The rosemary distillation industry produces a considerable amountof rosemary distillation residues (RRs), which can be an alternative forfeeding animals in harsh conditions and could enhance animal productquality. Given the meat quality is largely influenced by its fat content and fatty acid composition, the fatty acid (FA) profiles of longissimus thoracis et lumborum (LTL), semi-membranous (SM)muscles, and caudal (CFs) and omental fats (OFs) were determined using 21 Barbarine lambs fed with or without RRs. Diets contained 600 g of concentrate plus 600 g of forage. Foragerepresented oat hay, RR87 and RR60 pellets containing 87 % or 60 % of RR,respectively. At the end of the study, all lambs were slaughtered, and the fatty acid profile was studied.The inclusion of RR increased the polyunsaturated fatty acid (PUFA)contents and reduced saturated fatty acids (SFAs), and the thrombogenic andsaturation indexes in all tissues. The SM muscle was the richest tissue in PUFAs,  Consumers seek out natural and healthymeat, and they prefer to limit intake of saturated fatty acid (SFAs) andpolyunsaturated FA (PUFAs). Ruminant meat differs from that ofmonogastric animals and contains a high SFA level and low PUFA : SFA ratio asa consequence of the ruminal biohydrogenation of forage and concentrate PUFAsinto SFAs by rumen bacteria .Many factors such as diet and breed could strongly affect muscle FAcomposition . The use of nutritional strategiesrepresents a beneficial approach to improve meat FA composition by usingdifferent PUFA-rich feed ingredients, such as linseed and algae . However, an excessive fat unsaturation maycompromise meat oxidative stability, given its susceptibility to oxidation.Then, using dietary antioxidants is recommended . As a natural dietary strategy, the residues of aromaticand medicinal plants, naturally rich in antioxidants, could play this role.Among these, rosemary is one of the most aromatic plants used in thedistillation industry in the Mediterranean area. It covers a large area estimatedto be 346 000 ha in Tunisia , and its distillation industry generatedabout 5460 T yrOn the other hand, sheep accumulate an important level of fat in differentdepots as mesenteric, omental, perirenal and caudal fat due to their abilityto adapt to periods of food shortage . Asan old tradition in the Middle East and northern Africa, lamb fat depots areused in various dishes and meat products . Thereforetheir FA profile determination becomes important for human health, giventissues and organs are known to have varying FA composition , which reflect their differing physiological roles and metabolisms; therefore, their response to any dietary supply such as the RRwith concentrate should be different. Hence, the objective of the currentstudy was to evaluate the fatty acid profile in two adipose tissues and intwo intramuscular lipids for Barbarine lambs as affected by an RR-based diet.22.1Twenty-one fat-tailed Barbarine lambs (body weight (BW): 23.7 kg) wererandomly allocated to one of three basal diets. All diets contained 600 g ofconcentrate plus 600 g of forage. Forage was oat hay for the control group(C); pellets with 60 % of RR, 32 % of wheat bran and 8 % of soybeanfor RR60 group; and pellets containing 87 % of RR and 13 % of wheatbran for RR87 group. Chemical composition and FA profile of experimentaldiets are reported in Table 1.2.2At the end of the experimental period (77 d), all lambs were fasted for12 h with free access to water; then, they were weighed and slaughtered;the slaughter BW was 33 and 36 kg for C and both RR groups, respectively.After slaughter, a sample of omental fat was preserved. Then, the carcasseswere chilled at 4 2.3The FA profile in all tissues was determined following the Lee et al. (2012) methodand was expressed by % FAMEs (mg FAME per 100 mg FAMEs). For this, 0.4–0.8 gof lyophilized and minced samples were mixed with 1 mL of the internalstandard  and 2 mL of heptanes, and then 4 mL ofNaOH 2.4Experimental data were analyzed by conducting an analysis of variance(ANOVA) to test the effect of an RR-based diet on a FA profile of LTL and SM musclesand omental and caudal fats using the general linear model (GLM) procedure of the SAS Institute (2004). The differences between groups were compared with the Duncan's multiplerange test (DMRT). The significance was declared at C1: the RR presence effect (C vs. RR60C2: the RR inclusion rate effect (RR60 vs. RR87). However, this contrastwas tested and was not significant, for that it was not mentioned in tables.Furthermore, to compare the fatty acid profile of all tissue types (musclesand adipose tissue), the following contrasts were calculated:C3: muscles vs. fats (M vs. F),C4: LTL vs. SM (LTL vs. SM) andC5: omental fat vs. caudal fat (OM vs. CF).The following contrasts were used to compare the effects of the differentdiets:3As expected, the main FAs were palmitic (C16:0), stearic (C18:0) and oleic(C18:1) for all groups and all sites (muscles and adipose tissues). Theyaccounted for 70 %–80 % of the total FA. These results were consistent with valuescommonly accepted for fat-tailed  and thin-tailed sheep .3.1The determination of FA profile in SM muscle revealed 49 FA isomers. Palmiticand stearic acids were reduced with RR-based diets compared to the control(Table 2). However, oleic acid proportion was not altered . However, the oleic acid wassimilar among groups . It was reported that the FA composition depends onlocation of tissues in the carcass . According to ourstudy, the adipose tissues contained more individual SFAs (C14:0, Fat depots contained the higher proportions of short (The adipose tissues contained significantly lower total PUFA, 4Feeding sheep with rosemary residues resulted in different fatty acidprofiles that depend also on the tissue type. This study showed theexistence of a saturation gradient between tissues subject to theirlocation. Adipose tissues, especially the omental fat, contained the highestproportions of SFA; then, it is recommended to avoid the intake ofinternal and external fats, as it has been seen that they are rich in SFAs, which are undesirablefor human health. However, they can be used, in low amounts, to formulatemeat products to enhance their nutritional value.


Health literacy concerns the ability of citizens to meet the complex demands of health in modern society. Data on the distribution of health literacy in general populations and how health literacy impacts health behavior and general health remains scarce. The present study aims to investigate the prevalence of health literacy levels and associations of health literacy with socioeconomic position, health risk behavior, and health status at a population level.A nationwide cross-sectional survey linked to administrative registry data was applied to a randomly selected sample of 15,728 Danish individuals aged ≥25 years. By the short form HLS-EU-Q16 health literacy was measured for the domains of healthcare, disease prevention, and health promotion. Adjusted multinomial logistic regression analyses were used to estimate associations of health literacy with demographic and socioeconomic characteristics, health risk behavior , and health status .Overall, 9007 (57.3%) individuals responded to the survey. Nearly 4 in 10 respondents faced difficulties in accessing, understanding, appraising, and applying health information. Notably, 8.18% presented with inadequate health literacy and 30.94% with problematic health literacy. Adjusted for potential confounders, regression analyses showed that males, younger individuals, immigrants, individuals with basic education or income below the national average, and individuals receiving social benefits had substantially higher odds of inadequate health literacy. Among health behavior factors , only physical behavior [sedentary: OR: 2.31 ] was associated with inadequate health literacy in the adjusted models. The long-term health risk indicator body-weight showed that individuals with obesity [OR: 1.78 ] had significantly higher odds of lower health literacy scores. Poor self-assessed health [OR: 4.03 ] and payments of sickness absence compensation benefits [OR: 1.74 ] were associated with lower health literacy scores.Despite a relatively highly educated population, the prevalence of inadequate health literacy is high. Inadequate health literacy is strongly associated with a low socioeconomic position, poor health status, inactivity, and overweight, but to a lesser extent with health behavior factors such as smoking and high alcohol consumption. During the past decades, an unsolicited socioeconomic divide has intensified in Europe, and the population is increasingly challenged with the growing complexity of the modern healthcare system and the rising expectations of the individuals to participate actively in promoting and maintaining their health –3. The sIn recent years, health literacy has gained importance on the European health agenda, and there has been a growing interest in the concept, accompanied by the increased emphasis on the role and responsibilities of the individual in health and healthcare –7. HealtIn modern society, individuals with higher health literacy have advantages in health compared with those who have lower health literacy. Several studies have shown that limited health literacy is associated with adverse health-related outcomes, such as increased mortality , 13, morBeyond HLS-EU, the majority of previous research on health literacy is based on smaller samples, focusses on functional or specific dimensions of health literacy, or is centered on a specific population or patient groups , 21–25. To the best of our knowledge, the present study constitutes the largest national population-based study on health literacy using the short form (HLS-EU-Q16) of the comprehensive European Health Literacy Survey Questionnaire. Specifically, the study aims to examine the distribution of health literacy levels and investigate the association of health literacy with socioeconomic position, health risk behavior, and health status within a large representative sample of 15, 728 Danish adults.A national, cross-sectional, representative survey with a random stratified sampling design was conducted involving Danish residents aged 25 years and older. A minimum age of 25 years was set in order to obtain a more stable life situation of participants with respect to socioeconomic indicators. Participants were drawn from the Danish Civil Registration System, and the sample was stratified according to age, gender, and geographical location , leading to a probability of inclusion that was proportional to population size and density. Postal codes were included to ensure representative sampling from urban and rural residential areas. The sample was individually linked with information obtained from a variety of Danish administrative registries.N = 1082) were contacted by telephone to increase representation.Between December 2016 and February 2017, 15,728 citizens were contacted; of these, 9007 were willing to participate, leading to a response rate of 57.3%. Participants were recruited using an electronic email system administered by public authorities including a link to a self-administered web-based questionnaire. The wording of the material was provided only in Danish and was not specifically targeted to low-literate individuals. A random part of the study population  health literacy, (2) health behavior, (3) health risk indicators, and (4) perceived self-assessed health. Data on demographic characteristics, socioeconomic indicators, and health status were obtained from nationwide administrative registries.The HLS-EU-Q16 questionnaire developed by the HLS-EU Consortium for measuring health literacy in the general population was applied. Extensive information on the development and pre-testing is described elsewhere , 8. To eIn Denmark, all citizens are identified with a unique civil registration number that enables individual linkage of information across Danish administrative registries. We retrieved information on age (year of birth), gender , origin , marital status , and residence from the Danish Civil Personal Registration Registry . InformaDifferent measures of health behavior including smoking habits, alcohol consumption, and physical activity were included from the survey. Smoking was classified as “daily smoker”, “infrequent smoker”, “former smoker”, or “never smoker”. Alcohol consumption was measured according to official national recommendations stated by the Danish Health Authorities. Weekly alcohol consumption above 14 drinks for men and seven drinks for women is considered high-risk behavior. Physical activity was classified according to daily physical activity level as “sedentary behavior”, “light activities”, “moderate training”, or “hard training” within the last year before the survey completion.2), normal (BMI 18.5–24.9 kg/m2), overweight (BMI 25–30 kg/m2), or obese (BMI ≥30 kg/m2). Self-assessed general health stated as “How would you judge your current state of health?” included four categories ranging from very good to very poor. Information on sickness absence compensation benefits one year from the survey completion was obtained from the Dream database.Self-reported height and weight were obtained to allow calculation of body mass index (BMI). BMI was regarded as a health risk indicator and classified as underweight , and R statistical software package, version 3.3.2 (R Development Core Team) , slightly more women (54.5%) than men participated, and 7.2% were immigrants or descendants of immigrants. The majority of respondents were married (64.0%), approximately one-third (28.9%) had an annual income below the national average income, and more than one-fifth (21.9%) of participants received social welfare payments within the last year from the survey completion. Characteristics of respondents and non-respondents according to sociodemographic indicators are presented in Table Q1: 11.0, Q3: 15.0] on the 16-item scale. The health literacy scale was further classified into three levels described previously. Overall, 8.2% (N = 692) of the study population had inadequate health literacy, 30.9% (N = 2616) had problematic health literacy, and 60.9% (N = 5147) showed adequate health literacy. For individuals categorized within the inadequate health literacy category, the median health literacy score was 7.0 . Individuals within the problematic or adequate health literacy category presented with a median score of 11.0  and 14.0 , respectively. The Cronbach alpha coefficient indicated a high internal consistency of the assembled HLS-EU-Q16 questionnaire (α = 0.90) and good or acceptable internal consistency within each of the three health domains . For the competences of accessing, understanding, appraising, and applying information, the median score per item was highest for accessing health information  and lowest for appraising health information . When comparing all competences over the health domains of healthcare, disease prevention, and health promotion, the median score per item was highest within the domain of healthcare  and lowest in the domain of disease prevention   and ‘judge when you may need to get a second opinion from another doctor’ . In contrast, respondents experienced the least difficulty in relation to: ‘understand your doctor’s or pharmacist’s instruction on how to take a prescribed medicine’  and ‘understand health warnings about behavior such as smoking, low physical activity and drinking too much’ .The median health literacy score of respondents was 13.0 . Significant associations with health literacy and overweight (BMI > 25) were also found, demonstrating that individuals with higher health literacy scores tend to have a normal BMI.The association between health literacy and health behavior Fig.  showed n. Payment of sickness absence compensation benefits was used as a proxy for health status. In both univariable and multivariable regression models, significant associations between lower health literacy scores and payments of sickness absence compensation benefits were found .A large proportion of respondents reported their health as good (64.7%) or very good (19.0%), compared to individuals reporting their health as poor (14.9%) or very poor (1.4%). A strong association between both inadequate  and problematic [Adjusted OR: 1.99 ] health literacy with poor and very poor self-assessed health was found  and 75th (Q3) percentiles (age) or number of residents and percentage .


Atrial fibrillation/atrial flutter (AF/AFL) burden increases with increasing duration of AF/AFL history.Outcomes with dronedarone may also be impacted by duration of AF/AFL history.In this post hoc analysis of ATHENA, efficacy and safety of dronedarone vs placebo were assessed in groups categorized by time from first known AF/AFL episode to randomization : <3 months (short), 3 to <24 months (intermediate), and ≥ 24 months (long).Of 2859 patients with data on duration of AF/AFL history, 45.3%, 29.6%, and 25.1% had short, intermediate, and long histories, respectively. Patients in the long history group had the highest prevalence of structural heart disease and were more likely to be in AF/AFL at baseline. Placebo‐treated patients in the long history group also had the highest incidence of AF/AFL recurrence and cardiovascular (CV) hospitalization during the study. The risk of first CV hospitalization/death from any cause was lower with dronedarone vs placebo in patients with short  and intermediate (0.72 [0.56‐0.92]) histories; a trend favoring dronedarone was also observed in patients with long history (0.84 [0.66‐1.07]). A similar pattern was observed for first AF/AFL recurrence. No new drug‐related safety issues were identified.Patients with long AF/AFL history had the highest burden of AF/AFL at baseline and during the study. Dronedarone significantly improved efficacy vs placebo in patients with short and intermediate AF/AFL histories. While exploratory, these results support the potential value in initiating rhythm control treatment early in patients with AF/AFL. Patients were enrolled between June 2005 and December 2006 and followed up until December 2007 (minimum follow‐up duration of 12 months).2.2We evaluated patients in the ATHENA study who had investigator‐documented history of first known AF/AFL episode. Duration of AF/AFL history was defined as the time from the first known episode of AF/AFL to randomization, and was subsequently categorized as either short (<3 months), intermediate (3 months to <24 months), or long (≥24 months). Patient demographic and disease characteristics, CV history, and baseline medications were assessed among dronedarone‐ and placebo‐treated patients by duration of AF/AFL history. The impact of the duration of AF/AFL history on clinical outcomes and AF/AFL recurrences was assessed in patients treated with placebo to understand the natural history of the disease. Finally, the efficacy  and safety  of dronedarone vs placebo were analyzed by duration of AF/AFL history.First recurrence of AF/AFL was assessed in patients who were in sinus rhythm at baseline.2.3Baseline characteristics were summarized using descriptive statistics. For time‐to‐event outcomes, cumulative incidence functions in each treatment group were calculated using the Kaplan‐Meier method. Hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated using an unstratified Cox regression model with treatment group as the only factor. Inter‐treatment group comparisons were performed using the log‐rank test. For safety analyses, the frequency of patients with TEAEs, serious TEAEs, and TEAEs leading to discontinuation of study drug were summarized using descriptive statistics. Data were analyzed using SAS version 9.4 .3Among the 4628 patients randomized in the ATHENA study, 2859 (61.8%) had documentation of first known AF/AFL episode and constituted the study group for the present analysis (1441 of 2301 patients [62.6%] in the dronedarone treatment arm and 1418 of 2327 patients [60.9%] in the placebo arm). Of these patients, 1296 (45.3%), 845 (29.6%), and 718 (25.1%) had short, intermediate, and long AF/AFL histories, respectively. In the short AF history group, 18 patients (0.6%) had an AF/AFL history duration of <2 days and 219 (7.7%) had an AF/AFL history duration of 2 to <7 days. In all, 1942 patients (67.9%) had an AF/AFL history duration of <12 months.3.12DS2‐VASc scores  was balanced across AF/AFL history groups and treatment arms  and intermediate  AF/AFL histories, with a similar trend in patients with long AF/AFL history . This pattern was also observed for rates of CV hospitalization with dronedarone vs placebo in the short , intermediate , and long  AF/AFL history groups. AF/other supraventricular rhythm disorders were the most common reason for first CV hospitalization  in all AF/AFL history groups  and intermediate AF/AFL histories , and a similar trend was observed in patients with long history , CV hospitalization rate was only modestly greater with increased AF/AFL history duration. These findings may reflect the fact that in comparison to patients with persistent AF, patients with newly diagnosed AF and those with paroxysmal AF typically report more symptoms,Safety outcomes, including TEAEs, serious TEAEs, TEAEs leading to permanent discontinuation of study drug, and deaths, were comparable across AF/AFL history groups. The observation that no new safety concerns were identified with dronedarone treatment in any of the AF/AFL history groups is consistent with the overall results of the ATHENA study4.1Randomization in the ATHENA study was stratified by baseline AF/AFL status but not by duration of AF/AFL history5Duration of AF/AFL history is an important consideration in the evaluation and management of AF/AFL with regard to disease burden as well as response to therapy. The observations of this post hoc analysis of the ATHENA trial suggest that while dronedarone demonstrated efficacy vs placebo regardless of the duration of AF/AFL history, the effect was more robust among patients with short and intermediate AF/AFL history, suggesting that treatment with an AAD such as dronedarone should commence at an early stage of disease. Prospective trials are warranted to further investigate the impact of timing of AAD initiation on efficacy and safety among patients with AF/AFL.Outside the submitted work, Carina Blomström‐Lundqvist reports consulting fees from Bayer, Boehringer Ingelheim, Boston Scientific, Cardiome, Medtronic, MSD, Pfizer, and Sanofi‐Aventis. Nassir Marrouche reports grants and/or consulting fees from Abbott, Biosense, Boston Scientific, and Medtronic. Stuart Connolly reports consulting fees from Sanofi‐Aventis. Stefan Hohnloser reports consulting fees from Bayer, BMS, Boehringer Ingelheim, Daiichi Sankyo, Pfizer, Sanofi‐Aventis, and Zoll. Andrew Koren was a previous employee of Sanofi. Mattias Wieloch, and Valérie Corp dit Genti are employees of Sanofi.


Apis mellifera L. during the blossom seasons of different floral sources, to provide guidelines for the future rational application of royal jelly in cutaneous wounds, and to promote the further discovery of wound repair-promoting substances.Non-healing wounds have been a severe issue in the global healthcare system. Regrettably, royal jelly, a traditional remedy for various skin injuries, has not been widely applied in cutaneous wounds in clinical practice nowadays, which may be due to the confusion and the lack of knowledge about the efficacies of different types of royal jelly, the bioactive constituents, and the precise mechanisms underlying the wound repairing activity. Since the compositions and bioactivities of royal jelly are predominantly influenced by nectar plants, this study aims to explore the differences in the wound-healing properties of royal jelly produced by Castanea mollissima Bl. (chestnut) and Brassica napus L. (rapeseed) in South China, from which hydrophilic and lipophilic fractions were extracted. The in vivo wound-healing potential was preliminarily assessed in Wistar rats’ excisional full-thickness wounds, followed by investigating the mechanisms of action through in vitro assays with human epidermal keratinocytes and LPS-stimulated inflammation in macrophages.Royal jelly samples were harvested during flowering seasons of Castanea mollissima Bl. royal jelly was more potent. It sped up wound closure between day 2 and day 4 to 0.25 cm2/day (p < 0.05), and could accelerate wound repair by enhancing the proliferative and migratory capabilities of keratinocytes by 50.9% (p < 0.001) and 14.9% (p < 0.001), modulating inflammation through inhibiting nitric oxide production by 46.2% (p < 0.001), and promoting cell growth through increasing the secretion of transforming growth factor-β by 44.7% (p < 0.001). In contrast, Brassica napus L. royal jelly could regulate inflammation by reducing the amount of tumour necrosis factor-α by 21.3% (p < 0.001).The results indicated that different royal jelly samples exhibited distinct wound-healing potential, in which Castanea mollissima Bl. royal jelly promises the greatest potential. It also provides clues which may lead towards the identification of substances derived from royal jelly to treat wounds.The present study improves the application of royal jelly for curing difficult-to-heal wounds, in which the hydrosoluble extract of  Bee products, produced by honeybees and maintained in bee hives, including honey, royal jelly, propolis, pollen, venom and bee bread, have been important supplementary medicines since ancient times, due to their broad pharmacological efficacies. Amongst these natural products, royal jelly is known as a “superfood”, due to its powerful potency for queen-differentiation of honeybees and the longevity of the queen bee, as well as its nutritional and therapeutic values for human beings , 2. It iIn normal physiological conditions, wound healing can complete naturally in a sophisticated manner, within a certain period of time, in which predominant cells , cytokines , and mediators (nitric oxide and nitric oxide synthase) play critical roles –15. MoreRoyal jelly, a traditional remedy for many kinds of cutaneous injuries, may have potential applications in the therapy of abnormal wounds. Limited previous studies have revealed that royal jelly, or its derivatives, have the effectiveness of promoting wound repair at both animal and cellular levels , 19–24, Castanea mollissima Bl. (chestnut) and Brassica napus L. (rapeseed) by way of in vivo and in vitro wound healing assays. In the in vivo assay, excisional full-thickness wounds were created to preliminarily investigate the efficacy of different royal jelly in topical application. In the in vitro assays, human immortalised keratinocytes (HaCaT), resembling the normal keratinocytes making up the majority (95%) of epidermis, were employed to explore the proliferative and migratory activities of royal jelly extracts in wound healing. Moreover, anti-inflammatory effects of the extracts were evaluated in LPS-stimulated murine macrophages (RAW 264.7). This study will boost the development and application of particular royal jelly in wound healing, as well as the identification of biologically-active ingredients from royal jelly for use in different skin injury situations.Here, this is the first study to compare the wound-healing activities of royal jelly collected during flowering seasons of Apis mellifera L., was collected in the mid-term of flowering seasons of Castanea mollissima Bl. (chestnut) and Brassica napus L. (rapeseed), respectively, from apiaries in Zhejiang Province , P.R. China, and was named CmRJ-Zj and BnRJ-Zj. Immediately after harvesting, samples were preserved at − 20 °C prior to use. No permission was necessary for the collection of royal jelly. Castanea mollissima Bl. and Brassica napus L. were authenticated by Yan Lin and the voucher specimens were deposited in the herbarium in the College of Animal Sciences (College of Bee Science), Fujian Agriculture and Forestry University, with deposition numbers of CmZj-20180601 and BnZj-20190401.To ensure the relative purity and authenticity of botanical sources, raw royal jelly, produced by To preliminarily evaluate the differences in wound-healing activities between the raw royal jelly collected in flowering seasons of different floral sources, the in vivo wound healing assay was performed with samples CmRJ-Zj and BnRJ-Zj employing full-thickness wound models in rats. The experiments were performed according to the ARRIVE  guidelines  and the Castanea mollissima Bl. and Brassica napus L. royal jelly samples (10%) respectively every other day via topical administration and were left uncovered after application. Royal jelly and EGF were dissolved in sterile saline. Five rat replicates were included in the experiment. Wound areas were monitored on days 0, 2, 4, 6, 8, 10, 12 and 14, and photos were taken before each treatment. The open areas of wounds were calculated using Photoshop software . After the study, rats were killed by anesthetisation with 2% pentobarbital sodium  followed by cervical dislocation, in accordance with the Guidelines for the Care and Use of Medical Laboratory Animals. The researchers who were treating the wounds with agents and performing statistical analysis, were blinded. The area healed per day, between two adjacent observation time points (2 days), was calculated using the following formula:Adult female Wistar rats, approximately 12 weeks old, weight 180–250 g, were obtained from Animal Research Centre of Hubei Province. They were caged in a special room with a temperature of 25 ± 1 °C and a 12:12 light-dark cycle, and were supplied with a commercial pellet diet and water ad libitum. Before the experiment, the rats were maintained for 7 days to acclimatise to the laboratory environment. Five rats were randomly selected and anaesthetised by inhalation with 2% isoflurane in 98% air   prior to shaving the dorsal skin hair and creating four full-thickness excision wounds (1.5-cm-diameter) on the back of each rat using biopsy punch. After surgery and clearance of the blood on the wounds, the images were photographed with a digital camera  and recorded as day 0. The four wounds in each rat were treated with 200 μl of solvent sterile saline (negative control), epidermal growth factor , n-2” and “Arean” were the areas of wounds on day n-2 and day n; the “Dayn” and “Dayn-2” were the day n and day n-2 post-wounding. As wound healing is usually delayed, and it is a nonlinear dynamic process, expressing the wound healing rate as a percent change in the area would be inaccurate when applied to wounds with varying initial sizes [The “Areaal sizes , 28. To al sizes , 28.2 at 37 °C.The immortalised human epidermal keratinocyte cell line  possessing basic characteristics of normal epidermal keratinocytes was purchased from DSMZ, Germany, and the murine macrophage cell line  was obtained from the Cell Repository of the Chinese Academy of Sciences . They were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)  with 10% foetal bovine serum (FBS)  and 1% penicillin-streptomycin solution , and kept in a humidified incubator with 5% COHydrophilic and lipophilic components of royal jelly were extracted similarly to Gismondi’s work . In brie4 and 1 × 107 cells/ml, respectively, was added to each well of 96-well plates, followed by 24-h incubation. Royal jelly extracts were diluted from the stock solution (250 mg/ml) with serum-free DMEM. Before being treated with a series of extracts at different concentrations for 48 h (HaCaT cells) or 24 h (RAW 264.7 cells), cells were starved for 12 h in serum-free DMEM. Control cells were treated with serum-free DMEM containing the highest amount of PBS or DMSO present in test groups (0.1%). Then, 10 μl of 3--2,5-diphenyltetrazolium bromide  was added for reaction for about 4 h. After removal of the supernatants, formazan crystals were dissolved in 100 μl of DMSO, and the absorbance was measured at 492 nm in an Infinite F50 plate reader . Tests were repeated at least three times for each extract sample. Cell viability was calculated using the formula below:The proliferative and cytotoxic effects of royal jelly extracts on HaCaT and RAW 264.7 cells were evaluated with MTT colorimetric assay. It was conducted as described in our previous work with minor modification . Brieflyx” was the absorbance of royal jelly extracts treated cells; the “A0” was the absorbance of serum-free medium treated cells.The “A5 cells/ml) in each cell of the Inserts for around 18 h to form confluent cell monolayers. Culture-Inserts were removed discreetly and detached cells were washed off with PBS . Royal jelly extracts were diluted from the stock solution (250 mg/ml) with serum-free DMEM. Cells were then treated with 2 ml of sample solutions at specified concentrations for 24 h. Control cells were treated with serum-free DMEM containing the highest amount of PBS or DMSO present in test groups (0.0125%). The scratch wounds were observed under an inverted microscope . Images of three, randomly selected, cell-free regions in each well were captured at 0, 12 and 24 h following wounding, and analysed using Image J software . Three replicates were set for each concentration and three independent experiments were conducted. Wound closure rates were calculated using the following formula:The migratory effects of extract samples on HaCaT cells were assessed by scratch wound assay. Scratch wounds (500-μm-wide) were created by placing Culture-Inserts  into each well of 24-well plates before culturing 70 μl of HaCaT cells  were seeded into 24-well plates. After 12-h incubation, medium was replaced with serum-free DMEM with or without royal jelly extracts for a further 1-h incubation. Royal jelly extracts were diluted from the stock solution (8 g/ml) with serum-free DMEM. The final concentrations of DMSO present in control and experimental groups were lower than 0.05%. LPS from Escherichia coli  was added to a final concentration of 1 μg/ml [The production of nitric oxide (NO) by RAW 264.7 cells was determined based on the nitrite present in the culture supernatants using Griess reagent . RAW 264.7 cells  for a further 24 h [To measure the secretion of cytokines modulated by royal jelly extracts in RAW 264.7 cells, the culture supernatants collected above for the measurement of NO production was also tested for the generation of TNF-α (tumour necrosis factor-α) and TGF-β1 (transforming growth factor-β1) using ELISA kits . Briefly, 5.0 × 10her 24 h , 31. Thex” was the absorbance of cells exposed to royal jelly extracts and LPS; the “A0” was the absorbance of cells exposed to LPS alone.The “Ap < 0.05 was considered as statistically significant. Results were expressed as means ± SEM.Data were processed using GraphPad Prism 5.0 software . Comparisons of variance were performed using one-way ANOVA, in which Castanea mollissima Bl. royal jelly (CmRJ-Zj) and the epidermal growth factor (EGF) were larger than the initial sizes, and the corresponding areas healed per day in the initial 2 days were significantly lower than the control group. Surprisingly, in spite of the graver wounds after CmRJ-Zj treatment in the first 2 days, the areas healed per day of these wounds between day 2 and day 4 were statistically higher than the control (p < 0.05)  were healthy and no microbial infection was observed. As shown in Table 5) Table . Neverth5) Table , it seemCastanea mollissima Bl. and Brassica napus L. exhibited remarkably different wound healing activities. As the compositions and bioactivities of royal jelly may be affected by botanical sources, the hydrosoluble and liposoluble components were extracted from both royal jelly to further investigate the influence of different chemical components on their medical functions. As shown in Fig. Castanea mollissima Bl. royal jelly significantly promoted the proliferation of HaCaT cells without any cytotoxicity; in contrast, fractions from Brassica napus L. royal jelly could not enhance cell growth at the concentrations up to 250.00 μg/ml and might even cause cell death at some doses. It suggested that Castanea mollissima Bl. royal jelly might facilitate wound repair via its cell-growth promoting efficacy, while Brassica napus L. royal jelly might lack such potential.The preliminary in vivo wound healing study indicated that royal jelly harvested in the flowering seasons of Castanea mollissima Bl. and Brassica napus L. royal jelly were further investigated for the effects on cell migration. It is obvious that, at 24 h post-wounding, both hydrophilic and lipophilic extracts from Castanea mollissima Bl. royal jelly dramatically boosted the wound closure of human keratinocytes in vitro compared with control  at 7.81 μg/ml  between two adjacent observation time points [Castanea mollissima Bl. royal jelly enlarged on 2 days post-wounding, thus it further confirmed that traditional formulae could not reflect the detailed wound healing process, and instead, the wound healing should be evaluated separately at each time point. It was illustrated in Table Castanea mollissima Bl. royal jelly group was significantly lower than the control group (p < 0.001), whereas between day 2 and day 4  it was shown to be statistically higher than the control (p < 0.05). We speculated that the larger wounds on day 2 might be in the inflammatory stage of wound healing, in which no obvious contraction and scarring could be observed, as phagocytic cells were not responsible for filling in the defects [Castanea mollissima Bl. royal jelly might not have any potency on the early inflammation. The higher healing speed on the wounds exposed to Castanea mollissima Bl. royal jelly between day 2 and day 4 might be attributed to its anti-inflammatory effects on macrophages that usually infiltrated the wound sites at around 48 h after injury [Castanea mollissima Bl. royal jelly was confirmed (Fig. In the in vivo wound healing assay, excisional full-thickness wounds were created on the dorsal skin of Wistar rats. When evaluating the wound healing-promoting activity, −unlike many other studies measuring the wound healing with absolute changes of open wound areas, or with percentages of healed wounds relative to the initial areas , 27, 28, defects , and Casr injury ; the wear injury . In the Castanea mollissima Bl. and Brassica napus L. royal jelly exhibited slightly different wound healing effectiveness, the subjacent mechanisms of action were investigated with in vitro assays. It was found that both hydrophilic and lipophilic extracts from Castanea mollissima Bl. royal jelly could promote the proliferation and migration of keratinocytes, while both extracts from Brassica napus L. royal jelly were devoid of such effects (Figs. Castanea mollissima Bl. royal jelly might accelerate wound healing via facilitating re-epithelialisation.Since ts Figs.  and 3, iCastanea mollissima Bl. and Brassica napus L. royal jelly supressed the production of NO in LPS-induced RAW 264.7, and those from Castanea mollissima Bl. royal jelly were more potent (Fig. Brassica napus L. royal jelly could also repress LPS-stimulated production of TNF-α which was a kind of crucial pro-inflammatory cytokines promoting wound healing via inducing inflammation yet being surplus at chronic wound sites [Brassica napus L. royal jelly might regulate wound healing through controlling inflammation. Besides, hydrophilic extracts from both royal jelly could enhance the production of TGF-β1, while the lipophilic extracts displayed converse effects (Fig. In terms of anti-inflammatory effects, it is well-known that inflammation takes place immediately after skin injury, protecting wounds from infection and facilitating subsequent proliferation; however, prolonged inflammation may lead to delayed healing , 16, 34.nd sites  (Fig. 4cCastanea mollissima Bl. royal jelly possessed more potent wound-healing potential, through enhancing keratinocytes growth and migration, reducing NO production and increasing TGF-β1 generation. These modulatory effects might contribute to the wound healing potency observed in the animal assays in this study. Furthermore, it was also found that the anti-inflammatory efficacies of hydrophilic extracts of each royal jelly were stronger than those of lipophilic extracts. We speculate that it may be possible to treat non-healing wounds at different stages with different components from specific royal jelly and the bioactive substances are more likely to be water-soluble. In the future, it will be worthwhile to analyse the chemical components present in Castanea mollissima Bl. and Brassica napus L. royal jelly to verify the exact compounds evoking wound healing-promoting efficacy.Based on the in vivo and in vitro studies, we found that Castanea mollissima Bl. royal jelly and EGF were larger than the initial ones on day 2, greatly interfering with the analysis on the therapeutic effects, thus a greater number of rats should be tested in the future. Also, following the expansion of wounds on day 2 in the Castanea mollissima Bl. royal jelly group, there was a statistical difference in the wound healing speed compared with control between day 2 and day 4, thus histological and immunohistochemical analyses might be worth carrying out to explore the pathological and physiological changes in this period.This is the first study to compare the wound-healing potencies of royal jelly derived from blossom seasons of different nectar plants, and to preliminarily investigate the underlying mechanisms of their hydrophilic and lipophilic extracts, providing insights into the identification of bioactive compositions from particular royal jelly for the future of wound care. Nevertheless, there were several limitations to this study. The doses and dosing frequency administered to rats might have been too low to improve wound closure. The wounds treated with This study indicated that royal jelly, collected during blossom seasons of different nectariferous plants, modulated wound healing with distinct mechanisms. Over the past number of decades, chronic wounds have been a global healthcare issue, due to the aging population and the rise in the incidence of diabetes and obesity. Inspired by this study, some problematic wound models, such as diabetic foot ulcers, infected cutaneous wounds and even MRSA skin infections, could be established to explore the scope of application of specific royal jelly. This would facilitate the development of royal jelly for wound care, and the discovery of wound healing-promoting components to deal with skin injuries at different stages or with different symptoms.


Periprosthetic joint infection (PJI) is a catastrophic complication after total knee or hip arthroplasty. The diagnosis of PJI is very difficult, especially in the early postoperative period. The value of the neutrophil to lymphocyte ratio (NLR) is useful for diagnosing infectious diseases. The objective of this study was to investigate the accuracy of the NLR for the diagnosis of early PJI after total knee or hip arthroplasty.We retrospectively evaluated consecutive primary total knee or hip arthroplasty and identified the patients who readmitted within the first 90 days postoperatively between January 2011 and October 2018.There were 20 cases diagnosed early PJI and 101 uninfected cases on the basis of the modified Musculoskeletal Infection Society (MSIS) criteria. The serum parameters including C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), white blood-cell (WBC) count, NLR and interleukin-6 (IL-6) were compared between the two groups. Receiver operating characteristic curves were generated to estimate the optimal cutoff values for each parameter. The sensitivity, specificity, positive predictive value and negative predictive value for each parameter were calculated.p < 0.001). The median of ESR was 34.8 mm/hr. in the infected group and 17.4 mm/hr. in the uninfected group (p < 0.001). In the infected group and uninfected group, the median of WBC was 8.2X109 /L and 6.1 X109 /L (p = 0.002), respectively; while the median of NLR was 5.2 and 2.1 (p < 0.001). The median of IL-6 was 46 pg/ml and 6.4 pg/ml (p < 0.001),respectively. The best parameter for the diagnosis of early PJI was IL-6 (AUC = 0.814) followed by the NLR (AUC =0.802), CRP (AUC =0.793), ESR (AUC =0.744) and WBC (AUC = 0.632).The CRP, ESR, WBC, NLR and IL-6 values were all significally higher in the infected group than the uninfected group. The median of CRP was 66.6 mg/l in the infected group and 8.6 mg/l in the uninfected group (This study is the first to show that NLR values are more accurate than CRP and may be considered as useful parameters for the diagnosis of early PJI because it is a cheap and convenient parameter to be calculated in daily practice without extra costs. Periprosthetic joint infection (PJI) is a disastrous complication after total knee or hip arthroplasty with high morbidity and mortality. The early and accurate diagnosis of PJI is of great clinical significance for the treatment of PJI , 2. A leIt is essential to diagnose early PJI on the basis of a combination of clinical judgment, blood testing, synovial fluid aspiration, microbiologic and histopathologic inspections as well as imaging. There are various imaging techniques used in the assessment of PJI including radiographs, CT, MRI, ultrasound and nuclear medicine examinations. The ultrasonography (US) and MRI may be valuable tool for diagnosis of early PJI, which are characterized by soft tissue swelling, fluid collections and presence of sinus around the joint. However, there are very few literatures regarding for the diagnosis of early PJI because of lack of a suitable control group as the truly aseptic revisions are rare in the early postoperative period. Kim et al.  found thAmong the various kinds of tests, it is very critical to find a simple and practical marker for diagnosing early PJI. The serum WBC count has very little utility due to the very low sensitivity as markers of PJI . In receAfter Institutional Review Board approval for this study was obtained, we retrospectively reviewed the cases of consecutive primary total knee or hip arthroplasty in our institutions between January 2011 and October 2018. A total of 245 cases readmitted within the first 90 days postoperatively were identified. These patients were admitted to hospital again in 90 days for the following reasons: (1) staged bilateral total knee or hip arthroplasty, (2) dislocation, (3) patellar ligament rupture, (4) persistent wound drainage, (5) fever, (6) fracture, (7) erythema,(8) superficial infection, (9) postoperative infection was suspected.Blood samples for CRP, ESR, IL-6 and other markers were taken on the morning after readmission for all patients and were then sent to the medical laboratory center for testing as soon as possible. All data including CRP, ESR, white blood cell (WBC) count, IL-6, neutrophil count and lymphocyte count were obtained from electronic medical records by manual chart review. The NLR was obtained as the absolute neutrophil count divided by the absolute lymphocyte count.The aspirations were performed for these patients which resulted from symptoms such as persistent wound drainage,fever, erythema, superficial infection and postoperative infection being suspected. The synovial fluids were used for bacterial culture, a leukocyte esterase (LE) strip test and synovial fluid analysis (white blood-cell [WBC] count and polymorphonu-clear leukocyte [PMN] percentage).These inflammatory arthritis, such as rheumatoid arthritis, ankylosing spondylitisetc were excluded in order to ruling out interference with other possible preconditions associated with elevated inflammatory markers. Also excluded were 1) Patients with superficial infection (24cases); 2) postoperative fever ; 3) a history of malignancy (4cases); 4) missing critical data .These exclusions left 121 cases were analyzed. There were 20 infected cases and 101 uninfected according to the Musculoskeletal Infection Society (MSIS) criteria   and one case was managed with one-stage revision in the infected group. Three cases in the infected group failed and no cases were diagnosed and reoperated for PJI in the uninfected group with at least 1-year follow-up. Twenty four cases of superficial infection were also analyzed. There was no case that were diagnosed and reoperated for PJI with at least 1-year follow-up.Table .Table 2p < 0.05. The whole statistical analysis were performed with use of the statistical software packages R (The R Foundation) and Empower (X&Y Solution). The Receiver operating characteristic (ROC) curves and area under the curve (AUC) were calculated to examine each parameter for the evaluation of early PJI. The Youden index was used to determine the optimal threshold for each parameter as a diagnostic tool for early PJI. The sensitivity and specificity of each parameter were calculated.The categorical variables were assessed using chi-square tests and parametric data were analyzed using t tests. The results of the diagnostic tests in the two groups were compared using the Mann-Whitney test. Statistical significance was considered as a There were 20 cases in infected group and 101 cases in uninfected group based on the MSIS criteria. There was no significantly different in age, time interval, BMI, joint (hip or knee) between two groups. The characteristics of each cohort are shown in Table .Table 3p < 0.001). The median of ESR was 34.8 mm/hr.  in the infected group and 17.4 mm/hr.  in the uninfected group (p < 0.001). In the infected group and uninfected group, the median of WBC was 8.2X109 /L  and 6.1 X109 /L  (p = 0.002), respectively; while the median of NLR was 5.2  and 2.1 (p < 0.001). The median of IL-6 was 46 pg/ml  and 6.4 pg/ml  (p < 0.001), respectively.  in the infected group and 8.6 mg/l  in the uninfected group  followed by the NLR AUC =0.802), CRP (AUC =0.793), ESR (AUC =0.744) and WBC (AUC = 0.632)  in serum had the highest accuracy as a marker for diagnosing early PJI, followed by the NRL, the C-reactive protein, the erythrocyte sedimentation rate, and the white blood cell count.Interleukin-6 is an inflammatory cytokine produced by activated monocytes and macrophages and induces to product some major acute-phase proteins, including CRP . Some stThe serum IL-6 was not often detected because of expensive costs. Interestingly, another indicator, NLR performed very well and had the high AUC (0.802). The accuracy of the value of NLR was less than IL-6 but greater than CRP. It may be more suitable for screening to diagnose early PJI because it is convenient to be obtained in daily practice without extra costs.The value of the NRL had been shown a significant association with infection. Dogruel et al.  demonstrAlthough there were some literatures that elaborate possible explanations about the relation between elevated NLR and the development of –22, it rThere are some limitations of this study that should be considered. First, this is retrospective study and the inherent limitations exist. There may be some selection bias because several potential cases were excluded due to no record of the serum ESR, CRP, or IL-6. Although this study is a retrospective design, it may provide some useful information for diagnosing early PJI. Second, there is no consensus on definition of the early PJI. We defined 90 days as early PJI and shorter time needed to be detected. Finally, we recruited 121 cases to the study. Although the total sample size was relatively sufficient, there was a small sample size in infected cases that limited the statistical power of our conclusions. Therefore, our findings should be confirmed in a larger study and at multiple institutions.The findings of this study have shown that the IL-6 was the best test for diagnosis of early PJI. But it was not often detected because of expensive costs. However, the NLR that has the higher accuracy for the diagnosis of early PJI than CRP may be considered as a useful parameter for the diagnosis of early PJI because it is easy, cheap and convenient to be calculated in daily practice without extra costs. However, further studies evaluating the accuracy of NLR in more patients are needed to verify the findings of the present study.


Experiencing natural disasters is associated with common mental disorders including major depressive disorder (MDD). However, the latent structure of MDD is widely debated, and few studies tested the MDD factor structure in Chinese natural disaster survivors. Therefore, the aim of the current study was to evaluate the factorial validity of the Patient Health Questionnaire-9 (PHQ-9) for DSM-5 major depressive disorder (MDD) symptoms in Chinese earthquake survivors.Participants were 1058 Chinese earthquake survivors. Self-reported measures included the PHQ-9 and the Short-Form Health Survey (SF-36). Confirmatory factor analysis (CFA) and structural equation modelling (SEM) was used to examine the latent structure of MDD and the associations between latent factors of MDD and different domains of health-related quality of life (HRQoL), respectively.χ2 = 173.89, df = 26, CFI = 0.986, TLI = 0.981, RMSEA = 0.073, BIC = 18,091.13). Further SEM analyses indicated that the non-somatic factor was significantly related to both physical  and psychosocial HRQoL , while the somatic factor was a uniquely predictor of physical HRQoL . Furthermore, we found the somatic factor partially mediated the relationship between the cognitive/affective factor and physical HRQoL .In the current sample, the model consisted of somatic and cognitive/affective (non-somatic) factors demonstrated significantly better fit than the other competing MDD models (The MDD symptoms was best captured by a two-factor model comprised of somatic and cognitive/affective factors in Chinese natural disaster survivors. The two MDD factors were differentially associated with physical and psychosocial HRQoL, and the cognitive/affective factor associated physical HRQoL partially through the somatic factor. The current findings increase our understanding of latent structure of MDD symptoms, and carry implications for assessment and intervention of post-disaster mental health problems. DSM-5 [Due to urbanization, environmental degradation, climate change and other factors, the frequency of natural disasters has increased dramatically in recent years . NaturalDSM-5 . The relDSM-5 , 13. TheDSM-5 , howeverSeveral alternative models of MDD symptoms has been proposed and validated in previous studies , 15–17  suggested that external measures of psychopathology should be examined to clarify this issue . As highPrevious studies revealed that people who suffer from MDD often had physical problems , 42, andThe first aim of this study was to find the best fitting latent structural model of MDD; our second aim was to evaluate whether the somatic factor mediates the relationship between non-somatic factor and physical HRQoL. According to prior studies, our hypotheses were as follows: (1) the model comprised of a somatic factor and a cognitive/affective factor would provide a superior data fit over competing models; (2) the non-somatic factor would be associated with psychosocial health and physical health. Relatedly, we expected the somatic factor would correlate with physical health only; and, (3) the non-somatic factor would affect physical HRQoL through the somatic factor. Many previous study samples were restricted to medical patients , which might increase the endorsement rates of the MDD somatic symptoms and therefore might affect the factor structure of MDD , 25, 27.n = 14), leaving an effective sample of 1058.On May 12, 2008, a destructive earthquake measuring 8.0 on the Richter scale occurred in Sichuan Province. For the purposes of accessing disaster-related mental health problems, the sample was collected from 5 rebuilt communities of Hanwang town, Mianzhu City. Approximately 5000 people living in this town were killed in the earthquake. This survey was conducted in July, 2017. The procedures were as follows: (1) Selecting one family member randomly in each household as participants; (2) making sure that all participants were aged from 16 to 65 and experienced the 2008 earthquake; (3) excluding individuals with mental retardation or a major psychiatry history. This survey was conducted by professional clinical psychologists, psychology students. Before the participants completed questionnaires, the aim of the study was clearly explained. All participants have signed an informed consent. This study protocol was reviewed and approved by the Institutional Review Board of the Institute of Psychology, Chinese Academy of Sciences. A total of 1072 people participated in this study. People with missing data (> 20% items) were excluded . PHQ-9 is a brief, easily administered self-report questionnaire designed to capture MDD symptoms. Each item is rated on a 4-point scale using anchors ranging from 0  to 3 (extremely), reflecting the extent which symptom bothers a participant in the past 2 weeks. The reliability and validity of the PHQ-9 have been well-documented in a previous study . The PHQThe Short-Form Health Survey (SF-36) was used to assess health-related quality of life. There are 8 subscales in this instrument, 4 reflect Physical HRQoL , and 4 reflect Psychological HRQoL . Scores of all subscales range from 0 to 100, with higher scores indicating a better HRQoL. The Chinese version of SF-36 has been proven to have good reliability and validity, and widely used in Chinese populations . Wang  procedures. According to the scoring procedures of the SF-36, the missing values on the SF-36 were estimated with the average score of the same subscales.To evaluate the best-fitting factor model, five alternative CFA models , and was set as the dependent latent variable. In the second model, psychosocial quality of life was treated as a latent factor measured by the role-emotional and social functioning, and was set as the dependent latent variable. MDD factors of the best fitting model and covariates  were simultaneously included in the equation as predictors in all models. SEM was used to test all models, and regression coefficients were calculated to examine the different correlations between each MDD factor with physical and psychosocial HRQoL.A SEM model was built to examine the mediator role of MDD’s somatic factor between MDD’s non-somatic factor and physical HRQoL, standard errors of the mediating parameter were simultaneously estimated in the SEM model with bootstrap procedure (1000 times). Direct effects in this study include the relationship between MDD’s somatic and cognitive/affective factors, between MDD’s somatic factors and physical health, and between MDD’s non-somatic factors and physical health. Indirect effects included the effect of MDD’s cognitive/affective factor on physical health through MDD’s somatic factor.SD = 10.0), 911 (86.2%) were married, and 146 (13.8%) were unmarried . 1042 (98.6%) were Han people, and 5 (0.4%) were members of other ethnic groups . 732 (69.2%) did not complete high school, 240 (22.7%) completed high school, and 85 (8.0%) completed college.In the current sample, 347 (32.7%) men and 711 (67.3%) women with a mean age of 51.1 , and 236 (22.2%) participants were diagnosed with possible MDD .Descriptive statistics for the PHQ-9 and SF-36 total scores were estimated at first. Mean PHQ-9 score was 6.2 , “Role limitations due to physical health” was 64.4 , “Bodily Pain” was 76.4 , “General Health” was 60.7 , “Social Functioning” was 79.6 , and the “Role limitations due to emotional problems” was 60.3 .Mean scores on the SF-36 subscales were as follows: “Physical Functioning” was 79.2 (χ2 (1) = 45.653, p < 0.001). In terms of comparisons of non-nested models, the BIC value for model 2a was lower than the other models, and all △BIC were lower than 6 except the △BIC between Model 2a and other models. For these reasons, the Model 2a was the best fitting model  = 218.08, CFI = 0.99, TLI = 0.99, RMSEA = 0.03 (90% CI: 0.03–0.04) for the model with physical HRQoL as dependent variable, and χ2  = 243.98, CFI = 0.99, TLI = 0.98, RMSEA = 0.04 (90% CI: 0.04 ~ 0.05) for the model with psychosocial HRQoL as a dependent variable. Table β = − 0.336, p < .01), and the cognitive/affective factor was a significant predictor for both physical HRQoL  and psychosocial HRQoL .The models examining correlations between MDD factors and HRQoL demonstrated adequate fit. Fit statistics were β = − 0.296, p < .05)  and the somatic factor on physical HRQoL . The path from cognitive/affective factor to physical HRQoL was also significant . After 1000 bootstrapped tests, the somatic factor still significantly mediated the relationship .The indirect effect of the non-somatic factor on physical HRQoL was significant  proposed that MDD symptoms could be best captured by somatic and cognitive/affective factors, many studies supported two-factor MDD models in samples with somatic conditions  , 25, 27.To further verify the distinctiveness of the two MDD’s factors, the current study used HRQoL as an external measure, and found that HRQoL displayed specific associations with two factors of MDD. Previous studies also had similar findings. The results of Mordeno et al., (2018)‘s study revealed that somatic symptoms (measured by PHQ-15) were more related to the somatic factor than cognitive/affective factor . Elhai eFurthermore, based on previous theoretical evidence, we assessed the mediating role of somatic factor referencing the relationship between the non-somatic factor and physical HRQoL. Results indicated that the non-somatic factor of MDD probably affects the physical HRQoL via the MDD somatic factor in the present sample. This may have a cultural explanation. For example, expressing psychological distress is considered a dilemma in Chinese culture. People who experience psychological problems tend to attribute these problems to physical or external origins , 59. ThiFinding of this study has several implications for clinical applications. First, investigation of the factor structure of MDD could help us better understanding different components of MDD symptoms, which could guide clinically useful assessment and intervention for MDD symptoms. Second, findings of our study informed the different functional roles that distinct factors of MDD played in HRQoL. This would be helpful to structure and establish treatment which aims to improve the HRQoL of people with depression. Third, our findings benefit our understanding of the psychopathological process of MDD symptoms affecting physical HRQoL. Our findings illustrated that for Chinese people with significant somatic symptoms, their psychological problems need also to be recognized.There are several study limitations that need to be recognized. First, our findings might be limited because we used an earthquake-exposed sample. Thus, samples exposed to other trauma types should be used to further replicate our findings. Second, the limitation of self-reported symptoms has been acknowledged . TherefoSurvivors of natural disasters typically have a high MDD prevalence, and accurately assessing MDD symptoms among this population is very important for post-disaster mental health care. The present study found that MDD could be best represented by the two-factor model in Chinese natural disaster survivors, and the MDD factors have different relationships with HRQoL. Our findings could help to better understand the different components of MDD among natural disaster victims, which could guide more sophisticated assessment and intervention for their MDD symptoms. Moreover, findings of the current study are important in contributing to improving HRQoL of natural disaster victims.


Limited information exists about the nature of late-onset asthma (LOA) without medication intervention when compared to early-onset asthma (EOA). Our goal was to understand how EOA and LOA affect clinical and pathophysiological features.  A population-based cross-sectional study was carried out in Zhongshan Hospital . EOA and LOA were based on age of diagnosis . Clinical variables were collected with an emphasis on allergic features, analyzed, related, and compared using one-way ANOVA or Kruskal-Wallis test. Correlations between blood basophils and clinical data were evaluated by Spearman's rank test. Statistical analyses were conducted with SPSS v24.0. 1/FVC (%), FEV1% predicted, and FVC% predicted were all significantly lower in LOA compared to EOA (p < 0.01). The values of post-bronchodilator FEV1 in bronchodilator reversibility testing were higher in the LOA and EOA groups compared to subjects with COPD (p < 0.01). Among allergic features, mite sensitization was most common in EOA patients, followed by LOA and COPD, whereas mold sensitization was more prevalent in LOA than EOA. Moreover, blood eosinophils were a typical feature of asthma in both EOA and LOA compared to COPD and controls (p < 0.01), and there were no differences in blood neutrophils in LOA compared to controls. Interestingly, blood basophils were increased in both EOA (p < 0.01) and LOA (p < 0.05) compared to COPD and controls. This variable correlated with eosinophils in EOA  but not in LOA.  Of a total of 12,760 adults with cough, sputum, or chest tightness, 90 subjects with EOA (mean age ± standard deviation (SD):28.73 ± 5.89), 111 with LOA (mean age ± SD: 60.25 ± 9.85), and 106 with chronic obstructive pulmonary disease (COPD) (mean age ± SD: 61.58 ± 10.95) were selected. FEV LOA is a distinct clinical entity from EOA. In LOA, atopy was less frequent and spirometry values were lower when compared to EOA. In EOA, blood basophils and eosinophils were significantly correlated owing to pathophysiological differences between the two forms of the disease. Asthma is one of the major global health problems and contributes substantially to the worldwide burden of disease , 2. PrevDue to aging-related changes in respiratory pathology and immunity, LOA is frequently not diagnosed or is misdiagnosed . Unlike Most LOA patients enrolled in studies had been prescribed inhaled corticosteroids (ICS), oral corticosteroids (OCS), or leukotriene receptor blockers, which mask natural disease features and hamper research into the underlying mechanisms –15. It iA single-center, population-based, cross-sectional study was carried out in Zhongshan Hospital, Fudan University, Shanghai, China. This 2430-bed tertiary hospital is used by 10 million people in Shanghai and 150 million people in the surrounding Yangtze River Delta. More than 260,000 outpatients are referred to the Department of Pulmonary Medicine annually. All subjects signed informed consent forms and the study was approved by the Institutional Review Board, Human Subjects Research Protection Program Office (B2018-010R) at Zhongshan Hospital, Fudan University.Subjects were screened from the Screening and Monitoring for Obstructive Lung Disease project from February 2018 to October 2019. A total of 12,760 patients with cough, sputum, or chest tightness were referred to the Department of Pulmonary Medicine. We excluded 5085 patients with lung infection, lung shadow at chest xRay, or lung mass, and 3260 patients with previous diagnosis of asthma and/or COPD or already on medication with drugs for chronic obstructive airway disease. The remaining 1822 patients underwent pulmonary function and/or bronchodilator reversibility testing. If the spirometry results did not support the suspicion of asthma, the patients were subjected to peak expiratory flow (PEF) monitoring . Finally, 308 patients were diagnosed with asthma or COPD at the initial presentation by the pulmonologist or the attending physician according to the Global Initiative for Asthma (GINA) and the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria . The stu1) of >12% and >200 mL from baseline, 10–15 minutes after 200–400 mcg salbutamol  or a change in PEF of at least 20% from baseline after 4 weeks of anti-inflammatory treatment, were considered as positive for diagnosis of asthma [Before performing spirometry examination and collecting blood samples, patients under medication for other indications were asked to stop the related medication for at least 7–10 days . Briefly, adults with respiratory symptoms, as indicated by an increase in forced expiratory volume in 1 second  < 0.7 with chronic respiratory symptoms [COPD was defined as any incidence of dyspnoea, chronic cough, or sputum production, with a history of recurrent lower respiratory tract infections and/or a history of exposure to risk factors for the disease and post-bronchodilator FEVsymptoms , 19. All1 ≥ 80% pred; moderate (GOLD2), 50% ≤ FEV1 < 80% pred; severe (GOLD3), 30% ≤ FEV1 < 50% pred; and very severe (GOLD4), FEV1 < 30% pred, based on post-bronchodilator FEV1values in the GOLD guidelines [Clinical information of all patients was carefully collected, including age, gender, weight, height, body mass index (BMI), smoking history (smoking ≥ 1 cigarette per day for 1 year), family history, related disease history, and asthma severity. Subjects with asthma were classified as mild, well controlled with step 1 and 2 treatments , moderate, well controlled with step 3 treatment , or severe with step 4 or step 5 treatment  based on the GINA criteria and National Institutes of Health guidelines . Accordiidelines , 21.1% pred, FEV1 (L), FVC% pred, FVC (L), FEV1/FVC (%), and bronchodilator reversibility testing in accordance with the American Thoracic Society guideline [All subjects underwent allergy testing for specific immunoglobulin E (sIgE) in serum , and blood cell counts were performed . Pulmonauideline . Fractio1% pred, FEV1 (L), FVC% pred, FVC (L), FEV1/FVC (%), FeNO, allergens, and sIgE were evaluated by Spearman's rank tests [p values < 0.05 were considered statistically significant. Statistical analyses were conducted with SPSS for Windows, version 24.0 .Clinical characteristics are reported as means ± standard deviation or percentages. Continuous variables were analyzed by one-way analyses of variance, and categorical variables were assessed with Kruskal-Wallis tests . Correlank tests . All tesAfter enrollment and screening, a total of 308 patients were diagnosed with asthma or COPD at their initial presentation . Based op < 0.05). Subjects with LOA were less likely to report a diagnosis of allergic rhinitis compared to those with EOA (52.7% vs. 80%), and COPD patients had the highest rate of smoking history (62.3%) among the three groups. With regard to asthma severity, LOA patients were more likely to be categorized as moderate (52.3% vs. 46.7%%) or severe (25.2% vs. 14.4%) compared to those with EOA. In the COPD group, most of the patients belonged to GOLD1class (39.6%) compared to the other subgroups .There were no differences in BMI among the groups. However, the subgroup of female patients with LOA had a higher BMI compared to female patients in the EOA group (1/FVC (%), FEV1% pred, and FVC% pred were all significantly lower in LOA compared to EOA (p < 0.01). The values of post-bronchodilator FEV1 in bronchodilator reversibility testing were higher in the LOA and EOA groups compared to subjects with COPD (p < 0.01). Moreover, although FeNO was lower in LOA compared to EOA, both groups showed significantly higher FeNO compared to the COPD group (p < 0.01). Respiratory physiology information is shown in The lung function test and allergic features were depicted in n = 74, 82.2%, p < 0.01), followed by LOA  and COPD . However, more subjects with LOA showed sensitization to mold compared to EOA . Furthermore, the LOA group exhibited more sensitivity to Aspergillus fumigatus than the EOA group .There was a marked difference in allergic sensitization between the LOA and EOA groups . Sensitip < 0.01). Interestingly, blood basophils  were also increased in both LOA and EOA. Blood basophil levels were the highest in EOA patients (p < 0.01), followed by LOA (p < 0.05), compared to COPD and healthy controls. There were no differences in blood neutrophils  among the LOA, EOA, COPD, and healthy control groups.As shown in r = 0.549, p=0.002) and the absolute value of blood eosinophils . Although blood basophils were increased in LOA (p < 0.05) compared to COPD and healthy controls, this was not correlated with any clinical parameter in the LOA group  Atopy was less common in LOA compared to EOA, but the antigen sensitization profiles were different. Sensitization to mites was most frequent in EOA patients, followed by LOA and COPD. Subjects with LOA were significantly more likely than those with EOA to exhibit sensitization to mold. (2) Spirometry values were lower in patients with LOA compared to EOA at their initial presentation. (3) Blood eosinophils were a typical feature in both the LOA and EOA groups. Number of blood basophils were also increased and correlated with eosinophils in EOA but not with LOA. There were no differences in blood neutrophils among subjects with LOA or EOA and healthy controls.Traditionally, LOA was considered to have less of an allergic component, as an incomplete response to bronchodilators , and is Aspergillus fumigatus sensitivity was increased in LOA compared to EOA. This suggests that sensitization to mold in patients with LOA may be related to their living and work environment [Our findings showed a major difference in the atopic sensitization between LOA and EOA. The Epidemiology and Natural History of Asthma (TENOR) study found that subjects with LOA had less allergic rhinitis and fewer patients with sIgE to at least one allergen , which iironment , and epiironment , 32.1values in bronchodilator reversibility testing were higher in both LOA and EOA groups compared to COPD. A reasonable explanation for this is age-related physiological changes that may act synergistically with asthma to worsen airflow obstruction, including less elastic recoil, increased airway remodeling in smaller airways, increased thickness of the central airway wall, and increased inflammation [Compared to younger cohorts, subjects with LOA have more severe asthma and higher rates of AHR. In our study, LOA patients were more likely to be categorized as moderate (52.3% vs. 46.7%%) or severe (25.2% vs. 14.4%) compared to those with EOA. Spirometry values were also significantly lower in patients with LOA compared to EOA at their initial presentation, which is similar to a previous report . Howeverammation .Eosinophilic inflammation is a typical feature of asthma. Many studies reported that levels of blood eosinophils were similar to those of airway eosinophils in asthma , 35. ThiThere are limitations to our study. Airway inflammation could not be assessed as sputum induction was not performed due to the high concentration of saline, which could exacerbate asthma symptoms in patients without medication-controlled disease . LOA witLOA is a distinct clinical entity from EOA. In LOA, atopy was less frequent compared to EOA and antigen sensitization differences may be related to their living and work environments. Spirometry values were lower for LOA patients at their initial presentation than EOA patients. In EOA, blood basophils and eosinophils were significantly correlated.COPD, chronic obstructive pulmonary disease; EOA, early-onset asthma; LOA, late-onset asthma.


Klebsiella pneumoniae, a Gram-negative bacterium, is notorious for causing HAI, with many of these infections difficult to treat, as K. pneumoniae has become multidrug resistant. Epidemiological studies suggest that K. pneumoniae host-to-host transmission requires close contact and generally occurs through the fecal-oral route.An important yet poorly understood facet of the life cycle of a successful pathogen is host-to-host transmission. Hospital-acquired infections (HAI) resulting from the transmission of drug-resistant pathogens affect hundreds of millions of patients worldwide.  Klebsiella pneumoniae, a Gram-negative bacterium, is notorious for causing HAI, with many of these infections difficult to treat, as K. pneumoniae has become multidrug resistant. Epidemiological studies suggest that K. pneumoniae host-to-host transmission requires close contact and generally occurs through the fecal-oral route. Here, we describe a murine model that can be utilized to study mucosal  colonization, shedding within feces, and transmission of K. pneumoniae through the fecal-oral route. Using an oral route of inoculation, and fecal shedding as a marker for GI colonization, we showed that K. pneumoniae can asymptomatically colonize the GI tract in immunocompetent mice and modifies the host GI microbiota. Colonization density within the GI tract and levels of shedding in the feces differed among the clinical isolates tested. A hypervirulent K. pneumoniae isolate was able to translocate from the GI tract and cause hepatic infection that mimicked the route of human infection. Expression of the capsule was required for colonization and, in turn, robust shedding. Furthermore, K. pneumoniae carrier mice were able to transmit to uninfected cohabitating mice. Lastly, treatment with antibiotics led to changes in the host microbiota and development of a transient supershedder phenotype, which enhanced transmission efficiency. Thus, this model can be used to determine the contribution of host and bacterial factors toward K. pneumoniae dissemination.An important yet poorly understood facet of the life cycle of a successful pathogen is host-to-host transmission. Hospital-acquired infections (HAI) resulting from the transmission of drug-resistant pathogens affect hundreds of millions of patients worldwide.  Klebsiella pneumoniae, a member of the Enterobacteriaceae family that frequently causes pneumonia, bacteremia, pyogenic liver abscesses, and urinary tract infections  that can cause disease, such as community-acquired pyogenic liver abscesses in healthy individuals  tract , 17. The isolate –21. Besinew host , 22–24.K. pneumoniae to readily colonize a host. Studies also show that antibiotic treatment of mice predisposes them to a “supershedder” state in which they shed resident gut pathogens at a higher number, which enhances host-to-host transmission  (22–Our understanding of the fomites) , 22–24.K. pneumoniae GI colonization, shedding, and host-to-host transmission. Employing an oral route of K. pneumoniae inoculation in an inbred mouse population, we investigated K. pneumoniae gastric colonization and transmission. We demonstrate that K. pneumoniae can stably colonize the GI tract without treatment with antibiotics, and that these mice stay persistently colonized and can transmit K. pneumoniae to cagemates. Furthermore, antibiotic treatment of carrier mice induces gut dysbiosis and triggers a transient supershedder phenotype.Here, we describe a murine model to allow for the study of Klebsiella pneumoniae colonization that would mimic natural colonization in a host. Because of the difficulty in establishing K. pneumoniae GI colonization through gavage treatment, previous studies used antibiotic pretreatment to disrupt the host microbiota and allow for K. pneumoniae colonization via the gavage method , suggesting that a minimum dose of 105 is required (data not shown). Based upon these results, we chose 106 CFU as the minimum dose required to establish K. pneumoniae colonization. Based on our preliminary studies that suggest that poor K. pneumoniae fecal shedding correlates with reduced GI colonization, we used daily fecal shedding as a substitute for colonization density in the GI tract. Next, we determined how long K. pneumoniae colonizes the mouse GI tract. We followed K. pneumoniae shedding in feces of infected mice for either 15 or 30 days postinoculation (p.i.) and observed that K. pneumoniae was shed at similar levels throughout the study  that colonizes poorly to establish itself in the GI tract. However, whether antibiotic treatment affects colonization density of isolates that colonize robustly without requiring antibiotic treatment remains unknown. We determined whether treatment with antibiotics would lead to the development of a supershedder phenotype, in which an infected host sheds the pathogen at a much higher number than another infected host. This phenomenon has been observed in the natural setting and is considered a major source of host-to-host transmission (8 CFU/g (supershedder [SS] threshold) of the indicated pathogen in the feces is generally considered the threshold for the supershedder phenotype (SS phenotype)  , 26. Usihenotype  and B, whenotype .K. pneumoniae shedding. We supplemented the drinking water of mice with ampicillin 24 h before K. pneumoniae inoculation and continued for 10 days p.i. As K. pneumoniae is intrinsically resistant to ampicillin, the mice infected with MKP103 isolate displayed the K. pneumoniae supershedder phenotype . Fecal pellets were collected preinoculation to determine the baseline of the host GI microbiota. Samples were collected on days 7, 9, and 11 post-antibiotic treatment to determine changes in the host microbiota. At days 3 and 5 p.i., we were unable to detect K. pneumoniae 16S rRNA gene sequences, even though K. pneumoniae was shed at 106 CFU/g of fecal sample. This result suggests that K. pneumoniae comprises only a minor component of the host intestinal microbiota. The main component of a diverse microbial community of the host intestine included Bacteroidetes  and Firmicutes   led to dramatic changes in the intestinal microbiota. As detailed in K. pneumoniae, we tested shedding and colonization of the previously described capsule (CPS)-deficient mutant (ΔmanC) of the strain KPPR1S. As is evident from ΔmanC mutant shed and also colonized poorly  in comparison to the parental wild-type (WT) strain. To provide further evidence of the role of CPS in GI colonization, we also tested a mutant with a deletion of wcaJ , it behaved as the parental WT strain  biofilm in the GI tract, helping compensate for the capsule deficiency of the ΔmanC strain. However, coinfected mice still shed the ΔmanC strain poorly compared to the parental strain  and in vivo  (data not shown). To determine the requirement of the type 1 fimbria of KPPR1S in GI colonization, a deletion mutant of fimH, which encodes the type 1 fimbria tip adhesin, required for proper interaction with the host epithelial layer  with four infected mice (index). Fecal pellets were collected to enumerate colonization density and whether transmission from index to contact mice occurred. We observed 100% transmission efficiency with a ratio of 4:1, with transmission occurring within 24 h of cohousing the animals in all of our experiments  was observed, suggesting that not enough K. pneumoniae shedding events occurred for all uninfected mice to become colonized (n = 4), we observed at least one successful transmission event in each of the experiments.Transmission of enteric pathogens generally occurs through the fecal-oral route, and host-to-host transmission in a hospital setting is a major source of infection , 32. Thueriments  and C. Solonized  and C. HK. pneumoniae transmission dynamics. A single antibiotic treatment to the index mouse cohoused with four contact mouse led to >90% transmission, suggesting that high K. pneumoniae shedding in the fecal pellets can overcome colonization resistance of the contact mice .We hypothesize that l matter . Lastly,Klebsiella pneumoniae allows this pathogen to colonize a variety of host mucosal surfaces, which can dramatically impact the clinical outcome. Klebsiella pneumoniae disease manifestations in the respiratory and urinary tract have been extensively modeled in animals . However, orally feeding a similar dose allowed K. pneumoniae to colonize the GI tract and persist without disrupting the host microbiota. A recent study by Atarashi et al. showed that K. pneumoniae colonizing the oral cavity of patients can seed the GI tract . Our data suggest that K. pneumoniae (KPPR1S isolate) in the GI tract behaves in a manner that generally does not elicit an acute inflammatory response and carriage is considered an asymptomatic event.A hallmark of many GI pathogens is their ability to cause an acute host inflammatory response. Multiple reports also suggest that response , 42. Howividuals . In our K. pneumoniae, including its capsular polysaccharide (CPS), type 1 fimbriae, and others, have been extensively examined under both in vitro and in vivo conditions  and lower  GI tract. The role of the capsule possibly pertains to protection against host-mediated clearance and interactions with mucus . However, the type 1 fimbria might play a role in GI colonization for isolates that do express the structure.We also tested the requirement of the nization . Howevernization , 48. Furmembrane . HoweverKlebsiella pneumoniae. We observed transmission between K. pneumoniae-infected and contact mice, suggesting that K. pneumoniae shedding in this model is high enough to permit transmission, albeit at a lower frequency. We also observed that the contact mice were colonized at a lower density than the index mice, possibly because of the reduced K. pneumoniae dose in the fecal pellets. Thus, our data follow epidemiologic studies that suggest 5 to 25% carriage rates in the natural environment  (Lennox) broth, with constant agitation at 37°C. For all mouse infections, an overnight culture of K. pneumoniae was spun down at ∼27,000 × g for 15 min, and the resulting pellet was resuspended in a similar volume of 1× phosphate-buffered saline (PBS). To obtain the desired density for mouse infections (106 CFU/100 μl), the bacterial suspension in PBS was diluted into a 2% sucrose-PBS solution. Ten-fold serial dilutions were plated on selective media (LB agar with antibiotic) and incubated at 30°C overnight for quantitative culture. LB plates contained antibiotics to select for specific K. pneumoniae isolates. Antibiotics used for isolating various isolates included streptomycin , chloramphenicol (50 μg/ml), ampicillin (25 μg/ml), apramycin (50 μg/ml), spectinomycin (30 μg/ml), and rifampin (30 μg/ml). Strains used in the study are listed in wzi gene codes for a conserved outer membrane protein involved in the attachment of capsular polysaccharide to the outer membrane. Sequence polymorphism in the wzi gene has been used to identify and characterize different isolates. K. pneumoniae AZ10 (wzi 372), an antibiotic-sensitive isolate, was made Str resistant as described previously, subsequently mouse GI passaged, and named AZ99  with AZ101 genomic DNA as the template and primers fimH upstream (GGCGGTGATTAACGTCACCT) and fimH downstream (GATAGAGCAGCGTTTGCCAC), which give at least 500 bp of homology on either end of the transposon cassette. The PCR product was purified using the Qiagen MinElute kit. Lambda Red mutagenesis was carried out as described previously  from mice infected with K. pneumoniae. Broth culture was spun down as described above and resuspended in an equal volume of distilled water (dH2O) and boiled for 5 min. Also, a single colony was resuspended in 10 μl of dH2O and boiled for 5 min. DNA was isolated from fecal pellets (100 mg) using Quick-DNA fecal/soil microbe microprep (Zymo Research). Five microliters of sample was used in PCR with OneTaq polymerase (NEB) with primers Cas168 (GGGACAGATACGCGTTTGAT) and Cas169 (GCCTAACTGAACGGTTTGA) as described previously  mice obtained from Jackson Laboratory  were bred and maintained in a standard animal facility at Biotech Place, Wake Forest Baptist Medical Center. All animal work was done according to the guidelines provided by the American Association for Laboratory Animal Science (AALAS)  and with2 CFU/g. Each mouse was uniquely marked so that the fecal shedding of each individual mouse could be tracked for the duration of the experiment.To quantify daily bacterial shedding, mice were removed from their housing and placed into isolation containers. Fecal pellets  were collected and placed into a 2-ml screw-cap tube  along with at least 2 glass beads . Samples were diluted 1:10 (weight/volume) in PBS. A Bead Mill 24 homogenizer (Fisherbrand) was used to homogenize the fecal pellets . Afterwards, the tubes were spun in a minicentrifuge  to pellet out larger debris. Ten-fold serial dilutions were plated from the supernatant on appropriate antibiotic plates and incubated overnight at 30°C. Bacterial shedding was calculated in CFU per gram of feces. The limit of detection was 10K. pneumoniae supershedder phenotype, mice were infected orally with Str-resistant K. pneumoniae as described above. Four to 5 days postinoculation (p.i.), mice were treated by gavage with streptomycin (5 mg/200 μl) either once or on three consecutive days, and daily shedding was monitored post-antibiotic treatment. To determine the effect of neomycin treatment before K. pneumoniae infection, mice were treated by gavage with a single dose (5 mg/200 μl) 24 h before inoculation with MKP103, a derivative of the KPNIH1 isolate with a deletion of the KPC-3 carbapenemase-encoding gene  and Str (500 μg/ml) LB agar. The competitive index (CI) was calculated as described previously , using tA value of 0 would suggest that neither strain has an advantage. A value of >1 would suggest that the mutant has a competitive advantage, whereas a value of <1 would indicate that the WT has the advantage. Each CI experiment was carried out multiple times .2 (2 liters/min for 5 min) euthanasia of animals and subsequent cardiac puncture. The cecum, proximal colon, and a span of the terminal section of the ileum equal in length to the colon were removed from each animal. Organs were weighed and placed into individual 2-ml screw-cap tubes  with at least 2 glass beads . Samples were diluted 1:10 (weight/volume) in PBS and were homogenized and plated as described above. The limit of detection was 102 CFU/g.To determine colonization density in the GI tract, ileum, cecum, and colon were removed under sterile conditions immediately following CO2 CFU/ml.To determine colonization density in the kidneys, liver, and spleen, organs were removed under sterile conditions immediately following euthanasia as described above. Organs were weighed and placed in 15-ml conical tubes. For kidney and liver equal weight-to-volume PBS was added, and samples were homogenized using a PowerGen 700 homogenizer (power setting of 2 for 30 s), whereas for spleen, 10 times the volume of 1× PBS was added to the weight of the organ and homogenized as described above. The samples were plated as described above. The limit of detection for kidney and liver was 33 CFU/ml, and that for spleen was 10Oropharyngeal lavage was carried out with 200 μl of sterile PBS from a gavage needle inserted into the esophagus. The esophagus was exposed and cut transversely. A gavage needle, attached to a prefilled insulin syringe (BD) with 1× PBS, was then inserted into the cut esophagus, and PBS was collected from the mouth. The collected lavage was serially diluted and plated on appropriate antibiotic plates and incubated overnight at 30°C. The limit of detection for oral lavage was 33 CFU/ml.n = 1 or n = 4) at 5 to 7 weeks of age were infected with K. pneumoniae as described above, and feces were collected daily to determine colonization density of the GI tract. On day 4 p.i., contact mice (n = 4 or n = 1) were introduced to cages with the index mice. Feces of index and contact mice were collected and fecal shedding was quantified for at least 6 days postcohousing, for a total of 10 days for index mice and 6 days for contact mice. On day 10, the mice were euthanized as described above and the ileum, cecum, colon, and oral lavage samples of all mice were processed as described above to determine colonization density of K. pneumoniae.For 4:1 and 1:4 transmission experiments, C57BL/6J index mice  via gavage and then cohoused with contact mice. Fecal shedding was determined daily for index and contact mice. In a continuous antibiotic challenge transmission study, an index mouse was put on water containing ampicillin (1 g/liter) 24 h before infection. The contact mice were placed on water containing ampicillin (1 g/liter) 24 h before introduction of the index mouse. Once the mice were cohoused, feces were collected daily from both index and contact mice and shedding was quantified to determine if any transmission events occurred.In 1:4 transmission experiments, in which the administration of a single dose of antibiotic was assessed, index mice were infected with Str-resistant K. pneumoniae infections were carried out as described above. Four days postinfection, a contact mouse was introduced into the metabolic cage and feces were collected from both index and contact mice to determine transmission frequency. The metabolic cage’s metal wire floor allowed for fecal pellets and urine to fall through, separated through a special funnel and collected at the bottom. Therefore, the cage eliminated the ability of the mice to access fecal pellets for consumption (coprophagia). For each transmission study, ≥3 biologically independent experiments were carried out. Endpoint colonization of the lower GI tract  was considered a positive transmission event.To confirm that host-to-host transmission events occur through the fecal oral route, a metabolic cage  was used. K. pneumoniae-infected mice were treated by gavage with streptomycin  to induce the supershedder state at 5 days p.i. As a positive control, mice were put on 3% (wt/vol) dextran sodium sulfate (DSS)  in their drinking water ad libitum for 7 days. All the mice were euthanized at day 7 after initial treatment or infection. A total of 2.5 cm of colon immediately distal to the cecum was collected, washed with 1× PBS, and prepared using the Swiss roll method. Afterwards, the sample was preserved in 1:10 formalin  and after 24 h was transferred to 70% ethanol. The samples were embedded in paraffin before being sectioned, mounted, and stained with hematoxylin and eosin (H&E). The resulting slides were scored by the Wake Forest Baptist Medical Center pathology department.Mice were infected with either PBS (vehicle-only control) or the KPPR1S isolate. A subset of Liver was collected under sterile conditions from mock-infected mice and mice orally infected with hvKP1 and in extremis. Liver samples were cut in to sections about 6.5 mm and placed in 10% formalin . After 24 to 48 h, the samples were transferred to 70% ethanol and stored at 4°C until they were further processed for H&E and Gram staining, with scoring carried out as described above.https://www.r-project.org/) using the heatmap.2 and ggplots packages.Fecal microbiome was examined according to previously described methods –63. Brie–All statistical analyses were performed using GraphPad Prism 8.0 . Unless otherwise specified, differences were determined using the Mann-Whitney U test (comparing two groups) or the Kruskal-Wallis test with Dunn’s postanalysis (comparing multiple groups).