Gut microbiome resilience of green-lipped mussels, Perna canaliculus, to starvation.

Host gut microbiomes play an important role in animal health and resilience to conditions, such as malnutrition and starvation. These host-microbiome relationships are poorly understood in the marine mussel Perna canaliculus, which experiences significant variations in food quantity and quality in coastal areas. Prolonged starvation may be a contributory factor towards incidences of mass mortalities in farmed mussel populations, resulting in highly variable production costs and unreliable market supplies. Here, we examine the gut microbiota of P. canaliculus in response to starvation and subsequent re-feeding using high-throughput amplicon sequencing of the 16S rRNA gene. Mussels showed no change in bacterial species richness when subjected to a 14-day starvation, followed by re-feeding/recovery. However, beta bacteria diversity revealed significant shifts (PERMANOVA p-value < 0.001) in community structure in the starvation group and no differences in the subsequent recovery group (compared to the control group) once they were re-fed, highlighting their recovery capability and resilience. Phylum-level community profiles revealed an elevation in dominance of Proteobacteria (ANCOM-BC p-value <0.001) and Bacteroidota (ANCOM-BC p-value = 0.04) and lower relative abundance of Cyanobacteria (ANCOM-BC p-value = 0.01) in the starvation group compared to control and recovery groups. The most abundant genus-level shifts revealed relative increases of the heterotroph Halioglobus (p-value < 0.05) and lowered abundances of the autotroph Synechococcus CC9902 in the starvation group. Furthermore, a SparCC correlation network identified co-occurrence of a cluster of genera with elevated relative abundance in the starved mussels that were positively correlated with Synechococcus CC9902. The findings from this work provide the first insights into the effect of starvation on the resilience capacity of Perna canaliculus gut microbiota, which is of central importance to understanding the effect of food variation and limitation in farmed mussels.

Quantification of Photobacterium swingsii and characterisation of disease progression in the New Zealand Greenshell™ mussel, Perna canaliculus.

Greenshell™ mussels (Perna canaliculus) are endemic to New Zealand and support the largest aquaculture industry in the country. Photobacterium swingsii was isolated and identified from moribund P. canaliculus mussels following a mass mortality event. In this study, a challenge experiment was used to characterise, detect, and quantify P. swingsii in adult P. canaliculus following pathogen exposure via injection into the adductor muscle. A positive control (heat-killed P. swingsii injection) was included to account for the effects of injection and inactive bacterial exposure. Survival of control and infected mussels remained 100% during 72-hour monitoring period. Haemolymph was sampled for bacterial colony counts and haemocyte flow cytometry analyses; histology sections were obtained and processed for histopathological assessments; and adductor muscle, gill, digestive gland were sampled for quantitative polymerase chain reaction (PCR) analyses, all conducted at 12, 24, 48 h post-challenge (hpc). The most profound effects of bacterial injection on mussels were seen at 48 hpc, where mussel mortality, haemocyte counts and haemolymph bacterial colony forming were the highest. The quantification of P. swingsii via qPCR showed highest levels of bacterial DNA at 12 hpc in the adductor muscle, gill, and digestive gland. Histopathological observations suggested a non-specific inflammatory response in all mussels associated with a general stress response. This study highlights the physiological effects of P. swingsii infection in P. canaliculus mussels and provides histopathological insight into the tissue injury caused by the action of injection into the adductor muscle. The multi-technique methods used in this study can be applied for use in early surveillance programs of bacterial infection on mussel farms.

Editing the editors: Aims and priorities of health professions education journals.

PURPOSE: Although health professions education (HPE) scholarship has flourished in recent decades, the influence of HPE journals has received little attention. This study examines the editorial policies and priorities of leading HPE journals. METHODS: Fourteen HPE journals with the highest impact factors were reviewed for their editorial aims, scope, intended readership, and priority topic areas. Text from journal websites was coded using thematic analysis. RESULTS: 10/14 HPE journals included in this study were linked to regional or national education societies. Two focussed predominantly on medicine, one on dentistry, one on nursing, one on nutrition, and the remaining nine on general HPE. Although journals differed in their projected aims and proposed readerships, four overarching editorial themes were identified: (1) methodological and theoretical rigor; (2) impact on practice; (3) global relevance; (4) advancing knowledge. CONCLUSIONS: Leading HPE journals share a number of priority areas and principles, implying some cohesion and consensus amongst the HPE scholarly community. These journals prioritise impact at the level of individual practitioners. Given the importance of policy level change in the development and reform of HPE around the world, the relative lack of focus on policy impact in HPE journals is worthy of further exploration.

Sources of funding for research articles in medical education journals from 1999 to 2019.

BACKGROUND: Prior studies report that most published medical education research is unfunded. We sought to determine the extent and sources of funding for medical education research articles published in leading journals, and how these have changed in the last two decades. METHODS: All research articles published in Academic Medicine, Advances in Health Sciences Education, Medical Education and Medical Teacher in 1999, 2004, 2009, 2014, and 2019 were reviewed for funding declarations. Funding sources were categorised as: government; university; healthcare organisation; private not-for-profit organisation; and for-profit company. Time trends were analysed using the Cochran-Armitage test. RESULTS: 1822 articles were analysed. Over the aggregate 20-year period, 44% of all articles reported funding, with the proportion increasing from 30% in 1999 to 50% in 2019 (p < .001). The proportion of articles with government (10% to 16%, p = .049), university (6% to 17% p < .001), and not-for-profit funding sources (15% to 20%, p = .04) increased. Proportions of healthcare (3% to 4%, p = .45) and for-profit funding (2% to 1%, p = .25) did not significantly change with time. CONCLUSIONS: Over the last 20 years, the proportion of funded published medical education research has significantly increased, as has funding from government, universities, and not-for-profit sources. This may assist researchers in identifying funders with a track record of supporting medical education research, and enhances transparency of where research funding in the field originates.

Mapping the Green-Lipped Mussel (Perna canaliculus) Microbiome: A Multi-Tissue Analysis of Bacterial and Fungal Diversity.

Poor health and mortality events of the commercially important and endemic New Zealand green-lipped mussel (Perna canaliculus) pose a threat to its industry. Despite the known importance of microbiomes to animal health and environmental resilience, the host-associated microbiome is unexplored in this species. We conducted the first baseline characterization of bacteria and fungi within key host tissues (gills, haemolymph, digestive gland, and stomach) using high-throughput amplicon sequencing of 16S rRNA gene and ITS1 region for bacteria and fungi, respectively. Tissue types displayed distinctive bacterial profiles, consistent among individuals, that were dominated by phyla which reflect (1) a fluid exchange between the circulatory system (gills and haemolymph) and surrounding aqueous environment and (2) a highly diverse digestive system (digestive gland and stomach) microbiota. Gammaproteobacteria and Campylobacterota were mostly identified in the gill tissue and haemolymph, and were also found in high abundance in seawater. Digestive gland and stomach tissues were dominated by common gut bacterial phyla, such as Firmicutes, Cyanobacteria, Proteobacteria, and Bacteroidota, which reflects the selectivity of the digestive system and food-based influences. Other major notable taxa included the family Spirochaetaceae, and genera Endozoicomonas, Psychrilyobacter, Moritella and Poseidonibacter, which were highly variable among tissue types and samples. More than 50% of fungal amplicon sequence variants (ASVs) were unclassified beyond the phylum level, which reflects the lack of studies with marine fungi. However, the majority of those identified were assigned to the phylum Ascomycota. The findings from this work provide the first insight into healthy tissue microbiomes of P. canaliculus and is of central importance to understanding the effect of environmental changes on farmed mussels at the microbial level.

Acute thermal stress and endotoxin exposure modulate metabolism and immunity in marine mussels (Perna canaliculus).

Mass mortalities of New Zealand Green-lipped mussels (Perna canaliculus) are thought to be associated with increased water temperatures and immune challenges from opportunistic pathogens. However, the combined effects of acute thermal stress and immune stimulation on mussels are poorly understood. To investigate these responses, adult mussels were exposed to different temperatures (26 °C [thermal stress] vs 15 °C [ambient]) and a bacterial-derived endotoxin injection (with vs without) to mimic a pathogen infection. Various immunological and metabolic parameters were measured over two days via enzyme staining reactions, flow cytometry, and metabolomic profiling. None of the treatments impacted total and differential haemocyte counts, haemocyte viability or production of reactive oxygen species. Acid phosphatase and phenoloxidase activities were detected only within granulocytes (not in hyalinocytes), although their relative expressions also were not affected. Conversely, metabolite profiling exposed impacts of thermal stress and endotoxin exposure at a metabolic level, indicative of physiological changes in energy expenditure and partitioning. At the higher water temperature, free fatty acid and amino acid constituents increased and decreased, respectively, which supports an elevated energy demand and higher metabolic rate due to thermal stress. Ultimately, energy production is being sustained via multiple routes including the glycolysis pathway, TCA cycle, and ß-oxidation. Additionally, branched-chain amino acids, the urea cycle and the glutathione pathway were affected by the higher temperature. The metabolic response of mussels exposed to endotoxin exposure resulted in increased metabolite response largely linked to protein and lipid degradation. After 5 days of exposure, survival data confirmed a severe physiological impact of the higher temperature through incidences of mortality. However, the thermal challenge in combination with the specific endotoxin treatment applied did not lead to a synergistic effect on mortality. These findings provide new insights into the relationship between thermal stress and immunity to better understand the immune defence system in mussels.

VX-445-Tezacaftor-Ivacaftor in Patients with Cystic Fibrosis and One or Two Phe508del Alleles.

BACKGROUND: VX-445 is a next-generation cystic fibrosis transmembrane conductance regulator (CFTR) corrector designed to restore Phe508del CFTR protein function in patients with cystic fibrosis when administered with tezacaftor and ivacaftor (VX-445-tezacaftor-ivacaftor). METHODS: We evaluated the effects of VX-445-tezacaftor-ivacaftor on Phe508del CFTR protein processing, trafficking, and chloride transport in human bronchial epithelial cells. On the basis of in vitro activity, a randomized, placebo-controlled, double-blind, dose-ranging, phase 2 trial was conducted to evaluate oral VX-445-tezacaftor-ivacaftor in patients heterozygous for the Phe508del CFTR mutation and a minimal-function mutation (Phe508del-MF) and in patients homozygous for the Phe508del CFTR mutation (Phe508del-Phe508del) after tezacaftor-ivacaftor run-in. Primary end points were safety and absolute change in percentage of predicted forced expiratory volume in 1 second (FEV1) from baseline. RESULTS: In vitro, VX-445-tezacaftor-ivacaftor significantly improved Phe508del CFTR protein processing, trafficking, and chloride transport to a greater extent than any two of these agents in dual combination. In patients with cystic fibrosis, VX-445-tezacaftor-ivacaftor had an acceptable safety and side-effect profile. Most adverse events were mild or moderate. The treatment also resulted in an increased percentage of predicted FEV1 of up to 13.8 points in the Phe508del-MF group (P<0.001). In patients in the Phe508del-Phe508del group, who were already receiving tezacaftor-ivacaftor, the addition of VX-445 resulted in an 11.0-point increase in the percentage of predicted FEV1 (P<0.001). In both groups, there was a decrease in sweat chloride concentrations and improvement in the respiratory domain score on the Cystic Fibrosis Questionnaire-Revised. CONCLUSIONS: The use of VX-445-tezacaftor-ivacaftor to target Phe508del CFTR protein resulted in increased CFTR function in vitro and translated to improvements in patients with cystic fibrosis with one or two Phe508del alleles. This approach has the potential to treat the underlying cause of cystic fibrosis in approximately 90% of patients. (Funded by Vertex Pharmaceuticals; VX16-445-001 ClinicalTrials.gov number, NCT03227471 ; and EudraCT number, 2017-000797-11 .).

VX-659-Tezacaftor-Ivacaftor in Patients with Cystic Fibrosis and One or Two Phe508del Alleles.

BACKGROUND: The next-generation cystic fibrosis transmembrane conductance regulator (CFTR) corrector VX-659, in triple combination with tezacaftor and ivacaftor (VX-659-tezacaftor-ivacaftor), was developed to restore the function of Phe508del CFTR protein in patients with cystic fibrosis. METHODS: We evaluated the effects of VX-659-tezacaftor-ivacaftor on the processing, trafficking, and function of Phe508del CFTR protein using human bronchial epithelial cells. A range of oral VX-659-tezacaftor-ivacaftor doses in triple combination were then evaluated in randomized, controlled, double-blind, multicenter trials involving patients with cystic fibrosis who were heterozygous for the Phe508del CFTR mutation and a minimal-function CFTR mutation (Phe508del-MF genotypes) or homozygous for the Phe508del CFTR mutation (Phe508del-Phe508del genotype). The primary end points were safety and the absolute change from baseline in the percentage of predicted forced expiratory volume in 1 second (FEV1). RESULTS: VX-659-tezacaftor-ivacaftor significantly improved the processing and trafficking of Phe508del CFTR protein as well as chloride transport in vitro. In patients, VX-659-tezacaftor-ivacaftor had an acceptable safety and side-effect profile. Most adverse events were mild or moderate. VX-659-tezacaftor-ivacaftor resulted in significant mean increases in the percentage of predicted FEV1 through day 29 (P<0.001) of up to 13.3 points in patients with Phe508del-MF genotypes; in patients with the Phe508del-Phe508del genotype already receiving tezacaftor-ivacaftor, adding VX-659 resulted in a further 9.7-point increase in the percentage of predicted FEV1. The sweat chloride concentrations and scores on the respiratory domain of the Cystic Fibrosis Questionnaire-Revised improved in both patient populations. CONCLUSIONS: Robust in vitro activity of VX-659-tezacaftor-ivacaftor targeting Phe508del CFTR protein translated into improvements for patients with Phe508del-MF or Phe508del-Phe508del genotypes. VX-659 triple-combination regimens have the potential to treat the underlying cause of disease in approximately 90% of patients with cystic fibrosis. (Funded by Vertex Pharmaceuticals; VX16-659-101 and VX16-659-001 ClinicalTrials.gov numbers, NCT03224351 and NCT03029455 .).

Beyond relaxed: magnesium chloride anaesthesia alters the circulatory metabolome of a marine mollusc (Perna canaliculus).

BACKGROUND: The New Zealand Green-lipped mussel industry is well-established providing vastly to aquaculture exports. To assess mussel health and reproduction status, visual examination of organs and/or collection of haemolymph is commonly applied. Anesthetics, such as magnesium chloride (MgCl2) can be utilized to prevent muscle contraction and keep shells open during sampling. The specific effects of muscle relaxing agents on baseline metabolism in invertebrates is unknown, but it is evident that molecular, cellular and physiological parameters are altered with these chemical applications. To this end, metabolomics approaches can help elucidate the effects of relaxing agents for better assessment of their use as a research tool. METHODS: Adult Green-lipped mussels were anaesthetized for 3 h in a MgCl2 bath, whereafter haemolymph samples were collected and analyzed via gas chromatography-mass spectrometry applying methyl chloroformate alkylation derivatization. RESULTS: Anesthetized mussels were characterized as non-responsive to manual manipulation, with open valves, and limited siphoning function. Metabolite profiling revealed significant increases in the abundances of most metabolites with an array of metabolic activities affected, resulting in an energy imbalance driven by anaerobic metabolism with altered amino acids acting as neurotransmitters and osmolytes. CONCLUSION: This research is the first to use a metabolomics approach to identify the metabolic consequences of this commonly used bivalve relaxing technique. Ultimately the use of MgCl2 anesthetization as a sampling strategy should be carefully evaluated and managed when performing metabolomics-related research.

Nonaxial Traction Mechanisms of Nursemaid's Elbow.

OBJECTIVE: This study aims to better describe those patients who present with nonaxial traction mechanisms for nursemaid's elbow. METHODS: A retrospective review on patients with the International Statistical Classification of Diseases, Ninth/Tenth Revision, code for nursemaid's elbow was performed. Patients with the classic axial traction mechanism and unknown mechanism were excluded. Demographic information and mechanism of injury were collected, and statistical analysis on this data was performed. RESULTS: Sixty-nine subjects with a median age of 2.4 years (interquartile range, 1.5-3.6 years) were enrolled. There was no difference in sex or sidedness. The most common mechanisms of injury were fall (57%), direct hit to the elbow (16%), and rolling over (7%). An x-ray was obtained 49% of the time. Reduction was spontaneous 12% of the time and was successfully reduced on the first attempt 87% of the time. CONCLUSIONS: Nursemaid's elbow can occur in children with a reported nonaxial traction mechanism. They may present with history of other trauma, such as a fall, a direct blow to the elbow, or rolling over. For toddlers without the classic axial traction mechanism who refuse to move the elbow but do not have an examination consistent with fracture, it is still reasonable to suspect a nursemaid's elbow.

Effect of anticoagulants on farmed giant kokopu, Galaxias argenteus (Gmelin 1789) haematological parameters and erythrocyte fragility.

We sought to determine a compatible anticoagulant for routine haematological and physiological assessments with giant kokopu (Galaxias argenteus), an endemic New Zealand fish. We observed that blood treated with lithium heparin (LH) rapidly coagulated and haemolysed, making it unsuitable for G. argenteus. Dipotassium ethylenediaminetetraacetic acid (K2 EDTA) and trisodium citrate (citrate) effectively prevented blood coagulation. K2 EDTA-treated erythrocytes exhibited the least mean haemolysis and mean corpuscular fragility. Further studies into prolonged storage effects of citrate and K2 EDTA are recommended to find a compatible anticoagulant for use with G. argenteus blood.

Haematological and metabolic profiles associated with age and sex in giant kokopu (Galaxias argenteus) (Gmelin 1789) broodstock.

This study characterized selected peripheral blood (PB) haematological parameters, liver, serum and muscle metabolic features in 3- and 5-year-old male and female giant kokopu (Galaxias argenteus) broodstock reared indoor at 16°C. Sex and age did not affect PB total cell count and haematocrit values. Nonetheless, higher erythrocytes in 5-year-old fish, elevated thrombocyte and lymphocyte counts in 3-year-old fish indicate age-specific cellular regulation. Higher thrombocyte counts in female fish suggest sex-specific regulation. At a metabolic level, liver abundance for long chain saturated fatty acids (FAs) was higher in males, whereas females had elevated levels of polyunsaturated FAs. Essential and non-essential amino acids (AAs) in liver and serum were also elevated in females compared to males. These findings suggest differential allocation of FAs and AAs to reflect requirements for gonadal, development and provisioning. Similarly, age significantly resulted in higher liver and serum abundances of some non-essential AAs in 3-year-olds compared to 5-year-old fish, suggesting higher metabolism in younger fish. Overall, results enhance our understanding of sex- and age-based differences in fish haematology, muscle, liver, and serum metabolite profiles in healthy G. argenteus. Future studies should carefully consider potential age- and sex-specific differences in metabolic responses.

An integrated omics approach to investigate summer mortality of New Zealand Greenshell™ mussels.

BACKGROUND: Green-lipped mussels, commercially known as Greenshell™ mussels (Perna canaliculus Gmelin 1791), contribute > $300 million to New Zealand's aquaculture exports. However, mortalities during summer months and potential pathogenic outbreaks threaten the industry. Thermal stress mechanisms and immunological responses to pathogen infections need to be understood to develop health assessment strategies and early warning systems. METHODS: P. canaliculus were collected during a mortality event at a commercial aquaculture farm in Firth of Thames, New Zealand. Gill tissues from six healthy and six unhealthy mussels were excised and processed for metabolomic (GC-MS) and label-free proteomic (LC-MS) profiling. Univariate analyses were conducted separately on each data layer, with data being integrated via sparse multiple discriminative canonical correlation analysis. Pathway enrichment analysis was used to probe coordinated changes in functionally related metabolite sets. RESULTS: Findings revealed disruptions of the tricarboxylic acid (TCA) cycle and fatty acid metabolism in unhealthy mussels. Metabolomics analyses also indicated oxidative stress in unhealthy mussels. Proteomics analyses identified under-expression of proteins associated with cytoskeleton structure and regulation of cilia/flagellum in gill tissues of unhealthy mussels. Integrated omics revealed a positive correlation between Annexin A4 and CCDC 150 and saturated fatty acids, as well as a negative correlation between 2-aminoadipic acid and multiple cytoskeletal proteins. CONCLUSIONS: Our study demonstrates the ability of using integrative omics to reveal metabolic perturbations and protein structural changes in the gill tissues of stressed P. canaliculus and provides new insight into metabolite and protein interactions associated with incidences of summer mortality in this species.

Immune response in probiotic-fed New Zealand black-footed abalone (Haliotis iris) under Vibrio splendidus challenge.

Vibriosis disease is a major constraint for sustainable molluscan aquaculture. Development of strategies to enhance disease resistance during grow out would greatly reduce stock mortality and boost production yields. In this study, New Zealand black-footed abalone (Haliotis iris) were fed a commercial diet enhanced with multi-strain probiotics (Exiguobacterium JHEb1, Vibrio JH1 and Enterococcus JHLDc) for four months, then challenged with an injection of pathogenic Vibrio splendidus. Host immune responses in haemocytes were characterized using flow cytometry by measuring total haemocyte counts (THC) and viability, degree of apoptosis, and production of reactive oxygen species (ROS) 48 h post-challenge. Probiotic-fed abalone had significantly higher survival rates compared to control animals after the bacterial challenge. Infected probiotic-fed abalone also had significantly higher haemocyte viabilities, slightly lower proportions of haemocytes undergoing early apoptosis, and lower proportions of ROS-producing haemocytes compared to infected control-fed abalone. In addition, metabolite profiles of muscle tissues generated via gas chromatography-mass spectrometry (GC-MS) delivered complimentary evidence to support a perturbed ROS-regulatory system in infected abalone through changes in key metabolites associated with glutathione biosynthesis. The results of this study provide valuable information to assist in farm management practices, leading to enhance production and sustainability of the New Zealand abalone aquaculture industry.

Confirmation of the Cardioprotective Effect of MitoGamide in the Diabetic Heart.

PURPOSE: HFpEF (heart failure with preserved ejection fraction) is a major consequence of diabetic cardiomyopathy with no effective treatments. Here, we have characterized Akita mice as a preclinical model of HFpEF and used it to confirm the therapeutic efficacy of the mitochondria-targeted dicarbonyl scavenger, MitoGamide. METHODS AND RESULTS: A longitudinal echocardiographic analysis confirmed that Akita mice develop diastolic dysfunction with reduced E peak velocity, E/A ratio and extended isovolumetric relaxation time (IVRT), while the systolic function remains comparable with wild-type mice. The myocardium of Akita mice had a decreased ATP/ADP ratio, elevated mitochondrial oxidative stress and increased organelle density, compared with that of wild-type mice. MitoGamide, a mitochondria-targeted 1,2-dicarbonyl scavenger, exhibited good stability in vivo, uptake into cells and mitochondria and reactivity with dicarbonyls. Treatment of Akita mice with MitoGamide for 12 weeks significantly improved the E/A ratio compared with the vehicle-treated group. CONCLUSION: Our work confirms that the Akita mouse model of diabetes replicates key clinical features of diabetic HFpEF, including cardiac and mitochondrial dysfunction. Furthermore, in this independent study, MitoGamide treatment improved diastolic function in Akita mice.

Vena caval filters for the prevention of pulmonary embolism.

BACKGROUND: Pulmonary emboli (PE), or blood clots in the lungs,can be potentially fatal. Anticoagulation is the first line therapy to prevent PE. In some instances anticoagulation fails to prevent more emboli, or cannot be given because the person has a high risk of bleeding. Inferior vena caval filters (VCFs) are metal alloy devices that mechanically trap fragmented emboli from the deep leg veins en route to the pulmonary circulation. Retrievable filters are designed to be introduced and removed percutaneously. Although their deployment seems of theoretical benefit, their clinical efficacy and adverse event profile is unclear. This is the third update of a Cochrane Review first published in 2007. OBJECTIVES: To assess the evidence for the effectiveness and safety of vena caval filters (VCFs) in preventing pulmonary embolism (PE). SEARCH METHODS: For this review update, the Cochrane Vascular Information Specialist (CIS) searched the Specialised Register (last searched 10 September 2019) and the Cochrane Register of Controlled Trials (CENTRAL) (2019, Issue 8) via the Cochrane Register of Studies Online. The CIS also searched MEDLINE Ovid, EMBASE Ovid, CINAHL, and AMED (1 January 2017 to 10 September 2019) and trials registries to 10 September 2019. SELECTION CRITERIA: We included randomised controlled trials (RCTs) and controlled clinical trials (CCTs) that examined the efficacy of VCFs in preventing PE. DATA COLLECTION AND ANALYSIS: For this update, studies were assessed and data extracted independently. We assessed study quality with Cochrane's 'Risk of bias' tool and used the GRADE approach to assess the overall certainty of the evidence. The outcomes of interest were PE, mortality, lower limb venous thrombosis, filter-related complications and major bleeding. MAIN RESULTS: We identified four new studies for this update, bringing the total to six included studies involving 1388 participants. The six studies were clinically heterogeneous and we were unable to carry out meta-analysis. Only two studies were considered to be both applicable in current clinical settings and of good methodological quality. One was a randomised open-label trial studying the effect of a retrievable inferior vena caval filter plus anticoagulation versus anticoagulation alone on risk of recurrent pulmonary embolism (PE) in 399 participants over three months. There was no evidence of a difference in the rates of PE, death, lower extremity deep vein thrombosis (DVT), or bleeding at three and six months after the intervention (moderate-certainty evidence). A filter was inserted in 193 people, but could only be successfully retrieved from 153. Minor filter complications were noted at six months. The second clinically relevant study was a randomised open-label trial of 240 participants who had sustained multiple traumatic injuries, allocated to a filter or no filter, three days after injury, in conjunction with anticoagulation and intermittent pneumatic compression. Prophylactic anticoagulation was initiated in both groups when it was thought safe to do so. There was no evidence of a difference in symptomatic PE, death, or lower limb venous thrombosis rates (moderate-certainty evidence). The only major filter complication was that one person required surgical removal of the filter. We are unable to draw any conclusions from the remaining four included studies. One study showed an increased incidence of long-term lower extremity DVT at eight years. Three studies are no longer clinically applicable because they utilised permanent filters which are seldom used now, or they did not use routine prophylactic anticoagulation which is current standard practice. The fourth study compared two filter types and was terminated prematurely as one filter group had a higher rate of thrombosis compared to the other filter type. AUTHORS' CONCLUSIONS: Two of the six identified studies were relevant for current clinical settings. One showed no evidence of a benefit of retrievable filters in acute PE for the outcomes of PE, death, DVT and bleeding during the initial three months in people who can receive anticoagulation (moderate-certainty evidence). The other study did not show any benefit for prophylactic filter insertion in people who sustained multiple traumatic injuries, with respect to symptomatic PE, mortality, or lower extremity venous thrombosis (moderate-certainty evidence). We can draw no firm conclusions regarding filter efficacy in the prevention of PE from the remaining four RCTs identified in this review. Further trials are needed to assess vena caval filter effectiveness and safety, and clinical differences between various filter types.

Development stage of cryopreserved mussel (Perna canaliculus) larvae influences post-thaw impact on shell formation, organogenesis, neurogenesis, feeding ability and survival.

Cryopreservation of genetic material from farmed aquatic species is a valuable technique to advance selective breeding programs for stock improvement. In this study, effects of cryopreservation on development of trochophore and D-stage larvae of Greenshell™ mussel (Perna canaliculus) were evaluated through histology, light microscopy, scanning electron microscopy, and confocal microscopy. Larvae of both life stages were motile immediately post-thawing, but survival declined rapidly from 4 days post-fertilisation (dpf). At 18 dpf, ~23% of non-cryopreserved control larvae had progressed to the pediveliger stage, while <1% of cryopreserved larvae had survived. Control larvae grew faster and larger, and consumed more food than larvae cryopreserved at either life stage (trochophore or D-stage). Settlement competency was achieved in the control larvae at 21 days post-fertilization, with most remaining individuals developing eye spots. Organogenesis was delayed in all cryopreserved larvae, and eyespots did not appear at all. Neurogenesis was stunted in cryopreserved trochophore larvae but seemed to progress almost normally in their cryopreserved D-stage counterparts. Developing abnormalities in shell morphology rapidly became apparent in all mussels post-thaw, with trochophore larvae being most highly afflicted. These delays in organogenesis and overall development are indicative of cryo-injuries sustained at a cellular level. Our results show that D-stage larvae are somewhat more resilient to cryopreservation than trochophore larvae. D-larvae are good life-stage candidates for cryobanking genetic resources in this species because there is generally an excess of larvae from selective breeding family crosses and these can be banked and stored for later use. Further on-going research aims to improve the long-term viability of cryopreserved D-larvae for successful rearing.

Metabolic and immune responses of Chinook salmon (Oncorhynchus tshawytscha) smolts to a short-term poly (I:C) challenge.

Polyinosinic:polycytidylic acid [poly (I:C)] was administered in vivo to Chinook salmon (Oncorhynchus tshawytscha) post-smolts to determine the immune responses on haematological and cellular functional parameters, including spleen (SP), head kidney (HK) and red blood cell (RBC) cytokine expression, as well as serum metabolomics. Poly (I:C) in vivo (24 h exposure) did not affect fish haematological parameters, leucocyte phagocytic activity and phagocytic index, reactive oxygen species and nitric oxide production. Gas chromatography-mass spectrometry-based metabolomics revealed that poly (I:C) significantly altered the serum biochemistry profile of 25 metabolites. Metabolites involved in the branched-chain amino acid/glutathione and transsulphuration pathways and phospholipid metabolism accumulated in poly (I:C)-treated fish, whereas those involved in the glycolytic and energy metabolism pathways were downregulated. At cytokine transcript level, poly (I:C) induced a significant upregulation of antiviral ifnγ in HK and Mx1 protein in HK, SP and RBCs. This study provides evidence for poly (I:C)-induced, immune-related biomarkers at metabolic and molecular levels in farmed O. tshawytscha in vivo. These findings provide insights into short-term effects of poly (I:C) at haematological, innate and adaptive immunity and metabolic levels, setting the stage for future studies.

Advances in salmonid fish immunology: A review of methods and techniques for lymphoid tissue and peripheral blood leucocyte isolation and application.

Evaluating studies over the past almost 40 years, this review outlines the current knowledge and research gaps in the use of isolated leucocytes in salmonid immunology understanding. This contribution focuses on the techniques used to isolate salmonid immune cells and popular immunological assays. The paper also analyses the use of leucocytes to demonstrate immunomodulation following dietary manipulation, exposure to physical and chemical stressors, effects of pathogens and parasites, vaccine design and application strategies assessment. We also present findings on development of fish immune cell lines and their potential uses in aquaculture immunology. The review recovered 114 studies, where discontinuous density gradient centrifugation (DDGC) with Percoll density gradient was the most popular leucocyte isolation method. Fish head kidney (HK) and peripheral blood (PB) were the main sources of leucocytes, from rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). Phagocytosis and respiratory burst were the most popular immunological assays. Studies used isolated leucocytes to demonstrate that dietary manipulations enhance fish immunity, while chemical and physical stressors suppress immunity. In addition, parasites, and microbial pathogens depress fish innate immunity and induce pro-inflammatory cytokine gene transcripts production, while vaccines enhance immunity. This review found 10 developed salmonid cell lines, mainly from S. salar and O. mykiss HK tissue, which require fish euthanisation to isolate. In the face of high costs involved with density gradient reagents, the application of hypotonic lysis in conjunction with mico-volume blood methods can potentially reduce research costs, time, and using nonlethal and ethically flexible approaches. Since the targeted literature review for this study retrieved no metabolomics study of leucocytes, indicates that this approach, together with traditional technics and novel flow cytometry could help open new opportunities for in vitro studies in aquaculture immunology and vaccinology.

In vitro immune response of chinook salmon (Oncorhynchus tshawytscha) peripheral blood mononuclear cells stimulated by bacterial lipopolysaccharide.

We investigated cellular functional and targeted immune cytokine responses of farmed Chinook salmon (Oncorhynchus tshawytscha) peripheral blood mononuclear cells (PBMCs) in vitro to LPS from Escherichia coli (E. coli) serotypes O111: B4 and O55: B5, and a phorbol ester phorbol 12-myristate 13-acetate (PMA). Bacterial LPS and PMA significantly (p < 0.05) induced reactive oxygen species (ROS) production in O. tshawytscha PBMCs, and enhanced by interferon (IFN)-inducible cytokine production. Cellular phagocytosis was significantly enhanced with PMA and E. coli serotype O111: B4 LPS after 1 and 2 h respectively. At the molecular level, LPS and PMA significantly (p < 0.05) upregulated pro-inflammatory cytokine gene transcripts for IFNγ, TNF-α, and anti-inflammatory IL-10, 24 h post-stimulation. This response is postulated to be mediated via the MyD88 and TRIF pathways in TLR4, or synergistic TLR1 and TLR2 receptors. This is the first report of LPS induced immune related in vitro responses in farmed O. tshawytscha PBMCs.