Polymorphic parasitic larvae cooperate to build swimming colonies luring hosts.

Parasites have evolved a variety of astonishing strategies to survive within their hosts, yet the most challenging event in their personal chronicles is the passage from one host to another. It becomes even more complex when a parasite needs to pass through the external environment. Therefore, the free-living stages of parasites present a wide range of adaptations for transmission. Parasitic flatworms from the group Digenea (flukes) have free-living larvae, cercariae, which are remarkably diverse in structure and behavior.1,2 One of the cercariae transmission strategies is to attain a prey-like appearance for the host.3 This can be done through the formation of a swimming aggregate of several cercariae adjoined together by their tails.4 Through the use of live observations and light, electron, and confocal microscopy, we described such a supposedly prey-mimetic colony comprising cercariae of two distinct morphotypes. They are functionally specialized: larger morphotype (sailors) enable motility, and smaller morphotype (passengers) presumably facilitate infection. The analysis of local read alignments between the two samples reveals that both cercaria types have identical 18S, 28S, and 5.8S rRNA genes. Further phylogenetic analysis of these ribosomal sequences indicates that our specimen belongs to the digenean family Acanthocolpidae, likely genus Pleorchis. This discovery provides a unique example and a novel insight into how morphologically and functionally heterogeneous individuals of the same species cooperate to build colonial organisms for the purpose of infection. This strategy bears resemblance to the cooperating castes of the same species found among insects.5.

Introducing the Intergovernmental Policy Output Dataset (IPOD).

There is a growing recognition that international organizations (IOs) formulate and adopt policy in a wide range of areas. IOs have emerged as key venues for states seeking joint solutions to contemporary challenges such as climate change or COVID-19, and to establish frameworks to bolster trade, development, security, and more. In this capacity, IOs produce both extraordinary and routine policy output with a multitude of purposes, ranging from policies of historic significance like admitting new members to the more mundane tasks of administering IO staff. This article introduces the Intergovernmental Policy Output Dataset (IPOD), which covers close to 37,000 individual policy acts of 13 multi-issue IOs in the 1980-2015 period. The dataset fills a gap in the growing body of literature on the comparative study of IOs, providing researchers with a fine-grained perspective on the structure of IO policy output and data for comparisons across time, policy areas, and organizations. This article describes the construction and coverage of the dataset and identifies key temporal and cross-sectional patterns revealed by the data. In a concise illustration of the dataset's utility, we apply models of punctuated equilibria in a comparative study of the relationship between institutional features and broad policy agenda dynamics. Overall, the Intergovernmental Policy Output Dataset offers a unique resource for researchers to analyze IO policy output in a granular manner and to explore questions of responsiveness, performance, and legitimacy of IOs. Supplementary Information: The online version contains supplementary material available at 10.1007/s11558-023-09492-6.

Type I-E CRISPR-Cas System as a Defense System in Saccharomyces cerevisiae.

Defense against viruses and other mobile genetic elements (MGEs) is important in many organisms. The CRISPR-Cas systems found in bacteria and archaea constitute adaptive immune systems that can acquire the ability to target previously unrecognized MGEs. No CRISPR-Cas system is found to occur naturally in eukaryotic cells, but here, we demonstrate interference by a type I-E CRISPR-Cas system from Escherichia coli introduced in Saccharomyces cerevisiae. The designed CRISPR arrays are expressed and processed properly in S. cerevisiae. Targeted plasmids display reduced transformation efficiency, indicative of DNA cleavage. IMPORTANCE Genetic inactivation of viruses and other MGEs is an important tool with application in both research and therapy. Gene editing using, e.g., Cas9-based systems, can be used to inactivate MGEs in eukaryotes by introducing specific mutations. However, type I-E systems processively degrade the target which allows for inactivation without detailed knowledge of gene function. A reconstituted CRISPR-Cas system in S. cerevisiae can also function as a basic research platform for testing the role of various factors in the interference process.

Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition.

CRISPR-Cas systems are adaptive prokaryotic immune systems protecting against horizontally transferred DNA or RNA such as viruses and other mobile genetic elements. Memory of past invaders is stored as spacers in CRISPR loci in a process called adaptation. Here we developed a novel assay where spacer integration results in fluorescence, enabling detection of memory formation in single cells and quantification of as few as 0.05% cells with expanded CRISPR arrays in a bacterial population. Using this fluorescent CRISPR Adaptation Reporter (f-CAR), we quantified adaptation of the two CRISPR arrays of the type I-E CRISPR-Cas system in Escherichia coli, and confirmed that more integration events are targeted to CRISPR-II than to CRISPR-I. The f-CAR conveniently analyzes and compares many samples, allowing new insights into adaptation. For instance, we show that in an E. coli culture the majority of acquisition events occur in late exponential phase.

Exploring the ecological function of CRISPR-Cas virus defense.

Virus-host interaction is a key process in understanding the ecology and evolution of life. The study of the CRISPR-Cas RNA-guided adaptive immune systems of bacteria and archaea has added to our understanding of the virus defense mechanisms of microorganisms. The molecular details of the CRISPR-Cas systems are well explored and have allowed development a new generation of gene editing tools. However, the actual role and importance of CRISPR-Cas virus defense in nature is complex to study and have attracted less attention. Metagenomic analysis of microbial populations and the study of viruses-host systems in the laboratory have begun to unravel this question. Key findings in the field are described, with focus on recent developments.

The CRISPR-Cas immune system: biology, mechanisms and applications.

Viruses are a common threat to cellular life, not the least to bacteria and archaea who constitute the majority of life on Earth. Consequently, a variety of mechanisms to resist virus infection has evolved. A recent discovery is the adaptive immune system in prokaryotes, a type of system previously thought to be present only in vertebrates. The system, called CRISPR-Cas, provide sequence-specific adaptive immunity and fundamentally affect our understanding of virus-host interaction. CRISPR-based immunity acts by integrating short virus sequences in the cell's CRISPR locus, allowing the cell to remember, recognize and clear infections. There has been rapid advancement in our understanding of this immune system and its applications, but there are many aspects that await elucidation making the field an exciting area of research. This review provides an overview of the field and highlights unresolved issues.

New clues on the regulation of the CRISPR-Cas immune system.

Research into the CRISPR-Cas immune system of prokaryotes is progressing at a tremendous pace given both its important biological function and its role as a source of new genetic tools. However, a few areas of the field have remained largely unaddressed. A recent report provides information on one such overlooked area: how the cell regulates the CRISPR-Cas immune system. The processes, despite their importance, have remained illusive. In Pectobacterium atrosepticum regulation is, perhaps surprisingly, based on metabolic factors responding to glucose levels in the cell. Regulators include both activators and repressors of cas gene expression. It remains an open question why and how this regulatory system have evolved, and if it is a typical example of how CRISPR-as systems are regulated or not.

Efficient programmable gene silencing by Cascade.

Methods that permit controlled changes in the expression of genes are important tools for biological and medical research, and for biotechnological applications. Conventional methods are directed at individually changing each gene, its regulatory elements or its mRNA's translation rate. We demonstrate that the CRISPR-associated DNA-binding Cascade complex can be used for efficient, long-lasting and programmable gene silencing. When Cascade is targeted to a promoter sequence the transcription of the downstream gene is inhibited, resulting in dramatically reduced expression. The specificity of Cascade binding is provided by the integral crRNA component, which is easily designed to target virtually any stretch of DNA. Cascade targeted to the ORF sequence of the gene can also silence expression, albeit at lower efficiency. The system can be used to silence plasmid and chromosome targets, simultaneously target several genes and is active in different bacterial species and strains. The findings described here are an addition to the expanding range of CRISPR-based technologies and may be adapted to additional organisms and cell systems.

Rolf Bernander (1956-2014): pioneer of the archaeal cell cycle.

On 19 January 2014 Rolf ('Roffe') Bernander passed away unexpectedly. Rolf was a dedicated scientist; his research aimed at unravelling the cell biology of the archaeal domain of life, especially cell cycle-related questions, but he also made important contributions in other areas of microbiology. Rolf had a professor position in the Molecular Evolution programme at Uppsala University, Sweden for about 8 years, and in January 2013 he became chair professor at the Department of Molecular Biosciences, The Wenner-Gren Institute at Stockholm University in Sweden. Rolf was an exceptional colleague and will be deeply missed by his family and friends, and the colleagues and co-workers that he leaves behind in the scientific community. He will be remembered for his endless enthusiasm for science, his analytical mind, and his quirky sense of humour.

A novel bacterial enzyme with D-glucuronyl C5-epimerase activity.

Glycosaminoglycans are biologically active polysaccharides that are found ubiquitously in the animal kingdom. The biosynthesis of these complex polysaccharides involves complicated reactions that turn the simple glycosaminoglycan backbone into highly heterogeneous structures. One of the modification reactions is the epimerization of D-glucuronic acid to its C5-epimer L-iduronic acid, which is essential for the function of heparan sulfate. Although L-iduronic acid residues have been shown to exist in polysaccharides of some prokaryotes, there has been no experimental evidence for the existence of a prokaryotic D-glucuronyl C5-epimerase. This work for the first time reports on the identification of a bacterial enzyme with D-glucuronyl C5-epimerase activity. A gene of the marine bacterium Bermanella marisrubri sp. RED65 encodes a protein (RED65_08024) of 448 amino acids that has an overall 37% homology to the human D-glucuronic acid C5-epimerase. Alignment of this peptide with the human and mouse sequences revealed a 60% similarity at the carboxyl terminus. The recombinant protein expressed in Escherichia coli showed epimerization activity toward substrates generated from heparin and the E. coli K5 capsular polysaccharide, thereby providing the first evidence for bacterial D-glucuronyl C5-epimerase activity. These findings may eventually be used for modification of mammalian glycosaminoglycans.

The flame-retardant BDE-99 dose-dependently affects viral replication in CVB3-infected mice.

The flame retardant component 2,2',4,4',5-penta-BDE (BDE-99) is found in the environment and in human tissues and fluids. In mice the common human coxsackievirus B3 (CVB3) infection has been shown to change the tissue distribution of BDE-99. We now investigate how CVB3 infection in mice affects liver uptake of (14)C at two doses of radiolabelled BDE-99, and whether increased tissue levels are related to changed virus replication and gene expression of the proinflammatory chemokine monocyte chemoattractant protein-1 (MCP-1). Mice were infected on day 0, orally treated either with 200µg or 20mg (14)C-BDE-99/kgbw on day 1, and euthanised on day 3. Serum and liver levels of (14)C-BDE-99, as well as virus levels and gene expressions of MCP-1 in the liver, were measured. In non-infected mice, there was a dose-dependent uptake of BDE-99 in both liver and serum, and in infected animals the liver BDE-99 levels was further increased. When comparing infected mice exposed to the two BDE-99 doses, the higher BDE dose resulted in increased virus amounts in the liver, and decreased infection-induced expression of MCP-1. Consequently, a high enough dose/tissue concentration of BDE-99 may result in a disturbed mobilisation of immune cells into infected tissues that could explain higher virus titres and a possibly altered clinical course of the disease. Moreover, the fact that CVB3 infection increased the BDE-99 levels in liver but not in serum may impair the risk assessment of polybrominated diphenyl ethers (PBDEs) in subclinical and clinically infected individuals, as serum levels is the common marker of exposure.

Native tandem and ion mobility mass spectrometry highlight structural and modular similarities in clustered-regularly-interspaced shot-palindromic-repeats (CRISPR)-associated protein complexes from Escherichia coli and Pseudomonas aeruginosa.

The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) immune system of bacteria and archaea provides acquired resistance against viruses and plasmids, by a strategy analogous to RNA-interference. Key components of the defense system are ribonucleoprotein complexes, the composition of which appears highly variable in different CRISPR/Cas subtypes. Previous studies combined mass spectrometry, electron microscopy, and small angle x-ray scattering to demonstrate that the E. coli Cascade complex (405 kDa) and the P. aeruginosa Csy-complex (350 kDa) are similar in that they share a central spiral-shaped hexameric structure, flanked by associating proteins and one CRISPR RNA. Recently, a cryo-electron microscopy structure of Cascade revealed that the CRISPR RNA molecule resides in a groove of the hexameric backbone. For both complexes we here describe the use of native mass spectrometry in combination with ion mobility mass spectrometry to assign a stable core surrounded by more loosely associated modules. Via computational modeling subcomplex structures were proposed that relate to the experimental IMMS data. Despite the absence of obvious sequence homology between several subunits, detailed analysis of sub-complexes strongly suggests analogy between subunits of the two complexes. Probing the specific association of E. coli Cascade/crRNA to its complementary DNA target reveals a conformational change. All together these findings provide relevant new information about the potential assembly process of the two CRISPR-associated complexes.

Structural basis for CRISPR RNA-guided DNA recognition by Cascade.

The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and low-resolution structure of Cascade and show how it recognizes double-stranded DNA (dsDNA) targets in a sequence-specific manner. Cascade is a 405-kDa complex comprising five functionally essential CRISPR-associated (Cas) proteins (CasA(1)B(2)C(6)D(1)E(1)) and a 61-nucleotide CRISPR RNA (crRNA) with 5'-hydroxyl and 2',3'-cyclic phosphate termini. The crRNA guides Cascade to dsDNA target sequences by forming base pairs with the complementary DNA strand while displacing the noncomplementary strand to form an R-loop. Cascade recognizes target DNA without consuming ATP, which suggests that continuous invader DNA surveillance takes place without energy investment. The structure of Cascade shows an unusual seahorse shape that undergoes conformational changes when it binds target DNA.

Replication-biased genome organisation in the crenarchaeon Sulfolobus.

BACKGROUND: Species of the crenarchaeon Sulfolobus harbour three replication origins in their single circular chromosome that are synchronously initiated during replication. RESULTS: We demonstrate that global gene expression in two Sulfolobus species is highly biased, such that early replicating genome regions are more highly expressed at all three origins. The bias by far exceeds what would be anticipated by gene dosage effects alone. In addition, early replicating regions are denser in archaeal core genes (enriched in essential functions), display lower intergenic distances, and are devoid of mobile genetic elements. CONCLUSION: The strong replication-biased structuring of the Sulfolobus chromosome implies that the multiple replication origins serve purposes other than simply shortening the time required for replication. The higher-level chromosomal organisation could be of importance for minimizing the impact of DNA damage, and may also be linked to transcriptional regulation.

H-NS-mediated repression of CRISPR-based immunity in Escherichia coli K12 can be relieved by the transcription activator LeuO.

The recently discovered prokaryotic CRISPR/Cas defence system provides immunity against viral infections and plasmid conjugation. It has been demonstrated that in Escherichia coli transcription of the Cascade genes (casABCDE) and to some extent the CRISPR array is repressed by heat-stable nucleoid-structuring (H-NS) protein, a global transcriptional repressor. Here we elaborate on the control of the E. coli CRISPR/Cas system, and study the effect on CRISPR-based anti-viral immunity. Transformation of wild-type E. coli K12 with CRISPR spacers that are complementary to phage Lambda does not lead to detectable protection against Lambda infection. However, when an H-NS mutant of E. coli K12 is transformed with the same anti-Lambda CRISPR, this does result in reduced sensitivity to phage infection. In addition, it is demonstrated that LeuO, a LysR-type transcription factor, binds to two sites flanking the casA promoter and the H-NS nucleation site, resulting in derepression of casABCDE12 transcription. Overexpression of LeuO in E. coli K12 containing an anti-Lambda CRISPR leads to an enhanced protection against phage infection. This study demonstrates that in E. coli H-NS and LeuO are antagonistic regulators of CRISPR-based immunity.

Comparative and functional analysis of the archaeal cell cycle.

The temporal and spatial organization of the chromosome replication, genome segregation and cell division processes is less well understood in species belonging to the Archaea, than in those from the Bacteria and Eukarya domains. Novel insights into the regulation and key components of the Sulfolobus acidocaldarius cell cycle have been obtained through genome-wide analysis of cell cycle-specific gene expression, followed by cloning and characterization of gene products expressed at different cell cycle stages. Here, the results of the transcript profiling are further explored, and potential key players in archaeal cell cycle progression are highlighted in an evolutionary context, by comparing gene expression patterns and gene conservation between three selected microbial species from different domains of life. We draw attention to novel putative nucleases and helicases implicated in DNA replication, recombination and repair, as well as to potential genome segregation factors. Focus is also placed upon regulatory features, including transcription factors and protein kinases inferred to be involved in the execution of specific cell cycle stages, and regulation through metabolic coupling is discussed.

CRISPR-based adaptive and heritable immunity in prokaryotes.

The recently discovered CRISPR (clustered regularly interspaced short palindromic repeat) defense system protects bacteria and archaea against mobile genetic elements. This immunity system has the potential to continuously adjust its reach at the genomic level, implying that both gain and loss of information is inheritable. The CRISPR system consists of typical stretches of interspaced repetitive DNA (CRISPRs) and associated cas genes. Three distinct stages are recognized in the CRISPR defense mechanism: (i) adaptation of the CRISPR via the integration of short sequences of the invaders as spacers; (ii) expression of CRISPRs and subsequent processing to small guide RNAs; and (iii) interference of target DNA by the crRNA guides. Recent analyses of key Cas proteins indicate that, despite some functional analogies, this fascinating prokaryotic system shares no phylogenetic relation with the eukaryotic RNA interference system.

Sequential changes in serum cytokines reflect viral RNA kinetics in target organs of a coxsackievirus B infection in mice.

INTRODUCTION: The pattern of cytokine responses related to viral replication during the course of the common human coxsackievirus B3 (CVB3) infection is not known. METHODS: Serum levels of 21 cytokines and chemokines were studied (Luminex technique) in CVB3-infected in mice on days 3, 6, and 9 post-infection (p.i.). CVB3 was measured quantitatively (reverse transcriptase polymerase chain reaction) in the liver and pancreas. RESULTS: Virus levels peaked on day 3 in both the liver and pancreas, but were 1,000-fold higher in the pancreas. IL-17alpha, IFN-gamma, KC, MCP-1, MIP1beta, and RANTES were detected on all days. On day 3 p.i., IL-6, IL-12(p40), KC, MCP-1, RANTES, and TNF-alpha were found to peak. On day 6 p.i., IL-1beta, IL-9, IL-12(p70), IL-13, IL-17alpha, and IFN-gamma peaked. On day 9 p.i., MIP1beta, IL-1beta, MCP-1, and TNF-alpha were still increased. These changes in cytokines may be used to monitor the progress of enteroviral infections in clinical settings.

Polybrominated diphenyl ether exposure suppresses cytokines important in the defence to coxsackievirus B3 infection in mice.

Environmental pollutants can adversely affect the immune system. The host defence during infection depends on cytokine signalling and proper function of immune cells. However, no studies have addressed how polybrominated diphenyl ethers (PBDEs) affect cytokine responses. We investigated the combined effects in Balb/c mice of human coxsackievirus B3 (CVB3) infection and exposure to PBDEs (BDE-99 or Bromkal mixture) on 21 serum cytokines. The mice were infected (i.p.) on day 0, orally treated with BDE-99 or Bromkal on day 1 (20mg/kg bw) and put to death on day 3. CVB3 was quantitatively measured in the liver and pancreas by RT-PCR. The Luminex 200 multi-analyte system was used for cytokine analysis. High numbers of viral copies were found in the liver and pancreas. Infection increased TNF-alpha, IL-6, MCP-1, IL-12p40, KC and RANTES levels. Notably, PBDE-exposure resulted in a marked decrease, or even lack, of IL-13, MIP-1beta, RANTES, IFN-gamma and KC levels in non-infected mice. However, the effects of PBDE-exposure on cytokines did not affect viral replication during early CVB3 infection. In conclusion, PBDEs causes a selective block in immune signalling pathways but the consequences of this need to be further studied in different host resistance models of infection.

Small CRISPR RNAs guide antiviral defense in prokaryotes.

Prokaryotes acquire virus resistance by integrating short fragments of viral nucleic acid into clusters of regularly interspaced short palindromic repeats (CRISPRs). Here we show how virus-derived sequences contained in CRISPRs are used by CRISPR-associated (Cas) proteins from the host to mediate an antiviral response that counteracts infection. After transcription of the CRISPR, a complex of Cas proteins termed Cascade cleaves a CRISPR RNA precursor in each repeat and retains the cleavage products containing the virus-derived sequence. Assisted by the helicase Cas3, these mature CRISPR RNAs then serve as small guide RNAs that enable Cascade to interfere with virus proliferation. Our results demonstrate that the formation of mature guide RNAs by the CRISPR RNA endonuclease subunit of Cascade is a mechanistic requirement for antiviral defense.