Development of a novel glycoengineering platform for the rapid production of conjugate vaccines.

Conjugate vaccines produced either by chemical or biologically conjugation have been demonstrated to be safe and efficacious in protection against several deadly bacterial diseases. However, conjugate vaccine assembly and production have several shortcomings which hinders their wider availability. Here, we developed a tool, Mobile-element Assisted Glycoconjugation by Insertion on Chromosome, MAGIC, a novel biotechnological platform that overcomes the limitations of the current conjugate vaccine design method(s). As a model, we focused our design on a leading bioconjugation method using N-oligosaccharyltransferase (OTase), PglB. The installation of MAGIC led to at least twofold increase in glycoconjugate yield via MAGIC when compared to conventional N-OTase based bioconjugation method(s). Then, we improved MAGIC to (a) allow rapid installation of glycoengineering component(s), (b) omit the usage of antibiotics, (c) reduce the dependence on protein induction agents. Furthermore, we show the modularity of the MAGIC platform in performing glycoengineering in bacterial species that are less genetically tractable than the commonly used Escherichia coli. The MAGIC system promises a rapid, robust and versatile method to develop vaccines against serious bacterial pathogens. We anticipate the utility of the MAGIC platform could enhance vaccines production due to its compatibility with virtually any bioconjugation method, thus expanding vaccine biopreparedness toolbox.

Site-directed M2 proton channel inhibitors enable synergistic combination therapy for rimantadine-resistant pandemic influenza.

Pandemic influenza A virus (IAV) remains a significant threat to global health. Preparedness relies primarily upon a single class of neuraminidase (NA) targeted antivirals, against which resistance is steadily growing. The M2 proton channel is an alternative clinically proven antiviral target, yet a near-ubiquitous S31N polymorphism in M2 evokes resistance to licensed adamantane drugs. Hence, inhibitors capable of targeting N31 containing M2 (M2-N31) are highly desirable. Rational in silico design and in vitro screens delineated compounds favouring either lumenal or peripheral M2 binding, yielding effective M2-N31 inhibitors in both cases. Hits included adamantanes as well as novel compounds, with some showing low micromolar potency versus pandemic "swine" H1N1 influenza (Eng195) in culture. Interestingly, a published adamantane-based M2-N31 inhibitor rapidly selected a resistant V27A polymorphism (M2-A27/N31), whereas this was not the case for non-adamantane compounds. Nevertheless, combinations of adamantanes and novel compounds achieved synergistic antiviral effects, and the latter synergised with the neuraminidase inhibitor (NAi), Zanamivir. Thus, site-directed drug combinations show potential to rejuvenate M2 as an antiviral target whilst reducing the risk of drug resistance.

Building capacity for injury prevention: a process evaluation of a replication of the Cardiff Violence Prevention Programme in the Southeastern USA.

OBJECTIVES: Violence is a major public health problem in the USA. In 2016, more than 1.6 million assault-related injuries were treated in US emergency departments (EDs). Unfortunately, information about the magnitude and patterns of violent incidents is often incomplete and underreported to law enforcement (LE). In an effort to identify more complete information on violence for the development of prevention programme, a cross-sectoral Cardiff Violence Prevention Programme (Cardiff Model) partnership was established at a large, urban ED with a level I trauma designation and local metropolitan LE agency in the Atlanta, Georgia metropolitan area. The Cardiff Model is a promising violence prevention approach that promotes combining injury data from hospitals and LE. The objective was to describe the Cardiff Model implementation and collaboration between hospital and LE partners. METHODS: The Cardiff Model was replicated in the USA. A process evaluation was conducted by reviewing project materials, nurse surveys and interviews and ED-LE records. RESULTS: Cardiff Model replication centred around four activities: (1) collaboration between the hospital and LE to form a community safety partnership locally called the US Injury Prevention Partnership; (2) building hospital capacity for data collection; (3) data aggregation and analysis and (4) developing and implementing violence prevention interventions based on the data. CONCLUSIONS: The Cardiff Model can be implemented in the USA for sustainable violent injury data surveillance and sharing. Key components include building a strong ED-LE partnership, communicating with each other and hospital staff, engaging in capacity building and sustainability planning.

The role of infant life histories in the construction of identities in death: An incremental isotope study of dietary and physiological status among children afforded differential burial.

OBJECTIVES: Isotope ratio analyses of dentine collagen were used to characterize short-term changes in physiological status (both dietary status and biological stress) across the life course of children afforded special funerary treatment. MATERIALS AND METHODS: Temporal sequences of δ15 N and δ13 C isotope profiles for incrementally forming dentine collagen were obtained from deciduous teeth of 86 children from four early-medieval English cemeteries. Thirty-one were interred in child-specific burial clusters, and the remainder alongside adults in other areas of the cemetery. Isotope profiles were categorized into four distinct patterns of dietary and health status between the final prenatal months and death. RESULTS: Isotope profiles from individuals from the burial clusters were significantly less likely to reflect weaning curves, suggesting distinctive breastfeeding and weaning experiences. This relationship was not simply a factor of differential age at death between cohorts. There was no association of burial location neither with stage of weaning at death, nor with isotopic evidence of physiological stress at the end of life. DISCUSSION: This study is the first to identify a relationship between the extent of breastfeeding and the provision of child-specific funerary rites. Limited breastfeeding may indicate the mother had died during or soon after birth, or that either mother or child was unable to feed due to illness. Children who were not breastfed will have experienced a significantly higher risk of malnutrition, undernutrition and infection. These sickly and perhaps motherless children received care to nourish them during early life, and were similarly provided with special treatment in death.

A new multivariate method for determining sex of immature human remains using the maxillary first molar.

OBJECTIVES: This study investigated the use of sexually dimorphic metrics of the first permanent maxillary molar (M1 ) to determine sex in adult and immature individuals within and between populations. METHODS: Ten M1 dimensions were measured in 91 adults (19-55 years) and 58 immatures (5-18 years) from two English populations, one of documented sex (Spitalfields crypt) and another of morphologically-assigned sex (Black Gate). Preliminary statistical analysis was undertaken to explore bilateral differences and variation by age and sex, followed by multivariate analyses to predict sex from dental metrics. RESULTS: Both cross-validated linear discriminant analysis and binary logistic regression predicted biological sex consistent with known sex in 94.6% of adults and 90.9% of immatures. When functions extracted from the Spitalfields data were used to assign sex to Black Gate adults, consistency with morphological sex varied from 83.3% to 57.7%. A new function developed on Black Gate resulted in only a 4.8% increase in maximum accuracy but reduced bias. The immature cohort comprised 19 (52.8%) males and 17 (47.2%) females. CONCLUSIONS: This study demonstrates substantial sexual dimorphism in a single tooth which is commonly preserved in archaeological and forensic contexts. It successfully assigns biological sex to immatures from 5 years of age with substantially greater accuracy than any other morphological or metric method. We suggest that accurate cross-population functions based on dentition require a trade-off between accuracy and applicability, and that functions extracted from populations of documented sex can be used to assign sex to other archaeological and forensic remains.

Comparing apples and oranges: Why infant bone collagen may not reflect dietary intake in the same way as dentine collagen.

OBJECTIVES: Recent developments in incremental dentine analysis allowing increased temporal resolution for tissues formed during the first 1,000 days of life have cast doubt on the veracity of weaning studies using bone collagen carbon (δ13 C) and nitrogen (δ15 N) isotope ratio data from infants. Here, we compare published bone data from the well-preserved Anglo-Saxon site of Raunds Furnells, England, with co-forming dentine from the same individuals, and investigate the relationship of these with juvenile stature. The high-resolution isotope data recorded in dentine allow us to investigate the relationship of diet with juvenile stature during this critical period of life. MATERIALS AND METHODS: We compare incremental dentine collagen δ13 C and δ15 N data to published bone collagen data for 18 juveniles and 5 female adults from Anglo Saxon Raunds Furnells alongside new data for juvenile skeletal and dental age. An improvement in the method by sampling the first 0.5 mm of the sub-cuspal or sub-incisal dentine allows the isotopic measurement of dentine formed in utero. RESULTS AND DISCUSSION: δ13 C profiles for both dentine and bone are similar and more robust than δ15 N for estimating the age at which weaning foods are introduced. Our results suggest δ15 N values from dentine can be used to evaluate the maternal/in utero diet and physiology during pregnancy, and that infant dentine profiles may reflect diet PLUS an element of physiological stress. In particular, bone collagen fails to record the same range of δ15 N as co-forming dentine, especially where growth is stunted, suggesting that infant bone collagen is unreliable for weaning studies.

Community-Based Academic Level I Trauma Center Prepares for the 2016 Republican National Convention.

Disaster preparedness has come to the forefront for hospitals since the 9/11 attacks in 2001. Many improvements have been made in emergency management and planning for catastrophic events. Both urban and community hospitals have the same responsibilities and commitments to their patients and communities. When the announcement was made that the 2016 Republican National Convention was going to be held in Cleveland, OH, Cleveland Clinic Akron General (CCAG) had to be confident in its abilities to handle any situation that might arise not just as a community hospital but also as a Level I trauma center. Organizing and preparing for more than a year, CCAG developed a detailed and well-thought-out preparedness program, with senior leadership implementing a clear chain of command. Developing and maintaining a strong and steady defense through detailed preparation, communication, teamwork, and organization are the keys to success.

Investigation into the efficiency of diverse N-linking oligosaccharyltransferases for glycoengineering using a standardised cell-free assay.

The application of bacterial oligosaccharyltransferases (OSTs) such as the Campylobacter jejuni PglB for glycoengineering has attracted considerable interest in glycoengineering and glycoconjugate vaccine development. However, PglB has limited specificity for glycans that can be transferred to candidate proteins, which along with other factors is dependent on the reducing end sugar of glycans. In this study, we developed a cell-free glycosylation assay that offers the speed and simplicity of a 'yes' or 'no' determination. Using the assay, we tested the activity of eleven PglBs from Campylobacter species and more distantly related bacteria. The following assorted glycans with diverse reducing end sugars were tested for transfer, including Streptococcus pneumoniae capsule serotype 4, Salmonella enterica serovar Typhimurium O antigen (B1), Francisella tularensis O antigen, Escherichia coli O9 antigen and Campylobacter jejuni heptasaccharide. Interestingly, while PglBs from the same genus showed high activity, whereas divergent PglBs differed in their transfer of glycans to an acceptor protein. Notably for glycoengineering purposes, Campylobacter hepaticus and Campylobacter subantarcticus PglBs showed high glycosylation efficiency, with C. hepaticus PglB potentially being useful for glycoconjugate vaccine production. This study demonstrates the versatility of the cell-free assay in rapidly assessing an OST to couple glycan/carrier protein combinations and lays the foundation for future screening of PglBs by linking amino acid similarity to glycosyltransferase activity.

Weaning at Anglo-Saxon Raunds: Implications for changing breastfeeding practice in Britain over two millennia.

This study investigated stable-isotope ratio evidence of weaning for the late Anglo-Saxon population of Raunds Furnells, Northamptonshire, UK. δ(15)N and δ(13)C values in rib collagen were obtained for individuals of different ages to assess the weaning age of infants within the population. A peak in δ(15) N values at about 2-year-old, followed by a decline in δ(15) N values until age three, indicates a change in diet at that age. This change in nitrogen isotope ratios corresponds with the mortality profile from the site, as well as with archaeological and documentary evidence on attitudes towards juveniles in the Anglo-Saxon period. The pattern of δ(13) C values was less clear. Comparison of the predicted age of weaning to published data from sites dating from the Iron Age to the 19th century in Britain reveals a pattern of changing weaning practices over time, with increasingly earlier commencement and shorter periods of complementary feeding in more recent periods. Such a change has implications for the interpretation of socioeconomic changes during this period of British history, since earlier weaning is associated with decreased birth spacing, and could thus have contributed to population growth.

Assessing trauma readiness costs in level III and level IV trauma centers.

BACKGROUND: Readiness costs are expenses incurred by trauma centers to maintain essential infrastructure. Although the components for readiness are described in the American College of Surgeons' Resources for Optimal Care of the Injured Patient , the cost associated with each component is not well defined. Previous studies describe readiness costs for levels I and II trauma centers based on these criteria. The purpose of this study was to quantify the cost of levels III and IV trauma center readiness. METHODS: The state trauma commission, along with trauma medical directors, program managers, and trauma center financial staff, standardized definitions for each component of trauma center readiness costs and developed a survey tool for reporting. Readiness costs were grouped into four categories: Administrative/Program Support Staff, Clinical Medical Staff, and Education/Outreach. A financial auditor analyzed all data to verify consistent cost reporting. Trauma center outliers were evaluated to validate variances. All levels III and IV trauma centers (n = 14) completed the survey on 2019 data. RESULTS: Average annual readiness cost is $1,715,025 for a level III trauma center and $81,620 for level IV centers. Among the costliest components were clinical medical staff for level IIIs and administrative costs for level IVs, representing 54% and 97% of costs, respectively. Although education/outreach is mandated, levels III and IV trauma centers only spend approximately $8,000 annually on this category (0.8-3%). CONCLUSION: This study defines the cost associated with each readiness component outlined in the Resources for Optimal Care of the Injured Patient manual. The average readiness cost for a level III trauma center is $1,715,025 and $81,620 for a level IV, underscoring the need for additional trauma center funding to meet the requirements set forth by the American College of Surgeons. LEVEL OF EVIDENCE: Economic and Value-Based Evaluations; Level III.

Coagulopathy is prevalent and associated with adverse outcomes in transfused pediatric trauma patients.

OBJECTIVE: To evaluate coagulopathy in pediatric trauma patients on presentation to the emergency department, and to quantify the relationship with mortality. STUDY DESIGN: Pediatric trauma patients requiring a blood transfusion (red blood cells, fresh frozen plasma, platelets, or cryoprecipitate) within 24 hours of arrival were included. Coagulation values on emergency department arrival were analyzed, as were clinical details and outcome. RESULTS: A total of 102 children (mean age, 6 years; mean injury severity score 22, mean Glascow Coma Scale 7, 80% blunt trauma victims) were studied over a 4 year period. An abnormal prothrombin time was found in 72%, partial thromboplastin time in 38%, fibrinogen in 52%, hemoglobin in 58%, and platelet count in 23%. An abnormal prothrombin time, partial thromboplastin time, and platelet count were strongly associated with mortality (P=.005, .001, and <.0001, respectively) and remained significantly associated in multivariate analysis after adjusting for injury severity score. CONCLUSIONS: Coagulopathy is prevalent in pediatric trauma patients ill enough to require a transfusion and is strongly associated with mortality. Studies are needed to determine whether early coagulation factor replacement and the institution of massive transfusion protocols may improve outcomes in these patients.

Resistance mutations define specific antiviral effects for inhibitors of the hepatitis C virus p7 ion channel.

UNLABELLED: The hepatitis C virus (HCV) p7 ion channel plays a critical role during infectious virus production and represents an important new therapeutic target. Its activity is blocked by structurally distinct classes of small molecules, with sensitivity varying between isolate p7 sequences. Although this is indicative of specific protein-drug interactions, a lack of high-resolution structural information has precluded the identification of inhibitor binding sites, and their modes of action remain undefined. Furthermore, a lack of clinical efficacy for existing p7 inhibitors has cast doubt over their specific antiviral effects. We identified specific resistance mutations that define the mode of action for two classes of p7 inhibitor: adamantanes and alkylated imino sugars (IS). Adamantane resistance was mediated by an L20F mutation, which has been documented in clinical trials. Molecular modeling revealed that L20 resided within a membrane-exposed binding pocket, where drug binding prevented low pH-mediated channel opening. The peripheral binding pocket was further validated by a panel of adamantane derivatives as well as a bespoke molecule designed to bind the region with high affinity. By contrast, an F25A polymorphism found in genotype 3a HCV conferred IS resistance and confirmed that these compounds intercalate between p7 protomers, preventing channel oligomerization. Neither resistance mutation significantly reduced viral fitness in culture, consistent with a low genetic barrier to resistance occurring in vivo. Furthermore, no cross-resistance was observed for the mutant phenotypes, and the two inhibitor classes showed additive effects against wild-type HCV. CONCLUSION: These observations support the notion that p7 inhibitor combinations could be a useful addition to future HCV-specific therapies.

Implementation of a pediatric trauma massive transfusion protocol: one institution's experience.

BACKGROUND: Massive transfusion protocols (MTPs) with fixed ratios of blood products may improve outcomes in coagulopathic adult trauma patients. However, there is a paucity of data on transfusion support protocols for pediatric trauma patients, whose mechanisms of injury may differ from those seen in adults. We hypothesized that an MTP would improve outcomes in children, through a balanced blood product resuscitation. STUDY DESIGN AND METHODS: A pediatric trauma MTP, with a fixed ratio of red blood cells (RBCs):fresh-frozen plasma (FFP):platelets:cryoprecipitate in quantities based on the patient's weight, was initiated at a pediatric hospital. Data on clinical status, resuscitation volumes, and hospital course were collected and compared to data from pre-MTP trauma patients requiring transfusion. RESULTS: Fifty-three patients were enrolled over a 15-month period and compared to 49 pre-MTP patients. Seventy-two percent of MTP patients had at least one coagulation value outside of the normal range upon emergency department (ED) arrival, and the median time to FFP transfusion decreased fourfold after MTP implementation (p<0.0001). A total of 49% of MTP patients received greater than 70 mL/kg blood products, and the 24-hour median FFP:RBC transfusion ratio was twofold higher in these patients than the pre-MTP cohort (median, 1:1.8 vs. 1:3.6; p=0.002). No improvement in mortality was observed after MTP implementation, taking into consideration injury severity, prothrombin time, and partial thromboplastin time. CONCLUSIONS: A pediatric trauma MTP is feasible and allows for rapid provision of balanced blood products for transfusion to coagulopathic children. Larger studies are warranted to determine whether such protocols will improve outcomes for pediatric trauma patients.

Use of Precision-Cut Tissue Slices as a Translational Model to Study Host-Pathogen Interaction.

The recent increase in new technologies to analyze host-pathogen interaction has fostered a race to develop new methodologies to assess these not only on the cellular level, but also on the tissue level. Due to mouse-other mammal differences, there is a desperate need to develop relevant tissue models that can more closely recapitulate the host tissue during disease and repair. Whereas organoids and organs-on-a-chip technologies have their benefits, they still cannot provide the cellular and structural complexity of the host tissue. Here, precision cut tissue slices (PCTS) may provide invaluable models for complex ex-vivo generated tissues to assess host-pathogen interaction as well as potential vaccine responses in a "whole organ" manner. In this mini review, we discuss the current literature regarding PCTS in veterinary species and advocate that PCTS represent remarkable tools to further close the gap between target identification, subsequent translation of results into clinical studies, and thus opening avenues for future precision medicine approaches.

Needs Based Assessment of Trauma Systems 2, is it ready for primetime? A natural experiment testing its reliability.

INTRODUCTION: Needs Based Assessment of Trauma Systems 2 (NBATS-2) attempts to predict the impact on patient volume and travel time for patients when a new trauma center (TC) is added to the system. The purpose of this study was to examine NBATS-2 predictive accuracy regarding expected volume and travel times of trauma patients at a newly designated TC and nearby legacy TCs when compared with actual data. METHODS: Needs Based Assessment of Trauma Systems predictive model for volume of trauma patients at the new TC was run based on 25th, 50th, and 75th percentiles of both state and National Trauma Data Bank (NTDB) patients per 100 TC beds. This was compared with the actual number of trauma patients from the State Discharge Data set before (2011-2012) and after (2016-2017) designation of the TC. Analysis was then augmented using the geographic information system (ArcGIS) spatial modeling to characterize median travel times for actual trauma patients, before and after designation of the TC. RESULTS: Both state and NTDB 25th, 50th, and 75th percentiles resulted in significant overestimation of volume at the new TC in 2016. After another year of TC maturation (2017), overestimation decreased but was still present. The 25th percentile from state and NTDB data sets provided the most accurate predictions. For the legacy TCs, the model switched from under to overestimation as the state and NTDB percentiles increased. The geographic information system accurately showed patients traveling <40 minutes to a TC nearly doubled. CONCLUSION: Needs Based Assessment of Trauma Systems 2 provides an excellent template for state strategic planning; however, it overestimates new TC volume and under/overestimates volumes for legacy TCs depending on the state and NTDB percentiles used. This study shows that population density of the county in which the new or legacy TC is located should be considered when choosing the appropriate state or NTDB percentile. The geographic information system appropriately showed a decrease in trauma patient travel times after TC designation. LEVEL OF EVIDENCE: Care Management, level V.

Determinants of hepatitis C virus p7 ion channel function and drug sensitivity identified in vitro.

Hepatitis C virus (HCV) chronically infects 170 million individuals, causing severe liver disease. Although antiviral chemotherapy exists, the current regimen is ineffective in 50% of cases due to high levels of innate virus resistance. New, virus-specific therapies are forthcoming although their development has been slow and they are few in number, driving the search for new drug targets. The HCV p7 protein forms an ion channel in vitro and is critical for the secretion of infectious virus. p7 displays sensitivity to several classes of compounds, making it an attractive drug target. We recently demonstrated that p7 compound sensitivity varies according to viral genotype, yet little is known of the residues within p7 responsible for channel activity or drug interactions. Here, we have employed a liposome-based assay for p7 channel function to investigate the genetic basis for compound sensitivity. We demonstrate using chimeric p7 proteins that neither the two trans-membrane helices nor the p7 basic loop individually determines compound sensitivity. Using point mutation analysis, we identify amino acids important for channel function and demonstrate that null mutants exert a dominant negative effect over wild-type protein. We show that, of the three hydrophilic regions within the amino-terminal trans-membrane helix, only the conserved histidine at position 17 is important for genotype 1b p7 channel activity. Mutations predicted to play a structural role affect both channel function and oligomerization kinetics. Lastly, we identify a region at the p7 carboxy terminus which may act as a specific sensitivity determinant for the drug amantadine.

Correction: The dietary impact of the Norman Conquest: A multiproxy archaeological investigation of Oxford, UK.

[This corrects the article DOI: 10.1371/journal.pone.0235005.].

The dietary impact of the Norman Conquest: A multiproxy archaeological investigation of Oxford, UK.

Archaeology has yet to capitalise on the opportunities offered by bioarchaeological approaches to examine the impact of the 11th-century AD Norman Conquest of England. This study utilises an integrated multiproxy analytical approach to identify and explain changes and continuities in diet and foodways between the 10th and 13th centuries in the city of Oxford, UK. The integration of organic residue analysis of ceramics, carbon (δ13C) and nitrogen (δ15N) isotope analysis of human and animal bones, incremental analysis of δ13C and δ15N from human tooth dentine and palaeopathological analysis of human skeletal remains has revealed a broad pattern of increasing intensification and marketisation across various areas of economic practice, with a much lesser and more short-term impact of the Conquest on everyday lifestyles than is suggested by documentary sources. Nonetheless, isotope data indicate short-term periods of instability, particularly food insecurity, did impact individuals. Evidence of preferences for certain foodstuffs and cooking techniques documented among the elite classes were also observed among lower-status townspeople, suggesting that Anglo-Norman fashions could be adopted across the social spectrum. This study demonstrates the potential for future archaeological research to generate more nuanced understanding of the cultural impact of the Norman Conquest of England, while showcasing a method which can be used to elucidate the undocumented, everyday implications of other large-scale political events on non-elites.

NF-kappaB-mediated activation of the chemokine CCL22 by the product of the human cytomegalovirus gene UL144 escapes regulation by viral IE86.

The product of the human cytomegalovirus (HCMV) gene UL144, expressed at early times postinfection, is located in the UL/b' region of the viral genome and is related to members of the tumor necrosis factor receptor superfamily, but it does not bind tumor necrosis factor superfamily ligands. However, UL144 does activate NF-kappaB, resulting in NF-kappaB-mediated activation of the cellular chemokine CCL22. Consistent with this finding, isolates of HCMV lacking the UL/b' region show no such activation of CCL22. Recently, it has been suggested that activation of NF-kappaB is repressed by the product of the viral gene IE86: IE86 appears to block NF-kappaB binding to DNA but not nuclear translocation of NF-kappaB. Intriguingly, IE86 is detectable throughout an infection with the virus, so how UL144 is able to activate NF-kappaB in the presence of continued IE86 expression is unclear. Here we show that although IE86 does repress the UL144-mediated activation of a synthetic NF-kappaB promoter, it is unable to block UL144-mediated activation of the CCL22 promoter, and this lack of responsiveness to IE86 appears to be regulated by binding of the CREB transcription factor.

How much green does it take to be orange? Determining the cost associated with trauma center readiness.

BACKGROUND: Readiness costs are real expenses incurred by trauma centers to maintain essential infrastructure to provide emergent services on a 24/7 basis. Although the components for readiness are well described in the American College of Surgeons' Resources for Optimal Care of the Injured Patient, the cost associated with each component is not well defined. We hypothesized that meeting the requirements of the 2014 Resources for Optimal Care of the Injured Patient would result in significant costs for trauma centers. METHODS: The state trauma commission in conjunction with trauma medical directors, program managers, and financial officers of each trauma center standardized definitions for each component of trauma center readiness cost and developed a survey tool for reporting. Readiness costs were grouped into four categories: administrative/program support staff, clinical medical staff, in-house operating room, and education/outreach. To verify consistent cost reporting, a financial auditor analyzed all data. Trauma center outliers were further evaluated to validate variances. All level I/level II trauma centers (n = 16) completed the survey on 2016 data. RESULTS: Average annual readiness cost is US $10,078,506 for a level I trauma center and US $4,925,103 for level IIs. Clinical medical staff was the costliest component representing 55% of costs for level Is and 64% for level IIs. Although education/outreach is mandated, levels I and II trauma centers only spend approximately US $100,000 annually on this category (1%-2%), demonstrating a lack of resources. CONCLUSION: This study defines the cost associated with each component of readiness as defined in the Resources for Optimal Care of the Injured Patient manual. Average readiness cost for a level I trauma center is US $10,078,506 and US $4,925,103 for a level II. The significant cost of trauma center readiness highlights the need for additional trauma center funding to meet the requirements set forth by the American College of Surgeons. LEVEL OF EVIDENCE: Economic and value-based evaluations, level III.