Structure and function of factor X: properties, activation, and activity in prothrombinase. A retrospective in a historical context.

The evolution of our understanding of the formation of thrombin from the postulated thrombokinase of Morawitz to activated Factor X and prothrombinase occurred during a period of nearly 100 years. During this time structure-function relationships have emerged and the roles of phospholipid surfaces, the accessory factor, Factor V and its activated form have been clarified. This paper summarizes this story with particular acknowledgement of the seminal contributions of Haskell Milstone.

Temporal stability vs. community matrix measures of stability and the role of weak interactions.

Relationships between different measures of stability are not well understood in part because empiricists and theoreticians tend to measure different aspects and most studies only explore a single form of stability. Using time-series data from experimental plankton communities, we compared temporal stability typically measured by empiricists (coefficient of variation in biomass) to stability measures typically measured by theoreticians derived from the community matrix (asymptotic resilience, initial resilience and intrinsic stochastic invariability) using first-order multivariate autoregressive models (MAR). Community matrices were also used to derive estimates of interaction strengths between plankton groups. We found no relationship between temporal stability and stability measures derived from the community matrix. Weaker interaction strengths were generally associated with higher stability for community matrix measures of stability, but were not consistently associated with higher temporal stability. Temporal stability and stability measures derived from the community matrix stability appear to represent different aspects of stability reflecting the multi-dimensionality of stability.

An exploration into experiences and attitudes regarding risky health behaviours in an adult cystic fibrosis population.

Health risk behaviours (HRBs) are prevalent within the cystic fibrosis (CF) population, and there is a lack of research around what influences their engagement. This research explored beliefs associated with HRBs within an adult CF population using qualitative semi-structured interviews. Participants' beliefs towards their CF and its life impact were investigated to explore reasons for engaging in HRB. A desire for normalcy was evident, often accompanied by engagement in everyday HRB as a method of minimising the illness identity. Evidence of a life-orientated illness perspective was also prevalent, with participants engaging in some risky behaviours for fun. Overall, there was a lack of knowledge on the consequences of HRB, with many participants reporting not being informed of these by clinicians. This research highlights a dilemma between clinical recommendations and personal life strategies undertaken by individuals with CF to support their identity.

High mutational rates of large-scale duplication and deletion in Daphnia pulex.

Knowledge of the genome-wide rate and spectrum of mutations is necessary to understand the origin of disease and the genetic variation driving all evolutionary processes. Here, we provide a genome-wide analysis of the rate and spectrum of mutations obtained in two Daphnia pulex genotypes via separate mutation-accumulation (MA) experiments. Unlike most MA studies that utilize haploid, homozygous, or self-fertilizing lines, D. pulex can be propagated ameiotically while maintaining a naturally heterozygous, diploid genome, allowing the capture of the full spectrum of genomic changes that arise in a heterozygous state. While base-substitution mutation rates are similar to those in other multicellular eukaryotes (about 4 × 10(-9) per site per generation), we find that the rates of large-scale (>100 kb) de novo copy-number variants (CNVs) are significantly elevated relative to those seen in previous MA studies. The heterozygosity maintained in this experiment allowed for estimates of gene-conversion processes. While most of the conversion tract lengths we report are similar to those generated by meiotic processes, we also find larger tract lengths that are indicative of mitotic processes. Comparison of MA lines to natural isolates reveals that a majority of large-scale CNVs in natural populations are removed by purifying selection. The mutations observed here share similarities with disease-causing, complex, large-scale CNVs, thereby demonstrating that MA studies in D. pulex serve as a system for studying the processes leading to such alterations.

Genome-Wide Stress Responses to Copper and Arsenic in a Field Population of Daphnia.

Over the past decade, significant advances have been made to unravel molecular mechanisms of stress response in different ecotoxicological model species. Within this study, we focus on population level transcriptomic responses of a natural population of Daphnia magna Straus, (1820), to heavy metals. We aim to characterize the population level transcriptomic responses, which include standing genetic variation, and improve our understanding on how populations respond to environmental stress at a molecular level. We studied population level responses to two heavy metals, copper and arsenic, and their binary mixture across time. Transcriptomic patterns identified significantly regulated gene families and genes at the population level including cuticle proteins and resilins. Furthermore, some of these differentially regulated gene families, such as cuticle proteins, were also significantly enriched for genetic variations including SNPs and MNPs. In general, genetic variation was observed in specific gene families, many of which are known to be involved in stress response. Overall, our results indicate that molecular stress responses can be identified within natural populations and that linking molecular mechanisms with genetic variation at the population level could contribute significantly to adverse outcome frameworks.

The transcriptomic and proteomic responses of Daphnia pulex to changes in temperature and food supply comprise environment-specific and clone-specific elements.

BACKGROUND: Regulatory adjustments to acute and chronic temperature changes are highly important for aquatic ectotherms because temperature affects their metabolic rate as well as the already low oxygen concentration in water, which can upset their energy balance. This also applies to severe changes in food supply. Thus, we studied on a molecular level (transcriptomics and/or proteomics) the immediate responses to heat stress and starvation and the acclimation to different temperatures in two clonal isolates of the model microcrustacean Daphnia pulex from more or less stressful environments, which showed a higher (clone M) or lower (clone G) tolerance to heat and starvation. RESULTS: The transcriptomic responses of clone G to acute heat stress (from 20 °C to 30 °C) and temperature acclimation (10 °C, 20 °C, and 24 °C) and the proteomic responses of both clones to acute heat, starvation, and heat-and-starvation stress comprised environment-specific and clone-specific elements. Acute stress (in particular heat stress) led to an early upregulation of stress genes and proteins (e.g., molecular chaperones) and a downregulation of metabolic genes and proteins (e.g., hydrolases). The transcriptomic responses to temperature acclimation differed clearly. They also varied depending on the temperature level. Acclimation to higher temperatures comprised an upregulation of metabolic genes and, in case of 24 °C acclimation, a downregulation of genes for translational processes and collagens. The proteomic responses of the clones M and G differed at any type of stress. Clone M showed markedly stronger and less stress-specific proteomic responses than clone G, which included the consistent expression of a specific heat shock protein (HSP60) and vitellogenin (VTG-SOD). CONCLUSIONS: The expression changes under acute stress can be interpreted as a switch from standard products of gene expression to stress-specific products. The expression changes under temperature acclimation probably served for an increase in energy intake (via digestion) and, if necessary, a decrease in energy expenditures (e.g, for translational processes). The stronger and less stress-specific proteomic responses of clone M indicate a lower degree of cell damage and an active preservation of the energy balance, which allowed adequate proteomic responses under stress, including the initiation of resting egg production (VTG-SOD expression) as an emergency reaction.

Arsenic Reduces Gene Expression Response to Changing Salinity in Killifish.

Toxicogenomic approaches can detect and classify adverse interactions between environmental toxicants and other environmental stressors but require more complex experimental designs and analytical approaches. Here we use novel toxicogenomic techniques to analyze the effect of arsenic exposure in wild killifish populations acclimating to changing salinity. Fish from three populations were acclimated to full strength seawater and transferred to fresh water for 1 or 24 h. Linear models of gene expression in gill tissue identified 31 genes that responded to osmotic shock at 1 h and 178 genes that responded at 24 h. Arsenic exposure (100 µg/L) diminished the responses (reaction norms) of these genes by 22% at 1 h ( p = 1.0 × 10-6) and by 10% at 24 h ( p = 3.0 × 10-10). Arsenic also significantly reduced gene coregulation in gene regulatory networks ( p = 0.002, paired Levene's test), and interactions between arsenic and salinity acclimation were uniformly antagonistic at the biological pathway level ( p < 0.05, binomial test). Arsenic's systematic interference with gene expression reaction norms was validated in a mouse multistressor experiment, demonstrating the ability of these toxicogenomic approaches to identify biologically relevant adverse interactions between environmental toxicants and other environmental stressors.

Live Cell Imaging Reveals the Relocation of dsRNA Binding Proteins Upon Viral Infection.

Viral infection triggers a range of plant responses such as the activation of the RNA interference (RNAi) pathway. The double-stranded RNA binding (DRB) proteins DRB3 and DRB4 are part of this pathway and aid in defending against DNA and RNA viruses, respectively. Using live cell imaging, we show that DRB2, DRB3, and DRB5 relocate from their uniform cytoplasmic distribution to concentrated accumulation in nascent viral replication complexes (VRC) that develop following cell invasion by viral RNA. Inactivation of the DRB3 gene in Arabidopsis by T-DNA insertion rendered these plants less able to repress RNA viral replication. We propose a model for the early stages of virus defense in which DRB2, DRB3, and DRB5 are invasion sensors that relocate to nascent VRC, where they bind to viral RNA and inhibit virus replication.

Hybridization and the Origin of Contagious Asexuality in Daphnia pulex.

Hybridization plays a potentially important role in the origin of obligate parthenogenesis (OP) in many organisms. However, it remains controversial whether hybridization directly triggers the transition from sexual reproduction to obligate asexuality or a hybrid genetic background enables asexual species to persist. Furthermore, we know little about the specific genetic elements from the divergent, yet still hybridizing lineages responsible for this transition and how these elements are further spread to create other OP lineages. In this study, we address these questions in Daphnia pulex, where cyclically parthenogenetic (CP) and OP lineages coexist. Ancestry estimates and whole-genome association mapping using 32 OP isolates suggest that a complex hybridization history between the parental species D. pulex and D. pulicaria is responsible for the introgression of a set of 647 D. pulicaria single nucleotide polymorphism alleles that show perfect association with OP. Crossing experiments using males of OP lineages and females of CP lineages strongly support a polygenic basis for OP. Single-sperm analyses show that although normal meiotic recombination occurs in the production of haploid sperm by males of OP lineages, a significant proportion of such sperm are polyploid, suggesting that the spread of asexual elements through these males (i.e., contagious asexuality) is much less efficient than previously envisioned. Although the current Daphnia genome annotation does not provide mechanistic insight into the nature of the asexuality-associated alleles, these alleles should be considered as candidates for future investigations on the genetic underpinnings of OP.

Subclinical thiamine deficiency results in failed reproduction in Arctic foxes.

Thiamine deficiency can result in life-threatening physiological and neurological complications. While a thiamine-deficient diet may result in the onset of such symptoms, the presence of thiaminase - an enzyme that breaks down thiamine - is very often the cause. In such instances, thiaminase counteracts the bioavailability and uptake of thiamine, even when food-thiamine levels are adequate. Here, we report on a case of failed reproduction in seven Arctic fox (Vulpes lagopus) breeding pairs kept at a captive breeding facility, including the presentation of severe thiamine deficiency symptoms in two male foxes. Symptoms included ataxia, obtundation, truncal sway, star-gazing and visual impairment. Blood tests were inconclusive, yet symptoms resolved following treatment with a series of thiamine hydrochloride injections, thereby verifying the diagnosis. A fish-dominated feed, which for the first time had been frozen for a prolonged period, was identified as the likely source of thiaminase and subsequent deterioration in the animals' health. Symptoms in the two males arose during the annual mating period. All seven breeding pairs at the captive breeding station failed to reproduce - a phenomenon never recorded during the captive breeding facility's preceding 17-year operation. Relating our findings to peer-reviewed literature, the second part of this case report assesses how thiamine deficiency (due to thiaminase activity) likely resulted in subclinical effects that impaired the production of reproduction hormones, and thereby led to a complete breeding failure. While previous work has highlighted the potentially lethal effects of thiamine deficiency in farmed foxes, this is, to our knowledge the first study showing how subclinical effects in both males and females may inhibit reproduction in foxes in general, but specifically Arctic foxes. The findings from our case report are not only relevant for captive breeding facilities, but for the welfare and management of captive carnivorous animals in general.

Molecular cloning of doublesex genes of four cladocera (water flea) species.

BACKGROUND: The gene doublesex (dsx) is known as a key factor regulating genetic sex determination in many organisms. We previously identified two dsx genes (DapmaDsx1 and DapmaDsx2) from a freshwater branchiopod crustacean, Daphnia magna, which are expressed in males but not in females. D. magna produces males by parthenogenesis in response to environmental cues (environmental sex determination) and we showed that DapmaDsx1 expression during embryonic stages is responsible for the male trait development. The D. magna dsx genes are thought to have arisen by a cladoceran-specific duplication; therefore, to investigate evolutionary conservation of sex specific expression of dsx genes and to further assess their functions in the environmental sex determination, we searched for dsx homologs in four closely related cladoceran species. RESULTS: We identified homologs of both dsx genes from, D. pulex, D. galeata, and Ceriodaphnia dubia, yet only a single dsx gene was found from Moina macrocopa. The deduced amino acid sequences of all 9 dsx homologs contained the DM and oligomerization domains, which are characteristic for all arthropod DSX family members. Molecular phylogenetic analysis suggested that the dsx gene duplication likely occurred prior to the divergence of these cladoceran species, because that of the giant tiger prawn Penaeus monodon is rooted ancestrally to both DSX1 and DSX2 of cladocerans. Therefore, this result also suggested that M. macrocopa lost dsx2 gene secondarily. Furthermore, all dsx genes identified in this study showed male-biased expression levels, yet only half of the putative 5' upstream regulatory elements are preserved in D. magna and D. pulex. CONCLUSIONS: The all dsx genes of five cladoceran species examined had similar amino acid structure containing highly conserved DM and oligomerization domains, and exhibited sexually dimorphic expression patterns, suggesting that these genes may have similar functions for environmental sex determination in cladocerans.

Predation of endangered Arctic foxes by Golden eagles: What do we know?

Dedicated conservation efforts spanning the past two decades have saved the Fennoscandian Arctic fox (Vulpes lagopus) population from local extinction, and extensive resources continue to be invested in the species' conservation and management. Although increasing, populations remain isolated, small and are not yet viable in the longer term. An understanding of causes of mortality are consequently important to optimize ongoing conservation actions. Golden eagles (Aquila chrysaetos) are a predator of Arctic foxes, yet little information on this interaction is available in the literature. We document and detail six confirmed cases of Golden eagle depredation of Arctic foxes at the Norwegian captive breeding facility (2019-2022), where foxes are housed in large open-air enclosures in the species' natural habitat. Here, timely detection of missing/dead foxes was challenging, and new insights have been gained following recently improved enclosure monitoring. Golden eagle predation peaked during the winter months, with no cases reported from June to November. This finding contrasts with that which is reported from the field, both for Arctic and other fox species, where eagle depredation peaked at dens with young (summer). While the seasonality of depredation may be ecosystem specific, documented cases from the field may be biased by higher survey efforts associated with the monitoring of reproductive success during the summer. Both white and blue color morphs were housed at the breeding station, yet only white foxes were preyed upon, and mortality was male biased. Mitigation measures and their effectiveness implemented at the facility are presented. Findings are discussed in the broader Arctic fox population ecology and conservation context.

Novel candidate genes and regions for childhood apraxia of speech identified by array comparative genomic hybridization.

PURPOSE: The goal of this study was to identify new candidate genes and genomic copy-number variations associated with a rare, severe, and persistent speech disorder termed childhood apraxia of speech. Childhood apraxia of speech is the speech disorder segregating with a mutation in FOXP2 in a multigenerational London pedigree widely studied for its role in the development of speech-language in humans. METHODS: A total of 24 participants who were suspected to have childhood apraxia of speech were assessed using a comprehensive protocol that samples speech in challenging contexts. All participants met clinical-research criteria for childhood apraxia of speech. Array comparative genomic hybridization analyses were completed using a customized 385K Nimblegen array (Roche Nimblegen, Madison, WI) with increased coverage of genes and regions previously associated with childhood apraxia of speech. RESULTS: A total of 16 copy-number variations with potential consequences for speech-language development were detected in 12 or half of the 24 participants. The copy-number variations occurred on 10 chromosomes, 3 of which had two to four candidate regions. Several participants were identified with copy-number variations in two to three regions. In addition, one participant had a heterozygous FOXP2 mutation and a copy-number variation on chromosome 2, and one participant had a 16p11.2 microdeletion and copy-number variations on chromosomes 13 and 14. CONCLUSION: Findings support the likelihood of heterogeneous genomic pathways associated with childhood apraxia of speech.

Typical renal-coloboma syndrome phenotype in a patient with a submicroscopic deletion of the PAX2 gene.

We present a patient with optic nerve hypoplasia, secondary strabismus, mild deafness, abnormal external ear helices, and renal hypoplasia. The clinical phenotype was consistent with renal-coloboma syndrome, but no point mutation in the PAX2 gene could be identified. High-resolution array comparative genomic hybridization (aCGH) analysis showed that this patient has a submicroscopic deletion on chromosome 10, affecting the entire coding region of the PAX2 gene. This finding provided the molecular confirmation of the patient's clinical diagnosis and showed that, in addition to point mutations, deletions of the PAX2 gene contribute to the etiology of the renal-coloboma syndrome.

Array comparative genomic hybridization analysis in patients with anophthalmia, microphthalmia, and coloboma.

PURPOSE: The goal of our study was to determine whether genomic copy number abnormalities (deletions and duplications) affecting genes involved in eye development contributed to the etiology of anophthalmia, microphthalmia, and coloboma. METHODS: The affected individuals were evaluated for the presence of deletions and duplications in genomic DNA by a very high-resolution array comparative genomic hybridization. RESULTS: Array analysis of 32 patients detected one case with a deletion encompassing the renal-coloboma syndrome associated gene PAX2. Nonpolymorphic copy number changes were also observed at several candidate chromosomal regions, including 6p12.3, 8q23.1q23.2, 13q31.3, 15q11.2q13.1, 16p13.13, and 20q13.13. CONCLUSIONS: This study identified the first patient with the typical phenotype of the renal-coloboma syndrome caused by a submicroscopic deletion of the coding region of the PAX2 gene. The finding suggests that PAX2 deletion testing should be performed in addition to gene sequencing as a part of molecular evaluation for the renal-coloboma syndrome. Array comparative genomic hybridization testing of 32 affected individuals showed that genomic deletions and duplications are not a common cause of nonsyndromic anophthalmia, microphthalmia, or coloboma but undoubtedly contribute to the etiology of these eye anomalies. Therefore, array comparative genomic hybridization testing represents an important and valuable addition to candidate gene sequencing in research and diagnostics of ocular birth defects.

Low molecular weight heparins differ substantially: impact on developing biosimilar drugs.

Generic drugs are an important component for meaningful health-care reform currently being debated in the United States. Aside from defining the period of drug exclusivity, however, there is a critical need to ensure that generics of biologic medicines (biosimilars) are safe and effective. For low molecular weight heparins (LMWHs), the standard of care for management of venous thromboembolism, their complex structure and polypharmacological actions make producing a generic LMWH more challenging than a generic small molecule medicine. Because biosimilar LMWHs will be used interchangeably with their branded product, inherent variability between products could lead to important differences in potency, safety, or effectiveness, including unanticipated immune responses. Awareness of the specific problems associated with biosimilar LMWH development led to new recommendations from several expert bodies. This article discusses the implications of these differences for the production of biosimilar LMWHs and provides recommendations to address the limitations in the pending U.S. Congress legislation, a well-intentioned undertaking but one that must preserve the health and welfare of citizens who require these critical care medications.

Burn injuries among children from a region-wide paediatric burns unit.

Burns are known to be a cause of the most severe childhood injuries. The purpose of this retrospective study was to investigate socio-demographic and other factors involved in children being presented to a burns unit for treatment. This is the first reported comprehensive audit of burns admissions highlighting factors that may relate to the occurrence of burns in children. Raw data was obtained from the data service unit (DSU) and the ward registers of a paediatric burns unit. Of 1249 admissions, 1156 cases (92.5%) had clearly specified causes. The number of annual admissions ranged from 225 to 281 with a mean of 250 ± 25 per year. Eighty-eight percent of burns were superficial and covered less than 10% of body surface area. The majority of cases were males (744 cases; 60%). The mean age of cases was 4 years ± 1.8 years. The major causes of burns were 'spill' (765 cases; 61%) and 'contact' (150 cases; 12%). The largest group was white British (787 cases; 63%) followed by Asian (353 cases; 28%). Mixed and African population groups made up the remaining 9%. The risk of burns injuries is higher among younger children, during the early evening, and is disproportionately high in the Asian/Asian British groups. It is suggested that there is a need to increase injury prevention towards those at greatest risk.