We present novel insights into trade in amphorae-borne products over a 550-year period in Germania along the frontier of the Roman Empire, derived through probabilistic aoristic methods to study temporal changes in archaeological materials. Our data analysis reveals highly detailed differential patterns of consumption and production within the German market. We show how connections to far-flung regions such as the Eastern Mediterranean or the Iberian Peninsula wax and wane through time, and how the local German producers start to compete with these imported products. These chronological patterns provide important insight into a regional market within the larger Roman economy and provide an important case study in changing economic connections over a long period, demonstrating in a transparent and reproducible way a geographical and chronological pulsation in market activity that was otherwise unknown and undemonstrated. It is now widely accepted that many commodities moved over long distances throughout the Roman Empire (ca. 30 BC—AD 500), ranging from basic foodstuffs like grain to expensive luxuries such as ivory ,2. EfforWe demonstrate this for the case of Roman trade as evidenced by large ceramic transport vessels known as amphorae from the region of ancient Roman Germany, roughly equivalent with the parts of The Netherlands, Germany, France, and Switzerland that border the Rhine River in western Europe . This reThere are, however, many considerations when synthesizing data at this scale. Ceramic excavation, analysis and publication practices in this region reflect high heterogeneity of ceramic quantification methods, typologies used, temporal evidence and research agendas. For example, the site of Augusta Raurica  in Basel-Landschaft, Switzerland, is perhaps the most detailed chronological study available, reflecting the high-quality of excavation, study, and publication at the site –18, but This synthetic database forms a foundation for quantitative analysis of ceramic data at a geographical and chronological scale not yet otherwise achieved in Roman studies. The RAAD material allows for comprehensive analysis of the German frontier region’s consumption and, to some extent, production strategies at the regional level and allows us to move beyond site-based, anecdotal approaches that extrapolate single case studies to represent entire regions. This is achieved through the following wide range of exploratory data analysis queries. We will explore the impacts on data patterns between using production and consumption dates (respectively amphora type and site dates), and between ceramic quantification methods , Maximum Number of Individuals (MAX), and diagnostic Rim, Base, and Handles count (RBH)). We will additionally trace temporal patterns of amphorae from different regions , with different typical prime-use contents . Finally, we will explore differences between amphora data patterns at different site types , explore the exceptional case of Augustan period sites with a short occupation span, and closely examine the data pattern of the site of Augst that has a particularly rich, published material record.The regional, long-term, and replicable quantitative view on Roman trade patterns reflected through the amphorae data analysis results presented here is novel in its scope and scale. First, a synthetic view of Roman amphora consumption in one region through time has never been attempted at this scale–instead patterns are typically inferred through anecdotal use of one or several individual sites. Second, the heterogeneity of the amphora data has hindered efforts at quantification through time, especially since the individual quantification methodologies have differed so much by publication: this paper tackles the impact of this heterogeneity head-on. Third, the centuries-long patterns evident in these data provide new and important insights into the temporal fluctuations of Roman imperial commerce differentiated into regional markets. That is, the German frontier zone is used as a case study against which we can compare wider Roman imperial patterns.The Roman Amphorae Assemblage Database (RAAD), developed in conjunction with the Oxford Roman Economy Project’s (OXREP) aims to tackle Roman amphora data heterogeneity by taking a synthetic view of Roman trade ,20,21. IThe RAAD project synthesized diverse datasets into several standardized tables that accounted for information about amphora forms as well as the archaeological sites on which they were found . The “AmThe ‘Contents’ column records the most commonly carried product in each form: alum, fish sauce, fruit, olive oil, olives, wine, and unknown. While there are some hypotheses about some of the ‘unknown’ contents  to AD 450 at the settlement of Mainz, Germany (site 38). Thus, the database covers some 550 years of amphora consumption in the region. In contrast to production dates noted in the Forms table, these dates indicate the chronology of consumption. Finally, each site is noted for how its ceramic data are quantified: diagnostic rims, bases, and handles (RBH); minimum number of individuals (MNI), and maximum number of individuals (MAX) as well as how large each assemblage is. The sum of amphora frequency across all 79 sites and using these different quantification methods is 33,707 and it is important to note that some of the published amphora evidence concerns only diagnostic amphora sherds rather than a total quantification. These two datapoints are perhaps the most important details in the database, as they determine which sites are directly comparable in quantification method–while it is reasonable to compare the raw assemblages of Xanten, Germany (RAAD site 75) with Nijmegen Kops Plateau (RAAD site 50) because both use MNI, it is unreasonable to compare directly the raw assemblage from Xanten directly to Dangstetten, Germany (RAAD site 21) because it uses MAX. This issue of different quantification methods is central to our improved methodologies outlined below.The RAAD dataset is intended to act as a model for future study of other regions, and thus all of these attributes are easily replicated in other geographies. The dataset can also be extended to include other regions and standardized according to these tables. Thus, while this paper focuses on the German frontier, the data organization has a wide applicability and will facilitate future synthetic studies of amphorae.To enable our data analysis, the original RAAD database  was restsite names (see the full list in Appendix A) has been pre-processed: punctuation marks were removed, double spaces were replaced by single spaces, and all characters were lowercased. The motivation behind this strategy was to avoid inconsistency in spelling caused by manually filling out the original spreadsheets, and thus to prevent mistakes in calculations.It is worth noting that text data such as The analysis method aimed to study changes in production and consumption of amphorae over time is based on the probabilistic aoristic approach –42. ThisP(ti) of amphorae being present in a given year ti was calculated as,Fc is the frequency of an amphora item c of a particular form and Δτ = τend − τstart is the time range. The latter can be either a production date range of c (defined by the amphora type’s start and end dates) or a consumption period of an amphora (defined by site start and end dates). The summation runs over all c in case ti belongs to the interval Δτ.We focused our data analysis on identifying long-term change in three data patterns: (1) the frequency of amphorae in a given year, (2) the number of sites on which amphorae were found in a given year, and (3) the number of different amphora types present in the region in a given year. In accordance with the aoristic method, the probability c was found at a particular site and has a specific type number. Thus, in addition to the calculation described above for amphora frequency (data pattern 1), the count of unique sites at which c was present in a given ti (data pattern 2) as well as the count of unique type numbers per year (data pattern 3) were computed. The mentioned values were obtained by (i) forming a set of all unique sites/type numbers, for which ti ∈ Δτ and (ii) counting the number of elements in it.Each amphora item Much like the dataset creation, the data analysis method is intended to have a broad applicability outside this case study, and likewise can be used to expand this study into other regions of the Roman Empire where chronological uncertainty is equally problematic.The original RAAD database is openly available online . A GithuOur analysis reveals important patterns of trade in amphorae, chronological trends in the consumption of goods depending on both place of origin as well as contents. These chronological trends in turn reflect changing tastes among consumers as well as changing market connections. Both are significant for understanding trade at the local, regional, and Empire-wide level in the Roman Empire. In this section we describe the data patterns, and we interpret them in full detail in their historical and archaeological research contexts in the subsequent discussion section.Chronological trends differ, in some cases substantially, when dates are analyzed by the production chronologies of individual amphora forms (type dates) or by the chronologies of the sites on which they are consumed (site dates). Neither method necessarily fully captures the complete reality of ancient trade, but both contribute different aspects to our analyses when viewed side by side. A huge spike in amphora frequency around the turn of the era is only visible when using site dates , and cleIt must be emphasized that the type dates span a much longer period than the actual dataset, while the site dates are by definition tightly constrained within a historical reality. The 79 sites in the database fall between roughly 100 BC and AD 450, while the x-axes on these graphs range from 200 BC to AD 650. The plots of frequency generally respect the database timespan, though the type dates grow both earlier and later when plotted by site count and type count. This is again due to the broad production chronologies of some forms that are produced either earlier or later than the Roman occupation of Germany. It is clear, however, that most of the data from any of these figures falls within the correct date range, so even cutting out these temporally extraneous sections before 100 BC and after AD 450 does not change our results in any meaningful way.There are numerous ways to quantify ceramic assemblages, but this dataset is mainly composed of three different approaches: calculating a minimum number of individuals (MNI), counting the total sherds and hypothesizing a maximum number of individuals (MAX), or counting the main diagnostic sherds of rims, bases, and handles (RBH). Most modern studies will produce tables that contain all this information, plus weight and estimated vessel equivalents , but this is not always the case when material comes from curated collections or older excavations. It is worth noting that the MAX quantification method is used exclusively by Ulrike Ehmig, and thus the shape of these data is directly formed by the sites she has researched and published, i.e., Dangstetten (RAAD site 21), Mainz (RAAD site 38), and the central German region generally. Because these different methods count different things, they are not directly comparable in raw terms; thus, a site with 12 MNI is not the same as a site with 12 MAX or 12 RBH, and indeed may represent very different quantities of material. With this in mind, we split the dataset into sites by each quantification method in order to compare the influence that quantification methodology has on the data and graphed each according to each of our three methods by both dating chronologies (production and consumption).When considering the sum of frequency by production dates , we see When considering the sites per year by production dates , all thrFinally, when considering amphorae type count by production dates , we agaiThe total dataset can be broken down into two major avenues of investigation: product origin  and prodThese figures change dramatically when visualized using consumption chronologies. When looking at the count of sites using production chronologies , the IbeThe shapes of these visualizations are broadly similar when compared to those of the types of products carried within the amphorae. The frequency of contents by production chronology  shows a When visualized by consumption date, we again have substantial changes in chronological patterning. Turning to site counts per product contents , the samWhen visualized by site count per product contents using consumption chronologies , we agaiThis macro-view of amphora contents in the German region also allows for a more detailed view of specific products to better understand how things like wine and oil were consumed. Where wine was a dominant and sought-after commodity from many parts of the Roman Empire through time, olive oil shows a very different pattern. All of these investigations have focused on the German frontier at the macro-scale, presenting the full synthesis of data from 79 sites within the region. It is important to also understand how individual sites fit into this regional pattern, and so we also compare our methods of analysis with the well-studied and highly-resolved dataset from Augst, Switzerland (RAAD site 9) –18. The Our own investigation of the material from Augst was able to produce similar graphs for the Augst assemblages using the type dates  based on both contents and area of production origin but using the probabilistic aoristic method to calculate the sum of frequency. We were not able to include contextual or phased data within our database, and so here our methods diverge from the original publication. When viewed by origin , we see The RAAD database includes information on the types of site present in the dataset (21). Site types are categorized as either military sites (n = 41), civilian settlements (n = 30), villas (n = 6), or oppida (n = 2). The oppida site count is too low to be statistically relevant, so we have removed these from this case study analysis. Some of these sites contain areas that may fit into multiple categories, as they are not all mutually exclusive. We have paid close attention to where on the sites the amphora came from, however, so we make distinctions between the military site of Hofheim Steinkastell (RAAD site 29) and Hofheim Vicus (RAAD site 30). Augst presents a slightly different story, where amphorae from the civilian settlement of Augusta Raurica was published together with the amphorae from the late Roman fortification (which included civilians) of Kaiseraugst –18. To aThe dramatic spike in frequency evident in Augustan period sites is worth further exploration. We separated out the ten assemblages that date specifically to the period of Augustan conquest in Germany, roughly 19 BC—AD 9: Anreppen (RAAD site 5), Dangstetten (RAAD site 21), Höchst (RAAD site 28), Neuss Camps 1–4 (RAAD sites 41–44), Oberaden (RAAD site 51), Rödgen (RAAD site 58), and Waldgirmes (RAAD site 75), and compared their assemblages between the two different dating methods used in this paper. Our analytical methods have thus revealed a series of chronological patterns in the consumption of amphorae-borne products along the German frontier over the course of more than five centuries. While there is no single pattern consistent between these analytical methods and quantifications, there are enough similarities to draw new and important conclusions about the economic conditions of the Roman world that led to the creation of this archaeological record, as well as modern archaeological conditions that led to its publication.As our analysis is directly dependent on published data, the choices that went into those publications leading to heterogeneity of the dataset directly affect our outcomes. It is thus worth highlighting several places where modern scholarly practice has clearly influenced the shape of the amphora data patterns we have explored in this paper. Four cases are especially obvious: the dates of sites that have been excavated, the resolution of the ceramic data published after excavation, the date ranges created by scholars to bracket production chronologies of individual forms, and finally, quantification methods.The database contains a large number of sites that are discretely dated to the Augustan period, especially military sites linked to the earliest military campaigns in the region see . The subThe resolution of data published from these sites also has a strong influence on the patterns shown here. First, it is clear that recent publications such as Xanten (RAAD site 79) and Nijmegen Kops Plateau (RAAD site 50) ,49 preseOur methods and analyses outlined in this paper highlight the influence of this past research, especially evident with an assemblage such as Nijmegen Kops Plateau (RAAD site 50) where researchers identified and published 146 individual forms . Fig 12aIt must be remembered that all the dates used here, whether for amphorae forms, sites chronologies, or historical events are, to some extent, artificial brackets that have been created and reified by archaeological and historical research. The Augustan period sites are one part of this, but so too is the prominent end date of AD 260, the traditional date of the abandonment of the outer German limes as well as all Roman territory across the Rhine in Baden-Wurttemberg. Amphorae production chronologies are often given in 25-, 50-, or 100-year increments, and necessarily are estimates. The effect of this chronological blocking is perhaps most clearly seen in the calculation of RAAD type number count per year by type date , as rougFinally, when considering the quantification method, we can see the influence of particular researchers on the representation of data. Ulrike Ehmig’s extensive and invaluable work on amphorae in Germany is the most obvious case ,28,50,51We used two methods of establishing chronologies within our data: the first utilizing the ‘production chronologies’ of individual amphorae forms based on when the earliest and latest versions of forms are known, the second using the ‘consumption chronologies’ of individual sites where the start and end dates of occupation are known. We used both methods to account for some of the characterful ambiguity present in the many assemblages that are not published with phasing, as well as to fill in holes in knowledge such as when specific amphorae forms were produced and consumed. The strong contrast revealed by our results between the data patterns when using either typological or site dates should clearly caution against the exclusive use of typological dates of amphorae for interpreting when the foodstuffs contained in them were consumed. We demonstrate here that amphora production and consumption behaviour result in quantitatively different temporal patterning in published material culture, and we therefore treat them as separate but intertwined phenomena.Production chronologies have the potential to cover very large date ranges that sometimes extend outside the period of Roman rule in the German area. We pointed out above that Italian and Eastern Mediterranean wine amphorae especially illustrate this point , and theThe most problematic aspect of either method are amphorae forms that have a long production chronology but only a very specific chronology of consumption in Germany. Italian wine emphasizes this point , as alreOn the other hand, our methods do work well when amphorae production chronologies fall entirely within the period of Roman rule in the German region. This is especially evident in both Gallic wine and Spanish olive oil  arrived in very different patterns through time . Wine haWine and olive oil dominate assemblages in the region, and the other products are only ever ancillary to these two main imported staples. It is highly possible that Mediterranean fish sauce was not well received in local cuisines, but it is also probable that more localized fishing industries and subsequent production of fish sauces took over this market, possibly in the face of declining Mediterranean production . These lWine was imported to Germany from all over the Empire throughout the Roman period and, while most of it arrived in the first two and a half centuries AD, there was some sustained importation later, especially from the Eastern Mediterranean . Gallic similis (RAAD form 107), contained beer  for an Hispanic case study and [The rich archaeological record of the German region, as well its neighboring territories of northern Europe, however, provide ample material for such studies, and these regions should be included in economic history more often. Our focus on a frontier region has allowed for careful analysis of economic patterns on the edge of the Roman world, in a social context where economic activity is most often conceived by scholars as a command economy that was State-organized and isolated from the wealthier Mediterranean ,14. We h69tudy and  for compThe result of these innovations in synthesis and analysis is a comprehensive view of the market for amphorae-borne food commodities along the German frontier that reveals extensive patterns in long-distance trade connections with other regions of the Roman Empire, as well as changing patterns of taste and preference amongst local consumers. We reveal chronological and geographical pulsations of market connections that were highly heterogeneous and dynamic. These connections are important not only for understanding patterns of consumption in Germany, but also for understanding the regional productive economies of the Roman world–producers knew what regions provided demand, and they matched this demand with output.


Metal artifact reduction (MAR) algorithms are used with cone beam computed tomography (CBCT) during augmented reality surgical navigation for minimally invasive pedicle screw instrumentation. The aim of this study was to assess intra- and inter-observer reliability of pedicle screw placement and to compare the perception of baseline image quality (NoMAR) with optimized image quality (MAR). CBCT images of 24 patients operated on for degenerative spondylolisthesis using minimally invasive lumbar fusion were analyzed retrospectively. Images were treated using NoMAR and MAR by an engineer, thus creating 48 randomized files, which were then independently analyzed by 3 spine surgeons and 3 radiologists. The Gertzbein and Robins classification was used for screw accuracy rating, and an image quality scale rated the clarity of pedicle screw and bony landmark depiction. Intra-class correlation coefficients (ICC) were calculated. NoMAR and MAR led to similarly good intra-observer (ICC > 0.6) and excellent inter-observer (ICC > 0.8) assessment reliability of pedicle screw placement accuracy. The image quality scale showed more variability in individual image perception between spine surgeons and radiologists (ICC range 0.51–0.91). This study indicates that intraoperative screw positioning can be reliably assessed on CBCT for augmented reality surgical navigation when using optimized image quality. Subjective image quality was rated slightly superior for MAR compared to NoMAR. Over the course of the last two decades, the proportion of thoracolumbar Minimally Invasive Surgery (MIS) has increased for degenerative, traumatic, and metastatic indications of instrumented fusion techniques. The main benefits of MIS are reduction of blood loss, shorter hospital stay, improved short-term clinical outcomes, and cost-effectiveness ,2. PercuNevertheless, accurate intraoperative judgment can be challenging as metal artifacts, such as blooming around the screw shaft and streaks at the screw tip, may prevent the proper assessment of screw placement ,9. MetalWe hypothesized that the intraoperative CBCT imaging gathered from MIS-TLIF procedures using ARSN would allow accurate and reproducible pedicle screw placement assessment and that the perception of image quality might improve between NoMAR and MAR.The primary aim of this study was to assess intra- and inter-observer reliability of pedicle screw placement using intraoperative CBCT between spine surgeons and interventional radiologists with expertise in spinal procedures. The secondary aim was to compare the perception of image quality between NoMAR and MAR.Institutional review board approval (CE-2020-90) was obtained for this retrospective study evaluating demographic and intraoperative radiologic data. Twenty-four consecutive patients with degenerative spondylolisthesis who underwent single-level decompression and MIS-TLIF using ARSN fusion at L3-L4 or L4-L5 between January and December 2019 were included. Patients with previous surgery (except microdiscectomy) and implants in the lumbar spine or in the immediate neighborhood, as well as patients requiring more than single-level MIS fusion, were excluded.The surgeries were performed in a hybrid operating room. The patient was positioned prone on a carbon table . Percutaneous pedicle screw placement was performed using ARSN  equipped with a robotic C-arm comprising a flat panel detector for 2D and 3D X-ray imaging, and a video camera system for tracking adhesive fiducials on the skin in the surgical field ,6,7,8. AThe CBCT screw control images were retrospectively extracted in Digital Imaging and Communications in Medicine (DICOM) format and anonymized for further evaluation. The images of 24 patients were treated using NoMAR and MAR by an independent engineer, thus creating 48 randomized files. The images were then independently analyzed by 3 spine surgeons and 3 radiologists, with expertise in percutaneous interventional spinal applications, using the RadiAnt DICOM Viewer 2021.2.2 software. Each observer analyzed the images on multiplanar reconstructions twice, at an interval of one month, in random order.Grade 0: screw within the pedicle without cortical breach;Grade 1: 0–2 mm breach, minor perforation, including cortical encroachment;Grade 2: 2–4 mm breach, moderate breach;Grade 3: >4 mm breach, severe displacement, which was not found in this cohort.Screw positioning accuracy within the pedicle was rated using the Gertzbein and Robins classification :Grade 0:The screw diameter of 6.5 mm was used as a scale, which enabled the measurement of cortical breaches on axial and coronal views of each pedicle, as demonstrated in Grade 0: mild artifact, screw thread clearly visible, cortical bone clearly visible, bone soft tissue interface clearly visible, clear interpretation possible;Grade 1: moderate artifact, screw thread visible, cortical bone with unclear portions, bone soft tissue interface distinguishable, interpretation possible;Grade 2: strong artifact, screw thread unclear, cortical bone contours unclear, bone soft tissue not distinguishable, interpretation difficult.The perception of image clarity around the screw contours was rated using an image quality scale based on the following criteria :Grade 0:Statistical analysis was performed using R Software 3.6.0 . The reliability of rating screw positioning according to Gertzbein and Robins was compared between the observers, and for each observer, between both assessments. Likewise, the image quality scale was tested using Intraclass Correlation Coefficients (ICC) for categorical data. Observer agreement was then compared between NoMAR and MAR. The values of intraclass correlation coefficients were rated according to Landis and Koch : correla2. A total of 96 percutaneous pedicle screws, 4 per patient, were analyzed twice by 6 observers, which led to 1152 ratings for NoMAR and MAR imaging modalities, respectively.Twenty-four consecutive patients with degenerative spondylolisthesis were enrolled. L4-L5 was instrumented in 17 patients (70.8%) and L3-L4 in 6 patients (19.2%). There were 18 females and 6 males with an average age of 68.2 ± 9.1 years and Body Mass Index (BMI) of 25.9 ± 4.2 kg/mGrade 0, 20.2% versus 22.9% for Grade 1, and 5.9% versus 7.2% for Grade 2. The distribution of 1152 screw accuracy ratings comparing NoMAR versus MAR, respectively, was: 73.9% versus 69.9% for Grade 0, 56.5% versus 41.7% for Grade 1, and 22.3% versus 14.8% for Grade 2.The distribution of 1152 image quality ratings comparing NoMAR versus MAR, respectively, was: 21.2% versus 43.5% for The assessment of pedicle screw accuracy represents a major concern for spine surgeons, as inaccurate placement may result in injury to various anatomical structures surrounding the pedicle, including the nerve roots, the spinal cord, and the blood vessels. Intraoperative assessment is suitable during MIS, as the procedure usually relies on fluoroscopy ,20,21 orTwo main principles can be applied for improving intraoperative CBCT imaging of pedicle screws: optimized C-arm trajectories and MAR. Optimized C-arm orbits may be used to avoid collinearity between the X-ray beam and the direction of the screws to minimize metal artifacts in the 3D images. Klingler et al.  comparedSoftware-based MAR algorithms may be used to mitigate streak and blooming artifacts, typically by iterative reconstructions. Privalov et al.  acquiredThe present pilot study has limitations as it compares the intraoperative CBCT data collected retrospectively during a limited consecutive series of 24 patients operated on using ARSN. The imaging data focused on the operated and adjacent lumbar segments. The surrounding abdominal organs, paravertebral muscles, subcutaneous fat thickness, and bone mineral density may play a role, and the relationship between the patient’s BMI and image quality could not be evaluated retrospectively. An additional validation study might integrate these factors, which could influence image quality, aiming for an optimal patient-specific setting for image acquisition and evaluation.Intraoperative screw positioning can be reliably assessed on CBCT for ANSR when using optimized image quality, with good intra- and excellent inter-observer correlation for surgeons and radiologists. Subjective image quality was rated slightly superior for MAR compared to NoMAR.


Hospitals are required to have antibiotic stewardship programs (ASPs), but there are few models for implementing ASPs without the support of an infectious disease (ID) specialist, defined as an ID physician and/or ID pharmacist.In this study, we sought to understand ASP implementation at hospitals that lack on-site ID support within the Veterans’ Health Administration (VHA).Using a mandatory VHA survey, we identified acute-care hospitals that lacked an on-site ID specialist. We conducted semistructured interviews with personnel involved in ASP activities.The study was conducted across 7 VHA hospitals.In total, 42 hospital personnel were enrolled in the study.The primary responsibility for ASPs fell on the pharmacist champions, who were typically assigned multiple other non-ASP responsibilities. The pharmacist champions were more successful at gaining buy-in when they had established rapport with clinicians, but at some sites, the use of contract physicians and frequent staff turnover were potential barriers. Some sites felt that having access to an off-site ID specialist was important for overcoming institutional barriers and improving the acceptance of their stewardship recommendations. In general, stewardship champions struggled to mobilize institutional resources, which made it difficult to advance their programmatic goals.In this study of 7 hospitals without on-site ID support, we found that ASPs are largely a pharmacy-driven process. Remote ID support, if available, was seen as helpful for implementing stewardship interventions. These findings may inform the future implementation of ASPs in settings lacking local ID expertise. Although a variety of resources are needed to implement hospital-based ASPs, the support of infectious disease (ID) pharmacists and physicians seems to be particularly important. The involvement of an on-site ID pharmacist and/or ID physician was a common element in several randomized-controlled trials that demonstrated the effectiveness of antibiotic stewardship. In addition, a cluster-randomized trial found that only community hospitals with the highest level of off-site ID support were able to achieve reductions in total antibiotic use. Finally, observational data from the Veterans’ Health Administration (VHA) demonstrated that patients at hospitals with on-site ID support received antibiotics in a way more consistent with stewardship principles than patients at hospitals without such support.Antibiotic stewardship programs (ASPs) improve antibiotic prescribing and, in turn, reduce antibiotic resistance. In a 2016 survey of 244 US hospitals, the lack of ID or stewardship expertise was a common barrier to implementing prospective audit-and-feedback, which is one of the core stewardship strategies.Despite its benefits, many hospitals do not have access to ID expertise. Prior surveys have found that 41%–50% of US community hospitals lack an ID physician, whereas 93% lack an ID-trained pharmacist.It is unclear how hospitals without local ID support are implementing ASPs and whether these approaches are effective. In this VHA study, we sought to understand how antibiotic stewardship programs had been developed and maintained at VHA hospitals that lack on-site ID support.The Institutional Review Board (IRB) of the University of Iowa and Iowa City Veterans’ Health Care System approved this study. A waiver for written informed consent was granted. A key part of this directive was that each hospital was required to identify an antibiotic stewardship pharmacist and provider champion. Champions were expected to co-lead the ASP. In facilities without an ID physician, the provider champion could be a hospitalist, primary care provider, or advanced practice provider.This study was conducted within the national VHA system, which includes 130 acute-care hospitals. In 2014, the VHA mandated that all of its facilities develop and maintain an ASP. We have previously described the survey questions that informed whether we deemed a hospital to have an ID specialist.For this study, hospitals were eligible for inclusion if they had acute-care beds and lacked an on-site ID specialist. An ID specialist was defined as a pharmacist or physician who had completed a formal postgraduate residency or fellowship training program in ID. We identified eligible hospitals using data from a mandatory antibiotic stewardship survey, which had been administered by the VHA between December 30, 2015, and January 15, 2016, and was completed by a hospital staff member who was knowledgeable about local antibiotic stewardship activities.Once an eligible site was identified from the survey data, we used internal VHA resources to identify the designated antibiotic stewardship pharmacist and provider champion at each site. We contacted these champions by e-mail during March–April 2019 to confirm that the hospital still lacked an on-site ID specialist and to gauge their interest in participating in the study.Of the 18 sites that met our inclusion criteria, 5 had hired an ID specialist since the survey was conducted and no longer qualified for the study. An additional 6 sites refused to participate and 7 sites were eligible and willing to participate.In this study, 4 site visits were conducted in-person and, due to the COVID-19 pandemic, 2 visits were completed virtually. At the seventh site, only the ASP pharmacist champion agreed to be interviewed, so we chose to conduct a telephone interview rather than travel to the site. Data were collected between July 2019 and April 2020.During the on-site and virtual visits, we attempted to interview all members of the ASP team, inpatient physicians who frequently interacted with the ASP team, and members of the hospital’s leadership. A medical anthropologist (K.S.S.) and an infectious disease specialist with qualitative training (D.J.L.) conducted all interviews.1–4 online) with questions focused on the feasibility, appropriateness, and acceptability of implementing different antibiotic stewardship strategies. Interviews were recorded, transcribed, and reviewed for accuracy, except at the seventh site, where only notes were taken according to the lone participant’s request.Interviewers followed a semistructured interview guide  read 8 transcripts and shared their general impressions. Next, the analytic team independently coded transcripts, coding every third transcript in common. The team met weekly to review coded transcripts, discuss discrepancies, and reach consensus. After coding was complete, the team reviewed how each code intersected with the code for barriers or the code for facilitators. After reviewing the groups of coded segments separately, the 2 analysts shared their findings and identified common themes.We uploaded transcripts into MAXQDA, a management software program for qualitative data . We analyzed the data using thematic analysis; the code book was developed based on the interview guide (deductive) as well as interview responses and field notes (inductive).Table All 7 sites had a designated pharmacist champion, and 6 (86%) sites had named an ASP provider champion. At least 1 ASP pharmacist champion per site had completed an antibiotic stewardship certification course . At 6 sites (86%), the primary responsibility of the ASP pharmacist champions was to provide clinical pharmacy services to a specific area of the hospital, such as acute care (3 sites), the community living center  or the emergency department . The primary role of the provider champions was as follows: hospitalist physician (2 sites), hospital administrator (2 sites), hospitalist advanced practice provider (1 site), and geriatrician (1 site).All sites prepared annual antibiograms and used education and some form of prospective audit-and-feedback to improve inpatient antibiotic-prescribing. Five sites (71%) reported having inpatient antibiotic-prescribing orders sets. Several sites were also working to improve outpatient antibiotic use, largely through education and retrospective audit and review.Although sites reported using similar interventions to promote antibiotic stewardship, the shape of those interventions varied. The following 4 themes illustrate the ways in which hospitals without on-site ID support had implemented ASPs and the barriers they encountered Table .At 6 sites, being the ASP pharmacist champion was an added responsibility to their primary pharmacist role. Only 1 site had an ASP pharmacist champion whose only responsibility was antibiotic stewardship.Most of the ASP pharmacist champions spoke positively about filling their many roles. One ASP pharmacist champion explained: “One of the benefits of working at a small site is you wear lots of hats… It’s a good professional challenge for me” (site 7). Several others expressed having a passion for stewardship, sometimes even remembering the exact moment they agreed to take on the job. According to one champion, “Not many pharmacists like bugs and drugs, [but] for some reason that’s always been one of my passions” (site 3).Despite having several other responsibilities, the ASP pharmacist champion was consistently recognized as the daily leader of the program. Only a few sites had an engaged on-site ASP provider champion. At one site, the ASP pharmacist champion had been in place since the program’s inception but there had been 3 different provider champions during that same time. She complained, “Stewardship is seen as a pharmacy thing and not an everybody thing” (site 5).ASP pharmacist champions faced several challenges when trying to provide antibiotic recommendations to clinicians. Pharmacists recognized the importance of bedside patient care to informing antibiotic decisions, and they acknowledged the legal responsibility of the clinicians to manage patient care. Additionally, one ASP pharmacist champion said, “I have to build rapport with them to get them to listen to me but I have no recourse if they don’t…. I make a lot of suggestions and hope a couple of them stick” (site 4).To help develop rapport with clinicians, pharmacists spoke of the importance of having face-to-face conversations, providing evidence to support stewardship recommendations, and being persistent. The importance of taking a collaborative approach to stewardship was also well recognized. As one ASP pharmacist champion said, “I want it to be a positive approach, and I don’t want it to be condescending in any way to anybody that I talk to about it [antibiotic stewardship]” (site 6).Pharmacists who worked with clinicians on a range of clinical issues felt like these working relationships helped to build trust and specifically improved the physicians’ acceptance of their antibiotic recommendations. One ASP pharmacist champion whose primary role was in the ED explained, “ED physicians are very open to help especially when it comes to medications because they have so much going on at the same time. If I can say, ‘Hey, yeah, I can put in this medication,’ or I just tell them what to use, what dose, they really just love that support. So I think just kind of being down there [helps]” (site 3).In some situations, working at a smaller hospital facilitated good rapport, especially when a consistent group of physicians was staffing the facility. According to one ASP pharmacist champion, “That’s the nice thing with it being a smaller facility. I’m not talking to 20 physicians … I’m talking really to 5 … as far as the hospitalists and CLC go, I’m talking to 5 providers for the most part” (site 4).ASP pharmacist champions struggled to establish rapport with clinicians when a hospital relied heavily on contract physicians. One ASP pharmacist champion explained, “In the last year, we’ve had so many locums that it’s hard to track anything they do. There’s so many, a different person every week. We just got back in the last month to our standard [hospitalists], who have both been the hospitalists here for quite some time … and so it’s been nice to have a little bit of normalcy again” (site 6). Other sites struggled with the frequent turnover of outpatient providers and hospital leadership.At the sites we visited, a common sentiment was that the hospital was too small to justify an on-site ID specialist. All sites had access to ID physician electronic consultation for challenging cases, and 4 sites had access to an off-site ID specialist who assisted with at least some local ASP activities . These remote ID specialists helped give credibility to both the ASP pharmacists’ recommendations and to any stewardship initiatives the ASP team was trying to move forward. According to the ASP pharmacist champion at site 2, who was discussing prospective audit and feedback, “I’ve found that having the support of an infectious diseases physician really makes a huge difference in terms of acceptance of the recommendations, particularly if it’s to stop antibiotic therapy. So maximizing access to that, I think, is something that we need.” An off-site ID specialist could also help overcome institutional barriers to putting new protocols or order sets into place. At site 5, the ASP pharmacist champion would “love to have ID physician support” for stewardship but acknowledged that, given the size of the hospital, it would be “hard to justify.”Clostridioides difficile infection.The stewardship champions at 2 other sites (sites 4 and 6) did not perceive lack of ID support as a barrier to stewardship efforts. At site 4, the ASP pharmacist champion found it less helpful to consult with ID pharmacists at other sites as his facility was “very different” with a “completely different patient demographic.” However, a hospitalist at site 4 felt that ID consultation would have facilitated the application of stewardship principles: “At least from my standpoint, I can say ‘I consulted with infectious disease’ and then pass the buck there, but I can’t say, ‘Oh I consulted with pharmacy’ and then be totally left off the hook.” At site 6, the ASP provider and pharmacist champions had been working together for years. As the lead hospitalist, the provider champion was able to garner institutional buy-in for the ASP by demonstrating the overlap between excessive use of parenteral antibiotics and the metrics the hospital leadership cared about, such as length of stay and For ASPs that were using remote ID specialists, the manner in which this expertise was accessed varied. One hospital had an informal arrangement with an off-site ID VHA physician to conduct prospective audit and feedback with the on-site ASP pharmacist champion twice per week (site 2). At the time of our visit, this arrangement was becoming more formalized due to the ID physician’s need to capture her workload credit. Two other hospitals sought support as needed from either an ID-trained physician or ID pharmacist at another VHA site within their VISN (Veterans’ Integrated Service Network). At these 2 sites, ID support was sought either to provide feedback on current antimicrobial therapy in specific patients or to provide input on new stewardship protocols that were under development. Finally, at one hospital (site 1) an off-site ID physician received 0.2 full-time employment equivalent (FTEE) to provide remote ID support to the hospital’s physicians, infection control department, and ASP. This off-site ID physician clearly added value, as one hospitalist explained: “I think that the [remote ID physician’s] work with the lab has really improved the confidence in the information that’s received.” At the time of our visit, the ID physician had recently retired, and there was nobody to fill this void. The newly appointed ASP provider champion, who was a hospitalist, acknowledged that his lack of ID training made it difficult for him to audit and provide feedback on the antibiotic prescribing of his colleagues: “Since I don’t have any really additional training compared to my hospitalist colleagues, I try to stay out of their cases and so it’s not really appropriate for me to be trying to intervene on their management.”Most champions spoke of the importance and the difficulty of gaining institutional buy-in for their ASP. Hospital personnel were not always receptive to stewardship initiatives, and a common sentiment was that the ASP existed only to meet regulatory requirements . It was challenging for the ASP champions to get further guidance on how their efforts could assist the facility. In the words of one ASP pharmacist champion, “I would say our facility expects us to pass our new requirements. To have Joint Commission walk in and give us a thumbs up and walk out. That’s what our leadership wants” (site 7). An ASP provider champion at another site echoed this frustration, “I’ve stood in front of the medical staff multiple times and asked for their input on what they’d like to see from the [stewardship] program and what do they think about this and that, and it’s like crickets. Nobody says a word” (site 2).Lack of funding and lack of information technology support further hampered ASP efforts. However, funding a stewardship position was not a silver bullet, as shown by the one hospital that had assigned a full FTE to the ASP pharmacy champion: “I’ve got my FTE so I feel like a lot of the facility just thinks I’m just going to do it, but you know it’s hard for me to actually enact things…especially when we’ve had some turnover… I’ve had good advocates and then they leave” (site 4).Some ASPs reported success engaging leadership to overcome specific barriers. For example, at one hospital, there was a contract physician who wanted to frequently prescribe ceftaroline. When the ASP pharmacist champion went to the chief of medicine, she was able to gain support for changing this specific physician’s practice (site 5). At another hospital, the chief of staff authorized a peer review to be performed on a specific hospitalist with consistent guideline-discordant antibiotic prescribing; once this was done, the hospitalist agreed to change his practice (site 3).In this qualitative study of 7 hospitals without local ID support, we found that ASPs are largely an underfunded pharmacy-driven process. For some programs, having access to an off-site ID specialist was perceived to improve implementation of stewardship interventions, but other ASPs did not feel that the lack of this expertise was a barrier to their efforts. Furthermore, like other studies, we found that ASP teams found that collaborative, nonjudgmental approaches to stewardship were more effective and better received by clinicians. An organizational culture in which the full clinical team collaborates and is supportive of stewardship is also essential.In many ways, our findings at hospitals without on-site ID support are in line with prior qualitative work on ASPs at hospitals with ID support. At least 1 other US study of 46 hospitals reported that pharmacists were generally the leaders of day-to-day stewardship activities. Also, some hospitals valued regular access to outside ID expertise for ASP support. As shown in our study, hospitals can access remote ID expertise through a variety of means, including part-time contracts, their health system network, or telehealth. Although the Centers for Disease Control and Prevention acknowledges a benefit to having ID specialists involved in ASPs, they also recognize that non-ID personnel can be effective leaders of stewardship efforts at smaller hospitals. A few reports support this recommendation.Our qualitative study is exceptional in that it only included hospitals without local ID support. As described elsewhere, we found that personnel at these small, often rural, hospitals typically fulfill multiple different roles and struggle with the high turnover of clinical staff. Ultimately, these hospitals will need to tailor their antibiotic stewardship processes according to their local needs, resources and personnel.The findings from our work have broader implications, as a large proportion of US hospitals lack an on-site ID specialist but, based on regulatory requirements, are still expected to have an ASP. These hospitals, which tend to be small in size and rural in location, have been shown to have antibiotic use that is similar, if not higher, than hospitals with ID support.Our study has a several limitations. First, we only visited sites that were willing to participate in our study, and our findings may therefore have been biased toward hospitals that had active ASPs willing to share their success or those who were especially struggling and viewed participation as a possible avenue for help. Second, it is unknown whether the experience of these VHA hospitals is generalizable to nonintegrated healthcare systems.In conclusion, our study has provided a unique perspective on ASP implementation at 7 hospitals that lacked local ID support. Although many sites valued remote ID support for implementing stewardship processes, other sites did not feel that this expertise was necessary. Given the ongoing need to improve antibiotic use and expand the implementation of ASPs across the spectrum of healthcare, our findings could inform future work on ASP implementation in these settings.


Vaccinium macrocarpon Ait. buds explored the presence of an ice barrier at the bud base in September, January, and May. Light and confocal microscopy were used to evaluate the ice barrier region anatomy and cell wall composition related to their freezing tolerance. Buds had a temporal ice barrier at the bud base in September and January, although buds were only freezing tolerant in January. Lack of functionality of vascular tissues may contribute to the impedance of ice propagation. Pith tissue at the bud base had comparatively high levels of de-methyl-esterified homogalacturonan (HG), which may also block ice propagation. By May, the ice barrier was absent, xylogenesis had resumed, and de-methyl-esterified HG reached its lowest levels, translating into a loss of freezing tolerance. The structural components of the barrier had a constitutive nature, resulting in an asynchronous development of freezing tolerance between anatomical and metabolic adaptations.Bud freezing survival strategies have in common the presence of an ice barrier that impedes the propagation of lethally damaging ice from the stem into the internal structures of buds. Despite ice barriers’ essential role in buds freezing stress survival, the nature of ice barriers in woody plants is not well understood. High-definition thermal recordings of  Freezing temperatures are one most critical factors limiting woody plant geographical distribution. As buds sustain the development of new organs for the next growing season, their freezing survival mechanisms play a key role determining the low temperature threshold that each species can withstand . The curWoody plant species have developed multiple strategies for the survival of buds during freezing temperatures, namely deep supercooling and freeze-induced dehydration (extraorgan freezing). While deep supercooling and freeze dehydration have some mechanistic differences, they do have in common the presence of a barrier to the propagation of ice located at the base of the bud . This baPrunus spp. buds, cells near the primordium procambium do not complete their differentiation into vessel elements in the fall, thus preventing ice propagation to the inner bud structures, further allowing the sequestration of supercooled water inside the bud  were selected for all evaluations.Terminal shoots of Controlled freezing tests (CFT) were performed on the same days as sampling, based on the methodology described by Shoot preparation consisted of a rinse with tap water, followed by cutting underwater to 8 cm, and blotting dry with paper towels. Groups of five shoots, wrapped at their collective base with a small piece of moist paper towel, were placed in 50 ml capped plastic centrifuge tubes. Three replicate tubes were used for each test temperature. A set of three replicate tubes served as an unfrozen control and were kept on ice. The freezing chamber temperature program for each CFT started with a thermal equilibrium step at 1°C, followed by a temperature decrease to −1°C at a rate of 1°C/h. After 30 min at −1°C, the racks of tubes were firmly tapped to elicit ice nucleation, after which the temperature was held at −1°C for an additional hour. Afterward, three different freezing rates were used: 1, 2, and 4°C/h, until reaching −6°C, −12°C, and the lowest evaluated temperatures, respectively. For each CFT, nine test temperatures were used, spanning between 0 and −24°C in September 2018, 0 to −50°C in January 2019, and from 0 to −26°C in May 2019. Once each set of tubes was removed from the freezing chamber, tubes were kept on ice for 12 h, then transferred to a refrigerator at 4°C, and maintained in the dark for 3 days to allow any potential injury recovery. Next, tubes were kept in low light at room temperature for 24 h to facilitate damage expression of tissue browning.All 150 shoots from each CFT were assessed for freezing damage. Damage evaluation of dissected buds was performed according to the methodology described by From each shoot collection sampling date, four buds were randomly selected for histological and cytological evaluations. The fixation, dehydration, embedding, and cutting of the samples were performed as described by Section staining was performed according to the protocol developed by 2 for xylem and 15,000 μm2 for pith were anatomically characterized in each zone for each of the four repetitions and sampling date. Xylem conduits were independently evaluated according to both types present in the V. macrocarpon stem, tracheids and vessel elements (d) was calculated from the lumen area. The mean was calculated as: ∑d5/∑d4 . Samples were stored at 3°C in darkness upon receipt in North Carolina.via a USB connection, and videos were recorded using FLIR “Research IR” software . Shoots were stripped of most leaves to expose the terminal bud, while the base of the shoot was inserted into a moist soil mix [Farfard 4P soil mix ]. This soil spontaneously freezes between −0.5 and −2°C, so no inoculum was added to induce ice nucleation. Samples were placed against a black velvet cloth to provide a consistent background and contrast with the plant tissue. Freezing temperatures began at 0°C and ramped to −10°C at 1°C/h. For high-definition thermal recordings, a digital infrared camera FLIR SC8303  with a close-up lens 3–5 μm, 1X microscope f/4HD, and with <25 mK thermal sensitivity was used, following the same protocol as previously described.For thermal videography, ice nucleation patterns were monitored with a digital infrared camera FLIR T620  with a 45° lens with 0.05°C thermal sensitivity. For each sampling date, three videos were recorded using eight shoots each. The camera was placed inside a freezer and connected to a desktop computer p ≤ 0.05). When ANOVA assumptions were not satisfied, the non-parametric Kruskal-Wallis and subsequent Dunn test were performed. All statistical analyses were performed using R software .Results were compared within each evaluated zone and compared across sampling dates. Differences in mean values were tested through one-way ANOVA and Tukey test  and bud base (tracheids) did not reveal any seasonal differences for the hydraulic mean diameter . The aveThe average inner diameter of pith cells in the stem zone increased significantly among sampling dates, from 21.7 μm in September to 26.4 μm in January and was 24.1 μm in May; however no significant variations were observed in the bud base zone . Pith ce2 in January to 0.0018 mm2 in May samples  and winter (January), but not in spring (May; via IR thermography in intact shoots (V. macrocarpon terminal buds undergo freeze dehydration due to the existence of an ice barrier when exposed to low temperatures (V. macrocarpon terminal buds. These studies are consistent with our findings of a significant delay in ice propagation from the stem into the bud (The progression of freezing in ing May; . This det shoots  and endot shoots . Barriert shoots . A previeratures . Workmas the bud .To understand the nature of the ice barrier, we evaluated variations of methyl- and de-methyl-esterified HG in the pith and xylem of the bud base and the stem immediately below the bud base . The basBecause vascular tissues are the initial pathway for ice propagation  an inter2+ crosslinking to HG when present in its de-methyl-esterified form Demethylated pectins in the cell wall would limit water penetration, and could act as another type of barrier for ice propagation . Pith ceMalus domestica Borkh. the bark tissue around the buds reached lower osmotic potentials than the tissues inside the bud during midwinter. In order to avoid water migration from the meristematic tissues inside the bud toward the bark and stem, the bud cells increase their cell wall elasticity to adjust their water potential (V. macrocarpon cells, this idea would be in concordance with observed increases in plasma membrane unsaturated fatty acids during acclimation, resulting in increased membrane fluidity (We also found a decrease in the cell wall thickness of the pith cells at the base of the bud for the January sampling date when buds were fully acclimated to cold temperatures. Previous studies have suggested that an increase in cell wall thickness would increase rigidity, thus avoiding cell collapse under dehydrative strain resulting from prolonged exposure to freezing temperatures . Howeverotential . At the otential . The rolotential  and deseotential . In the fluidity .In May, when ice progressed uninterrupted into the bud, indicative of the absence of the barrier, there was also an increase in the number of tracheids by 66% . The losV. macrocarpon terminal buds apparently results from both anatomical and metabolic adaptations, although these do not necessarily occur concurrently (The development of cold hardiness in urrently . Adaptaturrently , raffinourrently , as wellurrently . These aurrently . AdaptatA potential explanation of the difference in the timing of these two acclimation factors could be associated with differences in the temperature thresholds that trigger acclimation and deacclimation patterns and their energetic costs. The anatomical component of the ice barrier, present since bud set, is of a constitutive nature, rather than an environmental response triggered by cold temperature, such as most cold acclimation responses . On the V. macrocarpon remains unclear. Different tissues can respond differentially to deacclimation temperatures (In the case of deacclimation, it is possible that the development of new xylem drives the fast rate of the loss of freezing tolerance seen in this species . The reaeratures , such thV. macrocarpon, where its deacclimation timing is tied to other growth stages, such as bud break and bloom. Non-precocious species, such as V. macrocarpon, display new leaf formation before reproductive structures during spring, allocating fewer resources to the formation of reproductive structures within buds during the year before, as compared to precocious species, which bloom before displaying leaf growth (It is also possible that the timing of xylogenesis is linked to the general phenology of f growth . Therefof growth . This ref growth , resultif growth .Several questions arise from our results regarding the cues influencing the timing and rates of these processes. Potential future research testing if catabolic processes related to deacclimation are a requirement for xylogenesis to begin would provide further insights. Determining the timing of xylogenesis initiation in comparison to the beginning of deacclimation would help define if xylogenesis and biochemical deacclimation processes respond to the same environmental cues. Answering these questions will contribute to a better understanding of the timing and influence of the processes conferring freezing tolerance in terminal buds, as well as the impact on their phenology.V. macrocarpon and we have described several of its anatomical characteristics. This barrier is partially responsible for the seasonal variations in freezing tolerance experienced by this woody evergreen perennial vine. The presence of the barrier is most likely a constitutive response to the bud’s development. In contrast, the timing of the development of new xylem observed during deacclimation is driven by environmental conditions, namely air temperature. Ice barriers in buds of species undergoing freeze dehydration contribute to the effectiveness of this freezing survival strategy. Our results also give insights into how components of freezing tolerance could be differentiated, such as metabolic and anatomical adaptations and how they relate to environmental cues. Future studies could explore the temperature thresholds and timing for spring xylogenesis and xylem activity resumption to indicate the loss of freezing tolerance. Knowing the different components involved in the development of freezing tolerance can provide breeders with screening tools targeting the selection of freezing tolerant genotypes.Our study identified a potential ice barrier in The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Design of the research and writing the manuscript were completed by AA, BW, and CV. Performance of the research was done by CV and DL. Data analysis, collection, or interpretation were performed by AA, BW, CV, and DL. All authors contributed to the article and approved the submitted version.This research is based upon work supported by the National Institute of Food and Agriculture, United States Department of Agriculture, through Hatch project 1009297, administered by the College of Agricultural and Life Sciences, University of Wisconsin–Madison, and by the Wisconsin Cranberry Research and Education Foundation.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


MYH6 variants that are present in >10% of HLHS patients, and which have been shown to be associated with decreased transplant-free survival in our previous studies. MYH6  variants have been reported in HLHS and numerous other CHDs, including LVOT malformations, and may provide a genetic link to these disorders. In this paper, we outline the MYH6 variants that have been identified, discuss how bioinformatic and functional studies can inform clinical decision making, and highlight the importance of genetic testing in HLHS.Hypoplastic left heart syndrome (HLHS) is a severe congenital heart disease (CHD) with complex genetic inheritance. HLHS segregates with other left ventricular outflow tract (LVOT) malformations in families, and can present as either an isolated phenotype or as a feature of a larger genetic disorder. The multifactorial etiology of HLHS makes it difficult to interpret the clinical significance of genetic variants. Specific genes have been implicated in HLHS, including rare, predicted damaging  MYH6 [NOTCH1 [NKX2.5 [ERBB4 [HAND1 [GJA1 [Hypoplastic left heart syndrome (HLHS) is a complex form of congenital heart disease (CHD) characterized by hypoplasia of the left ventricle and proximal aorta, as well as stenosis or atresia of the mitral and/or aortic valves . AlthougMYH6 ,9, NOTCH [NOTCH1 , NKX2.5  [NKX2.5 , ERBB4 [5 [ERBB4 , HAND1 [4 [HAND1 , and GJAD1 [GJA1 .MYH6 variants in a cohort of 190 unrelated HLHS subjects, comprising >10% of the cohort [MYH6 homologue, amhc/myh6, wherein loss-of-function mutations, along with amhc morpholino knockdown, disrupted atrial sarcomere assembly, impaired atrial contractility, and resulted in atrial dilation in zebrafish embryos. Mutant embryos also exhibited ventricular wall thickening and a narrowed ventricular lumen, mimicking the HLHS phenotype [myh6 mutants, compared to wild-type [myh6−/− Xenopus tropicalis hearts lacked cardiac contractility, which was accompanied by atrial and ventricular dilation, and impaired outflow tract development [MYH6-associated disease.We previously identified 19 distinct, rare, predicted damaging e cohort . These fhenotype . The addild-type . Similarelopment . AlthougMYH6 encodes for the alpha isoform of the cardiac myosin heavy chain (α-MHC), which is expressed throughout the myocardium during early cardiac development. As development proceeds, MYH6 expression decreases in the ventricles and is replaced with MYH7 (β-MHC) throughout gestation; α-MHC is the dominant atrial isoform postnatally [MYH6 and MYH7 have been linked to numerous human cardiac pathologies, including hereditary cardiomyopathies, arrhythmias, as well as CHD. While MYH7 variants have been characterized more extensively, the specific mechanisms underlying MYH6 variants are less understood. In this paper, we outline the MYH6 variants that have been reported in HLHS and other CHDs, discuss the benefits and limitations of biostatistical methods for interpreting variants, and emphasize the importance of mechanistic studies designed to improve personalized treatment strategies.tnatally ,19,20. GMYH6 was first reported more than 30 years ago in a family with hypertrophic cardiomyopathy (HCM). A disease locus, “FHC-1”, was initially identified in all affected but no unaffected members of this family [A possible pathogenic role for s family . Investis family . In the s family ,25,26,27s family ,29,30,31s family ,39,40,41s family ,44,45,46s family ,50,51,52s family , arrhyths family , and lefs family ,56. MYH6s family , coarctas family , and, ofs family ,25,26,59MYH6 variants to clinical phenotypes. In cohort studies, the designation of a variant as disease-causing relies on a combination of allele frequency and bioinformatic tools of pathogenicity, which are subject to change as new information is learned. There is not a single consensus on what constitutes a “rare” variant, or what is considered damaging when using a continuous variant scoring system, such as Combined Annotation Dependent Depletion (CADD). It is even more challenging to interpret the significance of variants when there are conflicting assessments between computational predictions and variant frequency, or between multiple predictive methods, for example, the MYH6 variants Q277H, M436V, I512T, V606I, D629N, R860H, A936V, R1151Q, A1298V, D1316E, and R1398Q , stage I surgical shunt type, age/era, and sex when possible. Patients with chromosomal abnormalities and those carrying MYH7 variants were excluded from this analysis. The difference in reaching the composite endpoint at 15 years between MYH6 variant and control groups did not reach statistical significance in this small study  to improve systolic and diastolic function, respectively. In phase III clinical trials, both drugs showed efficacy in the treatment of heart failure in adults [MYH6 variant, the cardiac specificity of omecamtiv mecarbil and mavacamten may offer a way to prevent disease progression. This treatment may be particularly important in variant carriers, given our previous report that HLHS patients with MYH6 variants have decreased cardiac transplant-free survival compared to HLHS patients without MYH6 variants [MYH7 variants, few studies have sought to understand MYH6 variant pathology at the molecular level; the findings from these studies are summarized in Understanding the specific mechanism of n adults ,70,71,72variants . HoweverMYH6 variants have utilized in vitro methods. The first variant to be functionally assessed was MYH6-I820N; this ASD-associated variant is located within the regulatory light chain (RLC) binding region of α-MHC and was found in cellular studies to decrease the binding affinity of α-MHC for RLC [MYH6-E933del variant enhanced binding to myosin-binding protein C (MyBP-C), an effect consistent with the location of E933del within the MyBP-C binding region of the α-MHC protein [MYH6-E933del also exhibited a slower electric propagation velocity compared to human MYH6-WT [Most of the mechanistic studies of  for RLC . However for RLC  and it i protein . HL-1 mo MYH6-WT , and neo MYH6-WT  variant  MYH6-WT . Togethe MYH6-WT , it is uMYH6 variants have shown similar changes in sarcomere structure. Cultured cardiomyocytes expressing the A230P [MYH6-V700M variant did not appear to impact sarcomere organization, despite its rare frequency (<0.001%) and being predicted as “likely damaging” by CADD, SIFT, and PolyPhen2. Our lab also assessed sarcomere organization in cardiac tissue from HLHS patients and found that atrial sarcomeres were disrupted with the R443P, K849del, and E1503V variants, while the ventricular sarcomere structure remained intact [MYH6 variant carriers and reported fibrosis in the conduction system [MYH6 variants in the patients studied.Many other he A230P , A1366D he A230P , E526K [he A230P , R1822_Ehe A230P , and HLHd intact , consistn system , and venn system . Given tMYH6-A1004S shortened at a slower rate, and consequently shortened less overall, when compared to NRVCMs expressing human MYH6-WT. In the same study, NRVCMs expressing human MYH6-P830L showed no difference in shortening rate, compared to MYH6-WT [MYH6-A1004S variant is also found at a frequency of 1.1% in the general population, which is more common than CHD. Meanwhile, MYH6-P830L is a novel variant and located near the RLC binding region and thus one would predict greater changes in function with P830L than A1004S. Our lab also found that the MYH6-R443P variant decreased the shortening rate, relaxation rate, extent of shortening, percent shortening, and calcium transient amplitude at the single CM level in patient-specific induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), without affecting action potentials. These MYH6-R443P iPSC-CMs also demonstrated sarcomere disorganization and the upregulation of MYH7, recapitulating the phenotype found in atrial tissue from an HLHS patient carrying the R443P variant [Some of the most informative in vitro studies are those that examine contractility at the cellular level. NRVCMs expressing human  MYH6-WT . This fi variant .MYH6 variants. Specifically, researchers evaluated the ability of the human MYH6-E933del and MYH6-R1252Q variants, which are associated with cardiac conduction disease, to rescue cardiac impairments resulting from myh6 knockdown. In both sets of experiments, the authors reported bradycardia in knockdown embryos at 48 hpf −137.7 ± 2.2 bpm in myh6−/− vs. 150.2 ± 1.6 in uninjected [myh6−/− vs. 153 ± 13 in uninjected [MYH6-WT and MYH6-R1252Q increased heart rate in knockdowns, human MYH6-E933del failed to rescue this phenotype.To date, zebrafish embryos are the only animal model that has been used to study human injected , and 144injected . While hMYH6 variants on the α-MHC structure are based on comparison to the solved structures of β-MHC. Mutational clustering analysis using population-level data found that pathologic variants in MYH7 cluster in certain regions [MYH6 , depending on the person [The structure of human α-MHC has not been solved, thus most hypotheses regarding the effect of  regions , which h regions . At presns [MYH6 ; howeverin mesa” ,81. Howee person .MYH6 variants. Molecular dynamics simulations predicted that the MYH6 variants E1207K and T1379M alter the helicity and flexibility of the tail domain, which would likely impact the rigidity and movement of the thick filament as a whole [MYH6-R1822_E1823dup likely increases the strength of the dimerized α-MHC tail domain, decreasing its flexibility [Some research groups have begun employing advanced in silico methods to model the effect of specific xibility . HoweverMYH6 variants are etiologic in a significant percentage of HLHS. Many of the studies cited in this paper identified additional candidate variants that may be contributing to cardiac disease development, including some patients carrying compound heterozygous MYH6 variants. New bioinformatic tools, such as Oligogenic Resource for Variant AnaLysis (ORVAL) [MYH6 variant pathogenicity and the reported cellular and clinical phenotypes. In any event, MYH6 variants will remain an important genetic risk factor for HLHS, having prognostic significance irrespective of other factors.HLHS is a complex and genetically heterogenous disease, and the origins of HLHS are likely multigenic. Evidence suggests  (ORVAL) , are desMYH6 variants on long-term cardiac function in HLHS.Our published work, ,75 and m


Aspergillus oryzae 001 to add functionality. Fermented abalone viscera exhibited increased angiotensin I-converting enzyme (ACE) inhibitory activity and enhanced inhibition of blood pressure elevation in spontaneously hypertensive rats (SHRs). Abalone viscera administration had no significant effect on body weight, food intake, liver and kidney weights, or serum components in SHRs. ACE inhibitors specific to fermented abalone viscera were identified through extraction, fractionation, purification, and analysis. The identified substance was L-m-tyrosine, which non-competitively inhibited ACE and, in a single oral administration, significantly reduced blood pressure in SHRs compared to that in the control. This study identified that abalone viscera fermented by A. oryzae 001 has an inhibitory effect on blood pressure elevation, suggesting its potential use as a functional food. In addition, L-m-tyrosine, a unique substance in fermented abalone viscera, was isolated for the first time as a single ACE-inhibitory amino acid.Abalone viscera, which accounts for more than 20% of the total weight of abalone, is generally regarded as waste in the food industry, and effective methods are required to utilize it productively. In this study, the viscera were fermented with  The food processing industry produces enormous amounts of organic residues and wastewater, most of which is left unused or untreated . BecauseAspergillus oryzae, one of the koji molds, has been used for over 2000 years for food fermentation and for over 50 years for the production of food enzymes [A. oryzae is enlisted in the U.S. Food and Drug Administration’s (FDAs) “Generally Recognized as Safe (GRAS)” list [idli increases gamma-aminobutyric acid content, angiotensin I-converting enzyme (ACE) inhibitory activity, and antioxidant activity through koji mold fermentation [idli has confirmed its blood pressure-lowering effect [ enzymes . TherefoS)” list . It prodS)” list . The ferentation . The in g effect . Koji mog effect .Hypertension is a major risk factor for cardiovascular disease, including coronary artery disease, left ventricular hypertrophy, valvular heart disease, and arrhythmias, such as atrial fibrillation, stroke, and renal failure . It is eLacticaseibacillus casei 001 and Lactiplantibacillus pentosus SN001 showed ACE-inhibitory activity [A. oryzae 001, and the inhibitory effect of the fermented product on elevated blood pressure was evaluated.With the increase in abalone production, the amount of abalone viscera that is discarded is expected to increase in the future. Therefore, more effective ways to utilize abalone viscera will be required than those at present. In previous studies, abalone viscera fermented with the lactic acid bacteria activity ,23. In aHaliotis ruber) and transported frozen. A. oryzae 001 is a proprietary fungus owned by the laboratory. L-tyrosine, soybean oil, isoflurane, tert-butylhydroquinone (TBHQ), L-cysteine, trifluoroacetic acid, sodium hydroxide, o-phthalaldehyde solution, phosphorus acid and kits for cholesterol, HDL-cholesterol, and triglyceride E tests as well as glucose and transaminase CII tests, were purchased from Wako Pure Chemical Industries . Potato dextrose broth (PDB) was purchased from Funakoshi Co., Ltd. . The ACE Kit-WST was purchased from Dojindo Laboratories . β-corn starch, casein, α-corn starch, sucrose, AIN76 mineral mixture, AIN76A vitamin mixture without choline deuterium tartrate, and cellulose were purchased from Oriental Yeast Co., Ltd. . Acetonitrile and distilled water were purchased from KOKUSAN CHEMICAL Co., Ltd. . ACE from rabbit lung was purchased from Sigma–Aldrich , and D-tyrosine was purchased from NACALAI TESQUE, Inc. . DL-o-tyrosine was purchased from Tokyo Chemical Industry Co., Ltd. . L-m-tyrosine was purchased from Cosmo Bio Co., Ltd. ; D-m-tyrosine was purchased from Santa Cruz Biotechnology, Inc. , Hip-His-Leu was purchased from Bachem AG , and His-Leu was purchased from Peptide Institute .Abalone viscera were sourced from Australian farmed blacklip abalone  in PDB medium. For fermentation, 1 mL of the pre-cultured bacterial solution was added to 100 mL of distilled water with 1 g of abalone viscera powder, and cultured with shaking . The culture supernatant of the ferment was subjected to measurement of ACE inhibitory activity. The ACE-inhibitory activity was determined using the ACE Kit-WST according to the manufacturer’s instructions.Abalone viscera were lyophilized, ground with a mixer, and sieved through a 500-mesh sieve. The abalone viscera powder was stored at −20 °C until use. Eighteen 14-week-old male SHR/Izm rats  were housed in a room at 25 ± 3 °C, with a humidity of 45 ± 5%, and a 12 h light/dark cycle (8:00–20:00 light period). Water (tap water) and feed were provided ad libitum. The rats were pre-reared for 1 week to acclimatize them to the environment. During the pre-rearing period, all rats were fed the same diet. The pre-reared rats were divided into control, fermented, and unfermented groups of 6 rats each and were fed the diets listed in g, 10 min). The aqueous extract was ultrafiltered using a centrifugal ultrafiltration unit Vivaspin 20  with molecular mass cut-off (MWCO) values of 3, 10, 30, and 100 kDa. Each fraction  was concentrated in a rotary evaporator, lyophilized, and measured for ACE-inhibitory activity. The fraction with high ACE-inhibitory activity was dissolved in distilled water, filtered through a 0.22 μm filter, and analyzed using reversed-phase high-performance liquid chromatography (RP-HPLC). ODS-120T , and liquid A (0.1% trifluoroacetic acid solution) and liquid B (0.1% trifluoroacetic acid solution/acetonitrile = 3:7 mixture) were used as the column and mobile phase, respectively. For elution, a concentration gradient of 0–50% ratio of solution B was applied over 40 min. The flow rate was set at 1.0 mL/min, and the detector at 220 nm. The peak with high ACE-inhibitory activity, unique to aqueous extracts of fermented abalone viscera, was collected and purified repeatedly. The peak with high ACE-inhibitory activity was subjected to a concentration gradient from 7% to 7.7% acetonitrile, and ACE-inhibitory activity was determined according to the manufacturer’s protocol for the ACE Kit-WST, and IC50 was calculated.Fermented and unfermented abalone viscera were extracted with water  and centrifuged  of the purified substance were determined, and the IC50 values were calculated. The structure of the purified product was determined by comparing the IC50 of the purified product with those of the various isomers. To eliminate foreign substances in the reagents, standards of all isomers were purified using HPLC and used for the measurement of ACE-inhibitory activity. HPLC conditions were the same as those used for the purification of ACE inhibitory components.The purified fractions were subjected to Edman degradation, and the purified products were identified using mass spectrometry. Phenylthiohydantoin derivatives produced by Edman degradation were separated and analyzed using RP-HPLC using Zaplous alpha, Pep C18 120A . The molecular weights of the purified materials were determined using an LTQ-Orbitrap XL mass spectrometer . The ACE-inhibitory activities of various isomers  and 100 μL of ACE (10 mU/mL) were mixed and incubated at 37 °C for 10 min. After incubation, 25 μL of Hip-His-Leu  was added, and the mixture was incubated at 37 °C for 40 min. Then, 50 μL of 1N NaOH was added, and after the reaction was stopped, 10 μL of 0.2% o-phthalaldehyde solution was added, and the reaction was carried out at room temperature for 15 min under light-shielding conditions. Then, 15 μL of 3.6 M phosphoric acid solution was added, and fluorescence intensity was measured at excitation and emission wavelengths of 360 and 460 nm, respectively. The Michaelis–Menten constant (Km) and the maximum reaction rate (Vmax) were calculated according to the Michaelis–Menten kinetic equation from the Lineweaver–Burk plot.The mode of inhibition was determined using Lineweaver–Burk plots ,24. Briem-tyrosine solution  or water (pH 3) was orally administered to each rat. The pH was adjusted to 3 because L-m-tyrosine is insoluble in water under neutral pH. Blood pressure was measured 6 times per animal before and 2, 4, 6, 8, and 24 h after administration using a non-observational blood pressure monitor.Male SHRs/Izm rats were purchased and housed as described in p < 0.05.The blood pressure measurements and weight changes are expressed as mean ± standard error, and other results are expressed as mean ± standard deviation. Rejection was performed using the Smirnov–Grubbs test. Multiple comparisons were performed using the Steel–Dwass test, and comparisons between two test intervals were performed using the t-test. Statistical significance was set at A. oryzae 001 was 56.9%. Fermentation of abalone viscera using lactic acid bacteria requires the addition of glucose [A. oryzae 001 fermented and unfermented abalone viscera administered to SHRs are shown in n = 5 due to hemolysis in the serum of one animal in the control group. There were no significant differences in kidney and liver weights between the study groups and no differences in appearance. Serum total cholesterol, HDL-cholesterol, glucose, triglyceride, ALT, and AST levels were also not significantly different between the groups. Rodents are commonly used as animal models of hypertension, with SHRs being the most commonly used model in studies of essential hypertension in humans [A. oryzae 001 imparted an antihypertensive effect to abalone viscera, and the fermented viscera was shown to suppress blood pressure elevation in vivo over the long term. In a previous study, L. casei 001 fermented abalone viscera mixed feed suppressed blood pressure elevation of SHRs after 28 days of administration [A. oryzae 001 fermented abalone viscera mixed feed suppressed blood-pressure elevation from day 12 of administration. Therefore, it was suggested that in A. oryzae 001 fermented abalone viscera suppressed blood pressure elevation more rapidly than L. casei 001 fermented abalone viscera in vivo. In a study on L. pentosus SN001 fermented abalone viscera, SHRs were reared for 9 weeks and L. pentosus SN001 fermented abalone viscera mixed feed suppressed blood pressure elevation from week 8 of rearing [A. oryzae 001 fermentation may have greatly enhanced the inhibition of blood pressure elevation in abalone viscera compared to L. pentosus SN001 fermentation. These results suggest that A. oryzae 001 fermentation enhanced the inhibition of blood pressure elevation in abalone viscera, and that the in vivo effect was stronger than that of lactic-acid fermentation. Significant differences in food intake and body weight between test groups have been used as an indicator of growth inhibition in rats [A. oryzae 001 fermented and unfermented abalone viscera did not inhibit SHR growth. Hypertensive patients tend to have lower HDL cholesterol levels and higher triglyceride levels, and total cholesterol levels above a certain level induce a greater increase in blood pressure [A. oryzae has lipolytic enzymes and has used fatty acids as a carbon source in previous reports [A. oryzae 001 may be degraded and reduced lipids in abalone viscera, resulting in lower total cholesterol levels in the fermented group than in the unfermented group. Hyperglycemia occurs due to abnormalities in glucose regulation, such as decreased glucose utilization, increased glucose production, and insulin secretion [A. oryzae produces α-amylase and glucoamylase [idli fermented with A. oryzae suppressed the increase in blood pressure in SHRs from at least day 14, and ALT and AST activities remained normal with no significant difference from control or unfermented for 10 weeks [A. oryzae did not affect ALT and AST activities.The ACE-inhibitory activity of abalone viscera fermented with  glucose ,23, whiln humans ,26,27. Istration , but A. pressure . Triglycpressure . A. oryz reports . Therefoecretion . Since toamylase . ALT andoamylase . These v50 values of each fraction separated by ultrafiltration are listed in 1–F7) and (N1–N2), respectively, with larger peaks detected in F1 and F4. F1 was similar in retention time and size to N1, so F4 was considered to be the fermentation product-specific peak; F4 was further purified using RP-HPLC to yield three peaks (F’1–F’3). The ACE-inhibitory activity was not observed in F’1 and F’2, but high ACE-inhibitory activity was observed in F’3 and it was subjected to Edman degradation and mass spectrometry. Protease activity involved in protein degradation was found to be enhanced during fermentation (Data not shown). Abalone viscera is rich in protein and fermentation is used as an effective means of protein hydrolysis [A. oryzae 001 fermented abalone viscera had high ACE-inhibitory activity in the low molecular weight fraction, suggesting that it is effective in vivo.The ICdrolysis . Fermentdrolysis ,36. Fermdrolysis . Those fdrolysis ,39. Thusm/z of 182.08122 and a composition of C9H12O3N. Thus, it was clear that the purified product was tyrosine. Since tyrosine has many isomers, the structure was determined by measuring the ACE-inhibitory activity of the various isomers and comparing the IC50 with that of the purified product. The IC50 values for each isomer were as follows: L-tyrosine and D-tyrosine showed less than 50% ACE inhibition at all concentrations. The IC50 values for the ACE inhibition of DL-o-tyrosine, L-m-tyrosine, and D-m-tyrosine were 0.62 mg/mL, 0.31 mg/mL, and 0.96 mg/mL, respectively. L-m-tyrosine was the most potent ACE inhibitor, with an IC50 value comparable to that of the isolated peak. Therefore, F’3 was determined to be L-m-tyrosine. Tyrosine is effective for mental health, and dietary tyrosine intake has been found to improve cognitive performance and physical performance tasks that are sensitive to it [Edman degradation analysis showed that only one tyrosine residue was present in the purified substance. Mass spectrometry results showed an ve to it . ACE inhve to it ,42,43. Pve to it ,44,45 anve to it ,47 ACE ive to it ,49. Tyrom-tyrosine was not determined. This study is the first to investigate the ACE-inhibitory activity of L-m-tyrosine. Lineweaver–Burk plots of ACE activity at various concentrations of L-m-tyrosine  are shown in max was 5.56, 2.20, and 0.78 mM/min, respectively, and was concentration dependent. Km was similar at 8.15, 8.24, and 8.14 mM, respectively. From the slope and y-axis intercept, Ki was 0.12 mM. Vmax was concentration-dependent while Km was relatively constant suggesting that ACE inhibition by L-m-tyrosine is a non-competitive inhibition. Similar to the present study, several tyrosine-containing dipeptides noncompetitively inhibited ACE [In previous studies, ACE-inhibitory activity and the mode of inhibition of peptides containing tyrosine were measured ,51, but ited ACE . In previted ACE ,53. Howeited ACE ,53. In mited ACE .m-tyrosine administration on the blood pressure of SHRs is shown in m-tyrosine. L-m-tyrosine was identified as the active component that acted as the ACE inhibitor in this study. Tyrosine isomers differ in structure, resulting in differences in ACE-inhibitory activity, behavior in the body, and digestibility [m-tyrosine may be the most potent inhibitor of elevated blood pressure in vivo.The effect of L-tibility . The smatibility ,39; therA. oryzae 001, its ACE-inhibitory activity was enhanced, and its inhibition of blood pressure elevation in vivo was confirmed. The ACE inhibitor unique to fermented abalone viscera was identified as L-m-tyrosine, which was found to inhibit ACE in a non-competitive manner. Furthermore, L-m-tyrosine showed antihypertensive effects in vivo. These results revealed that the fermentation of abalone viscera by A. oryzae 001 enhanced the antihypertensive effect of abalone viscera, suggesting that fermented abalone viscera can be utilized as a functional material to inhibit elevated blood pressure. In addition, L-m-tyrosine was found, for the first time, to be an amino acid with high ACE-inhibitory activity.In this study, abalone viscera, an underutilized resource, was fermented with