Transient association of MCM complex proteins with the nuclear matrix during initiation of mammalian DNA replication

The minichromosome maintenance complex (MCM2-7) is the putative DNA helicase in eukaryotes, and essential for DNA replication. By applying serial extractions to mammalian cells synchronized by release from quiescence, we reveal dynamic changes to the sub-nuclear compartmentalization of MCM2 as cells pass through late G1 and early S phase, identifying a brief window when MCM2 becomes transiently attached to the nuclear-matrix. The data distinguish 3 states that correspond to loose association with chromatin prior to DNA replication, transient highly stable binding to the nuclear-matrix coincident with initiation, and a post-initiation phase when MCM2 remains tightly associated with chromatin but not the nuclear-matrix. The data suggests that functional MCM complex loading takes place at the nuclear-matrix.     

Transitions in symbiosis: evidence for environmental acquisition and social transmission within a clade of heritable symbionts

A dynamic continuum exists from free-living environmental microbes to strict host-associated symbionts that are vertically inherited. However, knowledge of the forces that drive transitions in symbiotic lifestyle and transmission mode is lacking. Arsenophonus is a diverse clade of bacterial symbionts, comprising reproductive parasites to coevolving obligate mutualists, in which the predominant mode of transmission is vertical. We describe a symbiosis between a member of the genus Arsenophonus and the Western honey bee. The symbiont shares common genomic and predicted metabolic properties with the male-killing symbiont Arsenophonus nasoniae, however we present multiple lines of evidence that the bee Arsenophonus deviates from a heritable model of transmission. Field sampling uncovered spatial and seasonal dynamics in symbiont prevalence, and rapid infection loss events were observed in field colonies and laboratory individuals. Fluorescent in situ hybridisation showed Arsenophonus localised in the gut, and detection was rare in screens of early honey bee life stages. We directly show horizontal transmission of Arsenophonus between bees under varying social conditions. We conclude that honey bees acquire Arsenophonus through a combination of environmental exposure and social contacts. These findings uncover a key link in the Arsenophonus clades trajectory from free-living ancestral life to obligate mutualism, and provide a foundation for studying transitions in symbiotic lifestyle.Subject terms: Microbial ecology, Molecular evolution, Bacterial evolution, Bacterial genetics, Phylogenetics     

Resource partitioning by sympatric pagophilic seals in Alaska: monitoring effects of climate variation with fatty acids

Four species of pagophilic phocid seals in the Western Arctic—bearded (Erignathus barbatus), ringed (Phoca hispida), ribbon (Phoca fasciata), and spotted (Phoca largha)—are particularly vulnerable to arctic warming trends. Documenting diet composition over time is one way in which the flexibility of a species, in the face of broad ecosystem changes, can be measured. Because the fatty acid (FA) composition of depot lipids has long been known to reflect diet, we analysed the FA composition of blubber samples collected from bearded (n = 30), ringed (n = 15), ribbon (n = 32) and spotted seals (n = 24). All animals were taken near Little Diomede Island in May and June 2003, providing a unique opportunity to study interspecies differences with minimal effects of spatial and temporal variation. Bearded seal FA composition was significantly different from all other seals (P < 0.001 in all cases), ringed seal FA composition was significantly different from that of both ribbon and spotted seals (P < 0.001), but ribbon and spotted seal FA compositions could not be distinguished from each other. Overall, the blubber FA compositions of ribbon, ringed and spotted seals implied diets dominated by pelagic prey. Inferences we made about current diets of bearded and ringed seals suggest that certain prey important to them in this area in the 1970s remain important today, despite notable changes in the ecosystem. We believe that blubber FA analysis will be a useful tool in assessing the response of ice-associated pinnipeds and their ecosystem to changes associated with arctic warming.     

Management of plant health risks associated with processing of plant-based wastes: A review

The rise in international trade of plants and plant products has increased the risk of introduction and spread of plant pathogens and pests. In addition, new risks are arising from the implementation of more environmentally friendly methods of biodegradable waste disposal, such as composting and anaerobic digestion. As these disposal methods do not involve sterilisation, there is good evidence that certain plant pathogens and pests can survive these processes. The temperature/time profile of the disposal process is the most significant and easily defined factor in controlling plant pathogens and pests. In this review, the current evidence for temperature/time effects on plant pathogens and pests is summarised. The advantages and disadvantages of direct and indirect process validation for the verification of composting processes, to determine their efficacy in destroying plant pathogens and pests in biowaste, are discussed. The availability of detection technology and its appropriateness for assessing the survival of quarantine organisms is also reviewed.     

The effects of different decalcification protocols on TUNEL and general cartilage staining

Apoptosis is characterized by DNA strand breaks with a 3'-OH terminus, which are analyzed by terminal deoxy(d)-UTP nick end labeling (TUNEL). Proteinase K digestion is thought to be an essential step in the TUNEL procedure. The effects of decalcifying reagents on general staining and the TUNEL assay for cartilage sections are largely unknown. The effects of these reagents on retention and integrity of DNA in chondrocytes have not been described until now. We evaluated the effects of various decalcifying solutions, including 10% EDTA, 10% citric acid, 5% trichloroacetic acid, 5% acetic acid and a commercial hydrochloric acid-based reagent, on general cartilage staining and the TUNEL assay for cartilage. The effects of proteinase K on nucleus preservation were also examined. Decalcification with 10% EDTA gave the best result for general cartilage staining. Chondrocyte DNA was retained and intact after using this reagent. Decalcification with 10% EDTA is also the safest method of decalcification if the TUNEL assay is applied to cartilage. Proteinase K digestion may have adverse effects on nucleus preservation in cartilage. Awareness of these effects is important whenever the TUNEL assay is applied.     

A Dutch guideline for the treatment of scoliosis in neuromuscular disorders

Background

Children with neuromuscular disorders with a progressive muscle weakness such as Duchenne Muscular Dystrophy and Spinal Muscular Atrophy frequently develop a progressive scoliosis. A severe scoliosis compromises respiratory function and makes sitting more difficult. Spinal surgery is considered the primary treatment option for correcting severe scoliosis in neuromuscular disorders. Surgery in this population requires a multidisciplinary approach, careful planning, dedicated surgical procedures, and specialized after care.

Methods

The guideline is based on scientific evidence and expert opinions. A multidisciplinary working group representing experts from all relevant specialties performed the research. A literature search was conducted to collect scientific evidence in answer to specific questions posed by the working group. Literature was classified according to the level of evidence.

Results

For most aspects of the treatment scientific evidence is scarce and only low level cohort studies were found. Nevertheless, a high degree of consensus was reached about the management of patients with scoliosis in neuromuscular disorders. This was translated into a set of recommendations, which are now officially accepted as a general guideline in the Netherlands.

Conclusion

In order to optimize the treatment for scoliosis in neuromuscular disorders a Dutch guideline has been composed. This evidence-based, multidisciplinary guideline addresses conservative treatment, the preoperative, perioperative, and postoperative care of scoliosis in neuromuscular disorders.     

Early developmental influences on hepatic organogenesis

The liver is the largest of the body''s organs, with the greatest number of functions, playing a central role in coordinating metabolic homeostasis, nutrient processing and detoxification. The fetal liver forms during early gestation in response to a sequential array of distinct biological events, regulated by intrinsically programmed mechanisms and extracellular signals which instruct hepatic cells to either proliferate, differentiate or undergo apoptosis. A vast number of genes are involved in the initiation and control of liver development, many of which are sensitive to nutritional and hormonal regulation in utero. Moreover, liver mass is influenced by the gestational environment. Therefore, during periods of hepatic cell proliferation and differentiation, the developing fetal liver is sensitive to damage from both internal and external sources including teratogens, infection and nutritional deficiencies. For example, fetuses exposed to decreased materno-fetal nutrition during late gestation have a reduced liver mass, and/or perturbed liver function, which includes increased plasma LDL cholesterol and fibrinogen concentrations. These occur in conjunction with other risk factors present in the early stages of cardiovascular disease i.e. decreased glucose tolerance and insulin insensitivity in later life. Taken together, these findings suggest that liver mass, and later function, are essentially set in utero during fetal development—a process that is ultimately regulated by the intrauterine environment.Key Words: liver, development, maternal constraint, nutrition, placental insufficiency, parity, growth factor     

Differentiation of adult-type Leydig cells occurs in gonadotrophin-deficient mice

During mammalian testis development distinct generations of fetal and adult Leydig cells arise. Luteinising hormone (LH) is required for normal adult Leydig cell function and for the establishment of normal adult Leydig cell number but its role in the process of adult Leydig cell differentiation has remained uncertain. In this study we have examined adult Leydig cell differentiation in gonadotrophin-releasing hormone (GnRH)-null mice which are deficient in circulating gonadotrophins. Adult Leydig cell differentiation was assessed by measuring expression of mRNA species encoding four specific markers of adult Leydig cell differentiation in the mouse. Each of these markers (3β-hydroxysteroid dehydrogenase type VI (3βHSD VI), 17β-hydroxysteroid dehydrogenase type III (17βHSD III), prostaglandin D (PGD)-synthetase and oestrogen sulphotransferase (EST)) is expressed only in the adult Leydig cell lineage in the normal adult animal. Real-time PCR studies showed that all four markers are expressed in adult GnRH-null mice. Localisation of 3βHSD VI and PGD-synthetase expression by in situ hybridisation confirmed that these genes are expressed in the interstitial tissue of the GnRH-null mouse. Treatment of animals with human chorionic gonadotrophin increased expression of 3βHSD VI and 17βHSD III within 12 hours further indicating that differentiated, but unstimulated cells already exist in the GnRH-null mouse. Thus, while previous studies have shown that LH is required for adult Leydig cell proliferation and activity, results from the present study show that adult Leydig cell differentiation will take place in animals deficient in LH.