Calibration of replacement international standards of diphtheria and tetanus toxoids for use in flocculation test

The 1st International Reference Reagents (IRR) of Diphtheria and Tetanus Toxoids for Flocculation Test (DIFT and TEFT) were established by the WHO in 1988. These reagents are essential for the standardization of assays used to calculate Lf units of toxoids. Candidate replacement materials were provided by several European vaccine manufacturers and were formulated and freeze-dried at NIBSC. This paper provides a summary of the results of an international collaborative study including 18 laboratories from 16 countries, which examined the candidate replacement materials in a variety of methods. Materials 02/176 and 04/150 were proposed and adopted by the Expert Committee on Biological Standardization of WHO in October 2007 as 2nd WHO International Standards of Diphtheria and Tetanus Toxoid for use in Flocculation Test. The replacement standards were assigned the value of 1100 and 690Lf/ampoule, respectively, based on results of flocculation tests carried out using provided reagents. Material coded 02/176 fully complied with the WHO specifications for stability, residual moisture content, precision of fill and sterility. Stability of material coded 04/150 was slightly lower than expected but predictions were based only on 2-year data and were to be further monitored, post-adoption.     

Predicting the animal hosts of coronaviruses from compositional biases of spike protein and whole genome sequences through machine learning

The COVID-19 pandemic has demonstrated the serious potential for novel zoonotic coronaviruses to emerge and cause major outbreaks. The immediate animal origin of the causative virus, SARS-CoV-2, remains unknown, a notoriously challenging task for emerging disease investigations. Coevolution with hosts leads to specific evolutionary signatures within viral genomes that can inform likely animal origins. We obtained a set of 650 spike protein and 511 whole genome nucleotide sequences from 222 and 185 viruses belonging to the family Coronaviridae, respectively. We then trained random forest models independently on genome composition biases of spike protein and whole genome sequences, including dinucleotide and codon usage biases in order to predict animal host (of nine possible categories, including human). In hold-one-out cross-validation, predictive accuracy on unseen coronaviruses consistently reached ~73%, indicating evolutionary signal in spike proteins to be just as informative as whole genome sequences. However, different composition biases were informative in each case. Applying optimised random forest models to classify human sequences of MERS-CoV and SARS-CoV revealed evolutionary signatures consistent with their recognised intermediate hosts (camelids, carnivores), while human sequences of SARS-CoV-2 were predicted as having bat hosts (suborder Yinpterochiroptera), supporting bats as the suspected origins of the current pandemic. In addition to phylogeny, variation in genome composition can act as an informative approach to predict emerging virus traits as soon as sequences are available. More widely, this work demonstrates the potential in combining genetic resources with machine learning algorithms to address long-standing challenges in emerging infectious diseases.     

Butyrate-induced apoptosis in HCT116 colorectal cancer cells includes induction of a cell stress response

Short chain fatty acids (SCFA), principally butyrate, propionate, and acetate, are produced in the gut through the fermentation of dietary fiber by the colonic microbiotica. Butyrate in particular is the preferred energy source for the cells in the colonic mucosa and has been demonstrated to induce apoptosis in colorectal cancer cell lines. We have used proteomics, specifically 2D-DIGE and mass spectrometry, to identify proteins involved in butyrate-induced apoptosis in HCT116 cells and also to identify proteins involved in the development of butyrate insensitivity in its derivative, the HCT116-BR cells. The HCT116-BR cell line was characterized as being less responsive to the apoptotic effects of butyrate in comparison to its parent cell line. Our analysis has revealed that butyrate likely induces a cellular stress response in HCT116 cells characterized by p38 MAPK activation and an endoplasmic reticulum (ER) stress response, resulting in caspase 3/7 activation and cell death. Adaptive cellular responses to stress-induced apoptosis in HCT116-BR cells may be responsible for the development of resistance to apoptosis in this cell line. We also report for the first time additional cellular processes altered by butyrate, such as heme biosynthesis and dysregulated expression of nuclear lamina proteins, which may be involved in the apoptotic response observed in these cell lines.     

Characterization of the stop codon readthrough signal of Colorado tick fever virus segment 9 RNA

Termination codon readthrough is utilized as a mechanism of expression of a growing number of viral and cellular proteins, but in many cases the mRNA signals that promote readthrough are poorly characterized. Here, we investigated the readthrough signal of Colorado tick fever virus (CTFV) segment 9 RNA (Seg-9). CTFV is the type-species of the genus Coltivirus within the family Reoviridae and is a tick-borne, double-stranded, segmented RNA virus. Seg-9 encodes a 36-kDa protein VP9, and by readthrough of a UGA stop codon, a 65-kDa product, VP9'. Using a reporter system, we defined the minimal sequence requirements for readthrough and confirmed activity in both mammalian and insect cell-free translation systems, and in transfected mammalian cells. Mutational analysis revealed that readthrough was UGA specific, and that the local sequence context around the UGA influenced readthrough efficiency. Readthrough was also dependent upon a stable RNA stem-loop structure beginning eight bases downstream from the UGA codon. Mutational analysis of this stem-loop revealed a requirement for the stem region but not for substructures identified within the loop. Unexpectedly, we were unable to detect a ribosomal pause during translation of the CTFV signal, suggesting that the mechanism of readthrough, at least at this site, is unlikely to be dependent upon RNA secondary-structure induced ribosomal pausing at the recoded stop codon.