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Work was conducted to test the hypothesis that the nutritional composition of bakery meal varies depending on where in the United States the meal is produced due to different raw materials being used in the production of the meals. Forty-six samples of bakery meal were collected from feed mills located in the swine producing states in the United States. Based on the state where samples were collected, they were grouped into 5 regions: 1) AL, DE, GA, NC, PA, and VA (10 samples); 2) CO, MO, OK, and TX (10 samples); 3) IN, KY, OH, and TN (8 samples); 4) IA (11 samples); and 5) MN (7 samples). All samples were analyzed for proximate components, GE, AA, carbohydrates, and minerals, and IVDMD and in vitro energy digestibility (IVGED) were also determined. Results indicated that the average concentration of DM was (91.84 ± 1.29%) and there was no difference among regions. The concentration of ash in bakery meal from MN was greater (P < 0.05) than in meals from other regions, but for all other proximate components, no differences among sources were observed. The average concentration (DM basis) of CP (12.20 ± 2.16%), acid hydrolyzed ether extract (AEE, 9.38 ± 1.95%), starch (44.61 ± 5.47%), and NDF (13.77 ± 4.23%) indicated that bakery meal consists of a mixture of food ingredients originating from flour or whole cereal grains and with some high-fiber ingredients such as brans or canola coproducts also included. It also appears that oil or fats were added during production. With the exception of His, no differences among regions were observed for indispensable AA and the average concentrations (DM basis) of Lys, Met, Thr, and Trp were 0.35 ± 0.08%, 0.19 ± 0.03%, 0.38 ± 0.06%, and 0.13 ± 0.03%, respectively. The bakery meals from MN contained more (P < 0.05) Ca than bakery meals from other regions, indicating that limestone may have been added to bakery meal from MN to improve flowability. However, bakery meals from MN and IA contained less (P < 0.05) total P, phytate, and phytate-bound P than bakery meals produced in the states east of the Mississippi River. There were, however, no differences in IVDMD (79.06 ± 6.62%) or of IVGED (74.84 ± 8.20%) of bakery meals among regions. The present results indicate that variations in the chemical composition of bakery meal obtained from different regions in the United States are relatively small and likely without great impact on the nutritional value of the meals, but in vivo digestibility experiments are needed to confirm this hypothesis.

Breast cancer treatment often results in impaired shoulder function, in particular, decrements in muscular endurance and range of motion, which may lead to decreased quality of life. The purpose of this investigation was to determine the effects of walking pole use on shoulder function in female breast cancer survivors. Participants had previously been treated with 1 or a combination of the following: mastectomy, breast conservation therapy, axillary lymph node dissection, chemotherapy, or radiation. Participants were randomly placed in experimental (n = 6) and control (n = 6) groups and met with a cancer exercise specialist 2 times each week for 8 weeks. The experimental group used walking poles during the 20-minute aerobic portion of their workout, whereas the control group did not use walking poles but performed 20 minutes of aerobic exercise per workout session. Both groups participated in similar resistance training programs. Testing was done pre- and postexercise intervention to determine upper body muscular endurance and active range of motion at the glenohumeral joint. Repeated-measures analysis of variance (ANOVA) revealed significant improvements in muscular endurance as measured by the bench press (P = .046) and lat pull down (P = .013) in the walking pole group. No within-group improvements were found in the group that did not use walking poles. The data suggest that using a walking pole exercise routine for 8 weeks significantly improved muscular endurance of the upper body, which would clearly be beneficial in helping breast cancer survivors perform activities of daily living and regain an independent lifestyle. [ABSTRACT FROM AUTHOR]

Exercise for cancer patients is gaining support. In the current case study, a female breast cancer patient was diagnosed with breast cancer at the age of 29 years; she underwent a left modified radical mastectomy. She developed cancer again in the opposite breast at the age of 57 years and had a right modified radical mastectomy. After the second mastectomy (the right breast), the patient received chemotherapy and radiation. Following her cancer treatments, she participated in an exercise intervention for 6 months at the University of Northern Colorado's Rocky Mountain Cancer Rehabilitation Institute. A 6-month reassessment showed that she increased her muscular strength and cardiovascular function in addition to attenuating her cancer-related fatigue and depression. It is recommended that health professionals work together to ensure that a collaborative effort is undertaken to increase functional work capacity that will significantly improve patients' quality of life. [ABSTRACT FROM AUTHOR]

Local protein synthesis in axons and dendrites underpins synaptic plasticity. However, the composition of the protein synthesis machinery in distal neuronal processes and the mechanisms for its activity-driven deployment to local translation sites remain unclear. Here, we employed cryo-electron tomography, volume electron microscopy, and live-cell imaging to identify Ribosome-Associated Vesicles (RAVs) as a dynamic platform for moving ribosomes to distal processes. Stimulation via chemically-induced long-term potentiation causes RAV accumulation in distal sites to drive local translation. We also demonstrate activity-driven changes in RAV generation and dynamics in vivo , identifying tubular ER shaping proteins in RAV biogenesis. Together, our work identifies a mechanism for ribosomal delivery to distal sites in neurons to promote activity-dependent local translation., Competing Interests: Competing interests: The authors declare that they have no competing interests.

Bovine ischaemic teat necrosis (ITN) is a disease affecting the skin of the teats of dairy cows with an unknown aetiopathogenesis. Digital dermatitis (DD)-associated treponemes have previously been suggested as a potential aetiological agent in ITN, although the sample size was small. The current study, using established PCR techniques, aimed to examine the association with the presence of DD-associated treponemes in a large number of ITN samples from a wider geographical area, and surveyed the potential of milk as an infection reservoir. From 95 ITN lesions, 35.8% ( n = 34) were positive for at least one DD-associated treponeme compared with only 5.6% ( n = 1) of 18 non-lesioned teats from cows with ITN lesions on a different teat using a nested PCR approach. All 10 age- and production-matched control cows were negative for DD-associated treponemes via PCR. No DD-associated treponemes could be detected from foremilk of cows with ( n = 19) and without ( n = 31) a DD lesion on the hind feet. DD-associated treponemes could be detected via PCR after incubation in milk for up to 2 h. Therefore, milk does not appear to be a competent reservoir for transmission of DD-associated treponemes. Moreover, in the current study DD-associated treponemes were only detected in a subset of ITN samples, so it is unlikely these opportunistic skin-associated pathogens are the major or sole agent of ITN.

Leptospirosis is a zoonotic bacterial disease affecting mammalian species worldwide. Cattle are a major susceptible host; infection with pathogenic Leptospira spp. represents a public health risk and results in reproductive failure and reduced milk yield, causing economic losses. The characterisation of outer membrane proteins (OMPs) from disease-causing bacteria dissects pathogenesis and underpins vaccine development. As most leptospire pathogenesis research has focused on Leptospira interrogans , this study aimed to characterise novel OMPs from another important genomospecies, Leptospira borgpetersenii , which has global distribution and is relevant to bovine and human diseases. Several putative L. borgpetersenii OMPs were recombinantly expressed, refolded and purified, and evaluated for function and immunogenicity. Two of these unique, putative OMPs (rLBL0972 and rLBL2618) bound to immobilised fibronectin, laminin and fibrinogen, which, together with structural and functional data, supports their classification as leptospiral adhesins. A third putative OMP (rLBL0375), did not exhibit saturable adhesion ability but, together with rLBL0972 and the included control, OmpL1, demonstrated significant cattle milk IgG antibody reactivity from infected cows. To dissect leptospire host-pathogen interactions further, we expressed alleles of OmpL1 and a novel multi-specific adhesin, rLBL2618, from a variety of genomospecies and surveyed their adhesion ability, with both proteins exhibiting divergences in extracellular matrix component binding specificity across synthesised orthologs. We also observed functional redundancy across different L. borgspetersenii OMPs which, together with diversity in function across genomospecies orthologs, delineates multiple levels of plasticity in adhesion that is potentially driven by immune selection and host adaptation. These data identify novel leptospiral proteins which should be further evaluated as vaccine and/or diagnostic candidates. Moreover, functional redundancy across leptospire surface proteins together with identified adhesion divergence across genomospecies further dissect the complex host-pathogen interactions of a genus responsible for substantial global disease burden.

Objectives: Bovine digital dermatitis (BDD), a painful infectious foot disease in dairy cattle, endemic in many countries worldwide, causes substantial economic and welfare impacts. Treponema spp. are considered key to BDD pathogenesis. To aid infection reservoir identification and control measure development, survival of BDD treponemes was investigated in different temperatures (4, 12, 20, 37, 45 and 60 °C), pH values (5-9.0), dairy cattle faeces and bedding types: straw shavings, sand, sand containing 5% lime (w/w) and recycled manure solids (RMS)., Methods: A turbidity microplate methodology was adapted to measure pH impact on growth. Survival of BDD treponemes for the different conditions were assessed by sub-cultures of microcosms over different time points., Results: BDD treponemes remained viable between 4 and 37 °C and pH 5.5 and 9.0 under anaerobic conditions. In sterile faecal microcosms, incubated aerobically at 12 °C, BDD treponemes remained viable for a median of 1 day (15 min - 6 day range). Variation in duration of survival and ability to grow was observed between phylogroups and strains. In aerobic microcosms, T. phagedenis T320A remained viable for the full 7 days in sand, 6 days in sawdust, 5 days in RMS, but was not viable after 15 min in straw or sand containing 5% (w/w) lime., Conclusions: Treponeme survival conditions identified here should enhance future BDD infection reservoir surveys and enable control measures. Of note, straw or sand containing 5% (w/w) lime should be assessed in BDD field trials. Finally, these data indicate BDD treponemes exhibit characteristics of facultative anaerobes., 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 © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Bovine Digital Dermatitis (BDD) is a prevalent infectious disease, causing painful foot skin lesions and lameness in cattle. We describe herein the bovine foot skin microbiota and its associations with BDD using 16S rRNA gene amplicon and shotgun metagenomic sequencing on samples from 259 dairy cows from three UK dairy farms., Results: We show evidence of dysbiosis, and differences in taxonomy and functional profiles in the bovine foot skin microbiome of clinically healthy animals that subsequently develop BDD lesions, compared to those that do not. Our results suggest that taxonomical and functional differences together with alterations in ecological interactions between bacteria in the normal foot skin microbiome may predispose an animal to develop BDD lesions. Using genome-wide association and regional heritability mapping approaches, we provide first evidence for interactions between host genotype and certain members of the foot skin microbiota. We show the existence of significant genetic variation in the relative abundance of Treponema spp. and Peptoclostridium spp. and identify regions in the bovine genome that explain a significant proportion of this variation., Conclusions: Collectively this work shows early changes in taxonomic and functional profiles of the bovine foot-skin microbiota in clinically healthy animals which are associated with subsequent development of BDD and could be relevant to prevention of disease. The description of host genetic control of members of the foot skin microbiota, combined with the association of the latter with BDD development offer new insights into a complex relationship that can be exploited in selective breeding programmes. Video Abstract., (© 2023. The Author(s).)

Interactions between respiratory viruses during infection affect transmission dynamics and clinical outcomes. To identify and characterize virus-virus interactions at the cellular level, we coinfected human lung cells with influenza A virus (IAV) and respiratory syncytial virus (RSV). Super-resolution microscopy, live-cell imaging, scanning electron microscopy and cryo-electron tomography revealed extracellular and membrane-associated filamentous structures consistent with hybrid viral particles (HVPs). We found that HVPs harbour surface glycoproteins and ribonucleoproteins of IAV and RSV. HVPs use the RSV fusion glycoprotein to evade anti-IAV neutralizing antibodies and infect and spread among cells lacking IAV receptors. Finally, we show that IAV and RSV coinfection in primary cells of the bronchial epithelium results in viral proteins from both viruses co-localizing at the apical cell surface. Our observations define a previously unknown interaction between respiratory viruses that might affect virus pathogenesis by expanding virus tropism and enabling immune evasion., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Bovine ischaemic teat necrosis (ITN) is an emerging disease of unknown aetiology affecting mostly dairy cows in the early stages of first lactation and a substantial welfare concern frequently leading to premature culling and economic losses. Specific diagnostic criteria are lacking. The aims of this study were to develop an appropriate ITN grading system, describe the histopathological changes and investigate the potential aetiological role of several pathogens in 47 cows with 73 ITN lesions from 28 farms. ITN lesions were allocated to one of three broad macroscopic categories: presence of a non-proliferative lesion on the teat (type 1); proliferative teat lesion with crusting (type 2); severe purulent to eosinophilic, ulcerative and necrotising dermatitis and sloughing or total absence of the teat (type 3). Lesions were mostly observed on the medial aspect of the teat but there was no anatomical predisposition as to which teats were more frequently affected. In approximately 50% of the ITN teats reviewed, the lesions were continuous with the skin of the udder and 34.2% of cases had sloughed or partially sloughed teats. The main histological findings were: focally extensive severe purulent to eosinophilic, ulcerative and necrotizing dermatitis; serocellular crust formation; and epidermal hyperplasia with dyskeratosis. Some lesions also had leucocytoclastic to eosinophilic vasculitis and thrombosis with ischaemic necrosis. Macroscopic and histological analyses confirmed the suspected ischaemic nature of the lesions but the specific aetiopathogenesis was elusive with a wide range of bacteria present, probably as opportunistic infections. However, Treponema spp and Orthopox virus were excluded as major aetiological agents. This study establishes a foundation for further investigations of the pathogenesis of bovine ITN and a basis for consistency in diagnosis and classification of the stage of disease. The findings are also key to further understanding disease progression and prognosis., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The in vitro biological characterization of biomaterials is largely based on static cell cultures. However, for highly reactive biomaterials such as calcium-deficient hydroxyapatite (CDHA), this static environment has limitations. Drastic alterations in the ionic composition of the cell culture medium can negatively affect cell behavior, which can lead to misleading results or data that is difficult to interpret. This challenge could be addressed by a microfluidics-based approach ( i.e. on-chip), which offers the opportunity to provide a continuous flow of cell culture medium and a potentially more physiologically relevant microenvironment. The aim of this work was to explore microfluidic technology for its potential to characterize CDHA, particularly in the context of inflammation. Two different CDHA substrates (chemically identical, but varying in microstructure) were integrated on-chip and subsequently evaluated. We demonstrated that the on-chip environment can avoid drastic ionic alterations and increase protein sorption, which was reflected in cell studies with RAW 264.7 macrophages. The cells grown on-chip showed a high cell viability and enhanced proliferation compared to cells maintained under static conditions. Whereas no clear differences in the secretion of tumor necrosis factor alpha (TNF-α) were found, variations in cell morphology suggested a more anti-inflammatory environment on-chip. In the second part of this study, the CDHA substrates were loaded with the drug Trolox. We showed that it is possible to characterize drug release on-chip and moreover demonstrated that Trolox affects the TNF-α secretion and morphology of RAW 264.7 ​cells. Overall, these results highlight the potential of microfluidics to evaluate (bioactive) biomaterials, both in pristine form and when drug-loaded. This is of particular interest for the latter case, as it allows the biological characterization and assessment of drug release to take place under the same dynamic in vitro environment., Competing Interests: 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., (© 2022 The Authors.)

Cryo-electron tomography provides detailed views of macromolecules in situ. However, imaging a large field of view to provide more cellular context requires reducing magnification during data collection, which in turn restricts the resolution. To circumvent this trade-off between field of view and resolution, we have developed a montage data collection scheme that uniformly distributes the dose throughout the specimen. In this approach, sets of slightly overlapping circular tiles are collected at high magnification and stitched to form a composite projection image at each tilt angle. These montage tilt-series are then reconstructed into massive tomograms with a small pixel size but a large field of view. For proof-of-principle, we applied this method to the thin edge of HeLa cells. Thon rings to better than 10 Å were detected in the montaged tilt-series, and diverse cellular features were observed in the resulting tomograms. These results indicate that the additional dose required by this technique is not prohibitive to performing structural analysis to intermediate resolution across a large field of view. We anticipate that montage tomography will prove particularly useful for lamellae, increase the likelihood of imaging rare cellular events, and facilitate visual proteomics., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Bovine ischaemic teat necrosis (ITN) is an emerging disease of unknown aetiology that affects the teats of dairy cattle. It causes economic and animal welfare issues with many animals being culled. No effective treatments or epidemiological data to inform control strategies are currently available. The aim of this observational study was to investigate farmer-reported experiences and identify potential farm-level risk factors. In January 2018, a questionnaire was sent to a random sample of 1,855 Great Britain (GB) dairy farmers. A usable response rate of 12.3% was obtained. Fifty-one per cent [95% confidence interval (CI): 44.4-57.8%] of farmers reported having experienced ITN on their farm between 1985 and 2018. Rising numbers of farms indicated that ITN is an emerging disease with 46.3% of farmers reporting the first case in the 3 years up to 2018. At the animal level, 47.3% (95% CI: 38.7-55.9%) of the cases occurred during the first lactation and 78.9% (95% CI: 75.2-82.6%) within the first 90 days in milk. Only 20.8% (95% CI: 15.9-26.4%) of the cases were reported to recover, whereas 22.8% (95% CI: 17.8-28.5%) of the cases required culling. The remaining cases experienced complications such as loss of a teat and/or mastitis. From these data, the cost of ITN, through production losses and expenditure, was estimated to be £1,121 per farm per year. The costs were estimated at £720, £860 and £2,133 for recovered, complicated and culled cases, respectively. Univariable and multivariable logistic regression models were used to explore the associations between the presence of ITN on farm and various risk factors. The presence of udder cleft dermatitis (UCD) (odds ratio 2.80; 95% CI: 1.54-5.07; p < 0.01) and chapped teats (odds ratio 6.07; 95% CI: 1.96-18.76; p < 0.01) in the milking herd was associated with the presence of ITN at the farm level. This is the first national questionnaire of ITN within GB and highlights the association of UCD and chapped teats with ITN at the farm level. While there are many limitations and potential bias around farmer questionnaires, these findings highlight several key areas for further disease investigation and possible intervention., Competing Interests: AM was employed by Quality Milk Management Services Ltd. The remaining 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 © 2022 Crosby-Durrani, Blowey, Manning, Afonso, Carter, Evans and Angell.)

Viruses can be enveloped or non-enveloped, and require a host cell to replicate and package their genomes into new virions to infect new cells. To accomplish this task, viruses hijack the host-cell machinery to facilitate their replication by subverting and manipulating normal host cell function. Enveloped viruses can have severe consequences for human health, causing various diseases such as acquired immunodeficiency syndrome (AIDS), seasonal influenza, COVID-19, and Ebola virus disease. The complex arrangement and pleomorphic architecture of many enveloped viruses pose a challenge for the more widely used structural biology techniques, such as X-ray crystallography. Cryo-electron tomography (cryo-ET), however, is a particularly well-suited tool for overcoming the limitations associated with visualizing the irregular shapes and morphology enveloped viruses possess at macromolecular resolution. The purpose of this review is to explore the latest structural insights that cryo-ET has revealed about enveloped viruses, with particular attention given to their architectures, mechanisms of entry, replication, assembly, maturation and egress during infection. Cryo-ET is unique in its ability to visualize cellular landscapes at 3-5 nanometer resolution. Therefore, it is the most suited technique to study asymmetric elements and structural rearrangements of enveloped viruses during infection in their native cellular context., (© 2022 The Author(s).)

The signaling network of the unfolded protein response (UPR) adjusts the protein-folding capacity of the endoplasmic reticulum (ER) according to need. The most conserved UPR sensor, IRE1α, spans the ER membrane and activates through oligomerization. IRE1α oligomers accumulate in dynamic foci. We determined the in situ structure of IRE1α foci by cryogenic correlated light and electron microscopy combined with electron cryo-tomography and complementary immuno–electron microscopy in mammalian cell lines. IRE1α foci localized to a network of narrow anastomosing ER tubes (diameter, ~28 nm) with complex branching. The lumen of the tubes contained protein filaments, which were likely composed of arrays of IRE1α lumenal domain dimers that were arranged in two intertwined, left-handed helices. This specialized ER subdomain may play a role in modulating IRE1α signaling.

Contagious ovine digital dermatitis (CODD) is a severe and common infectious foot disease of sheep and a significant animal welfare issue for the sheep industry in the UK and some European countries. The etiology and pathogenesis of the disease are incompletely understood. In this longitudinal, experimental study, CODD was induced in 18 sheep, and for the first time, the clinical lesion development and associated microbiological changes in CODD affected feet are described over time, resulting in a completely new understanding of the etiopathogenesis of CODD. The majority of CODD lesions (83.9%) arose from pre-existing interdigital dermatitis (ID) and/or footrot (FR) lesions. All stages of foot disease were associated with high levels of poly-bacterial colonization with five pathogens, which were detected by quantitative PCR (qPCR): Treponema medium, Treponema phagedenis, Treponema pedis, Dichelobacter nodosus , and Fusobacterium necrophorum . Temporal colonization patterns showed a trend for early colonization by T. phagedenis , followed by F. necrophorum and D. nodosus, T. medium , and then T. pedis, D. nodosus was present at significantly higher predicted mean log 10 genome copy numbers in FR lesions compared to both ID and CODD, while Treponema species were significantly higher in CODD and FR lesions compared to ID lesions ( p < 0.001). Treatment of CODD-affected sheep with two doses of 10 mg/kg long acting amoxicillin resulted in a 91.7% clinical cure rate by 3 weeks post-treatment; however, a bacteriological cure was not established for all CODD-affected feet. The study found that in an infected flock, healthy feet, healed CODD feet, and treated CODD feet can be colonized by some or all of the five pathogens associated with CODD and therefore could be a source of continued infection in flocks. The study is an experimental study, and the findings require validation in field CODD cases. However, it does provide a new understanding of the etiopathogenesis of CODD and further supportive evidence for the importance of current advice on the control of CODD; namely, ensuring optimum flock control of footrot and prompt isolation and effective treatment of clinical cases., Competing Interests: JA is currently employed by Wern Vets CYF, at the time of carrying out the research he was employed by the University of Liverpool. The remaining 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 Staton, Angell, Grove-White, Clegg, Carter, Evans and Duncan.)

Biomaterials offer a promising approach to repair bone defects. Whereas traditional studies predominantly focused on optimizing the osteogenic capacity of biomaterials, less focus has been on the immune response elicited by them. However, the immune and skeletal systems extensively interact, a concept which is referred to as 'osteoimmunology'. This realization has fuelled the development of biomaterials with favourable osteoimmunomodulatory (OIM) properties, aiming to modulate the immune response and to support bone regeneration, thereby affecting the success of an implant. Given the plethora of in vitro assays used to evaluate the OIM properties of biomaterials, it may be challenging to select the right methods to produce conclusive results. In this review, we aim to provide a comprehensive and practical guide for researchers interested in studying the OIM properties of biomaterials in vitro. After a concise overview of the concept of osteoimmunology, emphasis is put on the methodologies that are regularly used to evaluate the OIM properties of biomaterials. First, a description of the most commonly used cell types and cell culture media is provided. Second, typical experimental set-ups and their relevant characteristics are discussed. Third, a detailed overview of the generally used methodologies and readouts, including cell type-specific markers and time points of analysis, is given. Finally, we highlight the promise of advanced approaches, namely microarrays, bioreactors and microfluidic-based systems, and the potential that these may offer to the osteoimmunology field. STATEMENT OF SIGNIFICANCE: Osteoimmunology focuses on the connection and communication between the skeletal and immune systems. This interaction has been recognized to play an important role in the clinical success of biomaterials, which has resulted in an increasing amount of research on the osteoimmunomodulatory (OIM) properties of biomaterials. However, the amount of literature makes it challenging to extract the information needed to design experiments from beginning to end, and to compare obtained results to existing work. This article intends to serve as a guide for those aiming to learn more about the commonly used experimental approaches in the field. We cover early-stage choices, such as cell types and experimental set-ups, but also discuss specific assays, including cell markers and time points of analysis., Competing Interests: Declaration of Competing Interest G. Mestres, S-S.D. Carter, and A. Diez-Escudero declare that they have no conflicts of interest. N.P. Hailer reports institutional support and lecturer's fees from two hip implant manufacturers, Waldemar Link GmbH Co KG, Hamburg, Germany, and Zimmer Biomet, Warsaw, Indiana, USA, and lecturer's fees from a bone cement manufacturer, Heraeus, Wehrheim, Germany., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Here, we report the first complete genomes of three cultivable treponeme species from bovine digital dermatitis (DD) skin lesions, two comparative human treponemes, considered indistinguishable from bovine DD species, and a bovine gastrointestinal (GI) treponeme isolate. Key genomic differences between bovine and human treponemes implicate microbial mechanisms that enhance knowledge of how DD, a severe disease of ruminants, has emerged into a prolific, worldwide disease. Bovine DD treponemes have additional oxidative stress genes compared to nearest human-isolated relatives, suggesting better oxidative stress tolerance, and potentially explaining how bovine strains can colonize skin surfaces. Comparison of both bovine DD and GI treponemes as well as bovine pathogenic and human non-pathogenic saprophyte Treponema phagedenis strains indicates genes encoding a five-enzyme biosynthetic pathway for production of 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, a rare di-N-acetylated mannuronic acid sugar, as important for pathogenesis. Bovine T. phagedenis strains further differed from human strains by having unique genetic clusters including components of a type IV secretion system and a phosphate utilisation system including phoU, a gene associated with osmotic stress survival. Proteomic analyses confirmed bovine derived T. phagedenis exhibits expression of PhoU but not the putative secretion system, whilst the novel mannuronic acid pathway was expressed in near entirety across the DD treponemes. Analysis of osmotic stress response in water identified a difference between bovine and human T. phagedenis with bovine strains exhibiting enhanced survival. This novel mechanism could enable a selective advantage, allowing environmental persistence and transmission of bovine T. phagedenis. Finally, we investigated putative outer membrane protein (OMP) ortholog families across the DD treponemes and identified several families as multi-specific adhesins capable of binding extra cellular matrix (ECM) components. One bovine pathogen specific adhesin ortholog family showed considerable serodiagnostic potential with the Treponema medium representative demonstrating considerable disease specificity (91.6%). This work has shed light on treponeme host adaptation and has identified candidate molecules for future diagnostics, vaccination and therapeutic intervention., Competing Interests: The authors have declared that no competing interests exist.

Background: Contagious Ovine Digital Dermatitis (CODD) is an emerging and common infectious foot disease of sheep which causes severe welfare and economic problems for the sheep industry. The aetiology of the disease is not fully understood and control of the disease is problematic. The aim of this study was to investigate the polybacterial aetiopathogenesis of CODD and the effects of antibiotic treatment, in a longitudinal study of an experimentally induced disease outbreak using a 16S rRNA gene amplicon sequencing approach., Results: CODD was induced in 15/30 experimental sheep. During the development of CODD three distinct phenotypic lesion stages were observed. These were an initial interdigital dermatitis (ID) lesion, followed by a footrot (FR) lesion, then finally a CODD lesion. Distinct microbiota were observed for each lesion in terms of microbial diversity, clustering and composition. Porphyromonadaceae, Family XI, Veillonellaceae and Fusobacteriaceae were significantly associated with the diseased feet. Veillonellaceae and Fusobacteriaceae were most associated with the earlier stages of ID and footrot rather than CODD. Following antibiotic treatment of the sheep, the foot microbiota showed a strong tendency to return to the composition of the healthy state. The microbiota composition of CODD lesions collected by swab and biopsy methods were different. In particular, the Spirochaetaceae family were more abundant in samples collected by the biopsy method, suggesting that these bacteria are present in deeper tissues of the diseased foot., Conclusion: In this study, CODD presented as part of a spectrum of poly-bacterial foot disease strongly associated with bacterial families Porphyromonadaceae, Family XI (a family in Clostridiales also known as Clostridium cluster XI), Veillonellaceae and Fusobacteriaceae which are predominately Gram-negative anaerobes. Following antibiotic treatment, the microbiome showed a strong tendency to return to the composition of the healthy state. The composition of the healthy foot microbiome does not influence susceptibility to CODD. Based on the data presented here and that CODD appears to be the severest end stage of sheep infectious foot disease lesions, better control of the initial ID and FR lesions would enable better control of CODD and enable better animal welfare.

This research communication reports the results from questionnaires used to identify the impact of recent research into the disinfection of cattle foot-trimming equipment to prevent bovine digital dermatitis (BDD) transmission on (a) biosecurity knowledge and (b) hygiene practice of foot health professionals. An initial questionnaire found that more than half of participating farmers, veterinary surgeons and commercial foot-trimmers were not considering hand or hoof-knife hygiene in their working practices. The following year, after the release of a foot-trimming hygiene protocol and a comprehensive knowledge exchange programme by the University of Liverpool, a second survey showed 35/80 (43.8%) farmers, veterinary surgeons and commercial foot-trimmers sampled considered they were now more aware of the risk of spreading BDD during foot- trimming. Furthermore, 36/80 (45.0%) had enhanced their hygiene practice in the last year, impacting an estimated 1383 farms and 5130 cows trimmed each week. Participants who reported having seen both the foot-trimming hygiene protocol we developed with AHDB Dairy and other articles about foot-trimming hygiene in the farming and veterinary press, were significantly more likely to have changed their working practices. Difficulties accessing water and cleaning facilities on farms were identified as the greatest barrier to improving biosecurity practices. Participants' preferred priority for future research was continued collection of evidence for the importance and efficacy of good foot-trimming hygiene practices.

Bovine digital dermatitis (BDD) is a common infectious disease of digital skin in cattle and an important cause of lameness worldwide, with limited treatment options. It is of increasing global concern for both animal welfare and food security, imposing a large economic burden on cattle farming industries each year. A polytreponemal etiology has been consistently identified, with three key phylogroups implicated globally: Treponema medium, Treponema phagedenis , and Treponema pedis. Pathogenic mechanisms which might enable targeted treatment/therapeutic development are poorly defined. This study used RNA sequencing to determine global differential mRNA expression in primary bovine foot skin fibroblasts following challenge with three representative BDD treponemes and a commensal treponeme, Treponema ruminis . A pro-inflammatory response was elicited by the BDD treponemes, mediated through IL-8/IL-17 signaling. Unexpectedly, the three BDD treponemes elicited distinct mechanisms of pathogenesis. T. phagedenis and T. pedis increased abundance of mRNA transcripts associated with apoptosis, while T. medium and T. pedis increased transcripts involved in actin rearrangement and loss of cell adhesion, likely promoting tissue invasion. The upregulation of antimicrobial peptide precursor, DEFB123, by T. phagedenis spirochaetes may present a microbial ecological advantage to all treponemes within BDD infected tissue, explaining their dominance within lesions. A commensal, T. ruminis , significantly dysregulated over three times the number of host mRNA transcripts compared to BDD treponemes, implying BDD treponemes, akin to the syphilis pathogen ( Treponema pallidum ), have evolved as "stealth pathogens" which avoid triggering substantial host immune/inflammatory responses to enable persistence and tissue invasion. Immunohistochemistry demonstrated increased IL-6, IL-8, RND1, and CFB protein expression in BDD lesions, confirming in vitro fibroblast observations and highlighting the system's value in modeling BDD pathogenesis. Several unique shared gene targets were identified, particularly RGS16 , GRO1 , MAFF , and ZC3H12A . The three key BDD Treponema phylogroups elicited both distinct and shared pathogenic mechanisms in bovine foot skin; upregulating inflammation whilst simultaneously suppressing adaptive immunity. The novel gene targets identified here should enable future vaccine/therapeutic approaches., 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 Newbrook, Carter, Crosby-Durrani and Evans.)

Electron cryo-tomography (cryo-ET) is a technique that allows the investigation of intact macromolecular complexes while they are in their cellular milieu. Over the years, cryo-ET has had a huge impact on our understanding of how large biomolecular complexes look like, how they assemble, disassemble, function, and evolve(d). Recent hardware and software developments and combining cryo-ET with other techniques, e.g., focused ion beam milling (FIB-milling) and cryo-light microscopy, has extended the realm of cryo-ET to include transient molecular complexes embedded deep in thick samples (like eukaryotic cells) and enhanced the resolution of structures obtained by cryo-ET. In this chapter, we will present an outline of how to perform cryo-ET studies on a wide variety of biological samples including prokaryotic and eukaryotic cells and biological plant tissues. This outline will include sample preparation, data collection, and data processing as well as hybrid approaches like FIB-milling, cryosectioning, and cryo-correlated light and electron microscopy (cryo-CLEM).

Subcellular neighborhoods, comprising specific ratios of organelles and proteins, serve a multitude of biological functions and are of particular importance in secretory cells. However, the role of subcellular neighborhoods in insulin vesicle maturation is poorly understood. Here, we present single-cell multiple distinct tomogram acquisitions of β cells for in situ visualization of distinct subcellular neighborhoods that are involved in the insulin vesicle secretory pathway. We propose that these neighborhoods play an essential role in the specific function of cellular material. In the regions where we observed insulin vesicles, a measurable increase in both the fraction of cellular volume occupied by vesicles and the average size (diameter) of the vesicles was apparent as sampling moved from the area near the nucleus toward the plasma membrane. These findings describe the important role of the nanometer-scale organization of subcellular neighborhoods on insulin vesicle maturation., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

Members of the tripartite motif (TRIM) protein family have been shown to assemble into structures in both the nucleus and cytoplasm. One TRIM protein family member, TRIM5α, has been shown to form cytoplasmic bodies involved in restricting retroviruses such as HIV-1. Here we applied cryogenic correlated light and electron microscopy, combined with electron cryo-tomography, to intact mammalian cells expressing YFP-rhTRIM5α and found the presence of hexagonal nets whose arm lengths were similar to those of the hexagonal nets formed by purified TRIM5α in vitro. We also observed YFP-rhTRIM5α within a diversity of structures with characteristics expected for organelles involved in different stages of macroautophagy, including disorganized protein aggregations (sequestosomes), sequestosomes flanked by flat double-membraned vesicles (sequestosome:phagophore complexes), sequestosomes within double-membraned vesicles (autophagosomes), and sequestosomes within multivesicular autophagic vacuoles (amphisomes or autolysosomes). Vaults were also seen in these structures, consistent with their role in autophagy. Our data 1) support recent reports that TRIM5α can form both well-organized signaling complexes and nonsignaling aggregates, 2) offer images of the macroautophagy pathway in a near-native state, and 3) reveal that vaults arrive early in macroautophagy., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

To bring novel biomaterials to clinical use, reliable in vitro models are imperative. The aim of this work was to develop a microfluidic tool to evaluate the biological properties of biomaterials for bone repair. Two approaches to embed medical grade titanium (Ti 6 Al 4 V) on-chip were explored. The first approach consisted of a polydimethylsiloxane microfluidic channel placed onto a titanium disc, held together by an additively manufactured fixture. In the second approach, a titanium disc was assembled onto a microscopic glass slide, using a double-sided tape microfluidic channel. Both approaches demonstrated potential for maintaining MC3T3-E1 preosteoblast-like cell cultures on-chip, as was shown by the vast majority of living cells after 1 day. In addition, the cells cultured on-chip showed a more elongated morphology compared to cells grown under static conditions and a tendency to align to the direction of the flow. For longer-term (i.e. 10 days) studies, the glass-based chip was selected. Assessment of cell viability showed a high number of living cells during the entire experimental period. Cell proliferation and differentiation studies revealed an increase in cell proliferation on-chip, suggesting that proliferation was the dominating process at the detriment of differentiation in this micrometric dynamic environment. These results illustrate the importance of optimizing in vitro cell culture conditions and how these may affect biomaterial testing outcomes. Overall, this work provides a step towards more in vivo-like microfluidic testing platforms, which are expected to provide more reliable in vitro screening of biomaterials.

Background: Bovine digital dermatitis (BDD) is an infectious foot disease found commonly in dairy herds. Foot-trimming is an important husbandry procedure for reducing the ensuing lameness; however, epidemiological, and microbiological studies have identified this as a risk activity for transmitting BDD. Three disinfectants have previously been identified in laboratory work as effective for removing viable BDD-associated Treponema spp., from hoof knife blades. The present study enrolled 133 dairy cattle with BDD lesions, and swabbed hoof knife blades before and after foot-trimming, and after knife disinfection with one of three disinfectants (1:100 FAM30®, 2% Virkon® and 2% sodium hypochlorite) to assess their efficacy under field conditions., Results: Detection of BDD treponeme phylogroup DNA was undertaken by direct PCR of swabs, and viable treponemes were detected by PCR of swab cultures after 6 weeks' incubation. Where hoof knives did not contact the lesion, BDD-associated treponemes were detected after foot-trimming in 12/22 (54.5%) cases by direct PCR and 1/22 (4.5%) cases by PCR of cultured organisms. Where contact was made with the lesion, 111/111 (100%) samples taken after trimming were positive by direct PCR and 47/118 (39.8%) were positive by culture PCR. Viable organisms were identified in cultures from lesion stages M2, M3, M4 and M4.1. No viable organisms were detected after disinfection of hoof knives., Conclusions: Hoof knives post-trimming were frequently contaminated with BDD-associated treponeme DNA. Viable organisms were identified in cultures whether contact had been made between hoof knife and lesion or not, although contact clearly increased the frequency of detection of viable organisms. The three disinfectants tested were effective for removing viable organisms. The disinfection protocol used in this study should therefore be considered reliable for adoption as standard industry practice.

Bovine digital dermatitis (BDD), an infectious disease of the bovine foot with a predominant treponemal etiology, is a leading cause of lameness in dairy and beef herds worldwide. BDD is poorly responsive to antimicrobial therapy and exhibits a relapsing clinical course; an effective vaccine is therefore urgently sought. Using a reverse vaccinology approach, the present study surveyed the genomes of the three BDD-associated Treponema phylogroups for putative β-barrel outer membrane proteins and considered their potential as vaccine candidates. Selection criteria included the presence of a signal peptidase I cleavage site, a predicted β-barrel fold, and cross-phylogroup homology. Four candidate genes were overexpressed in Escherichia coli BL21(DE3), refolded, and purified. Consistent with their classification as β-barrel OMPs, circular-dichroism spectroscopy revealed the adoption of a predominantly β-sheet secondary structure. These recombinant proteins, when screened for their ability to adhere to immobilized extracellular matrix (ECM) components, exhibited a diverse range of ligand specificities. All four proteins specifically and dose dependently adhered to bovine fibrinogen. One recombinant protein was identified as a candidate diagnostic antigen (disease specificity, 75%). Finally, when adjuvanted with aluminum hydroxide and administered to BDD-naive calves using a prime-boost vaccination protocol, these proteins were immunogenic, eliciting specific IgG antibodies. In summary, we present the description of four putative treponemal β-barrel OMPs that exhibit the characteristics of multispecific adhesins. The observed interactions with fibrinogen may be critical to host colonization and it is hypothesized that vaccination-induced antibody blockade of these interactions will impede treponemal virulence and thus be of therapeutic value., (Copyright © 2020 Staton et al.)

Background: Non-healing bovine foot lesions, including non-healing white line disease, non-healing sole ulcer and toe necrosis, are an increasingly important cause of chronic lameness that are poorly responsive to treatment. Recent studies have demonstrated a high-level association between these non-healing lesions and the Treponema phylogroups implicated in bovine digital dermatitis (BDD). However, a polymicrobial aetiology involving other gram-stain-negative anaerobes is suspected., Methods: A PCR-based bacteriological survey of uncomplicated BDD lesions (n=10) and non-healing bovine foot lesions (n=10) targeting Fusobacterium necrophorum , Porphyromonas endodontalis, Dichelobacter nodosus and Treponema pallidum/T. paraluiscuniculi was performed., Results: P. endodontalis DNA was detected in 80.0% of the non-healing lesion biopsies (p=<0.001) but was entirely absent from uncomplicated BDD lesion biopsies. When compared to the BDD lesions, F. necrophorum was detected at a higher frequency in the non-healing lesions (33.3% vs 70.0%, respectively), whereas D. nodosus was detected at a lower frequency (55.5% vs 20.0%, respectively). Conversely, T. pallidum/T. paraluiscuniculi DNA was not detected in either lesion type., Conclusion: The data from this pilot study suggest that P. endodontalis and F. necrophorum should be further investigated as potential aetiological agents of non-healing bovine foot lesions. A failure to detect syphilis treponemes in either lesion type is reassuring given the potential public health implications such an infection would present., Competing Interests: Competing interests: None declared., (© British Veterinary Association 2020. Re-use permitted under CC BY. Published by BMJ.)

The endoplasmic reticulum (ER) is a highly dynamic network of membranes. Here, we combine live-cell microscopy with in situ cryo-electron tomography to directly visualize ER dynamics in several secretory cell types including pancreatic β-cells and neurons under near-native conditions. Using these imaging approaches, we identify a novel, mobile form of ER, ribosome-associated vesicles (RAVs), found primarily in the cell periphery, which is conserved across different cell types and species. We show that RAVs exist as distinct, highly dynamic structures separate from the intact ER reticular architecture that interact with mitochondria via direct intermembrane contacts. These findings describe a new ER subcompartment within cells., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

Glucocorticoid hormones (GCs) play a pivotal role in many stress-related biological processes. In the hippocampus, GCs act through mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) to modify gene transcription. The involvement of GCs in biological processes has been investigated using the corticosterone (CORT)-synthesis blocker metyrapone. How metyrapone affects the action of GC at the genomic level still remains unclear. Therefore, we investigated the effects of this enzyme blocker on plasma CORT levels and hippocampal MR and GR binding to GC responsive elements (GREs) within the GC target genes Fkbp5 (FK506-binding protein 5), Per1 (Period 1) and Sgk1 (Serum- and glucocorticoid-activated kinase 1), as well as the transcriptional responses of these genes under control and acute stress conditions in rats. For comparison, we also investigated these endpoints in rats that had undergone adrenalectomy (ADX). Although metyrapone had no effect on baseline levels of CORT, the drug increased MR and GR to GRE binding within the GC target genes and the transcriptional activity of these genes. As expected, acute forced swim (FS) stress strongly increased plasma CORT levels, hippocampal MR and GR to GRE binding within Fkbp5, Per1 and Sgk1, and the transcriptional activity (mainly hnRNA levels) of these genes. Metyrapone attenuated, but did not abolish, these effects of stress on plasma CORT and MR and GR to GRE binding. The drug effects on FS-induced transcriptional activity were gene-dependent with a reduction seen in Fkbp5 hnRNA (but not Fkbp5 mRNA), an enhancement in Per1 hnRNA (but not Per1 mRNA), and no effect on both Sgk1 hnRNA and mRNA levels. ADX however completely abrogated the effects of FS on plasma CORT, as well as hippocampal MR and GR to GRE binding and transcriptional responses. Thus, in contrast to ADX, metyrapone produced inconsistent effects on GC-sensitive genomic endpoints that question its suitability as a tool in neuroendocrine and other research., (© 2019 British Society for Neuroendocrinology.)

Background: Bovine digital dermatitis (BDD) is a painful infectious foot disease of cattle, and much evidence implicates a pathogenic role for treponemes. This study measured the survival of BDD treponemes on hoof knife blades and tested the efficacy of relevant disinfectants under laboratory conditions., Methods: Two strains of BDD treponemes were applied to hoof knife blades under aerobic conditions. Swabs were taken at different time points (10 minutes, one hour, two hours, four hours and 18 hours) and again after 20-second disinfection time with one of five disinfectants. Swabs were used directly for nested PCR to detect treponemes or inoculated for anaerobic growth, and subsequently examined using phase contrast microscopy and PCR., Results: BDD treponeme DNA was detectable by nested PCR at all survival time points, and these organisms were culturable from hoof knives for two hours after exposure under aerobic conditions in the laboratory. Three of the five disinfectants-1 per cent volume per volume (v/v) FAM30 ® , 2 per cent weight per volume (w/v) Virkon ® or 2 per cent (v/v) sodium hypochlorite-were effective at preventing visible growth of treponemes following 20-seconds contact, and 1 per cent (v/v) FAM30 ® also prevented detection of treponemes by PCR., Conclusion: Treponeme viability of two hours under aerobic conditions suggests BDD treponemes could be transmitted between cows on hoof knives. It is therefore important to apply a disinfection protocol during foot-trimming; the authors have identified three common disinfectants that may be suitable., Competing Interests: Competing interests: None declared., (© British Veterinary Association 2019. Re-use permitted under CC BY. Published by BMJ.)

Over the last two decades, pancreatic islet transplantations have become a promising treatment for Type I diabetes. However, although providing a consistent and sustained exogenous insulin supply, there are a number of limitations hindering the widespread application of this approach. These include the lack of sufficient vasculature and allogeneic immune attacks after transplantation, which both contribute to poor cell survival rates. Here, these issues are addressed using a biofabrication approach. An alginate/gelatin-based bioink formulation is optimized for islet and islet-related cell encapsulation and 3D printing. In addition, a custom-designed coaxial printer is developed for 3D printing of multicellular islet-containing constructs. In this work, the ability to fabricate 3D constructs with precise control over the distribution of multiple cell types is demonstrated. In addition, it is shown that the viability of pancreatic islets is well maintained after the 3D printing process. Taken together, these results represent the first step toward an improved vehicle for islet transplantation and a potential novel strategy to treat Type I diabetes., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Integrative health is an expanding field that is increasingly called upon by conventional medicine to provide care for patients with chronic pain and disease. Although evidence has mounted for delivering integrative therapies individually, there is little consensus on how best to deliver these therapies in tandem as part of whole person care. While many models exist, few are financially sustainable., Methods and Results: This article describes a conceptual and logistical model for providing integrative outpatient health care within an academic medical center or hospital system to patients with chronic pain and disease. In hopes that the model will be replicated, administrative details are provided to explain how the model operates and has been maintained over nine years. The details include the intentional building of a particular work culture., Conclusion: This whole person care model that addresses chronic pain and disease in an outpatient integrative clinic has been successful, sustainable and can be replicated in other academic medical centers or hospital clinics.

The fibroblast growth factor FGF21 was labeled with molecularly defined gold nanoparticles (AuNPs), applied to human adipocytes, and imaged by cryo-electron tomography (cryo-ET). Most AuNPs were in pairs about 80 Å apart, on the outer cell surface. Pairs of AuNPs were also abundant inside the cells in clathrin-coated vesicles and endosomes. AuNPs were present but no longer paired in multivesicular bodies. FGF21 could thus be tracked along the endocytotic pathway. The methods developed here to visualize signaling coupled to endocytosis can be applied to a wide variety of cargo and may be extended to studies of other intracellular transactions., Competing Interests: MA, SC, GJ, RK No competing interests declared, JW, JZ, YL Employee of Amgen at the time the study was conducted. There are no other competing financial interests to declare., (© 2019, Azubel et al.)

Mitochondrial diseases produce profound neurological dysfunction via mutations affecting mitochondrial energy production, including the relatively common Leigh syndrome (LS). We recently described an LS case caused by a pathogenic mutation in USMG5, encoding a small supernumerary subunit of mitochondrial ATP synthase. This protein is integral for ATP synthase dimerization, and patient fibroblasts revealed an almost total loss of ATP synthase dimers. Here, we utilize in situ cryoelectron tomography (cryo-ET) in a clinical case-control study of mitochondrial disease to directly study mitochondria within cultured fibroblasts from a patient with LS and a healthy human control subject. Through tomographic analysis of patient and control mitochondria, we find that loss of ATP synthase dimerization due to the pathogenic mutation causes profound disturbances of mitochondrial crista ultrastructure. Overall, this work supports the crucial role of ATP synthase in regulating crista architecture in the context of human disease., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Since the first report of bovine digital dermatitis (DD) in 1974, there is a large body of the literature published; however, effective prevention and control of the disease remain elusive. Although many aspects of the pathogenesis of DD have been investigated, even some of the most basic questions such as the aetiology of this disease remain under debate. Treponema spp. have been strongly associated with DD lesions and occur in abundance in advanced lesions; however, efforts to induce disease with pure cultures of these organisms have been largely underwhelming and inconsistent. Furthermore, although the disease has been presented for several decades, there is limited scientific evidence regarding effective treatment of DD. Apparent discrepancies between effectiveness in vitro and in vivo have challenged the scientific community to identify new potential treatment options. With no treatment resulting in a 100% cure rate, the current expectation is manageable control, but prospects for the eradication of the disease are unlikely using current approaches. In order to develop more effective approaches to control DD on-farm, there is a critical need for a deeper understanding regarding the causation, ecology, transmission and treatment of this disease. In this article, we attempt to provide insights into specific research needs related to DD in order to assist the industry, researchers, pharmaceutical companies and research sponsors with decision-making and identified research gaps., (© 2017 Blackwell Verlag GmbH.)

Objectives: We compared rates of analytical outliers, and percent of emergency department (ED) patients with cardiac troponin (cTn) values above the 99th percentile upper reference limit (URL), for two conventional and one high sensitivity cTn assay., Methods: We measured 3008 samples from 1931 ED patients by Roche e411 4th generation Troponin T (cTnT); and Abbott STAT Troponin I (cTnI) and high sensitivity troponin I (hscTnI) on an Architect i2000. Within 24h of initial measurement, samples were aliquoted, re-centrifuged, and repeated in duplicate by all methods. Outliers were defined as one or both replicates exceeding initial value by a critical difference (CD): where CD=z×2×SDanalytical (z=3.29 at a probability of 0.0005), and at least one replicate on a different side of 99th percentile URL compared to initial value. We also assessed percent of ED patients with values >99th percentile by all methods (excluding outliers), using both sex-neutral and sex-specific hscTnI URL., Results: The outlier rate for cTnI (3.66%) was significantly higher than the outlier rate for either cTnT (0.33%) or hscTnI (0.47%) (p<0.0001). More ED patients (33%) had elevated cTnT values compared to either cTnI (25%) or hscTnI (29%). Application of sex-specific URL did not change the percent of ED patients with >99th percentile hscTnI values., Conclusion: Abbott STAT cTnI had more analytic outliers than Roche cTnT or Abbott hscTnI. Compared to cTnT, use of hscTnI will significantly decrease the percent of ED patients with elevated cTn values without increasing analytical outliers., (Copyright © 2018 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

The objective was to assess sustainability of a statewide program of HIV rapid testing (RT) for pregnant women presenting for delivery with unknown HIV status. This is a population-based retrospective cohort study of women delivered in Illinois hospitals (2012-15). Deidentified data on RT metrics from state-mandated surveillance reports were compared using descriptive statistics and non-parametric tests of trend. Over 95% of the 608,408 women delivered had documented HIV status at presentation. The rate of undocumented HIV status rose from 4.19 to 4.75% (p < 0.001). However, overall 99.60% of women with undocumented status appropriately received RT and the proportion who did not receive RT declined (p = 0.003). The number of neonates discharged with unknown HIV status declined (p = 0.011). RT identified 23 new HIV diagnoses, representing 4.62% of maternal HIV diagnoses. In conclusion, statewide perinatal HIV RT resulted in nearly 100% of Illinois mother-infant dyads with known HIV status. Sustained RT completion represents an important prevention safety net.

In cryogenic correlated light and electron microscopy (cryo-CLEM), frozen targets of interest are identified and located on EM grids by fluorescence microscopy and then imaged at higher resolution by cryo-EM. Whilst working with these methods, we discovered that a variety of mammalian cells exhibit strong punctate autofluorescence when imaged under cryogenic conditions (80 K). Autofluorescence originated from multilamellar bodies (MLBs) and secretory granules. Here we describe a method to distinguish fluorescent protein tags from these autofluorescent sources based on the narrower emission spectrum of the former. The method is first tested on mitochondria and then applied to examine the ultrastructural variability of secretory granules within insulin-secreting pancreatic beta-cell-derived INS-1E cells., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The immediate early genes (IEGs) c-Fos and Egr-1 are rapidly and transiently induced in sparse neurons within the hippocampus after exposure to an acute stressor. The induction of these genes is a critical part of the molecular mechanisms underlying successful behavioral adaptation to stress. Our previous work has shown that transcriptional activation of c-Fos and Egr-1 in the hippocampus requires formation of a dual histone mark within their promoter regions, the phosphorylation of serine 10 and acetylation of lysine 9/14 of histone H3. In the present study, using chromatin immuno-precipitation (ChIP), we found that an increase in the formation of H3K9ac-S10p occurs within the c-Fos and Egr-1 promoters after FS stress in vivo and that these histone modifications were located to promoter regions containing cAMP Responsive Elements (CREs), but not in neighboring regions containing only Serum Responsive Elements (SREs). Surprisingly, however, subsequent ChIP analyses showed no changes in the binding of pCREB or CREB-binding protein (CBP) to the CREs after FS. In fact, pCREB binding to the c-Fos and Egr-1 promoters was already highly enriched under baseline conditions and did not increase further after stress. We suggest that constitutive pCREB binding may keep c-Fos and Egr-1 in a poised state for activation. Possibly, the formation of H3K9ac-S10p in the vicinity of CRE sites may participate in unblocking transcriptional elongation through recruitment of additional epigenetic factors.

A Gram-stain-negative, obligatory anaerobic spirochete, CHPAT, was isolated from the rectal tissue of a Holstein-Friesian cow. On the basis of 16S rRNA gene comparisons, CHPAT was most closely related to the human oral spirochete, Treponema parvum, with 88.8 % sequence identity. Further characterisation on the basis of recA gene sequence analysis, cell morphology, pattern of growth and physiological profiling identified marked differences with respect to other recognised species of the genus Treponema. Microscopically, the helical cells measured approximately 1-5 µm long and 0.15-0.25 µm wide, with two to five irregular spirals. Transmission electron microscopy identified four periplasmic flagella in a 2 : 4 : 2 arrangement. CHPAT grew independently of serum, demonstrated no evidence of haemolytic activity and possessed an in vitro enzyme activity profile that is unique amongst validly named species of the genus Treponema, exhibiting C4 esterase, α-galactosidase and β-galactosidase activity. Taken together, these data indicate that CHPAT represents a novel species of the genus Treponema, for which the name Treponema rectale is proposed. The type strain of Treponema rectale is CHPAT (=DSM 103679T=NCTC 13848T).

A novel bacterium, strain Ru1T, was encountered during a survey of spirochaetes living in the gastrointestinal tract of ruminants. Comparative analysis of 16S rRNA gene sequence data indicated that strain Ru1T clustered within the genus Treponemabut shared at most 86.1 % sequence similarity with other recognised species of the genus Treponema. Further phylogenetic analysis based on partial recombinase A (recA) gene sequence comparisons, together with phenotypic characterization, also demonstrated the divergence of strain Ru1T from other recognised species of the genus Treponema. Microscopically, strain Ru1T appeared as a very small, highly motile, helical spirochaete with eight periplasmic flagella in a 4 : 8 : 4 arrangement. It exhibited C8 esterase lipase, leucine arylamidase, β-galactosidase and β-glucosidase activity. A distinctive, serum-independent growth pattern was also observed, characterized by colonies with an absence of the local haemolysis that is typical of many pathogenic treponemes. On the basis of these data, strain Ru1T is considered to represent a novel species of the genus Treponema, for which the name Treponema ruminis sp. nov. is proposed. The type strain is Ru1T (=DSM 103462T=NCTC 13847T).