To prevent rereplication of genomic segments, the eukaryotic cell cycle is divided into two nonoverlapping phases. During late mitosis and G1 replication origins are “licensed” by loading MCM2-7 ...double hexamers and during S phase licensed replication origins activate to initiate bidirectional replication forks. Replication forks can stall irreversibly, and if two converging forks stall with no intervening licensed origin—a “double fork stall” (DFS)—replication cannot be completed by conventional means. We previously showed how the distribution of replication origins in yeasts promotes complete genome replication even in the presence of irreversible fork stalling. This analysis predicts that DFSs are rare in yeasts but highly likely in large mammalian genomes. Here we show that complementary strand synthesis in early mitosis, ultrafine anaphase bridges, and G1-specific p53-binding protein 1 (53BP1) nuclear bodies provide a mechanism for resolving unreplicated DNA at DFSs in human cells. When origin number was experimentally altered, the number of these structures closely agreed with theoretical predictions of DFSs. The 53BP1 is preferentially bound to larger replicons, where the probability of DFSs is higher. Loss of 53BP1 caused hypersensitivity to licensing inhibition when replication origins were removed. These results provide a striking convergence of experimental and theoretical evidence that unreplicated DNA can pass through mitosis for resolution in the following cell cycle.
The soil bacterium
is a model organism to investigate the formation of biofilms, the predominant form of microbial life. The secreted protein BslA self-assembles at the surface of the biofilm to give ...the
biofilm its characteristic hydrophobicity. To understand the mechanism of BslA self-assembly at interfaces, here we built a molecular model based on the previous BslA crystal structure and the crystal structure of the BslA paralogue YweA that we determined. Our analysis revealed two conserved protein-protein interaction interfaces supporting BslA self-assembly into an infinite 2-dimensional lattice that fits previously determined transmission microscopy images. Molecular dynamics simulations and in vitro protein assays further support our model of BslA elastic film formation, while mutagenesis experiments highlight the importance of the identified interactions for biofilm structure. Based on this knowledge, YweA was engineered to form more stable elastic films and rescue biofilm structure in
deficient strains. These findings shed light on protein film assembly and will inform the development of BslA technologies which range from surface coatings to emulsions in fast-moving consumer goods.
Kinesin-13 proteins have a critical role in animal cell mitosis, during which they regulate spindle microtubule dynamics through their depolymerisation activity. Much of what is known about ...Kinesin-13 function emanates from a relatively small sub-family of proteins containing MCAK and Kif2A/B. However, recent work on kinesins from the much more widely distributed, ancestral Kinesin-13 family, which includes human Kif24, have identified a second function in flagellum length regulation that may exist either alongside or instead of the mitotic role.
The African trypanosome Trypanosoma brucei encodes 7 distinct Kinesin-13 proteins, allowing scope for extensive specialisation of roles. Here, we show that of all the trypanosomal Kinesin-13 proteins, only one is nuclear. This protein, TbKIN13-1, is present in the nucleoplasm throughout the cell cycle, but associates with the spindle during mitosis, which in trypanosomes is closed. TbKIN13-1 is necessary for the segregation of both large and mini-chromosomes in this organism and reduction in TbKIN13-1 levels mediated by RNA interference causes deflects in spindle disassembly with spindle-like structures persisting in non-mitotic cells. A second Kinesin-13 is localised to the flagellum tip, but the majority of the Kinesin-13 family members are in neither of these cellular locations.
These data show that the expanded Kinesin-13 repertoire of trypanosomes is not associated with diversification of spindle-associated roles. TbKIN13-1 is required for correct spindle function, but the extra-nuclear localisation of the remaining paralogues suggests that the biological roles of the Kinesin-13 family is wider than previously thought.
In late mitosis and G1, origins of DNA replication must be “licensed” for use in the upcoming S phase by being encircled by double hexamers of the minichromosome maintenance proteins MCM2–7. A ...“licensing checkpoint” delays cells in G1 until sufficient origins have been licensed, but this checkpoint is lost in cancer cells. Inhibition of licensing can therefore kill cancer cells while only delaying normal cells in G1. In a high-throughput cell-based screen for licensing inhibitors we identified a family of 2-arylquinolin-4-amines, the most potent of which we call RL5a. The binding of the origin recognition complex (ORC) to origin DNA is the first step of the licensing reaction. We show that RL5a prevents ORC forming a tight complex with DNA that is required for MCM2–7 loading. Formation of this ORC-DNA complex requires ATP, and we show that RL5a inhibits ORC allosterically to mimic a lack of ATP.
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•Arylquinolin-amines identified as small-molecule inhibitors of replication licensing•Inhibitor prevents tight ORC-DNA interaction required for MCM2–7 loading•ORC-DNA binding inhibited allosterically to mimic a lack of ATP
Gardner et al. describe the identification of a family of arylquinolin-amines as inhibitors of “replication licensing”, the loading of replication origins with double hexamers of MCM2–7. Arylquinolin-amines prevent the tight ORC-DNA interaction required for licensing, mimicking a lack of ATP.
Migratory CD103+ and lymphoid-resident CD8+ dendritic cells (DCs) share many attributes, such as dependence on the same transcription factors, cross-presenting ability and expression of certain ...surface molecules, such that it has been proposed they belong to a common sub-lineage. The functional diversity of the two DC types is nevertheless incompletely understood. Here we reveal that upon skin infection with herpes simplex virus, migratory CD103+ DCs from draining lymph nodes were more potent at inducing Th17 cytokine production by CD4+ T cells than CD8+ DCs. This superior capacity to drive Th17 responses was also evident in CD103+ DCs from uninfected mice. Their differential potency to induce Th17 differentiation was reflected by higher production of IL-1β and IL-6 by CD103+ DCs compared with CD8+ DCs upon stimulation. The two types of DCs from isolated lymph nodes also differ in expression of certain pattern recognition receptors. Furthermore, elevated levels of GM-CSF, typical of those found in inflammation, substantially increased the pool size of CD103+ DCs in lymph nodes and skin. We argue that varied levels of GM-CSF may explain the contrasting reports regarding the positive role of GM-CSF in regulating development of CD103+ DCs. Together, we find that these two developmentally closely-related DC subsets display functional differences and that GM-CSF has differential effect on the two types of DCs.
Survival of various immune cell populations has been proposed to preferentially rely on a particular anti-apoptotic BCL-2 family member, for example, naive T cells require BCL-2, while regulatory T ...cells require MCL-1. Here we examined the survival requirements of multiple immune cell subsets in vitro and in vivo, using both genetic and pharmacological approaches. Our findings support a model in which survival is determined by quantitative participation of multiple anti-apoptotic proteins rather than by a single anti-apoptotic protein. This model provides both an insight into how the sum of relative levels of anti-apoptotic proteins BCL-2, MCL-1 and A1 influence survival of T cells, B cells and dendritic cells, and a framework for ascertaining how these different immune cells can be optimally targeted in treatment of immunopathology, transplantation rejection or hematological cancers.
Expression of HLA-C varies widely across individuals in an allele-specific manner. This variation in expression can influence efficacy of the immune response, as shown for infectious and autoimmune ...diseases. MicroRNA binding partially influences differential HLA-C expression, but the additional contributing factors have remained undetermined. Here we use functional and structural analyses to demonstrate that HLA-C expression is modulated not just at the RNA level, but also at the protein level. Specifically, we show that variation in exons 2 and 3, which encode the α1/α2 domains, drives differential expression of HLA-C allomorphs at the cell surface by influencing the structure of the peptide-binding cleft and the diversity of peptides bound by the HLA-C molecules. Together with a phylogenetic analysis, these results highlight the diversity and long-term balancing selection of regulatory factors that modulate HLA-C expression.
Dendritic cells (DCs) are heterogeneous, comprising subsets with functional specializations that play distinct roles in immunity as well as immunopathology. We investigated the molecular control of ...cell survival of two main DC subsets: plasmacytoid DCs (pDCs) and conventional DCs (cDCs) and their dependence on individual antiapoptotic BCL-2 family members. Compared with cDCs, pDCs had higher expression of BCL-2, lower A1, and similar levels of MCL-1 and BCL-XL. Transgenic overexpression of BCL-2 increased the pDC pool size in vivo with only minor impact on cDCs. With a view to immune intervention, we tested BCL-2 inhibitors and found that ABT-199 (the BCL-2 specific inhibitor) selectively killed pDCs but not cDCs. Conversely, genetic knockdown of A1 profoundly reduced the proportion of cDCs but not pDCs. We also found that conditional ablation of MCL-1 significantly reduced the size of both DC populations in mice and impeded DC-mediated immune responses. Thus, we revealed that the two DC types have different cell survival requirements. The molecular basis of survival of different DC subsets thus advocates the antagonism of selective BCL-2 family members for treating diseases pertaining to distinct DC subsets.
Significance Dendritic cells (DCs) are pivotal for immune responses as they present antigens to T cells. Different DC subsets have different functions and are associated with different diseases. For example, plasmacytoid DCs (pDCs) produce type 1 interferons and are associated with the autoimmune disease, systemic lupus erythematosus. Understanding control of survival/apoptosis in different DC subsets may not only provide a molecular basis for their homeostasis but also guide therapeutic intervention of immunopathology. We revealed that two major DC subsets (pDCs and conventional DCs) express distinct BCL-2 family proteins at different levels and this correlated with their survival requirements. Accordingly, clinically applicable antagonist drugs killed the appropriate DC subsets, informing on the future use of these compounds for treating immune-mediated damage.
Emergency departments (EDs) are often first to feel the intra-hospital effects of disasters. Compromised care standards during disasters eventuate from increased demands on health resources; the ...facilities, supplies, equipment and manpower imperative for a functioning healthcare facility. Emergency departments must understand the effect of disasters on their health resources. This paper examines the impact on resources within the ED as a result of a disaster and provides a review against the United Nations Office for Disaster Risk Reduction’s Sendai Framework for Disaster Risk Reduction 2015–2030 priorities.
An integrative literature review design was utilised. Articles were extracted from databases and search engines. The Preferred Reporting Items of Systematic reviews and Meta-Analysis Guidelines for systematic literature reviews were used.
Seven papers met inclusion criteria. Disaster consumable stocking was used to mitigate disaster risk and improve resilience. Logistical challenges were exacerbated by poor building design. Ineffective human resource management, communications failure, insufficient ED space, diminished equipment and supplies and unreliable emergency power sources were described.
Disaster planning and preparedness strategies can address health resource deficits, increasing ED resilience. Further retrospective case studies are required to greater understand the effects of disasters on ED health resources.
The objective of this study was to evaluate the seroprevalence and identify the strains of swine influenza virus (SwIV), as well as the seroprevalence of porcine parvovirus (PPV), transmissible ...gastroenteritis virus (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine respiratory coronavirus (PRCV), porcine circovirus type 2 (PCV-2), and classical swine fever virus (CSFV) in pigs in Trinidad and Tobago (T&T). Blood samples (309) were randomly collected from pigs at farms throughout T&T. Serum samples were tested for the presence of antibodies to the aforementioned viruses using commercial ELISA kits, and the circulating strains of SwIV were identified by the hemagglutination inhibition test (HIT). Antibodies against SwIV were detected in 114 out of the 309 samples (37%). Out of a total of 26 farms, 14 tested positive for SwIV antibodies. HI testing revealed high titers against the A/sw/Minnesota/593/99 H3N2 strain and the pH1N1 2009 pandemic strain. Antibodies against PPV were detected in 87 out of the 309 samples (28%), with 11 out of 26 farms testing positive for PPV antibodies. Antibodies against PCV-2 were detected in 205 out of the 309 samples tested (66%), with 25 out of the 26 farms testing positive for PCV-2 antibodies. No antibodies were detected in any of the tested pigs to PRRSV, TGEV, PRCV, or CSFV.
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