Viral vaccine target discovery requires understanding the diversity of both the virus and the human immune system. The readily available and rapidly growing pool of viral sequence data in the public ...domain enable the identification and characterization of immune targets relevant to adaptive immunity. A systematic bioinformatics approach is necessary to facilitate the analysis of such large datasets for selection of potential candidate vaccine targets.
This work describes a computational methodology to achieve this analysis, with data of dengue, West Nile, hepatitis A, HIV-1, and influenza A viruses as examples. Our methodology has been implemented as an analytical pipeline that brings significant advancement to the field of reverse vaccinology, enabling systematic screening of known sequence data in nature for identification of vaccine targets. This includes key steps (i) comprehensive and extensive collection of sequence data of viral proteomes (the virome), (ii) data cleaning, (iii) large-scale sequence alignments, (iv) peptide entropy analysis, (v) intra- and inter-species variation analysis of conserved sequences, including human homology analysis, and (vi) functional and immunological relevance analysis.
These steps are combined into the pipeline ensuring that a more refined process, as compared to a simple evolutionary conservation analysis, will facilitate a better selection of vaccine targets and their prioritization for subsequent experimental validation.
Most urinary tract infections (UTIs) are caused by uropathogenic
(UPEC), which depends on an extracellular organelle (type 1 pili) for adherence to bladder cells during infection. Type 1 pilus ...expression is partially regulated by inversion of a piece of DNA referred to as
, which contains the promoter for the
operon encoding type 1 pili.
inversion is regulated by up to five recombinases collectively known as Fim recombinases. These Fim recombinases are currently known to regulate two other switches: the
and
switches. A long-standing question has been whether the Fim recombinases regulate the inversion of other switches, perhaps to coordinate expression for adhesion or virulence. We answered this question using whole-genome sequencing with a newly developed algorithm (structural variation detection using relative entropy SVRE) for calling structural variations using paired-end short-read sequencing. SVRE identified all of the previously known switches, refining the specificity of which recombinases act at which switches. Strikingly, we found no new inversions that were mediated by the Fim recombinases. We conclude that the Fim recombinases are each highly specific for a small number of switches. We hypothesize that the unlinked Fim recombinases have been recruited to regulate
, and
only, as a secondary locus; this further implies that regulation of type 1 pilus expression (and its role in gastrointestinal and/or genitourinary colonization) is important enough, on its own, to influence the evolution and maintenance of multiple additional genes within the accessory genome of
UTI is a common ailment that affects more than half of all women during their lifetime. The leading cause of UTIs is UPEC, which relies on type 1 pili to colonize and persist within the bladder during infection. The regulation of type 1 pili is remarkable for an epigenetic mechanism in which a section of DNA containing a promoter is inverted. The inversion mechanism relies on what are thought to be dedicated recombinase genes; however, the full repertoire for these recombinases is not known. We show here that there are no additional targets beyond those already identified for the recombinases in the entire genome of two UPEC strains, arguing that type 1 pilus expression itself is the driving evolutionary force for the presence of these recombinase genes. This further suggests that targeting the type 1 pilus is a rational alternative nonantibiotic strategy for the treatment of UTI.
Since the sequencing of the human reference genome, many human disease-related genes have been discovered. However, understanding the functions of all the genes in the genome remains a challenge. The ...biological activities of these genes are usually investigated in model organisms such as mice and zebrafish. Large-scale mutagenesis screens to generate disruptive mutations are useful for identifying and understanding the activities of genes. Here, we report a multifunctional mutagenesis system in zebrafish using the maize Ds transposon. Integration of the Ds transposable element containing an mCherry reporter for protein trap events and an EGFP reporter for enhancer trap events produced a collection of transgenic lines marking distinct cell and tissue types, and mutagenized genes in the zebrafish genome by trapping and prematurely terminating endogenous protein coding sequences. We obtained 642 zebrafish lines with dynamic reporter gene expression. The characterized fish lines with specific expression patterns will be made available through the European Zebrafish Resource Center (EZRC), and a database of reporter expression is available online (http://fishtrap.warwick.ac.uk/). Our approach complements other efforts using zebrafish to facilitate functional genomic studies in this model of human development and disease.
Repetitive DNA sequences cause genomic instability and are important genetic markers. Identification of repeats is a critical step in genome annotation and analysis. On the other hand, repeats also ...pose a technical challenge for genome assembly and alignment programs using NGS data. RFGR is a comprehensive tool that can find exact repetitive sequences in complete genomes and assembled genomes, as well as NGS reads of prokaryotes. For complete genomes, RFGR uses a suffix trees to find seed repeats of repetitive sequences of fixed length with indels. For assembled genomes, RFGR uses a modified Bowtie aligner to find seed repeats of exact repetitive sequences in the contigs/ scaffolds, which are then extended to maximal repeats. The repeats are classified and for repeats near a gene, RFGR reports the gene as well. For the control dataset of E. coli UTI89 and E. coli K12, RFGR reports 35,141 and 49,352 repeats, respectively. For NGS reads, RFGR uses the frequency of the repetitive k-mers to determine FASTQ reads containing repetitive sequences and removes them from the dataset. An E. coli K12 NGS dataset pre-processed using RFGR, on comparison with the original dataset, gives an improved assembly. The N50 value improves by 22.86% with a decrease in size of the assembly graph by nearly 50%. Thus, with RFGR, we achieve a better assembly with reduced computation. RFGR can be improved in terms of the length of the minimum repeat found, extending to find approximate repeats and to be applicable to Eukaryotes as well.
Human respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infections in children ,2 years of age. Little is known about RSV intra-host genetic diversity over the course of ...infection or about the immune pressures that drive RSV molecular evolution. We performed whole-genome deep-sequencing on 53 RSV-positive samples (37 RSV subgroup A and 16 RSV subgroup B) collected from the upper airways of hospitalized children in southern Vietnam over two consecutive seasons. RSV A NA1 and RSV B BA9 were the predominant genotypes found in our samples, consistent with other reports on global RSV circulation during the same period. For both RSV A and B, the M gene was the most conserved, confirming its potential as a target for novel therapeutics. The G gene was the most variable and was the only gene under detectable positive selection. Further, positively selected sites inG were found in close proximity to and in some cases overlapped with predicted glycosylation motifs, suggesting that selection on amino acid glycosylation may drive viral genetic diversity. We further identified hotspots and coldspots of intra-host genetic diversity in the RSV genome, some of which may highlight previously unknown regions of functional importance.
The identification of three scorpionfish species, the black scorpionfish (
Scorpaena porcus
Linnaeus, 1758), the large-scaled scorpionfish (
S. scrofa
Linnaeus, 1758) and the small red scorpionfish (
...S. notata
Rafinesque, 1810) is possible in adults by morphometry, but often problematic in juveniles due to their similar phenotypes. To develop a molecular species identification tool, first, we have analyzed the genetic similarity of the three species by a PCR-based ‘blind method’ that amplified bands from various locations of the genome. We found high levels of nucleotide similarity between
S. porcus
and
S. scrofa,
whereas
S. notata
showed a higher level of divergence from the other two species. Then, we have searched these patterns for differences between the genomes of Adriatic specimen of these three species and identified several species-specific products in two of them. For the third one a species-specific primer pair amplifying from the 16S ribosomal DNA was designed. One marker for each species was cloned, sequenced and converted into Sequence Characterized Amplified Region (SCAR) markers amplified by specific primer pairs. The SCAR markers amplified robust bands of limited variability from the target species, while no or only occasional weak products were obtained from the other two, proving that they can be used for molecular identification of these three species. These markers can help the conservation and future analysis of these three species as well as their possible selection programs for aquaculture purposes.
Room‐temperature phosphorescence of metal and heavy atom‐free organic molecules has emerged as an area of great potential in recent years. A rational design played a critical role in controlling the ...molecular ordering to impart efficient intersystem crossing and stabilize the triplet state to achieve room‐temperature ultralong phosphorescence. However, in most cases, the strategies to strengthen phosphorescence efficiency have resulted in a reduced lifetime, and the available nearly degenerate singlet‐triplet energy levels impart a natural competition between delayed fluorescence and phosphorescence, with the former one having the advantage. Herein, an organic helical assembly supports the exhibition of an ultralong phosphorescence lifetime. In contrary to other molecules, 3,6‐phenylmethanone functionalized 9‐hexylcarbazole exhibits a remarkable improvement in phosphorescence lifetime (>4.1 s) and quantum yield (11 %) owing to an efficient molecular packing in the crystal state. A right‐handed helical molecular array act as a trap and exhibits triplet exciton migration to support the exceptionally longer phosphorescence lifetime.
A right‐handed helical molecular array of phenylmethanone functionalized carbazole acts as a trap and exhibits triplet exciton migration to support an exceptionally long phosphorescence lifetime.
Organic room temperature phosphorescent materials are known for their synthetic feasibility, tunable phosphorescence wavelength and lifetime, etc. Hence new design strategies have been applied on ...various fluorophores to improve phosphorescence features. Among those, triphenylamines are phosphorescence active due to the presence of nitrogen atom, propeller molecular structure, and intermolecular interactions in the crystal state. Here we have studied the room temperature phosphorescence of a series of triphenylamines with various functional groups. Detailed studies have shown that the phosphorescence can be fine-tuned by functional group modification. A long phosphorescence lifetime around 100 ms at room temperature in air can be achieved by the interplay of intermolecular interactions, singlet-triplet energy gap and extent of intersystem crossing using functional group variation. Interestingly, an exciplex assisted ultralong phosphorescence lifetime (more than 20 times) is observed for a combination of triphenylamine and naphthalenemonoimide in air.
•Phosphorescence features of TPA are fine-tuned by functional group modification.•Even though reported TPAs employ polymer support or vacuum, our TPAs exhibit long phosphorescence lifetime in air at room temperature.•A phosphorescence lifetime around 100 ms at room temperature in air can be achieved via functional group variation.•TPA with least lifetime (8 ms) forms an exciplex assisted ultralong phosphorescence along with tunable emission color and improved quantum yield.
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