Analyze Diet

PloS one.

Periodical
Medicine
Science
Publisher:
Public Library of Science
Frequency: Monthly
Country: United States
Language: English
Author(s):
Public Library of Science.
Start Year:2006 -
Identifiers
ISSN:1932-6203 (Electronic)
1932-6203 (Linking)
NLM ID:101285081
(OCoLC):70662135
LCCN:2006214532
Focal experimental injury leads to widespread gene expression and histologic changes in equine flexor tendons.
PloS one    April 2, 2015   Volume 10, Issue 4 e0122220 doi: 10.1371/journal.pone.0122220
Jacobson E, Dart AJ, Mondori T, Horadogoda N, Jeffcott LB, Little CB, Smith MM.It is not known how extensively a localised flexor tendon injury affects the entire tendon. This study examined the extent of and relationship between histopathologic and gene expression changes in equine superficial digital flexor tendon after a surgical injury. One forelimb tendon was hemi-transected in six horses, and in three other horses, one tendon underwent a sham operation. After euthanasia at six weeks, transected and control (sham and non-operated contralateral) tendons were regionally sampled (medial and lateral halves each divided into six 3 cm regions) for histologic (scoring and ...
Intermediate hosts of Angiostrongylus cantonensis in Tenerife, Spain.
PloS one    March 24, 2015   Volume 10, Issue 3 e0120686 doi: 10.1371/journal.pone.0120686
Martin-Alonso A, Abreu-Yanes E, Feliu C, Mas-Coma S, Bargues MD, Valladares B, Foronda P.The nematode Angiostrongylus cantonensis is the causative agent of human angiostrongyliasis, the main clinical manifestation of which is eosinophilic meningitis. Although this parasite has been found recently in its definitive rat host in Tenerife (Canary Islands, Spain), showing a widespread distribution over the north-east part of the island, there are no available data regarding which snail and/or slug species are acting as intermediate hosts on this island. Consequently, the objective of this work was to determine the possible role of three mollusc species, Plutonia lamarckii, Cornu aspers...
Regulatory T cells in early life: comparative study of CD4+CD25high T cells from foals and adult horses.
PloS one    March 19, 2015   Volume 10, Issue 3 e0120661 doi: 10.1371/journal.pone.0120661
Hamza E, Mirkovitch J, Steinbach F, Marti E.The immune system of mammals is subject to continuous development during the postnatal phase of life. Studies following the longitudinal development of the immune system in healthy children are limited both by ethical considerations and sample volumes. Horses represent a particular valuable large animal model for T regulatory (Treg) cells and allergy research. We have recently characterised Treg cells from horses, demonstrated their regulatory capability and showed both their expansion and induction in vitro. Insect bite hypersensitivity (IBH) is a common allergy in horses resembling atopic de...
The transcriptome of equine peripheral blood mononuclear cells.
PloS one    March 19, 2015   Volume 10, Issue 3 e0122011 doi: 10.1371/journal.pone.0122011
Pacholewska A, Drögemüller M, Klukowska-Rötzler J, Lanz S, Hamza E, Dermitzakis ET, Marti E, Gerber V, Leeb T, Jagannathan V.Complete transcriptomic data at high resolution are available only for a few model organisms with medical importance. The gene structures of non-model organisms are mostly computationally predicted based on comparative genomics with other species. As a result, more than half of the horse gene models are known only by projection. Experimental data supporting these gene models are scarce. Moreover, most of the annotated equine genes are single-transcript genes. Utilizing RNA sequencing (RNA-seq) the experimental validation of predicted transcriptomes has become accessible at reasonable costs. To...
Mares prefer the voices of highly fertile stallions.
PloS one    February 25, 2015   Volume 10, Issue 2 e0118468 doi: 10.1371/journal.pone.0118468
Lemasson A, Remeuf K, Trabalon M, Cuir F, Hausberger M.We investigated the possibility that stallion whinnies, known to encode caller size, also encoded information about caller arousal and fertility, and the reactions of mares in relation to type of voice. Voice acoustic features are correlated with arousal and reproduction success, the lower-pitched the stallion's voice, the slower his heart beat and the higher his fertility. Females from three study groups preferred playbacks of low-pitched voices. Hence, females are attracted by frequencies encoding for large male size, calmness and high fertility. More work is needed to explore the relative i...
Development of an equine groove model to induce metacarpophalangeal osteoarthritis: a pilot study on 6 horses.
PloS one    February 13, 2015   Volume 10, Issue 2 e0115089 doi: 10.1371/journal.pone.0115089
Maninchedda U, Lepage OM, Gangl M, Hilairet S, Remandet B, Meot F, Penarier G, Segard E, Cortez P, Jorgensen C, Steinberg R.The aim of this work was to develop an equine metacarpophalangeal joint model that induces osteoarthritis that is not primarily mediated by instability or inflammation. The study involved six Standardbred horses. Standardized cartilage surface damage or "grooves" were created arthroscopically on the distal dorsal aspect of the lateral and medial metacarpal condyles of a randomly chosen limb. The contralateral limb was sham operated. After 2 weeks of stall rest, horses were trotted 30 minutes every other day for 8 weeks, then evaluated for lameness and radiographed. Synovial fluid was analyzed ...
Functional locomotor consequences of uneven forefeet for trot symmetry in individual riding horses.
PloS one    February 3, 2015   Volume 10, Issue 2 e0114836 doi: 10.1371/journal.pone.0114836
Wiggers N, Nauwelaerts SL, Hobbs SJ, Bool S, Wolschrijn CF, Back W.Left-right symmetrical distal limb conformation can be an important prerequisite for a successful performance, and it is often hypothesized that asymmetric or uneven feet are important enhancing factors for the development of lameness. On a population level, it has been demonstrated that uneven footed horses are retiring earlier from elite level competition, but the biomechanical consequences are not yet known. The objectives of this study were to compare the functional locomotor asymmetries of horses with uneven to those with even feet. Hoof kinetics and distal limb kinematics were collected ...
Recombinant antigens rLipL21, rLoa22, rLipL32 and rLigACon4-8 for serological diagnosis of leptospirosis by enzyme-linked immunosorbent assays in dogs.
PloS one    December 19, 2014   Volume 9, Issue 12 e111367 doi: 10.1371/journal.pone.0111367
Ye C, Yan W, Xiang H, He H, Yang M, Ijaz M, Useh N, Hsieh CL, McDonough PL, McDonough SP, Mohamed H, Yang Z, Chang YF.Animal leptospirosis is one of the most common zoonotic diseases in the United States and around the world. In a previous study, we applied four recombinant antigens, rLipL21, rLoa22, rLipL32 and rLigACon4-8 of Leptospira interrogans (L. interrogans) for the serological diagnosis of equine leptospirosis (Ye et al, Serodiagnosis of equine leptospirosis by ELISA using four recombinant protein markers, Clin. Vaccine. Immunol. 21:478-483). In this study, the same four recombinant antigens were evaluated for their potential to diagnose canine leptospirosis by ELISA. A total of 305 canine sera that ...
RNA-seq analysis of equine conceptus transcripts during embryo fixation and capsule disappearance.
PloS one    December 16, 2014   Volume 9, Issue 12 e114414 doi: 10.1371/journal.pone.0114414
Tachibana Y, Sakurai T, Bai H, Shiota K, Nambo Y, Nagaoka K, Imakawa K.Extensive studies have been conducted to characterize the unique phenomena of equine pregnancy. Most studies have focused on embryo transmigration when the embryo is covered with a mucin-like glycoprotein capsule and on the characterization of the chorionic girdle and chorionic gonadotropin (CG) secretion. However, the events preceding and following capsule disappearance have not been well studied. In this study, the mRNA expression in conceptus membranes at days 19, 21, and 25 (day 0 = day of ovulation) was analyzed by RNA-seq (SOLiD3), and transcript levels on these three days and day 13...
Behavioral and Transcriptomic Fingerprints of an Enriched Environment in Horses (Equus caballus).
PloS one    December 10, 2014   Volume 9, Issue 12 e114384 doi: 10.1371/journal.pone.0114384
Lansade L, Valenchon M, Foury A, Neveux C, Cole SW, Layé S, Cardinaud B, Lévy F, Moisan MP.The use of environmental enrichment (EE) has grown in popularity over decades, particularly because EE is known to promote cognitive functions and well-being. Nonetheless, little is known about how EE may affect personality and gene expression. To address this question in a domestic animal, 10-month-old horses were maintained in a controlled environment or EE for 12 weeks. The control horses (n = 9) lived in individual stalls on wood shaving bedding. They were turned out to individual paddocks three times a week and were fed three times a day with pellets or hay. EE-treated horses (n =â€...
Idiopathic focal eosinophilic enteritis (IFEE), an emerging cause of abdominal pain in horses: the effect of age, time and geographical location on risk.
PloS one    December 2, 2014   Volume 9, Issue 12 e112072 doi: 10.1371/journal.pone.0112072
Archer DC, Costain DA, Sherlock C.Idiopathic focal eosinophilic enteritis (IFEE) is an emerging cause of abdominal pain (colic) in horses that frequently requires surgical intervention to prevent death. The epidemiology of IFEE is poorly understood and it is difficult to diagnose pre-operatively. The aetiology of this condition and methods of possible prevention are currently unknown. The aims of this study were to investigate temporal and spatial heterogeneity in IFEE risk and to ascertain the effect of horse age on risk. Results: A retrospective, nested case-control study was undertaken using data from 85 IFEE cases and 848 ...
Post-thaw non-cultured and post-thaw cultured equine cord blood mesenchymal stromal cells equally suppress lymphocyte proliferation in vitro.
PloS one    December 1, 2014   Volume 9, Issue 12 e113615 doi: 10.1371/journal.pone.0113615
Williams LB, Tessier L, Koenig JB, Koch TG.Multipotent mesenchymal stromal cells (MSC) are receiving increased attention for their non-progenitor immunomodulatory potential. Cryopreservation is commonly used for long-term storage of MSC. Post-thaw MSC proliferation is associated with a lag-phase in vitro. How this lag-phase affect MSC immunomodulatory properties is unknown. We hypothesized that in vitro there is no difference in lymphocyte suppression potential between quick-thawed cryopreserved equine cord blood (CB) MSC immediately included in mixed lymphocyte reaction (MLR) and same MSC allowed post-thaw culture time prior to inclus...
The effect of five different wetting treatments on the nutrient content and microbial concentration in hay for horses.
PloS one    November 26, 2014   Volume 9, Issue 11 e114079 doi: 10.1371/journal.pone.0114079
Moore-Colyer MJ, Lumbis K, Longland A, Harris P.Five different hays were used to determine the effect of 5 different soaking and steaming treatments on the water soluble carbohydrate and microbial (bacteria and mould) contents of UK hay. Hays were subjected to the following 5 treatments: 1. Dry; 2. Steamed for 50 minutes in the Haygain- 600 steamer; 3. Soaked in water at 16°C for 9 hours; 4. Steamed then soaked and 5. Soaked then steamed. Post treatment hays were tested for water soluble carbohydrates, bacteria and mould contents. Differences between means were determined using ANOVA and least significant difference with hay (5), bale (3) ...
Heterologous expression of equine CYP3A94 and investigation of a tunable system to regulate co-expressed NADPH P450 oxidoreductase levels.
PloS one    November 21, 2014   Volume 9, Issue 11 e113540 doi: 10.1371/journal.pone.0113540
Dettwiler R, Schmitz AL, Plattet P, Zielinski J, Mevissen M.The activity of cytochrome P450 enzymes depends on the enzyme NADPH P450 oxidoreductase (POR). The aim of this study was to investigate the activity of the equine CYP3A94 using a system that allows to regulate the POR protein levels in mammalian cells. CYP3A94 and the equine POR were heterologously expressed in V79 cells. In the system used, the POR protein regulation is based on a destabilizing domain (DD) that transfers its instability to a fused protein. The resulting fusion protein is therefore degraded by the ubiquitin-proteasome system (UPS). Addition of "Shield-1" prevents the DD fusion...
Presence and function of dopamine transporter (DAT) in stallion sperm: dopamine modulates sperm motility and acrosomal integrity.
PloS one    November 17, 2014   Volume 9, Issue 11 e112834 doi: 10.1371/journal.pone.0112834
Urra JA, Villaroel-Espíndola F, Covarrubias AA, Rodríguez-Gil JE, Ramírez-Reveco A, Concha II.Dopamine is a catecholamine with multiple physiological functions, playing a key role in nervous system; however its participation in reproductive processes and sperm physiology is controversial. High dopamine concentrations have been reported in different portions of the feminine and masculine reproductive tract, although the role fulfilled by this catecholamine in reproductive physiology is as yet unknown. We have previously shown that dopamine type 2 receptor is functional in boar sperm, suggesting that dopamine acts as a physiological modulator of sperm viability, capacitation and motility...
Spatial and temporal distribution of West Nile virus in horses in Israel (1997-2013)–from endemic to epidemics.
PloS one    November 17, 2014   Volume 9, Issue 11 e113149 doi: 10.1371/journal.pone.0113149
Aharonson-Raz K, Lichter-Peled A, Tal S, Gelman B, Cohen D, Klement E, Steinman A.With the rapid global spread of West Nile virus (WNV) and the endemic state it has acquired in new geographical areas, we hereby bring a thorough serological investigation of WNV in horses in a longstanding endemic region, such as Israel. This study evaluates the environmental and demographic risk factors for WNV infection in horses and suggests possible factors associated with the transition from endemic to epidemic state. West Nile virus seroprevalence in horses in Israel was determined throughout a period of more than a decade, before (1997) and after (2002 and 2013) the massive West Nile f...
The aggregation of four reconstructed zygotes is the limit to improve the developmental competence of cloned equine embryos.
PloS one    November 14, 2014   Volume 9, Issue 11 e110998 doi: 10.1371/journal.pone.0110998
Gambini A, De Stefano A, Bevacqua RJ, Karlanian F, Salamone DF.Embryo aggregation has been demonstrated to improve cloning efficiency in mammals. However, since no more than three embryos have been used for aggregation, the effect of using a larger number of cloned zygotes is unknown. Therefore, the goal of the present study was to determine whether increased numbers of cloned aggregated zygotes results in improved in vitro and in vivo embryo development in the equine. Zona-free reconstructed embryos (ZFRE's) were cultured in the well of the well system in four different experimental groups: I. 1x, only one ZFRE per microwell; II. 3x, three per microwell;...
Preliminary investigation into a potential role for myostatin and its receptor (ActRIIB) in lean and obese horses and ponies.
PloS one    November 12, 2014   Volume 9, Issue 11 e112621 doi: 10.1371/journal.pone.0112621
Morrison PK, Bing C, Harris PA, Maltin CA, Grove-White D, Argo CM.Obesity is a widespread problem across the leisure population of horses and ponies in industrialised nations. Skeletal muscle is a major contributor to whole body resting energy requirements and communicates with other tissues through the secretion of myokines into the circulation. Myostatin, a myokine and negative regulator of skeletal muscle mass, has been implicated in obesity development in other species. This study evaluated gene and protein expression of myostatin and its receptor, ActRIIB in adipose tissues and skeletal muscles and serum myostatin concentrations in six lean and six obes...
Faecal microbiota of forage-fed horses in New Zealand and the population dynamics of microbial communities following dietary change.
PloS one    November 10, 2014   Volume 9, Issue 11 e112846 doi: 10.1371/journal.pone.0112846
Fernandes KA, Kittelmann S, Rogers CW, Gee EK, Bolwell CF, Bermingham EN, Thomas DG.The effects of abrupt dietary transition on the faecal microbiota of forage-fed horses over a 3-week period were investigated. Yearling Thoroughbred fillies reared as a cohort were exclusively fed on either an ensiled conserved forage-grain diet ("Group A"; n = 6) or pasture ("Group B"; n = 6) for three weeks prior to the study. After the Day 0 faecal samples were collected, horses of Group A were abruptly transitioned to pasture. Both groups continued to graze similar pasture for three weeks, with faecal samples collected at 4-day intervals. DNA was isolated from the faeces and microbial 16S ...
Calcium-sensing receptor-mediated osteogenic and early-stage neurogenic differentiation in umbilical cord matrix mesenchymal stem cells from a large animal model.
PloS one    November 7, 2014   Volume 9, Issue 11 e111533 doi: 10.1371/journal.pone.0111533
Umbilical cord matrix mesenchymal stem cells (UCM-MSCs) present a wide range of potential therapeutical applications. The extracellular calcium-sensing receptor (CaSR) regulates physiological and pathological processes. We investigated, in a large animal model, the involvement of CaSR in triggering osteogenic and neurogenic differentiation of two size-sieved UCM-MSC lines, by using AMG641, a novel potent research calcimimetic acting as CaSR agonist. Results: Large (>8 µm in diameter) and small (<8 µm) equine UCM-MSC lines were cultured in medium with high calcium (Ca2+) concentration (...
Non-avian animal reservoirs present a source of influenza A PB1-F2 proteins with novel virulence-enhancing markers.
PloS one    November 4, 2014   Volume 9, Issue 11 e111603 doi: 10.1371/journal.pone.0111603
Alymova IV, York IA, McCullers JA.PB1-F2 protein, expressed from an alternative reading frame of most influenza A virus (IAV) PB1 segments, may possess specific residues associated with enhanced inflammation (L62, R75, R79, and L82) and cytotoxicity (I68, L69, and V70). These residues were shown to increase the pathogenicity of primary viral and secondary bacterial infections in a mouse model. In contrast to human seasonal influenza strains, virulence-associated residues are present in PB1-F2 proteins from pandemic H1N1 1918, H2N2 1957, and H3N2 1968, and highly pathogenic H5N1 strains, suggesting their contribution to viruses...
Using Bayes’ rule to define the value of evidence from syndromic surveillance.
PloS one    November 3, 2014   Volume 9, Issue 11 e111335 doi: 10.1371/journal.pone.0111335
Andersson MG, Faverjon C, Vial F, Legrand L, Leblond A.In this work we propose the adoption of a statistical framework used in the evaluation of forensic evidence as a tool for evaluating and presenting circumstantial "evidence" of a disease outbreak from syndromic surveillance. The basic idea is to exploit the predicted distributions of reported cases to calculate the ratio of the likelihood of observing n cases given an ongoing outbreak over the likelihood of observing n cases given no outbreak. The likelihood ratio defines the Value of Evidence (V). Using Bayes' rule, the prior odds for an ongoing outbreak are multiplied by V to obtain the post...
Amniotic membrane-derived mesenchymal cells and their conditioned media: potential candidates for uterine regenerative therapy in the horse.
PloS one    October 31, 2014   Volume 9, Issue 10 e111324 doi: 10.1371/journal.pone.0111324
Corradetti B, Correani A, Romaldini A, Marini MG, Bizzaro D, Perrini C, Cremonesi F, Lange-Consiglio A.Amniotic membrane-derived mesenchymal cells (AMCs) are considered suitable candidates for a variety of cell-based applications. In view of cell therapy application in uterine pathologies, we studied AMCs in comparison to cells isolated from the endometrium of mares at diestrus (EDCs) being the endometrium during diestrus and early pregnancy similar from a hormonal standpoint. In particular, we demonstrated that amnion tissue fragments (AM) shares the same transcriptional profile with endometrial tissue fragments (ED), expressing genes involved in early pregnancy (AbdB-like Hoxa genes), pre-imp...
Genome-wide association study identifies phospholipase C zeta 1 (PLCz1) as a stallion fertility locus in Hanoverian warmblood horses.
PloS one    October 29, 2014   Volume 9, Issue 10 e109675 doi: 10.1371/journal.pone.0109675
Schrimpf R, Dierks C, Martinsson G, Sieme H, Distl O.A consistently high level of stallion fertility plays an economically important role in modern horse breeding. We performed a genome-wide association study for estimated breeding values of the paternal component of the pregnancy rate per estrus cycle (EBV-PAT) in Hanoverian stallions. A total of 228 Hanoverian stallions were genotyped using the Equine SNP50 Beadchip. The most significant association was found on horse chromosome 6 for a single nucleotide polymorphism (SNP) within phospholipase C zeta 1 (PLCz1). In the close neighbourhood to PLCz1 is located CAPZA3 (capping protein (actin filam...
Intramuscular administration of a synthetic CpG-oligodeoxynucleotide modulates functional responses of neutrophils of neonatal foals.
PloS one    October 15, 2014   Volume 9, Issue 10 e109865 doi: 10.1371/journal.pone.0109865
Cohen ND, Bourquin JR, Bordin AI, Kuskie KR, Brake CN, Weaver KB, Liu M, Felippe MJ, Kogut MH.Neutrophils play an important role in protecting against infection. Foals have age-dependent deficiencies in neutrophil function that may contribute to their predisposition to infection. Thus, we investigated the ability of a CpG-ODN formulated with Emulsigen to modulate functional responses of neutrophils in neonatal foals. Eighteen foals were randomly assigned to receive either a CpG-ODN with Emulsigen (N = 9) or saline intramuscularly at ages 1 and 7 days. At ages 1, 3, 9, 14, and 28, blood was collected and neutrophils were isolated from each foal. Neutrophils were assessed for basal and R...
Development of a likelihood of survival scoring system for hospitalized equine neonates using generalized boosted regression modeling.
PloS one    October 8, 2014   Volume 9, Issue 10 e109212 doi: 10.1371/journal.pone.0109212
Dembek KA, Hurcombe SD, Frazer ML, Morresey PR, Toribio RE.Medical management of critically ill equine neonates (foals) can be expensive and labor intensive. Predicting the odds of foal survival using clinical information could facilitate the decision-making process for owners and clinicians. Numerous prognostic indicators and mathematical models to predict outcome in foals have been published; however, a validated scoring method to predict survival in sick foals has not been reported. The goal of this study was to develop and validate a scoring system that can be used by clinicians to predict likelihood of survival of equine neonates based on clinica...
Congenital hepatic fibrosis in the Franches-Montagnes horse is associated with the polycystic kidney and hepatic disease 1 (PKHD1) gene.
PloS one    October 8, 2014   Volume 9, Issue 10 e110125 doi: 10.1371/journal.pone.0110125
Drögemüller M, Jagannathan V, Welle MM, Graubner C, Straub R, Gerber V, Burger D, Signer-Hasler H, Poncet PA, Klopfenstein S, von Niederhäusern R....Congenital hepatic fibrosis has been described as a lethal disease with monogenic autosomal recessive inheritance in the Swiss Franches-Montagnes horse breed. We performed a genome-wide association study with 5 cases and 12 controls and detected an association on chromosome 20. Subsequent homozygosity mapping defined a critical interval of 952 kb harboring 10 annotated genes and loci including the polycystic kidney and hepatic disease 1 (autosomal recessive) gene (PKHD1). PKHD1 represents an excellent functional candidate as variants in this gene were identified in human patients with autosoma...
Assessment of intraocular measurements in neonatal foals and association with gender, laterality, and body weight: a clinical study.
PloS one    October 8, 2014   Volume 9, Issue 10 e109491 doi: 10.1371/journal.pone.0109491
Valentini S, Castagnetti C, Musella V, Spinella G.Objective of this study was to describe intraocular measurements in newly born foals (1-7 days of age) and assess the association between globe measurements and gender, laterality, and body weight. B-scan ultrasonographic biometry was performed on both eyes of 22 healthy foals (44 eyes) ages 1-7 days using a 10-MHz transducer. Intraocular measurements (anterior chamber depth, central lens thickness, vitreous chamber depth, axial globe length, longitudinal globe length, lens poles distance) were carried out using the ultrasound internal calipers. The influence of gender (male or female), latera...
UTF1, a putative marker for spermatogonial stem cells in stallions.
PloS one    October 1, 2014   Volume 9, Issue 10 e108825 doi: 10.1371/journal.pone.0108825
Jung H, Roser JF, Yoon M.Spermatogonial stem cells (SSCs) continuously undergo self-renewal and differentiation to sustain spermatogenesis throughout adulthood in males. In stallions, SSCs may be used for the production of progeny from geldings after cryopreservation and therapy for infertile and subfertile stallions. Undifferentiated cell transcription factor 1 (UTF1) is a putative marker for undifferentiated spermatogonia in humans and rats. The main purposes of this study are to determine the following: 1) changes in the expression pattern of UTF1 at various reproductive stages of stallions, 2) subpopulations of sp...
Testing VHF/GPS collar design and safety in the study of free-roaming horses.
PloS one    September 8, 2014   Volume 9, Issue 9 e103189 doi: 10.1371/journal.pone.0103189
Collins GH, Petersen SL, Carr CA, Pielstick L.Effective and safe monitoring techniques are needed by U.S. land managers to understand free-roaming horse behavior and habitat use and to aid in making informed management decisions. Global positioning system (GPS) and very high frequency (VHF) radio collars can be used to provide high spatial and temporal resolution information for detecting free-roaming horse movement. GPS and VHF collars are a common tool used in wildlife management, but have rarely been used for free-roaming horse research and monitoring in the United States. The purpose of this study was to evaluate the design, safety, a...
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