Salmonella enterica, the RND pump is translated from the acrAB gene, which is regulated by the activator RamA. Many MEP-caused AMR strains have high ramA gene expression due to mutations in RamR, which has a homodimeric structure comprising the dimerization domain and DNA-binding domain (DBD). Three mutations on the dimerization domain, namely Y59H, M84I, and E160D, are far from the DBD; the molecular mechanism through which they influence RamR’s binding affinity to the ramA gene promoter and consequently disrupt RamA remains unclear. The present study conducted molecular dynamics simulations, binding free energy calculations, and normal mode analysis to investigate the mechanism through which Y59H, M84I, and E160D mutations on the dimerization domain influence the binding affinity of RamR to the ramA promoter. The present results suggest that the three mutations alter the RamR structure, resulting in decreased DNA-binding affinity.Antimicrobial resistance (AMR) in pathogenic microorganisms with multidrug resistance (MDR) constitutes a severe threat to human health. A major causative mechanism of AMR is mediated through the multidrug efflux pump (MEP). The resistance-nodulation-division superfamily (RND family) of Gram-negative bacteria is usually the major cause of MDR in clinical studies. In  Klebsiella pneumonia1 and colistin-resistant Enterobacteriaceae2 can pose severe threats to human life.In recent years, antimicrobial resistance (AMR) has become a global concern. Pathogens with AMR can threaten human life; if pathogens with AMR infect inpatients, their treatment periods tend to be prolonged, thereby increasing healthcare costs. The abuses of antimicrobial drugs in clinics and agricultural settings imperceptibly culture many AMR strains in nature. For example, carbapenem-resistant 3. Multidrug resistance (MDR) is an important aspect of AMR, and the multidrug efflux pump (MEP)4 is one of the essential mechanisms that mediate MDR. Several MEP-caused MDR pathogens include Salmonella enterica5, Escherichia coli6, Staphylococcus aureus7, and K. pneumonia8. The bacterial MEP can be classified into five families9: resistance-nodulation-division superfamily (RND family)10, major facilitator superfamily11, ATP-binding cassette superfamily12, small MDR family13, and multidrug and toxic compound extrusion family14. Of the five MEP families, the RND family is the major cause of MDR in clinical studies15; for instance, the acrAB gene in E. coli and S. enterica16. Several studies on RND family-caused bacterial MDR18 have reported that RamA, the activator of the acrAB gene, is an essential target for elucidating the MDR mechanism, particularly in S. enterica19 and K. pneumonia21. In 2008, Abouzeed et al.22 identified ramR, a TetR family of transcriptional repressor gene, which is located upstream of the ramA gene. RamR binds to the promoter region of the ramA gene to repress ramA gene transcription, thereby resulting in MDR. Several studies have reported different RamR mutations in the MDR strains of S. enterica and K. pneumonia that can reduce the repression of the ramA gene24. However, the molecular mechanism through which mutations, such as Y59H, M84I, and E160D26, on the dimerization domain of RamR reduce its ability to repress ramA gene expression remains unclear. The present study conducted molecular dynamics (MD) simulations and binding free energy calculations to investigate the mechanism through which Y59H, M84I, and E160D mutations in the dimerization domain cause MDR by assessing the binding events between the RamR protein and the ramA promoter.The mechanism of AMR includes target alteration, drug inactivation, decreased permeability, and increased effluxramA promoter DNA. To further investigate the mutation-induced changes in interaction properties, the total free energy of the MT and WT complexes was calculated to obtain the binding free energy. In addition, normal mode analysis (NMA) was performed to determine whether the mutations alter the dynamics networks to influence large-scale domain motion. The binding free energy calculations and NMA results were in accordance with the observations from MD simulations.We constructed four structures, specifically, the wild-type RamR (WT-RamR) and three mutants (MTs), namely Y59H-RamR, M84I-RamR, and E160D-RamR. The root mean square deviation (RMSD) and RMS fluctuation (RMSF) simulation results suggested that mutations in the dimerization domain of RamR might stabilize the overall structure but make structural changes in the DNA-binding domain (DBD), which could influence RamR’s binding affinity to the 27 as the template to run MODELLER (9v8)28 for predicting the structures of Y59H-, M84I-, and E160D-RamR. Three mutant RamR models were constructed on the basis of the sequence and structural alignment of the 3VVX template. The qualities of all modeled structures were assessed through Ramachandran plots29. The steepest descent algorithm30 was used for structural optimization (energy minimization), and PyMOL31 was used for all three-dimensional (3D) structures and charge distribution presentation.We selected 3VVX–DNA complex is yet to be solved, we predicted its structure. Previous studies have demonstrated that RamR acts as a repressor and binds to the promoter region (TATAATGAGTGCTTACTCACTCATAATC) of the ramA gene to inhibit ramA gene expression32. Therefore, we used 3D-DART33 to construct a 3D model of the ramA promoter. Furthermore, we used HADDOCK (v2.2)35 to perform the docking of the promoter DNA molecule to the RamR protein for constructing the RamR–DNA complex structural model.Because the structure of the RamR36. The Amber99 force field was used for energy calculations. The models were solvated with TIP4P water molecules and simulated in a cubic box with periodic boundary conditions. The energy of the models was first minimized using the steepest descent algorithm. The simulations were performed in the canonical NVT  and NPT  ensembles through the position-restrained MD simulation for 100 ps, respectively. The linear constraint solver algorithm was used to constrain the bonds37. The MD simulations were performed at constant pressure and temperature for 20 ns using an integration time step of 2 fs. The nonbonded interactions were cutoff at 10 Å. The coordinates from the MD simulations were saved at every 100 time steps.Molecular dynamics (MD) simulations were performed using GROMACS software (ver. 5.1.4)rref, was calculated as i at time t after last square fitting of the structure to the reference structure; (B) the RMSF of the backbone Cα atoms with respect to the starting conformation was calculated as follows. The RMSF is a measure of the deviation between the position of particle i and some reference position: T is the time over which one wants to average and i. Typically, this reference position will be the time-averaged position of the same particle, that is, To understand the structural and functional implications of the amino acid substitutions in the dimerization domain on the DBD, the trajectories of the WT and MT structures were analyzed for several structural properties as a function of time: (A) The RMSD of the Cα atoms of the DBD with respect to the starting conformation, 39. This tool automatically generates hybrid structures and topologies for amino acid mutations that represent the two physical states of the system. After obtaining this hybrid structure, free energy simulations were performed using GROMACS. From the 10-ns equilibrated trajectories, 100 snapshots were extracted, and a rapid 100-ps simulation was performed starting from each frame. The lambda (λ) ranged from 0 to 1 and from 1 to 0 for the forward and backward integrations, respectively, thus describing the interconversion of the WT to MT systems and the MT to WT systems, respectively. Finally, the pmx tool was used to integrate over the multiple curves and subsequently estimate free energy differences using the fast-growth thermodynamic integration approach, which relies on the Crooks–Gaussian Intersection (CGI) protocol40. The ΔG values for the mutations in the DNA-free and DNA-bound structures were calculated to construct a double free energy difference (ΔΔG). The final ΔΔG values indicate whether mutations stabilize or destabilize the protein in the DNA-bound state.The pmx tool was used to construct the hybrid topologies of the system used in the free energy calculations44. A low-frequency large-scale motion usually reflects real-world biological functional motions45. Although NMA is powerful, it is very time-consuming. In this study, we used Elastic Network Model (ENM)48, a coarse-grained NMA method, to compute the low-frequency motions of the RamR protein using the web-service DynOmics49. In addition, pair-wise residue correlation maps were constructed.Normal mode analysis (NMA) is commonly adopted to estimate dynamics based on structuresS. typhimurium as the template; it is an all-alpha helix structure consisting of two dimerization domains and two DBDs  from BDs Fig. . The sub33 to build the 3D coordinates of the RamR binding promoter (TATAATGAGTGCTTACTCACTCATAATC), followed by the HADDOCK35 service tool. Figure et al.27, the fragment from residues 2 to 42 of RamR was defined as the DBD region. In our predicted complex structure, residues A31, V32, A34, R35, A37, G38, V39, E41, T43, and R46 in both chain A and chain B were involved in DNA-binding. Figure To construct the RamR–DNA complex structures, we first used 3D-DARTThe trajectories from MD simulations of the WT- and MT-RamR–DBDs in the explicit solvent were calculated. The RMSD values of the Cα atoms for WT- and MT-RamR–DBDs with regard to the initial structures were plotted Fig. . The quaThe overall trend of the RMSF profiles of Y59H-, M84I-, and E160D-RamR was similar to that of WT-RamR, except for their DBDs, which exhibited different values than that of WT-RamR Figure . We calc40. The DNA-binding affinity differences (ΔΔGbind) were calculated according to ΔG1 (DNA-bound)–ΔG2 (DNA-free), as shown in the thermodynamic cycle  between WT- and Y59H-, M84I-, and E160D-RamR were 1.22, 8.55, and 23.82 kJ/mol, respectively .The RamR–DNA binding free energy differences between WT and MT structures were calculated in a series of alchemical free energy simulations and using the CGI protocolcle Fig. . Notablymol Fig. , for M84mol Fig. , and formol Fig. , respectely Fig. . The DNA48. The normal modes of WT-RamR and WT-RamR–DNA structures were calculated. For investigating the motional correlations between residues, we combined the 10 slowest modes to calculate the correlation heat maps for WT-RamR  is very time-consuming, we used the coarse-grained ENMamR Fig.  and WT-RamR Fig. . The covamR Fig. , which damR Fig. , particu26, might affect RamR’s binding affinity to the ramA promoter by altering the dynamics properties of the DBD. Previous study have suggested that amino acid residues Y59, M84, and E160 are considered to be involved in RamR substrate recognition and amino acid substitutions at these positions might reduce the DNA-binding affinity according to expand the distance between the N-terminal helix-turn-helix motifs in the DBD27. This inference could be also observed in our dynamics and binding free energy analysis, our studies demonstrate that these mutations could disrupt the RamR–DNA binding affinity through changing the dynamics networks of the RamR structure    Fig.  but this 0) Fig. . The E16Gdimer) between WT- and Y59H-, M84I-, and E160D-RamR were −3.03, −2.33, and 22.54 kJ/mol, respectively  (Fig. In addition, we also performed a simulation to analyze the RamR–RamR duplex dimerization free-energy changes upon these three mutations Figure . For Ramy Figure . Comparey Figure , but theIn this study, we have applied computational methods to understand the structural implications of Y59H, M84I, and E160D in RamR that are related to induce bacterial drug resistance. Our findings underscore that the Y59H, M84I, and E160D mutations may exhibit different behavior as compared to RamR WT, especially in DNA binding domain. In summary, our simulated results can properly in accordance with current study results, which demonstrated that amino acid substitutions of Y59H, M84I, and E160D in RamR might reduce its DNA-binding affinity.Supplementary figures


In 2009, a common set of questions addressing handwashing behavior was introduced into nationally representative Demographic and Health Surveys (DHS) and Multiple Indicator Cluster Surveys (MICS), providing large amounts of comparable data from numerous countries worldwide. The objective of this analysis is to describe global handwashing patterns using two proxy indicators for handwashing behavior from 51 DHS and MICS surveys conducted in 2010–2013: availability of soap anywhere in the dwelling and access to a handwashing place with soap and water. Data were also examined across geographic regions, wealth quintiles, and rural versus urban settings. We found large disparities for both indicators across regions, and even among countries within the same World Health Organization region. Within countries, households in lower wealth quintiles and in rural areas were less likely to have soap anywhere in the dwelling and at designated handwashing locations than households in higher wealth quintiles and urban areas. In addition, disparities existed among various geographic regions within countries. This analysis demonstrates the need to promote access to handwashing materials and placement at handwashing locations in the dwelling, particularly in poorer, rural areas where children are more vulnerable to handwashing-preventable syndromes such as pneumonia and diarrhea. Repeated episodes of diarrhea have been associated with poor growth outcomes and neurocognitive deficits.4–6 Recent evidence suggests that children living in households with relatively poor environmental conditions are more likely to have signs consistent with environmental enteropathy and growth faltering.7 Handwashing with soap can reduce the risk of diarrhea episodes by 30–47% and respiratory infections by 23%.8–12 The available evidence using structured observation methods in households in several low- and middle-income countries suggests that handwashing behavior must be improved substantially.13,14 However, in most countries where high child morbidity results from handwashing-preventable infections, there is little data on handwashing behavior. Though studies of household handwashing behavior have been conducted sporadically worldwide, there has been no systematic method of data collection to allow for comparison of handwashing behavior across regions, and identification of national and subnational populations where child mortality and morbidity remain high and where particular need exists with respect to handwashing promotion.15 Without meaningful and globally comparable data, it is difficult for governments and international organizations to prioritize handwashing as a public health tool.Pneumonia and diarrheal disease are leading causes of postneonatal child mortality, accounting for approximately 1.6 million child deaths worldwide in 2013.16,17 In surveys from the initial rounds (starting in 1985 for DHS and 1995 for MICS) questions on household indicators of handwashing were sporadically included in the core questionnaire, but these questions were not consistently included with standardized wording in every survey until 2009. Also, prior to 2009, questions addressed a range of self-reported and proxy measures, which were worded inconsistently, making it difficult to make comparisons regarding handwashing behaviors across countries and over time.18The Multiple Indicator Cluster Surveys (MICS) and Demographic and Health Surveys (DHS) are nationally representative household surveys supported by United Nations Children’s Fund, U.S. Agency for International Development, other development partners, and national governments in low- and middle-income countries. Administered about every 3–5 years in more than 100 countries, MICS and DHS collect information on a variety of indicators related to health, population, and nutrition. The use of core questionnaires allows for comparable data on such indicators across numerous countries and over serial surveys.19–22 and direct measurement of behavior by structured observation is infeasible in large surveys such as MICS and DHS because of the personnel time required.23 The available evidence suggests that the presence of a fixed place for washing hands with water and soap present at that place is a feasibly collected measure to describe handwashing behavior in large, nationally representative household surveys such as MICS or DHS.23–28 Members of households with soap and water available in a visible, convenient handwashing location wash their hands more frequently than those without handwashing materials at fixed locations.24Measurement of handwashing behavior by self-report risks substantial overestimation of the behavior,In 2009, a common set of questions was introduced to the core MICS and DHS questionnaires to document indicators of handwashing behavior: availability of soap anywhere in the dwelling (MICS), and access to water and soap at a place for handwashing (MICS and DHS). With the introduction of a standard set of handwashing questions in the MICS and DHS core questionnaires, we have for the first time a large set of comparable data on handwashing from numerous countries. Routine reporting from both survey networks includes disaggregation of data by potential sources of disparity and geographic regions within countries, allowing novel insight into differences affecting handwashing behavior and revealing potential targets for intensive handwashing promotion. Using data from 51 MICS and DHS surveys with comparable handwashing questions administered between 2010 and 2013, we describe global patterns in handwashing using two indicators: availability of soap anywhere in the dwelling and access to a handwashing place with soap and water. We describe handwashing across geographic regions, wealth quintiles, and rural and urban settings.In household MICS and DHS surveys, interviewers recorded the presence of soap and water at a place for handwashing by asking the respondent to “Please show me where members of your household most often wash their hands.” For households in which a place for handwashing was observed, interviewers recorded the presence of soap , other cleansing agents , and water. If a handwashing place could not be observed, interviewers recorded the reason for the nonobservation, including whether the household refused permission to observe the place. In households in which soap was not observed at a place for handwashing, interviewers in MICS surveys assessed the availability of soap anywhere in the dwelling by asking the respondent “Do you have any soap or detergent  in your household for washing hands?” If yes, the respondent was asked to show the soap to the interviewer.http://mics.unicef.org/tools#analysis). Handwashing-related data from MICS and DHS surveys conducted between 2010 and 2013 were compiled by UNICEF from published survey reports that applied the standard tabulation plans for those indicators. For the availability of soap anywhere in the dwelling, the proportion was calculated based on the entire set of households included in the sample. For the indicator of soap and water at the handwashing location, the standard tabulation plan excludes from the analysis households in which the respondent denied permission to see the place where household members wash their hands, households which do not have one specific place where members wash their hands, and households which have no handwashing place. For the analysis presented in this paper, we included in the denominator all households irrespective of whether the household had an observed handwashing location to portray a more representative picture of overall access to handwashing facilities, since some households simply did not have observable handwashing locations  region: Africa Region, Eastern Mediterranean Region, southeast Asia Region, European Region, Western Pacific Region, and the Region of the Americas.29 Wealth indices were used to categorize households within each survey population to wealth quintiles.We describe each indicator at the individual country level and by WHO region to understand differences within broader geographic areas. We also provide information on each indicator according to the following potential sources of disparity within each survey: wealth quintile, area . Among nationally representative surveys, we evaluated geographic disparities . As part of the standard analyses by the DHS and MICS programs, a wealth index was created for households within each survey dataset using data on household ownership of goods , housing characteristics , and basic services .Disaggregated data are not presented in the figures for countries in which fewer than 10% of households overall had soap and water at a handwashing location due to lack of heterogeneity in the total sample  and 4A–CBetween 2010 and 2013, 28 MICS and 23 DHS with standard handwashing indicators became available for analysis. Of the 51 surveys, three were from the Western Pacific Region, five from the southeast Asian Region, six from the Eastern Mediterranean Region, seven from the European Region, 25 from the Africa Region, and five from the Region of the Americas. We identified seven surveys that were conducted among subpopulations and, thus, were not nationally representative: the Roma population of Serbia , the Roma population of Bosnia and Herzegovina , south Madagascar , the Nyanza Province of Kenya , mid and far western Nepal , the Balochistan Region of Pakistan , and the Punjab Region of Pakistan . The total number of households in nationally representative surveys ranged from 4,223 in Equatorial Guinea to 43,852 in Indonesia, whereas the total number of households in subpopulation surveys ranged from 1,544 in the Roma population of Bosnia and Herzegovina to 95,238 in the Punjab Region of Pakistan .Supplemental Table 1).For households with a handwashing place observed by the interviewer, information was collected on whether soap and water were available at that place . In seveIn MICS surveys that included information on whether soap was available anywhere in the dwelling , availability of soap in the household ranged from 20.8% in Senegal to 99.1% in Iraq and Serbia . Eleven Across all surveys, Serbia had the highest proportion of households surveyed with soap and water at a handwashing place (96.4%) and Ethiopia had the lowest (less than 0.1%) . In fourIn almost every country, households in higher wealth quintiles were much more likely to have soap in the dwelling and places for handwashing with soap and water than those in lower wealth quintiles  and 4A, Within countries, households in urban areas were much more likely to have soap in the dwelling and a handwashing place with soap and water than households in rural areas  and 4B. The Gambia has the greatest disparity between geographic regions within a single country; 11.8% of households in the Kuntaur Region of the country were found to have soap anywhere in the household compared with 67.9% of households in the West Region  and 4C. There were also differences in disparities among countries within the same WHO region. For example, within the Western Pacific Region, the disparity in access to a handwashing place with soap and water across wealth quintiles was more evident in Cambodia (30.4–84.6%) than in Mongolia (78.1–99.2%) . Similar14 highlight the need to improve handwashing with soap globally to reduce the continued high burden of diarrhea and pneumonia morbidity and mortality. The overwhelming majority of under-five deaths occur in low- and middle-income countries, with about three-quarters of all child deaths occurring in sub-Saharan Africa and South Asia.30 Under-five mortality is highest in rural areas and among poorer and less educated communities.31 In particular, children in sub-Saharan Africa are nearly 14 times as likely to die before the age of five as children in high-income countries.30 A sizable proportion of these deaths are due to preventable causes including diarrhea and pneumonia, both of which can be reduced by handwashing with soap.1 The DHS and MICS surveys indicate that soap for handwashing is least likely to be found in households in the Africa Region. Moreover, fixed handwashing places with soap and water were infrequently observed in most African countries, along with some countries in southeast Asia, Western Pacific, and the Americas, indicating that promotion of handwashing as a preventive measure is not only necessary for lower-income countries, but also in higher-income countries where disparities still exist and could also have adverse sequelae.Our analyses of proxy measures of handwashing from 51 DHS and MICS household surveys, together with information from an analysis of 42 studies demonstrating that only 19% of people wash their hands after fecal contact when observed,Availability of soap for handwashing in the dwelling is a crude indicator of handwashing behavior, since the sheer presence of soap in a household does not mean that its residents actually wash their hands with that soap. In addition, since the MICS survey enumerators asked specifically to observe soap for handwashing, it is possible that households may have had soap available for purposes such as laundry and dishwashing but they have not prioritized its use for handwashing, or identified it as such. However, handwashing with soap is not possible if the material is entirely absent from the dwelling. The poorest households, and those in rural areas, may not have soap for handwashing for a variety of reasons, including cost of materials, access to materials in local shops and markets, and dynamic of decision-making power within household members that may determine purchase of soap and other health-related goods above other expenditures. Distribution of commercial goods to rural areas with poor road networks may be challenging and, thus, rural households might be less able to purchase soap for handwashing than those in urban areas. Additionally, rural regions may be not prioritized and given equal consideration by the companies controlling handwashing materials.32Low- or no-cost hand cleansing agents, such as ash, were rarely observed in households in any of the countries included in this analysis. Ash has been shown to be microbiologically effective in removing organisms from hands, although there are no data supporting or refuting the impact of ash use for handwashing on health outcomes.33 may close the gap in access to soap for handwashing in low-income households. The ease of soapy water preparation, its low cost, and equivalent antimicrobial efficacy may facilitate uptake, especially since these materials may be less likely to be stolen than bar soap.33 Soapy water is used at relatively small scale currently but should be considered for possible inclusion as a type of soap in future administrations of MICS and DHS.33,34 In addition, social marketing approaches with public–private partnerships and hygiene promotion may be useful to generate demand and increase affordability within the areas of greatest need.35 Successful behavior change interventions have yielded increases in handwashing behavior at the promoted times without the specific promotion of maintenance of handwashing materials36 at designated locations; however, adherence to the promoted behavior is predicated on having the materials available in the home.35,37Increasing the availability and promotion of affordable alternatives to bar soap, such as soapy water,13,38 The data supporting this indicator as a marker of handwashing behavior are largely observational in nature; multiple studies to validate proxies of handwashing behavior have shown that presence of water, soap, or both materials at the handwashing place were associated with observed increased handwashing behavior.23,24 The presence of water and a handwashing place was associated with a decrease in respiratory illness outcomes in one study in Bangladesh.27 Also in Bangladesh, better hand cleanliness was observed among members of households with soap available in the handwashing place used after defecation.28 In an analysis of similarly collected data on various self-reported and proxy measures of handwashing from Peru, Senegal, and Vietnam, the only indicator associated with observed handwashing with soap during structured observation in all three countries was the presence of soap and water at a handwashing location.24 However, promotion of maintenance of a handwashing station with materials may not result in substantial gains in handwashing behavior in the absence of other behavior change communication . Several randomized controlled trials of interventions combining the provision of handwashing materials with behavior change communication, promoting maintenance of materials at a fixed place along with handwashing with soapy water after defecation and before food preparation, are underway in Bangladesh and Kenya, and they should be informative regarding the behavioral and health effects of such a comprehensive behavior change approach.39Households may maintain a fixed location for handwashing and ensure the presence of soap and water at that location because they prioritize handwashing, or household members may wash their hands more frequently as a result of those materials being visible or conveniently located where and when they are needed, as evidenced by qualitative research in numerous countries.40 In our experience, in some countries in Africa, many households rely on mobile devices for handwashing. When hands need to be washed, the individual may move a jug, basin, and soap from inside the home to the outdoor courtyard to wash hands. There is no published information on the frequency of such mobile device use, nor is there information on whether the concepts of convenience and visual cueing operate differently in the societies in which the social norm is to keep handwashing materials at fixed locations where hands are washed. But given that the countries in which these fully stocked fixed handwashing locations were infrequently found also share high rates of child mortality from handwashing-preventable diseases such as diarrhea and pneumonia, gaining further understanding of the physical and social norm-related barriers to handwashing with soap in Africa is vital.The MICS and DHS data demonstrate broad global disparities in the maintenance of a fixed handwashing location with the materials needed to support handwashing. Although such fixed and fully stocked handwashing locations were found nearly universally in dwellings in most of the survey countries in the Eastern Mediterranean and European Regions, they were rarely confirmed in dwellings in most African countries included in the analysis. Similar to the general pattern shown here for Africa, Kamm and others found that fewer than 1% of households in Western Kenya were observed to have soap and water at a fixed location.19–23The handwashing indicators now included in the MICS and DHS describe household-level factors. Neither informs us directly about the behavior of individuals and when that behavior is practiced. From these existing indicators, we cannot know whether soap maintained in the home or at a fixed handwashing location is used for handwashing by the relatively healthy adolescent or by the mother of a particularly vulnerable neonate. From the household survey data, we cannot elucidate within-household disparities nor can we assess factors that may prevent some members of the household, for example, young children or the disabled, from accessing soap for handwashing. Structured observations can directly reveal the behaviors of individuals without proxy measures and describe within-household disparities, which are largely unstudied. But observation of handwashing behavior over multiple hours is simply not feasible within the already lengthy data collection involved in the MICS and DHSs.Our study has a few limitations. First, if the handwashing place was not in the dwelling/plot/yard, it was not observed and no information was collected on the presence of soap and water. Public places for handwashing with soap and water were, thus, not captured, potentially underestimating the availability of fixed handwashing locations. However, a fixed location for handwashing placed at some distance outside the household’s immediate environment could be inconvenient enough to discourage handwashing. Other reasons for lack of observation were not fully delineated in the survey instruments. The proportion of households not observed to have a handwashing station due to these “other reasons” was as high as 53% in Rwanda. Potential explanations could include use of portable devices for handwashing , miscommunication between the interviewer and respondent, and physical constraints . It is important to conduct confirmatory studies in such settings to understand the limitations of the indicator. For the purposes of this study, we deviated from standard tabulation plans which excluded households where handwashing locations were not observed. This study instead measured proportions out of the total number of households, which provides a more realistic representation of access to handwashing materials in a population, since in some cases, a high proportion of handwashing stations were not observed. Ideally, we recommend all nationally representative surveys include reasons for unobserved handwashing locations to parse out those households that did not have a fixed place for handwashing from households with places to wash hands, but for which permission was not granted for observation.The individual sources of disparity may be interconnected. The poorest households may also have the least educated heads of household and are more likely to live in rural areas. In this descriptive analysis, we are not adjusting for potential confounding among these various sources of disparity and availability of soap, or observation of soap and water at fixed handwashing locations. Further, estimating wealth principally on the basis of household goods risks masking other sources of disparity; however, the wealth index is universally applied in MICS and DHS analyses and represents a widely recognized metric for evaluating within-country inequities.41Handwashing with soap can substantially reduce the prevalence of pneumonia and diarrhea, two leading causes of child morbidity and mortality worldwide. Our analysis of data from 51 recent household surveys worldwide indicates a substantial need to increase availability of soap for handwashing, and to promote placement of soap and water at fixed handwashing places for many households to increase handwashing with soap. The need is particularly pressing among poorer households and households in rural areas where children may be at greatest risk for preventable mortality. However, this analysis also shows that disparities persist even in higher-income countries. The preventive potential of handwashing with soap was not addressed in the Millennium Development Goals, which expired in 2015. With their focus on equity and the incorporation of handwashing into Sustainable Development Goal target 6.2, the post-2015 Sustainable Development Goals have the potential to substantively level the access to preventive measures, such as soap for handwashing, to promote child survival, health, and growth.Supplemental Table.


Natural Killer cells (NK) are lymphocytes with the potential to recognize and lyse cells which escaped T-cell mediated lysis due to their aberrant HLA expression profiles. Killer cell immunoglobulin-like receptors (KIR) influence NK-cell activity by mediation of activating or inhibitory signals upon interaction with HLA-C  ligands. Therefore, absence of ligands for donor inhibitory KIRs following hematopoietic stem cell transplantation (HSCT) may have an influence on its outcome. Previous studies showed that C1 negative patients have a decreased HSCT outcome. Our study, based on a cohort of 200 C1-negative patients, confirmed these findings for the endpoints: overall survival , disease free survival , treatment related mortality , and relapse incidence  all being inferior when compared to C1-positive patients (n = 1246). Subsequent analysis showed that these findings applied for patients with myeloid malignancies but not for patients with lymphoproliferative diseases . Interestingly, within the C1-negative patient group, transplantation with KIR2DS2 resulted in better OS  as well as DFS , and transplantation with KIR2DS1 positive donors was associated with a decreased RI . TRM was increased when the donor was positive for KIR2DS1 . Our findings suggest that inclusion of KIR2DS1/2/5 and KIR3DS1-genotyping in the unrelated donor search algorithm of C1-ligand negative patients with myeloid malignancies may prove to be of clinical relevance. Haematopoietic stem cell transplantation (HSCT) has been established as a potentially curative treatment for a variety of haematologic diseases. However, malignancy relapse after HSCT remains the most frequent cause of treatment failure. TherefoPossibly, the most important KIR-ligand interaction is that between HLA-C antigens and their corresponding KIRs, in which both, inhibitory and activating, receptors are involved . HoweverIn this multicentre study, we evaluated the C-ligand impact on unrelated HSCT outcome in a large cohort of 1446 KIR & 5-loci-high resolution HLA-typed HSCT transplant pairs and thereby confirmed the C1-ligand negative patient cohort to be at higher risk than C1-positive patients. However, the incidence of patient C2 homozygosity is relatively low (in our cohort 14%). Transplantation with KIR B-haplotype positive donors has already been shown to increase HSCT success –31. KIR The study population included a total of 1446 patients transplanted for malignant disorders with T-cell repleted grafts from unrelated donors between 2000 and 2009. Benign diseases of the hematopoietic system as well as inborn genetic diseases were excluded. Patients with AML (acute myeloid leukemia), ALL (acute lymphoblastic leukemia), AL (unclassified acute leukemia), CML (chronic myeloid leukemia), CLL (chronic lymphoblastic leukemia), MDS (myelodysplastic syndrome), and MM (multiple myeloma) were eligible. Disease stages were adopted from a previous report published by the European Society for Blood and Marrow Transplantation (EBMT) study group: Early d2, and/or busulfan equal or greater than 16 mg/kg[Graft source was either peripheral blood stem cells or bone marrow. Only first allogeneic transplantations were included in the analysis. Transplant pairs were either 10/10 matched or single HLA-A, -B, or -C mismatched. All patients received myeloablative or reduced intensity conditioning. Myeloablative conditioning regimen was defined according to the EBMT recommendations as treatment with total body irradiation equal or above 10 Gy and/or cyclophosphamide equal or greater than 120 mg/m 16 mg/kg;40. TreaDonor-recipient pairs from unrelated donor searches completed before May 2005 were low resolution typed for HLA-A and -B, and high resolution typed for HLA-DRB1 and -DQB1. Stored DNA material of these individuals was retrospectively high resolution typed for HLA-A, -B, and -C. For unrelated donor searches which were initiated after 05/2005 5-loci high resolution HLA-typing was carried out upfront.High resolution HLA-typing was performed by sequence based typing (SBT) using a CE-certified in-house Kit for HLA-class I  and by in-house SBT  or Luminex®  PCR-sequence specific oligonucleotide probe (PCR-SSO) method for HLA-class II. Exons 2 and 3 were sequenced for HLA-class I typing, and exon 2 was analysed for HLA-class II testing. Additional typing for relevant non-expressed alleles was performed according to the National Marrow Donor Program confirmatory typing requirements.KIR-typing was performed using the commercially available “KIR Genotyping SSP Kit” from Thermo Fisher Scientific . The Kit uses sequence specific primer PCR to assign presence or absence, respectively, of a given KIR gene. The method allows the identification of the following KIR-genes: KIR2DL1/2/3/4/5A/5B, KIR2DS1/2/3/4/5, KIR3DL1/2/3, KIR3DS1, KIR2DP1, and KIR3DP1.KIR ligand status of patients was obtained from high resolution HLA-typing. HLA-C molecules were grouped according to the presence of epitopes recognized by KIRs: HLA-C antigens with an asparagine residue at amino acid sequence position 80 possess the C1 epitope, which acts as ligand for the inhibitory KIR2DL2 and KIR2DL3 as well as for the activating KIR2DS2. Accordingly, HLA-C antigens with a lysine residue at this position possess the C2 epitope, which acts as ligand for the inhibitory KIR2DL1 as well as for the activating KIR2DS1.The absence of C1 or C2 in patients who were transplanted with a KIR2DL2/3 or KIR2DL1 positive donor, respectively, was considered as “missing ligand”. Control patient groups carried at least one ligand for any donor KIR2DL1 or KIR2DL2/3, respectively.All statistical analyses were performed using the open source program for statistical computing “R”, version 3.0.2. Cumulative estimates of overall survival (OS) and disease free survival (DFS) were obtained using Kaplan-Meier analysis. Extended Cox regression models allowing for time-dependent covariates were used to conduct multivariate analyses of OS and DFS. Univariate analyses of relapse incidence (RI) and treatment related mortality (TRM) were performed using competing risk analysis. Competing risk regression models for stratified data were used for multivariate analysis of RI and TRM. The categorical variable diagnosis showed different baselines of hazard for the respective patient sub-groups. Hence, for both multivariate models stratification for diagnosis was accomplished rather than integrating diagnosis as a covariate;43. CentGeneral patient characteristics are shown in Distribution of KIR2DL1/2/3 and HLA-C-ligands among patients and donors is shown in Patients who did not possess the C1 ligand (i.e. C2 homozygous) showed significantly inferior OS and DFS after 1, 3 and 5 years post transplantation  when comA significant impact of the missing ligand model described in previous studies was solely found for myeloid malignancies. The four endpoints were therefore re-analysed in subgroups of patients with myeloid and lymphoblastic diseases, respectively.The protective effect of C1 could be observed only in the myeloid group , where aC1-negative patients with myeloid malignancies were thus identified as being at risk and therefore this patient group is referred as such in subsequent analyses.Missing C2-ligand does not affect HSCT outcome as shown by Kaplan-Meier analysis Figs –8 and muSince C1-deficiency was shown to negatively affect HSCT outcome in patients with myeloid, but not lymphoblastic, malignancies, following analyses were conducted in the myeloid patient group, only.As KIR2DS4 is present in almost all individuals and is expressed on both, A- and B-Haplotypes, this KIR gene was not taken into consideration for any analyses in our study. Additionally, OS and DFS analyses were conducted in subgroups of 10/10 and 9/10 matched transplants.OS and DFS were significantly improved when the donor was positive for KIR2DS2 and the transplant pair was mismatched at a single HLA-class I gene: The OS rates were twice as high as in the 9/10 matched patient group with a KIR2DS2 negative donor  or with stratification for HLA matching .Transplantation with KIR2DS1 positive donors significantly reduced RI in C1-negative patients  , Table 8The results of the multivariate TRM analyses are summarised in et al, that C1-positive, but not C1-negative patients benefit from transplantation with donors who carry two or more B-motifs. We used the B-content calculator that can be found at http://www.ebi.ac.uk/ipd/kir/donor_b_content.html to assign the donors of our cohort accordingly to one of three groups based on KIR B-content—neutral (n = 142), better (n = 40) or best (n = 18)—and analysed the impact of this classification on the transplantation outcome of the C1-negative patients. As case numbers in the “best” group were low, we decided to combine the “better” and the “best” donors into the “better/best” group. Multivariate analyses showed that in 9/10 matched transplantations, “better/best” donors may improve DFS , but had no significant effect on OS , RI , or TRM . In 10/10 matched transplant pairs, however, the “better/best” KIR donor status had no effect on transplantation outcome .The KIR locus is divided into a centromeric and a telomeric region, each bearing variable KIR gene content motifs designated as cen-A, cen-B, tel-A, and tel-B. The B-motifs contain one or more of the seven KIR B-haplotype specific genes. It has been shown by Cooley If our findings prove to be correct, and can be confirmed by others, then a possible selection algorithm of donors for C1-negative patients with myeloid malignancies could be as displayed in allelefrequencies.net[et al.[et al.[et al.[The missing ligand model is based on the premise that underrepresentation of inhibiting signals will increase donor NK-alloreactivity against remaining malignant cells in the patient. As already described in the results section, all patients and donors within our cohort where positive for at least one inhibitory KIR that binds to HLA-C ligands. These results are consistent with the KIR-frequencies in Caucasoid and German populations provided by ncies.net. We subset[et al. . In this context, small subgroups were inevitable. However, while our study was limited by small sample sizes it nonetheless demonstrated statistically significant findings with a robust effect size.S1 FigDashed red line: C1-negative patients (n = 200), fine red lines: corresponding confidence intervals. Solid black line: C1-positive patients (n = 1239), fine black lines: corresponding confidence intervals. p = 0.0014.(TIFF)Click here for additional data file.S2 FigDashed red line: C1-negative patients (n = 200), fine red lines: corresponding confidence intervals. Solid black line: C1-positive patients (n = 1239), fine black lines: corresponding confidence intervals. p = 0.006.(TIFF)Click here for additional data file.S3 FigDashed red line: C1-negative patients (n = 200), fine red lines: corresponding confidence intervals. Solid black line: C1-positive patients (n = 1239), fine black lines: corresponding confidence intervals. p = 0.04.(TIFF)Click here for additional data file.S4 FigDashed red line: C1-negative patients (n = 200), fine red lines: corresponding confidence intervals. Solid black line: C1-positive patients (n = 1239), fine black lines: corresponding confidence intervals. p = 0.06.(TIFF)Click here for additional data file.S5 FigDashed red line: C2-negative patients (n = 823), fine red lines: corresponding confidence intervals. Solid black line: C2-positive patients (n = 558), fine black lines: corresponding confidence intervals. p = 0.08.(TIFF)Click here for additional data file.S6 FigDashed red line: C2-negative patients (n = 823), fine red lines: corresponding confidence intervals. Solid black line: C2-positive patients (n = 558), fine black lines: corresponding confidence intervals. p = 0.11.(TIFF)Click here for additional data file.S7 FigDashed red line: C2-negative patients (n = 823), fine red lines: corresponding confidence intervals. Solid black line: C2-positive patients (n = 558), fine black lines: corresponding confidence intervals. p = 0.47.(TIFF)Click here for additional data file.S8 FigDashed red line: C2-negative patients (n = 823), fine red lines: corresponding confidence intervals. Solid black line: C2-positive patients (n = 558), fine black lines: corresponding confidence intervals. p = 0.30.(TIFF)Click here for additional data file.S9 FigSolid black line: donor KIR2DS2-negative, fine black lines: corresponding confidence intervals. Dashed red line: donor KIR2DS2-positive, fine red lines: corresponding confidence intervals. 10/10 matched transplant pairs, donor KIR2DS2-negative (n = 32) vs. donor KIR2DS2-positive (n = 45), p = 0.86.(TIFF)Click here for additional data file.S10 FigSolid black line: donor KIR2DS2-negative, fine black lines: corresponding confidence intervals. Dashed red line: donor KIR2DS2-positive, fine red lines: corresponding confidence intervals. 9/10 matched transplant pairs, donor KIR2DS2-negative (n = 22) vs. donor KIR2DS2-positive (n = 18), p = 0.04.(TIFF)Click here for additional data file.S11 FigSolid black line: donor KIR2DS2-negative, fine black lines: corresponding confidence intervals. Dashed red line: donor KIR2DS2-positive, fine red lines: corresponding confidence intervals. 10/10 matched transplant pairs, donor KIR2DS2-negative (n = 32) vs. donor KIR2DS2-positive (n = 45), p = 0.88.(TIFF)Click here for additional data file.S12 FigSolid black line: donor KIR2DS2-negative, fine black lines: corresponding confidence intervals. Dashed red line: donor KIR2DS2-positive, fine red lines: corresponding confidence intervals. 9/10 matched transplant pairs, donor KIR2DS2-negative (n = 22) vs. donor KIR2DS2-positive (n = 18), p = 0.05.(TIFF)Click here for additional data file.S13 FigSolid black line: donor KIR2DS1 negative, fine black lines: corresponding confidence intervals. Dashed red line: donor KIR2DS1 positive, fine red lines: corresponding confidence intervals. Donor KIR2DS1-negative (n = 72) vs. donor KIR2DS1-positive (n = 45), p = 0.008.(TIFF)Click here for additional data file.S14 FigSolid black line: donor KIR2DS2 negative, fine black lines: corresponding confidence intervals. Dashed red line: donor KIR2DS2 positive, fine red lines: corresponding confidence intervals. Donor KIR2DS2-negative (n = 54) vs. donor KIR2DS2-positive (n = 63), p = 0.33.(TIFF)Click here for additional data file.S15 FigSolid black line: donor KIR2DS5 negative, fine black lines: corresponding confidence intervals. Dashed red line: donor 5 positive, fine red lines: corresponding confidence intervals. Donor KIR2DS5-negative (n = 83) vs. donor KIR2D5-positive (n = 34), p = 0.05.(TIFF)Click here for additional data file.S16 FigSolid black line: donor KIR3DS1 negative, fine black lines: corresponding confidence intervals. Dashed red line: donor KIR3DS1 positive, fine red lines: corresponding confidence intervals. Donor KIR3DS1-negative (n = 72) vs. donor KIR3DS1-positive (n = 45), p = 0.06.(TIFF)Click here for additional data file.S17 FigSolid black line: donor KIR2DS1 negative, fine black lines: corresponding confidence intervals. Dashed red line: donor KIR2DS1 positive, fine red lines: corresponding confidence intervals. Donor KIR2DS1-negative (n = 72) vs. donor KIR2DS1-positive (n = 45), p = 0.06.(TIFF)Click here for additional data file.S18 FigSolid black line: donor KIR2DS5 negative, fine black lines: corresponding confidence intervals. Dashed red line: donor KIR2DS5 positive, fine red lines: corresponding confidence intervals. Donor KIR2DS5-negative (n = 83) vs. donor KIR2D5-positive (n = 34), p = 0.45.(TIFF)Click here for additional data file.S19 FigSolid black line: donor KIR2DS2 negative, fine black lines: corresponding confidence intervals. Dashed red line: donor KIR2DS2 positive, fine red lines: corresponding confidence intervals. Donor KIR2DS2-negative (n = 54) vs. donor KIR2DS2-positive (n = 63), p = 0.001.(TIFF)Click here for additional data file.S20 FigSolid black line: donor KIR3DS1 negative, fine black lines: corresponding confidence intervals. Dashed red line: donor KIR3DS1 positive, fine red lines: corresponding confidence intervals. Donor KIR3DS1-negative (n = 72) vs. donor KIR3DS1-positive (n = 45), p = 0.35.(TIFF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Several recent reports have demonstrated that the preoperative sensitivity and accuracy of identifying and locating non-palpable testes increases with the use of conventional MRI, in addition to diffusion-weighted imaging (DWI). Therefore, pre-operative prediction of the presence and location of testes using imaging techniques may guide management of intra-abdominal testis. Fowler-Stephens orchiopexy is effective for treating patients with intra-abdominal testis; however, long-term testicular function after this procedure has not been clarified. We present a case of a high intra-abdominal testis located below the kidney, and discuss the usefulness of fusion view with T2-weighted and DWI images to make a diagnosis of high intra-abdominal testis and the pathological findings to predict future fertility potential.A 10-month-old boy was referred to the urology department for the management of non-palpable testis. We employed not only conventional MRI, but also DWI, to improve the diagnostic accuracy of non-palpable testes by MRI examination. The high-intensity mass-like structure below the kidney on the T2-weighted image and the markedly high signal intensity mass on the DWI image completely matched, which suggested that the mass below the kidney was the right testis. The patient underwent diagnostic and therapeutic laparoscopy. A testis was found under the ascending colon, 1 cm below the right kidney. We performed 2-stage Fowler-Stephens orchiopexy. The testis could be delivered to the scrotum without any tension. We examined expression patterns of the stem cell marker, undifferentiated embryonic cell transcription factor 1 (UTF1) in the testicular biopsy sample, and demonstrated that the UTF1-positive Ad spermatogonia / negative Ad spermatogonia ratio was lower in this patient than in boys his age with descended and inguinal undescended testes, indicating that spermatogonial stem cell activity may decrease remarkably in this boy.Fusion view with T2-weighted and DWI images may be a useful diagnostic modality for high intra-abdominal testes. Fowler-Stephens orchiopexy may provide blood supply to the testis but that might not be enough to achieve spermatogenesis. The advent of laparoscopy has dramatically changed the diagnosis and treatment of non-palpable testis in children. Laparoscopy is an accepted procedure to investigate the presence and location, and to perform subsequent treatment, of intra-abdominal testis. Several recent reports have demonstrated that the preoperative sensitivity and accuracy of identifying and locating non-palpable testes increases with the use of conventional MRI, in addition to diffusion-weighted imaging (DWI) , 2. TherA 10-month-old boy was referred to the urology department for the management of non-palpable testis. There was no significant familial or past history. A clinical examination demonstrated a left, well-positioned testis, 16 × 11 × 11 mm in size, and an empty right scrotum. No abnormality of the external genitalia was found. Physical examination could not confirm the presence of the right testis.Ultrasonography could not identify the right testis in the abdomen or inguinal region. We employed not only conventional MRI, but also DWI, to improve the diagnostic accuracy of non-palpable testes by MRI examination. The T2-weighted image showed a high-intensity mass-like structure, which was difficult to distinguish from the surrounding fat tissue, below the right kidney. Furthermore, DWI showed a mass, at that position, of markedly high signal intensity. The T2-weighted image was fused with the DWI image, using medical image viewer software , to identify the anatomical location. The high-intensity mass-like structure on the T2-weighted image and the markedly high signal intensity mass on the DWI image completely matched Fig. , which sn = 5; Fig. n = 5) and in inguinal undescended testes   Fig. . This waTen months after the second surgery, the right testis, 13 × 8 × 7 mm in size, was located in the scrotum, and good vascularization was detected on echo color Doppler ultrasound.Diagnostic modalities, such as ultrasound and MRI, are nonspecific in the intra-abdominal testis, and the accuracy of these examinations was not satisfactory. The only effective diagnostic procedure is considered to be laparoscopy. However, there is a possibility of being unable to locate a testis on diagnostic laparoscopy . An accuPrevious reports demonstrated that the success rate of Fowler-Stephens orchiopexy, which was assessed by whether the testis was returned to the scrotum or the testicular size, was 69–95% , 9. A reWe presented a case of a high intra-abdominal testis located below the kidney. Fusion view with T2-weighted and DWI images may be a useful diagnostic modality for high intra-abdominal testes. Fowler-Stephens orchiopexy may provide blood supply to the testis but that might not be enough to achieve spermatogenesis; therefore, further studies are needed.


Autophagy removes both functional and damaged intracellular macromolecules from cells via lysosomal degradation. Three autophagic mechanisms, namely macroautophagy, chaperone-mediated autophagy (CMA), and microautophagy, have been described in mammals. Studies in experimental systems have found macroautophagy and CMA to decrease with normal aging, despite the fact that oxidative stress, which can activate both processes, increases with normal aging. Whether autophagic mechanisms decrease in the human brain during normal aging is unclear. The primary objective of this study was to examine the association of a major autophagy protein, lysosome-associated membrane glycoprotein (lamp2), with age in cerebrospinal fluid (CSF) samples from healthy subjects. Lamp2 consists of three isoforms, lamp2a, 2b and 2c, all of which participate in autophagy. Lamp2’s CSF concentration decreases in Parkinson’s disease (PD) and increases in Alzheimer’s disease (AD), but whether its CSF concentration changes during normal aging has not been investigated. Our secondary objectives were to examine the associations of lamp2’s CSF concentration with CSF levels of the molecular chaperone heat shock 70-kDa protein (HSPA8), which interacts with lamp2a in CMA, and oxidative stress markers 8-hydroxy-2′-deoxyguanosine (8-OHdG), 8-isoprostane (8-ISO) and Total Antioxidant Capacity (TAC) in healthy subjects. We found lamp2’s observed associations with these variables to be weak, with all Kendall’s tau-b absolute values ≤0.20. These results suggest that CSF lamp2 concentration changes little during normal aging and does not appear to be associated with HSPA8 or oxidative stress. Further studies are indicated to determine the relationship between CSF lamp2 concentration and brain autophagic processes. Proteostasis involves regulation of protein transcription, translation, folding, trafficking, processing, assembly/disassembly, localization, and degradation  for monitoring brain autophagy. Beclin-1, p62 and LC3-II have been suggested as macroautophagy biomarkers  biospecimen collection procedures  from Cloud-Clone Corp. . The lower limit of detection for lamp2 in the ELISA kit was stated by the manufacturer to be 26.2 pg/mL. The standard curve in the assay ranged from 62.5 pg/mL to 4000 pg/mL. The lamp2 concentration of each sample was measured in duplicate after diluting the sample with an equal volume of 0.01 M PBS buffer, pH 7.2; the lamp2 concentrations used in statistical analyses were the means of these duplicate measurements. The standard curve was generated using Softmax Pro software  using the log-log function as recommended by the manufacturer, and the concentration of lamp2 in each sample was calculated by Softmax. One sample produced a mean optical density value which was below the value for the lowest point on the standard curve; for statistical purposes, the sample was assigned a concentration of 26.2 pg/mL, the lower limit of sensitivity for the assay. After accounting for the dilution factor, the concentration of the sample was listed as 52.4 pg/mL.t-tests. Original (non-transformed) lamp2 values were used for Kendall’s tau-b calculations, while log-transformed lamp2 values were used for scatterplots and gender comparisons. Statistical analysis used The SAS System for Windows version 9.3  and Minitab Release 14  was used for graphs.Measures were summarized with means ± SDs for variables which were normally distributed, and with medians and ranges for variables which were not normally distributed. Lamp2 values were distributed abnormally (strongly skewed right with extreme outliers); natural-log transformation produced a reasonably normal distribution. Kendall’s rank-correlation coefficient  with associated 95% confidence intervals (CI), and scatterplots with best-fit regression lines and locally weighted scatterplot smoother (LOWESS) curves were used to examine lamp2’s associations with age, HSPA8, 8-OHdG, 8-ISO and TAC. Differences for lamp2 concentrations between male and female subjects were examined with b = 0.16; 95% CI for tau-b = ) and with the other variables . The 95% CIs for lamp2’s associations with age  and with 8-OHdG  do not rule out the possibility of moderate positive associations.Numeric summaries for age and lamp2, HSPA8, 8-OHdG, 8-ISO, and TAC concentrations are shown in Table The scatterplot of log-transformed lamp2 vs. age Figure  indicatep = 0.80). Because the male study subjects tended to be younger than the female study subjects, the gender comparison was limited to subjects ≥40 years old, namely 16 females (mean age 60.3) and nine males (mean age 60.1).No significant differences were found for CSF lamp2 concentration between genders (R2 = 1.4%). Whether the lack of age-related changes in CSF lamp2 reflects a similar lack of change in brain lamp2 and/or brain autophagic processes during normal aging is unknown; such a determination would require measurement of lamp2 on lysosomal membranes, as well as assessment of CMA and macroautophagy activities, in normal brain specimens across a wide age range.The main finding in this study was that the association between CSF lamp2 concentration and age appeared to be weak in healthy subjects. The scatter plot for the distribution of log-transformed lamp2 vs. age Figure  indicateBecause the concentration of lamp2 is the sum of the concentrations of all three lamp2 isoforms, the lack of change that we found in CSF lamp2 concentration during normal aging does not necessarily indicate a lack of change in the concentrations of its isoforms. A study measuring mRNA for lamp2’s isoforms in normal human anterior cingulate and occipital cortex  might have increased our ability to detect age-related changes in lamp2.Recent studies have indicated a relationship between autophagic processes and lipid metabolism. In experimental models, lipid loading exerts inhibitory effects on both macroautophagy and CMA in hepatocytes (Koga et al., We conclude that CSF lamp2 concentration appears to change little during normal aging, and it appears to be poorly associated with CSF concentrations of HSPA8 and of oxidative stress markers. Further studies are indicated to clarify the effects of aging on autophagic processes in the human brain and to identify CSF biomarkers for these processes.DAL directed the study and prepared the original and revised manuscript. ACK performed the assays, collated the data and reviewed the manuscript. MPC performed the statistical analysis and assisted with manuscript revisions. JOA recruited the patients, collected the CSF samples and reviewed 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.


A recent study showed that posterior circulation plaques have a greater capacity for positive remodeling in a non-Asian population. We aimed to investigate if the features of plaques in the middle cerebral artery (MCA) were different from those in the basilar artery (BA) in a northern Chinese population. We retrospectively analysed the records of 71 consecutive patients with acute ischemic stroke. All patients had at least one MCA or BA plaque with early or mild (<50% stenosis) atherosclerosis identified using vessel wall magnetic resonance imaging. The remodeling ratio, eccentricity index, and plaque range were compared between MCA and BA plaques using multilevel analysis. A total of 101 plaques were included. There were 70 plaques located in the MCA and 31 plaques located in the BA. The features of non-advanced atherosclerotic plaques did not differ between the MCA and BA when accounting for the degree of stenosis or plaque burden in a northern Chinese population. Symptomatic plaques were associated with a higher eccentricity index and smaller plaque range than asymptomatic plaques under the same plaque burden. Further studies are warranted to investigate the progression of atherosclerosis in different intracranial arteries. Intracranial atherosclerotic disease is one of the important causes of ischemic stroke or transient ischemic attack, especially in Asian, Hispanic and African populations14567Atherosclerosis is a systemic disease that may involve several vascular beds, including coronary, peripheral, and renal arteries in addition to the cerebral arteries. However, a previous study showed that the carotid artery has a pattern of remodeling that was distinct from that of the femoral artery, which may be caused by differences in anatomy and blood flowA total of 71 patients with 101 plaques were included in this study. There were 70 plaques located in the MCA and 31 plaques located in the BA. Among the 70 MCA plaques, 20 were classified as symptomatic, 6 were indeterminate, and 44 were asymptomatic. Among the 31 BA plaques, 11 were symptomatic, 12 were indeterminate, and 8 were asymptomatic . The inter-observer agreements (intraclass correlation coefficient) for wall area (WA), lumen area (LA), minimum wall thickness, maximum wall thickness, and plaque range were 0.93, 0.92, 0.89, 0.90, and 0.86, respectively.The results of the null model showed that about 38% of the variance in remodeling ratio stems from differences between patients, clearly indicating that a multilevel model was required. The presence of significant between-group heterogeneity (P = 0.035) also indicated that a multilevel model was required see .The level 2 explanatory variables of age, sex, hypertension, diabetes mellitus, dyslipidaemia, smoking, and history of coronary heart disease were added to the null model respectively. When these variables were added to the null model, the intercept kept almost unchanged, which suggested that the between-group variation could not be explained by the level 2 explanatory variables.Level 1 explanatory variables, including the classification of plaque, location of plaque (MCA or BA), and degree of stenosis, were added to the null model. The results showed that the degree of stenosis was significantly associated with the remodeling ratio (P < 0.001) . The locThe results of the null model indicated that a multilevel model was required for the outcome measures of the eccentricity index see . The samThe results of the null model indicated that a multilevel model was required for the plaque range see . The levEight MCA plaques and two BA plaques were excluded because the degree of stenosis was ≥ 50%. An additional analysis was performed including these 10 plaques. By using multilevel model, this study found that the remodeling ratio, eccentricity index, and plaque range were not significantly different between MCA and BA plaques with the same degree of stenosis or plaque burden in a northern Chinese population. Symptomatic plaques were associated with a higher eccentricity index and smaller plaque range than asymptomatic plaques.The multilevel model, also referred to as mixed-effects models, random coefficient models, random effects models, or variance component models, has become a popular method in many fields of research that have nested data structures11et al. recently reported that posterior circulation plaques have a higher remodeling ratio than anterior circulation plaques in a non-Asian populationBy using multilevel model, Qiao The formula deduced from the remodeling model see  and data9et al. reported that culprit plaques were associated with positive remodeling when compared with both non-culprit and indeterminate plaques or non-culprit plaques alone. However, they reported that the association became non-significant after adjusting for the plaque burdenPrevious studies showed that positive remodeling is associated with symptomatic plaque; however, this was not confirmed in our study14in vitro study showed that eccentric plaques were associated with a non-significant higher risk of cerebrovascular events compared with concentric plaques Eccentricity is another important feature of atherosclerotic plaques. Previous studies showed that for carotid plaques, larger maximum wall thickness and eccentric plaques were associated with the occurrence of subsequent cerebrovascular events1618We also introduced a new parameter related to plaques, the plaque range. We found that symptomatic plaques had a smaller plaque range than asymptomatic plaques after adjusting for the plaque burden. However, a recent published study showed that plaques with <50% wall involvement were significantly associated with a higher possibility of asymptomatic plaques than plaques with >50% wall involvementThere are several limitations of this study. First, a 2D technique may lead to overestimation of thickness measurements because of partial volume effectsIn conclusion, by using multilevel model, this study found that the remodeling ratio, eccentricity index, and plaque range were not significantly different between non-advanced MCA and BA plaques after controlling for the degree of stenosis or plaque burden in a northern Chinese population. Symptomatic plaques were associated with a higher eccentricity index and smaller plaque range than asymptomatic plaques. A prospective study to investigate the progression of MCA and BA atherosclerosis may further answer the question of whether plaque features are different between MCA and BA plaques.We retrospectively collected consecutive patients who were admitted to the Department of Neurology of the First Hospital of Jilin University between January 2015 and January 2016 with the following characteristics: (1) patients with acute ischemic stroke confirmed by diffusion-weighted imaging within 7 days of symptom onset; (2) patients with atherosclerotic plaques in the MCA or BA that were identified using VWMRI; (3) patients with at least two vascular risk factors18Since almost all of the VWMRI examinations were performed on a 3 T Siemens Trio MR scanner  between January 2015 and January 2016, the only qualifying patient that was examined using the Achieva 3 T scanner  was excluded. The VWMRI protocol included 3D time-of-flight MR angiography , 3D T1-weighted SPACE (Sampling Perfection with Application optimized Contrast using different angle Evolutions) , and 2D T2-weighted VWMRI . Two-dimensional T2-weighted VWMRI was obtained perpendicular to the long axis of the BA (approximately 13 slices) or the M1 segments of the relevant MCA (11 slices) and contralateral MCA (11 slices). A 12-channel head coil was used for the above sequences.Each plaque was classified as either symptomatic, indeterminate, or asymptomatic according to criteria adapted from previous studies10Images from 2D T2-weighted VWMRI were used to perform all measurements. For each atherosclerotic lesion, the cross-sectional slice with the highest degree of stenosis was selected to measure the parameters. For patients with more than one plaque in the same artery, all of the plaques were analyzed. A normal slice (i.e. without plaque on VWMRI) proximal to the selected slice was chosen as the reference site. If there was no such slice, a normal slice distal to the selected slice was chosen as the reference site1415The plaque measurements were performed by two experienced neuroradiologists, each with approximately 3 years of VWMRI reading experience. Quantitative plaque measurement software  was used to measure the parameters including LA, WA, outer wall area (OWA = LA + WA), minimum wall thickness, and maximum wall thickness  and 2. Tlesion/WAreference was used as the plaque burden in our study (23lesion) to the OWA of the reference site (OWAreference)  . The eccference) 5.We loaded the images of lesions to a DICOM viewer . The distribution range of the plaque was calculated using the angle tool  and 2. TQuantitative data are expressed as mean ± SD. Qualitative data are expressed as the number and percentages. For quantitative data, two-sample Student’s t  or Mann–Whitney U tests were used. The chi-squared or Fisher’s exact tests were used for qualitative data. The eccentricity index, remodeling ratio, and plaque range were compared between the MCA and BA plaques using multilevel model to adjust for the effect of including more than one lesion from a single patientHow to cite this article: Niu, P.-P. et al. Vessel wall differences between middle cerebral artery and basilar artery plaques on magnetic resonance imaging. Sci. Rep.6, 38534; doi: 10.1038/srep38534 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Corynebacterium striatum is an opportunistic pathogen, often multidrug-resistant, which has been associated with serious infections in humans. Aminoglycosides are second-line or complementary antibiotics used for the treatment of Corynebacterium infections. We investigated the susceptibility to six aminoglycosides and the molecular mechanisms involved in aminoglycoside resistance in a collection of 64 Corynebacterium striatum isolated in our laboratory during the period 2005–2009. Antimicrobial susceptibility was determined using E-test. The mechanisms of aminoglycoside resistance were investigated by PCR and sequencing. The 64 C. striatum were assessed for the possibility of clonal spreading by Pulsed-field Gel Electrophoresis (PFGE). Netilmicin and amikacin were active against the 64 C. striatum isolates . Twenty-seven of the 64 C. striatum strains showed a MIC90 for kanamycin > 256 mg/L, and 26 out the 27 were positive for the aph(3’)-Ic gene. Thirty-six out of our 64 C. striatum were streptomycin resistant, and 23 out of the 36 carried both the aph(3”)-Ib and aph(6)-Id genes. The gene aac(3)-XI encoding a new aminoglycoside 3-N acetyl transferase from C. striatum was present in 44 out of the 64 isolates, all of them showing MICs of gentamicin and tobramycin > 1 mg/L. CS4933, a C. striatum showing very low susceptibility to kanamycin and streptomycin, contains an aminoglycoside resistance region that includes the aph(3’)-Ic gene, and the tandem of genes aph(3”)-Ib and aph(6)-Id. Forty-six major PFGE types were identified among the 64 C. striatum isolates, indicating that they were mainly not clonal. Our results showed that the 64 clinical C. striatum were highly resistant to aminoglycosides and mostly unrelated. Corynebacterium consists of more than 100 species . Corynebacterium spp. are widely distributed in the environment. Pathogenic Corynebacterium species include Corynebacterium diphtheriae and nondiphtheroid Corynebacterium. Nondiphtheroid corynebacteria are part of the normal skin and mucous membranes microbiota of humans. They have traditionally been considered as sample contaminants when isolated from clinical stuffs. However, nondiphtheroid Corynebacterium are actually recognized as causing opportunistic disease , or Enterobacteriae  by PCR using primers specific for each gene , 1.5 mM MgCl2, 5 μl of 10× PCR amplification buffer , 40 pmol of each primer, 0.2 mM each deoxynucleoside triphosphate (Bioline), and double-distilled water to a final volume of 50 μl. DNA was first denatured at 95°C for 5 min and then subjected to 30 cycles of amplification using a Perkin Elmer model 9600 thermal cycler under the following conditions: denaturation at 95°C for 1 min, annealing for 1 min, and extension at 72°C for 1 min. After the final cycle, the reactions were terminated by an extra run at 72°C for 10 min, and the reaction products were then kept at 4°C until analysis. Amplification products were characterized by electrophoresis in 1.5% agarose using 0.5x TBE running buffer. PCR products were cleaned of amplification primer using the QIAquick PCR Purification kit  following the manufacturer’s instructions. Purified DNA was sequenced with the primers outlined in The 64 ach gene  and by sC. striatum CS4933 whole DNA was prepared from cultures grown on blood agar plates overnight. Bacterial cells were mixed with 2 ml of distilled water poured on the plate, transferred to a 2-ml Eppendorff tube, collected by centrifugation at 10,000g for 4 min, re-suspended in 0.25 ml of Tris-EDTA buffer containing 20 mg of lysozyme/ml and 50 mg of proteinase K/ml, and incubated at 37°C for 1 h. Bacterial cells were then lysed by the addition of 0.25 ml of 0.1 M Tris containing 1% sodium dodecyl sulfate (SDS) and 400 μg/ml of proteinase K and incubated at 55°C for 1 h. The lysate was mixed with 0.1 ml of 5 M NaCl and 100 μl of cetyltrimethylammonium bromide-NaCl and incubated at 65°C for 10 min. DNA was then extracted with chloroform-isoamyl alcohol and phenol-chloroform, precipitated with isopropanol, and re-suspended gently in distilled water. For genomic sequencing genomic libraries were prepared using the Illumina TruSeq DNA Sample Prep Kit and size-selected to a 400 bp mean insert size using Sage Pippin Blue. Final libraries were sequenced on a MiSeq platform using 250 base paired-end reads (v2 chemistry). Contigs were assembled with the MacVector tool (http://macvector.com), and analysed using the NCBI ORF finder (www.ncbi.nlm.nih.gov/gorf) and BLAST (www.ncbi.nlm.nih.gov/BLAST) tools. The sequence of the C. striatum CS4933 region including aph(3’)-Ic, aph(3”)-Ib and aph(6)-Id genes has been deposited in GenBank under accession number KP119857.C. striatum included in this study were resistant to at least one of the six aminoglycosides, whereas the remaining 12 isolates were aminoglycoside-susceptible. The MIC50 and MIC90 distributions of the different aminoglycosides for the 64 C. striatum are presented in According to the breakpoints previously indicated, 52 of the 64 90, netilmicin was the more active aminoglycoside against the C. striatum we tested (MIC90 = 0.38 mg/L), followed by amikacin (MIC90 = 0.5 mg/L). All the isolates were susceptible to these two compounds. Martínez-Martínez et al  and aminoglycoside-6-phosphotransferase [APH (6)-Id], respectively [C. striatum were streptomycin resistant. In 23 streptomycin resistant C. striatum PCR amplification resulted in the expected aph(3”)-Ib (0.55 Kb) and aph(6)-Id (0.51 Kb) amplicons. However, twelve out of the 24 C. striatum streptomycin resistant isolates did not give aph(3”)-Ib neither aph(6)-Id amplicons, suggesting that in these strains streptomycin resistance is mediated by allelic variants of these genes or by another mechanism. One C. striatum (CS6865) was susceptible to streptomycin (MIC = 1 mg/L) but gave the aph(3”)-Ib and aph(6)-Id amplicons, which might be due to mutations affecting the aph(3”)-Ib-aph(6)-Id genes or their promoter region.In ectively . Thirty-C. striatum were positive for the gene aac(3)-XI, encoding AAC(3)-XI, a new aminoglycoside 3-N acetyl transferase from C. striatum [C. striatum carrying the aac(3)-XI gene showed MICs of both gentamicin and tobramycin > 1 mg/L. Nineteen out the 20 isolates negative for the aac(3)-XI gene were susceptible to both gentamicin and tobramycin (MICs < 0.064 mg/L) whereas one isolate showed a MIC = 1.5 mg/L for both compounds. Therefore, there is a good correlation between resistance to these two compounds and the presence of the aac(3)-XI gene.Forty-four out of 64 striatum . AAC (3)aac(3)-XI have a gentamicin MIC range of 1–16 mg/L, supporting the EUCAST clinical breakpoint of > 1 mg/L. If the gentamicin breakpoint recommended by the CLSI was used (>16 mg/L) [aac(3)-XI gene are not considered as resistant. This difference in interpretation between the European and the US recommendations could result in an underestimate of gentamicin resistant C. striatum if the CLSI breakpoint instead of the EUCAST breakpoint was used, with possible serious consequences for patients.The 44 isolates positive for the gene C. striatum CS4933, which was chosen for its clinical relevance (it was isolated from soft tissue abscess) and its resistance profile (it was resistant to kanamycin and streptomycin). Annotation of the genome sequence of C. striatum CS4933 revealed that the aph(3’)-Ic gene is flanked by two divergently oriented IS26-like elements (Tn5715 but with the aph(3’)-Ic gene and the IS26 modules inversely oriented [aph(3’)-Ic gene is part of a larger DNA region containing the aph(3”)-Ib—aph(6)-Id tandem pair of resistance genes conferring streptomycin resistance. The aph(3”)-Ib—aph(6)-Id coding regions of C. striatum CS4933 are located downstream of the resolvase gene from a Tn5393-like transposon that is disrupted by the Tn5715 element containing the aph(3’)-Ic gene. Essentially identical aminoglycoside resistance regions were found in the plasmid pTP10 from C. striatum [C. urealyticum DSM1709 [C. resistens DSM 45100 [In order to know the genomic context of the aminoglycoside resistance genes and inquiry about their transfer mechanisms we sequenced the genome of the isolate elements , formingoriented . The aphstriatum , in the  DSM1709  and in tSM 45100  Fig 1)..C. striaC. striatum isolates into forty-six distinct PFGE types (The PFGE method displayed a high tipability (100% of the isolates were clearly assigned to one pulsotype), and discriminatory power. PFGE delineated the 64 GE types . On thesGE types .aph(3”)-Ib and aph(6)-Id, whereas the 3 strains of the type 23 were sensitive to all aminoglycosides. The 3 strains of the pulsotype 28 were resistant to four aminoglycosides  and contain the aph(3’)-Ic, aph(3”)-Ib, aph(6)-Id and aac(3)-XI genes.Most of the pulsotypes include strains showing the same resistance properties. The 3 strains of the PFGE type 4 were resistant to streptomycin and positive for the genes C. striatum indicated a high genetic diversity in this species. PFGE analysis revealed that our 64 C. striatum are mostly unrelated, producing sporadic infections.High number of pulsotypes in S1 File(DOCX)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


An active packaging system has been designed for the shelf life extension of ready to eat meat products. The package included an inner surface coated with a chitosan film with thyme essential oil  not in direct contact with the meat. Our aim was to reduce the impact of thyme essential oil (EO) on meat sensory properties by using a chemotype with low odor intensity. The pH, color parameters, microbial populations, and sensory properties were assessed during 4 weeks of refrigerated storage. The presence of EO films reduced yeast populations, whereas aerobic mesophilic bacteria, lactic acid bacteria, and enterobacteria were not affected by the presence of the EO in the films. Meat color preservation (a *) was enhanced in the presence of EO, giving a better appearance to the packaged meat. The presence of the chitosan-EO layer reduced water condensation inside the package, whereas packages containing only chitosan had evident water droplets. Thyme odor was perceived as desirable in cooked meat, and the typical product odor intensity decreased by increasing the EO concentration. Further studies should point towards developing oil blends or combinations with natural antimicrobial agents to be incorporated into the film to improve its antimicrobial properties. Active food packaging may include oxygen scavengers, moisture absorbers, ultraviolet barriers, or compounds that deliver flavoring, antioxidant, or antimicrobial agents . In the The use of natural products, such as essential oils (EOs), as food preserving agents is being promoted given the current trend towards green consumerism. Several essential oils (EO) have shown antioxidant properties as well as antimicrobial effects against mold, yeasts, bacteria, and viruses, mainly due to their bioactive components such as flavonoids, terpenes, carotenes, etc. ,8. HowevThe antimicrobial activity of EOs is very difficult to compare due to the high variability of the chemical composition of EOs within the same species, affected by many factors  . EO compChitosan edible films containing mild odor thyme EO may be an innovative preservation technique to extend the commercial shelf life of ready to eat meat products, and even replace the use of artificial chemical preservation agents. Although a large number of studies on the antimicrobial effectiveness of EOs are available, very few studies are available on food products. The aim of this study was to evaluate an active packaging system including a layer of chitosan film containing the EO of mild flavored thyme  for sliced ready to eat (RTE) cooked pork and evaluate the pH, color, evolution of microbial populations, and sensory changes on the RTE meat during refrigerated storage. The main purpose was to evaluate if a layer of chitosan film with thyme EO could be successfully incorporated in a packaging system for RTE meat products to provide an active effect to the packaging and promote an increase in the shelf-life of the cooked meat.Thymus vulgaris L.) commercial essential oil (EO) was purchased from Herbes del Molí . The company reported that the EO was extracted from the whole plant of organic grown autochthonous thyme by hydro distillation. The company is certified by the Comité de Agricultura Ecológica de la Comunidad Valenciana.Essential oil: Thyme (8–C32); and (b) mass spectra . Identification was considered to be tentative when it was based on mass spectral data only. Semi-quantification of the compounds was run in a Shimadzu GC-2100 equipped with an FID detector and the same column and conditions as the GC-MS. Quantitative data were obtained electronically from FID area data without using correction factors. All the tests were performed in triplicate.GC-MS and GC-FID Analytical Conditions for oil analysis: The volatile compounds were isolated, identified, and quantified as described in a previous work ,11, withFilm preparation: High molecular weight chitosan of analytical grade was obtained from Sigma Aldrich . The chitosan solution was prepared as follows: 1% chitosan, 1% lactic acid, 0.1% tween, and 0.25% glycerol with different concentrations of EO . To prepare the active packaging, 20 g of each solution were poured into the inner surface of the cover of a Petri dish (90 mm diameter) and were allowed to dry for 72 h at 37 °C, and then the plates were stored at 53% relative humidity until they were used.Meat material: Commercially cooked ham  was used to obtain slices under hygienic conditions. Overall composition, as indicated in the label, was: 18.5% proteins, 1.5% carbohydrates, 1.5% fat, and <0.2% fiber.Package design: The basic design was a Petri dish in which the cover had a chitosan film, and one slice of RTE cooked pork was introduced in the opposite cover (no film), and each individual plate was packed in a plastic bag under 50% vacuum (to avoid the collapse of the Petri dish). The package was sealed to avoid uncontrolled release of EO compounds outside of it . The volMicrobial analysis and sampling of meat: Samples were taken weekly for the determination of counts of total aerobic mesophilic bacteria in Plate Count Agar (PCA), incubated at 37 °C for 48 h ,12. MesopH determination of meat: The pH was measured in a GLP21 pH-meter  with a punction electrode, and 3 measurements were taken per sample.Commission internationale de l'éclairage, L*, a* and b*) color space was used to provide L *, a *, and b * values. A spectrocolorimeter Minolta CM-2022  with illuminant D65 and 10° observer was used. A CR-A51 glass  was put between the sample and the equipment following the American Meat Science Association recommendations . Ten replicates per sample were taken: measurements were taken on both sides of the slices. Hue, Chroma, and ΔE were calculated.Color determination of meat: CIELAB . Regarding sensory data, a Kruskal-Wallis H test was carried out on the medians.Statistical analysis: The whole experiment was run in duplicate, and triplicate analyses were run for all determinations (10 for color). The IBM SPSS statistics package was used for analysis . Microbial counts, color, and pH data were analyzed by an ANOVA with two factors. Thyme EO concentration , storage time , and their interactions were considered. Tukey’s test was used for mean comparison , accounting for 62.93% of the total profile. There are several chemotypes of thyme, and the present oil was selected based on its mild flavor and because it was a common autochthonous T. vulgaris. As can be seen in Thymus mastichina rich in 1,8-cineole , and compared to the other thymes it showed less antimicrobial effectiveness that the others which were higher in thymol and carvacrol. Ruiz-Navajas et al. [Thymus piperella and Thymus moroderi) on several spoilage bacteria. They reported that T. piperella had a higher effect than T. moroderi, probably due to the higher concentration of carvacrol in T. piperella. The thyme used in the present study was not of the same or similar composition to those reported in both studies. The main antimicrobial essential oil compounds are carvacrol, thymol, cinnamaldehyde, methylchavicol, and linalool [Ballester  studied s et al.  studied linalool . The prelinalool  reportedSeveral food related factors may affect EO antimicrobial activity such as fat content, water activity, and pH, among others. Reduced pH and storage temperature may enhance antimicrobial activity, whereas other factors such as fat content may reduce it . TherefoTotal aerobic mesophilic bacteria (MAB), mesophilic lactic acid bacteria (MLAB), enterobacteria, and molds followed , Table 2Aerobic Mesophilic Bacteria (AMB) counts were under 2 log cfu/g during the first day of the study, reached about 6 log cfu/g after one week of storage, and ended at 7 log cfu/g at the end of the refrigerated storage . AlthougSeveral authors studied chitosan films with thyme EO obtaining similar results to ours , howeverLactobacillus plantarum was scarcely affected by chitosan films containing thyme and oreganum EOs [Scarce or little effect of thyme EO presence has been observed on mesophilic LAB counts, although counts tended to be higher in the control packages. After only seven days of storage, MLAB populations were significantly lower in RTE packaged with EO at all concentrations. As storage time increased, MLAB populations became similar for all packaging conditions. Other studies have reported that anum EOs . In a stanum EOs . In contanum EOs  reportedEnterobacteria were not detected during the first day of the study, were detected after one week of storage around 1 log cfu/g, and at about 5 log cfu/g at the end of the study . RegardiMold initial counts were 3 log cfu/g in all batches and were not significantly affected by EO presence . Mold coGiven that yeasts were inhibited in the presence of EO, it may be assumed that EO was in fact released from the film, so the present results point to a scarce antibacterial activity but a promising activity against yeast from this 1,8-chemotype thyme. The reduction of mold and yeast counts in active packages containing EOs has been previously reported in mangos in packages containing sachets with oregano and lemongrass , and in The pH of RTE meat packaged in the presence of EO  was loweAs can be seen in Regarding color parameters, no differences were reported for L *  due to ERedness preservation due to the use of edible films with thyme EO has been previously reported in ground beef patties , and it The sensory panel detected significant color changes with storage time and no significant differences between slices packaged with different EO content , howeverp > 0.01), whereas off-flavors and thyme flavor were scarcely affected (p < 0.05). The presence of thyme EO affected ham odor, thyme odor, and the presence of exudates, whereas no significant differences were detected for off-flavors and perceived color intensity. Ham odor decreased as EO presence in the package increased, whereas thyme odor increased with increased presence of EO. Panelists indicated that the perceived thyme odor was desirable, and was not considered an off-flavor for this type of product, which is commonly available with fine herbs and similar odor intensity. The evolution of the odor was expected as related to meat ageing and the presence of an EO that poses high aromatic intensity, however the most relevant result is the reduced presence of exudates in those packaging systems containing EO  in RTE cooked pork, higher antimicrobial activity of the EO was demonstrated against yeast, whereas the other studied microbial populations were not affected by the presence of the EO. The presence of the chitosan-EO layer avoided water condensation, whereas the packages containing only chitosan had evident water droplets. The most remarkable sensory effects were observed for odor, with ham odor intensity decreasing with increased EO concentration, as a result of a masking process produced by thyme odor. However, thyme odor was perceived as desirable for this RTE. Other issues need to be addressed in future works such as EO migration kinetics, duration of the activity of the films, and, when brought to the industrial scale, the packaging processing effect on film retention and properties. In the present work, our aim was to reduce the impact of EO on meat sensory properties by using thyme from a chemotype with mild odor, which happened to have low content of antimicrobial compounds (thymol and carvacrol). Even under such conditions, yeast populations were controlled, color preservation was enhanced, and exudates in the package were reduced, which gave a better appearance to the packaged meat. In future studies, combining EOs with organic acids or salts, or even with isolated active compounds, should also be explored.