model to find adversarial examples in the search space. However, existing approaches prioritize query efficiency by reducing the search space, thereby overlooking hard-to-attack textual instances. To address this issue, we propose BFS2Adv, a brute force algorithm that generates adversarial examples for both easy-to-attack and hard-to-attack textual inputs. BFS2Adv, starting with an original text, employs word-level perturbations and synonym substitution to construct a comprehensive search space, with each node representing a potential adversarial example. The algorithm systematically explores this space through a breadth-first search, combined with queries to the target model, to effectively identify qualified adversarial examples. We implemented and evaluated a prototype of BFS2Adv against renowned models such as ALBERT and BERT, utilizing the SNLI and MR datasets. Our results demonstrate that BFS2Adv outperforms state-of-the-art algorithms and effectively improves the success rate of short-text adversarial attacks. Furthermore, we provide detailed insights into the robustness of BFS2Adv by analyzing those hard-to-attack examples.

Abstract 1075: Buccal mucosal remodeling predicts esophageal cancer risk

Abstract Lifestyle habits, including alcohol consumption, tobacco smoking, and germline variants of ALDH2 and ADH1B, increase the risk of developing esophageal squamous cell carcinoma (ESCC). However, current prediction methods that rely on self-reported lifestyle information lack accuracy. We previously demonstrated that driver-mutated clones remodel the esophageal epithelium with age and that this remodeling is accelerated in heavy drinkers and smokers. Nonetheless, predicting cancer from clonal expansion in normal tissues remains challenging, due to limited sample availability beyond the blood. The squamous epithelium continuously covers the esophagus, throat, and oral cavity. Therefore, we evaluated buccal mucosal remodeling by mutant clones and its potential as a predictive biomarker of ESCC. A total of 387 buccal mucosa samples were collected from 110 subjects, including 62 with ESCC and 48 without, using swabs of three different sizes, followed by error-corrected sequencing targeting 26 driver genes that showed positive selection in samples from ESCC and physiologically normal esophageal tissues, as well as ALDH2 and ADH1B. Somatic mutations were detected in all samples (median, 27 (1-134)). In total, 13,235 mutations, including 12,085 nonsynonymous and 1,150 synonymous mutations, with VAFs ranging from 0.0016-0.12 (median, 0.0025), were detected. Ten of the 26 genes, TP53, CHEK2, NOTCH1, CDKN2A, NOTCH2, FAT1, AJUBA, PPM1D, ARID1A, and ZNF750 showed positive selection (dN/dS &amp;gt;1, q&amp;lt;0.1). Correlation analysis demonstrated that driver mutations in TP53, FAT1, and PPM1D were positively correlated with not only germline risk variants of ALDH2 but also alcohol consumption. The impact of alcohol consumption on the number and sum of VAFs of mutations in each of the three genes was analyzed and stratified by the presence or absence of the germline risk variant of ALDH2, and positive correlations were observed only among subjects with risk variants. Compared to subjects without ESCC, patients with early-stage or advanced ESCC displayed significantly higher numbers and sums of VAFs of driver mutations (p&amp;lt;0.05). These results indicated that buccal mucosal remodeling driven by positively selected mutations is associated with lifestyle and germline risk factors for ESCC, thus motivating the establishment of a predictive model for ESCC using genetic swab data. The prediction model for ESCC using sequencing data outperformed a model using self-reported lifestyle and alcohol flushing reaction and a model using lifestyle and germline risk variants (AUC of 0.94, 0.72, and 0.88, respectively). In conclusion, this is the first study to use clonal expansion in normal tissues to predict the presence of solid organ cancer, and our study successfully established a prediction model for ESCC based on buccal mucosal remodeling and germline variants. Citation Format: Akira Yokoyama, Tomonori Hirano, Koichi Watanabe, Masashi Tamaoki, Kenshiro Hirohashi, Yoshihiro Ishida, Yoshikage Inoue, Yasuhide Takeuch, Yo Kishimoto, Soo Ki Kim, Chikatoshi Katada, Yasuhito Nanya, Hiroshi Seno, Seishi Ogawa, Manabu Muto, Nobuyuki Kakiuchi. Buccal mucosal remodeling predicts esophageal cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1075.

Abstract 148: Multiomic sequencing reveals diversity in clonal landscape and genomic alterations in a lung metastatic PDX osteosarcoma model

Abstract Introduction: Osteosarcoma is a primary malignant bone tumor characterized by the production of spindle cells resulting in immature bone formation. The lung is the most frequent site of metastatic disease and relapse occurs in more than 30 % of patients. Recent results show that relapse is due to a subset of cells with different phenotypic and genetic signatures conferring advantage to drive progression and drug resistance within the intra-tumorally heterogeneous population. Thus, developing a defined in vivo model that can identify the rare genetic subpopulation and recapitulate clonal evolution is crucial. In this study, we used whole exome, barcode and RNA sequencing to characterize the landscape of genomic alterations and also track and identify the clonal subpopulations, clonal and genetic drivers of lungs metastasis. Methods: We injected barcoded PDX (OS17) cells intravenously in ten SCID mice to track the clonal subpopulation of cells that metastasize to the lungs. Metastasized lung nodules were collected following death or euthanasia. All samples collected were snap frozen in liquid nitrogen and stored at -80°C. DNA and RNA was extracted and analyzed by PCR, NGS, WES and RNA sequencing to map clonality, mutational and evolutionary profiles. Results: Four (40%) of the mice developed lung metastases with three mice having multiple metastatic lung nodules. Surgery to remove the metastatic nodules was performed 216 ± 55.5 days following tumor cell injection. Nine metastatic lung nodules from three mice were used for sequencing and analysis. A Shannon-Weaver and Jaccard similarity index show a diversity in clonal architecture between the lung nodules. A total of 15,394 somatic variants was identified including 12,547 single nucleotide variants (SNV), 4054 synonymous, 2662 missense, 28 nonsense mutations and 1639 somatic indels. The SNVs accounted for 81% of all variants followed by CNV 11 % and indels 8% respectively. Synonymous and missense mutations were the most frequently observed in all samples. Copy number loss was two-fold compared to copy number gain across all tumors. Furthermore, the deletions span longer genomic distances compared to amplifications. The mutational spectrum showed that the highest substitutions were C&amp;gt;T/G&amp;gt;A followed by T&amp;gt;C/A&amp;gt;G; the least substitutions were T&amp;gt;A/A&amp;gt;T. Also, we identified a large set of genes with genomic aberrations. These include amplifications in VEGF, RB1, RUNX, PARP4, ICAM3, EGFR, BRCA2, COLA6A1, COLA6A2, CCND3, CDKN2D etc. Conclusions: In the current model, we identified multiple tumorigenic seeding clones and the diverse set of genes mutated in osteosarcoma. Further analysis of the mutational signature resulted in the identification of potential genes that act as drivers in the lung metastatic process. Acknowledgments: Swim Across America, the Foster Foundation and the Barbara Epstein Foundation. Citation Format: Sylvester Jusu, Wendong Zhang, Zhongting Zhang, Xu Zhaohui, Sankaranarayanan Kannan, Yifei Wang, Zhou Xin, Yi Yanhua, Michael Roth, Jonathan Gill, Richard Gorlick. Multiomic sequencing reveals diversity in clonal landscape and genomic alterations in a lung metastatic PDX osteosarcoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 148.

Abstract 7292: Surveying the landscape of somatic variation in healthy breast tissue for cancer risk prediction

Abstract Carcinogenesis centers on principles of somatic evolution, where a cell gradually accumulates mutations, eventually transforming into a tumor cell. Exploring somatic variation in the healthy population offers insights for predicting cancer risk and facilitating early detection. In breast cancer (BC), the emergence of the malignant clone occurs within a broader spectrum of abnormal cells, often referred to as the 'field effect.' BC development is intricately linked to hormonal regulation, with prior research indicating that the initial changes manifest during puberty and pregnancy. Although around 10% of diagnosed BCs affect young women (&amp;lt;45 years), with 35-55% of these cases arising within a decade after completed pregnancy (postpartum BC), effective screening tools are lacking. We propose a novel approach for the individualized quantification of somatic variation and the field effect using breast milk (BM) as a liquid biopsy. We conducted a retrospective analysis of 272 BM samples obtained from both the left and right breasts of 125 healthy donors, including 62 donors with elevated cancer risk due to a germline predisposition involving BRCA. Given the heterogeneous cellular composition of BM, encompassing epithelial cells and lymphocytes, we initially implemented a comprehensive analysis to reliably detect somatic mutations in whole BM, as well as in magnetically activated cell-sorted (MACS) epithelial-enriched (EE) and -depleted (ED) samples from a subset of donors. DNA extracted from all samples was used for NanoSeq, a single-molecule sequencing methodology, focusing on a targeted panel of 84 genes recognized as drivers in either BC or normal tissue. Our assessment of mutation burden estimates across all sample types revealed consistent results. Generally, cells accumulated somatic mutations linearly with age, but with a higher rate in individuals with a BRCA2 cancer predisposition. We found several critical driver genes, including PIK3CA, PIK3R1, TP53, to be under significant positive selection using the ratio of non-synonymous to synonymous mutations. When we assessed the fraction of cells in each sample carrying mutations in these genes, we however detected a significantly higher fraction of mutated cells in individuals with germline predisposition - possibly representing a genetic manifestation of the epidemiologically higher risk of BRCA germline carriers. As we expand our understanding of the interplay between the somatic mutation landscape and cancer risk, our efforts provide evidence for the utility of BM as an easily accessible, non-invasive sample. The proposed methodology, involving BM with precise genetic analysis through NanoSeq, presents a promising avenue for elucidating personalized somatic variation patterns. We anticipate that BM holds potential as a valuable tool for risk stratification, particularly for individuals under 45 and postpartum women. Citation Format: Moritz Jakob Przybilla, Andrew R. Lawson, Pantelis Nicola, Brian T. Pentecost, Inigo Martincorena, Kathleen F. Arcaro, Peter J. Campbell. Surveying the landscape of somatic variation in healthy breast tissue for cancer risk prediction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7292.

Abstract 1540: The dendritic cell receptor DNGR-1 shapes immunity to cancer

Abstract Background: Central to the success of CD8+ T cell-mediated anti-cancer response are type 1 conventional dendritic cells (cDC1s), acting as professional antigen presenting cells. In both mouse and human, DNGR-1 (a.k.a. CLEC9A) is a dedicated cDC1 receptor that, upon binding to its physiological ligand filamentous-actin (F-actin) exposed upon cell death, prime cytotoxic CD8+ T cells specifically against dead cell-associated antigens. Here, we interrogate the role of DNGR-1 in shaping the tumor microenvironment during carcinogenesis. Methods: We utilized two models of chemical carcinogenesis: 3-methylcholanthrene (MCA)-induced sarcoma, and azoxymethane (AOM) and dextran sodium sulfate colitis (DSS)-associated carcinoma. Rag1−/- (no B and T cells), Clec9agfp/gfp, Clec9acre/cre and WT (wildtype) mice on C57BL/6 background were used. Primary MCA-sarcoma cell lines were generated and subsequently tested in a series of transplantation experiments in naïve syngeneic WT mice. Whole exome and RNA sequencing were performed to elucidate the mutational landscape and decipher the immunopeptidome of these primary cell lines. Results: Upon challenge with MCA, there is shorter latency for tumorigenesis in DNGR-1-deficient mice (n=46), like in Rag1−/− mice (n=43), in contrast to immunocompetent WT hosts (n=59) (median: 95 vs 113 days, hazard ratio: 1.72, 95% CI 1.11-2.68, p=0.008). DNGR-1-deficient mice also display increased susceptibility to AOM-DSS carcinogenesis compared to co-housed WT mice. Majority (n=16/24, 68%) of MCA-induced primary sarcoma cell lines derived from DNGR-1-deficient hosts are highly immunogenic and were controlled/rejected at a similar rate, upon challenge in secondary naïve WT recipients, like those derived from Rag1−/− mice (n=13/19, 68%). Whole-exome sequencing of cell lines derived from DNGR-1-deficient hosts revealed that they harbor high mutational burden, including mutations in F-actin-binding proteins that have been previously associated with better survival outcomes in patients with cancer (Giampazolias et al. Cell, 2021). Collectively, these data argue that DNGR-1 protects against carcinogenesis and selects for less immunogenic tumor cells. To evaluate the degree of immunoediting which occurred in the respective primary host mice, we applied the evolutionary metric based on the ratio of nonsynonymous to synonymous mutations (dN/dS), and found that they are less immunoedited compared to cell lines derived from WT hosts. Conclusion: Our findings highlight DNGR-1 as a novel immune checkpoint that couples cell death sensing and (neo-)antigen cross-presentation to functional tumor-specific CD8+ T cell responses during cancer immunoediting. Understanding the mechanism by which DNGR-1 sculpts the mutational landscape of tumors may further inform the dynamics of cancer evolution and may have translational therapeutic implications in directing effective immunotherapy strategies. Citation Format: Kok Haw Lim, Evangelos Giampazolias, Bruno Frederico, Neil C. Rogers, Probir Chakravarty, Gavin Kelly, Ana Cardoso, Emma Nye, Alejandro Suárez-Bonnet, Simon Priestnall, Robert Goldstone, Cell Services Science Technology Platform, Sonia Lee, Oliver Schulz, Jessica Strid, Luis Zapata, Caetano Reis e Sousa. The dendritic cell receptor DNGR-1 shapes immunity to cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1540.

Effect of polymorphisms in the FAM151B gene on testis-related trait parameters in Hu sheep

The family with sequence similarity 151 member B (FAM151B) gene family is widely distributed in animals. This gene family encodes proteins belonging to the phosphodiesterase superfamily which plays a role in hydrolyzing intracellular second messengers, and affects the proliferation of Sertoli cells in the testis by degrading intracellular cAMP or cGMP content, thereby affecting the sperm production capacity of the testis. In this study, mutations in the FAM151B gene were screened in 491 Hu sheep to analyze the association among FAM151B gene polymorphisms testicular traits, and sperm-related parameters. A synonymous mutation (5: g.77976636 C>T), was detected in FAM151B, resulting in three genotypes, namely, CC, TT, and TC, with genotypic frequencies of 0.33, 0.05, and 0.62, respectively. This synonymous mutation was found to be significantly correlated with testicular weight, long testicular circumference, short testicular circumference, epididymal weight, and sperm count (p <0.05). In addition, individuals with CC genotypes had significant reproductive performance advantages compared with those with TC and TT genotypes. The current study shows that the FAM151B gene may play important roles in testicular development, and its SNPs are associated with testicular parameters, and sperm count, which provides important indicators for ram selection at early stage.

Modifications of the 5' region of the CASPONTM tag's mRNA further enhance soluble recombinant protein production in Escherichia coli.

Escherichia coli is one of the most commonly used host organisms for the production of biopharmaceuticals, as it allows for cost-efficient and fast recombinant protein expression. However, challenging proteins are often produced with low titres or as inclusion bodies, and the manufacturing process needs to be developed individually for each protein. Recently, we developed the CASPONTM technology, a generic fusion tag-based platform process for high-titer soluble expression including a standardized downstream processing and highly specific enzymatic cleavage of the fusion tag. To assess potential strategies for further improvement of the N-terminally fused CASPONTM tag, we modified the 5'UTR and 5' region of the tag-coding mRNA to optimize the ribosome-mRNA interactions. In the present work, we found that by modifying the 5'UTR sequence of a pET30acer plasmid-based system, expression of the fusion protein CASPONTM-tumour necrosis factor α was altered in laboratory-scale carbon-limited fed-batch cultivations, but no significant increase in expression titre was achieved. Translation efficiency was highest for a construct carrying an expression enhancer element and additionally possessing a very favourable interaction energy between ribosome and mRNA (∆Gtotal). However, a construct with comparatively low transcriptional efficiency, which lacked the expression enhancer sequence and carried the most favourable ∆Gtotal tested, led to the highest recombinant protein formation alongside the reference pET30a construct. Furthermore, we found, that by introducing synonymous mutations within the nucleotide sequence of the T7AC element of the CASPONTM tag, utilizing a combination of rare and non-rare codons, the free folding energy of the nucleotides at the 5' end (-4 to + 37) of the transcript encoding the CASPONTM tag increased by 6kcal/mol. Surprisingly, this new T7ACrare variant led to improved recombinant protein titres by 1.3-fold up to 5.3-fold, shown with three industry-relevant proteins in lab-scale carbon limited fed-batch fermentations under industrially relevant conditions. This study reveals some of the complex interdependencies between the ribosome and mRNA that govern recombinant protein expression. By modifying the 5'UTR to obtain an optimized interaction energy between the mRNA and the ribosome (ΔGtotal), transcript levels were changed, highlighting the different translation efficiencies of individual transcripts. It was shown that the highest recombinant titre was not obtained by the construct with the most efficient translation but by a construct with a generally high transcript amount coupled with a favourable ΔGtotal. Furthermore, an unexpectedly high potential to enhance expression by introducing silent mutations including multiple rare codons into the 5'end of the CAPONTM tag's mRNA was identified. Although the titres of the fusion proteins were dramatically increased, no formation of inclusion bodies or negative impact on cell growth was observed. We hypothesize that the drastic increase in titre is most likely caused by better ribosomal binding site accessibility. Our study, which demonstrates the influence of changes in ribosome-mRNA interactions on protein expression under industrially relevant production conditions, opens the door to the applicability of the new T7ACrare tag in biopharmaceutical industry using the CASPONTM platform process.

Fitness difference between two synonymous mutations of Phytophthora infestansATP6 gene

BackgroundSequence variation produced by mutation provides the ultimate source of natural selection for species adaptation. Unlike nonsynonymous mutation, synonymous mutations are generally considered to be selectively neutral but accumulating evidence suggests they also contribute to species adaptation by regulating the flow of genetic information and the development of functional traits. In this study, we analysed sequence characteristics of ATP6, a housekeeping gene from 139 Phytophthora infestans isolates, and compared the fitness components including metabolic rate, temperature sensitivity, aggressiveness, and fungicide tolerance among synonymous mutations.ResultsWe found that the housekeeping gene exhibited low genetic variation and was represented by two major synonymous mutants at similar frequency (0.496 and 0.468, respectively). The two synonymous mutants were generated by a single nucleotide substitution but differed significantly in fitness as well as temperature-mediated spatial distribution and expression. The synonymous mutant ending in AT was more common in cold regions and was more expressed at lower experimental temperature than the synonymous mutant ending in GC and vice versa.ConclusionOur results are consistent with the argument that synonymous mutations can modulate the adaptive evolution of species including pathogens and have important implications for sustainable disease management, especially under climate change.

Distribution of human papillomavirus among Vietnamese women with cervical cancer and unusual genetic variability of HPV16

HPV16, with typical mutations that differ in geographical distribution and carcinogenic potency, has implications for cervical cancer screening, clinical diagnosis, and treatment. DNASTAR and MEGA were used to identify HPV16 variants and construct a phylogenetic tree. The most prevalent HPV genotypes were HPV16 (63.9%), HPV18 (26.7%), and other HPV (6.9%). HPV16 alterations were found in all E6, E7, and L1 genes, including 15 missense and 18 synonymous mutations. Missense mutations include R10G, Q14H, D25E, H78Y, L83V (E6); M29V, R35K, L78R, L95P (E7); H73Y, T176 N, N178T, T317P, T386S, L472F/I (L1). HPV16 sublineages include A1 (17.2%), A2 (0.9%), A3 (56.0%), A4 (19.0%), D1 (4.3%), and D3 (2.6%). Although several mutations in the oncoproteins E6, E7, and L1 have been detected, mutations known to be associated with cervical cancer risk, such as D25E and L83V, occur at a relatively low frequency. This suggests that HPV16 mutations are associated with cervical cancer through a complicated mechanism.

Trends of Plasmodium falciparum molecular markers associated with resistance to artemisinins and reduced susceptibility to lumefantrine in Mainland Tanzania from 2016 to 2021

BackgroundTherapeutic efficacy studies (TESs) and detection of molecular markers of drug resistance are recommended by the World Health Organization (WHO) to monitor the efficacy of artemisinin-based combination therapy (ACT). This study assessed the trends of molecular markers of artemisinin resistance and/or reduced susceptibility to lumefantrine using samples collected in TES conducted in Mainland Tanzania from 2016 to 2021.MethodsA total of 2,015 samples were collected during TES of artemether-lumefantrine at eight sentinel sites (in Kigoma, Mbeya, Morogoro, Mtwara, Mwanza, Pwani, Tabora, and Tanga regions) between 2016 and 2021. Photo-induced electron transfer polymerase chain reaction (PET-PCR) was used to confirm presence of malaria parasites before capillary sequencing, which targeted two genes: Plasmodium falciparum kelch 13 propeller domain (k13) and P. falciparum multidrug resistance 1 (pfmdr1).ResultsSequencing success was ≥ 87.8%, and 1,724/1,769 (97.5%) k13 wild-type samples were detected. Thirty-seven (2.1%) samples had synonymous mutations and only eight (0.4%) had non-synonymous mutations in the k13 gene; seven of these were not validated by the WHO as molecular markers of resistance. One sample from Morogoro in 2020 had a k13 R622I mutation, which is a validated marker of artemisinin partial resistance. For pfmdr1, all except two samples carried N86 (wild-type), while mutations at Y184F increased from 33.9% in 2016 to about 60.5% in 2021, and only four samples (0.2%) had D1246Y mutations. pfmdr1 haplotypes were reported in 1,711 samples, with 985 (57.6%) NYD, 720 (42.1%) NFD, and six (0.4%) carrying minor haplotypes (three with NYY, 0.2%; YFD in two, 0.1%; and NFY in one sample, 0.1%). Between 2016 and 2021, NYD decreased from 66.1% to 45.2%, while NFD increased from 38.5% to 54.7%.ConclusionThis is the first report of the R622I (k13 validated mutation) in Tanzania. N86 and D1246 were nearly fixed, while increases in Y184F mutations and NFD haplotype were observed between 2016 and 2021. Despite the reports of artemisinin partial resistance in Rwanda and Uganda, this study did not report any other validated mutations in these study sites in Tanzania apart from R622I suggesting that intensified surveillance is urgently needed to monitor trends of drug resistance markers and their impact on the performance of ACT.

Complete Mitochondrial Genome and Phylogenetic Position of Nurudea zhengii Ren (Insecta, Hemiptera, Aphididae).

Nurudea zhengii Ren was identified by aphid morphological characteristics as well as the gall shape and host plant species, and placed in the tribe Fordini (Hemiptera, Aphididae, Eriosomatinae). Here, its whole genome was firstly sequenced by a genome-skimming method and its mitochondrial genome (mitogenome) was assembled to examine its genetic variation and phylogenetic position. The complete mitogenome of Nurudea zhengii is 15,392bp in length, and consists of 13 protein-coding genes, 22 tRNAs, two rRNAs and one D-loop region. The gene order follows the mitogenomes of the other Rhus gall aphids, and similarly has an AT bias with the content of 83.9%. The majority strand is A-skewed and C-skewed, and shows opposite skewness for G-skewed in the minority strands. The ratios of nonsynonymous to synonymous substitution rates of protein-coding genes are lower than one except for ATP8, which indicated that ATP8 was undergoing positive selection. Phylogenetic analysis among the Rhus gall aphids based on 13 protein-coding genes and two rRNA genes showed that N. zhengii was sister to N. shiraii, and then clustered with N. yanoniella as a group with high support value. The two species, N. shiraii and N. yanoniella, share the same host plant Rhus chinensis, while the host of N. zhengii is R. hypoleuca. However, the phylogenetic relationship indicated that the taxa sharing the same host plant were not absolutely clustered as the closest taxa at least at species level.

Characterization of the complete mitochondrial genome of Paecilomyces variotii and comparative evolutionary mitochondriomics of 36 fungi.

Paecilomyces variotii has important economic value in stimulating crop growth, biodegradation, and other aspects. Up to now, there are no research reports on its mitochondrial genome. The mitochondrial genome of Paecilomyces variotii was determined with the next-generation sequencing method (Illumina, NovaSeq), and its characteristics were analyzed using various bioinformatics approaches. The length of complete mitochondrial genome sequence of P. variotii is 40,965bp and consists of 14 protein-coding genes, 2 ribosomal RNA genes, 1 ribosomal protein S3 gene, 26 transport RNA genes. The results of phylogenetics analysis using Bayesian inference and Maximum likelihood methods showed that P. variotii belongs to the Eurotiales order in the Thermoascaceae family, and 9 genera within the Eurotiomycetes class were effectively distinguished with high support rates (bootstrap value > 92% and posterior probabilities > 99%). The analysis of synonymous substitution rates and nonsynonymous substitution rates indicated that the Ka/Ks values of the 14 PCGs in the mitochondrial genomes of the two orders in the Eurotiomycetes class ranged from 0 to 0.4333. This study revealed the structural and sequence information characteristics of the mitochondrial genome of P. variotii, and the phylogenetic results strongly support its classification within the family Thermoascaceae, consistent with traditional morphological taxonomy studies. The 14 PCGs in the mitochondrial genomes of the two orders in the Eurotiomycetes class are subject to strong purifying (negative) selection. The results of this research provides an important molecular basis for the development of genomics, evolutionary genetics and molecular markers of P. variotii in the future.

From cyanobacteria and cyanophages to chloroplasts: the fate of the genomes of oxyphototrophs and the genes encoding photosystem II proteins.

Chloroplasts are the result of endosymbiosis of cyanobacterial organisms with proto-eukaryotes. The psbA, psbD and psbO genes are present in all oxyphototrophs and encode the D1/D2 proteins of photosystem II (PSII) and PsbO, respectively. PsbO is a peripheral protein that stabilizes the O2-evolving complex in PSII. Of these genes, psbA and psbD remained in the chloroplastic genome, while psbO was transferred to the nucleus. The genomes of selected cyanobacteria, chloroplasts and cyanophages carrying psbA and psbD, respectively, were analysed. The highest density of genes and coding sequences (CDSs) was estimated for the genomes of cyanophages, cyanobacteria and chloroplasts. The synonymous mutation rate (rS) of psbA and psbD in chloroplasts remained almost unchanged and is lower than that of psbO. The results indicate that the decreasing genome size in chloroplasts is more similar to the genome reduction observed in contemporary endosymbiotic organisms than in streamlined genomes of free-living cyanobacteria. The rS of atpA, which encodes the α-subunit of ATP synthase in chloroplasts, suggests that psbA and psbD, and to a lesser extent psbO, are ancient and conservative and arose early in the evolution of oxygenic photosynthesis. The role of cyanophages in the evolution of oxyphototrophs and chloroplastic genomes is discussed.

Identification, expression and molecular polymorphism of T-cell receptors α and β from the glacial relict Hucho bleekeri

The T-cell receptor (TCR) is a specific molecule on the surface of all T cells that mediates cellular adaptive immune responses to antigens. Hucho bleekeri is a critically endangered species and is regarded as a glacial relict that has the lowest-latitude distribution compared with any Eurasian salmonid. In the present study, two TCR genes, namely, TCR α and β, were identified and characterized in H. bleekeri. Both TCR α and TCR β have typical TCR structures, including the IgV domain, IgC domain, connecting peptide, transmembrane and cytoplasmic domains. The two TCR genes were constitutionally expressed in various tissues, with the highest expression found in the spleen for TCR α and in the trunk kidney for TCR β. Challenge of H. bleekeri with LPS or poly(I:C) resulted in significant upregulation of both TCR α and β expression in headkidney and spleen primary cells, indicating their potential roles in the immune response. Molecular polymorphism analysis of the whole ORF regions of TCR α and β in different individuals revealed high diversity of IgV domains of these two genes, especially in complementarity-determining region (CDR) 3. The ratio of nonsynonymous substitution occurred at a significantly higher frequency than synonymous substitution in the CDR of TCR α and β, demonstrating the existence of positive selection. The results obtained in the present study enhance our understanding of TCR roles in regulating immune mechanisms and provide new information for the study of TCR lineage diversity in fish.