Low codon bias and high rates of synonymous substitution in Drosophila hydei and D. melanogaster histone genes

We have evaluated codon usage bias in Drosophila histone genes and have obtained the nucleotide sequence of a 5,161-bp D. hydei histone gene repeat unit. This repeat contains genes for all five histone proteins (H1, H2a, H2b, H3, and H4) and differs from the previously reported one by a second EcoRI site. These D. hydei repeats have been aligned to each other and to the 5.0-kb (i.e., long) and 4.8-kb (i.e., short) histone repeat types from D. melanogaster. In each species, base composition at synonymous sites is similar to the average genomic composition and approaches that in the small intergenic spacers of the histone gene repeats. Accumulation of synonymous changes at synonymous sites after the species diverged is quite high. Both of these features are consistent with the relatively low codon usage bias observed in these genes when compared with other Drosophila genes. Thus, the generalization that abundantly expressed genes in Drosophila have high codon bias and low rates of silent substitution does not hold for the histone genes.      

The uptake and stability of simian-virus-40 DNA after calcium phosphate transfection of CV-1 cells.

The uptake and fate of purified SV40 (Simian virus 40) DNA, transfected into permissive CV-1 cells by calcium phosphate precipitates, was examined. By using a viral plaque assay, optimum conditions for transfection were established and transfection efficiencies of up to 10(6) plaque-forming units/micrograms of SV40 DNA were obtained. After a 2h exposure to 3H-labelled SV40 DNA-calcium phosphate co-precipitates under basal conditions, up to 7% of the input DNA became cell-associated, with approx. 4% reaching the nuclear fraction. This value was never exceeded, even under conditions known to enhance significantly the ultimate transfection efficiency, such as increased exposure time, addition of carrier DNA or treatment with DMSO (dimethyl sulphoxide) or glycerol. Substantial degradation of this SV40 DNA occurred within a further 4h, apparently in both nucleus and cytoplasm. Degradation of form-II and form-III SV40 DNA, which have lower transfection efficiencies than form-I DNA, was no more rapid than degradation of form-I DNA. The results indicate that less than 0.5% of the transfected DNA which reached the nucleus is protected from nuclease attack. The mechanism of action of agents such as glycerol, DMSO or carrier DNA remains obscure, but they may be involved in conferring greater stability to the intracellular SV40 DNA rather than merely affecting its rate of entry into the cell.     

Germination and production ofMacrophomina phaseolina microsclerotia as affected by oxygen and carbon dioxide concentration

Summary Germination of microsclerotia ofMacrophomina phaseolina was observed at O₂ concentrations of 16% or higher in autoclaved soil. Germination was delayed but otherwise unaffected as O₂ decreased from 21 to 16% and was in all cases complete in 32 hours. Laboratory-produced microsclerotia consistently germinated more rapidly and seemed more independent of O₂ concentrations within the range that permitted germination than naturallyproduced microsclerotia.Population changes in soil as measured by microsclerotial counts were inversely correlated with depth of interment and reduced O₂ concentration. Our inability to detect significantly growth responses ofM. phaseolina in non autoclaved soil was apparently related to limited O₂ although other possibilities are discussed.Contribution of the Missouri Agricultural Experiment Station Scientific Journal Series No. 9124.     

Analysis and discrimination of necrosis and apoptosis (programmed cell death) by multiparameter flow cytometry.

Necrosis and apoptosis are two distinct modes of cell death which differ in morphology, mechanism and incidence. Membrane disruptants, respiratory poisons and hypoxia cause ATP depletion, metabolic collapse, cell swelling and rupture leading to inflammation. These are typical features of necrosis. Apoptosis plays a crucial role in embryogenesis and development and is also prevalent in tumours. It is characterised by cell shrinkage, chromatin condensation and systematic DNA cleavage. Apoptotic cells are rapidly engulfed by phagocytes, thus preventing inflammatory reaction to degradative cell contents. In vivo, apoptosis is almost impossible to quantify due to problems of heterogeneity and the short half-life of an apoptotic cell. In vitro, mechanistic studies are further complicated by a late phase of apoptosis where the cell membrane becomes permeable to vital dyes and which occurs in the absence of phagocytes. Here we describe a novel and rapid multiparameter flow cytometric assay which discriminates and quantifies viable, apoptotic and necrotic cells via measurement of forward and side light scatter (proportional to cell diameter and internal granularity, respectively) and the DNA-binding fluorophores Hoechst 33342 and propidium. It is anticipated that mechanistic studies of apoptosis in a variety of cell types will greatly benefit from this mode of analysis.     

Prediction of gall-stone pancreatitis by computer.

The clinical features at presentation of 53 patients admitted with primary acute pancreatitis due to gall stones were compared with those of 31 patients in whom the disease was due to other causes. Between these two groups 10 significant differences existed. By listing the frequency of symptoms and signs for each group a computer data base was prepared and incorporated into a program used in the differential diagnosis of acute abdominal pain. A program written to predict the presence of gall stones in patients with acute pancreatitis was accurate in 92% of the patients studied. A predictive index devised from the presence of three of the significantly differing clinical features correctly identified 82% of patients with gall-stone pancreatitis. Predicting the presence of gall stones on admission by analysing the presenting symptoms and signs with a computer had an accuracy comparable to that of ultrasonography or radiology and may be of value in the management of patients with acute pancreatitis.     

Superiority of Trans-Oral over Trans-Nasal Sampling in Detecting Streptococcus pneumoniae Colonization in Adults

The human nasopharynx is the main reservoir for Streptococcus pneumoniae. We applied conventional and molecular methods to determine the prevalence of S. pneumoniae nasopharyngeal colonization in adults. Paired trans-orally and trans-nasally obtained nasopharyngeal samples from 268 parents of 24-month-old children were assessed for pneumococcal presence. Parents were classified as colonized when live pneumococci were recovered from either sample cultured on medium selective for S. pneumoniae. Of the 52 (19%) colonized parents 49 (18%) were culture-positive in trans-nasal and 10 (4%) in trans-oral samples. Bacterial growth was harvested from these cultures, DNA isolated and tested by quantitative-PCR (qPCR) targeting lytA and piaA genes specific for S. pneumoniae. A sample was considered positive if signals for both genes were detected. Altogether 105 (39%) individuals were classified as positive for pneumococcus by qPCR including 50 (19%) in trans-nasal and 94 (35%) in trans-oral settings. Although significantly more trans-nasal compared to trans-oral samples were culture-positive for S. pneumoniae at the primary diagnostic step (p<0.001) the opposite was observed in qPCR results (p<0.001). To confirm the presence of live pneumococcus in samples positive by qPCR but negative at the initial diagnostic step, we serially-diluted cell harvests, re-cultured and carefully examined for S. pneumoniae presence. Live pneumococci were recovered from an additional 43 parents including 42 positive in trans-oral and 4 in trans-nasal samples increasing the number of individuals culture- and qPCR-positive to 93 (35%) and positive by either of two methods to 107 (40%). There were significantly more trans-oral than trans-nasal samples positive for pneumococcus by both culture and qPCR (n = 71; 27%; vs. n = 50; 19%; p<0.05). Our data suggest that pneumococcal colonization is more common in adults than previously estimated and point towards the superiority of a trans-oral over a trans-nasal approach when testing adults for colonization with S. pneumoniae.     

A Modified RNA-Seq Approach for Whole Genome Sequencing of RNA Viruses from Faecal and Blood Samples

To date, very large scale sequencing of many clinically important RNA viruses has been complicated by their high population molecular variation, which creates challenges for polymerase chain reaction and sequencing primer design. Many RNA viruses are also difficult or currently not possible to culture, severely limiting the amount and purity of available starting material. Here, we describe a simple, novel, high-throughput approach to Norovirus and Hepatitis C virus whole genome sequence determination based on RNA shotgun sequencing (also known as RNA-Seq). We demonstrate the effectiveness of this method by sequencing three Norovirus samples from faeces and two Hepatitis C virus samples from blood, on an Illumina MiSeq benchtop sequencer. More than 97% of reference genomes were recovered. Compared with Sanger sequencing, our method had no nucleotide differences in 14,019 nucleotides (nt) for Noroviruses (from a total of 2 Norovirus genomes obtained with Sanger sequencing), and 8 variants in 9,542 nt for Hepatitis C virus (1 variant per 1,193 nt). The three Norovirus samples had 2, 3, and 2 distinct positions called as heterozygous, while the two Hepatitis C virus samples had 117 and 131 positions called as heterozygous. To confirm that our sample and library preparation could be scaled to true high-throughput, we prepared and sequenced an additional 77 Norovirus samples in a single batch on an Illumina HiSeq 2000 sequencer, recovering >90% of the reference genome in all but one sample. No discrepancies were observed across 118,757 nt compared between Sanger and our custom RNA-Seq method in 16 samples. By generating viral genomic sequences that are not biased by primer-specific amplification or enrichment, this method offers the prospect of large-scale, affordable studies of RNA viruses which could be adapted to routine diagnostic laboratory workflows in the near future, with the potential to directly characterize within-host viral diversity.     

Whole genome sequencing and de novo assembly identifies Sydney-like variant noroviruses and recombinants during the winter 2012/2013 outbreak in England

Background

Influenza B viruses can cause morbidity and mortality in humans but due to the lack of an animal reservoir are not associated with pandemics. Because of this, there is relatively limited genetic sequences available for influenza B viruses, especially from developing countries. Complete genome analysis of one influenza B virus and several gene segments of other influenza B viruses isolated from Uganda from May 2009 through December 2010 was therefore undertaken in this study.

Methods

Samples were collected from patients showing influenza like illness and screened for influenza A and B by PCR. Influenza B viruses were isolated on Madin-Darby Canine Kidney cells and selected isolates were subsequently sequenced and analyzed phylogenetically.

Findings

Of the 2,089 samples collected during the period, 292 were positive by PCR for influenza A or B; 12.3% of the PCR positives were influenza B. Thirty influenza B viruses were recovered and of these 25 that grew well consistently on subculture were subjected to further analysis. All the isolates belonged to the B/Victoria-lineage as identified by hemagglutination inhibition assay and genetic analysis except one isolate that grouped with the B-Yamagata-lineage. The Ugandan B/Victoria-lineage isolates grouped in clade 1 which was defined by the N75K, N165K and S172P substitutions in hemagglutinin (HA) protein clustered together with the B/Brisbane/60/2008 vaccine strain. The Yamagata-like Ugandan strain, B/Uganda/MUWRP-053/2009, clustered with clade 3 Yamagata viruses such as B/Bangladesh/3333/2007 which is characterized by S150I and N166Y substitutions in HA.

Conclusion

In general there was limited variation among the Ugandan isolates but they were interestingly closer to viruses from West and North Africa than from neighboring Kenya. Our isolates closely matched the World Health Organization recommended vaccines for the seasons.