Application of TraDIS to define the core essential genome of Campylobacter jejuni and Campylobacter coli.

Campylobacter species are the major cause of bacterial gastroenteritis. As there is no effective vaccine, combined with the rapid increase in antimicrobial resistant strains, there is a need to identify new targets for intervention. Essential genes are those that are necessary for growth and/or survival, making these attractive targets. In this study, comprehensive transposon mutant libraries were created in six C. jejuni strains, four C. coli strains and one C. lari and C. hyointestinalis strain, allowing for those genes that cannot tolerate a transposon insertion being called as essential. Comparison of essential gene lists using core genome analysis can highlight those genes which are common across multiple strains and/or species. Comparison of C. jejuni and C. coli, the two species that cause the most disease, identified 316 essential genes. Genes of interest highlighted members of the purine pathway being essential for C. jejuni whilst also finding that a functional potassium uptake system is essential. Protein-protein interaction networks using these essential gene lists also highlighted proteins in the purine pathway being major 'hub' proteins which have a large number of interactors across the network. When adding in two more species (C. lari and C. hyointestinalis) the essential gene list reduces to 261. Within these 261 essential genes, there are many genes that have been found to be essential in other bacteria. These include htrB and PEB4, which have previously been found as core virulence genes across Campylobacter species in other studies. There were 21 genes which have no known function with eight of these being associated with the membrane. These surface-associated essential genes may provide attractive targets. The essential gene lists presented will help to prioritise targets for the development of novel therapeutic and preventative interventions.

Genomic surveillance of SARS-CoV-2 in COVID-19 vaccinated healthcare workers in Lebanon.

BACKGROUND: The emergence of SARS-CoV-2 variants including the Delta and Omicron along with waning of vaccine-induced immunity over time contributed to increased rates of breakthrough infection specifically among healthcare workers (HCWs). SARS-CoV-2 genomic surveillance is an important tool for timely detection and characterization of circulating variants as well as monitoring the emergence of new strains. Our study is the first national SARS-CoV-2 genomic surveillance among HCWs in Lebanon. METHODS: We collected 250 nasopharyngeal swabs from HCWs across Lebanon between December 2021 and January 2022. Data on the date of positive PCR, vaccination status, specific occupation, and hospitalization status of participants were collected. Extracted viral RNA from nasopharyngeal swabs was converted to cDNA, library prepped using the coronaHIT method, followed by whole genome sequencing on the Illumina NextSeq 500 platform. RESULTS: A total of 133 (57.1%) samples belonging to the Omicron (BA.1.1) sub-lineage were identified, as well as 44 (18.9%) samples belonging to the BA.1 sub-lineage, 28 (12%) belonging to the BA.2 sub-lineage, and only 15 (6.6%) samples belonging to the Delta variant sub-lineage B.1.617.2. These results show that Lebanon followed the global trend in terms of circulating SARS-CoV-2 variants with Delta rapidly replaced by the Omicron variant. CONCLUSION: This study underscores the importance of continuous genomic surveillance programs in Lebanon for the timely detection and characterization of circulating variants. The latter is critical to guide public health policy making and to timely implement public health interventions.

Genome-Wide Analysis of Innate Susceptibility Mechanisms of Escherichia coli to Colistin.

Colistin is an antibiotic that has seen increasing clinical use for the treatment of human infections caused by Gram-negative pathogens, particularly due to the emergence of multidrug-resistant pathogens. Colistin resistance is also a growing problem and typically results from alterations to lipopolysaccharides mediated by phosphoethanolamine (pETn) transferase enzymes which can be encoded on the chromosome, or plasmids. In this study, we used 'TraDIS-Xpress' (Transposon Directed Insertion site Sequencing with expression), where a high-density transposon mutant library including outward facing promoters in Escherichia coli BW25113 identified genes involved in colistin susceptibility. We examined the genome-wide response of E. coli following exposure to a range of concentrations of colistin. Our TraDIS-Xpress screen confirmed the importance of overexpression of the two-component system basSR (which regulates pETn transferases) but also identified a wider range of genes important for survival in the presence of colistin, including genes encoding membrane associated proteins, DNA repair machinery, various transporters, RNA helicases, general stress response genes, fimbriae and phosphonate metabolism. Validation experiments supported a role in colistin susceptibility for novel candidate genes tested. TraDIS-Xpress is a powerful tool that expands our understanding of the wider landscape of genes involved in response to colistin susceptibility mechanisms.

Prey localization in spider orb webs using modal vibration analysis.

Spider webs are finely tuned multifunctional structures, widely studied for their prey capture functionalities such as impact strength and stickiness. However, they are also sophisticated sensing tools that enable the spider to precisely determine the location of impact and capture the prey before it escapes. In this paper, we suggest a new mechanism for this detection process, based on potential modal analysis capabilities of the spider, using its legs as distinct distributed point sensors. To do this, we consider a numerical model of the web structure, including asymmetry in the design, prestress, and geometrical nonlinearity effects. We show how vibration signals deriving from impacts can be decomposed into web eigenmode components, through which the spider can efficiently trace the source location. Based on this numerical analysis, we discuss the role of the web structure, asymmetry, and prestress in the imaging mechanism, confirming the role of the latter in tuning the web response to achieve an efficient prey detection instrument. The results can be relevant for efficient distributed impact sensing applications.

Replacement of the Alpha variant of SARS-CoV-2 by the Delta variant in Lebanon between April and June 2021.

The COVID-19 pandemic continues to expand globally, with case numbers rising in many areas of the world, including the Eastern Mediterranean Region. Lebanon experienced its largest wave of COVID-19 infections from January to April 2021. Limited genomic surveillance was undertaken, with just 26 SARS-CoV-2 genomes available for this period, nine of which were from travellers from Lebanon detected by other countries. Additional genome sequencing is thus needed to allow surveillance of variants in circulation. In total, 905 SARS-CoV-2 genomes were sequenced using the ARTIC protocol. The genomes were derived from SARS-CoV-2-positive samples, selected retrospectively from the sentinel COVID-19 surveillance network, to capture diversity of location, sampling time, sex, nationality and age. Although 16 PANGO lineages were circulating in Lebanon in January 2021, by February there were just four, with the Alpha variant accounting for 97 % of samples. In the following 2 months, all samples contained the Alpha variant. However, this had changed dramatically by June and July 2021, when all samples belonged to the Delta variant. This study documents a ten-fold increase in the number of SARS-CoV-2 genomes available from Lebanon. The Alpha variant, first detected in the UK, rapidly swept through Lebanon, causing the country's largest wave to date, which peaked in January 2021. The Alpha variant was introduced to Lebanon multiple times despite travel restrictions, but the source of these introductions remains uncertain. The Delta variant was detected in Gambia in travellers from Lebanon in mid-May, suggesting community transmission in Lebanon several weeks before this variant was detected in the country. Prospective sequencing in June/July 2021 showed that the Delta variant had completely replaced the Alpha variant in under 6 weeks.

Long-read sequencing for identification of insertion sites in large transposon mutant libraries.

Transposon insertion site sequencing (TIS) is a powerful method for associating genotype to phenotype. However, all TIS methods described to date use short nucleotide sequence reads which cannot uniquely determine the locations of transposon insertions within repeating genomic sequences where the repeat units are longer than the sequence read length. To overcome this limitation, we have developed a TIS method using Oxford Nanopore sequencing technology that generates and uses long nucleotide sequence reads; we have called this method LoRTIS (Long-Read Transposon Insertion-site Sequencing). LoRTIS enabled the unique localisation of transposon insertion sites within long repetitive genetic elements of E. coli, such as the transposase genes of insertion sequences and copies of the ~ 5 kb ribosomal RNA operon. We demonstrate that LoRTIS is reproducible, gives comparable results to short-read TIS methods for essential genes, and better resolution around repeat elements. The Oxford Nanopore sequencing device that we used is cost-effective, small and easily portable. Thus, LoRTIS is an efficient means of uniquely identifying transposon insertion sites within long repetitive genetic elements and can be easily transported to, and used in, laboratories that lack access to expensive DNA sequencing facilities.

Random versus regular square lattice experimental comparison for a subwavelength resonant metasurface.

An experimental comparison is reported here between two equivalent resonant subwavelength metasurfaces made of long aluminum beams glued closely together on a thin aluminum plate. One metasurface has a random distribution of the resonator beams, and the other has a regular square lattice of pitch 1.5 cm. The random lattice shows the "resonant" behavior of a typical metasurface, with a wide full bandgap for the first A0 Lamb mode. Instead, the regular square lattice combines Fano resonance with Bragg scattering at the edges of the passband, thus creating anisotropy and a pseudo bandgap. Comparisons with numerical simulations are performed, with good agreement with the experimental data. The multimodal response of the beams is also responsible for double negativity in a narrow frequency band, and the event of a pseudo bandgap around this same flexural resonance. In addition, the scattering regimes for both the random and regular metasurfaces are characterized using coherent and incoherent signal analysis.

Negative index metamaterial through multi-wave interactions: numerical proof of the concept of low-frequency Lamb-wave multiplexing.

We study numerically the potential of a multimodal elastic metamaterial to filter and guide Lamb waves in a plate. Using a sub-wavelength array of elongated beams attached to the plate, and combining the coupling effects of the longitudinal and flexural motion of these resonators, we create narrow transmission bands at the flexural resonances of the beams inside the wide frequency bandgap induced by their longitudinal resonance. The diameter of the beams becomes the tuning parameter for selection of the flexural leakage frequency, without affecting the main bandgap. Finally, by combination of the monopolar and dipolar scattering effects associated with the coupled beam and plate system, we create a frequency-based multiplexer waveguide in a locally resonant metamaterial.

From local to global measurements of nonclassical nonlinear elastic effects in geomaterials.

In this letter, the equivalence between local and global measures of nonclassical nonlinear elasticity is established in a slender resonant bar. Nonlinear effects are first measured globally using nonlinear resonance ultrasound spectroscopy (NRUS), which monitors the relative shift of the resonance frequency as a function of the maximum dynamic strain in the sample. Subsequently, nonlinear effects are measured locally at various positions along the sample using dynamic acousto elasticity testing (DAET). After correcting analytically the DAET data for three-dimensional strain effects and integrating numerically these corrected data along the length of the sample, the NRUS global measures are retrieved almost exactly.

Reliable, verifiable and efficient monitoring of biodiversity via metabarcoding.

To manage and conserve biodiversity, one must know what is being lost, where, and why, as well as which remedies are likely to be most effective. Metabarcoding technology can characterise the species compositions of mass samples of eukaryotes or of environmental DNA. Here, we validate metabarcoding by testing it against three high-quality standard data sets that were collected in Malaysia (tropical), China (subtropical) and the United Kingdom (temperate) and that comprised 55,813 arthropod and bird specimens identified to species level with the expenditure of 2,505 person-hours of taxonomic expertise. The metabarcode and standard data sets exhibit statistically correlated alpha- and beta-diversities, and the two data sets produce similar policy conclusions for two conservation applications: restoration ecology and systematic conservation planning. Compared with standard biodiversity data sets, metabarcoded samples are taxonomically more comprehensive, many times quicker to produce, less reliant on taxonomic expertise and auditable by third parties, which is essential for dispute resolution.

Sulforaphane can protect lens cells against oxidative stress: implications for cataract prevention.

PURPOSE: Protecting the lens against oxidative stress is of great importance in delaying the onset of cataract. Isothiocyanates, such as sulforaphane (SFN), are proposed to provide cytoprotection against oxidative stress. We therefore tested the ability of SFN to perform this role in lens cells and establish its ability to delay the onset of cataract. METHODS: The human lens epithelial cell line FHL124 and whole porcine lens culture systems were used. The ApoTox-Glo Triplex Assay was used to assess FHL124 cell survival, cytotoxicity, and apoptosis. The MTS assay was used to assess cell populations. To determine levels of DNA strand breaks, the alkaline comet assay was performed and quantified. Lactate dehydrogenase levels in the medium were evaluated to reflect cell damage/death. To assess level of gene expression, an Illumina whole-genome HT-12 v4 beadchip was used. Protein expression was determined by Western blot and immunocytochemistry. RESULTS: Exposures of 30 µM H2O2 to FHL124 cells caused a reduction in cell viability and increased cytotoxicity/apoptosis; these effects were significantly inhibited by 24-hour pretreatment with 1 µM SFN. In addition, 1 µM SFN significantly reduced H2O2-induced DNA strand breaks. When applied to cultured porcine lenses, SFN protected against H2O2-induced opacification. Illumina whole-genome HT-12 v4 beadchip microarray data revealed eight genes upregulated following 24-hour exposure to 1- and 2-µM SFN, which included NQO1 and TXNRD1. This pattern was confirmed at the protein level. Nrf2 translocated to the nucleus in response to 0.5- to 2.0-µM SFN exposure CONCLUSIONS: The dietary component SFN demonstrates an ability to protect human lens cells against oxidative stress and thus could potentially delay the onset of cataract.

Activation of the innate immune response and interferon signalling in myotonic dystrophy type 1 and type 2 cataracts.

Myotonic dystrophy (DM) is caused by a triplet repeat expansion in the non-coding region of either the DMPK (DM1) or CNBP (DM2) gene. Transcription of the expanded region causes accumulation of double-stranded RNA (dsRNA) in DM cells. We sought to determine how expression of triplet repeat RNA causes the varied phenotype typical of DM. Global transcription was measured in DM and non-DM cataract samples using Illumina Bead Arrays. DM samples were compared with non-DM samples and lists of differentially expressed genes (P≤ 0.05) were prepared. Gene set enrichment analysis and the Interferome database were used to search for significant patterns of gene expression in DM cells. Expression of individual genes was measured using quantitative real-time polymerase chain reaction. DMPK and CNBP expression was confirmed in native lens cells showing that a toxic RNA gain of function mechanism could exist in lens. A high proportion, 83% in DM1 and 75% in DM2, of the significantly disregulated genes were shared by both forms of the disease, suggesting a common mechanism. The upregulated genes in DM1 and DM2 were highly enriched in both interferon-regulated genes (IRGs) and genes associated with the response to dsRNA and the innate immune response. The characteristic fingerprint of IRGs and the signalling pathways identified in lens cells support a role for dsRNA activation of the innate immune response in the pathology of DM. This new evidence forms the basis for a novel hypothesis to explain the complex mechanism of DM.

RDP3: a flexible and fast computer program for analyzing recombination.

UNLABELLED: RDP3 is a new version of the RDP program for characterizing recombination events in DNA-sequence alignments. Among other novelties, this version includes four new recombination analysis methods (3SEQ, VISRD, PHYLRO and LDHAT), new tests for recombination hot-spots, a range of matrix methods for visualizing over-all patterns of recombination within datasets and recombination-aware ancestral sequence reconstruction. Complementary to a high degree of analysis flow automation, RDP3 also has a highly interactive and detailed graphical user interface that enables more focused hands-on cross-checking of results with a wide variety of newly implemented phylogenetic tree construction and matrix-based recombination signal visualization methods. The new RDP3 can accommodate large datasets and is capable of analyzing alignments ranging in size from 1000 × 10 kilobase sequences to 20 × 2 megabase sequences within 48 h on a desktop PC. AVAILABILITY: RDP3 is available for free from its web site http://darwin.uvigo.es/rdp/rdp.html.

Inferring polyploid phylogenies from multiply-labeled gene trees.

BACKGROUND: Gene trees that arise in the context of reconstructing the evolutionary history of polyploid species are often multiply-labeled, that is, the same leaf label can occur several times in a single tree. This property considerably complicates the task of forming a consensus of a collection of such trees compared to usual phylogenetic trees. RESULTS: We present a method for computing a consensus tree of multiply-labeled trees. As with the well-known greedy consensus tree approach for phylogenetic trees, our method first breaks the given collection of gene trees into a set of clusters. It then aims to insert these clusters one at a time into a tree, starting with the clusters that are supported by most of the gene trees. As the problem to decide whether a cluster can be inserted into a multiply-labeled tree is computationally hard, we have developed a heuristic method for solving this problem. CONCLUSION: We illustrate the applicability of our method using two collections of trees for plants of the genus Silene, that involve several allopolyploids at different levels.

Identifying recombinants in human and primate immunodeficiency virus sequence alignments using quartet scanning.

BACKGROUND: Recombination has a profound impact on the evolution of viruses, but characterizing recombination patterns in molecular sequences remains a challenging endeavor. Despite its importance in molecular evolutionary studies, identifying the sequences that exhibit such patterns has received comparatively less attention in the recombination detection framework. Here, we extend a quartet-mapping based recombination detection method to enable identification of recombinant sequences without prior specifications of either query and reference sequences. Through simulations we evaluate different recombinant identification statistics and significance tests. We compare the quartet approach with triplet-based methods that employ additional heuristic tests to identify parental and recombinant sequences. RESULTS: Analysis of phylogenetic simulations reveal that identifying the descendents of relatively old recombination events is a challenging task for all methods available, and that quartet scanning performs relatively well compared to the triplet based methods. The use of quartet scanning is further demonstrated by analyzing both well-established and putative HIV-1 recombinant strains. In agreement with recent findings, we provide evidence that the presumed circulating recombinant CRF02_AG is a 'pure' lineage, whereas the presumed parental lineage subtype G has a recombinant origin. We also demonstrate HIV-1 intrasubtype recombination, confirm the hybrid origin of SIV in chimpanzees and further disentangle the recombinant history of SIV lineages in a primate immunodeficiency virus data set. CONCLUSION: Quartet scanning makes a valuable addition to triplet-based methods for identifying recombinant sequences without prior specifications of either query and reference sequences. The new method is available in the VisRD v.3.0 package http://www.cmp.uea.ac.uk/~vlm/visrd.

PADRE: a package for analyzing and displaying reticulate evolution.

UNLABELLED: Recent advances in gene sequencing for polyploid species, coupled with standard phylogenetic tree reconstruction, leads to gene trees in which the same species can label several leaves. Such multi-labeled trees are then used to reconstruct the evolutionary history of the polyploid species in question. However, this reconstruction process requires new techniques that are not available in current phylogenetic software packages. Here, we describe the software package PADRE (Package for Analyzing and Displaying Reticulate Evolution) that implements such techniques, allowing the reconstruction of complex evolutionary histories for polyploids in the form of phylogenetic networks. AVAILABILITY: PADRE is an open-source Java program freely available from http://www.uea.ac.uk/cmp/research/cmpbio/PADRE.

The complexity of deriving multi-labeled trees from bipartitions.

Recently, multi-labeled trees have been used to help unravel the evolutionary origins of polyploid species. A multi-labeled tree is the same as a phylogenetic tree except that more than one leaf may be labeled by a single species, so that the leaf set of a multi-labeled tree can be regarded as a multiset. In contrast to phylogenetic trees, which can be efficiently encoded in terms of certain bipartitions of their leaf sets, we show that it is NP-hard to decide whether a collection of bipartitions of a multiset can be represented by a multi-labeled tree. Even so, we also show that it is possible to generalize to multi-labeled trees a well-known condition that characterizes when a collection of bipartitions encodes a phylogenetic tree. Using this generalization, we obtain a fixed-parameter algorithm for the above decision problem in terms of a parameter associated to the given multiset.

Reconstructing the evolutionary history of polyploids from multilabeled trees.

In recent studies, phylogenetic networks have been derived from so-called multilabeled trees in order to understand the origins of certain polyploids. Although the trees used in these studies were constructed using sophisticated techniques in phylogenetic analysis, the presented networks were inferred using ad hoc arguments that cannot be easily extended to larger, more complicated examples. In this paper, we present a general method for constructing such networks, which takes as input a multilabeled phylogenetic tree and outputs a phylogenetic network with certain desirable properties. To illustrate the applicability of our method, we discuss its use in reconstructing the evolutionary history of plant allopolyploids. We conclude with a discussion concerning possible future directions. The network construction method has been implemented and is freely available for use from http://www.uea.ac.uk/ approximately a043878/padre.html.