Tocochromanols are organic compounds mostly produced by photosynthetic organisms that exhibit vitamin E activity in animals. They result from the condensation of homogentisate with four different polyprenyl side chains derived all from geranylgeranyl pyrophosphate. The core tocochromanol biosynthesis has been investigated in several photosynthetic organisms and is now well-characterized. In contrast, our current knowledge of the biosynthesis and transport of tocochromanol biosynthetic precursors is much more limited. While tocochromanol synthesis occurs in plastids, converging genetic data in Arabidopsis and soybean demonstrate that the synthesis of the polar precursor homogentisate is located in the cytoplasm. These data implies that tocochromanol synthesis involves several plastidic membrane transporter(s) that remain to be identified. In addition, the metabolic origin of the lipophilic isoprenoid precursor is not fully elucidated. While some genetic data exclusively attribute the synthesis of the prenyl component of tocochromanols to the plastidic methyl erythritol phosphate pathway, multiple lines of evidence provided by feeding experiments and metabolic engineering studies indicate that it might partially originate from the cytoplasmic mevalonate pathway. Although this question is still open, these data demonstrate the existence of membrane transporter(s) capable of importing cytosolic polyprenyl pyrophosphate such as farnesyl pyrophosphate into plastids. Since the availability of both homogentisate and polyprenyl pyrophosphates are currently accepted as major mechanisms controlling the type and amount of tocochromanols produced in plant tissues, we summarized our current knowledge and research gaps concerning the biosynthesis, metabolic origins and transport of tocochromanol biosynthetic precursors in plant cells. Figure 1). To date, four types of tocochromanols, namely tocopherols, tocotrienols, plastochromanol-8 (PC-8) and tocomonoenols, have been identified in higher plants. These organic compounds drew the attention because tocopherols and tocotrienols exhibit vitamin E activity that is notably essential for animal reproduction . Following its synthesis by the plastidic shikimate pathway, tyr is converted into 4-hydroxyphenylpruvate (HPP) by tyrosine aminotransferases (TATs), a class of enzymes that catalyze the transamination between tyr and 2-oxoglutarate, and HPP and L-glutamate . The TAT isoform(s) involved in seed tocochromanol synthesis remains to be identified.Homogentisate is an aromatic compound resulting from the degradation of lutamate . Based os genome . Howeverd AtTAT2 . AtTAT1 d AtTAT2 . The othd AtTAT2 . It was d AtTAT2 . This daSpinacia oleracea and Lemna gibba localized the HPPD activity in chloroplasts  gene degrading HGA into 4-maleyl acetoacetate, overaccumulated free HGA and tocochromanols in seeds . These data further support the existence of a mechanism transferring cytoplasmic HGA into plastids where it is notably used for tocochromanol synthesis. To date, the identity of this membrane transporter(s) remains to be identified. Interestingly, the Arabidopsis HGO gene is also predicted to be localized in the cytoplasm (TAIR10).Hydroxyphenylpruvate is converted into HGA by the 4-hydroxyphenylpyruvate dioxygenase (HPPD), an Fe(II)-containing non-heme oxygenase encoded by a single gene in Arabidopsis . Severalroplasts . In toma peptide . In maizroplasts . In controplasts . Besidesytoplasm . The recin seeds . InteresFigure 2). Among the 10 GGPPS identified in Arabidopsis, seven are predicted to be localized in plastids, notably GGPPS11 that is the main paralog responsible for the synthesis of GGPP used for tocochromanol production  derive from GGPP produced by GERANYLGERANYL PYROPHOSPHATE SYNTHASEs . Seed and leaf tocopherols of vte5 mutants are reduced by 80 and 65%, respectively, while vte6 leaves fully lack tocopherols  or dimethylallyl pyrophosphate (DMAPP) showed that both enzymes poorly utilize geranyl pyrophosphate and DMAPP  1 and 2 . In vitrnd DMAPP , 2005. Ia and GGPP into phytylated chlorophyll a and PPP, respectively  3:1 and 3:2 that both stabilize GGR . To date, this transporter(s) remains to be identified.Most articles on plant tocochromanol metabolism attribute the origin of the prenyl component of tocochromanols exclusively to the plastidic methylerythritol phosphate (MEP) pathway. These references cite two significant review articles published right after the discovery of this non-mevalonate isoprenoid pathway that did not support the exchange of isoprenoid precursor(s) between the plastidic MEP and cytoplasmic mevalonate (MVA) pathways at that time , 1999. Rcopherol . This sue chains . These r feeding . Since t. This suggests that MVA pathway-derived radiolabeled IPP is concatenated in the cytoplasm into radiolabeled FPP that is imported into plastids where it is condensed with non-labeled MEP pathway-derived IPP. This biosynthetic model is supported by data showing the import of cytosolic FPP into plastids and its incorporation into the phytyl side chain of chlorophylls and into β-carotene .Recent feeding experiments with radiolabeled MVA combined to NMR analysis further refined our understanding of the import of cytoplasmic MVA pathway-derived isoprenoid precursors into plastids. It was independently shown that three quarters of the prenyl units composing the phytyl side chain of chlorophylls, β-carotene or other plastidic isoprenoids produced after [2-carotene , 1997. Mcarotene . CollectBrassica juncea 3-HYDROXY-3-METHYLGLUTARYL-COA SYNTHASE1, an MVA pathway biosynthetic gene, did not only increase the accumulation of cytoplasmic phytosterols in transgenic tomato fruits, but also strongly increased the synthesis of plastidic isoprenoids such as α-tocopherol (fivefold) and carotenoids . Thus, if both pathways are not fully functional at a given physiological stage, they cannot complement the one disrupted by the mutation, and this, despite the possible exchange of isoprenoid precursor(s) showed by feeding experiments and metabolic engineering. Importantly, there is currently no report in the literature demonstrating the dual origin of the prenyl side chain of tocochromanols in wild-type plants.Several metabolic engineering studies overexpressing cytoplasmic isoprenoid biosynthetic genes further support the utilization of cytoplasmic MVA-derived isoprenoid precursors for the synthesis of plastidic isoprenoids. It was shown for instance that overexpression of twofold; . Overexp pathway . Similar pathway . TogetheFrom the cloning of the first tocopherol biosynthetic genes to the recent identification of the tocomonoenol ones, substantial progress has been made in understanding the core tocochromanol biosynthetic pathways in plants . In contSP and LM-S designed and wrote the manuscript.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.


However, these effects were unrelated to disease severity because they were seen both in uninfected controls and in infants hospitalized with different levels of RSV disease severity. We conclude that ADE and reduction of neutralization are unlikely to be involved in RSV disease in infants with neutralizing matAbs.Respiratory syncytial virus (RSV) is the leading cause of severe respiratory illness in infants. At this young age, infants typically depend on maternally transferred antibodies (matAbs) and their innate immune system for protection against infections. RSV-specific matAbs are thought to protect from severe illness, yet severe RSV disease occurs mainly below 6 months of age, when neutralizing matAb levels are present. To investigate this discrepancy, we asked if disease severity is related to antibody properties other than neutralization. Some antibody effector functions are mediated via their Fc binding region. However, it has been shown that this binding may lead to antibody-dependent enhancement (ADE) of infection or reduction of neutralization, both possibly leading to more disease. In this study, we first showed that high levels of ADE of RSV infection occur in monocytic THP-1 cells in the presence of RSV antibodies and that neutralization by these antibodies was reduced in Vero cells when they were transduced with Fc gamma receptors. We then demonstrated that antibodies from cotton rats with formalin-inactivated (FI)-RSV-induced pulmonary pathology were capable of causing ADE. Human matAbs also caused ADE and were less neutralizing IMPORTANCE It is unclear why severity of RSV disease peaks at the age when infants have neutralizing levels of maternal antibodies. Additionally, the exact reason for FI-RSV-induced enhanced disease, as seen in the 1960s vaccine trials, is still unclear. We hypothesized that antibodies present under either of these conditions could contribute to disease severity. Antibodies can have effects that may lead to more disease instead of protection. We investigated two of those effects: antibody-dependent enhancement of infection (ADE) and neutralization reduction. We show that ADE occurs in vitro with antibodies from FI-RSV-immunized RSV-infected cotton rats. Moreover, passively acquired maternal antibodies from infants had the capacity to induce ADE and reduction of neutralization. However, no clear association with disease severity was seen, ruling out that these properties explain disease in the presence of maternal antibodies. Our data contribute to a better understanding of the impact of antibodies on RSV disease in infants. Human respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in young children . Hospita6–Maternal antibody levels are high during the first 6 months after birth. Strikingly, severe RSV disease most frequently occurs in this period of life , 3, indiin vitro (Antibody-dependent enhancement (ADE) of RSV infection has been demonstrated in vitro  in monocin vitro , 11. FCGin vitro , allowinin vitro . TherefoWe assessed two FCGR-mediated effects of RSV-specific antibodies: antibody-dependent enhancement of infection in monocytic THP-1 cells and reduction of neutralization capacity in cells transduced with Fc gamma receptors. Both were investigated for passively transferred matAbs by titration series of plasma from human cord blood and for actively acquired antibodies from immunized cotton rats, a small-animal model that is highly susceptible to RSV and shows vaccine-enhanced pulmonary pathology , 15. To in vitro: antibody-dependent enhancement (ADE) and reduction of RSV neutralization .Infants that encounter RSV in the first months of life have maternal antibodies. Despite the presence of maternal antibodies, a subset of infants that encounter RSV become severely ill. The antibodies of severely diseased infants are not neutralizing enough to prevent infection, but they can induce multiple other effector functions through FCGR interactions. In this paper, we investigate the impact of these Fc receptor-mediated functions of maternal antibodies on the severity of disease symptoms during RSV infection. We assessed two antibody-Fc receptor-mediated effects ADE of RSV infection in the presence of human serum or RSV-specific monoclonal antibodies has been demonstrated previously in monocytic cell lines, which carry Fc receptors , 11. ADEThe second effect we investigated is the ability of the FCGR to reduce the neutralization capacity (PRNT50) of antibodies. Antibodies have higher neutralization potential on cells lacking FCGR than the same cell type expressing FCGR, as is shown for wild-type Vero cells compared to Vero cells expressing FCGR . We callADE of RSV infection in the presence of human serum has been demonstrated previously . To inveEliciting RSV-neutralizing antibodies is the primary goal of RSV vaccination efforts. Intriguingly, the presence of FCGRs in target cells mostly reduces the RSV neutralization capacity of monoclonal antibodies . PreventTo further analyze ADE or ΔPRNT50 and any link to disease symptoms, we first assessed whether it would be feasible to distinguish between individuals with different capacities of ADE and ΔPRNT50. We tested the two Fc-mediated effects in cord blood from 15 individual donors. Most samples showed about 400% ADE compared to infection in the absence of antibodies . The ΔPRTo investigate a possible contribution of classical antibody characteristics to ADE or ΔPRNT50, both phenotypes were compared to two classical serological parameters: RSV neutralization in Vero cells  and B anin vivo, but the inoculation of mice with RSV in complex with antibodies has been shown to reshape the immune response compared to RSV alone  for protective immunity. First, RSV-specific antibodies in cotton rats were characterized. RSV neutralization in Vero cells demonstrated low, dose-dependent titers of neutralizing antibodies in all FI-RSV-immunized animals, whereas neutralization capacity of plasma from animals immunized with rRSV was high . CorrespThe ability of cotton rat antibodies to cause ADE in the human THP-1 cell model was assessed next. Plasma of all immunized animals caused ADE in THP-1 cells . InfectiTo assess the relationship between ADE of RSV infection and severity of pulmonary pathology in cotton rats, pathology after RSV challenge of FI-RSV- or rRSV-immunized cotton rats was studied. Affirming previous studies, immunization with rRSV protected the animals and resulted in little pathology after challenge . Both FIin vitro.The ADE and RSV neutralization titers of cotton rat sera next were compared to the sum of pathology scores (cumulative pathology score). Sera from mock-immunized animals were excluded. The capacity to induce ADE was present in all sera that contained RSV-specific antibodies independent of the pathology score. More ADE appears to be linked to lower levels of pathology, although the difference in ADE over the range of pathology was small . A lowerin vitro might relate to severe disease in vivo in the absence of neutralization, a possible association between severity of symptoms in infants and ADE or ΔPRNT50 was investigated. Plasma from hospitalized patients with an acute primary RSV infection was analyzed. As this is not a prospective study, the ideal control group of RSV-infected children with mild symptoms was not available, because blood of these children is not routinely sampled. A common practice in RSV research is to use age-matched uninfected infants as controls. One assumes that this group is not protected and if healthy infants become RSV infected, a vast majority (98 to 99%) would develop only mild symptoms  of infection or to reduction of neutralization (ΔPRNT50). We performed this study because we were intrigued by the fact that most severe RSV cases occur in RSV patients under the age of 6 months, when relatively high levels of RSV-neutralizing matAbs can be found. Moreover, infants that experienced vaccine-enhanced disease after the 1960s FI-RSV trials had an RSV-specific antibody response, albeit nonprotective. Therefore, both of these maternal and FI-RSV-induced antibodies could have a detrimental role in RSV-mediated disease when not neutralizing enough to protect infants from infection.in vitro, including an increase of ADE after RSV infection activation to shutting down cellular effector functions. Any of these effects could lead to the known contribution of immune cells to immunopathology in severe RSV-mediated disease. Determining whether ADE of RSV infection is possible in these cells in vivo and how these cells react may provide clues to why severe RSV disease occurs in certain cases and how we can prevent the burden of disease in these young infants.This report shows that ADE should not be ignored as a possible player in RSV infection. Most importantly, the induction of nonneutralizing antibodies or subneutralizing antibody titers should be avoided. It remains a challenge to find out whether immune cells are infected through ADE of RSV infection Plasma samples from healthy controls and hospitalized infants with RSV infections used in this study have been described before . HospitaMonkey kidney epithelial Vero-CCL81 cells were propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal calf serum (FCS) and 1% penicillin–streptomycin–GlutaMAX (PSG). The Vero cell-derived cell line expressing FCGR2a (Vero2a) was described earlier , and FCGLive-attenuated recombinant RSV-X (rRSV) , recombiSigmodon hispidus) were held at the animal facilities of Intravacc (Netherlands), and experiments were approved by the Animal Ethical Committee of RIVM. Animals were immunized intranasally with 105 infectious units of rRSV or with uninfected Vero cell supernatant (mock) or intramuscularly with a high- or low-dose FI-RSV  on days 0 and 21. Animals were challenged intranasally with 105 infectious units of RSV-A2 on day 49. Animals were sacrificed, blood plasma was collected, and lungs were fixed on day 53. Histopathologic analysis of lung sections using hematoxylin and eosin staining was done by Paul Roholl  as described previously . RSV neutralization assays were carried out on Vero and Vero2a cells. Assays were performed in triplicate as described earlier  against RSV particles as described previously . IVIg at


Equine inflammatory airway disease (IAD) and recurrent airway obstruction (RAO) represent a spectrum of chronic inflammatory disease of the airways in horses resembling human asthma in many aspects. Therefore, both are now described as severity grades of equine asthma. Increasing evidence in horses and humans suggests that local pulmonary inflammation is influenced by systemic inflammatory processes and the other way around. Inflammation, coagulation, and fibrinolysis as well as extracellular remodeling show close interactions. Cytology of bronchoalveolar lavage fluid and tracheal wash is commonly used to evaluate the severity of local inflammation in the lung. Other mediators of inflammation, like interleukins involved in the chemotaxis of neutrophils, have been studied. Chronic obstructive pneumopathies lead to remodeling of bronchial walls and lung parenchyma, ultimately causing fibrosis. Matrix metalloproteinases (MMPs) are discussed as the most important proteolytic enzymes during remodeling in human medicine and increasing evidence exists for the horse as well. A systemic involvement has been shown for severe equine asthma by increased acute phase proteins like serum amyloid A and haptoglobin in peripheral blood during exacerbation. Studies focusing on these and further possible inflammatory markers for chronic respiratory disease in the horse are discussed in this review of the literature. Disorders of the respiratory system, particularly the lower airways, are the most frequently diagnosed conditions in sport horses evaluated for poor performance , 2. EquiSince bronchoalveolar lavage (BAL) using fiber-optic endoscopy was first described in horses , cytologIn these cases, commonly used examination techniques may be insufficient for diagnosis and evaluation of treatment success. Therefore, multiple studies have been performed to establish further inflammatory markers for equine respiratory disease. Evidence exists that systemic involvement may exist in severe equine asthma as has been shown for human asthma  and chro2O caused by bronchoconstriction, mucosal swelling and mucus accumulation [Neutrophilia in TW and BALF is a predominant cytological feature of inflammation in mild-to-moderate and in particular in severe equine asthma –22, in wmulation , and infmulation . The defmulation , which imulation –32 and lmulation , 33, 34 mulation , 36 or emulation .γ expression support a Th-2 base [α on IL-8 expression by pulmonary artery endothelial cells. Receptors for IL-4, however, were not increased on neutrophils in severe equine asthma compared to healthy horses, but a genetic linkage of polymorphisms in IL-4-receptor-α on chromosome 13 with severe equine asthma was found and increased expression of this receptor in one high-prevalence family of horses, but not in another one [γ production in BAL cells, which supports a Th1-based background for RAO, while still others suggest no involvement of either type response in this disease [β and IL-23 also play a role [The immunologic background of severe equine asthma remains not fully elucidated despite many studies on the pathogenesis –41 and th-2 base . Lavoie h-2 base  showed tther one . On the ther one  demonstr disease . Inhalat disease . IL-8 is disease . In chro disease  and IL-1y a role .While the percentage of neutrophils in BALF decreases to reference levels in phases of remission, which hinders cytological diagnosis of the disease, myeloperoxidase (MPO) concentration in BALF was significantly higher in severe equine asthma during either crisis or remission compared to control horses . TherefoκB (NF-κB) activity are observable in asymptomatic horses affected with severe equine asthma [κB is a specific transcription factor that has a key role in inflammatory processes. It also plays a role in airway smooth muscle (ASM) cells phenotype modulation, characterized by reversible switching between contractile and proliferative phenotypes, which is considered to contribute to airway proliferation in human asthma [κB has also been reported as a key regulator for the occurrence and development of equine asthma. During disease exacerbation, peripheral blood leucocyte activation and increased concentration of circulatory inflammatory mediators have been observed in affected horses, suggesting that the inflammatory process might not be limited to the lung [Pulmonary neutrophilic inflammation during exacerbation has been the focus of many studies. It is largely reversible by antigen withdrawal in a low-dust environment , 28. Howe asthma , 71, 72.the lung –77. Humathe lung . These ithe lung –80. Systthe lung , 81–84.α und interferon-γ) in serum of healthy individuals and horses affected by severe equine asthma over 30 days of exposition to hay and straw. Haptoglobin was found to be a suitable marker for both acute and chronic systemic inflammation, whereas high concentrations of serum amyloid A indicated acute inflammation. There was no difference in serum concentrations of the evaluated cytokines between affected horses and controls. In another study though, increased TNF-α concentrations were found after ex vivo stimulation with bacterial products in severe equine asthma [Literature on systemic inflammatory processes in equine asthma is rare. Lavoie-Lamoureux et al.  comparede asthma . Niedźwie asthma  found inSince the 1970s a correlation of sepsis and hypocalcemia has gained increasing attention in research. In sepsis, procalcitonin (PCT) is found in high concentration in the peripheral blood and is expressed by many organ tissues . PCT is Rieger et al.  establisIn the long term, chronic obstructive pneumopathies lead to chronic remodeling of bronchial walls and lung parenchyma, which is accompanied by fibrosis formation –104. ThiAfter mechanical and inflammatory defects, which destroy the capillary endothelium and in particular the alveolar epithelium, plasma proteins transudate into the alveolar space and activate coagulation, of which fibrin is the end product. This is part of the natural healing and repair process and serves as a primary occlusion of the membranous defect . On the κB, which increases proinflammatory cytokine production [Apart from favoring fibrosis formation, fibrin and its derivates influence pathomechanisms of inflammation and the further course of the disease and repair of affected lung tissue . Fibrin oduction . In addioduction . Interacoduction .α2-AP  and on d-dimers (a fibrin-degradation product containing two D fragments of the fibrin protein joined by a cross-link) have demonstrated an increase in coagulation in bronchoalveolar lavage fluid (BALF) in patients suffering from chronic respiratory disease [It has been hypothesized early in human medicine that respiratory disease damaging the alveolar membrane must cause a misbalance between coagulatory and fibrinolytic activity in the extravascular space. Studies on different parameters of fibrinolysis like u-PA (urokinase-type plasminogen activator), PAI-1 (plasminogen activator inhibitor 1), and  disease . Coagula disease .Increased coagulatory activity also influences airway smooth muscle (ASM) proliferation. During asthma exacerbation, plasma circulating coagulant factor X (FX) enters the inflamed airways and is activated (FXa). FXa, but not FX, stimulated increases in ASM IL-6 production and cell number .In veterinary medicine and in particular in the equine lung, literature in this field is rare so far. Nevertheless, thickened alveolar interstitial spaces by the accumulation of fibrin and fibrinogen have been found in horses affected by chronic pulmonary disease . In seveRemodeling of the extracellular matrix (ECM) of pulmonary connective tissue is a continuous process allowing growth and regeneration. To maintain the tissue's stability, a balance between degradation, in which matrix metalloproteinases are the most important proteolytic enzymes, and resynthesis of extracellular matrix structures is required . The pulThe function of matrix metalloproteinases (MMPs) has originally been described over fifty years ago . MMPs plMMPs are very likely to have a central role in destructive pulmonary disease, characterized by fibrosis formation and the loss of elastin and collagen fibers. Type I collagen is very resistant against enzymatic degradation, which is possible only by few specific MMPs at physiologic pH . Some MMSeveral studies have been performed in horses, in which MMPs were evaluated by zymography in BALF samples. In severe equine asthma increased MMP-2 and MMP-9 concentrations were found compared to healthy controls . In partIt seems desirable to develop therapeutic approaches that protect the lung against overwhelming MMP activity but allow physiologic cell remodeling, which is important in the immunology of healing processes . To prevTissue inhibitors of metalloproteinases (TIMPs) are natural, endogenous MMP-inhibitors that regulate MMP-induced turnover of the ECM, tissue remodeling, and cellular behavior , 148. NeASM hyperresponsiveness and hyperplasia are a well-known phenomena in severe equine asthma . In vitrκß and FXa have been discussed already. Further key mediators in human asthma include CXC chemokines, Th-17 derived cytokines, and semaphorins [The roles of NF-aphorins . CXCL1, aphorins –158. Th1aphorins –162. Disaphorins , 163. A aphorins . Th17 cyaphorins . Semaphoaphorins  but are aphorins , 168.In conclusion, the results of the studies reviewed here show the complexity in the pathogenesis of respiratory disease in the horse. Many parallels can be drawn to human asthma, but species specific differences have also been found. Therefore, it is a challenge to find new therapeutic approaches to these pathologies with a high economic impact in veterinary medicine. Nevertheless, the numerous inflammatory markers that have been established for horses so far may help to understand the pathogenesis of lower airway disease in more detail and to diagnose the different diseases correctly even in subclinical cases. Further studies should focus on the course of the evaluated markers during therapy to evaluate their use in follow-up after therapy or during long-term therapy and to evaluate the success of not only established, but also new therapeutic approaches for chronic respiratory disease.


Forested settings present challenges for understanding the full extent of past human landscape modifications. Field-based archaeological reconnaissance in forests is low-efficiency and most remote sensing techniques are of limited utility, and together, this means many past sites and features in forests are unknown. Archaeologists have increasingly used light detection and ranging (lidar), a remote sensing tool that uses pulses of light to measure reflecting surfaces at high spatial resolution, to address these limitations. Archaeology studies using lidar have made significant progress identifying permanent structures built by large-scale complex agriculturalist societies. Largely unaccounted for, however, are numerous small and more practical modifications of landscapes by smaller-scale societies. Here we show these may also be detectable with lidar by identifying remnants of food storage pits (cache pits) created by mobile hunter-gatherers in the upper Great Lakes during Late Precontact (ca. AD 1000–1600) that now only exist as subtle microtopographic features. Years of intensive field survey identified 69 cache pit groups between two inland lakes in northern Michigan, almost all of which were located within ~500 m of a lakeshore. Applying a novel series of image processing techniques and statistical analyses to a high spatial resolution DTM we created from commercial-grade lidar, our detection routine identified 139 high potential cache pit clusters. These included most of the previously known clusters as well as several unknown clusters located >1500 m from either lakeshore, much further from lakeshores than all previously identified cultural sites. Food storage is understood to have emerged regionally as a risk-buffering strategy after AD 1000 but our results indicate the current record of hunter-gatherer cache pit food storage is markedly incomplete and this practice and its associated impact on the landscape may be greater than anticipated. Our study also demonstrates the potential of harnessing commercial-grade lidar for other fine-grained archaeology applications. Forested landscapes pose significant challenges for understanding the full extent of past human activities and modifications of the landscape. Field-based archaeological surveys in forests are often arduous and time-intensive, resulting in restricted spatial coverage. Most available remote sensing survey techniques are also of limited utility for finding archaeological features under forest canopies . As a result, many archaeological sites and formations in forested settings are simply unknown –3. LidarLidar, a remote sensing tool that uses pulses of light to measure the elevation of reflecting surfaces, has increasingly served to further archaeological research. It has been suggested that its expanded application could transform the discipline in ways comparable to the advent of radiocarbon dating . This reNot all past societies were complex, however, and over the course of human history a diverse range of relatively low-density, egalitarian, mobile hunter-gatherer societies existed across the globe. While early research conceived of these as simple societies on a low rung of a progressive social evolutionary ladder, more contemporary research recognizes that mobile hunter-gatherers were dynamic and had, if not complex, still complicated cultural systems ,14. EvenRecent studies have presented cases where mobile hunter-gatherers overtly modified the landscape, like through the building of mounds and earthworks , but morIn order to better grasp their importance, we would benefit from a more complete record of cache pit features across local and regional landscapes. Ethnohistoric accounts indicate cache pits in active use were subterranean u-shaped features, ranging from 1 to 2 meters wide and deep, lined with birch bark, filled with multiple foodstuffs in containers with grass-type fill in between, and capped with mounded dirt . Cache pThe State of Michigan recently launched a lidar collection program, Michigan Statewide Authoritative Imagery & Lidar (MiSAIL), which provided an opportunity to test the utility of lidar to expand insight on the extent of these fine-scale microtopographic features in one inland lake landscape within the upper Great Lakes, Douglas and Burt Lake . Our fieThe results of this work indicate these cache pits date to the Late Precontact period (ca. AD 1000–1600) which is when cache pits became a key scarcity mitigation strategy across the upper Great Lakes . If we hDiscrete return lidar data were collected during leaf-off conditions in the spring of 2015 as part of the Michigan Statewide Authoritative Imagery & Lidar (MiSAIL) program. The Sanborn Map Company was tasked by the State of Michigan to deliver high point density returns with Nominal Point Spacing of 0.71 m. Data were collected using a Leica ALS80 sensor flown on board an aircraft at an altitude of approximately 1200 m with a maximum scan angle of 32 degrees. Lidar acquisition was in compliance with Quality Level 2 of the United States Geologic Survey (USGS) National Geospatial Program (NGP) Base Lidar Specifications ; horizonClassified point clouds were processed in ArcGIS Desktop 10.3.1  using the LAS Dataset and Conversion toolsets. A digital terrain model (DTM) was derived by creating a new LAS Dataset layer consisting of a filtered subset of points classified as American Society for Photogrammetry and Remote Sensing (ASPRS) Class 2 (Ground) . These bWe developed an automated routine to delineate candidate caches in DTMs via a two-step process . First whttp://www.R-project.org/). Using the probability calculated from the results of the logistics regression and the area of each cache, we performed a pseudo accuracy assessment on the manually interpreted sample of caches. Accuracy was calculated as:For each candidate cache, we calculated a series of parameters to describe its shape and size, including perimeter-area ratio (L/S), corrected perimeter-area (CPA) and related circumscribing circle (RCC), the radius of the circumscribing circle (radius), and the perimeter and area of the polygon . We alsosessment  Tables.To better understand the distribution and clustering of individual cache pits, we calculated the kernel density using the Kernel Density tool in the Spatial Analyst Toolbox in ArcGIS 10.1, using a 25 foot (7.62 meters) search radius weighted by cache pit area with an output cell size of 1 foot (0.305 meters). We used a threshold of 0.06 to identify distinct areas of relatively high density, which we considered as possible discrete cache pit clusters. We selected a subset of the identified candidate clusters for a logistic regression, using the locations of the cache clusters previously identified in our team’s archaeological field survey to confirm delineated clusters as cache pits and manual interpretation to identify false positives. Logistic regression was performed using a set of variables describing the shape, size, and terrain variability of each candidate cluster and the cache pits within. Using the results of the logistic regression, we performed a pseudo accuracy assessment to reduce the candidate clusters to a set of likely cache pits.One of the surest ways to rot food in a Great Lakes subterranean storage pit is saturation by water. Previous work used a Compound Topographic Index (CTI), which is an index of water accumulation in soils, and found locating cache pits where they had reduced likelihoods of becoming saturated by water was a critical factor in storage success . FollowiWe resampled our 1-foot (0.305 meter) DTM to 10 m, 30 m, and 90 m, and calculated CTI at each spatial resolution. We then compared CTI values of pixels that overlapped with cache clusters to CTI values of pixels coinciding with 350 randomly generated locations throughout the extent of the DTM using two nonparametric tests: the Kolmogorov-Smirnov, which allows for testing of differences in distributions of two samples of continuous observations, and Mood’s median test, which allows for testing of the equality of medians from two or more populations. Lower CTI scores indicate improved drainage. In every iteration of the comparison of CTI values of pixels containing cache pits and pixels underlying randomly selected coordinate pairs, cache pits were statistically significantly located on lower CTI scores . Using t2, respectively . By performing a pseudo accuracy assessment, we found that high accuracy and high percentage of true caches retained was by using probability and area thresholds of 0.58 and 13 ftectively . ApplyinFrom the kernel density analysis, we identified 13,686 candidate clusters of cache pits. The logistic regression resulted in a significant relationship with variables describing the mean perimeter of caches within the delineated cluster (perimeter), the mean value of DTMmod within caches (cache DTMmod), the mean value of DTMmod within the delineated cluster (cluster DTMmod), and the mean TRI within the delineated cluster (cluster TRI) . Following the pseudo accuracy assessment , we filtUsing the Kolmogorov-Smirnov test, we determined that there is a significant difference (p<0.001) between lidar-derived CTI for cache clusters at all three scales , with increasing disparity between the distributions as pixel size increases . In addiWe visually identified 139 of these 543 candidate clusters as having a high potential to be anthropogenic cache pit features refer to . We haveOur study indicates that cache pits, relatively subtle archaeological features produced by mobile hunter-gatherers, can be identified through advanced analyses of commercial-grade lidar data. Very few archaeological research programs can acquire research-grade lidar, but access to commercial-grade lidar is increasing globally as state-sponsored programs collect lidar data for other applications. Our case not only demonstrates the potential of lidar for fine-grained archaeology applications, but also corroborates the notion that DTM generation should be parameterized for individual use-cases. Indeed, without end-user involvement in the processing of point-cloud data, subtle archaeological features like cache pits can be filtered out of output DTMs entirely , as theyAs lidar becomes available across the upper Great Lakes, our work demonstrates a capability to examine vast geographic swaths for microtopographical archaeological features, such as cache pits. Field survey can then be focused on areas with higher probability of such features, rather than time consuming walk-over survey in forests. Given that we have found several new cache pit clusters in a landscape that has seen some of the most intensive field surveys ever done for cache pits, the possibility of finding numerous previously unknown cache pit clusters across the upper Great Lakes region is high. As more past anthropogenic microtopographic features are found, it raises the question of the impacts such modifications may carry for ecological processes, which may warrant consideration not just by archaeological research programs. What this study makes clear is that, at best, we have a markedly incomplete record of the ways mobile hunter-gatherers of the upper Great Lakes regularly modified the natural world to ensure their own well-being. As we utilize tools like lidar to expand this record, our interpretations of past social and economic developments among these relatively low-density, mobile societies will have to change and with this, the remnant influence of early conceptualizations of such societies as simple will be further dissolved.S1 FigThe lidar DTM (left) shows slight depressions, accentuated in DTMmod (middle), which was used to generate a binary image of pit and non-pit pixels (right). Pits shown in red were filtered from the candidate cache data set using probability and area thresholds determined from logistic regression and a pseudo accuracy assessment.(JPG)Click here for additional data file.S2 Fig(JPG)Click here for additional data file.S3 FigThe lidar DTM (left) shows locations of cache pit clusters after automated and statistical filtering. Clusters shown in red were manually filtered based on visual assessment of each individual cluster, while those shown in green were included in the final count of cache pit clusters.(JPG)Click here for additional data file.S1 Tablebold) for high accuracy as well as high proportion of pits (positives) to non-pits .For each area threshold, the probability threshold with the highest accuracy is reported. Thresholds of area 13 and probability 0.58 were selected (in (PDF)Click here for additional data file.S2 Tablebold) was selected because of high accuracy, high proportion of positively identified clusters to false positive clusters, and few omission errors.A probability threshold of 0.51 (in (PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


Sepsis-induced myocardial dysfunction is a common complication in septic patients and is associated with increased mortality. In the clinical setting, it was once believed that myocardial dysfunction was not a major pathological process in the septic patients, at least in part, due to the unavailability of suitable clinical markers to assess intrinsic myocardial function during sepsis. Although sepsis-induced myocardial dysfunction has been studied in clinical and basic research for more than 30 years, its pathophysiology is not completely understood, and no specific therapies for this disorder exist. The purpose of this review is to summarize our current knowledge of sepsis-induced myocardial dysfunction with a special focus on pathogenesis and clinical characteristics. Sepsis is a systemic deleterious host response to infection or injury resulting in severe sepsis and septic shock. It is a leading cause of morbidity and mortality in intensive care units , 2. AlthThe cardiovascular system plays an important role in the pathogenesis of sepsis. Over the last 50 years, a large number of studies have demonstrated that myocardial dysfunction is a common finding in septic patients, and approximately 50 % of septic patients exhibit signs of myocardial dysfunction. Nevertheless, the exact clinical significance of sepsis-induced myocardial dysfunction (SIMD) is still elusive. Because the heart, as only one part of the circulatory system, constantly responds to changing peripheral hemodynamics, it is difficult to distinguish between cardiac responses to alterations in preload, afterload or/and neurohumoral activity during sepsis and the direct influence of sepsis on the heart in the clinical setting . RecentlThe aim of the present review is to discuss the pathophysiology of SIMD, with a special focus on its clinical characteristics and pathogenesis.Although numerous studies have demonstrated evidence of cardiovascular impairments in patients with sepsis over the last 50 years, there is no universally accepted definition of SIMD . The iniHemodynamic alterations during sepsis have been investigated for 60 years. Early animal studies performed by Weil et al. in 1956 showed that an injection of endotoxin could cause a sudden decrease in venous return, arterial blood pressure and cardiac output (CO) with increased systemic vascular resistance (SVR), leading to animal death . ClinicaIn 1984, Parker and Parrillo et al. provided the first valuable clue for the SIMD . Using sDuring the same historical period, two-dimensional echocardiography was performed to evaluate myocardial function in septic patients , 23. BecTo detect subtle myocardial dysfunction during sepsis, some investigators examined myocardial function during sepsis using advanced echocardiographic techniques, such as speckle tracking and Doppler tissue echocardiography. Speckle tracking echocardiography is more sensitive than the conventional echocardiographic technique and is able to detect ventricular strain that reflects segmental myocardial deformation. It was demonstrated that peak left ventricular global longitudinal systolic strain, determined by using speckle tracking echocardiography at the time of admission in septic patients, correlated with mortality rate, whereas left ventricular EF had no prognostic relevance . AnotherIn contrast to systolic dysfunction, diastolic dysfunction is often ignored, and its role in determining early mortality from sepsis has not been adequately investigated. Recently, it has been demonstrated that the mitral annular early diastolic peak velocity (e’ wave) obtained by tissue Doppler imaging is one of the most load-independent measures of diastolic dysfunction. The ratio of the early mitral inflow velocity (E), recorded with pulsed-wave Doppler, to the e’ wave (E/e’) correlates with left ventricular end-diastolic pressure, and a high E/e’ ratio represents low left ventricular compliance in numerous cardiac conditions , 29. UsiIn 1982, Terradellas et al. reported the acute elevation of the ST segment in bacterial shock patients with no history of heart disease . Other iB-type natriuretic peptide (BNP) is a hormone synthesized in the myocardium. It is produced in the prohormone form, and before secretion is split into the inactive NT-proBNP and the active BNP . NumerouHuman autopsies and animal experimental studies have revealed that sepsis-induced myocardial changes are classified within inflammatory cardiomyopathy. The major cardiac pathological changes during sepsis include myocardial infiltration by immune cells , subendocardial hemorrhage, interstitial and intracellular edema, endothelial cell edema, microcirculatory fibrin deposition, as well as focal myofibrillar dissolution, cardiomyocyte necrosis and interstitial fibrosis. Intracytoplasmic lipid accumulation in cardiomyocytes is also observed in septic hearts. Immunohistochemical examination shows that high diffuse expression of tumor necrosis factor-α (TNF–α) is localized to cardiomyocytes, macrophages, smooth muscle cells and endothelial cells , 47–49. Based on an animal study, the first hypothesis on the mechanism of SIMD was global myocardial ischemia resulting from inadequate coronary blood flow. However, coronary blood flow was later found to be either preserved or increased in septic shock patients with myocardial dysfunction, disproving the above hypothesis. Although some studies demonstrated that impairment in cardiac microcirculation was present during sepsis due to significant maldistribution of coronary blood flow, endothelial damage, intravascular fibrin depositions and neutrophil infiltration, which might result in focal myocardial ischemia and decreased cardiac function, no myocardial hypoxia was confirmed in septic animals. It is now suggested that the elevation of plasma cardiac troponins may be attributed to an increase in cardiomyocyte membrane permeability rather than myocardial ischemic necrosis. These findings indicate that coronary circulation alterations are less important in the mechanisms of SIMD , 50. AccIn the 1960s, many investigators reported the presence of myocardium-depressing factors that could cause SIMD . During 1-adrenoceptor promoted p38MAPK, JNK and NF-kB activation and subsequent TNF-α expression in LPS-treated cardiomyocytes [1-adrenoceptor can suppress LPS-induced cardiomyocyte TNF-α expression and improve cardiac dysfunction during endotoxemia [2-adrenoceptor suppressed myocardial TNF-α and inducible nitric oxide synthase (iNOS) expression and cardiomyocyte apoptosis and cardiac dysfunction in endotoxemic animals [Indeed, it is likely that circulating myocardium-depressing factors are the initial stimuli and driving forces of septic myocardial dysfunction. It is well known that cardiomyocytes express Toll-like receptors such as TLR2 and TLR4 , 60. LPSmyocytes . Activatotoxemia . We also animals . Thus, iIn addition to cardiomyocytes, cardiac fibroblasts and endothelial cells are involved in SIMD. Cardiac fibroblasts make up 60 %–70 % of the total cell number in the heart. Tomita et al. demonstrated that LPS significantly increased the expression of TNF-α and matrix metalloproteinase (MMP)-9 in cultured cardiac fibroblasts. CLP induced cardiac MMP-9 expression, cardiac fibrosis and cardiac dysfunction in mice, and treatment with a broad-spectrum MMP inhibitor significantly alleviated these histological and functional changes during sepsis . In addi1-adrenoceptors, reduced levels of stimulatory G-proteins and increased expression of inhibitory G-proteins in cardiomyocytes during sepsis. These results indicate that impaired myocardial responsiveness to catecholamines in sepsis can be attributed to the downregulation of adrenergic receptors and/or post-receptor signaling [3-adrenoceptors, which mediate an increased negative inotropic response to agonists, were found to be upregulated during sepsis, suggesting that activation of β3-adrenoceptors by catecholamines may contribute to SIMD [The autonomic nervous system plays an important role in sepsis. Some evidence indicates that sepsis induces autonomic dysregulation, including neuronal and glial apoptosis within the autonomic centers of the heart, high plasma levels of catecholamines, reduced heart rate variability and decreased cardiac responsiveness to intrinsic catecholamines, which may contribute to SIMD. A number of studies showed decreased densities of βignaling , 75. In  to SIMD .2+ sensitivity due to a sustained increase in cardiac troponin I phosphorylation at Ser23/24 [2+ transporters in cardiomyocytes underlies SIMD. During sepsis, downregulated L-type calcium channels and a suppressed sarcoplasmic reticulum (SR) pump lead to a decrease in the amplitude of cellular Ca2+ transients and SR calcium load in cardiomyocytes [2+ transport in the cardiac SR [2+ extrusion pathways by depressing the function of the Na+/Ca2+exchanger and the plasmalemmal Ca2+ ATPase, which in turn resulted in intracellular diastolic Ca2+ overload [2+ homeostasis in cardiomyocytes may contribute to SIMD. However, the mechanism underlying the reduction in transient systolic Ca2+ is not well established. A recent study demonstrated that sepsis induced a decrease in sodium current in cardiomyocytes, which reduced cardiac excitability. This reduction in the density of Na+ channels might lessen the transient Ca2+ action potential in cardiomyocytes by decreasing the number of Ca2+ channels that open during the action potential due to the reduction in peak depolarization or/and by reducing Ca2+ entry due to a shortening of the action potential [In addition to a decrease in myofilament CaSer23/24 , a dysfumyocytes . CLP incrdiac SR . LPS alsoverload . This diotential .Although oxygenation of the myocardium does not appear to be altered during sepsis, accumulating evidence indicates that impaired metabolism and reduced energy production in cardiomyocytes play a critical role in SIMD. Under normal conditions, approximately 70 % of adenosine triphosphate (ATP) in cardiomyocytes is produced via fatty acid oxidation, and the remainder is produced via glucose oxidation. A small amount of ATP is also derived from the catabolism of lactate and ketone bodies . During Although early myocardial dysfunction during sepsis is associated with myocardial inflammation rather than mitochondrial injury , enzyme 1-adrenoceptors almost completely abolished cardiomyocyte apoptosis in LPS-challenged mice [1-adrenoceptor activation promotes LPS-induced cardiomyocyte apoptosis [1-adrenoceptor activation appears to be more important than cytokines in LPS-induced cardiomyocyte apoptosis. In fact, a randomized clinical trial observed that therapy with the β-blocker esmolol reduced heart rate, increased left ventricular stroke work index and decreased 28-day mortality in septic shock patients [In addition to leading to DNA fragmentation, activated caspases can directly induce the breakdown of myofibrillar proteins, decrease ATPase activity and force development in cardiomyocytes . Althougged mice . We furtpoptosis . Therefopatients . The reapatients .SIMD refers to the intrinsic myocardial systolic and diastolic dysfunction of both the left and right sides of the heart during sepsis. Early recognition of intrinsic myocardial dysfunction is critical for the administration of the most appropriate therapy for septic patients. However, traditional insensitive parameters, such as EF, cannot accurately assess sepsis-induced intrinsic myocardial dysfunction. It is now suggested that the routine use of speckle tracking and tissue Doppler echocardiography may be valuable in the identification of SIMD in septic patients. The circulating myocardium-depressing factors are only initial stimulators that induce cardiac structure and function damage, in which cardiomyocytes, cardiac endothelial cells and even fibroblasts are involved. A deeper understanding of the effects of immuno-metabolic and neuroendocrine factors on cardiomyocytes, endothelial cells and fibroblasts at molecular and subcellular levels will expand our knowledge of the mechanisms contributing to SIMD. The elucidation of these mechanisms should help identify new cardiac-specific therapeutic targets and improve the prognosis of septic patients.


Reduce Avoidable Imaging Initiative, designed to “reduce testing and imaging with low risk patients through the implementation of Choosing Wisely recommendations.” However, it is unknown whether New England emergency departments (ED) have already implemented evidence-based interventions to improve adherence to ACEP Choosing Wisely recommendations related to imaging after their initial release in 2013. Our objective was to determine this, as well as whether provider-specific audit and feedback for imaging had been implemented in these EDs.In June 2016, the American College of Emergency Physicians (ACEP) Emergency Quality Network began its Choosing Wisely-focused interventions in 2014. Initial mailings included cover letters denoting the endorsement of each state’s ACEP chapter, and we followed up twice with repeat mailings to non-responders. Data analysis included descriptive statistics and a comparison of state differences using Fisher’s exact test.This survey study was exempt from institutional review board review. In 2015, we mailed surveys to 195 hospital-affiliated EDs in all six New England states to determine whether they had implemented Choosing Wisely imaging scenario; 57% reported implementing a specific guideline/policy/clinical pathway and 28% reported implementing a computerized decision support system. The most common interventions were for chest computed tomography (CT) in patients at low risk of pulmonary embolism (47% of EDs) and head CT in patients with minor trauma (45% of EDs). In addition, 40% of EDs had implemented provider-specific audit and feedback, without significant interstate variation (range: 29–55%).A total of 169/195 (87%) of New England EDs responded, with all individual state response rates >80%. Overall, 101 (60%) of responding EDs had implemented an intervention for at least one Choosing Wisely recommendations, most New England EDs had a guideline/policy/clinical pathway related to at least one of the recommendations. However, only a minority of them were using provider-specific audit and feedback or computerized decision support. Few EDs have embraced the opportunity to implement the multiple evidence-based interventions likely to advance the national goals of improving patient-centered and resource-efficient care.One year after release of the ACEP  Choosing Wisely® recommendations for emergency department (ED) use of diagnostic tests and treatments,Choosing Wisely recommendations, a number of tools, including clinical pathways,In 2013, the American College of Emergency Physicians (ACEP) published 10 evidence-based Reduce Avoidable Imaging InitiativeChoosing Wisely recommendations.” The initiative is a laudable endeavor meant to emphasize the many tools available to assist with adherence to these recommendations. However, there are sparse data on whether EDs have implemented any interventions to improve adherence to guidelines since their initial publication in 2013. Our objective was to investigate whether New England EDs implemented evidence-based interventions to improve adherence to ACEP Choosing Wisely recommendations after their release, and also whether provider-specific audit and feedback for imaging had been implemented in these EDs.In June 2016, ACEP’s Emergency Quality Network (E-QUAL) began its Choosing Wisely recommendations). These initial mailings included cover letters denoting the endorsement of each state’s ACEP chapter, and were followed up twice with repeat mailings to non-responders.This survey study was exempt from institutional review board review. In 2015 we used the 2012 National Emergency Department InventoryChoosing Wisely imaging scenarios: head computed tomography (CT) studies (for minor traumatic brain injury [MTBI] and in asymptomatic adults with syncope); chest CT for low-risk pulmonary embolism (PE); lumbar spine magnetic resonance imaging (MRI) for atraumatic low back pain; and abdominal CT for renal colic. For each of the scenarios, respondents were asked dichotomous yes/no subquestions regarding whether they had implemented either a guideline/policy/clinical pathway and/or computerized decision support. Pediatric EDs and EDs without CT/MRI capability to which individual questions might not apply were asked to indicate “NA” (not applicable). “Guideline/policy/clinical pathway” and “computerized decision support” were not further defined to allow respondents flexibility in deciding which of their interventions fell into each category. Respondents were also asked whether their clinicians received provider-specific audit and feedback regarding use of advanced imaging .The survey included a total of 30 questions, of which two focused on interventions that EDs had implemented targeting the five What do we already know about this issue?ACEP has recommended a number of tests that can be avoided as part of the Choosing Wisely initiative.What was the research question?Do EDs have guidelines, policies, pathways, decision support, or feedback regarding ACEP Choosing Wisely Initiatives?What was the major finding of the study?57% have guidelines, policies, or pathways, 40% have decision support, and only 28% provide feedback regarding Choosing Wisely.How does this improve population health?Adherence to Choosing Wisely requires more than just education - the use of the evidence-based tools we studied should improve adherence to Choosing Wisely.Choosing Wisely imaging scenarios, as well as the use of provider-specific audit and feedback. Data analysis included descriptive statistics and a comparison of state differences using Fisher’s exact test.The outcomes were the presence or absence of at least one reported intervention for each of the five Choosing Wisely imaging scenario; a guideline/policy/clinical pathway (57% of EDs) was more frequently reported than CDS (28%) (Responses were received from 169/195 (87%) of New England EDs; all individual state response rates were >80%. Overall, 101 (60%) of responding EDs had implemented an intervention for at least one DS (28%) . In addiChoosing Wisely targets had interventions implemented for them.The most common interventions were for chest CT in patients at low risk of PE (47% of EDs), and head CT in patients with MBTI (45% of EDs) . By statChoosing Wisely recommendations, most New England EDs focused their interventions on only two imaging scenarios: patients with suspected PE and those with MTBI. While most EDs had a guideline/policy/clinical pathway related to at least one of the Choosing Wisely recommendations, only a minority had implemented CDS related to one of the recommendations. In addition, fewer than half of New England EDs were providing provider-specific audit and feedback about imaging utilization to their clinicians.One year after release of the ACEP Choosing Wisely recommendations are largely evidence-based and meant to target likely unnecessary and overused imaging studies. Translating these recommendations into clinical practice to reduce low-value care is the next needed step. A recent analysis of the effectiveness of the publication of several Choosing Wisely recommendations on outcomes (including head and lumbar spine imaging) found mixed results.Reduce Avoidable Imaging Initiative will be key to broad implementation of tools targeting the established Choosing Wisely targets.The Reducing Avoidable Imaging Initiative to succeed, understanding current ED interest and practice in imaging re-education is essential to guiding future efforts. From our data, it is evident that a number of EDs, at least in New England, are already focusing on reducing imaging in patients with suspected PE and suspected MTBI. Both conditions have a broader evidence base to guide imaging decisions, including widely disseminated ACEP clinical policies, likely making provider engagement in quality improvement (QI) easier. Conversely, clear evidence gaps remain for two of the other targets —CT for syncope and abdominal CT for renal colic —for which no clinical practice guidelines or many large clinical trials exist. In the case of lumbar spine MRI for back pain, which is supported by the Choosing Wisely campaigns of numerous medical specialty societies and clinical practice guidelines, lower rates of ED QI interventions may reflect that many of the guidelines are still based on expert consensus rather than evidence-based decision instruments. As national efforts such as E-QUAL continue to expand, resources must be dedicated to developing an evidence base and the associated clinical practice guidelines necessary to engender physician trust in recommendations to reduce imaging use historically considered necessary to exclude high-risk, life-threatening diagnoses.For campaigns such as the E-QUAL Choosing Wisely recommendations. Poor adoption of CDS is surprising given the rapid adoption of electronic health records (EHR) in the ED as a result of the CMS Meaningful Use program. However, not all EHRs have easily-customized CDS capabilities, and ED staff may not have had the opportunity to readily implement CDS to address Choosing Wisely recommendations. As our survey preceded implementation of the 2014 Protecting Access to Medicare Act (PAMA) that mandates physician use of CDS, increased adoption of CDS is likely to be reflected in future surveys of EDs.Our work also demonstrates wide variability in the implementation of evidence-based QI strategies, with a notable lack of CDS and provider-specific audit and feedback. There is evidence that both of these interventions can improve the appropriateness of imaging use in the EDThis study has two main limitations. The first is that it was conducted only in New England, and therefore results may not generalize nationally. We used a survey methodology that relies on self-reporting and we did not assess any potential differences in actual “on the ground” implementation. However, we have no reason to believe that respondents were untruthful, particularly as they were told in the survey instructions that no identifying information would be used and responses would be reported only in aggregate.Choosing Wisely recommendations shows broad interest in reducing avoidable imaging. However, the QI practices are largely limited to select interventions and certain clinical scenarios. Few EDs have embraced the opportunity to implement multiple evidence-based interventions likely to yield synergistic gains necessary for emergency care to advance the national goals of improving patient-centered and resource-efficient care.10Our assessment of initial ED efforts undertaken after publication of the


Health improvements realized through sanitation are likely achieved through high levels of facilities utilization by all household members. However, measurements of sanitation often rely on either the presence of latrines, which does not guarantee use, or respondent-reported utilization of sanitation facilities, which is prone to response bias. Overstatement of sanitation metrics limits the accuracy of program outcome measures, and has implications for the interpretation of related health impact data.We conducted a cross-sectional study of 213 households in 14 village water, sanitation and hygiene committee clusters throughout rural Bangladesh and used a combined data- and relationship-scale approach to assess agreement between respondent-reported latrine utilization and sensor-recorded measurement.r=0.35) and thus only weak concordance between measures (ρc=0.29 [95% BCa CI 0.15 to 0.43]).Four-day household-level respondent-reported defecation averaged 28 events (inter-quartile range [IQR] 20–40), while sensor-recorded defecation averaged 17 events (IQR 11–29). Comparative analyses suggest moderately high accuracy (bias correction factor=0.84), but imprecision in the data (broad scatter of data, Pearson’s Respondent-reported latrine utilization data should be interpreted with caution, as evidence suggests use is exaggerated. Coupling reported utilization data with objective measures of use may aid in the estimation of latrine use. In response, governments, nongovernmental organizations and others have undertaken large-scale programs to increase latrine coverage. Bangladesh has achieved success with such programs, as 95% of rural households have access to some form of sanitation facility.2 The country has observed progress over time, as the proportion of households  with an improved sanitation facility not shared with other households has increased from 34% in 2011 to 45% in 2014.2Globally, an estimated 2.4 billion people lack access to improved sanitation.5 Even these studies, however, may exaggerate actual latrine utilization.International monitoring of sanitation relies principally on self-reported data. For example, the Joint Monitoring Programme on Water and Sanitation (JMP) recommends that national surveys assess sanitation by asking informants about the facilities that their household uses (JMP Core question 2006). While this may yield helpful estimates of sanitation access (coverage) and type of facilities, there is increasing evidence that it does not capture actual use. In neighboring India, for example, several studies have now shown that even amongst households with latrines, use is low or inconsistent, especially among men and children.10 Sensor-monitored use of cookstoves and water filters also suggests respondents tend to over-estimate environmental health behaviors that programs are designed to promote.11 Gaps between latrine coverage and actual use have also been reported in Ethiopia12 and Ghana.13 Deficiencies in use may at least partially explain why recent studies of sanitation interventions have failed to detect reductions in diseases such as diarrhea and soil-transmitted helminthiasis.15Assessments of latrine use typically rely on respondent-reported practices, a measurement method that can be unreliable due to courtesy, social desirability and recall biases. Studies of respondent-reported handwashing, for example, have reported exaggerated compliance.16 PLUMs have been used in India to determine methods of assessing latrine use that minimize bias.19 The objective of this research was to determine agreement between respondent-reported and PLUM-recorded latrine use and to ascertain whether reported latrine use data indicate evidence of bias relative to PLUM-recorded use.In order to overcome potential bias associated with respondent reporting of latrine utilization, researchers developed the passive latrine use monitor (PLUM), a battery-operated device that can be discretely mounted inside latrines to detect use by recording motion.upazilas) in rural Bangladesh. Data were collected during June–August 2014, monsoon season in Bangladesh.This research was conducted within a cross-sectional verification study assessing the accuracy of implementer-reported sanitation outcomes from a water, sanitation and hygiene (WASH) program implemented throughout rural Bangladesh. The verification was a post-only study conducted in 52 village WASH committee (VWC) clusters from 177 program subdistricts (upazilas) were eligible for inclusion in the verification study. Additional inclusion criteria required that households have at least one adult  who consented to participate in the verification study and serve as the primary survey respondent; this individual had to be capable of understanding and providing informed consent.Data for this study were collected in 14 of the 52 verification VWC clusters. These clusters were eligible for inclusion in this latrine utilization study if they were included in the verification and had at least one household with a functional household latrine  with walls or a roof suitable for PLUM installation. A household was defined as a person or group of related or unrelated persons who usually live together in the same dwelling(s), have common cooking and eating arrangements and acknowledge one adult member as the head of household. All VWCs and households residing within the WASH program’s targeted subdistricts (zilas), subdistricts (upazilas), unions and VWCs targeted by the WASH program. We selected primary sampling units using probability proportional to size. During the next sampling stage, one VWC was selected from each union using simple random sampling. In the final sampling stage we identified potential study households by obtaining the household register from each selected VWC and stratifying the household sampling frame by wealth category, as defined by the VWC, to select eight households from each stratum. From the list of VWCs selected for the verification study, we used a random number generator to identify 14 VWCs in which we installed PLUMs in all functional and suitable latrines in each household selected for inclusion in the latrine utilization study.We employed a multilevel sampling strategy to randomly select VWCs and households for inclusion in the larger verification study, and subsequently this latrine utilization study. For the verification study, our sampling frame consisted of a line list of districts  .Sensor-recorded latrine utilization monitoring data captured via PLUM deployments in household latrines.Enumerators collected data on latrine utilization using different measurement methods, includingIn households randomly selected for inclusion in the study, enumerators sought out adult respondents, with a preference for the primary female caretaker of the youngest child within the household, who likely is most knowledgeable about the latrine use and defecation practices of most members of her household. Enumerators administered the household survey using electronic data capture on password-protected mobile devices that allowed for range and consistency checks. The survey instrument was designed to collect data on household demographics, wealth and socioeconomic status; latrine construction, maintenance and repair; latrine spot check indicators and reported latrine use and defecation practices. Within this survey instrument, enumerators administered a structured household latrine use schedule, which systematically captured reported data regarding latrine use and defecation practices for each regular user of the household’s latrine(s), including household members and other regular users. Enumerators asked all latrine users present during survey administration to provide self-reports of their own usual latrine utilization and defecation practices and asked the survey respondent to report for those who were not present during survey administration.Each respondent was asked about his or her primary place of defecation, whether the primary place of defecation changes during the year and whether the latrine was always exclusively used for defecation. Enumerators subsequently asked about the number of times each household latrine user usually uses the latrine during four specific periods of the day—morning , afternoon , evening , and night .http://www.pdx.edu/sweetlab). Details regarding earlier generations of similar devices, field testing, validation and the algorithm are described elsewhere.16 PLUMs collected electronic sensing data in the latrines over the course of a 1-week installation period.After administering the survey, field staff installed one PLUM device in each functional and suitable (see above-mentioned definitions) household latrine in all consenting selected households. We used a fourth-generation PLUM device developed by co-authors from Portland State University  to compare latrine utilization metrics from the subset of households with successful PLUM installations. To assess agreement between latrine utilization measurements, we quantified latrine use count data generated from the respective measurement methods, as indicated below. For both measures, latrine utilization was calculated via the quantification of 4-day, household-level ‘likely defecation’ events. For the PLUM-recorded metric, we define the 4-day, household-level ‘likely defecation’ event total as a sum of all events recorded during days 3–6 of the PLUM installation period . In order to minimize reactivity observed during the first days of a PLUM installation period , we dropped the first 2 days of PLUM data, analyzing data from these 4 days. For respondent-reported events, we defined this 4-day, household-level metric by summing the reported individual-level ‘usual’ latrine use events across the four specific periods of the day and then summing the individual-level totals for all latrine users older than 3 years of age. In order to generate data that were comparable to a 4-day PLUM-recorded measure, we multiplied the household-level ‘usual’ event total by 4.Respondent-reported use measures reflect a 4-day, household-level reported latrine use total for all household latrine users over the age of 3 years. Reported use for children under 3 years of age was excluded from the household-level total number of reported daily ‘likely defecation’ events because the PLUM algorithm has not been validated to capture child feces disposal and latrine training events, and inclusion of these events in the reported use total could threaten the comparability of utilization measurements between the two methods.PLUM-recorded use measures reflect a 4-day, household-level, sensor-detected latrine use total. We generated these data by employing a validated algorithm to assess raw PLUM signal data and detect and quantify signal patterns indicative of ‘likely defecation’ events.Households with a malfunctioning PLUM that registered faulty data on one or more days;Households with a malfunctioning PLUM in one household latrine but functioning PLUMs in the remaining household latrines. These households were dropped from the analytical sample because data loss from one or more PLUM sensors would prevent us from generating an accurate PLUM-recorded use measure for those respective households; andHouseholds that accepted PLUM installation in one latrine but refused PLUM installation in one or more subsequent latrines. These households were also dropped from the analytical sample because we would be unable to generate an accurate PLUM-recorded use measure for these respective households as well.During the data collection period we experienced several technical issues with the PLUM sensors that resulted in data loss. Any household meeting one or more of the following criteria were dropped from the analytical PLUM sample:batplot and concord packages.17 To assess agreement between respondent-reported and PLUM-recorded methodologies, we combined the data-scale approach recommended by Bland and Altman’s limits of agreement (LoA)18 with the complementary relationship-scale approach suggested by Lin’s concordance correlation coefficient (CCC), a scaled index of agreement.20 The magnitude of the CCC reflects both the precision and accuracy of the data and can be used to determine whether the observed data deviate from the line of perfect concordance. This method allowed for an analysis of the accuracy and precision of the data that is not provided by the limits of agreement approach in the absence of a scaled index of agreement. We performed a model II, least-products regression to fit a reduced major axis to the data in a concordance plot. We employed a bootstrap resampling technique (1000 replications) to generate a bias-corrected and accelerated bootstrap confidence interval (BCa) for the CCC presented herein.21 Details related to the steps executed to generate relevant statistics and produce modified Bland–Altman and concordance plots are outlined in the We analyzed data in Stata 13.1 , using user-written Arguably, no ‘gold standard’ latrine utilization measurement method exists; therefore, we used a data structure that consisted of discrete utilization measures, without replications, and no assumed ‘gold standard’ measurement method.The field team was given consent to install PLUMs in 250 of 319 households targeted for PLUM installation, resulting in an installation success rate of 78%. The final analytical sample included data from 213 households in the 14 VWC clusters, from which respondent-reported demographic and utilization data were captured along with PLUM-recorded utilization data. This translated to an 85% (213/250) data extraction rate among successful installations. Table Data on defecation and latrine utilization practices were captured from a total of 1363 household latrine members from the 213 latrine utilization study households. Of those latrine users, 27%  provided self-reported data on latrine use and defecation practices; the remaining data were provided via proxy. The average household-level proportion of self-reporting latrine users was similar (25% [interquartile range {IQR} 17–40]).The 4-day, household-level, respondent-reported ‘likely defecation’ events averaged 28 events (IQR 20–40) and the number of PLUM-recorded ‘likely defecation’ events averaged 17 events (IQR 11–29). Given that the median number of household latrine users was 5 (IQR 4–7), this translates to 1.4 respondent-reported ‘likely defecation’ events per person per day and 0.85 PLUM-recorded ‘likely defecation’ events per person per day. Utilization data generated by the two measurement methods were found to have similar distributions , meaningSee the When we assessed the difference between and average of latrine utilization measurement methods, we found an upward bias in the difference between measurements relative to the average, indicating overestimation of respondent-reported utilization relative to PLUM-recorded utilization. The modified Bland–Altman plot indicated a relatively wide 95% LoA, which suggests poor agreement between the two latrine utilization measures from the data-scale perspective Figure , panel Ar and the ratio of the standard deviations . This CCC indicates that respondent-reported ‘likely defecation’ events are only weakly correlated with PLUM-recorded ‘likely defecation’ events. The reduced major axis was moderately close to the line of perfect concordance, and the bias-correction factor was 0.84, which indicates a moderately high level of accuracy in the measures. However, the data were imprecise, as indicated by Pearson’s r=0.35, and the broad spread of observations from the reduced major axis. These findings help explain the relatively weak concordance correlation between reported and recorded latrine utilization measures.The CCC between respondent-reported and PLUM-recorded utilization was Findings from both data- and relationship-scale agreement assessments indicate an upward bias in respondent-reported latrine utilization measures, suggesting overreporting of latrine use relative to PLUM-recorded use. These findings are demonstrated through the upward slope of the mean measurement line relative to zero in Figure As indicated in our methods, we assumed no gold standard measurement approach, thus our rationale for employing a type II modeling approach to carry out our comparative analyses. When selecting language to describe the results of our analyses, we had to choose one metric as a referent. We chose to use the more objective measure as the referent. As such, we describe respondent-reported use relative to sensor-recorded use and indicate that these data are overreported relative to sensor-recorded use. The reverse could also be said—that PLUM-recorded latrine use is under-estimated relative to respondent-reported use.16 it is possible that the PLUM did not appropriately distinguish between ‘likely defecation’ events that occurred in immediate succession of each other. In addition to being prone to recall bias, respondent-reports may have been prone to courtesy bias, as defecating in a latrine reflects a behavior promoted by the WASH program that was under evaluation. As such, some respondents may have exaggerated latrine defecation events. However, socially-influenced factors  may have caused some respondents to curtail reports of defecation events.Although PLUM-recorded data represent more objective measures of latrine utilization compared with subjective reports, both metrics are susceptible to different types of bias. Given prior evidence of undercounting bias of sensor-recorded ‘likely defecation’ events in high-traffic scenarios,While reported latrine utilization is subject to reporting bias, when these measures are captured via the household use schedule, the data that are produced facilitate examination of more nuanced utilization trends within and across household settings. In other words, while respondent-reported use may be biased, it allows for individual-level assessments of latrine use patterns across age, sex and position within the household as well as household-level utilization assessments not possible with sensor-recorded data. These considerations are important when weighing options for latrine utilization measurement metrics, particularly for programs that intend to target utilization across late-adopting age and sex cohorts.22 This is important, as programs and policies that are informed by reported data may not be on target to address sanitation uptake among different household members. These data also suggest it is necessary to obtain objective measures of latrine utilization, such as lower-cost sensors or latrine spot check indicators, to triangulate respondent-reported data and provide a more comprehensive assessment of latrine use. Employing sensors in a sub-set of program households can help determine whether and to what extent bias in reported latrine utilization exists, which may allow for adjustment and interpretation of respondent-reported utilization measures. Such bias may vary by population, setting, and cultural practices.Our findings corroborate results from prior investigation that suggest levels of adoption of improved sanitation practices are at least moderately exaggerated.22 That study found higher concordance between PLUM-recorded latrine use data and data captured on a 48 h recall as opposed to usual daily defecation and latrine use data. Similarly, a study of cookstove and water filter use also suggest overreporting of the utilization of these materials relative to sensor-recorded use.11 Other studies of respondent-reported handwashing indicate exaggeration of reported versus directly observed or instrument-monitored compliance as well.10 Unlike the handwashing literature, which indicates more severe overreporting of handwashing with soap relative to other more objective measures of the behavior,23 our results indicate relatively moderate bias in reported latrine utilization. The nature of the bias is indicated by the magnitude of the gradient of the mean measurement line relative to zero on the data-scale assessment, and the relative proximity of the reduced major axis to the line of perfect concordance on the relationship-scale assessment.Our findings are consistent with results from several other studies that suggest respondent-reported data are susceptible to response bias. For example, our findings correspond with those of a recent study that found poor concordance between usual daily latrine use and average daily PLUM-recorded events and evidence of exaggerated reporting of household latrine use.6 In addition, our analytical sample was relatively small, with respondent-reported and PLUM-recorded data provided from 213 households, which may have contributed to the relatively wide limits of agreement. Furthermore, the cross-sectional study design prevented us from being able to assess temporal changes in agreement between measurement methods. Our data were captured during monsoon season and therefore may not represent usual defecation and latrine use practices carried out during other seasons of the year. To the greatest extent possible, PLUMs were installed on the same day enumerators obtained reported data. However, given time and resource constraints, we were unable to ensure contemporaneous collection of respondent-reported and PLUM-recorded data at all study households. This may not be an important limitation of this study, given the dearth of information regarding the impact of the timing of sensor placement and survey administration on the level of agreement between the two metrics. In some cases, it may even be preferable to avoid contemporaneous collection of reported and recorded latrine use measures. The algorithm used to detect defecation events was validated against direct observations of ‘likely defecation’ events. It is uncertain whether the algorithm detects child feces disposal, child latrine training, and menstrual hygiene management events as ‘likely defecation’ events, as the validation exercise did not explore these distinctions. However, respondent-reported utilization for defecation was higher than PLUM utilization rates, and one would expect that if these non-defecation-related sanitation events were included in PLUM-recorded measures they would serve to increase PLUM-recorded events and thus improve agreement between measures. Further research is needed to determine whether these latrine use practices are captured by the PLUM algorithm and whether respondents typically consider these events when responding to questions regarding latrine utilization. Irrespective of these limitations, our study findings provide important insights into assessments of latrine utilization and related measurement metrics.There are several limitations of this study. While the authors do not assume that day-to-day variation of latrine use events is uniform—thus the rationale for using language regarding ‘usual’ practices as opposed to ‘yesterday’s’ practices—inquiring about ‘usual’ practices does draw on an inherent assumption that there is some uniformity in practices. As a result, this underlying assumption may constrain, to some degree, the comparability of these respondent-reported and PLUM-recorded latrine use data. A relatively high proportion of defecation and latrine use data were obtained by having informants report on other household latrine users’ practices. These respondent reports may not be completely accurate. That being said, the use of proxies for the provision of reported defecation and latrine use measures is an efficient way of collecting data, and is a commonplace practice for assessing sanitation outcomes.Given that respondent-reported latrine use is overreported relative to PLUM-recorded use measures, these data may not provide an accurate picture of sanitation program uptake. However, at the lower range of measurement values, overreporting is only modest, which indicates that reported data do not always immensely inflate sanitation utilization. This is worth considering, especially in contexts where latrine access is relatively high. Coupling reported utilization data with objective measures of use may further aid the estimation of latrine use. Program managers and policymakers should be mindful of moderate overreporting when using respondent-reported latrine use data to assess sanitation program progress.In order to continue advancing the measurement of latrine utilization and defecation practices, and improving the evaluation of sanitation programming, the sector should continue pursuing several avenues. Further work is needed to transition from high-cost sensors used for research to simple, low-cost and acceptable sensors that can monitor latrine utilization amongst all household members, including the safe disposal of child feces in a sanitation facility. The availability of low-cost sensors could facilitate their incorporation into routine program monitoring, further increasing demand and lowering unit cost, thereby increasing opportunities for more objective measures of latrine use. Such sensors, or any other objective and reliable metrics of latrine use monitoring, could have the potential to transform donor funding to a pay-for-performance approach that reimburses implementing entities based on metrics of open defecation averted as opposed to the number of latrines built or the prevalence of latrine access. An approach such as this would further justify the cost of the sensors and their incorporation into sanitation programming. Similar work with sensors on handpumps and boreholes is currently under way.As indicated above, PLUM signal data can be relatively imprecise, particularly in high-traffic scenarios. The sensors work well for monitoring household latrine utilization, but they do not work well for monitoring utilization at community, public or school facilities due to the limitations in the algorithm’s ability to distinguish between individual latrine events when there is a line of users. That being said, there may be an opportunity to investigate whether devices that take digitally blurred infrared photographs can be employed to distinguish between latrine use events while producing only anonymized ‘chalk drawing’ outlines.Supplementary DataClick here for additional data file.