Restoring FAS Expression via Lipid-Encapsulated FAS DNA Nanoparticle Delivery Is Sufficient to Suppress Colon Tumor Growth In Vivo

A hallmark of human colorectal cancer is lost expression of FAS, the death receptor for FASL of cytotoxic T lymphocytes (CTLs). However, it is unknown whether restoring FAS expression alone is sufficient to suppress csolorectal-cancer development. The FAS promoter is hypermethylated and inversely correlated with FAS mRNA level in human colorectal carcinomas. Analysis of single-cell RNA-Seq datasets revealed that FAS is highly expressed in epithelial cells and immune cells but down-regulated in colon-tumor cells in human colorectal-cancer patients. Codon usage-optimized mouse and human FAS cDNA was designed, synthesized, and encapsulated into cationic lipid to formulate nanoparticle DOTAP-Chol-mFAS and DOTAP-Chol-hFAS, respectively. Overexpression of codon usage-optimized FAS in metastatic mouse colon-tumor cells enabled FASL-induced elimination of FAS+ tumor cells in vitro, suppressed colon tumor growth, and increased the survival of tumor-bearing mice in vivo. Overexpression of codon-optimized FAS-induced FAS receptor auto-oligomerization and tumor cell auto-apoptosis in metastatic human colon-tumor cells. DOTAP-Chol-hFAS therapy is also sufficient to suppress metastatic human colon tumor xenograft growth in athymic mice. DOTAP-Chol-mFAS therapy exhibited no significant liver toxicity. Our data determined that tumor-selective delivery of FAS DNA nanoparticles is sufficient for suppression of human colon tumor growth in vivo.

The non-neutral, multi-level, phenotypic impact of synonymous mutations revealed through heterologous gene expression in human cells.

Redundancy in the genetic code allows for differences in transcription and/or translation efficiency between mRNA molecules carrying synonymous polymorphisms, with potential phenotypic impact at the molecular and at the organismal level. A combination of neutral and selective processes determines the global genome codon usage preferences, as well as local differences between genes within a genome and between positions along a single gene. The relative contribution of evolutionary forces at shaping codon usage bias in eukaryotes is a matter of debate, especially in mammals. The main riddle remains understanding the sharp contrast between the strong molecular impact of gene expression differences arising from codon usage preferences and the thin evidence for codon usage selection at the organismal level. Here we report a multiscale analysis of the consequences of alternative codon usage on heterologous gene expression in human cells. We generated synonymous versions of the shble antibiotic resistance gene, fused to a fluorescent reporter, and expressed independently them in human HEK293 cells. We analysed: i) mRNA-to-DNA and protein-to-mRNA ratios for each shble version; ii) cellular fluorescence, using flow cytometry, as a proxy for single cell-level construct expression; and iii) real-time cell proliferation in absence or presence of antibiotic, as a proxy for the cellular fitness. Our results show that differences in codon usage preferences in our focal gene strongly impacted the molecular and the cellular phenotype: i) they elicited large differences in mRNA and in protein levels, as well in mRNA-to-protein ratio; ii) they introduced splicing events not predicted by current algorithms; iii) they lead to reproducible phenotypic heterogeneity as different multimodal distributions of cellular fluorescence EGFP; iv) they resulted in a trade-off between burden of heterologous expression and antibiotic resistance. While certain codon usage-related variables monotonically correlated with protein expression, other variables (e.g. CpG content or mRNA folding energy) displayed a bell-like behaviour. We interpret that codon usage preferences strongly shape the molecular and cellular phenotype in human cells through a direct impact on gene expression.

Comparative studies of codon usage profile of Anisakis simplex (Nematoda) and Carassius gibelio (Prussian carp)

Aim: The aim of the present study was to understand the molecular relationship between nematode (parasite) and fish (host) through codon usage bias (CUB) analysis. Methodology: The Codon usage bias analysis has been performed in fish Carassius gibelio (Prussian carp) and nematode fish parasite Anisakis simplex. The complete coding sequences (CDS) of C. gibelio (Prussian carp) and A. simplex (Nematode) were retrieved from National Center for Biotechnology Information and followed to that we have performed bioinformatics analysis to understand the codon usage pattern between host and parasite. Results: Different CUB indices like Relative synonymous codon usage (RSCU), Effective number of codons (ENC), Codon adaptation index (CAI) and Codon bias index (CBI) revealed a similar pattern in the codon usage in C. gibelio and A. simplex. In addition, inclusive analysis using different plots (ENC, parity, neutrality) had shown the influence of both the evolutionary forces i.e mutational and translational selection on codon usage pattern. This describes the role of evolutionary forces in determining the conserved genome to establish species-specific function-level differences for efficient survival. Interpretation: The present study elucidated the association between Carassiusgibelio (host) and Anisakis simplex (parasite) based on the similar pattern of codon usage bias between both the species.

Distinct responses to rare codons in select Drosophila tissues

Codon usage bias has long been appreciated to influence protein production. Yet, relatively few studies have analyzed the impacts of codon usage on tissue-specific mRNA and protein expression. Here, we use codon-modified reporters to perform an organism-wide screen in Drosophila melanogaster for distinct tissue responses to codon usage bias. These reporters reveal a cliff-like decline of protein expression near the limit of rare codon usage in endogenously expressed Drosophila genes. Near the edge of this limit, however, we find the testis and brain are uniquely capable of expressing rare codon-enriched reporters. We define a new metric of tissue-specific codon usage, the tissue-apparent Codon Adaptation Index, to reveal a conserved enrichment for rare codon usage in the endogenously expressed genes of both Drosophila and human testis. We further demonstrate a role for rare codons in restricting protein expression of an evolutionarily young gene, RpL10Aa, to the Drosophila testis. Rare codon-mediated restriction of this testis-specific protein is critical for female fertility. Our work highlights distinct responses to rarely used codons in select tissues, revealing a critical role for codon bias in tissue biology.

Comprehensive Analysis of Codon Usage Patterns in Chinese Porcine Circoviruses Based on Their Major Protein-Coding Sequences

Porcine circoviruses (PCVs) are distributed in swine herds worldwide and represent a threat to the health of domestic pigs and the profits of the swine industry. Currently, four PCV species, including PCV-1, PCV-2, PCV-3 and PCV-4, have been identified in China. Considering the ubiquitous characteristic of PCVs, the new emerged PCV-4 and the large scale of swine breeding in China, an overall analysis on codon usage bias for Chinese PCV sequences was performed by using the major proteins coding sequences (ORF1 and ORF2) to better understand the relationship of these viruses with their host. The data from genome nucleotide frequency composition and relative synonymous codon usage (RSCU) analysis revealed an overrepresentation of AT pair and the existence of a certain codon usage bias in all PCVs. However, the values of an effective number of codons (ENC) revealed that the bias was of low magnitude. Principal component analysis, ENC-plot, parity rule two analysis and correlation analysis suggested that natural selection and mutation pressure were both involved in the shaping of the codon usage patterns of PCVs. However, a neutrality plot revealed a stronger effect of natural selection than mutation pressure on codon usage patterns. Good host adaptation was also shown by the codon adaptation index analysis for all these viruses. Interestingly, obtained data suggest that PCV-4 might be more adapted to its host compared to other PCVs. The present study obtained insights into the codon usage pattern of PCVs based on ORF1 and ORF2, which further helps the understanding the molecular evolution of these swine viruses.