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Processing of a viral glycoprotein in the endoplasmic reticulum for class II presentation

Endogenous processing of viral glycoproteins for presentation to CD4(+) T cells is a poorly investigated aspect of antigen processing and presentation. This pathway may involve not only pathogens, but also self proteins, and may thus be involved in self‐tolerance. We have characterized the processing of the endoplasmic reticulum‐restricted glycoprotein (G) of vesicular stomatitis virus, termed poison tail (Gpt), biochemically and enzymatically, and by T cell recognition assays. Expressed with a vaccinia vector, Gpt remains endoglycosidase H‐sensitive and does not mature to endoglycosidase D sensitivity. The protein is degraded in the ER with a T1/2 of 4 h. Gpt peptides are not secreted since Gpt‐infected cells are unable to sensitize uninfected antigen‐presenting cells in an innocent bystander assay. Using flow cytometry, Gpt is undetectable on the plasma membrane; in contrast, wild‐type G is readily found on the surface or secreted into the milieu as soluble G following infection of A20 cells with a vaccinia recombinant expressing G. The degradation of Gpt is sensitive to the thiol reagent diamide and occurs optimally at physiological pH. A series of proteolytic inhibitors were tested: 3,4‐dichloroisocoumarin and 1‐chloro‐3‐tosylamido‐7‐amino‐2‐heptanone inhibited degradation, which suggests the involvement of a serine protease. The degradation does not require transport to the Golgi complex, and is not sensitive to a variety of lysosomotropic agents. We show that the degradation products include the immunogenic epitopes recognized by a panel of T cell clones and hybridomas.

Development and implementation of a system for the early identification of emerging risks in food and feed

According to EFSA's Founding Regulation, the Authority is required to “undertake action to identify and characterise emerging risks” in the field of food and feed safety. EFSA provides scientific advice to the risk manager, at both European and Member State level, for the identification of risks present in the food chain. In the area of currently unrecognised but potentially significant risks for public health, EFSA has set up a dedicated unit on emerging risks (EMRISK). Through the identification of drivers of emerging risks, EFSA also intends to anticipate future risks derived from changes in current food/feed production practices or factors impinging on food/feed production or changes in human exposure through food consumption. EFSA aims to establish a data monitoring capacity, data filtering methodology and networking structures to identify emerging risks and drivers of emerging risks in a timely fashion and to communicate these to the risk manager. To date, the first step of this process (data monitoring) is in place. The following steps, that is, filtering and communication, are being rapidly established. Whilst the current data sources monitored are limited, they have been sufficient to enable the elaboration of the procedures for the next steps in the emerging risks identification process. As more data sources become accessible, the process will become more effective. All processes should be in place by mid −2010 and reported on in EFSA's first annual report on emerging risks in 2011. By the end of the second year of operation (2012), the soundness and utility of this approach will be given an initial review.

Scientific Opinion on the public health hazards to be covered by inspection of meat from sheep and goats

A risk ranking process identified Toxoplasma gondii and pathogenic verocytotoxin‐producing Escherichia coli (VTEC) as the most relevant biological hazards for meat inspection of sheep and goats. As these are not detected by traditional meat inspection, a meat safety assurance system using risk‐based interventions was proposed. Further studies are required on T. gondii and pathogenic VTEC. If new information confirms these hazards as a high risk to public health from meat from sheep or goats, setting targets at carcass level should be considered. Other elements of the system are risk‐categorisation of flocks/herds based on improved Food Chain Information (FCI), classification of abattoirs according to their capability to reduce faecal contamination, and use of improved process hygiene criteria. It is proposed to omit palpation and incision from post‐mortem inspection in animals subjected to routine slaughter. For chemical hazards, dioxins and dioxin‐like polychlorinated biphenyls were ranked as being of high potential concern. Monitoring programmes for chemical hazards should be more flexible and based on the risk of occurrence, taking into account FCI, which should be expanded to reflect the extensive production systems used, and the ranking of chemical substances, which should be regularly updated and include new hazards. Control programmes across the food chain, national residue control plans, feed control and monitoring of environmental contaminants should be better integrated. Meat inspection is a valuable tool for surveillance and monitoring of animal health and welfare conditions. Omission of palpation and incision would reduce detection effectiveness for tuberculosis and fasciolosis at animal level. Surveillance of tuberculosis at the slaughterhouse in small ruminants should be improved and encouraged, as this is in practice the only surveillance system available. Extended use of FCI could compensate for some, but not all, the information on animal health and welfare lost if only visual post‐mortem inspection is applied.

The distribution, abundance and host plant relationships of Salix‐ feeding psyllids (Homoptera: Psylloidea) in arctic Alaska

Abstract. 1. Five species of psyllid occurred on seven species of Salix at Meade River, Alaska. Studies were made on the two common species Psylla pclmeni Löw and P.phlebophyllae Hodkinson. The former feeds on the phanerophy tes Salix pulchra, S.lanata, S.alaxensis and S.glauca, the latter on the chamaephytes S.phlebophylla and S.reticulata. 2. Both P.palmeni and P.phlebophyllae had a 1‐year life cycle and nymphal development took place on the female Salix catkin. The life cycle was generally closely synchronized with the period of catkin development. However, only a few eggs were laid on S.glauca 3. Seasonal perturbation of the host plant by flooding, ice movement and blown sand prevented psyllids breeding in certain areas colonized by the host plant. 4. In P.palmenidensities and ‘feeding pressure’, measured as biomass of psyllids per gram of catkin, on the different host plants followed the sequence S.pulchra>S.lanata> S.alaxensis > S.glauca. In P.phlebophyllae densities and feeding intensities were similar onS.phlebophyllaandS.reticulataand grazing intensity was comparable withP.palmenion S.pulchra. 5. A highly significant negative correlation was found between psyllid density and catkin dry weight in S.pulchra, S.phlebophylla and S.reticulata, suggesting that psyllid feeding is affecting catkin growth. 6. Predation of psyllid nymphs by syrphid larvae was heavy but there was no evidence of parasitism. 7. The life history strategies of the five psyllid species are discussed within the context of the constraints imposed by the arctic environment.

Species prioritization for monitoring and management in regional multiple species conservation plans

Successful conservation plans are not solely achieved by acquiring optimally designed reserves. Ongoing monitoring and management of the biodiversity in those reserves is an equally important, but often neglected or poorly executed, part of the conservation process. In this paper we address one of the first and most important steps in designing a monitoring program – deciding what to monitor. We present a strategy for prioritizing species for monitoring and management in multispecies conservation plans. We use existing assessments of threatened status, and the degree and spatial and temporal extent of known threats to link the prioritization of species to the overarching goals and objectives of the conservation plan. We consider both broad and localized spatial scales to capture the regional conservation context and the practicalities of local management and monitoring constraints. Spatial scales that are commensurate with available data are selected. We demonstrate the utility of this strategy through application to a set of 85 plants and animals in an established multispecies conservation plan in San Diego County, California, USA. We use the prioritization to identify the most prominent risk factors and the habitats associated with the most threats to species. The protocol highlighted priorities that had not previously been identified and were not necessarily intuitive without systematic application of the criteria; many high‐priority species have received no monitoring attention to date, and lower‐priority species have. We recommend that in the absence of clear focal species, monitoring threats in highly impacted habitats may be a way to circumvent the need to monitor all the targeted species.

Politics of monitoring and evaluation: Lessons from the AIDS epidemic

Monitoring and evaluation programs must strike a balance between generating meaningful tactical information for program managers while taking steps to ensure that public data use does not worsen discrimination and stigma toward people who are positive for the human immunodeficiency virus.

Host–parasite interaction: multiple sites in the Plasmodium vivax tryptophan‐rich antigen PvTRAg38 interact with the erythrocyte receptor band 3

Tryptophan‐rich antigens of malarial parasites interact with host molecules and play an important role in parasite survival. Merozoite expressed Plasmodium vivax tryptophan‐rich antigen PvTRAg38 binds to human erythrocytes and facilitates parasite growth in a heterlologous Plasmodium falciparum culture system. Recently, we identified band 3 in human erythrocytes as one of its receptors, although the receptor‐ligand binding mechanisms remain unknown. In the present study, using synthetic mutated peptides of PvTRAg38, we show that multiple amino acid residues of its 12 amino acid domain (KWVQWKNDKIRS) at position 197–208 interact with three different ectodomains of band 3 receptor on human erythrocytes. Our findings may help in the design of new therapeutic approaches for malaria.

Components and regulation of nuclear transport processes

The spatial separation of DNA replication and gene transcription in the nucleus and protein translation in the cytoplasm is a uniform principle of eukaryotic cells. This compartmentalization imposes a requirement for a transport network of macromolecules to shuttle these components in and out of the nucleus. This nucleo‐cytoplasmic transport of macromolecules is critical for both cell physiology and pathology. Consequently, investigating its regulation and disease‐associated alterations can reveal novel therapeutic approaches to fight human diseases, such as cancer or viral infection. The characterization of the nuclear pore complex, the identification of transport signals and transport receptors, as well as the characterization of the Ran system (providing the energy source for efficient cargo transport) has greatly facilitated our understanding of the components, mechanisms and regulation of the nucleo‐cytoplasmic transport of proteins in our cells. Here we review this knowledge with a specific emphasis on the selection of disease‐relevant molecular targets for potential therapeutic intervention.

Seasonal changes in distribution and abundance of euphausiids in the coastal area of north‐eastern Japan

ABSTRACT: Seasonal changes in distribution and abundance of euphausiids off south‐eastern Hokkaido (41°−43°N), Sanriku (38°−41°N), and Joban (36°−38°N) were investigated using cylindrical‐conical nets every two months from March 1997 to February 1998. Twenty‐six species of seven genera of euphausiids occurred during the survey. Among them, subarctic‐transitional Euphausia pacifica was the most abundant throughout the year in coastal waters, as their relative contribution to the total abundance of euphausiids was 89–92%. This species occurred in each coastal water throughout the survey and was abundant from winter to early summer (February–June) off Sanriku and Joban and in autumn in south‐eastern Hokkaido. Thysanoessa inspinata occurred off south‐eastern Hokkaido and Sanriku throughout the survey, mainly in spring (April) but rarely occurred off Joban. Three other subarctic Thysanoessa species occurred mainly off south‐eastern Hokkaido from winter to spring. Conversely, warm‐ and transitional‐water epipelagic species occurred exclusively off Sanriku and Joban in autumn. The characteristics of seasonal distributional patterns of euphausiids are discussed in relation to the spatial and temporal changes of oceanographic conditions and several predators off north‐eastern Japan.

Arabidopsis HY5 protein functions as a DNA‐binding tag for purification and functional immobilization of proteins on agarose/DNA microplate

Protein microarray is considered to be one of the key analytical tools for high‐throughput protein function analysis. Here, we report that the Arabidopsis HY5 functions as a novel DNA‐binding tag (DBtag) for proteins. We also demonstrate that the DBtagged proteins could be immobilized and purified on a newly designed agarose/DNA microplate. Furthermore, we show three applications using the microarray: (1) detection of autophosphorylation activity of DBtagged human protein kinases and inhibition of their activity by staurosporine, (2) specific cleavage of DBtagged proteins by a virus protease and caspase 3, and (3) detection of a protein–protein interaction between the DBtagged UBE2N and UBE2v1. Thus, this method may facilitate rapid functional analysis of a wide range of proteins.

Aquatic bryophytes in Himalayan streams: testing a distribution model in a highly heterogeneous environment

1. Aquatic bryophytes were sampled from 108 streams spanning over 3000 m of altitude in four regions of Nepal. Richness, cover and community composition were related to physicochemistry using multiple regression, DECORANA ordination and TWINSPAN. The performance of a hierarchically scaled descriptive model, developed in New Zealand for predicting bryophyte distribution, was examined in this highly heterogeneous Himalayan region. 2. Community composition and cover varied highly significantly with altitude, streambed stability and alkalinity, with evidence of effects of riparian land use on bryophyte cover. Cover was greatest in streams at low to middle altitudes with steep slopes (> 15°), high stability and low conductivity (< 60 μS cm(–1)), where communities were dominated by two Isopterygium spp., two Philonotis spp., Mnium punctatum and Lejeuneaceae. 3. Richness, by contrast, increased significantly but weakly at high altitude and moderate stability, where streams were dominated by Eurynchium praelongum, Rhynchostegium spp., Fissidens grandifrons and Hygroamblystegium spp. Richness and cover were lowest in unstable streams at the lowest altitude, where no single taxon was consistently most abundant. 4. Although these results were similar to those in the descriptive model developed for bryophytes in New Zealand, subtle differences were apparent. Substrate size, although influencing the presence of bryophytes in New Zealand streams, appeared to be unimportant in Nepal. By contrast, streambed stability was more important in Nepal than New Zealand, perhaps reflecting pronounced monsoonal floods, and subsequent increased frequency of bed movement in the former. A suggested habitat template indicates that large plant size and vegetative reproduction may be responsible for the widespread distribution of some species, even into unstable Himalayan streams.

Template‐assisted rational design of peptide inhibitors of furin using the lysine fragment of the mung bean trypsin inhibitor

Highly active, small‐molecule furin inhibitors are attractive drug candidates to fend off bacterial exotoxins and viral infection. Based on the 22‐residue, active Lys fragment of the mung bean trypsin inhibitor, a series of furin inhibitors were designed and synthesized, and their inhibitory activity towards furin and kexin was evaluated using enzyme kinetic analysis. The most potent inhibitor, containing 16 amino acid residues with a K (i) value of 2.45 × 10(−9) m for furin and of 5.60 × 10(−7) m for kexin, was designed with three incremental approaches. First, two nonessential Cys residues in the Lys fragment were deleted via a Cys‐to‐Ser mutation to minimize peptide misfolding. Second, residues in the reactive site of the inhibitor were replaced by the consensus substrate recognition sequence of furin, namely, Arg at P(1), Lys at P(2), Arg at P(4) and Arg at P(6). In addition, the P(7) residue Asp was substituted with Ala to avoid possible electrostatic interference with furin inhibition. Finally, the extra N‐terminal and C‐terminal residues beyond the doubly conjugated disulfide loops were further truncated. However, all resultant synthetic peptides were found to be temporary inhibitors of furin and kexin during a prolonged incubation, with the scissile peptide bond between P(1) and P(1)′ being cleaved to different extents by the enzymes. To enhance proteolytic resistance, the P(1)′ residue Ser was mutated to d‐Ser or N‐methyl‐Ser. The N‐methyl‐Ser mutant gave rise to a K (i) value of 4.70 × 10(−8) m for furin, and retained over 80% inhibitory activity even after a 3 h incubation with the enzyme. By contrast, the d‐Ser mutant was resistant to cleavage, although its inhibitory activity against furin drastically decreased. Our findings identify a useful template for the design of potent, specific and stable peptide inhibitors of furin, shedding light on the molecular determinants that dictate the inhibition of furin and kexin.

The role of ecological theory and practice in poverty alleviation and environmental conservation

The fight against global poverty has gained momentum following the creation of the Millennium Development Goals, which aim to halve extreme poverty by 2015. Traditionally, ecologists have not played leading roles in poverty alleviation. Yet, knowledge of ecosystem functions and processes can be applied to improve the lives of millions of people, suffering from hunger, lacking clean drinking water and reliable, efficient energy sources, dying from preventable diseases, and suffering disproportionately from natural disasters. Here, we describe ways in which ecologists can apply ecological theory and tools to help improve the efficacy of poverty alleviation programs.

Thylakoid protein phosphorylation in evolutionally divergent species with oxygenic photosynthesis

Phosphothreonine antibody was used to explore reversible thylakoid protein phosphorylation in vivo in evolutionally divergent organisms with oxygenic photosynthesis. Three distinct groups of organisms were found. Cyanobacteria and red algae, both with phycobilisome antenna system, did not show phosphorylation of any of the photosystem II (PSII) proteins and belong to group 1. Group 2 species, consisting of a moss, a liverwort and a fern, phosphorylated both the light‐harvesting chlorophyll a/b proteins (LHCII) and the PSII core proteins D2 and CP43, but not the D1 protein. Reversible phosphorylation of the D1 protein seems to be the latest event in the evolution of PSII protein phosphorylation and was found only in seed plants, in group 3 species. Light‐intensity‐dependent regulation of LHCII protein phosphorylation was similar in group 2 and 3 species, with maximal phosphorylation of LHCII at low light and nearly complete dephosphorylation at high light.

Characterization of inhibitory mechanism and antifungal activity between group‐1 and group‐2 phytocystatins from taro (Colocasia esculenta)

Tarocystatin from Colocasia esculenta, a group‐2 phytocystatin, is a defense protein against phytopathogenic nematodes and fungi. It is composed of a highly conserved N‐terminal region, which is homological to group‐1 cystatin, and a repetitive peptide at the C‐terminus. The purified recombinant proteins of tarocystatin, such as full‐length (FL), N‐terminus (Nt) and C‐terminus (Ct) peptides, were produced and their inhibitory activities against papain as well as their antifungal effects were investigated. Kinetic analysis revealed that FL peptide exhibited mixed type inhibition (K (ia) = 0.098 μm and K (ib) = 0.252 μm) and Nt peptide showed competitive inhibition (K (i) = 0.057 μm), whereas Ct peptide possessed weak papain activation properties. A shift in the inhibitory pattern from competitive inhibition of Nt peptide alone to mixed type inhibition of FL peptide implied that the Ct peptide has an regulatory effect on the function of FL peptide. Based on the inhibitory kinetics of FL (group‐2) and Nt (group‐1) peptides on papain activity, an inhibitory mechanism of group‐2 phytocystatins and a regulatory mechanism of extended Ct peptide have each been proposed. By contrast, the antifungal activity of Nt peptide appeared to be greater than that of FL peptide, and the Ct peptide showed no effect on antifungal activity, indicating that the antifungal effect is not related to proteinase inhibitory activity. The results are valid for most phytocystatins with respect to the inhibitory mechanism against cysteine proteinase.

An insect picornavirus may have genome organization similar to that of caliciviruses

Computer‐assisted analysis of the amino acid sequence of the product encoded by the sequenced 3′ portion of the cricket paralysis virus (CrPV), an insect picornavirus, genome showed that this protein is homologous not to the RNA‐directed RNA polymerases, as originally suggested, but to the capsid proteins of mammalian picornaviruses. Alignment of the CrPV protein sequence with those of picornavirus and calicivirus capsid proteins demonstrated that the sequenced portion of the insect picornavirus genome encodes the C‐terminal part of VP3 and the entire VP1. Thus CrPV seems to have a genome organization distinct from that of other picornaviruses but closely resembling that of caliciviruses, with the capsid proteins encoded in the 3′ part of the genome. On the other hand, the tentative phylogenetic trees generated from the VP3 alignment revealed grouping of CrPV with hepatitis A virus, a true picornavirus, not with caliciviruses. Thus CrPV may be a picornavirus with a calicivirus‐like genome organization. Different options for CrPV genome expression are discussed.

Prokaryotic and eukaryotic DNA helicases: Essential molecular motor proteins for cellular machinery

DNA helicases are ubiquitous molecular motor proteins which harness the chemical free energy of ATP hydrolysis to catalyze the unwinding of energetically stable duplex DNA, and thus play important roles in nearly all aspects of nucleic acid metabolism, including replication, repair, recombination, and transcription. They break the hydrogen bonds between the duplex helix and move unidirectionally along the bound strand. All helicases are also translocases and DNA‐dependent ATPases. Most contain conserved helicase motifs that act as an engine to power DNA unwinding. All DNA helicases share some common properties, including nucleic acid binding, NTP binding and hydrolysis, and unwinding of duplex DNA in the 3′ to 5′ or 5′ to 3′ direction. The minichromosome maintenance (Mcm) protein complex (Mcm4/6/7) provides a DNA‐unwinding function at the origin of replication in all eukaryotes and may act as a licensing factor for DNA replication. The RecQ family of helicases is highly conserved from bacteria to humans and is required for the maintenance of genome integrity. They have also been implicated in a variety of human genetic disorders. Since the discovery of the first DNA helicase in Escherichia coli in 1976, and the first eukaryotic one in the lily in 1978, a large number of these enzymes have been isolated from both prokaryotic and eukaryotic systems, and the number is still growing. In this review we cover the historical background of DNA helicases, helicase assays, biochemical properties, prokaryotic and eukaryotic DNA helicases including Mcm proteins and the RecQ family of helicases. The properties of most of the known DNA helicases from prokaryotic and eukaryotic systems, including viruses and bacteriophages, are summarized in tables.

Unfolding of in planta activity of anti‐rep ribozyme in presence of a RNA silencing suppressor

Antisense RNA ribozymes have intrinsic endonucleolytic activity to effect cleavage of the target RNA. However, this activity in vivo is often controlled by the dominance of antisense or other double‐stranded RNA mechanism. In this work, we demonstrate the in planta activity of a hammerhead ribozyme designed to target rep‐mRNA of a phytopathogen Mungbean Yellow Mosaic India virus (MYMIV) as an antiviral agent. We also found RNA‐silencing is induced on introduction of catalytically active as well as inactive ribozymes. Using RNA‐silencing suppressors (RSS), we demonstrate that the endonucleolytic activity of ribozymes is a true phenomenon, even while a mutated version may demonstrate a similar down‐regulation of the target RNA. This helps to ease the confusion over the action mechanism of ribozymes in vivo.

Spatial modelling of the infestation indices of Aedes aegypti: an innovative strategy for vector control actions in developing countries

BACKGROUND: Larval indices such as the house index (HI), Breteau index (BI) and container index (CI) are widely used to interpret arbovirus vector density in surveillance programmes. However, the use of such data as an alarm signal is rarely considered consciously when planning programmes. The present study aims to investigate the spatial distribution pattern of the infestation of Aedes aegypti, considering the data available in the Ae. aegypti Infestation Index Rapid Survey (LIRAa) for the city of Campina Grande, Paraíba State in Brazil. METHODS: The global and local Moranʼs indices were used in spatial analysis to measure the effects of spatial dependencies between neighbourhoods, using secondary data related to HI and BI gathered from surveillance service. RESULTS: Our analysis shows that there is a predominance of high rates of mosquito infestation, placing Campina Grande at a near-constant risk of arbovirus outbreaks and epidemics. A highly significant Moranʼs index value (P < 0.001) was observed, indicating a positive spatial dependency between the neighbourhoods in Campina Grande. Using the Moran mapping and LISA mapping, the autocorrelation patterns of Ae. aegypti infestation rates among neighbourhoods have revealed hotpots that should be considered a priority to preventive actions of the entomological surveillance services. Predominance of high infestation rates and clearer relationships of these between neighbourhoods were observed between the months of May and July, the period with the highest rainfall in the city. CONCLUSIONS: This analysis is an innovative strategy capable of providing detailed information on infestation locations to the relevant public health authorities, which will enable a more efficient allocation of resources, particularly for arbovirus prevention. [Image: see text]

Use of antimalarials in dermatology

The antimalarials chloroquine and hydroxychloroquine have been used for the treatment of inflammatory diseases for more than 60 years. Even today new indications evolve due to the complex mode of action of these compounds. Due to the fear of side effects, especially irreversible retinopathy, their use is often limited. These side‐effects, however, are a consequence of excessive daily dosages. An effective, safe therapy needs correct dosing, i. e. adherence to maximal daily dosages of 3.5(–4) mg chloroquine or 6(–6.5) mg hydroxychloroquine per kilogram ideal body weight. If the actual body weight is lower than the ideal body weight, this actual weight is used for the calculation of the dosage. Observing these limits allows a rather safe therapy of the diseases like lupus erythematosus, REM syndrome, porphyria cutanea tarda (2 × 125 mg chloroquine/week), cutaneous sarcoidosis and dermatomyositis. If standard therapies fail, then antimalarials can be tried to treat Sjögren syndrome, granu‐loma annulare or erosive lichen planus. If therapy fails, either can be combined with quinacrine to increase their effectiveness. Chloroquine and hydroxychloroquine are indispensable and well‐tolerated essential drugs in dermatology and especially suited as part of a combination scheme, for example with corticosteroids, as they act synergistically and reduce side‐effects.

Mechanopharmacology and Synergistic Relaxation of Airway Smooth Muscle

Asthmatic airways are stiffer than normal. We have shown that the cytoskeletal passive stiffness of airway smooth muscle (ASM) can be regulated by intracellular signaling pathways, especially those associated with Rho kinase (ROCK). We have also shown that an oscillatory strain reduces the passive stiffness of ASM and its ability to generate force. Here, we investigated the combined effect of inhibiting the ASM contraction with β(2) agonist and decreasing the ASM cytoskeletal stiffness with ROCK inhibitor and/or force oscillation (FO) on the relaxation of contracted ASM. We hypothesize that the ASM relaxation can be synergistically enhanced by the combination of these interventions, because drug-induced softening of the cytoskeleton enhances the FO-induced relaxation and vice versa. Sheep tracheal strips were isotonically contracted to acetylcholine (3 × 10(−5) M). At the plateau of shortening, β(2) agonist salbutamol (10(−7) M), ROCK inhibitor H1152 (10(−7) M), and FO (square wave, 1 Hz, amplitude 6% maximal active force) were applied either alone or in combination. After adjusting for nonspecific time-dependent variation, relengthening by individual interventions with low-dose salbutamol or H1152, or small amplitude FO was not significantly different from zero. However, significant relengthening was observed in all combination treatments. The relengthening was greater than the mathematical sum of relengthening caused by individual treatments thereby demonstrating synergistic relaxation. The ASM stiffness did not change with salbutamol or H1152 treatments, but was lower with FO in combination with H1152. The results suggest that the mechanopharmacological treatment can be an effective therapy for asthma.

Preliminary Development and Engineering Evaluation of a Novel Cricothyrotomy Device

Cricothyrotomy is one of the procedures used to ventilate patients with upper airway blockage. This paper examines the most regularly used and preferred cricothyrotomy devices on the market, suggests critical design specifications for improving cricothyrotomy devices, introduces a new cricothyrotomy device, and performs an engineering evaluation of the device’s critical components. Through a review of literature, manufacturer products, and patents, four principal cricothyrotomy devices currently in clinical use were identified. From the review, the Cook™ Melker device is the preferred method of clinicians but the device has acknowledged problems. A new emergency needle cricothyrotomy device (ENCD) was developed to address all design specifications identified in literature. Engineering, theoretical, and experimental assessments were performed. In situ evaluations of a prototype of the new device using porcine specimens to assess insertion, extraction, and cyclic force capabilities were performed. The device was very successful in its evaluation. Further discussion focuses on these aspects and a comparison of the new device with established devices. The proposed emergency needle cricothyrotomy device performed very well. Further work will be pursued in the future with in-vitro and in-vivo with canine models demonstrates the capabilities of the ENCD.

The Effect of Chemical Cleaning on Mechanical Properties of Three-Dimensional Printed Polylactic Acid

Three-dimensional (3D) printing may be a solution to shortages of equipment and spare parts in the healthcare sector of low- and middle-income countries (LMICs). Polylactic acid (PLA) for 3D printing is widely available and biocompatible, but there is a gap in knowledge concerning its compatibility with chemical disinfectants. In this study, 3D-printed PLA tensile samples were created with six different printer settings. Each of these six batches consisted of five sets with five or six samples. The first set remained untreated, the others were soaked in Cidex OPA or in a chlorine solution. These were applied for seven consecutive days or in 25 short cycles. All samples were weighed before and after treatment and subjected to a tensile test. Results showed that a third of the treatments led to an increase of the median weight with a maximum of 8.3%, however, the samples with the best surface quality did not change. The median strength increase was 12.5% and the largest decrease was 8.8%. The median stiffness decreased 3.6% in one set and increased in three others up to 13.6%. When 3D printing PLA medical tools, surface porosity must be minimized to prevent transfer of disinfectants to people. The wide variability of mechanical properties due to 3D printing itself and as a consequence of disinfection must be considered when designing medical tools by selecting appropriate printer settings. If these conditions are met, reusing 3D-printed PLA medical tools seems safe from a mechanical point of view.

Design and Prototyping of a Low-Cost Portable Mechanical Ventilator

This paper describes the design and prototyping of a low-cost portable mechanical ventilator for use in mass casualty cases and resource-poor environments. The ventilator delivers breaths by compressing a conventional bag-valve mask (BVM) with a pivoting cam arm, eliminating the need for a human operator for the BVM. An initial prototype was built out of acrylic, measuring [Formula: see text] and weighing 9 lbs. It is driven by an electric motor powered by a 14.8 VDC battery and features an adjustable tidal volume up to a maximum of 750 ml. Tidal volume and number of breaths per minute are set via user-friendly input knobs. The prototype also features an assist-control mode and an alarm to indicate overpressurization of the system. Future iterations of the device will include a controllable inspiration to expiration time ratio, a pressure relief valve, PEEP capabilities, and an LCD screen. With a prototyping cost of only $420, the bulk-manufacturing price for the ventilator is estimated to be less than $200. Through this prototype, the strategy of cam-actuated BVM compression is proven to be a viable option to achieve low-cost, low-power portable ventilator technology that provides essential ventilator features at a fraction of the cost of existing technology.

Custom-Fit Three-Dimensional-Printed BiPAP Mask to Improve Compliance in Patients Requiring Long-Term Noninvasive Ventilatory Support

Noninvasive ventilator support using bi-level positive airway pressure/continuous positive airway pressure (BiPAP/CPAP) is commonly utilized for chronic medical conditions like sleep apnea and neuromuscular disorders like amyotrophic lateral sclerosis (ALS) that lead to weakness of respiratory muscles. Generic masks come in standard sizes and are often perceived by patients as being uncomfortable, ill-fitting, and leaky. A significant number of patients are unable to tolerate the masks and eventually stop using their devices. The goal of this project is to develop custom-fit masks to increase comfort, decrease air leakage, and thereby improve patient compliance. A single-patient case study of a patient with variant ALS was performed to evaluate the custom-fit masks. His high nose bridge and overbite of lower jaw caused poor fit with generic masks, and he was noncompliant with his machine. Using desktop Stereolithography three-dimensional (3D) printing and magnetic resonance imaging (MRI) data, a generic mask was extended with a rigid interface such that it was complementary to the patient's unique facial contours. Patient or clinicians interactively select a desired mask shape using a newly developed computer program. Subsequently, a compliant silicone layer was applied to the rigid interface. Ten different custom-fit mask designs were made using computer-aided design software. Patient evaluated the comfort, extent of leakage, and satisfaction of each mask via a questionnaire. All custom-fit masks were rated higher than the standard mask except for two. Our results suggest that modifying generic masks with a 3D-printed custom-fit interface is a promising strategy to improve compliance with BiPAP/CPAP machines.

Saponins of Dioscorea Nipponicae Inhibits IL-17A-Induced Changes in Biomechanical Behaviors of In Vitro Cultured Human Airway Smooth Muscle Cells

Airway hyperresponsiveness (AHR) is one of the main pathologic features of bronchial asthma, which is largely attributable to enhanced contractile response of asthmatic airway smooth muscle. Although β2 adrenergic receptor agonists are commonly used to relax airway smooth muscle for treating AHR, there are side effects such as desensitization of long-term use. Therefore, it is desirable to develop alternative relaxant for airway smooth muscle, preferably based on natural products. One potential candidate is the inexpensive and widely available natural herb saponins of Dioscorea nipponicae (SDN), which has recently been reported to suppress the level of inflammatory factor IL-17A in ovalbumin-induced mice, thereby alleviating the inflammation symptoms of asthma. Here, we evaluated the biomechanical effect of SDN on IL-17A-mediated changes of cultured human airway smooth muscle cells (HASMCs) in vitro. The stiffness and traction force of the cells were measured by optical magnetic twisting cytometry (OMTC), and Fourier transform traction microscopy (FTTM), respectively. The cell proliferation was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetry, the cell migration was measured by cell scratch test, and the changes of cell cytoskeleton were assessed by laser confocal microscopy. We found that the stiffness and traction force of HASMCs were enhanced along with the increases of IL-17A concentration and exposure time, and SDN treatment dose-dependently reduced these IL-17A-induced changes in cell mechanical properties. Furthermore, SDN alleviated IL-17A-mediated effects on HASMCs proliferation, migration, and cytoskeleton remodeling. These results demonstrate that SDN could potentially be a novel drug candidate as bronchodilator for treating asthma-associated AHR.

Airway Transmural Pressures in an Airway Tree During Bronchoconstriction in Asthma

Airway transmural pressure in healthy homogeneous lungs with dilated airways is approximately equal to the difference between intraluminal and pleural pressure. However, bronchoconstriction causes airway narrowing, parenchymal distortion, dynamic hyperinflation, and the emergence of ventilation defects (VDefs) affecting transmural pressure. This study aimed to investigate the changes in transmural pressure caused by bronchoconstriction in a bronchial tree. Transmural pressures before and during bronchoconstriction were estimated using an integrative computational model of bronchoconstriction. Briefly, this model incorporates a 12-generation symmetric bronchial tree, and the Anafi and Wilson model for the individual airways of the tree. Bronchoconstriction lead to the emergence of VDefs and a relative increase in peak transmural pressures of up to 84% compared to baseline. The highest increase in peak transmural pressure occurred in a central airway outside of VDefs, and the lowest increase was 27% in an airway within VDefs illustrating the heterogeneity in peak transmural pressures within a bronchial tree. Mechanisms contributing to the increase in peak transmural pressures include increased regional ventilation and dynamic hyperinflation both leading to increased alveolar pressures compared to baseline. Pressure differences between intraluminal and alveolar pressure increased driven by the increased airway resistance and its contribution to total transmural pressure reached up to 24%. In conclusion, peak transmural pressure in lungs with VDefs during bronchoconstriction can be substantially increased compared to dilated airways in healthy homogeneous lungs and is highly heterogeneous. Further insights will depend on the experimental studies taking these conditions into account.

The Aftermath of Bronchoconstriction

Asthma is characterized by chronic airway inflammation, airway remodeling, and excessive constriction of the airway. Detailed investigation exploring inflammation and the role of immune cells has revealed a variety of possible mechanisms by which chronic inflammation drives asthma development. However, the underlying mechanisms of asthma pathogenesis still remain poorly understood. New evidence now suggests that mechanical stimuli that arise during bronchoconstriction may play a critical role in asthma development. In this article, we review the mechanical effect of bronchoconstriction and how these mechanical stresses contribute to airway remodeling independent of inflammation.

Myosin Crossbridge, Contractile Unit, and the Mechanism of Contraction in Airway Smooth Muscle: A Mechanical Engineer's Perspective

Muscle contraction is caused by the action of myosin motors within the structural confines of contractile unit arrays. When the force generated by cyclic interactions between myosin crossbridges and actin filaments is greater than the average load shared by the crossbridges, sliding of the actin filaments occurs and the muscle shortens. The shortening velocity as a function of muscle load can be described mathematically by a hyperbola; this characteristic force–velocity relationship stems from stochastic interactions between the crossbridges and the actin filaments. Beyond the actomyosin interaction, there is not yet a unified theory explaining smooth muscle contraction, mainly because the structure of the contractile unit in smooth muscle (akin to the sarcomere in striated muscle) is still undefined. In this review, functional and structural data from airway smooth muscle are analyzed in an engineering approach of quantification and correlation to support a model of the contractile unit with characteristics revealed by mathematical analyses and behavior matched by experimental observation.

The Role of Airway Shunt Elastance on the Compartmentalization of Respiratory System Impedance

An inverse model consisting of two elastic compartments connected in series and served by two airway conduits has recently been fit to measurements of respiratory impedance in obese subjects. Increases in the resistance of the distal conduit of the model with increasing body mass index have been linked to peripheral airway compression by mass loading of the chest wall. Nevertheless, how the two compartments and conduits of this simple model map onto the vastly more complicated structure of an actual lung remain unclear. To investigate this issue, we developed a multiscale branching airway tree model of the respiratory system that predicts realistic input impedance spectra between 5 and 20 Hz with only four free parameters. We use this model to study how the finite elastances of the conducting airway tree and the proximal upper airways affect impedance between 5 and 20 Hz. We show that progressive constriction of the peripheral airways causes impedance to appear to arise from two compartments connected in series, with the proximal compartment being a reflection of the elastance of upper airway structures proximal to the tracheal entrance and the lower compartment reflecting the pulmonary airways and tissues. We thus conclude that while this simple inverse model allows evaluation of overall respiratory system impedance between 5 and 20 Hz in the presence of upper airway shunting, it does not allow the separate contributions of central versus peripheral pulmonary airways to be resolved.

Internet-of-Things-Enabled Dual-Channel Iontophoretic Drug Delivery System for Elderly Patient Medication Management

Wireless controllers have found its application in the supervision of the patients in the hospitals. It is not only a valid issue for the developing countries but also for the developed countries. For this reason, scientists are working on the advancement of medical devices that are capable of decreasing the workload of health caregivers. In this study, the development of an iontophoretic drug delivery device that could be controlled using a mobile is described. For the purpose, hardware and a software module were developed. The hardware module consisted of a two-channel voltage-controlled constant current sources that were used for driving the iontophoretic device. A mobile app was developed to control the two-channel iontophoretic device and to monitor the loose lead of the active and the passive patches. In the case of detection of the loose lead, the specific iontophoretic channel was stopped. Further, the audio-visual indicator was developed for the detection of the detachment of the patches (loose lead). The device was tested in vitro by performing the drug release study using drug-loaded emulsion gels that were formulated.

The Strain on Airway Smooth Muscle During a Deep Inspiration to Total Lung Capacity

The deep inspiration (DI) maneuver entices a great deal of interest because of its ability to temporarily ease the flow of air into the lungs. This salutary effect of a DI is proposed to be mediated, at least partially, by momentarily increasing the operating length of airway smooth muscle (ASM). Concerningly, this premise is largely derived from a growing body of in vitro studies investigating the effect of stretching ASM by different magnitudes on its contractility. The relevance of these in vitro findings remains uncertain, as the real range of strains ASM undergoes in vivo during a DI is somewhat elusive. In order to understand the regulation of ASM contractility by a DI and to infer on its putative contribution to the bronchodilator effect of a DI, it is imperative that in vitro studies incorporate levels of strains that are physiologically relevant. This review summarizes the methods that may be used in vivo in humans to estimate the strain experienced by ASM during a DI from functional residual capacity (FRC) to total lung capacity (TLC). The strengths and limitations of each method, as well as the potential confounders, are also discussed. A rough estimated range of ASM strains is provided for the purpose of guiding future in vitro studies that aim at quantifying the regulatory effect of DI on ASM contractility. However, it is emphasized that, owing to the many limitations and confounders, more studies will be needed to reach conclusive statements.

Medical Devices for Low- and Middle-Income Countries: A Review and Directions for Development

The development of diagnostics and medical devices has historically been concentrated in high-income countries, despite a significant need to expand healthcare services to low- and middle-income countries (LMIC). Poor quality healthcare extends beyond LMIC to underserved communities in developed countries. This paper reviews diseases and conditions that have not received much attention in the past despite imposing a significant burden on healthcare systems in these circumstances. We review the underlying mechanism of action of these conditions and current technology in use for diagnosis or surgical intervention. We aim to identify areas for technological development and review policy considerations that will enable real-world adoption. Specifically, this review focuses on diseases prevalent in sub-Saharan Africa and south Asia: melioidosis, infant and maternal mortality, schistosomiasis, and heavy metal and pesticide poisoning. Our aim with this review is to identify problems facing the world that require the attention of the medical device community and provide recommendations for research directions for groups interested in this field.

Structured Tree Impedance Outflow Boundary Conditions for 3D Lung Simulations

In this paper, we develop structured tree outflow boundary conditions for modeling the airflow in patient specific human lungs. The utilized structured tree is used to represent the nonimageable vessels beyond the 3D domain. The coupling of the two different scales (1D and 3D) employs a Dirichlet–Neumann approach. The simulations are performed under a variety of conditions such as light breathing and constant flow ventilation (which is characterized by very rapid acceleration and deceleration). All results show that the peripheral vessels significantly impact the pressure, however, the flow is relatively unaffected, reinforcing the fact that the majority of the lung impedance is due to the lower generations rather than the peripheral vessels. Furthermore, simulations of a hypothetical diseased lung (restricted flow in the superior left lobe) under mechanical ventilation show that the mean pressure at the outlets of the 3D domain is about 28% higher. This hypothetical model illustrates potential causes of volutrauma in the human lung and furthermore demonstrates how different clinical scenarios can be studied without the need to assume the unknown flow distribution into the downstream region.

Reorganization of the Vimentin Network in Smooth Muscle

Vimentin intermediate filaments (IFs) link to desmosomes (intercellular junctions) on the membrane and dense bodies in the cytoplasm, which provides a structural base for intercellular and intracellular force transmission in smooth muscle. There is evidence to suggest that the vimentin framework plays an important role in mediating smooth muscle mechanical properties such as tension and contractile responses. Contractile activation induces vimentin phosphorylation at Ser-56 and vimentin network reorientation, facilitating contractile force transmission among and within smooth muscle cells. p21-activated kinase 1 and polo-like kinase 1 catalyze vimentin phosphorylation at Ser-56, whereas type 1 protein phosphatase dephosphorylates vimentin at this residue. Vimentin filaments are also involved in other cell functions including migration and nuclear positioning. This review recapitulates our current knowledge how the vimentin network modulates mechanical and biological properties of smooth muscle.

Diagnostics as the Key to Advances in Global Health: Proposed Methods for Making Reliable Diagnostics Widely Available

This paper proposes a structure and method for the development of an AI diagnostic system as a highly leveraged step toward improvements in delivery of healthcare in underserved regions. First, the paper provides a high-level, general review of the current efforts to provide healthcare services in underserved areas and the many efforts being made to impact health outcomes by various international, governmental, and NGO entities. We also very briefly review university programs and research institutions that have specific technical and institutional assets with significant potential to carry out research or to partially implement such a plan. Our review uses weighted values in a decision-system that takes in a variety of assets we consider fundamental to successful engagement in delivery of new, innovative, technology-enabled healthcare systems for under-resourced settings. We then review nine factors that hinder the advancement in healthcare in under-resourced settings, some of which are well described in current literature and some that may bring new perspectives. The paper then attempts to review how a proposed system can manage to operate successfully within the context of the nine named hindrance factors. The primary focus of the paper is in the description of a system which can increase the availability of diagnostics through technology-enabled systems. Such a system would impact the outcomes of persons in underserved regions. The paper then describes why making diagnostics available is a critical priority among efforts for improvements in global health.

Flow and Particle Dispersion in Lung Acini: Effect of Geometric and Dynamic Parameters During Synchronous Ventilation

The human lung comprises about 300 million alveoli which are located on bronchioles between the 17th to 24th generations of the acinar tree, with a progressively higher population density in the deeper branches (lower acini). The alveolar size and aspect ratio change with generation number. Due to successive bifurcation, the flow velocity magnitude also decreases as the bronchiole diameter decreases from the upper to lower acini. As a result, fluid dynamic parameters such as Reynolds (Re) and Womersley (α) numbers progressively decrease with increasing generation number. In order to characterize alveolar flow patterns and inhaled particle transport during synchronous ventilation, we have conducted measurements for a range of dimensionless parameters physiologically relevant to the upper acini. Acinar airflow patterns were measured using a simplified in vitro alveolar model consisting of a single transparent elastic truncated sphere (representing the alveolus) mounted over a circular hole on the side of a rigid circular tube (representing the bronchiole). The model alveolus was capable of expanding and contracting in-phase with the oscillatory flow through the bronchiole thereby simulating synchronous ventilation. Realistic breathing conditions were achieved by exercising the model over a range of progressively varying geometric and dynamic parameters to simulate the environment within several generations of the acinar tree. Particle image velocimetry was used to measure the resulting flow patterns. Next, we used the measured flow fields to calculate particle trajectories to obtain particle transport and deposition statistics for massless and finite-size particles under the influence of flow advection and gravity. Our study shows that the geometric parameters (β and ΔV/V) primarily affect the velocity magnitudes, whereas the dynamic parameters (Re and α) distort the flow symmetry while also altering the velocity magnitudes. Consequently, the dynamic parameters have a greater influence on the particle trajectories and deposition statistics compared to the geometric parameters. The results from this study can benefit pulmonary research into the risk assessment of toxicological inhaled aerosols, and the pharmaceutical industry by providing better insight into the flow patterns and particle transport of inhalable therapeutics in the acini.

The effect of inhaled and intranasal sodium cromoglyeate on symptoms of upper respiratory tract infections

Background A well established drug for the treatment of asthma and allergy, sodium cromoglycale, was found in open trials to be useful as a symptomalic treatment for upper respiratory tract infections. Objective To compare the efficacy of inhaled and intranasal sodium cromoglyeate and matching placebos on the symptoms of upper respiratory tract infections. Methods Adult subjects with symptoms of runny nose, throat pain, or cough for less than 24 h were recruited. They were treated for 7 days using a randomized, double‐blind, placebo‐controlled, group comparative design. The medication given was: sodium cromoglyeate dry powder 20mg per inhalation in spincaps; sodium cromoglycate aqueous nasal spray delivering 5.2mg per dose; or matching placebo as dry powder and nasal spray. One spincap and one spray per nostril were taken every 2h during waking hours on days 1 and 2 and then four times daily on days 3–7. Severity of nine symptoms (general malaise, body aches and pains, chills and shivering, snzeening, nasal running, nasal blocking, sore throat, cough and voice disturbance) was recorded twice daily by subjects on diary cards, using a scale of 0 (absent) to 3 (severe). Results The sludy was conducted between February and April 1993. One hundred and eighteen patients aged 21–63 years (mean 41 years) were included. Symptoms resolved faster (P < 0.001) and the severity in the last three days of treatment was significantly less in patients treated with sodium cromoglycate than with placebo (P < 0.05‐day 5; P < 0.01‐day 6; P < 0.001‐day 7). Side‐effects were local and mild and did not differ between the treatment groups. Conclusion Sodium cromoglyeate administered both by inhalation and intranasally is an effective treatment for the symptoms of upper respiratory tract infection. Its combined safety and efficacy would make it an acceptable form of treatment for these conditions.

Amplified rhinovirus colds in atopic subjects

Abstract. Evidence suggests that atopic individuals may be predisposed to more severe rhinoviral colds coupled to a worsening of existing airway disease than those with asthma. The role of atopy and IgE levels, as well as their relationship to clinical disease expression have not been defined. We hypothesized that an allergic diathesis modulates rhinoviral colds and have initiated studies of normal, atopic and asthmatic subjects employing experimental rhinoviral infection, with measurements of symptom scores, viral shedding and cultures, albumin in nasal washes and serological responses. Twenty‐two subjects (11 normal, 5 atopic, 6 atopic and asthmatic) participated and were inoculated with human rhinovirus serotype 16 (HRV 16). Measurements of neutralizing antibody and viral culture were performed at screening, pre‐inoculation, during the cold and at 8–10 weeks convalescence. Daily symptoms were noted, nasal washes done, IgE measured and atopy was diagnosed by skin tests. Seventeen volunteers developed clinical colds as assessed by symptom scores, virus shedding was demonstrated (with positive culture) in all subjects and a fourfold or higher seroconversion occurred in 11/22. Neutralizing HRV antibody developed unexpectedly in 10 subjects between screening and inoculation and the presence or absence of this pre‐inoculation antibody determined subsequent severity of colds in normal but not in atopic subjects. Atopic antibody positive individuals developed severe clinical colds that were independent of preinoculation antibody in contrast to normal subjects who developed mild colds in the presence of a neutralizing antibody (.P= 0.01). Both atopic and normal antibody negative subjects developed severe colds. This differential response was matched by nasal wash albumin levels which were significantly increased (P= 0–01) during the cold in atopic (but not in normal) volunteers with pre‐inoculation antibody. Levels of IgE were not correlated with severity of clinical disease or viral shedding. Our studies of HRV disease in atopic subjects suggest heightened susceptibility to the detrimental effects of colds; additional studies are needed to clarify the relevant mechanisms.

Effect of experimental rhinovirus 16 colds on airway hyperresponsiveness to histamine and interleukin‐8 in nasal lavage in asthmatic subjects in vivo

Background Asthma exacerbations are closely associated with respiratory virus infections. However, the pathophysiological consequences of such infections in asthma are largely unclear. Objective To examine the effect of rhinovirus 16 (RV16) infection on airway hypersensitivity to histamine. and on interleukin‐8 (IL‐8) in nasal lavage. Objective Twenty‐seven non‐smoking atopic, mildly asthmatic subjects participated in a placebo‐controlled, parallel study. A dose of 0.5–2.9 ± 10(4) TCID50 RV16 or placebo was nasally administered. Cold symptoms were recorded by questionnaire throughout the study. Histamine challenges were performed at entry, and on days 4 and 11 after inoculation. Nasal lavages were obtained at entry, and on days 2 and 9. The response to histamine was measured by PC(20) (changes expressed as doubling doses: DD). IL‐8 levels were obtained by ELISA, and were expressed in ng/ml. Results RV infection was confirmed by culture of nasal lavage and/or by antibody titre rise in each of the RV16‐treated subjects. Among the 19 RV16‐treated subjects, eight developed severe cold symptoms. Baseline FEV(1) did not change significantly during the study in either treatment group (P= 0.99). However, in the RV16‐treated subjects there was a decrease in PC(20) at day 4, which was most pronounced in those with a severe cold (mean change ± SEM: –1.14 ± 0.28 DD, P= 0.01). In addition. IL‐8 levels increased in tbe RV16 group at days 2 and 9 (P < 0.001). The increase in nasal IL‐8 at day 2 correlated significantly with the change in PC(20) at day 4 (r=–0.48, P= 0.04). Conclusion We conclude that the severity of cold, as induced by experimental RV16 infection, is a determinant of the increase in airway hypersensitivity to histamine in patients with asthma. Our results suggest that this may be mediated by an infiammatory mechanism, involving the release of chemokines such as IL‐8.

Bronchial inflammation and the common cold: a comparison of atopic and non‐atopic individuals

Background Cold virus infections are associated with asthma attacks and with increased bronchial responsiveness even in normal subjects. Possible mechanisms include epithelial damage, interaction with adhesion molecules or with T‐helper cell subsets. Objective To determine whether colds increase lower airway inflammation, comparing atopic with non‐atopic normal subjects. Methods Thirty healthy volunteers (15 atopic) took part. Basehne tests included viral serology. microbiological culture and polymerase chain reaction for rhinovirus infection (HRV‐PCR), histamine bronchial provocation and bronchoscopy. Twenty subjects (eight atopic) underwent repeat tests when they developed a cold. Results Forced expiratory volume in one second (FEV(1)) was significantly lower during colds (‐0.19L [95% confidence mterval ‐0.10, ‐0.29], P= 0,0004) and there was a significant increase in bronchial responsiveness (+0.62 doublings of the dose‐response slope [+0.24, +1.00], P=0.003). Eight subjects (two atopic) had a diagnosed viral infection: two HRV. three coronavirus (HCV), one HRV + HCV, one parainfluenza III(PI) and one respiratory syncytial virus (RSV) (also Haemophilus influenzae). In biopsies, during colds, total eosinophils (EG1(+)) increased significantly (geometric mean 6.73‐fold [1.12,40.46], P=O.04). Activated eosinophils (EG2(+)) only increased significantly in the subgroup without diagnosed viral infection and particularly in atopic rhinitics. T‐suppressor (CD8(+)) cells also increased significantly (median +178.3 cells mm(2), P= 0.004). Epithelial expression of intercellular adhesion molecule‐1 (ICAM‐1) expression increased in four atopic rhinitics during colds. Bronchial washings showed a significant increase in neutrophils (GM 1.53‐fold [1.04,2.25], P= 0.02). Conclusion Lower airway inflammation was present in atopic and non‐atopic normal subjects with colds. Atopic subjects differed in that they were less likely to have positive virological tests and were more likely to show activated eosinophilia in the lower airway, despite a similar spectrum of symptoms.

A Single Ventilator for Multiple Simulated Patients to Meet Disaster Surge

Objectives To determine if a ventilator available in an emergency department could quickly be modified to provide ventilation for four adults simultaneously. Methods Using lung simulators, readily available plastic tubing, and ventilators (840 Series Ventilator; Puritan‐Bennett), human lung simulators were added in parallel until the ventilator was ventilating the equivalent of four adults. Data collected included peak pressure, positive end‐expiratory pressure, total tidal volume, and total minute ventilation. Any obvious asymmetry in the delivery of gas to the lung simulators was also documented. The ventilator was run for almost 12 consecutive hours (5.5 hours of pressure control and more than six hours of volume control). Results Using readily available plastic tubing set up to minimize dead space volume, the four lung simulators were easily ventilated for 12 hours using one ventilator. In pressure control (set at 25 mm H(2)O), the mean tidal volume was 1,884 mL (approximately 471 mL/lung simulator) with an average minute ventilation of 30.2 L/min (or 7.5 L/min/lung simulator). In volume control (set at 2 L), the mean peak pressure was 28 cm H(2)O and the minute ventilation was 32.5 L/min total (8.1 L/min/lung simulator). Conclusions A single ventilator may be quickly modified to ventilate four simulated adults for a limited time. The volumes delivered in this simulation should be able to sustain four 70‐kg individuals. While further study is necessary, this pilot study suggests significant potential for the expanded use of a single ventilator during cases of disaster surge involving multiple casualties with respiratory failure.

Isozymes, and the status of Taraxacum (Asteraceae) agamospecies

HUGHES, L. & RICHARDS, A. J., 1989. Isozymes and the status of Taraxacum (Asteraceae) agamospecies. Genetic identities I and Similarity Indices SI are calculated between 12 samples of Taraxacum, on the basis of 40 isozymes at 15 loci for 10 enzyme systems. Samples included three polyploid agamospermous populations from northern England (group 1), three sexual diploid populations from south‐central France (group 2), and six accessions of ‘primitive’ diploid self‐compatible sexual taxa from southern Europe and Australia. Samples could be assigned to eight species, classified in seven sections of the genus. Two clusters of high relationship were evident. All the group 1 and group 2 species were very closely related, with pairwise comparisons for I in excess of 0.93. The three group 3 accessions identified as T. bessarabicum showed pairwise comparisons for SI in excess of 0.71. Comparisons for SI between the other group 3 species, and between all the group 3 species and the group 1 and 2 species were all very low, not exceeding 0.45. It is concluded that dissimilarity between samples as assessed by isozymes is probably related to the time of evolutionary divergence of those samples. Although allopolyploid, and morphologically very diverse, the group 1 agamospecies may have very recently diverged asexually from a common stock. The group 2 diploids may have resulted from rediploidization and regained sexuality from the same originally agamospermous stock. In areas of Europe in which such ‘modern’ sexuality is common, it is probable that all ‘modern’ Taraxaca, including at least five sections of the genus, should be included within a single taxon. In contrast, ‘primitive’ self‐compatible sexual species in group 3 appear to have diverged from each other several million years ago, and with the exception of the disjunct accessions of T. bessarabicum, are genetically highly distinct. Such species should be maintained in the taxonomies of all areas. It is probable that an agamospecies classification of ‘modern’Taraxacum will continue to convey much useful information in areas, such as northern Europe, in which sexuality is absent.

VENTILatOry strategies in patients with severe traumatic brain injury: the VENTILO Survey of the European Society of Intensive Care Medicine (ESICM)

BACKGROUND: Severe traumatic brain injury (TBI) patients often develop acute respiratory failure. Optimal ventilator strategies in this setting are not well established. We performed an international survey to investigate the practice in the ventilatory management of TBI patients with and without respiratory failure. METHODS: An electronic questionnaire, including 38 items and 3 different clinical scenarios [arterial partial pressure of oxygen (PaO(2))/inspired fraction of oxygen (FiO(2)) > 300 (scenario 1), 150–300 (scenario 2), < 150 (scenario 3)], was available on the European Society of Intensive Care Medicine (ESICM) website between November 2018 and March 2019. The survey was endorsed by ESICM. RESULTS: There were 687 respondents [472 (69%) from Europe], mainly intensivists [328 (48%)] and anesthesiologists [206 (30%)]. A standard protocol for mechanical ventilation in TBI patients was utilized by 277 (40%) respondents and a specific weaning protocol by 198 (30%). The most common tidal volume (TV) applied was 6–8 ml/kg of predicted body weight (PBW) in scenarios 1–2 (72% PaO(2)/FIO(2) > 300 and 61% PaO(2)/FiO(2) 150–300) and 4–6 ml/kg/PBW in scenario 3 (53% PaO(2)/FiO(2) < 150). The most common level of highest positive end-expiratory pressure (PEEP) used was 15 cmH(2)O in patients with a PaO(2)/FiO(2) ≤ 300 without intracranial hypertension (41% if PaO(2)/FiO(2) 150–300 and 50% if PaO(2)/FiO(2) < 150) and 10 cmH(2)O in patients with intracranial hypertension (32% if PaO(2)/FiO(2) 150–300 and 33% if PaO(2)/FiO(2) < 150). Regardless of the presence of intracranial hypertension, the most common carbon dioxide target remained 36–40 mmHg whereas the most common PaO(2) target was 81–100 mmHg in all the 3 scenarios. The most frequent rescue strategies utilized in case of refractory respiratory failure despite conventional ventilator settings were neuromuscular blocking agents [406 (88%)], recruitment manoeuvres [319 (69%)] and prone position [292 (63%)]. CONCLUSIONS: Ventilatory management, targets and practice of adult severe TBI patients with and without respiratory failure are widely different among centres. These findings may be helpful to define future investigations in this topic.

Comparison of ELISA with electro-chemiluminescence technology for the qualitative and quantitative assessment of serological responses to vaccination

BACKGROUND: Profiling immune responses induced by either infection or vaccination can provide insight into identification of correlates of protection. Furthermore, profiling of serological responses can be used to identify biomarkers indicative of exposure to pathogens. Conducting such immune surveillance requires readout methods that are high-throughput, robust, and require small sample volumes. While the enzyme-linked immunosorbent assay (ELISA) is the classical readout method for assessing serological responses, the advent of multiplex assays has significantly increased the throughput and capacity for immunoprofiling. This report describes the development and assay performance (sensitivity, linearity of detection, requirement for multiple dilutions for each sample, intra- and inter-assay variability) of an electro-chemiluminescence (ECLIA)-based multiplex assay. METHODS: The current study describes the development of a multiplex ECLIA-based assay and characterizes the sensitivity, linear range, and inter- and intra-assay variability of the ECLIA platform and its agreement with the traditional ELISA. Special emphasis was placed on potential antigenic competition when testing closely related antigens in the multiplex format. RESULTS: Multiplexing of antigens in ECLIA provides significant practical benefits in terms of reducing sample volume requirements and experimental time. Beyond the practical advantages of multiplexing, the ECLIA provides superior assay performance when compared to the ELISA. Not only does ECLIA show good agreement with the ELISA assay, but the linear range of ECLIA is also sufficiently wide to permit single-dilution measurements of concentration without the need to do serial dilutions. The lack of antigenic competition allows the simultaneous testing of closely related antigens, such as plate antigens representing different alleles of the same protein, which can inform about cross-reactivities—or lack thereof—of serological responses. CONCLUSION: The advantages of the newly developed tool for assessing the antigen profiles of serological responses may ultimately lead to the identification of biomarkers associated with various disease stages and or protection against disease.

Enteric viral infections as a cause of diarrhoea in the acquired immunodeficiency syndrome

Background and aims The role of non‐cytomegalovirus (CMV) enteric viral infection in causing diarrhoea in patients with human immunodeficiency virus (HIV) is poorly understood. We aimed to investigate the prevalence of these infections in acute and chronic diarrhoea. Methods Stool specimens from 377 HIV‐infected patients presenting with diarrhoea were studied prospectively for evidence of non‐CMV enteric viral infection. Patients with diarrhoea underwent investigation for gastrointestinal pathogens, including electron microscopic examination of stool for enteric viruses. We collected data on patients in whom enteric virus was identified and examined the association of enteric virus infection with diarrhoeal symptomatology. Results Eighty‐nine (10.3%) stool specimens from 60 (15.9%) HIV(+) individuals were positive for coronavirus (n = 13, 22%), rotavirus (n = 11, 18%), adenovirus (n = 30, 50%) and small round structured viruses (n = 5, 8%) or dual infection (n = 2, 3%). Thirty‐four of 52 (65%) patients available for analysis had acute diarrhoea, and 18/52 (35%) had chronic diarrhoea. Twenty‐three of 52 (44%) patients had a concurrent gut pathogen. After exclusion of concurrent pathogens enteric viral infections were found to be significantly associated with acute as opposed to chronic diarrhoea (P = 0.004). The presence of adenovirus colitis was significantly more likely to be associated with chronic diarrhoea (15/21 cases) than adenovirus isolated from stool alone (9/23 cases) (P = 0.03). There was a trend towards an association between adenovirus colitis and colonic cytomegalovirus infection (P = 0.06). Conclusion Enteric viral infection is strongly associated with acute diarrhoea in patients with HIV. Light microscopic examination of large bowel biopsies can identify adenovirus colitis which is significantly associated with chronic diarrhoea, and in addition may facilitate gastrointestinal co‐infection with CMV.

Preventing healthcare‐associated infection: risks, healthcare systems and behaviour

More than 177 000 potentially preventable healthcare‐associated infections (HAIs) occur per annum in Australia with sizable attributable mortality. Organizational systems to protect against HAI in hospitals in Australia are relatively poorly developed. Awareness and practice of infection control by medical and other healthcare staff are often poor. These lapses in practice create significant risk for patients and staff from HAI. Excessive patient exposure to antimicrobials is another key factor in the emergence of antibiotic‐resistant bacteria and Clostridium difficile infection. Physicians must ensure that their interactions with patients are safe from the infection prevention standpoint. The critical preventative practice is hand hygiene in accord with the World Health Organization 5 moments model. Improving the use of antimicrobials, asepsis and immunization also has great importance. Hospitals should measure and feed back HAI rates to clinical teams. Physicians as leaders, role models and educators play an important part in promoting adherence to safe practices by other staff and students. They are also potentially effective system engineers who can embed safer practices in all elements of patient care and promote essential structural and organizational change. Patients and the public in general are becoming increasingly aware of the risk of infection when entering a hospital and expect their carers to adhere to safe practice. Poor infection control practice will be regarded in a negative light by patients and their families, regardless of any other manifest skills of the practitioner.

Integrated bus transit scheduling for the Beijing bus group based on a unified mode of operation

This paper presents an applied study of scheduling buses and drivers for the Beijing Bus Group (BJBUS), the largest bus company in China. This is pioneering research in China for bus transit scheduling using computers based on a unified mode of operation. It is anticipated that the research fruits and experiences obtained would be of benefit to other bus operators in China. The bus transit scheduling problem in BJBUS is first presented in the paper. The particular characteristics are pointed out, which are different from those in developed countries, while many are common in China. After a brief review and analysis of the appropriateness of some currently successful approaches, the paper focuses on reporting the solution ideas and methods for BJBUS, especially those developed to solve the specific problems of BJBUS, such as scheduling buses with built‐in meal periods, multi‐type bus scheduling, restricting drivers to one or two particular buses, etc. Finally, the implementation and computational results are reported before the concluding remarks.

Antiviral Antibodies in Dogs in the Netherlands

SUMMARY: Antiviral antibodies in dogs in the Netherlands A collection of more than 700 canine sera, coming from open and closed populations and from kennels with frequent neonatal mortality, were screened for the prevalence of antibodies to canine adenoviruses, canine herpesvirus, polyoma virus, REOviruses, parainfluenza viruses, equine influenza viruses and vomiting and wasting disease virus of pigs. The data from the different groups were compared and related to the results of similar studies carried out in other countries. ZUSAMMENFASSUNG: Virus‐Antikörper bei Hunden in den Niederlanden Antivirale Antikörper in Hunden in den Niederlanden. Über 700 Sera von Hunden aus der offenen Population, isolierten Kollektiven sowie aus Zwingern mit hoher Welpensterblichkeit wurden auf Antikörper gegen die folgenden Viren untersucht: canine Adeno‐ und Herpesviren, Polyomavirus, REOviren, Parainfluenzaviren, Pferdeinfluenzaviren und das porcine „vomiting and wasting disease”‐Virus. Die aus den verschiedenen Kollektiven erhaltenen Werte wurden untereinander verglichen wie auch mit den Ergebnissen ähnlicher Erhebungen im Ausland. RÉSUMÉ: Anticorps antiviraux chez des chiens aux Pays‐Bas On a recherché des anticorps contre les virus suivants sur plus que 700 sérums de chiens pris dans la population ouverte, dans des collectivités isolées et dans des chenils avec forte mortalité des chiots: Adeno‐ et Herpesvirus canins, Polyomavirus, REOvirus, Parainfluenzavirus, Influenzavirus du cheval et le virus du «vomiting ans wasting disease» du porc. Les valeurs obtenues dans les différentes collectives ont été comparées entre elles et également avec les résultats de recherches semblables faites à l'éranger. RESUMEN: Anticuerpos antivirales en perros de los Países Bajos Se examinaron más de 700 sueros sanguíneos de perros de la población abierta, colectivos aislados y perreras con mortalidad elevada de cachorros en cuanto a la presencia de anticuerpos contra los virus siguientes: adeno y herpes caninos, polioma, REO, parainfluenza, influenza equina y de la enfermedad del vómito y extenuación en porcinos. Se compararon entre sí los valores obtenidos en los diversos colectivos, así como también con los resultados de pesquisas semejantes Ilevadas a cabo en el extranjero.

Structural and Functional Characterization of Gene Products Encoded in the Human Genome by Homology Detection

Availability of the human genome data has enabled the exploration of a huge amount of biological information encoded in it. There are extensive ongoing experimental efforts to understand the biological functions of the gene products encoded in the human genome. However, computational analysis can aid immensely in the interpretation of biological function by associating known functional/structural domains to the human proteins. In this article we have discussed the implications of such associations. The association of structural domains to human proteins could help in prioritizing the targets for structure determination in the structural genomics initiatives. The protein kinase family is one of the most frequently occurring protein domain families in the human proteome while P‐loop hydrolase, which comprises many GTPases and ATPases, is a highly represented superfamily. Using the superfamily relationships between families of unknown and known structures we could increase structural information content of the human genome by about 5%. We could also make new associations of domain families to 33 human proteins that are potentially linked to genetically inherited diseases. IUBMB Life, 56: 317‐331, 2004

Acute and chronic changes in the microcirculation of the liver in inbred strains of mice following infection with mouse hepatitis virus type 3

The acute and chronic effects of mouse hepatitis virus type 3 on the microcirculation of the liver in both semisusceptible C3HeB/FeJ and fully resistant A/J mice were studied. In the C3HeB/FeJ mice, abnormalities of microcirculatory flow were noted as early as 12 hr after infection and by 24 hr, localized avascular foci appeared. Disturbances were characterized by granular blood flow, sinusoidal microthrombi, distortion of sinusoids by edematous hepatocytes and necrotic lesions. Following the acute infection, Day 10, two patterns of chronic disease were observed. Eighty percent of the mice developed chronic granulomatous hepatitis whereas in the remaining 20% a more severe chronic aggressive hepatitis was observed which was characterized by ongoing hepatocellular necrosis and a marked mononuclear cell infiltrate. In both cases, in vivo microcirculatory abnormalities were found predominantly around visible lesions. Onset of the microcirculatory abnormalities was found to be concomitant with a rise in monocyte related procoagulant activity. Procoagulant activity rose acutely and remained elevated throughout the chronic phase but was higher in animals with severe disease. In contrast to the above, normal blood flow and histology were seen in the resistant A/J mice at all times following infection, and procoagulant activity remained at basal levels despite active viral replication as demonstrated by immunofluorescence studies and recovery of infectious virus. These observations suggest a role for monocyte procoagulant activity in the development of microcirculatory abnormalities following mouse hepatitis virus type 3 infection which may be important in the pathogenesis of the disease.

VERTICAL MIGRATION AND AVOIDANCE CAPABILITY OF EUPHAUSIIDS IN THE CALIFORNIA CURRENT

Comparisons of day and night vertical distributions of euphausiids at the same stations make it possible to estimate 1) the extent of vertical migration of the furcilia, juveniles, and adults of the Euphausiacea (Crustacea) species, or 2) their ability to avoid the net in the daytime, or both. Seven species of Euphausia and one of Thysanopoda migrated more than 300 m, rising to near the surface at night. Adults of Nematoscelis (three species) migrated vertically but were bounded at upper limits by the thermocline at all but the most inshore stations. Nyctiphanes simplex and Stylocheiron carinatum were short‐distance (ca. 150–0 m) migrants. The extent of vertical migration was greatest in the most offshore central water, intermediate for midcurrent species, and least for coastal. Nonmigrants included all Stylocheiron species (except S. carinatum), Thysanoessa gregaria, and two Nematobrachion species. S. affine, S. suhmi, and T. gregaria inhabited the homogeneous layer. Apparent increases with depth in their daytime densities arise from avoidance of nets in the upper layers. This is minimal at ca. 100 m, where day and night densities are nearly equal.

Some chronic rhinosinusitis patients have elevated populations of fungi in their sinuses,

OBJECTIVES/HYPOTHESIS: To measure the populations of 36 fungi in the homes and sinuses of chronic rhinosinusitis (CRS) and non‐CRS patients. STUDY DESIGN: Single‐blind cross‐sectional study. METHODS: Populations of 36 fungi were measured in sinus samples and in the home vacuum cleaner dust of CRS (n = 73) and non‐CRS patients (n = 16) using quantitative polymerase chain reaction. Etest strips containing amphotericin B, anidulafungin, caspofungin, fluconazole, and voriconazole were used to test the susceptibility of seven potentially relevant fungi. RESULTS: Seven fungi (Alternaria alternata, Cladosporium cladosporioides types 1 and 2, Cladosporium herbarum, Penicillium brevicompactum, Penicillium crustosum, and Penicillium chrysogenum type 2) were discovered at very high concentrations in some CRS patients. In vitro antifungal susceptibility testing of these seven fungi demonstrated species specific sensitivities. Four CRS patients with marked elevations of fungal populations in their sinus samples underwent endoscopic sinus surgery. After surgical treatment, the fungal populations were reduced by several orders of magnitude. CONCLUSIONS: Seven fungi were found in very high concentrations in the sinuses of some CRS patients. Not one of the five common antifungal agents could control all seven of these fungi based on in vitro tests.

Parental behaviour in Maned wolf Chrysocyon brachyurus at Belo Horizonte Zoo

There is limited information on the reproductive behaviour of the Maned wolf Chrysocyon brachyurus. Although it is a monogamous species, little is known about the ♂'s role in the rearing of pups. In 1994 a comparative study of the behaviour of two ♀♀ with new‐born pups was carried out at Belo Horizonte Zoo to try to establish the ♂'s role in rearing: one ♀ was housed alone with her pups throughout the observation period; the other ♂ was housed with the ♀ and pups for the first month and alone with the pups for the second month of observation. The applied methodology for behaviour evaluation was instantaneous sampling (scan). The behaviour of each adult was recorded at one‐minute intervals. Location in the enclosure and spatial proximity were also recorded. The results suggest that Maned wolves are inactive for large parts of the day and that they prefer secluded areas within an enclosure. The results also suggest that only the ♂ is directly involved in rearing the offspring and that ♀ participation is limited to protection only. The time that the ♂ stays with the pups is at a maximum on the days immediately following birth, but this time decreases considerably as the pups grow. At around day 50, when lactation has ceased, contact between the ♂ and pups is minimal and the pups have their own resting places within the enclosure. These resting places, inside the burrows or thick vegetation, are rarely shared with others.

Extending the Latent Dirichlet Allocation model to presence/absence data: A case study on North American breeding birds and biogeographical shifts expected from climate change

Understanding how species composition varies across space and time is fundamental to ecology. While multiple methods having been created to characterize this variation through the identification of groups of species that tend to co‐occur, most of these methods unfortunately are not able to represent gradual variation in species composition. The Latent Dirichlet Allocation (LDA) model is a mixed‐membership method that can represent gradual changes in community structure by delineating overlapping groups of species, but its use has been limited because it requires abundance data and requires users to a priori set the number of groups. We substantially extend LDA to accommodate widely available presence/absence data and to simultaneously determine the optimal number of groups. Using simulated data, we show that this model is able to accurately determine the true number of groups, estimate the underlying parameters, and fit with the data. We illustrate this method with data from the North American Breeding Bird Survey (BBS). Overall, our model identified 18 main bird groups, revealing striking spatial patterns for each group, many of which were closely associated with temperature and precipitation gradients. Furthermore, by comparing the estimated proportion of each group for two time periods (1997–2002 and 2010–2015), our results indicate that nine (of 18) breeding bird groups exhibited an expansion northward and contraction southward of their ranges, revealing subtle but important community‐level biodiversity changes at a continental scale that are consistent with those expected under climate change. Our proposed method is likely to find multiple uses in ecology, being a valuable addition to the toolkit of ecologists.

Discovery of sympatric cryptic species within Gekko hokouensis (Gekkonidae: Squamata) from the Okinawa Islands, Japan, by use of allozyme data

An electrophoretic survey of samples of the gekkonid lizard, Gekko hokouensis, from the East Asian islands demonstrated that two genetically divergent, but morphologically almost identical, entities occur on five islands of the Okinawa Group, Ryukyu Archipelago, Japan. These entities, while sharing all of the external character states diagnostic of G. hokouensis, exhibited fixed allele differences at six to eight out of 30 loci examined and great overall genetic distances [Nei’s (1978) D = 0.489–0.654]. On Kumejima and Tonakijima Islands of the Okinawa Group, the two entities were collected together from identical microhabitats. These results indicate that the two entities represent separate biological species. Genetic comparisons of these two cryptic species from the Okinawa Group with ‘G. hokouensis’ from other island groups revealed that one occurs broadly in the insular region of East Asia, whereas the other is restricted to the Okinawa Group. Implications of the present findings for the morphological evolution of ‘G. hokouensis’ are also discussed.

Seroepidemiology of Feline Infectious Peritonitis Virus Infections Using Transmissible Gastroenteritis Virus as Antigen

SUMMARY: By indirect immunofluorescence on pig thyroid cells infected with transmissible gastroenteritis (TGE) virus of swine, antibodies were detected in sera from cats after experimental infection with feline infectious peritonitis (FIP) virus material. Also antibodies could be demonstrated in body fluids from 21 field cases where FIP had been diagnosed by clinical and post mortem examination and in most sera (91%) from catteries with a FIP history. In randomly selected open population samples 16% of seropositive animals were detected whereas none of 109 sera from a barrier‐contained closed breeding colony gave a positive reaction. No TGE neutralizing antibodies could be found in our sera and peritoneal fluids. The possibility of an antigenic relationship between FIP and TGE viruses is discussed. ZUSAMMENFASSUNG: Seroepidemiologie der Infektionen mit dem Virus der felinen infektiösen Peritonitis unter Verwendung des transmissiblen Gastroenteritisvirus als Antigen Mit Hilfe der indirekten Immunofluoreszenz auf TGE‐virusinfizierten Schweineschilddrüsenzellen wurden in den Seren von experimentell mit FIP‐Virusmaterial infizierten Katzen Antikörper nachgewiesen. Auch in den Körperflüssigkeiten von 21 Spontanfällen, in denen FIP klinisch und pathologischanatomisch diagnostiziert worden war und in den meisten Seren (91%) aus Katzenzuchten mit einer FIP‐Anamnese konnten Antikörper angezeigt werden. In Stichproben von klinisch unauffälligen Katzen befanden sich 16% seropositive Reagenten, wohingegen alle 109 Seren von Katzen aus einer geschlossenen SPF‐Zucht negativ ausfielen. Neutralisierende Antikörper gegen das TGE‐Virus konnten in unseren Seren und Aszitesflüssigkeiten nicht gefunden werden. Die Möglichkeit einer antigenen Verwandtschaft zwischen den FIP und TGE Viren wird diskutiert. RÉSUMÉ: Séroépidémiologie des infections avec le virus de la péritonite infectieuse du chat utilisant le virus de la gastroentérite transmissible du porc comme antigène Dans des sérums felins aprés infection expérimentale avec le virus de la FIP on a démontré des anticorps par immunofluorescence indirecte sur cellules de la thyroïde porcine infectées avec le virus de la TGE. Aussi dans 21 sérums des chats où la FIP a été diagnostiquée par examens cliniques et anatomo‐pathologiques et dans la plupart (91%) des sérums provenant des élevages avec une anamnése FIP, des anticorps ont été démontré. Dans la population féline normale il y avait 16% des animaux séropositifs tandis qu'aucun sérum des 109 échantillons provenant d'une colonie SPF ne donnait pas de réaction positive. Des anticorps neutralisants contre le virus de la TGE n'étaient pas démontrable dans nos sérums et liquides ascitiques. La possibilité d'une relation antigénique entre les virus de la FIP et de la TGE est discutée. RESUMEN: Sueroepidemiología de infecciones con el virus de la peritonitis infecciosa del gato utilizando el virus de la gastroenteritis transmisible porcina como antígeno Por medio de inmunofluorescencia indirecta en células tiroideas del cerdo infectadas con el virus de la gastroenteritis transmisible porcina (TGE) se mostraron anticuerpos en sueros de gato después de una infección experimental con el virus de la peritonitis infecciosa (FIP). También en los líquidos ascíticos de 21 animales en los cuales la FIP fué diagnosticada mediante exámenes clínicos y postmortales y en la mayoría (91%) de sueros obtenidos de varias gaterías con problemas de FIP se pudo evidenciar la presencia de anticuerpos. En la población abierta se logró mostrar 16% de animales sueropositivos mientras que todos los 109 sueros obtenidos de un colectivo aislado SPF eran negativos. No se pudo evidenciar anticuerpos neutralizantes contra el virus de la TGE en nuestros sueros y líquidos ascéticos. Se discute la posibilidad de una relación antigénica entre los virus de la FIP y de la TGE.

An immunoprobe to measure Rubisco concentrations and maximal photosynthetic rates of individual phytoplankton cells

The cross‐reactivity of an immunological probe to the key photosynthetic enzyme Rubisco (ribulose‐1,5‐bisphosphate carboxylase/oxygenase) was characterized as part of a larger effort to determine maximal photosynthetic rates of individual phytoplankton cells. Polyclonal antiserum was produced against purified Rubisco from the marine diatom Chaetoceros gracilis. The results of western immunoblotting demonstrated that the antiserum reacted positively with Rubisco from 38 species of algae and higher plants and failed to react with only three species of dinoflagellates and one prochlorophyte species. However, the binding affinity or the strength of the cross‐reaction for the polyclonal antiserum with purified Rubisco varied among species. The antiserum was then affinity purified against spinach Rubisco and its binding affinity for purified Rubisco determined by ELISA. Two taxonomic groupings resulted: one with high‐binding affinity (these species included chrysophytes, bacillariophytes, prymnesiophytes, and chlorophytes) and the other with low‐binding affinity (dinophytes and cyanophytes). Rubisco concentration per cell and light‐saturated rates of photosynthesis were highly correlated for cultures of the diatom Thalassiosira weissflogii. These results indicate that affinity‐purified antiserum can be rigorously characterized for use in quantifying Rubisco concentration and for assessing the maximal photosynthetic potential of individual phytoplankton cells.

Mucosal immunity and tolerance: relevance to vaccine development

Summary: The mucosal immune system of mammals consists of an integrated network of lymphoid cells which work in concert with innate host factors to promote host defense. Major mucosal effector immune mechanisms include secretory antibodies, largely of immunoglobulin A (IgA) isotype, cytotoxic T cells, as well as cytokines, chemokines and their receptors. Immunologic unresponsiveness (tolerance) is a key feature of the mucosal immune system, and deliberate vaccination or natural immunization by a mucosal route can effectively induce immune suppression. The diverse compartments located in the aerodigestive and genitourinary tracts and exocrine glands communicate via preferential homing of lymphocytes and antigen‐presenting cells. Mucosal administration of antigens may result in the concomitant expression of secretory immunoglobulin A (S‐IgA) antibody responses in various mucosal tissues and secretions, and under certain conditions, in the suppression of immune responses. Thus, developing formulations based on efficient delivery of selected anti‐gens/tolerogens, cytokines and adjuvants may impact on the design of future vaccines and of specific immunotherapeutic approaches against diseases associated with untoward immune responses, such as autoimmune disorders, allergic reactions, and tissue‐damaging inflammatory reactions triggered by persistent microorganisms.

Molecular mimicry: Can epitope mimicry induce autoimmune disease?

Mimicry of host antigens by infectious agents may induce cross‐reactive autoimmune responses to epitopes within host proteins which, in susceptible individuals, may tip the balance of immunological response versus tolerance toward response and subsequently lead to autoimmune disease. Epitope mimicry may indeed be involved in the pathogenesis of several diseases such as post‐viral myocarditis or Chagas disease, but for many other diseases in which it has been implicated, such as insulin‐dependent diabetes mellitis or rheumatoid arthritis, convincing evidence is still lacking. Even if an epitope mimic can support a cross‐reactive T or B cell response in vitro, its ability to induce an autoimmune disease in vivo will depend upon the appropriate presentation of the mimicked host antigen in the target tissue and, in the case of T cell mimics, the ability of the mimicking epitope to induce a proliferative rather than anergizing response upon engagement of the MHC‐peptide complex with the T cell receptor. B cell presentation of mimicking foreign antigen to T cells is a possible mechanism for instigating an autoimmune response to self antigens that in turn can lead to autoimmune disease under particular conditions of antigen presentation, secondary signalling and effector cell repertoire. In this review evidence in support of epitope mimicry is examined in the light of the necessary immunological considerations of the theory.

Evapotranspiration and water source of a tropical rainforest in peninsular Malaysia

To evaluate water use and the supporting water source of a tropical rainforest, a 4‐year assessment of evapotranspiration (ET) was conducted in Pasoh Forest Reserve, a lowland dipterocarp forest in Peninsular Malaysia. The eddy covariance method and isotope signals of rain, plant, soil, and stream waters were used to determine forest water sources under different moisture conditions. Four sampling events were conducted to collect soil and plant twig samples in wet, moderate, dry, and very dry conditions for the identification of isotopic signals. Annual ET from 2012 to 2015 was quite stable with an average of 1,182 ± 26 mm, and a substantial daily ET was observed even during drought periods, although some decline was observed, corresponding with volumetric soil water content. During the wet period, water for ET was supplied from the surface soil layer between 0 and 0.5 m, whereas in the dry period, approximately 50% to 90% was supplied from the deeper soil layer below 0.5‐m depth, originating from water precipitated several months previously at this forest. Isotope signatures demonstrated that the water sources of the plants, soil, and stream were all different. Water in plants was often different from soil water, probably because plant water came from a different source than water that was strongly bound to the soil particles. Plants showed no preference for soil depth with their size, whereas the existence of storage water in the xylem was suggested. The evapotranspiration at this forest is balanced and maintained using most of the available water sources except for a proportion of rapid response run‐off.

Comparative morphology of the venom apparatus in the braconid wasp subfamily Rogadinae (Insecta, Hymenoptera, Braconidae) and related taxa

Zaldivar‐Riverón, A., Areekul, B., Shaw, M. R. & Quicke, D. L. J. (2004). Comparative morphology of the venom apparatus in the braconid wasp subfamily Rogadinae (Insecta, Hymenoptera, Braconidae) and related taxa. —Zoologica Scripta, 33, 223–237. The morphology of the venom apparatus intima in representatives of 38 genera of the problematic braconid wasp subfamily Rogadinae and other cyclostome braconids was investigated and a preliminary phylogenetic analysis for the group was performed with the information obtained. Despite the limited number of characters, the data suggest several relationships at various taxonomic levels. The venom apparatus in the Clinocentrini and the Stiropiini is relatively unmodified and similar to that found in other genera previously placed within a broader concept of the Rogadinae (e.g. genera of Lysitermini, Pentatermini, Tetratermini, Hormiini) and also to that of the Betylobraconinae. The presence of a cone of filaments located inside the secondary venom duct near to its insertion on the venom reservoir/primary venom duct is proposed as a synapomorphy for the tribe Rogadini to the exclusion of Stiropiini, Clinocentrini and Yeliconini. Other features of the secondary venom duct and its insertion on the venom reservoir/primary venom duct support a number of relationships between the genera of the Rogadini and also within the large genus Aleiodes. A clade containing 15 Rogadini genera (Bathoteca, Bathotecoides, Bulborogas, Canalirogas, Colastomion, Conspinaria, Cystomastacoides, Macrostomion, Megarhogas, Myocron, Pholichora, Rectivena, Rogas, Spinaria and Triraphis) is supported by the presence of a thickened and short secondary venom duct, whereas the different members of Aleiodes (excluding members of the subgenus Heterogamus) and Cordylorhogas are distinguished by having a recessed secondary venom duct with well‐defined and numerous internal filaments. New World Rogas species exhibit a unique venom apparatus and may not be closely related to the Old World ones. Features of the venom apparatus of the enigmatic genus Telengaia and the exothecine genera Shawiana and Colastes suggest that the Telengainae and Exothecinae are both closely related to the Braconinae, Gnamptodontinae, and possibly to the Opiinae and Alysiinae. An unsculptured venom reservoir was found in one specimen of the type species of Avga, A. choaspes, which is consistent with it occupying either a very basal position within the cyclostome braconids or belonging to a recently recognized ‘Gondwanan’ clade that also includes the Aphidiinae.

The Effect of Mouse Hepatitis Virus Infection on the Microcirculation of the Liver

Mouse hepatitis virus type 3 infection results in strain‐dependent liver disease. The effects of mouse hepatitis virus type 3 on the microcirculation of the liver in both fully susceptible (Balb/cJ) and fully resistant (A/J) mice were studied. In Balb/cJ mice, 6 to 12 hr following infection, abnormalities in liver blood flow were observed which consisted of granular blood flow in both terminal hepatic and terminal portal venules. In addition, sinusoidal microthrombi were present predominantly in periportal areas. By 24 to 48 hr, liver cell edema and small focal lesions were prominent. At 48 hr, thrombi and hepatocellular necrosis were widespread, and blood was shunted from damaged areas into patent sinusoids. In sharp contrast to these abnormal findings, normal streamlined blood flow was present in the resistant A/J animals at all time points following infection. Since large amounts of virus were demonstrated by immunofluorescene in and by recovery and growth from livers of both resistant and susceptible strains, the presence of the virus per se cannot explain the abnormalities observed.

The role of the neutral amino acid transporter B(0)AT1 (SLC6A19) in Hartnup disorder and protein nutrition

Hartnup disorder (OMIM 234500) is an autosomal recessive disorder, which was first described in 1956 as an aminoaciduria of neutral amino acids accompanied by a variety of symptoms, such as a photo‐sensitive skin‐rash and cerebellar ataxia. The disorder is caused by mutations in the neutral amino acid transporter B(0)AT1 (SLC6A19). To date 21 mutations have been identified in more than twenty families. SLC6A19 requires either collectrin or angiotensin‐converting enzyme 2 for surface expression in the kidney and intestine, respectively. This ties SLC6A19 together with more complex functions such as blood‐pressure control, glomerular structure, and exocytosis. © 2009 IUBMB IUBMB Life, 61(6): 591–599, 2009

PERICARDITIS ASSOCIATED WITH TICK‐BORNE Q FEVER

A case of pericarditis associated with Q fever is described. Transmission was probably via an arthropod vector, which was most likely the kangaroo tick Amblyomma triguttatum. Complete recovery occurred in association with the use of non‐steroidal anti‐inflammatory agents only. This is a rare presentation of Q fever implicating transmission by a novel vector.

Promising Trends in Access to Medicines

It is a vast understatement to say that the problem of access to medicines in developing countries is complex. Access is limited by a range of factors including inability to pay, a lack of infrastructure, and corruption in some countries. Surrounding and exacerbating these structural and technological problems is the layer of legal rights created by patents and their licensing that complicate and render more expensive the preparation and delivery of needed medicines, particularly those that need to be adapted to the social, health and cultural environment of developing countries. This article provides a survey of innovative strategies that aim at maximizing the potential of patents to facilitate the development and delivery of medicines against diseases, the burden of which falls principally on developing country populations. To understand the context in which these strategies are being proposed and implemented, the article reviews the battles over access to medicines beginning in the late 1980s. It then surveys some of the principal suggestions put forward to better direct innovation systems in addressing the critical health needs of the world’s majority including advance market commitments, patent buy‐outs, prize funds, public–private partnerships and patent pools.

Plant species richness of nature reserves: the interplay of area, climate and habitat in a central European landscape

Aim To detect regional patterns of plant species richness in temperate nature reserves and determine the unbiased effects of environmental variables by mutual correlation with operating factors. Location The Czech Republic. Methods Plant species richness in 302 nature reserves was studied by using 14 explanatory variables reflecting the reserve area, altitude, climate, habitat diversity and prevailing vegetation type. Backward elimination of explanatory variables was used to analyse the data, taking into account their interactive nature, until the model contained only significant terms. Results A minimal adequate model with reserve area, mean altitude, prevailing vegetation type and habitat diversity (expressed as the number of major habitat types in the reserve) accounted for 53.9% of the variance in species number. After removing the area effect, habitat diversity explained 15.6% of variance, while prevailing vegetation type explained 29.6%. After removing the effect of both area and vegetation type, the resulting model explained 10.3% of the variance, indicating that species richness further increased with habitat diversity, and most obviously towards warm districts. After removing the effects of area, habitat diversity and climatic district, the model still explained 9.4% of the variance, and showed that species richness (i) significantly decreased with increasing mean altitude and annual precipitation, and with decreasing January temperature in the region of the mountain flora, and (ii) increased with altitudinal range in regions of temperate and thermophilous flora. Main conclusions We described, in quantitative terms, the effects of the main factors that might be considered to be determining plant species richness in temperate nature reserves, and evaluated their relative importance. The direct habitat effect on species richness was roughly equal to the direct area effect, but the total direct and indirect effects of area slightly exceeded that of habitat. It was shown that the overall effect of composite variables such as altitude or climatic district can be separated into particular climatic variables, which influence the richness of flora in a context‐specific manner. The statistical explanation of richness variation at the level of families yielded similar results to that for species, indicating that the system of nature conservation provides similar degrees of protection at different taxonomic levels.

Initial Lung Lesions in Two Calves Experimentally Infected with Haemophilus somnus

The initial lung lesions in two calves intrabronchially inoculated with Haemophilus somnus are described. The animals were euthanized within 7 h after challenge. The in situ location of H. somnus and accompanying lesions were examined by light microscopy, immunohistochemistry and transmission electron microscopy (TEM). Inoculation with H. somnus resulted in the development of acute pulmonary lesions within 3.5 h. H. somnus antigen was demonstrated only within the luminal spaces of the airways and in one area of bronchio‐associated lymphoid tissue (BALT). As observed by TEM, the bacteria were phagocytized by both neutrophils and alveolar macrophages. Antigen was never demonstrated in the pulmonary intravascular macrophages.

Major vault protein plays important roles in viral infection

Viral replication and related protein expression inside the host cells, and host antiviral immune responses can lead to the occurrence of diverse diseases. With the outbreak of viral infection, a large number of newly diagnosed and died patients infected with various viruses are still reported every year. Viral infection has already been one of the major global public health issues and lead to huge economic and social burdens. Studying of viral pathogenesis is a very important way to find methods for prevention, diagnosis, and cure of viral infection; more evidence has confirmed that major vault protein (MVP) is closely associated with viral infection and pathogenesis, and this review is intended to provide a broad relationship between viruses and MVP to stimulate the interest of related researchers.

Safety procedures of coagulation factors

Abstract Two main types of safety procedures must be applied to biological products, including plasma derivatives: (i) preventive procedures and (ii) elimination procedures.Prevention includes epidemiological control of donor populations; checks on each donor’s health condition; analysis of each donation for the main pathogens using serological methods; additional analysis of all plasma for human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis A virus (HAV) and the B19 virus, using nucleic acid amplification techniques (NAT). A 60 days or longer inventory hold of all plasma donations is applied, to allow additional time to discard previous donations from potential seroconverting or otherwise rejectable donors.Elimination procedures minimize the low residual risk of transmitting pathogens, including unknown or previously undetected ones. Since the introduction 20 years ago of solvent‐detergent treatment, very effective against enveloped viruses (HIV, HBV, HCV, West Nile virus, SARS, avian influenza virus etc), there have been no known cases of transmission of this type of pathogens by products manufactured according to this procedure. Other inactivation procedures such as pasteurization, dry‐heat or nanofiltration may prove equally effective. In addition, dry‐heat treatment and nanofiltration are capable of effectively eliminating non‐enveloped viruses (HAV, B19 virus). Recent studies show that the B19 virus is much more sensitive to heat (in lyophilized state or by pasteurization) and acid pH than previously thought.Although there is no evidence for the transmission of classic transmissible spongiform encephalopathies (TSEs) through blood or blood‐products transfusion, four possible cases have been reported in the United Kingdom involving transmission by non‐leukoreduced blood components of the agent that causes variant Creutzfeldt‐Jakob Disease (vCJD), a disease linked to the outbreak of bovine spongiform encephalopathy (BSE) which took place in that country. However, there are no cases of human TSE (classic or variant) transmission by plasma‐derived products. Analytical methods capable of detecting the vCJD agent in patients’ brains (where high titres are found) and other tissues (such as the spleen, appendix and lymph nodes, where much lower concentrations are found) are unable to detect the agent in blood or plasma from patients with vCJD, even in the clinical phase of the disease. Experiments by Grifols and other groups show that the capacity of the production processes to eliminate vCJD agent models is many orders of magnitude greater than the maximum expected load of the agent. In this regard, the efficacy of precipitation, affinity chromatography, depth filtration and nanofiltration are particularly notable.

Construction of a multiplex allele‐specific PCR‐based universal array (ASPUA) and its application to hearing loss screening

We demonstrate a new method, using a universal array approach termed multiplex allele‐specific PCR‐based universal array (ASPUA), and applied it to the mutation detection of hereditary hearing loss. Mutations in many different genes may be the cause of hereditary hearing loss, a sensory defect disorder. Effective methods for genetic diagnosis are clearly needed to provide clinical management. Owing to the broad genetic basis of this condition, clinical assay of such a highly heterogeneous disorder is expensive and time consuming. In ASPUA, the allele discrimination reaction is carried out in solution by multiplex allele‐specific PCR and a universal solid phase array with different tag probes is used to display the PCR result. The purpose of developing the ASPUA platform was to utilize the rapidity and simplicity of the amplification refractory mutation system (ARMS) with the detection power of microarray hybridization. This is the first report of the combination of these two technologies, which allow for the completion of allele‐specific detection of 11 of the most frequent mutations causing hereditary hearing loss in under 5 hr. The ASPUA platform was validated by accurately analyzing 141 patient samples that had been previously genotyped for GJB2, GJB3, SLC26A4, and MTRNR1. In addition, we also developed a simplified assay by using streptavidin‐coated magnetic beads instead of fluorescence for signal display that can be assessed through a conventional light microscope. We demonstrate that the ASPUA platform is rapid, cost‐effective, and easily‐used, and is especially appropriate for mutation detection in clinical genetic diagnostics. Hum Mutat 29(2), 306–314, 2008. © 2007 Wiley‐Liss, Inc.

Prioritizing Zoonotic Diseases: Differences in Perspectives Between Human and Animal Health Professionals in North America

Zoonoses pose a significant burden of illness in North America. Zoonoses represent an additional threat to public health because the natural reservoirs are often animals, particularly wildlife, thus eluding control efforts such as quarantine, vaccination and social distancing. As there are limited resources available, it is necessary to prioritize diseases in order to allocate resources to those posing the greatest public health threat. Many studies have attempted to prioritize zoonoses, but challenges exist. This study uses a quantitative approach, conjoint analysis (CA), to overcome some limitations of traditional disease prioritization exercises. We used CA to conduct a zoonoses prioritization study involving a range of human and animal health professionals across North America; these included epidemiologists, public health practitioners, research scientists, physicians, veterinarians, laboratory technicians and nurses. A total of 699 human health professionals (HHP) and 585 animal health professionals (AHP) participated in this study. We used CA to prioritize 62 zoonotic diseases using 21 criteria. Our findings suggest CA can be used to produce reasonable criteria scores for disease prioritization. The fitted models were satisfactory for both groups with a slightly better fit for AHP compared to HHP (84.4% certainty fit versus 83.6%). Human‐related criteria were more influential for HHP in their decision to prioritize zoonoses, while animal‐related criteria were more influential for AHP resulting in different disease priority lists. While the differences were not statistically significant, a difference of one or two ranks could be considered important for some individuals. A potential solution to address the varying opinions is discussed. The scientific framework for disease prioritization presented can be revised on a regular basis by updating disease criteria to reflect diseases as they evolve over time; such a framework is of value allowing diseases of highest impact to be identified routinely for resource allocation.

Respiratory inflammation and infections in high‐performance athletes

Upper respiratory illness is the most common reason for non‐injury‐related presentation to a sports medicine clinic, accounting for 35–65% of illness presentations. Recurrent or persistent respiratory illness can have a negative impact on health and performance of athletes undertaking high levels of strenuous exercise. The cause of upper respiratory symptoms (URS) in athletes can be uncertain but the majority of cases are related to common respiratory viruses, viral reactivation, allergic responses to aeroallergens and exercise‐related trauma to the integrity of respiratory epithelial membranes. Bacterial respiratory infections are uncommon in athletes. Undiagnosed or inappropriately treated asthma and/or allergy are common findings in clinical assessments of elite athletes experiencing recurrent URS. High‐performance athletes with recurrent episodes of URS should undergo a thorough clinical assessment to exclude underlying treatable conditions of respiratory inflammation. Identifying athletes at risk of recurrent URS is important in order to prescribe preventative clinical, training and lifestyle strategies. Monitoring secretion rates and falling concentrations of salivary IgA can identify athletes at risk of URS. Therapeutic interventions are limited by the uncertainty of the underlying cause of inflammation. Topical anti‐inflammatory sprays can be beneficial for some athletes. Dietary supplementation with bovine colostrum, probiotics and selected antioxidants can reduce the incidence or severity of URS in some athletes. Preliminary studies on athletes prone to URS indicate a genetic predisposition to a pro‐inflammatory response and a dysregulated anti‐inflammatory cytokine response to intense exercise as a possible mechanism of respiratory inflammation. This review focuses on respiratory infections and inflammation in elite/professional athletes.

Clinical assessment of Optivate(®), a high‐purity concentrate of factor VIII with von Willebrand factor, in the management of patients with haemophilia A

Summary. Factor VIII (FVIII) concentrates have revolutionized the treatment of patients with haemophilia A. Concerns over the transmission of viral infections through these products have been addressed through stringent, donor‐screening procedures and robust antiviral manufacturing steps. Bio Products Laboratory has developed a high‐purity FVIII product with von Willebrand factor, Optivate(®). Its safety, tolerability and efficacy as prophylaxis and treatment of bleeds have been established in long‐term studies. Seventy previously treated patients with severe haemophilia A, with ≥20 exposure days, were recruited into two long‐term, multicentre, open‐label studies. The protocols were virtually identical. Patients received Optivate(®) either prophylactically or on‐demand. A mean of 159.0 EDs were experienced over 11 320 infusions. Under both conditions, Optivate(®) was well tolerated. Only 10% of patients experienced a treatment‐related adverse event; the most commonly reported were headache (4% of patients) and dizziness (3% of patients). The mean number of bleeds/patient over the 2 year treatment period was 23.5 during prophylactic use and 70.4 during on‐demand use. In patients treated prophylactically, clinical responses to breakthrough bleeds were rated by physicians as excellent or good and as very helpful or helpful by patients in 95% of bleeds. Clinical responses for on‐demand patients were rated as excellent or good by physicians and helpful or very helpful by the patients for 91% of bleeds. There were no viral transmissions or inhibitors. The studies confirm the clinical efficacy and safety of Optivate(®) in both prophylactic and on‐demand management of patients with haemophilia A.

Pathogenicity of Hemagglutinating Encephalomyelitis (Vomiting and Wasting Disease) Virus of Pigs, using Different Routes of Inoculation

SUMMARY: Forty‐eight pigs were inoculated by different routes with the VW 572 isolate of the hemagglutinating encephalomyelitis (vomiting and wasting disease) virus. All piglets inoculated by the combined oral — nasal route (16) or into the infraorbital nerve (3) became sick after an incubation period of 5 days. Six of the 7 pigs inoculated into the stomach wall, 6 of the 8 pigs inoculated intramuscularly and 3 of the 5 pigs inoculated intracerebrally became ill after an incubation period of 3–3.5 days. None of the pigs inoculated either intravenously or into the abdominal cavity or into the stomach lumen became sick. All diseased pigs showed the vomiting and wasting syndrome. In oronasally inoculated pigs, killed during the early stages of disease, the virus was reisolated consistently from the tonsils and respiratory tract but irregularly from the pons + medulla and the stomach wall. Pigs inoculated by other routes were positive for virus when sick. All except one of the pigs which remained healthy had seroconverted. The site of virus replication which gives rise to the vomition could not be determined. It was concluded from the present studies that virus spread within the body occurs along nerve pathways. ZUSAMMENFASSUNG: Die Pathogenität von hämagglutinierendem Enzephalomyelitis‐Virus (Kümmern und Erbrechen) bei Shweinen nach Infektion über vershiedene Inokulationswege Achtundvierzig Schweine wurden übegr verschiedene Inokulationswege mit dem VW 572‐Isolat des hämagglutinierendtn Enzephalomyelitis‐Virus (Kümmern und Erbrechen) infiziert. Alle Schweine, die entweder kombiniert oro‐nasal (16) oder über den Infraorbitalnerv (3) infiziert wurden, erkrankten nach einer Inkubationszeit von 5 Tagen. Sechs der sieben über die Magenwand inokulierten, 6 oder 8 intramuskulär und 3 der 5 intrazerebral infizierten Tiere wurden nach einer Inkubationszeit von 3–3,5 Tagen krank. Bei den Schweinen, die intravenös oder in die Bauchhöhle bzw. direkt in den Magen inokuliert wurden, kamen Erkrankungsfälle nicht vor. Alle erkrankten Schweine zeigten das Syndrom des Kümmerns und Erbrechens. Von oro‐nasal infizierten Schweinen, die während des Frühstadiums der Erkrankung getötet wurden, konnte das Virus regelmäßig von den Tonsillen und dem Respirationstrakt und gelegentlich vom Gewebe des Pons‐Medulla‐Bereiches sowie aus der Magenwand reisoliert werden. Von Schweinen, die nach Infektion über andere Routen erkrankten, ließ sich immer Virus isolieren. Alle Tiere, die nicht erkrankten (mit Ausnahme eines Ferkels) bildeten jedoch Antikörper. Der Ort der Virusvermehrung, mit dem das Erbrechen zusammenhängt, ließ sich nicht ermitteln. Die Ergebnisse der vorgelegten Untersuchungen lassen den Schluß zu, daß die Virusausbreitung im Körper über die Nervenbahnen erfolgt. RÉSUMÉ: Pathogénicité du virus hémagglutinant de l'encéphalomyélite du porc (dépérissement et vomissement) après infection par différents modes d'inoculation 48 porcs ont été infectés selon différents procédés d'inoculation avec l'isolement VW 572 du virus hémagglutinant de l'encéphalomyélite (dépérissement et vomissement). Tous les porcs infectés soit par la voie combinée oronasale (16) soit par le nerf infraorbital (3) tombèrent malades après une incubation de 5 jours. 6 des 7 animaux infectés par la paroi stomacale, 6 des 8 par voie intramusculaire et 3 des 5 intracérébralement tombèrent malades après un temps d'incubation de 3–3,5 jours. Il n'y a pas eu de maladie chez les porcs inoculés par voie intraveineuse, dans l'abdomen ou directement dans l'estomac. Tous les porcs malades ont présenté le syndrome de dépérissement et de vomissement. Chez les animaux infectés par voie oro‐nasale et sacrifiés au début de la maladie, on a pu régulièrement réisoler le virus à partir des amygdales et de l'appareil respiratoire, parfois du tissu de la région «Pons‐Medulla» et de la paroi stomacale. Le virus a toujours été isolé chez les porcs tombés malades après un mode d'infection différent. Tous les animaux qui ne furent pas malades (à l'exception d'un porcelet) formèrent des anticorps. L'endroit de multiplication du virus lié au syndrome de vomissement n'a pas été déterminé. Les résultats de ces essais permettent de conclure que la propagation du virus dans le corps se fait par voie nerveuse. RESUMEN: La patogeneidad del virus hemoaglutinante de la encéfalomielitis (hipotrepsia y vómitos) en el cerdo tras infección a través de vías diversas de inoculación Se infectaron cuarenta y ocho cerdos a través de diferentes vías de inoculación con el aislamiento VW 572 del virus hemoaglutinante de la encéfalomielitis (hipotrepsia y vómitos). Todos los cerdos infectados bien con arreglo al procedimiento combinado buco‐nasal (16) o bien a través del nervio infraorbitario (3) enfermaron tras un tiempo de incubación de 5 días. Seis de siete animales inoculados a través de la pared gástrica, 6 de 8 por vía intramuscular y 3 de 5 por vía intracerebral enfermaron tras un tiempo de incubación de 3–3,5 días. No se registraron casos de enfermedad en los cerdos inoculados por vía intravenosa o en la cavidad abdominal resp. directamente en el estómago. Todos los cerdos que enfermaron presentaban el síndrome de hipotrepsia y vómitos. De los cerdos infectados por vía buco‐nasal, que se sacrificaron durante el estadio precoz de la enfermedad, se pudo reaislar el virus con regularidad de las tonsilas y del tracto respiratorio y, en ocasiones, del tejido correspondiente al ámbito puente de Varolio‐medula, así como de la pared gástrica. Se logró siempre aislar virus de cerdos que enfermaron tras infección por otras vías. Sin embargo, todos los animales que no enfermaron (excepción hecha por un lechón) produjeron anticuerpos. No se logró descubrir el lugar en donde se multiplicaba el virus, hecho relacionado con los vómitos. Los resultados de los estudios presentados permiten llegar a la conclusión de que la propagación del virus en el organismo acontece a través de las vías nerviosas.

Vulnerability of rat and mouse brain cells to murine hepatitis virus (JHM‐strain): Studies in vivo and in vitro

The pathogenicity and cell tropism of mouse hepatitis virus (MHV‐JHM‐strain) in the developing mouse (Balb/c) and rat (Wistar and Lewis) brain were analysed. Intracranial infection of Balb/c mice at postnatal day 5 induced a lethal encephalitis in all animals. Of Wistar rats infected at day 2 or 5 after birth, 30 to 70%, respectively, survived. The distribution of viral antigen was studied in frozen brain sections of animals that died after infection; astrocytes were found to be the major virus‐infected cell type throughout the central nervous system. More than 75% of the surviving rat pups developed paralysis, but viral antigen was detected in only few brain cells and not in astrocytes. The cell tropism of MHV‐JHM was examined further in virus‐infected glial cell cultures derived from brains of rats or mice. In the glial cultures derived from Wistar rats, only oligodendrocytes were infected, whereas in cultures derived from mouse or Lewis rat brain viral antigen was detected in both astrocytes and oligodendrocytes. Infection of astrocytes led to the formation of syncytia and degradation of the cytoskeleton. Infected rat oligodendrocytes gradually disappeared from the cultures because of cell death. These phenomena indicate that, besides an indirect autoimmune response triggered by infected astrocytes, direct virus‐induced injury to astrocytes or to oligodendrocytes can have a dominant role in the neuropathogenicity of mouse hepatitis virus. The present results underscore the importance of species and developmental stage of experimental animals in the neurotropism and pathogenicity of MHV‐JHM.

An historical overview of selected rare ruminants in captivity

In assessing the situation revealed by the survey, we concluded that in general when sufficient numbers of founders were available and attention paid to the animals general and specific needs, including preventative medicine, the captive populations have done reasonably well. Is is obvious that some small groups, such as the small African antelope and the duikers, need highly specialised care and there is a great deal of work still to be done on their husbandry. The mountain or alpine species have been fairly successful with the goats showing the greatest adaptability. We feel there are grounds for optimism for the future of such animals as the Rocky mountain goat, the Japanese serow and even the Blue sheep. Of those species whose captive future is still in doubt, particularly those which are threatened in the wild, our researches indicate that concentrated efforts should be made to obtain sufficient founder stock to establish self‐sustaining populations as a hedge against total disaster in the wild. We felt that the evidence was strong that any of the ungulate species reviewed could be successfully maintained in captivity given the right circumstances. Basically, the husbandry procedures of all the reporting zoos were similar, although, of course, each zoo had its own variations. In some cases these measurably affected the success of the animal population. In the last 20 to 25 years inter‐zoo co‐operation and sharing of information has greatly expanded. Nevertheless we would now urge that even more emphasis should be put on the exchange of information to provide each zoo with up‐ to‐date data which would ensure that all zoos are kept abreast with the 'state of the art. With such a spirit of co‐operation the prospect of managing species as total captive populations appears to be practical and promising.

Distribution of Trichoptera communities in the Hozgargantacatchment (Los Alcornocales Natural Park, SW Spain)

The distribution of Trichoptera of the Hozgarganta River (Los Alcornocales Natural Park, SW Spain) in relation with environmental factors was examined. Three groups of species were recognised according to the altitudinal gradient. In the headwaters the caddisflies Rhyacophila fonticola, Lepidostoma hirtum, Silonella aurata, Allogamus gibraltaricus, Hydropsyche infernalis and Diplectrona felix predominated; in the constrained section of the tributaries Polycentropus kingi, Chimarra marginata, Hydropsyche iberomaroccana, R. fonticola and Tinodes sp. prevailed; finally, in the main channel H. iberomaroccana, C. marginata, Hydropsyche lobata, Leptocerus lusitanicus and Rhyacophila munda were the most important species. A direct ordination analysis (CCA) was used to describe assemblage changes among sites and corroborated that conductivity and temperature were the variables that best explained Trichoptera distribution. The temporal analysis showed changes in the Trichoptera diversity and richness in permanent stretches, as well as variations in the structure of the communities according to the season. We identified autumn‐winter species (H. infernalis, H. siltalai, H. lobata, R. fonticola and R. munda ) and summer ones (Ithytrichia sp, Oxyethira unidentata, Mystacides azurea and Setodes argentipunctellus ). In the basin we distinguished permanent, intermittent and ephemeral reaches with similar caddisfly richness and diversity, however the species composition associated with each one was different. (© 2006 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Isolation of a Virus Closely Related to Feline Panleukopenia Virus from Dogs with Diarrhea

SUMMARY: Parvovirus‐like particles were demonstrated by negative contrast electron microscopy in feces of dogs involved in outbreaks of a contagious diarrhea in the Netherlands. The virus was isolated in a continuous cell line in which it produced intranuclear inclusion bodies. It showed a close antigenic relationship with feline panleukopenia virus: a two‐way cross‐reaction was observed in hemagglutination inhibition, immune electron microscopy, immunofluorescence and serum neutralization tests. ZUSAMMENFASSUNG: Isolierung eines mit dem Virus der felinen Panleukopenie verwandten Virus von Hunden mit Diarrhoe Parvovirus‐ähnliche Partikel wurden mit Hilfe der Negativkontrast‐Elektronenmikroskopie während eines Ausbruches von kontagiöser Diarrhoe in Fäces von Hunden in den Niederlanden nachgewiesen. Das Virus wurde in einer permanenten Zell‐Linie isoliert, in der intranukleäre Einschlußkörperchen nachgewiesen werden konnten. Es zeigte eine nahe Antigenverwandt‐schaft mit dem Virus der felinen Panleukopenie. Eine beiderseitige Kreuzreaktion wurde beobachtet in der Hämagglutinationshemmung, Immunelektronenmikroskopie, Immunofluoreszenz und im Serumneutralisations‐Tests. RÉSUMÉ: Isolement d'un virus très proche au virus de la panleucopénie féline chez des chiens diarhéiques Des particules ressemblant aux parvovirions ont été démontrées au microscope électronique (contraste‐négatif) dans les matières fécales des chiens diarrhéiques aux Pays‐Bas. Le virus était isolé sur une ligne cellulaire continue dans laquelle il produisait des corpuscules d'inclusion intranucléairs. Le virus manifestait un parenté antigénique avec le virus de la panleucopénie féline: des réactions croisées dans les deux sens étaient observées, utilisant des méthodes d'inhibition d'hémagglutination, immunomicroscopie électronique, immunofluorescence et séroneutralisation. RESUMEN: Aislamiento de un virus estrechamente relacionado con el virus de la panleucopenia felina en perros con diarrea Partículas semejantes a parvoviriones fueron demostrados por medio de microscopía electrónica (contraste negativo) en muestras fecales de perros durante endemias de diarrea infecciosa en los Paises Bajos. Se mostró el aislamiento del virus en una línea celular continua en donde producía corpúsculos de inclusión intranucleares. El virus tiene una relación antigénica estrecha con el virus de la panleucopenia felina: reacciones cruzadas en ambos sentidos fueron observadas por medio de pruebas de inhibición de la hemaglutinación, inmunomicroscopía electrónica, inmunofluorescencia y sueroneutralización.

Indoor bioaerosol dynamics

Inhaling indoor air is the primary means by which humans are exposed to bioaerosols. Considering bacteria, fungi, and viruses, this study reviews the dynamic processes that govern indoor concentrations and fates of biological particulate material. Bioaerosol behavior is strongly coupled to particle size; this study emphasizes the range 0.1–10 μm in aerodynamic diameter. The principle of material balance allows concentrations to be determined from knowledge of important source and removal processes. Sources reviewed here include outdoor air introduced by air exchange plus indoor emission from occupants, occupant activities, and moldy materials. Important mechanisms that remove bioaerosols from indoor air include air exchange, deposition onto indoor surfaces, and active filtration. The review summarizes knowledge about size‐dependent particle deposition in different regions of the respiratory tract, techniques for measuring indoor bioaerosols, and evidence for diseases caused by airborne exposure to bioaerosols. Future research challenges and opportunities are highlighted.

Molecular systematics of the Philippine forest skinks (Squamata: Scincidae: Sphenomorphus): testing morphological hypotheses of interspecific relationships

Skinks of the genus Sphenomorphus are the most diverse clade of squamates in the Philippine Archipelago. Morphological examination of these species has defined six phenotypic groups that are commonly used in characterizations of taxonomic hypotheses. We used a molecular phylogeny based on four mitochondrial and two nuclear genes to assess the group's biogeographical history in the archipelago and examine the phylogenetic validity of the currently recognized Philippine species groups. We re‐examined traditional characters used to define species groups and used multivariate statistics to quantitatively evaluate group structure in morphometric space. Clustering analyses of phenotypic similarity indicate that some (but not all) members of previously defined species groups are phenotypically most similar to other members of the same group. However, when species group membership was mapped on our partitioned Bayesian phylogenetic hypothesis, only one species group corresponds to a clade; all other species group arrangements are strongly rejected by our phylogeny. Our results demonstrate that (1) previously recognized species group relationships were misled by phenotypic convergence; (2) Sphenomorphus is widely paraphyletic; and (3) multiple lineages have independently invaded the Philippines. Based on this new perspective on the phylogenetic relationships of Philippine Sphenomorphus, we revise the archipelago's diverse assemblage of species at the generic level, and resurrect and/or expand four previously recognized genera, and describe two new genera to accommodate the diversity of Philippine skinks of the Sphenomorphus group. © 2011 The Linnean Society of London, Zoological Journal of the Linnean Society, 2011, 163, 1217–1243.

Comparative genome organization of the major histocompatibility complex: lessons from the Felidae

Summary: The mammalian major histocompatibility complex (MHC) has taught both immunologists and evolutionary biologists a great deal about the patterns and processes that have led to immune defenses. Driven principally by human and mouse studies, comparative MHC projects among other mammalian species offer certain advantages in connecting MHC genome characters to natural situations. We have studied the MHC in the domestic cat and in several wild species of Felidae. Our observations affirm class I and class II homology with other mammalian orders, derivative gene duplications during the Felidae radiation, abundant persistent trans‐species allele polymorphism, recombination‐derived amino acid motifs, and inverted ratios of non‐synonymous to silent substitutions in the MHC peptide‐binding regions, consistent with overdominant selection in class I and II genes. MHC diversity as quantified in population studies is a powerful barometer of historic demographic reduction for several endangered species including cheetahs, Asiatic lions, Florida panthers and tigers. In two cases (Florida panther and cheetah), reduced MHC variation may be contributing to uniform population sensitivity to emerging infectious pathogens. The Felidae species, nearly all endangered and monitored for conservation concerns, have allowed a glimpse of species adaptation, mediated by MHC divergence, using comparative inferences drawn from human and mouse models.

Characterization of an animal model of metastatic colon carcinoma

Although numerous animal tumor models have been used to study colon carcinoma, few display metastatic properties. We have characterized an animal tumor model that has 3 properties essential for the study of metastasis of colon carcinoma cells: epithelial cell origin; a reproducible pattern of metastatic behavior and the ability to be propagated both in vitro and in vivo to facilitate identification of biochemical correlates of metastasis. The KI2/TR cell line was derived from a transplantable colon carcinoma induced by dimethylhydrazine in the BD‐IX rat strain. Transmission electron microscopy of KI2/TR cells demonstrated junctional complexes, desmosomes and surface microvilli characteristic of gastrointestinal epithelial cells. The epithelial cell origin of KI2/TR was confirmed by demonstrating the presence of keratin, a marker of epithelial cells, but not vimentin, a constituent of mesenchymal cells. Secretion of CEA and Ca19‐9 antigens by KI2/TR cells in vitro was below the sensitivity of the assays (1 ng/ml and 6 U/ml respectively). KI2/TR cells produced tumors following s.c. injection into syngeneic BD‐IX rats, allogeneic RNU/rnuDF rats and xenogeneic CRL:nu/nuBR mice. Macroscopic lung metastases were observed in animals from all 3 groups. Distal lymph node metastases were more frequent in BD‐IX rats than in nude rats or mice. The histological appearances of all tumors and metastases were similar, showing a moderate to poorly differentiated glandular carcinoma. Intrasplenic injections of KI2/TR cells in nude mice resulted in liver colonization. Preferential growth of tumor cells at sites of trauma was also observed. The results show that the KI2/TR system can be used as a model to study metastasis of colon carcinoma cells and may find utility in the testing of chemotherapeutic agents against metastatic lesions.

Revision of the coral genus Acropora (Scleractinia: Astrocoeniina: Acroporidae) in Indonesia

The coral genus Acropora is reviewed from Indonesia for the first time, following detailed collections made at 131 sites and additional material collected from approximately 40 sites throughout the archipelago during the period 1993–6. Eighty‐three species are recorded, four of these (Acropora halmaherae, A. awi, A. plumosa and A. simplex) new to science, six first described in 1994 and six in 1997. Records are compared with specimen‐based records from localities worldwide. The species of Acrokora occurring in Indonesian waters include five recorded only from the Indian Ocean and Indonesia, seven recorded only from the Pacific Ocean, South China Sea and Indonesia, and a further 10 species apparently endemic to Indonesia, as well as widespread Indo‐Pacific species. Two species (A. jacquelineae Wallace, 1994 and A. batunai Wallace, 1996) are recorded only from north central Indonesia and Papua New Guinea, and two species (A. russelli Wallace, 1994 and A. turaki Wallace, 1994) only from north central Indonesia and north western Australia. The findings contribute to a new view of the corals of the Indo‐Pacific ‘centre of diversity’ as a composite fauna with origins in a number of events in space and time.

Tolerance and autoimmunity in TCR transgenic mice specific for myelin basic protein

Summary: T‐cell receptor (TCR) transgenic mice provide the ability to follow the maturation and fate of T cells specific for self‐antigens in vivo. This technology represents a major breakthrough in the study of autoimmune diseases in which specific antigens have been implicated. Proteins expressed within the central nervous system are believed to be important autoantigens in multiple sclerosis, TCR transgenic models specific for myelin basic protein (MBP) allowed us to assess the role of tolerance in providing protection from T cells with this specificity Our studies demonstrate that T cells specific for the immunodominant epitope of MBP do not undergo tolerance in vivo and that TCR transgenic mice are susceptible to spontaneous autoimmune disease. The susceptibility to spontaneous disease is dependent on exposure to microbial antigens, MBP TCR transgenic models expressing TCRs specific for the same epitope of MBP but utilizing different V(α) genes exhibit differing susceptibilities to, spontaneous disease. These data support the idea that genetic and environmental differences play a role in susceptibility to autoimmunity MBP TCR transgenic models are playing an important role in defining mechanisms by which infectious agents trigger autoimmune disease as well as defining mechanisms by which tolerance is induced to distinct epitopes within self‐antigens.

Assessment of hazard metrics for predicting field benthic invertebrate toxicity in the Detroit River, Ontario, Canada

Numerical sediment quality guidelines (SQGs) are frequently used to interpret site‐specific sediment chemistry and predict potential toxicity to benthic communities. These SQGs are useful for a screening line of evidence (LOE) that can be combined with other LOEs in a full weight of evidence (WOE) assessment of impacted sites. Three common multichemical hazard quotient methods (probable effect concentration [PEC]‐Q(avg), PEC‐Q(met), and PEC‐Q(sum)) and a novel (hazard score [HZD]) approach were used in conjunction with a consensus‐based set of SQGs to evaluate the ability of different scoring metrics to predict the biological effects of sediment contamination under field conditions. Multivariate analyses were first used to categorize river sediments into distinct habitats based on a set of physicochemical parameters to include gravel, low and high flow sand, and silt. For high flow sand and gravel, no significant dose–response relationships between numerically dominant species and various toxicity metric scores were observed. Significant dose–response relationships were observed for chironomid abundances and toxicity scores in low flow sand and silt habitats. For silt habitats, the HZD scoring metric provided the best predictor of chironomid abundances compared to various PEC‐Q methods according to goodness‐of‐fit tests. For low flow sand habitats, PEC‐Q(sum) followed by HZD, provided the best predictors of chironomid abundance. Differences in apparent chironomid toxicity between the 2 habitats suggest habitat‐specific differences in chemical bioavailability and indicator taxa sensitivity. Using an IBI method, the HZD, PEC‐Q(avg), and PEC‐Q(met) approaches provided reasonable correlations with calculated IBI values in both silt and low flow sand habitats but not for gravel or high flow sands. Computation differences between the various multi‐chemical toxicity scoring metrics and how this contributes to bias in different estimates of chemical mixture toxicity scores are discussed and compared. Integr Environ Assess Manag 2017;13:410–422. © 2016 SETAC

Context of diversification of the viviparous Gyrodactylidae (Platyhelminthes, Monogenoidea)

Using four criteria proposed a decade ago by Brooks & McLennan to identify a case of adaptive radiation indicates that the evolutionary history of the viviparous clade of the Gyrodactylidae is dominated by nonvicariant processes. The viviparous clade, with 446 species, has significantly more species than its sister clade (one species), and high species richness was shown to be an apomorphic trait of only the viviparous gyrodactylids within the Gyrodactylidae. Reconciliation of the phylogenetic tree of the viviparous Gyrodactylidae with that of its hosts showed a low probability for cospeciation suggesting that adaptive modes of speciation and not vicariance were predominant during the historical diversification of the clade. The proposed hypothesis suggests that the Gyrodactylidae originated on the South American continent about 60 Mya after geographical dispersal and host switching of its common ancestor to demersal freshwater catfishes by a marine ancestor. Development of hyperviviparity and the consequent loss of ‘sticky’ eggs in conjunction with other symplesiomorphic and apomorphic features allowed rapid diversification coupled with high dispersal to new host groups and geographical areas by viviparous members of the Gyrodactylidae.