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Amoebic gill disease (AGD) is the primary health concern for Atlantic salmon Salmo salar farmed in Tasmania, Australia. Neoparamoeba perurans is the aetiological agent of AGD; however, a diversity of other amoebae colonise the gills, and their role in AGD is unknown. Previous studies which document these accompanying amoebae on AGD-affected farmed Atlantic salmon relied on culture-based techniques which do not accurately determine the prevalence and abundance of these species nor whether they correlate with AGD pathology. Drawing on our previous culture-based study, here we develop and apply 5 new Taqman quantitative PCR (qPCR) assays to profile the prevalence of multiple amoeba species on the gills of AGD-affected Atlantic salmon held at 2 Tasmanian farm sites over a 1 yr period. The prevalence and abundance of N. perurans was also assessed using a previously established qPCR method. N. perurans was the dominant species, and its abundance positively correlated with the progression of gross gill pathology. Only a small number of sporadic detections of Pseudoparamoeba and Vannellida species were observed. Nolandella spp. was the notable exception, as it was the most prevalent amoeba (92%) at 1 site at 1 sample time, during which no N. perurans were detected on gills but low levels of gross gill pathology were observed. N. perurans is the predominant species and primary pathogen of AGD; however, there were instances when they were not detected on diseased gills and Nolandella spp. were highly prevalent. The significance of Nolandella spp. in relation to AGD is not yet understood.

"MightyLev," a new multi-emitter ultrasonic acoustic levitation device capable of extremely stable levitation of materials of density up to at least 11.3 g cm-3, is described. The exceptional stability of medium- to high-density samples levitated in MightyLev makes the device highly suitable for chemical and structural analysis using micro-focused spectroscopic and x-ray scattering techniques. In combination with mid-infrared laser heating, MightyLev is capable of levitating metallic and oxide materials during high-temperature cycling and melting above 1500 K. Instabilities in particle confinement during heating were investigated by directly visualizing the acoustic field using schlieren imaging. The results reveal jets of hot-air directed along the anti-nodes of the acoustic field. The reaction force on the sample from the jet, coupled with the restoring force of the acoustic trap, generates a parametric lateral oscillation of the sample. This result provides valuable insight for future optimization and wider application of acoustic levitation for high-temperature containerless material processing., (© 2024 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC) license (https://creativecommons.org/licenses/by-nc/4.0/).)

This article focuses on the research and scholarship of leadership training. We begin by defining and giving context to the concept of leadership training. Then, we provide a high-level literature review of the history and components of leadership training (e.g., content, pedagogy, and assessment). Next, we connect leadership training with the leadership learning framework (LLF) to address common critiques of leadership training. The article concludes with projections of leadership training in the future and ethical questions for leadership educators to consider when designing leadership training programs., (© 2024 The Author(s). New Directions for Student Leadership published by Wiley Periodicals LLC.)

The long-read sequencing platform MinION, developed by Oxford Nanopore Technologies, enables the sequencing of bacterial genomes in resource-limited settings, such as field conditions or low- and middle-income countries. For this purpose, protocols for extracting high-molecular-weight DNA using nonhazardous, inexpensive reagents and equipment are needed, and some methods have been developed for gram-negative bacteria. However, we found that without modification, these protocols are unsuitable for gram-positive Streptococcus spp., a major threat to fish farming and food security in low- and middle-income countries. Multiple approaches were evaluated, and the most effective was an extraction method using lysozyme, sodium dodecyl sulfate, and proteinase K for lysis of bacterial cells and magnetic beads for DNA recovery. We optimized the method to consistently achieve sufficient yields of pure high-molecular-weight DNA with minimal reagents and time and developed a version of the protocol which can be performed without a centrifuge or electrical power. The suitability of the method was verified by MinION sequencing and assembly of 12 genomes of epidemiologically diverse fish-pathogenic Streptococcus iniae and Streptococcus agalactiae isolates. The combination of effective high-molecular-weight DNA extraction and MinION sequencing enabled the discovery of a naturally occurring 15 kb low-copy number mobilizable plasmid in S. iniae, which we name pSI1. We expect that our resource-limited settings-adapted protocol for high-molecular-weight DNA extraction could be implemented successfully for similarly recalcitrant-to-lysis gram-positive bacteria, and it represents a method of choice for MinION-based disease diagnostics in low- and middle-income countries., (© 2024 The Author(s). MicrobiologyOpen published by John Wiley & Sons Ltd.)

Cotton is an important agricultural crop to many regions across the globe but is sensitive to low-temperature exposure. The activity of the enzyme SENSITIVE TO FREEZING 2 (SFR2) improves cold tolerance of plants and produces trigalactosylsyldiacylglycerol (TGDG), but its role in cold sensitive plants, such as cotton remains unknown. Recently, it was reported that cotton SFR2 produced very little TGDG under normal and cold conditions. Here, we investigate cotton SFR2 activation and TGDG production. Using multiple approaches in the native system and transformation into Arabidopsis thaliana , as well as heterologous yeast expression, we provide evidence that cotton SFR2 activates differently than previously found among other plant species. We conclude with the hypothesis that SFR2 in cotton is not activated in a similar manner regarding acidification or freezing like Arabidopsis and that other regions of SFR2 protein are critical for activation of the enzyme than previously reported.

Background: Viscoelastic assays have widely been used for evaluating coagulopathies but lack the addition of shear stress important to in vivo clot formation. Stasys technology subjects whole blood to shear forces over factor-coated surfaces. Microclot formation is analyzed to determine clot area (CA) and platelet contractile forces (PCFs). We hypothesize the CA and PCF from this novel assay will provide information that correlates with trauma-induced coagulopathy and transfusion requirements., Methods: Blood samples were collected on adult trauma patients from a single-institution prospective cohort study of high-level activations. Patient and injury characteristics, transfusion data, and outcomes were collected. Thromboelastography, coagulation studies, and Stasys assays were run on paired samples collected at admission. Stasys CA and PCFs were quantified as area under the curve calculations and maximum values. Normal ranges for Stasys assays were determined using healthy donors. Data were compared using Kruskal-Wallis tests and simple linear regression., Results: From March 2021 to January 2023, 108 samples were obtained. Median age was 37.5 (IQR 27.5-52) years; patients were 77% male. 71% suffered blunt trauma, 26% had an Injury Severity Score of ≥25. An elevated international normalized ratio significantly correlated with decreased cumulative PCF (p=0.05), maximum PCF (p=0.05) and CA (p=0.02). Lower cumulative PCF significantly correlated with transfusion of any products at 6 and 24 hours (p=0.04 and p=0.05) as well as packed red blood cells (pRBCs) at 6 and 24 hours (p=0.04 and p=0.03). A decreased maximum PCF showed significant correlation with receiving any transfusion at 6 (p=0.04) and 24 hours (p=0.02) as well as transfusion of pRBCs, fresh frozen plasma, and platelets in the first 6 hours (p=0.03, p=0.03, p=0.03, respectively)., Conclusions: Assessing coagulopathy in real time remains challenging in trauma patients. In this pilot study, we demonstrated that microfluidic approaches incorporating shear stress could predict transfusion requirements at time of admission as well as requirements in the first 24 hours., Level of Evidence: Level II., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: In DiRECT, a randomised controlled effectiveness trial, weight management intervention after 2 years resulted in mean weight loss of 7·6 kg, with 36% of participants in remission of type 2 diabetes. Of 36 in the intervention group who maintained over 10 kg weight loss at 2 years, 29 (81%) were in remission. Continued low-intensity dietary support was then offered up to 5 years from baseline to intervention participants, aiming to maintain weight loss and gain clinical benefits. This extension study was designed to provide observed outcomes at 5 years., Methods: The DiRECT trial took place in primary care practices in the UK. Participants were individuals aged 20-65 years who had less than 6 years' duration of type 2 diabetes, a BMI greater than 27 kg/m 2 , and were not on insulin. The intervention consisted of withdrawal of antidiabetic and antihypertensive drugs, total diet replacement (825-853 kcal per day formula diet for 12-20 weeks), stepped food reintroduction (2-8 weeks), and then structured support for weight-loss maintenance. After sharing the 2-year results with all participants, UK National Health Service data were collected annually until year 5 from remaining intervention participants who received low-intensity dietary support, intervention withdrawals, and the original randomly allocated groups. The primary outcome was remission of type 2 diabetes; having established in the DiRECT trial that sustained weight loss was the dominant driver of remission, this was assumed for the Extension study. The trial is registered with the ISRCTN registry, number 03267836., Findings: Between July 25, 2014, and Aug 5, 2016, 149 participants were randomly assigned to the intervention group and 149 were assigned to the control group in the original DiRECT study. After 2 years, all intervention participants still in the trial (101 [68%] of 149) were approached to receive low-intensity support for a further 3 years. 95 (94%) of 101 were able to continue and consented and were allocated to the DiRECT extension group. 54 participants were allocated to the non-extension group, where intervention was withdrawn. At 5 years, DiRECT extension participants (n=85) lost an average of 6·1 kg, with 11 (13%) of 85 in remission. Compared with the non-extension group, DiRECT extension participants had more visits with HbA 1c <48 mmol/mol (<6·5%; 36% vs 17%, p=0·0004), without glucose-lowering medication (62% vs 30%, p<0·0001), and in remission (34% vs 12%, p<0·0001). Original control participants (n=149) had mean weight loss 4·6 kg (n=82), and 5 (5%) of 93 were in remission. Compared with control participants, original intervention participants had more visits with weight more than 5% below baseline (61% vs 29%, p<0·0001), HbA 1c below 48 mmol/mol (29% vs 15%, p=0·0002), without antidiabetic medication (51% vs 16%, p<0·0001), and in remission (27% vs 4%, p<0·0001). Of those in remission at year 2, 26% remained in remission at 5 years. Serious adverse events in the original intervention group (4·8 events per 100 patient-years) were under half those in the control group (10·2 per 100 patient-years, p=0·0080)., Interpretation: The extended DiRECT intervention was associated with greater aggregated and absolute weight loss, and suggested improved health status over 5 years., Funding: Diabetes UK., Competing Interests: Declaration of interests MEJL reports grants and personal fees unconnected with the present work from Counterweight, Novo Nordisk, Novartis, and Eli Lilly. RT has received lecture fees from Novartis and Eli Lilly, and has served on an advisory panel for Wilmington Healthcare. NB reports employment and shareholding in Counterweight, and PhD fees funded by Cambridge Weight Plan (outside the submitted work). GT received funding for PhD fees and conference attendance from Cambridge Weight Plan outside the submitted work. WSL reports support for conference attendance from Cambridge Weight Plan outside the submitted work. NS reports grants and personal fees from AstraZeneca, Boehringer Ingelheim, Novartis, and Roche Diagnostics; and personal fees from Abbott Laboratories, Amgen, Eli Lilly, Hanmi Pharmaceuticals, Janssen, Merck Sharp & Dohme, Novo Nordisk, Pfizer, and Sanofi, outside the submitted work. LM reports employment by Counterweight during the conduct of study, was previously a shareholder in Counterweight, and previous employment from Cambridge Weight Plan outside the submitted work. All other authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

The structure of aerodynamically levitated liquid [Formula: see text] was measured by neutron diffraction with isotope substitution (NDIS). Classical and ab initio molecular dynamics simulations were performed and their results were found to be in close agreement with each other and the NDIS data. The results reveal that molten [Formula: see text] may be considered as an ionic liquid without any preference for particular short-range structural motifs. This article is part of the theme issue 'Exploring the length scales, timescales and chemistry of challenging materials (Part 1)'.

Severe cold, defined as a damaging cold beyond acclimation temperatures, has unique responses, but the signaling and evolution of these responses are not well understood. Production of oligogalactolipids, which is triggered by cytosolic acidification in Arabidopsis (Arabidopsis thaliana), contributes to survival in severe cold. Here, we investigated oligogalactolipid production in species from bryophytes to angiosperms. Production of oligogalactolipids differed within each clade, suggesting multiple evolutionary origins of severe cold tolerance. We also observed greater oligogalactolipid production in control samples than in temperature-challenged samples of some species. Further examination of representative species revealed a tight association between temperature, damage, and oligogalactolipid production that scaled with the cold tolerance of each species. Based on oligogalactolipid production and transcript changes, multiple angiosperm species share a signal of oligogalactolipid production initially described in Arabidopsis, namely cytosolic acidification. Together, these data suggest that oligogalactolipid production is a severe cold response that originated from an ancestral damage response that remains in many land plant lineages and that cytosolic acidification may be a common signaling mechanism for its activation., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Weight loss in overweight or obese individuals with Type 2 diabetes (T2D) can normalize hepatic fat metabolism, decrease fatty acid oversupply to β cells and restore normoglycaemia. One in six people has BMI <27 kg/m2 at diagnosis, and their T2D is assumed to have different aetiology. The Personal Fat Threshold hypothesis postulated differing individual thresholds for lipid overspill and adverse effects on β-cell function. To test this hypothesis, people with Type 2 diabetes and body mass index <27kg/m2 (n = 20) underwent repeated 5% weight loss cycles. Metabolic assessments were carried out at stable weight after each cycle and after 12 months. To determine how closely metabolic features returned to normal, 20 matched normoglycemic controls were studied once. Between baseline and 12 months: BMI fell (mean ± SD), 24.8 ± 0.4 to 22.5 ± 0.4 kg/m2 (P<0.0001) (controls: 21.5 ± 0.5); total body fat, 32.1 ± 1.5 to 27.6 ± 1.8% (P<0.0001) (24.6 ± 1.5). Liver fat content and fat export fell to normal as did fasting plasma insulin. Post-meal insulin secretion increased but remained subnormal. Sustained diabetes remission (HbA1c < 48 mmol/mol off all glucose-lowering agents) was achieved by 70% (14/20) by initial weight loss of 6.5 (5.5-10.2)%. Correction of concealed excess intra-hepatic fat reduced hepatic fat export, with recovery of β-cell function, glycaemic improvement in all and return to a non-diabetic metabolic state in the majority of this group with BMI <27 kg/m2 as previously demonstrated for overweight or obese groups. The data confirm the Personal Fat Threshold hypothesis: aetiology of Type 2 diabetes does not depend on BMI. This pathophysiological insight has major implications for management., (© 2023 The Author(s).)

Gene inactivation studies are critical in pathogenic bacteria, where insights into species biology can guide the development of vaccines and treatments. Allelic exchange via homologous recombination is a generic method of targeted gene editing in bacteria. However, generally applicable protocols are lacking, and suboptimal approaches are often used for nonstandard but epidemiologically important species. Photobacterium damselae subsp. piscicida (Pdp) is a primary pathogen of fish in aquaculture and has been considered hard to transform since the mid-1990s. Consequently, conjugative transfer of RK2/RP4 suicide vectors from Escherichia coli S17-1/SM10 donor strains, a system prone to off-target mutagenesis, was used to deliver the allelic exchange DNA in previous studies. Here we have achieved efficient electrotransformation in Pdp using a salt-free highly concentrated sucrose solution, which performs as a hypertonic wash buffer, cryoprotectant, and electroporation buffer. High-efficiency transformation has enabled vector-free mutagenesis for which we have employed circular minimalistic constructs (knockout minicircles) containing only allelic exchange essentials that were generated by Gibson assembly. Preparation of competent cells using sucrose and electroporation/integration of minicircles had virtually no detectable off-target promutagenic effect. In contrast, a downstream sacB selection apparently induced several large deletions via mobilization of transposable elements. Electroporation of minicircles into sucrose-treated cells is a versatile broadly applicable approach that may facilitate allelic exchange in a wide range of microbial species. The method permitted inactivation of a primary virulence factor unique to Pdp, apoptogenic toxin AIP56, demonstrating the efficacy of minicircles for difficult KO targets located on the high copy number of small plasmids., (© 2023 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Aims: As sustained weight loss is vital for achieving remission of type 2 diabetes, we explored whether randomisation to weight loss plus maintenance in the DiRECT trial was associated with physical activity, inactivity or sleep., Methods: Participants were randomised to either a dietary weight management programme or best-practice care. The weight management group were encouraged to increase daily physical activity to their sustainable maximum. Objective measurement was achieved using a wrist-worn GENEActiv accelerometer for 7 days at baseline, 12 and 24 months in both groups., Results: Despite average weight loss of 10 kg at 12 months in the intervention (n = 66) group, there were no differences in total physical activity or inactivity compared with the control (n = 104) at any time point. However, in our exploratory analysis, those who lost more than 10% of their baseline body weight performed on average 11 mins/day more light activity than the <10% group at 24 months (p = 0.033) and had significantly lower bouts of Inactivity 30min (interaction, p = 0.005) across 12 and 24 months. At 24 months, the ≥10% group had higher daily acceleration (38.5 ± 12.1 vs. 33.2 ± 11.1 mg, p = 0.020), and higher accelerations in the most active 5-hour period (59.4 ± 21.8 vs. 50.6 ± 18.3 mg, p = 0.023). Wakefulness after sleep onset decreased in the intervention group compared with the control group and also in the ≥10% weight loss group at 12 and 24 months., Conclusions: Randomisation to a successful intensive weight loss intervention, including regular physical activity encouragement, was not associated with increased physical activity although sleep parameters improved. Physical activity was greater, and night-time waking reduced in those who maintained >10% weight loss at 12 and 24 months. TRIAL REGISTRATION ISRCTN03267836., (© 2022 The Authors. Diabetic Medicine published by John Wiley & Sons Ltd on behalf of Diabetes UK.)

Quantification of specific antibodies underpins the assessment of adaptive immunity in response to vaccination or infection and is performed by enzyme-linked immunosorbent assay (ELISA). A biolayer interferometry (BLI) assay was recently developed that simultaneously quantifies IgM antibodies and their avidity in giant grouper (Epinephelus lanceolatus) sera and proved to be a robust, repeatable and more high-throughput alternative to ELISA [1]. Here we attempted to optimise a similar single-step BLI assay using an Octet HTX instrument to quantify IgM specific to the hapten 2,4-dinitrophenol (DNP) in serum from Atlantic salmon (Salmo salar) primed and boosted with DNP conjugated to keyhole limpet hemocyanin., Competing Interests: Declaration of competing interest No potential competing interest was reported by the authors., (Crown Copyright © 2023. Published by Elsevier Ltd. All rights reserved.)

Mixed culture purple phototrophic bacteria (PPB) is a rapidly emerging technology for resource recovery from wastewaters. PPB biomass can be used as single-cell protein, with a high protein content complemented by value-added components (e.g. pigments and polyhydroxyalkanoates), merging functionalities within a single product. This has the potential to increase the value and impact the economic feasibility, justifying higher capital costs for PPB photobioreactors for real-life applications. Artificial illumination is prohibitively expensive, and naturally illuminated, outdoor units are a critical next step. However, information required for informed technoeconomic assessment of single-cell protein from PPB is still missing and can only be determined in dedicated larger-scale, outdoor systems. Larger scale units are also required to supply feed for larger cohort trials. Although data from microalgae research can be used as starting point to estimate costs, they cannot be translated directly for PPB, as the organisms and metabolic growth are fundamentally different., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Photobacterium damselae comprises two subspecies, P. damselae subsp. damselae and P. damselae subsp. piscicida , that contrast remarkably despite their taxonomic relationship. The former is opportunistic and free-living but can cause disease in compromised individuals from a broad diversity of taxa, while the latter is a highly specialized, primary fish pathogen. Here, we employ new closed curated genome assemblies from Australia to estimate the global phylogenetic structure of the species P. damselae. We identify genes responsible for the shift from an opportunist to a host-adapted fish pathogen, potentially via an arthropod vector as fish-to-fish transmission was not achieved in repeated cohabitation challenges despite high virulence for Seriola lalandi . Acquisition of ShdA adhesin and of thiol peroxidase may have allowed the environmental, generalist ancestor to colonize zooplankton and to occasionally enter in fish host sentinel cells. As dependence on the host has increased, P. damselae has lost nonessential genes, such as those related to nitrite and sulfite reduction, urea degradation, a type 6 secretion system (T6SS) and several toxin-antitoxin (TA) systems. Similar to the evolution of Yersinia pestis, the loss of urease may be the crucial event that allowed the pathogen to stably colonize zooplankton vectors. Acquisition of host-specific genes, such as those required to form a sialic acid capsule, was likely necessary for the emergent P. damselae subsp. piscicida to become a highly specialized, facultative intracellular fish pathogen. Processes that have shaped P. damselae subsp. piscicida from subsp. damselae are similar to those underlying evolution of Yersinia pestis from Y. pseudotuberculosis. IMPORTANCE Photobacterium damselae subsp. damselae is a ubiquitous marine bacterium and opportunistic pathogen of compromised hosts of diverse taxa. In contrast, its sister subspecies P. damselae subsp. piscicida ( Pdp ) is highly virulent in fish. Pdp has evolved from a single subclade of Pdd through gene loss and acquisition. We show that fish-to-fish transmission does not occur in repeated infection models in the primary host , Seriola lalandi, and present genomic evidence for vector-borne transmission, potentially via zooplankton. The broad genomic changes from generalist Pdd to specialist Pdp parallel those of the environmental opportunist Yersinia pseudotuberculosis to vector-borne plague bacterium Y. pestis and demonstrate that evolutionary processes in bacterial pathogens are universal between the terrestrial and marine biosphere.

Native Americans domesticated maize ( Zea mays ssp. mays ) from lowland teosinte parviglumis ( Zea mays ssp. parviglumis) in the warm Mexican southwest and brought it to the highlands of Mexico and South America where it was exposed to lower temperatures that imposed strong selection on flowering time. Phospholipids are important metabolites in plant responses to low-temperature and phosphorus availability and have been suggested to influence flowering time. Here, we combined linkage mapping with genome scans to identify High PhosphatidylCholine 1 ( HPC1 ), a gene that encodes a phospholipase A1 enzyme, as a major driver of phospholipid variation in highland maize. Common garden experiments demonstrated strong genotype-by-environment interactions associated with variation at HPC1, with the highland HPC1 allele leading to higher fitness in highlands, possibly by hastening flowering. The highland maize HPC1 variant resulted in impaired function of the encoded protein due to a polymorphism in a highly conserved sequence. A meta-analysis across HPC1 orthologs indicated a strong association between the identity of the amino acid at this position and optimal growth in prokaryotes. Mutagenesis of HPC1 via genome editing validated its role in regulating phospholipid metabolism. Finally, we showed that the highland HPC1 allele entered cultivated maize by introgression from the wild highland teosinte Zea mays ssp. mexicana and has been maintained in maize breeding lines from the Northern United States, Canada, and Europe. Thus, HPC1 introgressed from teosinte mexicana underlies a large metabolic QTL that modulates phosphatidylcholine levels and has an adaptive effect at least in part via induction of early flowering time.

Purpose: To present a case of refractory systemic lupus erythematosus (SLE)-associated retinal vasculitis that responded to the anti-tumor necrosis factor (TNF)-alpha inhibitor adalimumab as corticosteroid-sparing therapy., Methods: Descriptive case report of a patient with SLE with retinal vasculitis complicated by an ischemic retinal vein occlusion and cystoid macular edema., Results: A 30-year-old female patient with a history of SLE presented with retinal vasculitis and an ischemic, branch retinal vein occlusion with macular edema in the left eye. Oral corticosteroid was administered along with mycophenolate mofetil (MMF) as a corticosteroid-sparing agent. Despite MMF therapy, the patient developed an exacerbation of her vasculitis with the involvement of both eyes. Adalimumab was initiated with a resultant resolution of retinal vasculitis as a corticosteroid-sparing strategy with over 2 years of follow-up., Conclusion: Anti-TNF-alpha therapy with adalimumab may be effective as a corticosteroid-sparing agent in select patients with ocular inflammation associated with SLE.

Fish mortality caused by Streptococcus iniae is a major economic problem in aquaculture in warm and temperate regions globally. There is also risk of zoonotic infection by S. iniae through handling of contaminated fish. In this study, we present the complete genome sequence of S. iniae strain QMA0248, isolated from farmed barramundi in South Australia. The 2.12 Mb genome of S. iniae QMA0248 carries a 32 kb prophage, a 12 kb genomic island and 92 discrete insertion sequence (IS) elements. These include nine novel IS types that belong mostly to the IS 3 family. Comparative and phylogenetic analysis between S. iniae QMA0248 and publicly available complete S. iniae genomes revealed discrepancies that are probably due to misassembly in the genomes of isolates ISET0901 and ISNO. Long-range PCR confirmed five rRNA loci in the PacBio assembly of QMA0248, and, unlike S. iniae 89353, no tandemly repeated rRNA loci in the consensus genome. However, we found sequence read evidence that the tandem rRNA repeat existed within a subpopulation of the original QMA0248 culture. Subsequent nanopore sequencing revealed that the tandem rRNA repeat was the most prevalent genotype, suggesting that there is selective pressure to maintain fewer rRNA copies under uncertain laboratory conditions. Our study not only highlights assembly problems in existing genomes, but provides a high-quality reference genome for S. iniae QMA0248, including manually curated mobile genetic elements, that will assist future S. iniae comparative genomic and evolutionary studies.

Objective: To evaluate whether pentosan polysulfate maculopathy manifests distinctive imaging features that can be differentiated from those found in age-related macular degeneration (AMD)., Methods: Local databases were queried to identify patients with a diagnosis of interstitial cystitis who were seen at the Emory Eye Center between May 2014 and January 2019 and who had fundus imaging available for review. Ninety patients met the eligibility criteria. Masked graders categorized patients based on imaging characteristics as follows: category 1: pentosan polysulfate maculopathy; category 2: AMD or drusen; category 3: neither; and category 4: unsure. Pentosan polysulfate exposure characteristics were compared among groups., Results: Of the 90 subjects evaluated, 79 (88%) were female and the median age was 61.5 years (range, 30-89). Seventeen patients were placed in category 1; 25 in category 2; 47 in category 3, and; 1 in category 4. Among categories 1 to 4, respectively, 17 (100%), 15 (60%), 28 (60%), and 0 patients had exposure to pentosan polysulfate (p = 0.007). Mean cumulative exposure to pentosan polysulfate across the four categories was 2.1, 0.36, 0.34, and 0 kg, respectively (p < 0.00001). Eyes with pentosan polysulfate maculopathy did not have typical drusen in the macula., Conclusion: Although pentosan polysulfate maculopathy resembles some aspects of AMD, the two conditions can be differentiated with the use of multimodal fundus imaging., (Copyright © 2021 Canadian Ophthalmological Society. Published by Elsevier Inc. All rights reserved.)

Introduction: The Diabetes REmission Clinical Trial (DiRECT) has shown that sustained remission of type 2 diabetes in primary care is achievable through weight loss using total diet replacement (TDR) with continued behavioural support. Understanding participants' experiences can help optimise the intervention, support implementation into healthcare, and understand the process of behaviour change., Methods: Thirty-four DiRECT participants were recruited into this embedded qualitative evaluation study. In-person and telephone interviews were conducted before the TDR; at week 6-8 of the TDR; 2 weeks into food reintroduction (FR); and at 1 year, to learn about participant experiences with the programme. Transcribed narratives were analysed thematically, and we used interpretation to develop overarching themes., Results: Initiation of the TDR and transition to FR were challenging and required increased behavioural support. In general, adhering to TDR proved easier than the participants had anticipated. Some participants chose the optional extension of TDR. Rapid weight loss and changes in diabetes markers provided ongoing motivation. Further weight loss, behavioural support and occasional use of TDR facilitated weight loss maintenance (WLM). A process of behaviour adaptation to change following regime disruption was identified in three stages: (1) expectations of the new, (2) overcoming difficulties with adherence, and (3) acceptance of continuous effort and establishment of routines., Conclusions: The DiRECT intervention was acceptable and regularity, continuity, and tailoring of behavioural support was instrumental in its implementation in primary care. The adaptation process accounts for some of the individual variability of experiences with the intervention and highlights the need for programme flexibility., (© 2021 Diabetes UK.)

Aims/hypothesis: Type 2 diabetes confers a greater relative increase in CVD risk in women compared with men. We examined sex differences in intraorgan fat and hepatic VLDL1-triacylglycerol (VLDL1-TG) export before and after major dietary weight loss., Methods: A group with type 2 diabetes (n = 64, 30 male/34 female) and a group of healthy individuals (n = 25, 13 male/12 female) were studied. Intraorgan and visceral fat were quantified by magnetic resonance and VLDL1-TG export by intralipid infusion techniques., Results: Triacylglycerol content of the liver and pancreas was elevated in people with diabetes with no sex differences (liver 16.4% [9.3-25.0%] in women vs 11.9% [7.0-23.1%] in men, p = 0.57, and pancreas 8.3 ± 0.5% vs 8.5 ± 0.4%, p = 0.83, respectively). In the absence of diabetes, fat levels in both organs were lower in women than men (1.0% [0.9-1.7%] vs 4.5% [1.9-8.0%], p = 0.005, and 4.7 ± 0.4% vs 7.6 ± 0.5%, p< 0.0001, respectively). Women with diabetes had higher hepatic VLDL1-TG production rate and plasma VLDL1-TG than healthy women (559.3 ± 32.9 vs 403.2 ± 45.7 mg kg -1  day -1 , p = 0.01, and 0.45 [0.26-0.77] vs 0.25 [0.13-0.33] mmol/l, p = 0.02), whereas there were no differences in men (548.8 ± 39.8 vs 506.7 ± 29.2 mg kg -1  day -1 , p = 0.34, and 0.72 [0.53-1.15] vs 0.50 [0.32-0.68] mmol/l, p = 0.26). Weight loss decreased intraorgan fat and VLDL1-TG production rates regardless of sex, and these changes were accompanied by similar rates of diabetes remission (65.4% vs 71.0%) and CVD risk reduction (59.8% vs 41.5%) in women and men, respectively., Conclusions/interpretation: In type 2 diabetes, women have liver and pancreas fat levels as high as those of men, associated with raised hepatic VLDL1-TG production rates. Dynamics of triacylglycerol turnover differ between sexes in type 2 diabetes and following weight loss. These changes may contribute to the disproportionately raised cardiovascular risk of women with diabetes., (© 2021. The Author(s).)

People living in the Balsas River basin in southwest México domesticated maize from the bushy grass teosinte. Nine thousand years later, in 2021, Ms. Deb Haaland - a member of the Pueblo of Laguna tribe of New Mexico - wore a dress adorned with a cornstalk when she was sworn in as the Secretary of Interior of the United States of America. This choice of garment highlights the importance of the coevolution of maize and the farmers who, through careful selection over thousands of years, domesticated maize and adapted the physiology and shoot architecture of maize to fit local environments and growth habits. Some traits such as tillering were directly selected on (arches), and others such as tassel size are the by-products (spandrels) of maize evolution. Here, we review current knowledge of the underlying cellular, developmental, physiological, and metabolic processes that were selected by farmers and breeders, which have positioned maize as a top global staple crop., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Quantification of specific antibody responses is critical in determining activation of MHCII-dependent immune memory and is generally performed by enzyme-linked immunosorbent assay (ELISA). Antibody avidity for a particular antigen is also informative of the quality of the adaptive immune response following vaccination. Avidity can be determined by chaotropic elution ELISA, pre-absorption ELISA, or surface plasmon resonance (SPR), although multimeric antibodies such as IgM are problematic for SPR. ELISA-based assays are very time consuming, require secondary antibody reagents, and are poorly repeatable. Here we demonstrate that biolayer interferometry (BLI) using an Octet HTX instrument can robustly and reproducibly quantify and determine avidity of specific IgM for an antigen directly from fish serum in a single step. We collected sera from giant grouper (Epinephelus lanceolatus) that had been vaccinated with the hapten 2,4-dinitrophenol conjugated to keyhole limpet hemocyanin (DNP-KLH) and from control fish injected with phosphate buffered saline. The specific IgM in the serum and its avidity for DNP were quantified via ELISA and BLI. BLI was precise and highly repeatable for determination of the quantity and avidity of antibody in the serum compared to ELISA. The wet-lab preparation and machine running time for BLI was 3-5 times faster than ELISA to generate the same amount of data. The ELISA inter-plate variation significantly affected reproducibility while BLI was consistent and repeatable between samples and plates. Indeed, the consistency of BLI data indicated that technical triplicates were redundant. Biological replication alone was sufficient to elucidate the effect of treatments. However, BLI required a lower serum dilution than ELISA for similar sensitivity, and thus more serum was required to produce high resolution data. BLI is an extremely high-throughput assay, providing teleost serum IgM quantification and avidity data as a single-step, agile alternative to ELISA., (Crown Copyright © 2021. Published by Elsevier Ltd. All rights reserved.)

Streptococcus iniae is an emerging zoonotic pathogen of increasing concern for aquaculture and has caused several epizootics in reef fishes from the Caribbean, the Red Sea and the Indian Ocean. To study the population structure, introduction pathways and evolution of S. iniae over recurring epizootics on Reunion Island, we developed and validated a Multi Locus Sequence Typing (MLST) panel using genomic data obtained from 89 isolates sampled during epizootics occurring over the past 40years in Australia, Asia, the United States, Israel and Reunion Island. We selected eight housekeeping loci, which resulted in the greatest variation across the main S. iniae phylogenetic clades highlighted by the whole genomic dataset. We then applied the developed MLST to investigate the origin of S . iniae responsible for four epizootics on Reunion Island, first in inland aquaculture and then on the reefs from 1996 to 2014. Results suggest at least two independent S . iniae emergence events occurred on the island. Molecular data support that the first epizootic resulted from an introduction, with inland freshwater aquaculture facilities acting as a stepping-stone. Such an event may have been facilitated by the ecological flexibility of S. iniae , able to survive in both fresh and marine waters and the ability of the pathogen to infect multiple host species. By contrast, the second epizootic was associated with a distinct ST of cosmopolitan distribution that may have emerged as a result of environment disturbance. This novel tool will be effective at investigating recurrent epizootics occurring within a given environment or country that is despite the fact that S. iniae appears to have low genetic diversity within its lineage., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Irion, Silayeva, Sweet, Chabanet, Barnes, Tortosa and Séré.)

Aims/hypothesis: Our aim was to evaluate the safety and efficacy of a planned therapeutic withdrawal of all antihypertensive and diuretic medications, on commencing a formula low-energy diet replacement, targeting remission of type 2 diabetes., Methods: Post hoc analysis of changes in BP, antihypertensive medication prescriptions and symptoms during the initial total diet replacement phase was performed in the intervention arm of the Diabetes Remission Clinical Trial (n = 143) and in the subset (n = 69) who discontinued antihypertensive medications at the start of total diet replacement. The Counterweight-Plus total diet replacement provided about 3470 kJ/day (830 kcal) with automatic reductions in all nutrients, including sodium, to achieve marked negative energy balance and rapid weight loss over 12-20 weeks, with regular BP monitoring and an antihypertensive reintroduction protocol based on current clinical guidelines., Results: Of 143 intervention group participants who commenced total diet replacement, 78 (55%) were on treatment for hypertension at baseline. The overall mean BP fell significantly from the start of total diet replacement (week 1) and was significantly lower at week 20, after total diet replacement finished, and also at 12 and 24 months. Of the 78 participants previously on treatment for hypertension, 65 (83%) stopped all antihypertensive and diuretic medications as per protocol, and four (5%) stopped some drugs. These 69 participants experienced no immediate (within the first week) change in BP, but their mean BP fell significantly from 9 weeks. No excessive rises in BP were recorded in individuals, but antihypertensive medications were reintroduced during total diet replacement to manage raised BP for 19/69 (27.5%) participants, mostly within the first 3-7 weeks, despite some weight loss. Reintroduction of antihypertensive medications was necessary for 5/19 participants previously on one drug, and for 14/19 previously on two or more drugs. Of the 69 who stopped antihypertensives, 19 (28%) remained off medications at 24 months. Among the 53 participants who achieved sustained remissions of diabetes at 24 months (with a mean weight loss of 11.4 kg), 31 had been previously treated for hypertension. Twenty-seven stopped medication at baseline, and 15/27 required reintroduction of antihypertensive medications. Mild to moderate dizziness, suggesting some postural hypotension, was reported during total diet replacement by 51 participants, 15 of whom had recorded dizziness at baseline prior to starting total diet replacement, with nine of these on antihypertensive or diuretic medications., Conclusions/interpretation: Replacing antihypertensive medications with a 3470 kJ/day (830 kcal) diet to induce weight loss reduces BP substantially and may increase mild dizziness. It is safe to stop antihypertensives, but BP should be monitored regularly, particularly for those taking two or more antihypertensives, as over two-thirds will require reintroduction of some medications. Long-term support to maintain weight loss is vital., Trial Registration: ISRCTN registry, number 03267836., (© 2021. The Author(s).)

Aim: To identify predictors of type 2 diabetes remission in the intervention arm of DiRECT (Diabetes Remission Clinical Trial)., Methods: Participants were aged 20-65 years, with type 2 diabetes duration of <6 years and BMI 27-45 kg/m 2 , and were not receiving insulin. Weight loss was initiated by total diet replacement (825-853 kcal/day, 3-5 months, shakes/soups), and weight loss maintenance support was provided for 2 years. Remissions (HbA 1c <48 mmol/mol [<6.5%], without antidiabetes medications) in the intervention group (n = 149, mean age 53 years, BMI 35 kg/m 2 ) were achieved by 68/149 participants (46%) at 12 months and by 53/149 participants (36%) at 24 months. Potential predictors were examined by logistic regression analyses, with adjustments for weight loss and effects independent of weight loss., Results: Baseline predictors of remission at 12 and 24 months included being prescribed fewer antidiabetes medications, having lower triglyceride and gamma-glutamyl transferase levels, and reporting better quality of life with less anxiety/depression. Lower baseline HbA 1c was a predictor at 12 months, and older age and male sex were predictors at 24 months. Being prescribed antidepressants predicted non-remission. Some, but not all effects were explained by weight loss. Weight loss was the strongest predictor of remission at 12 months (adjusted odds ratio per kg weight loss 1.24, 95% CI 1.14, 1.34; P < 0.0001) and 24 months (adjusted odds ratio 1.23, 95% CI 1.13, 1.35; P <0.0001). Weight loss in kilograms and percentage weight loss were equally good predictors. Early weight loss and higher programme attendance predicted more remissions. Baseline BMI, fasting insulin, fasting C-peptide and diabetes duration did not predict remission., Conclusions: Other than weight loss, most predictors were modest, and not sufficient to identify subgroups for which remission was not a worthwhile target., (© 2020 The Authors. Diabetic Medicine published by John Wiley & Sons Ltd on behalf of Diabetes UK.)

Two IgM heavy (H) chain sub-isotypes (80 and 40 kDa) and two light (L) chain variants (25 and 30 kDa) were detected in the serum of giant grouper (Epinephelus lanceolatus), purified by ammonium sulphate precipitation followed by protein A affinity chromatography. This method yielded 5.6 mg/mL high purity IgM from grouper serum, with efficiency estimated at 39.5% recovery from crude serum. The H and L chains were identified by SDS-PAGE and mass spectrometry (MS). Nanopore long-read sequencing was used to generate a genomic contig (MW768935), containing Cμ, Cδ loci, V H regions, and a H chain Joining segment. cDNA sequencing of Cμ transcripts (MW768933 and MW768934) were used to polish the genomic contig and determine the exons and introns of the corresponding locus. MS peptide mapping revealed that the 80 kDa H chain consisted of C H 1-4 domains while peptides from the 40 kDa H chain only mapped to C H 1-2 domains. Our genomic contig showed the Cμ locus has a Cμ1-Cμ2-Cμ3-Cμ4 arrangement on the same strand as the other Ig loci identified in this genomic sequence. Our study corrects the NCBI annotations of the opposing Cμ loci (LOC117268697 and LOC117268550) in chromosome 16 (NC&#95;047006). Further, we identified both κ and λ L chain isotypes in serum IgM. The molecular weight differences observed may result from different combinations of C L and V L genes. Putative IgM sub-isotypes have also been reported in Epinephelus itajara and Epinephelus coioides. The presence of IgM sub-isotypes may be a conserved trait among Epinephelus species., (Crown Copyright © 2021. Published by Elsevier Ltd. All rights reserved.)

This article explores the ethical use of assessments in leadership training, education, and development. From the importance of having well-trained facilitators to the consideration of power and social identity in the interpretation of individual results, this article advocates for approaching the use of leadership assessments and inventories with a critical ethic of care. This practical focus on ethics serves as a compliment to the other articles in this text by helping leadership educators and leadership professionals apply this critical lens to every facilitation., (© 2021 Wiley Periodicals, LLC.)

Despite the recent advances in sequencing technologies, the complete assembly of multi-chromosome genomes of the Vibrionaceae , often containing several plasmids, remains challenging. Using a combination of Oxford Nanopore MinION long reads and short Illumina reads, we fully sequenced, closed and curated the genomes of two strains of a primary aquatic pathogen Photobacterium damselae subsp. piscicida isolated in Australia. These are also the first genome sequences of P. damselae subsp. piscicida isolated in Oceania and, to our knowledge, in the Southern hemisphere. We also investigated the phylogenetic relationships between Australian and overseas isolates, revealing that Australian P. damselae subsp. piscicida are more closely related to the Asian and American strains rather than to the European ones. We investigated the mobilome and present new evidence showing that a host specialization process and progressive adaptive evolution to fish are ongoing in P. damselae subsp. piscicida , and are largely mediated by transposable elements, predominantly in chromosome 2, and by plasmids. Finally, we identified two novel potential virulence determinants in P. damselae subsp. piscicida - a chorismate mutase gene, which is ubiquitously retained and co-localized with the AIP56 apoptogenic toxin-encoding gene on the pPHDP10 plasmid, and transfer-messenger RNA gene ssrA located on the main chromosome, homologous to a critical-to-virulence determinant in Yersinia pseudotuberculosis . Our study describes, to our knowledge, the only fully closed and manually curated genomes of P. damselae subsp. piscicida available to date, offering new insights into this important fish pathogen and its evolution.

Background: The management of an outbreak of endophthalmitis associated with intravitreal bevacizumab represents a challenging real-time process involving identification of cases, treatment and mitigation measures during the outbreak. We summarize the clinical presentation and management of a cluster of endophthalmitis cases from contaminated bevacizumab, in addition to mathematical probabilistic assessment of the number of cases that define an outbreak., Methods: A retrospective study was conducted to assess the management of an endophthalmitis outbreak after intravitreal bevacizumab (IVB) administration. Demographic data, clinical information, individual patient management and public health reporting measures were reviewed. Outcomes of patients who received prophylactic antibiotics for endophthalmitis prevention were also reviewed. Binomial tail probability calculations were performed to determine the likelihood of clusters of endophthalmitis that could inform when an outbreak was evolving that would warrant more public health notification measures and communication., Results: Forty-five eyes of 42 patients who received IVB from a single batch were reviewed. Four cases of endophthalmitis from Granulicatella adiacens, a nutritionally-variant Streptococcus species, were treated successfully with intravitreal antibiotics ± vitrectomy. Thirty-four of the remaining 41 eyes were treated with prophylactic intravitreal vancomycin with no additional cases of endophthalmitis. Outbreak management also included CDC, ASRS and public health authority notification. Binominal tail probabilities demonstrated the rarity of clusters from a single batch (i.e. ~ 1/10,000 for 2 cases; 1/2 million for 3 cases). However, given the U.S. scale of IVB administration, there is an 87% chance of a cluster ≧ 2 and a 1% chance of a cluster ≧ 3 cases annually, which may guide outbreak management. A process diagram was developed to incorporate patient management and public health measures when an outbreak is suspected., Conclusion: Intravitreal antibiotics and vitrectomy were effective in the individual management of cases of endophthalmitis, and no serious adverse events occurred with prophylactic intravitreal vancomycin for at-risk eyes. Best practices for outbreaks should be evaluated, given their likelihood within the U.S. and the sight-threatening consequences of endophthalmitis.

Aim: To investigate whether appetite-related hormones were predictors of weight regain in the Diabetes Remission Clinical Trial (DiRECT)., Materials and Methods: DiRECT is a cluster-randomized clinical trial, designed to assess the effect of weight loss on type 2 diabetes remission. For this post hoc analysis, data were available for 253 (147 interventions, 106 controls) individuals with type 2 diabetes (age 53.6 ± 7.5 years, body mass index 34.7 ± 4.4 kg/m 2 , 59% men). Intervention participants received a 24-month weight management programme, and controls remained on usual diabetes care. Fasting plasma concentrations of leptin, ghrelin, glucagon-like peptide-1 and peptide YY were measured at baseline, 12 months and 24 months in all participants, and at 5 months in a subset of participants in the intervention (n = 56) and control groups (n = 22). Potential predictors were examined using multivariable linear regression models., Results: The intervention group lost 14.3 ± 6.0% body weight at 5 months but regained weight over time, with weight losses of 10.0 ± 7.5% at 12 months and 7.6 ± 6.3% at 24 months. Weight loss in controls was 1.1 ± 3.7% and 2.1 ± 5.0% at 12 and 24 months, respectively. Body weight increased by 2.3% (95% confidence interval [CI] 0.4, 4.1; P = 0.019) between 12 and 24 months for every 1-ng/mL increase in ghrelin between baseline and 12 months, and weight regain between 12 and 24 months was increased by 1.1% (95% CI 0.2, 2.0; P = 0.023) body weight for every 1-ng/mL increase in ghrelin at 12 months., Conclusion: The rise in ghrelin (but not any other measured hormone) during diet-induced weight loss was a predictor of weight regain during follow-up, and concentrations remained elevated over time, suggesting a small but significant compensatory drive to regain weight. Attenuating the effects of ghrelin may improve weight-loss maintenance., (© 2020 John Wiley & Sons Ltd.)

Amoebic gill disease (AGD) is one of the main health issues impacting farmed Atlantic salmon. Neoparamoeba perurans causes AGD; however, a diversity of other amoeba species colonizes the gills and there is little understanding of whether they are commensal or potentially involved in different stages of gill disease development. Here, we conduct in vivo challenges of naïve Atlantic salmon with cultured Nolandella sp. and Pseudoparamoeba sp. to investigate their pathogenicity to Atlantic salmon gills. Additionally, we assessed whether the presence of Nolandella sp. and Pseudoparamoeba sp. influences the onset and/or severity of N. perurans-induced AGD. All three strains attached and multiplied on the gills according to qPCR analysis. Furthermore, minor gross gill lesions and histological changes were observed post-exposure. While N. perurans was found associated with classical AGD lesions, Nolandella sp. and Pseudoparamoeba sp. were not found associated with lesion sites and these lesions did not meet the expected composite of histopathological changes for AGD. Moreover, the presence of these non-N. perurans species did not significantly increase the severity of AGD. This trial provides evidence that cultured Nolandella sp. and Pseudoparamoeba sp. do not induce AGD and do not influence the severity of AGD during the early stages of development., (© 2020 John Wiley & Sons Ltd.)

Jeune syndrome is a rare skeletal dysplasia with an associated retinal dystrophy. The authors describe a case of progressive bilateral macular atrophy (with multimodal imaging) in a patient with Jeune syndrome who was followed over 13 years. This case, confirmed with genetic testing, highlights the importance of characterizing the relationship between phenotype and genotype in this genetically heterogenous condition. [Ophthalmic Surg Lasers Imaging Retina. 2021;52:107-109.]., (Copyright 2021, SLACK Incorporated.)

Purpose: To evaluate whether pentosan polysulfate (PPS) maculopathy manifests distinctive characteristics that permit differentiation from hereditary maculopathies with multimodal fundus imaging., Design: Retrospective review., Participants: Emory Eye Center databases were queried for the following International Classification of Diseases codes from May 20, 2014, through October 22, 2019: 362.70 (unspecified hereditary retinal dystrophy), 362.74 + H35.52 (pigmentary retinal dystrophy), 362.76 + H35.54 (dystrophies primarily involving the retinal pigment epithelium), and H35.50 (unspecified macular degeneration)., Methods: Fundus images for each patient were evaluated, including color fundus photographs, fundus autofluorescence images, and spectral-domain OCT images. Cases with imaging sufficient for diagnostic classification were analyzed. Masked graders classified patient images as follows: highly suggestive of PPS maculopathy; some features resembling PPS maculopathy, but not classic disease; and clearly distinct from PPS maculopathy., Main Outcome Measures: Sensitivity and specificity for identification of PPS maculopathy by masked reviewers., Results: A total of 1394 patients were evaluated, and 1131 had imaging sufficient for classification. Fifteen patients were categorized as having findings highly suggestive of PPS maculopathy; 25 patients showed some features resembling PPS maculopathy but not classic disease; and 1091 patients showed evidence of disease clearly distinct from PPS maculopathy. All 10 patients with PPS maculopathy in this dataset were correctly categorized as having PPS maculopathy. Five patients without PPS exposure were categorized incorrectly as having PPS maculopathy. This represented a 100% sensitivity and 99.6% specificity for identification of PPS maculopathy by masked review of fundus imaging in this dataset., Conclusions: The imaging characteristics of PPS maculopathy allow for differentiation from hereditary maculopathies even in the absence of known exposure to the drug., (Copyright © 2020 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Pathogens continuously adapt to changing host environments where variation in their virulence and antigenicity is critical to their long-term evolutionary success. The emergence of novel variants is accelerated in microbial mutator strains (mutators) deficient in DNA repair genes, most often from mismatch repair and oxidized-guanine repair systems (MMR and OG respectively). Bacterial MMR/OG mutants are abundant in clinical samples and show increased adaptive potential in experimental infection models, yet the role of mutators in the epidemiology and evolution of infectious disease is not well understood. Here we investigated the role of mutation rate dynamics in the evolution of a broad host range pathogen, Streptococcus iniae, using a set of 80 strains isolated globally over 40 years. We have resolved phylogenetic relationships using non-recombinant core genome variants, measured in vivo mutation rates by fluctuation analysis, identified variation in major MMR/OG genes and their regulatory regions, and phenotyped the major traits determining virulence in streptococci. We found that both mutation rate and MMR/OG genotype are remarkably conserved within phylogenetic clades but significantly differ between major phylogenetic lineages. Further, variation in MMR/OG loci correlates with occurrence of atypical virulence-associated phenotypes, infection in atypical hosts (mammals), and atypical (osseous) tissue of a vaccinated primary host. These findings suggest that mutators are likely to facilitate adaptations preceding major diversification events and may promote emergence of variation permitting colonization of a novel host tissue, novel host taxa (host jumps), and immune-escape in the vaccinated host., (Crown Copyright © 2020. Published by Elsevier B.V. All rights reserved.)

Competing Interests: The author declares no competing interest.

Acoustic standing wave devices offer excellent potential applications in biological sciences for drug delivery, cell manipulation and tissue engineering. However, concerns have been raised about possible destructive effects on cells due to the applied acoustic field, in addition to other produced secondary factors. Here, we report a systematic study employing a 1D resonant acoustic trapping device to evaluate the cell viability and cell metabolism for a healthy cell line (Human Dermal Fibroblasts, HDF) and a cervical cancer cell line (HeLa), as a function of time and voltages applied (4-10 V pp ) under temperature-controlled conditions. We demonstrate that high cell viability can be achieved reliably when the device is operated at its minimum trapping voltage and tuned carefully to maximise the acoustic standing wave field at the cavity resonance. We found that cell viability and reductive metabolism for both cell lines are kept close to control levels at room temperature and at 34 °C after 15 minutes of acoustic exposure, while shorter acoustic exposures and small changes on temperature and voltages, had detrimental effects on cells. Our study highlights the importance of developing robust acoustic protocols where the operating mode of the acoustic device is well defined, characterized and its temperature carefully controlled, for the application of acoustic standing waves when using live cells and for potential clinical applications.

Higher education and student affairs are fields of study that focus on the institution and student experience in post-secondary education at the graduate level. Located in colleges of education, these departments often offer academic leadership minors often taught by student affairs leadership educators., (© 2020 Wiley Periodicals, Inc.)

The role of hepatic lipoprotein metabolism in diet-induced remission of type 2 diabetes is currently unclear. Here, we determined the contributions of hepatic VLDL1-triglyceride production rate and VLDL1-palmitic acid content to changes in intra-pancreatic fat and return of first phase insulin response in a subgroup of the Diabetes Remission Clinical Trial. Liver fat, VLDL1-triglyceride production, and intra-pancreatic fat decreased after weight loss and remained normalized after 24 months of remission. First-phase insulin response remained increased only in those maintaining diabetes remission. Compared with those in remission at 24 months, individuals who relapsed after initial remission had a greater rise in the content of VLDL1-triglyceride and VLDL1-palmitic acid, re-accumulated intra-pancreatic fat, and lost first-phase response by 24 months. Thus, we observed temporal relationships between VLDL1-triglyceride production, hepatic palmitic acid flux, intra-pancreatic fat, and β-cell function. Weight-related disordered fat metabolism appears to drive development and reversal of type 2 diabetes., Competing Interests: Declaration of Interests R.T. reports grants from Diabetes UK, and lecture fees from Novartis, Lilly, and Jansen during the conduct of the study. A.A.-M. reports a grant from Diabetes UK to conduct the Re-TUNE study. N.S. reports grants and personal fees from Boehringer Ingelheim and personal fees from, Astrazenica, Eli Lilly, Sanofi, and NovoNordisk, and M.E.J.L. reports personal fees from Counterweight and Cambridge Weight Plan not related to the present work. All other authors declare no competing interests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

This study describes antibiotic use by small-scale freshwater aquaculture farmers in the upper Mekong Delta in southwestern Vietnam and the knowledge and practices surrounding the cause and prevention of aquaculture disease in that region. Forty five farmers were included in the study, of which 19 (42%) cultivated tilapia Oreochromis spp., 13 (29%) Striped Catfish Pangasianodon hypophthalmus and 13 (29%) giant river prawns Macrobrachium rosenbergii. Antibiotics were used by farmers of tilapia and Striped Catfish (84% and 69% of farmers, respectively), but not by any of the prawn farmers. Most farmers (72%) used antibiotics for around 3 d when treating diseases, depending on the farmers' economic means and whether the fish recovered, as judged by the farmer. If farmers perceived that the antibiotic treatment had failed, the most common response was to change to another type of antibiotic. Some farmers also used antibiotics in the absence of clinical symptoms as a preventive measure. In the absence of rapid, cost-effective diagnostics, the likelihood for the incorrect use of antibiotics is high, which has implications for antibiotic resistance. Moreover, the sequential use of different antibiotics following therapeutic failure is a risk factor for the emergence of resistance. All farmers that were surveyed were aware of the risks associated with antibiotic use. This may lead to successful intervention toward reduced antibiotic use in freshwater fish farming in Vietnam., (© 2019 The Authors. Journal of Aquatic Animal Health published by Wiley Periodicals, Inc. on behalf of American Fisheries Society.)

The inflammatory response of fish to LPS is subdued, attributed to absence of TLR4, a key pro-inflammatory receptor for LPS in mammals. Nevertheless, LPS is processed in fish in a T-independent manner and is a protective antigen in fish vaccines, yet pathways for processing LPS in fish remain to be elucidated. Here, we report that caspases and NOD-like receptor inflammasomes typically responsible for LPS recognition and processing in mammals lack critical domains or are absent in barramundi (Lates calcarifer). On the other hand, leucocyte integrins MAC-1 and LFA-1 were detected on the surface of neutrophil- and lymphocyte-like cells respectively in the barramundi spleen by immunocytochemistry, and leucocytes displaying MAC-1 or LFA-1 bound to Factor X and ESM-1 respectively. Exposure to MAC-1 and LFA-1 induced significant IL-1β expression post-stimulation with LPS compared to unstimulated and isotype controls, but the differences observed in TNF-α expression were inconclusive. Our findings implicate MAC-1 and LFA-1 involvement in immune processing of LPS in barramundi and in antigen processing in fish., (Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.)

Streptococcus agalactiae (Group B Streptococcus, GBS) is emerging as a genetically diverse species infecting farmed and wild fish, including commercially and culturally important groupers. To better understand how S. agalactiae are pathogenic in fish, we investigated interactions between isolates from fish and terrestrial hosts and the cellular immune system of Queensland grouper Epinephelus lanceolatus using flow cytometry. Adherent head-kidney leucocytes (HKL) from Queensland grouper displayed two main cell populations with distinct forward and side scatter by flow cytometry. The population of smaller and less complex cells (P1) was composed of monocytes, lymphocytes and thrombocytes, while the population of primarily larger and more complex cells (P2) comprised predominantly of macrophages and neutrophils. The cells in P2 had higher phagocytic index and capacity when incubated with fluorescent latex beads. HKL were activated by phorbol myristate acetate (PMA) but were unresponsive to lipopolysaccharide (LPS) and peptidoglycan (PTG), suggesting the absence of specific receptors on the surface of these cells for these ligands or a requirement for intermediates. In in vitro phagocytosis assays, all fish isolates of GBS activated a respiratory burst in P2 indicated by significant production of intracellular reactive oxygen species (ROS). Similarly, dog and cat isolates of different serotype and sequence type also induced ROS production in grouper HKL. However, human, crocodile and bovine isolates of GBS did not elicit significant ROS in HKL although they coincided with the highest phagocytic index. This suggests that these strains are capable of quenching ROS production. Terrestrial isolates significantly increased mortality of Queensland grouper leucocytes in vitro, aligned with a more diverse repertoire of cellular toxins in these strains. Opsonisation of a marine strain and terrestrial strain of GBS with antiserum raised against the marine strain resulted in an increase in ROS production by HKL in both cases although there was low antigenic cross reactivity between the two strains by flow cytometry, reflecting their diverse serotypes (Ib vs III). However, pre-incubation of either strain with normal serum from grouper also increased ROS production of HKL suggesting other opsonins may be involved. Based on these results it appears that piscine and terrestrial GBS isolates have contrasting strategies when interacting with the cellular immune system of Queensland grouper; the former seemingly evading phagocytosis, whilst the latter are readily phagocytosed but counteract ROS production., (Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.)