Representativeness of whole-genome sequencing approaches in England: the importance for understanding inequalities associated with SARS-CoV-2 infection.

Whole-genome sequencing (WGS) information has played a crucial role in the SARS-CoV-2 (COVID-19) pandemic by providing evidence about variants to inform public health policy. The purpose of this study was to assess the representativeness of sequenced cases compared with all COVID-19 cases in England, between March 2020 and August 2021, by demographic and socio-economic characteristics, to evaluate the representativeness and utility of these data in epidemiological analyses. To achieve this, polymerase chain reaction (PCR)-confirmed COVID-19 cases were extracted from the national laboratory system and linked with WGS data.

Regulators of male and female sexual development are critical for the transmission of a malaria parasite.

Malaria transmission to mosquitoes requires a developmental switch in asexually dividing blood-stage parasites to sexual reproduction. In Plasmodium berghei, the transcription factor AP2-G is required and sufficient for this switch, but how a particular sex is determined in a haploid parasite remains unknown. Using a global screen of barcoded mutants, we here identify genes essential for the formation of either male or female sexual forms and validate their importance for transmission.

Genomic reconstruction of the SARS-CoV-2 epidemic in England.

The evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus leads to new variants that warrant timely epidemiological characterization. Here we use the dense genomic surveillance data generated by the COVID-19 Genomics UK Consortium to reconstruct the dynamics of 71 different lineages in each of 315 English local authorities between September 2020 and June 2021. This analysis reveals a series of subepidemics that peaked in early autumn 2020, followed by a jump in transmissibility of the B.

The antimalarial efficacy and mechanism of resistance of the novel chemotype DDD01034957.

New antimalarial therapeutics are needed to ensure that malaria cases continue to be driven down, as both emerging parasite resistance to frontline chemotherapies and mosquito resistance to current insecticides threaten control programmes. Plasmodium, the apicomplexan parasite responsible for malaria, causes disease pathology through repeated cycles of invasion and replication within host erythrocytes (the asexual cycle). Antimalarial drugs primarily target this cycle, seeking to reduce parasite burden within the host as fast as possible and to supress recrudescence for as long as possible.

An enhanced toolkit for the generation of knockout and marker-free fluorescent .

The rodent parasite is an important model of malaria. The ability to produce chronic infections makes it particularly useful for investigating the development of anti- immunity, as well as features associated with parasite virulence during both the acute and chronic phases of infection. also undergoes asexual maturation (schizogony) and erythrocyte invasion in culture, so offers an experimentally-amenable to model for studying gene function and drug activity during parasite replication.

Genome-Scale Identification of Essential Metabolic Processes for Targeting the Plasmodium Liver Stage.

Plasmodium gene functions in mosquito and liver stages remain poorly characterized due to limitations in the throughput of phenotyping at these stages. To fill this gap, we followed more than 1,300 barcoded P. berghei mutants through the life cycle.

A forward genetic screen reveals a primary role for Plasmodium falciparum Reticulocyte Binding Protein Homologue 2a and 2b in determining alternative erythrocyte invasion pathways.

Invasion of human erythrocytes is essential for Plasmodium falciparum parasite survival and pathogenesis, and is also a complex phenotype. While some later steps in invasion appear to be invariant and essential, the earlier steps of recognition are controlled by a series of redundant, and only partially understood, receptor-ligand interactions. Reverse genetic analysis of laboratory adapted strains has identified multiple genes that when deleted can alter invasion, but how the relative contributions of each gene translate to the phenotypes of clinical isolates is far from clear.

Functional Profiling of a Plasmodium Genome Reveals an Abundance of Essential Genes.

The genomes of malaria parasites contain many genes of unknown function. To assist drug development through the identification of essential genes and pathways, we have measured competitive growth rates in mice of 2,578 barcoded Plasmodium berghei knockout mutants, representing >50% of the genome, and created a phenotype database. At a single stage of its complex life cycle, P.

A genome-scale vector resource enables high-throughput reverse genetic screening in a malaria parasite.

The genome-wide identification of gene functions in malaria parasites is hampered by a lack of reverse genetic screening methods. We present a large-scale resource of barcoded vectors with long homology arms for effective modification of the Plasmodium berghei genome. Cotransfecting dozens of vectors into the haploid blood stages creates complex pools of barcoded mutants, whose competitive fitness can be measured during infection of a single mouse using barcode sequencing (barseq).

PlasmoGEM, a database supporting a community resource for large-scale experimental genetics in malaria parasites.

The Plasmodium Genetic Modification (PlasmoGEM) database (http://plasmogem.sanger.ac.

Phosphoinositide metabolism links cGMP-dependent protein kinase G to essential Ca²⁺ signals at key decision points in the life cycle of malaria parasites.

Many critical events in the Plasmodium life cycle rely on the controlled release of Ca²⁺ from intracellular stores to activate stage-specific Ca²⁺-dependent protein kinases. Using the motility of Plasmodium berghei ookinetes as a signalling paradigm, we show that the cyclic guanosine monophosphate (cGMP)-dependent protein kinase, PKG, maintains the elevated level of cytosolic Ca²⁺ required for gliding motility. We find that the same PKG-dependent pathway operates upstream of the Ca²⁺ signals that mediate activation of P.

Comparative genomics in Chlamydomonas and Plasmodium identifies an ancient nuclear envelope protein family essential for sexual reproduction in protists, fungi, plants, and vertebrates.

Fertilization is a crucial yet poorly characterized event in eukaryotes. Our previous discovery that the broadly conserved protein HAP2 (GCS1) functioned in gamete membrane fusion in the unicellular green alga Chlamydomonas and the malaria pathogen Plasmodium led us to exploit the rare biological phenomenon of isogamy in Chlamydomonas in a comparative transcriptomics strategy to uncover additional conserved sexual reproduction genes. All previously identified Chlamydomonas fertilization-essential genes fell into related clusters based on their expression patterns.

A Plasmodium calcium-dependent protein kinase controls zygote development and transmission by translationally activating repressed mRNAs.

Calcium-dependent protein kinases (CDPKs) play key regulatory roles in the life cycle of the malaria parasite, but in many cases their precise molecular functions are unknown. Using the rodent malaria parasite Plasmodium berghei, we show that CDPK1, which is known to be essential in the asexual blood stage of the parasite, is expressed in all life stages and is indispensable during the sexual mosquito life-cycle stages. Knockdown of CDPK1 in sexual stages resulted in developmentally arrested parasites and prevented mosquito transmission, and these effects were independent of the previously proposed function for CDPK1 in regulating parasite motility.

The UEA sRNA workbench: a suite of tools for analysing and visualizing next generation sequencing microRNA and small RNA datasets.

Summary: RNA silencing is a complex, highly conserved mechanism mediated by small RNAs (sRNAs), such as microRNAs (miRNAs), that is known to be involved in a diverse set of biological functions including development, pathogen control, genome maintenance and response to environmental change. Advances in next generation sequencing technologies are producing increasingly large numbers of sRNA reads per sample at a fraction of the cost of previous methods. However, many bioinformatics tools do not scale accordingly, are cumbersome, or require extensive support from bioinformatics experts.

PfSET10, a Plasmodium falciparum methyltransferase, maintains the active var gene in a poised state during parasite division.

A major virulence factor of the malaria parasite Plasmodium falciparum is erythrocyte membrane protein 1 (PfEMP1), a variant protein expressed on the infected erythrocyte surface. PfEMP1 is responsible for adherence of infected erythrocytes to the endothelium and plays an important role in pathogenesis. Mutually exclusive transcription and switched expression of one of 60 var genes encoding PfEMP1 in each parasite genome provides a mechanism for antigenic variation.

A scalable pipeline for highly effective genetic modification of a malaria parasite.

In malaria parasites, the systematic experimental validation of drug and vaccine targets by reverse genetics is constrained by the inefficiency of homologous recombination and by the difficulty of manipulating adenine and thymine (A+T)-rich DNA of most Plasmodium species in Escherichia coli. We overcame these roadblocks by creating a high-integrity library of Plasmodium berghei genomic DNA (>77% A+T content) in a bacteriophage N15-based vector that can be modified efficiently using the lambda Red method of recombineering. We built a pipeline for generating P.

mRNA expression profiling reveals conserved and non-conserved miR-140 targets.

microRNAs are non-coding RNAs that regulate gene expression. A significant proportion of microRNAs is perfectly conserved across the vertebrate clade, including miR-140, which is specifically expressed in cartilage. Although it has been computationally predicted that a large majority of microRNA targets are conserved, experimental evidence for this hypothesis remains scarce.

Profiling of short RNAs during fleshy fruit development reveals stage-specific sRNAome expression patterns.

Plants feature a particularly diverse population of short (s)RNAs, the central component of all RNA silencing pathways. Next generation sequencing techniques enable deeper insights into this complex and highly conserved mechanism and allow identification and quantification of sRNAs. We employed deep sequencing to monitor the sRNAome of developing tomato fruits covering the period between closed flowers and ripened fruits by profiling sRNAs at 10 time-points.

The Arabidopsis RNA-directed DNA methylation argonautes functionally diverge based on their expression and interaction with target loci.

Argonaute (AGO) effectors of RNA silencing bind small RNA (sRNA) molecules and mediate mRNA cleavage, translational repression, or epigenetic DNA modification. In many organisms, these targeting mechanisms are devolved to different products of AGO multigene families. To investigate the basis of AGO functional diversification, we characterized three closely related Arabidopsis thaliana AGOs (AGO4, AGO6, and AGO9) implicated in RNA-directed DNA methylation.

Deciphering the diversity of small RNAs in plants: the long and short of it.

RNA silencing is a complex and highly conserved regulatory mechanism that is now known to be involved in such diverse processes as development, pathogen control, genome maintenance and response to environmental changes. Since its recent discovery, RNA silencing has become a fast moving key area of research in plant and animal molecular biology. Research in this field has greatly profited from recent developments in novel sequencing technologies that allow massive parallel sequencing of small RNA (sRNA) molecules, the key players of all RNA silencing phenomena.

Highly specific gene silencing by artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii.

MicroRNAs (miRNAs) are small RNAs, 21 to 22 nucleotides long, with important regulatory roles. They are processed from longer RNA molecules with imperfectly matched foldback regions and they function in modulating the stability and translation of mRNA. Recently, we and others have demonstrated that the unicellular alga Chlamydomonas reinhardtii, like diverse multicellular organisms, contains miRNAs.

Experimental identification of microRNA-140 targets by silencing and overexpressing miR-140.

MicroRNAs (miRNAs) are short noncoding RNA molecules regulating the expression of mRNAs. Target identification of miRNAs is computationally difficult due to the relatively low homology between miRNAs and their targets. We present here an experimental approach to target identification where the cartilage-specific miR-140 was overexpressed and silenced in cells it is normally expressed in separate experiments.

A toolkit for analysing large-scale plant small RNA datasets.

Unlabelled: Recent developments in high-throughput sequencing technologies have generated considerable demand for tools to analyse large datasets of small RNA sequences. Here, we describe a suite of web-based tools for processing plant small RNA datasets. Our tools can be used to identify micro RNAs and their targets, compare expression levels in sRNA loci, and find putative trans-acting siRNA loci.

Deep sequencing of tomato short RNAs identifies microRNAs targeting genes involved in fruit ripening.

In plants there are several classes of 21-24-nt short RNAs that regulate gene expression. The most conserved class is the microRNAs (miRNAs), although some miRNAs are found only in specific species. We used high-throughput pyrosequencing to identify conserved and nonconserved miRNAs and other short RNAs in tomato fruit and leaf.