Evidence-based practice (EBP) is integral to the delivery of high-quality health care. Chiropractic has been a licensed health profession in Sweden since 1989, but little is known of the uptake of EBP in this professional group. This study explored the self-reported skills, attitudes and uptake of EBP, and the enablers and barriers of EBP uptake, among licensed chiropractors in Sweden.n = 172) of the Swedish Chiropractic Association (Legitimerade Kiropraktorers Riksorganisation) were invited to participate in an anonymous online questionnaire, using the Evidence-Based Practice Attitude and Utilisation Survey (EBASE) in February 2019.Licensed chiropractors (Fifty-six (33%) chiropractors completed the survey. Participants were predominantly male, aged 30–49 years, held a Master’s degree, and had received their highest qualification and practiced chiropractic for over a decade. Chiropractors rated their EBP skill-level mostly in the moderate to moderate-high range. The majority of chiropractors reported positive attitudes towards EBP, with most agreeing or strongly agreeing that EBP is necessary in the practice of chiropractic, and that EBP assists in making decisions about patient care. Chiropractors reported an average level of engagement in EBP activities. All participants indicated professional literature and research findings were useful in their day-to-day chiropractic practice. The main perceived enabler of EBP uptake was internet access in the workplace, whereas the main barrier to EBP uptake was lack of clinical evidence in chiropractic.Participating chiropractors of the Swedish Chiropractic Association were generally favourable of EBP, though only reported modest levels of EBP-related skills and engagement in EBP activities. Our findings suggest future studies investigating interventions focussed on improving chiropractors’ skills and uptake of EBP are warranted. Contemporary health care requires practitioners to make clinical decisions that are patient-centred, collaborative, individualised for the patient, and informed by research evidence , 2. SincChiropractic can be defined as a health care profession focussed on diagnosing, treating and preventing mechanical disorders of the musculoskeletal system, predominantly relying on manual therapy interventions including spinal manipulation . Recent In other physical and manual therapies, such as physiotherapy and osteopathy, there have been calls for these professions to incorporate EBP into their practice models and philosophies –11. In cThe Swedish National Board of Health and Welfare issues licences for health professions that meet the requirements of the state to practice health care . ChiroprPrevious studies exploring perceptions and uptake of EBP among chiropractors in the US, Canada and Australia have revealed positive attitudes towards EBP and satisfactory self-reported skills in EBP. However, these same studies also uncovered important barriers to EBP implementation, such as lack of time, perceived lack of evidence and research relevance, with many chiropractors reporting that they neither used clinical practice guidelines nor routinely applied evidence in clinical practice –33. DespWhat is the perceived level of skill in EBP among the chiropractors?What is the attitude toward EBP among the chiropractors?What is the level of engagement in EBP activities among the chiropractors?What do the chiropractors identify as enablers of, and barriers to EBP uptake?Are there any associations between EBP skill-level, use and attitude, and participant demographic factors?The study presented in this paper was developed in direct response to this knowledge gap, with the aim to describe the self-reported skill-level, uptake and attitudes toward EBP, as well as the enablers and barriers to EBP uptake, among chiropractors in the Swedish Chiropractic Association. To address these aims, the study set out to answer the following research questions:Cross-sectional survey.The Swedish Chiropractic Association, representing 172 licensed chiropractors as of February 2019, was invited and agreed to participate in the survey. To achieve a response distribution of 50%, margin of error of 12% and confidence interval of 95% for any individual item in the survey, we needed to survey at least 49 of the 172 licensed chiropractors of the Swedish Chiropractic Association.The Evidence-Based practice Attitude and utilization SurvEy (EBASE) is a 83-item, multidimensional instrument designed to gather information on the following constructs: level of skill in EBP, attitude toward EBP, uptake of EBP, training and education in EBP, enablers of EBP uptake and barriers to the uptake of EBP. A series of demographic items is also integrated into EBASE . EBASE has been administered to more than 8 distinct health professions to date , 34–38, To ensure EBASE was suitable to administer to a Swedish chiropractic population, we adapted the WHO process for translating instruments : we trann = 172) in February 2019. The survey was open for 2 months (February to April 2019) during which the licensed chiropractors received two reminders to participate at one and 3 weeks after the first invite. The survey was hosted online by SUNET Artologik, a secure web based survey system used in Swedish higher education [The Swedish Chiropractic Association emailed a web link to the anonymous EBASE survey to their licensed chiropractor members  response rate.Participants were predominantly male 57.1%), aged between 30 and 49 years (59.0%), and held a Master’s degree qualification or higher (71.4%). Most participants had received their highest qualification 11 or more years ago (71.4%) and had practiced in the field of chiropractic for ≥11 years (76.8%). Almost two-thirds (62.5%) of participants reported working 31–45 h per week in clinical practice. More than one-third (37.5%) of chiropractors participated in research, with only 3.6% reporting teaching in higher education. Most participants practiced in an urban location (76.8%) with a group of conventional providers (26.8%) or a group of conventional and CM providers (23.2%)  related to the identification of precise clinical questions 73.2%), and the areas of evidence acquisition , and evidence application  , hours per week participating in research  and practice setting . There was a moderate negative correlation between the skill subscore and years since receiving the highest qualification . No statistically significant associations were found between the skill subscore and other demographic factors.The median skill subscore was 44.5 . This reflected a perceived skill level mostly in the moderate to moderate-high range, as defined by an overall value ranging between 39.1 and 51.9. A moderate positive correlation was observed between the skill subscore (categorised by quartiles) and highest qualification , but not with any other demographic factors.The median attitude subscore  was indicative of mostly agree to strongly agree responses, as defined by an overall score ranging between 32.0 and 40.0. There was a weak positive correlation between the attitude subscore (categorised by quartiles) and highest qualification  at least once in the past month (75.0–92.9%), typically between 1 and 5 times (37.5–60.7%), but generally no more than 10 times 62.4–82.2%)  of participants indicated that a large proportion (76–99%) of their practice was based on evidence from clinical research, with 37.5% reporting a moderate proportion (51–75%), 8.9% a small proportion (26–50%) and 7.1% a very small proportion (1–25%). No participants reported their practice as being 100% based on clinical research evidence.p = 0.019; with higher use observed among females) and hours per week participating in research . There were no statistically significant associations between the skill subscore and other demographic factors.The median use subscore  reflected an average level of engagement in EBP activities between 1 and 10 times in the preceding month . A moderate positive correlation was found between the use subscore (categorised by quartiles) and gender , the application of research evidence to clinical practice (96.4%), critical analysis/critical thinking (92.9%), conducting clinical research (87.5%) and conducting systematic reviews and meta-analyses (83.9%). In most cases (50.0–60.7%), the training was undertaken as a component/course/module of a multi-year undergraduate program.Most (50.0–91.1%) participants believed that 9 of the 10 listed factors were ‘very useful’ enablers of EBP uptake. The factors favoured the most were: access to the internet in the workplace (91.1% reported this factor as ‘very useful’), access to free online databases in the workplace (73.2%), access to critical reviews of research evidence relevant to chiropractic (67.9%), access to critically appraised topics relevant to chiropractic (64.3%) and ability to download full-text journal articles (60.7%). The factor favoured the least was access to online tools to assist with conducting critical appraisals of multiple research papers, with 55.4% of respondents identifying this factor as not/slightly/moderately useful in improving EBP uptake.The majority (60.7–92.9%) of participants indicated that all 13 potential barriers listed in EBASE were either not a barrier or only a minor barrier to EBP uptake. Those factors considered by most to not be a barrier to EBP uptake were: lack of colleague support for EBP (75.0%), lack of relevance to chiropractic practice (67.9%), lack of resources (67.9%), lack of interest in EBP (64.3%), lack of industry support for EBP (62.5%) and lack of incentive to participate in EBP (58.9%). Factors largely reported as minor or moderate barriers to EBP uptake included: lack of clinical evidence in chiropractic (78.5%), insufficient skills to critically appraise the literature (62.5%), lack of time (59.0%), insufficient skills for interpreting research (59.0%), insufficient skills to apply research findings to practice (58.9%), insufficient skills for locating research (55.4%) and patient preference for a particular treatment (53.5%).The findings of this study provide insights into the self-reported skills, attitudes, and uptake of EBP among Swedish chiropractors who are members of the Swedish Chiropractic Association. Participating chiropractors reported a moderate to moderate-high level of perceived skill in EBP, an overall positive attitude towards EBP, and an average level of engagement in EBP activities. Workplace internet access was the most favoured enabler of EBP uptake, whereas a lack of clinical evidence in chiropractic was considered the main barrier for such uptake.Participating chiropractors reported a relatively high level of perceived skill in performing several aspects of the EBP process; in particular, asking precise clinical questions, acquiring evidence and applying evidence. By contrast, chiropractors were less confident regarding their level of skill in conducting clinical research and systematic reviews. This is consistent with findings from studies involving other health professions –37, 44.It has been suggested that clinical health care providers are “consumers” rather than “producers” of scientific knowledge ; thus, cAttitudes toward EBP were generally positive among participating chiropractors. This corroborates the favourable perceptions of EBP reported among Canadian and Australian chiropractors , 33, as Our study revealed a positive association between the participants’ attitude toward EBP and their highest qualification. This concurs with previous study findings, suggesting that holding a higher educational qualification, such as a Master’s degree, may contribute to a better understanding of evidence-based practice, and in turn, lead to a more favourable attitude towards EBP –53. ThusParticipating Swedish chiropractors reported a low to moderate level of engagement in EBP activities. This is consistent with the level of EBP uptake observed in other health professions , 36, 38.Chiropractic associations and unions may play a pivotal role in propelling policy and practice recommendations on the importance of EBP uptake, promoting advances in chiropractic research, and communicating evidence-based chiropractic to the profession and wider community, including government, patients and other health care providers. The European Chiropractors’ Union has recently attempted such communication at the European Parliament level . SomewhaThere were several limitations to the current study. While the participant response rate of 33% was higher than that reported in previous chiropractic studies utilising EBASE , 32, it We acknowledge that the EBASE survey is long, and that this could potentially contribute to survey fatigue. However, all 56 chiropractors that participated in the survey completed all survey items; as such, there was no indication of survey fatigue in this cohort of participants. Additional limitations of this study, such as self-selection bias, cannot be excluded due to the use of self-selection sampling. Meaning that it cannot be excluded that participants that had more interest in EBP may have been more likely to participate in the survey and vice versa. Potential implications of such selection bias, if present, could for example be that some participants’ EBP attitudes were more positive, and their levels of reported EBP engagement more frequent, than across all chiropractors of the Swedish Chiropractic Association. Also, as the Swedish version of EBASE was not psychometrically tested, it is uncertain if the Swedish version of the survey retained the same psychometric properties as the English  version of EBASE. These limitations should be taken into consideration when interpreting the findings of the study.The EBASE survey did not include questions about the participants’ chiropractic institutional background nor their philosophical chiropractic orientation, which may be useful to include in future studies to inform additional perspectives of chiropractic in relation to EBP. Furthermore, it is likely that the barriers and facilitators to chiropractors’ uptake of EBP may be influenced by a range of personal values, professional norms, and clinical/working environments, e.g. multidisciplinary clinics where the use of EBP is established and expected, such as in medicine and physiotherapy, , and mayConsidering the current findings, it is suggested that important directions for future action should be to develop and evaluate interventions focussed on improving EBP skill level and EBP uptake among chiropractors. A better understanding of barriers and facilitators to EBP skill development and uptake is an important next step to overcoming the obstacles to EBP use in chiropractic. The use of observational data collection methods to explore how chiropractic attitudes toward EBP and perceptions of EBP skill level relate to real world chiropractic practice behaviour and decision-making, represent another key focus for future research.This is the first study to examine the skills, attitude and uptake of EBP, as well as the barriers and enablers of EBP uptake, among Swedish chiropractors who are members of the Swedish Chiropractic Association. Although participating chiropractors reported a generally positive attitude towards EBP, their level of engagement in EBP activities was modest. A number of perceived barriers to EBP uptake may have contributed to this modest level of engagement, including a lack of clinical evidence in chiropractic, lack of time and insufficient skills in locating, appraising and translating evidence into practice. Notwithstanding, a deeper exploration of the barriers and facilitators to EBP utilisation is warranted. The findings of such work would help inform innovative strategies aimed at improving EBP skill development and supporting EBP uptake among Swedish chiropractors.


The present study aimed to determine the immunoadjuvant efficacy of mixed cross-linked dextran microspheres (CDM) and tetanus toxoid (TT)-loaded trimethyl chitosan (TMC) nanospheres in dry powder form. The TMC nanoparticles (NPs) containing TT were produced using the ionic gelation method. Co-administration of TT-loaded TMC NPs and CDM as an absorption enhancer was performed to improve immunity against the antigen. Dry powder formulations were delivered via the nasal route in a rabbit model. Among immunization groups, mixing of CDM with TT encapsulated in TMC NPs could elicit the highest titer of systemic IgG antibody. Furthermore, the addition of CDM to TT-loaded TMC enhanced the sIgA response relative to the TT solution. The TMC NPs had a considerable effect on mucosal and systemic immunity against the TT antigen. Therefore, the CDM excipient can be utilized for nasal immunization to elevate systemic and mucosal responses.  Most of the present vaccines are developed to deliver the formulation in a systemic manner such as intravenous (IV), intramuscular (IM), subcutaneous (SC), or intradermal (ID). The current vaccine formulations are administered parenterally to promote cellular and humoral immunity against pathogens. The parenteral vaccine cannot contact with a mucosal barrier to induce mucosal immunity due to the functional difference between the mucosal and systemic immune systems. On the other hand, the administration of vaccines via nasal route stimulates systemic immunity and robust mucosal immune response -3. ThereChitosan (CHT) is a non-toxic and hydrophilic cationic polysaccharide chemically derived by the partial deacetylation of chitin with biodegradable and biocompatible properties. CHT nanoparticles (NPs) and derivatives have potential applications as the controlled release drug and antigen delivery systems -12. CHT The N-trimethyl CHT (TMC) is a quaternized CHT derivative that improves physicochemical properties such as water solubility in a broader pH range relative to the CHT polymer , 24. TheThe purpose of this investigation was to assess the immune efficacy of encapsulated tetanus toxoid (TT) antigen as a model antigen in TMC NPs with CDM as a penetration enhancer to induce the mucosal and systemic responses followed by the nasal administration in a rabbit model. MaterialsThe alum-adsorbed TT (50 Lf/m) and TT (2750 Lf/ml) solutions were provided by Razi Institute (Iran). CHT  was purchased from Primex (Norway) to synthesize the TMC polymer. Additionally, CDM (Sephadex G-150) was obtained from Biogen (Sweden). Tripolyphosphate (TPP), Lactose, Span 80, and Tween 80 were used from the Merck group (Germany). Anti-rabbit secretory immunoglobulin A (sIgA) and immunoglobulin G (IgG) antibodies were purchased from Bethyl Laboratories Inc. (USA) and Sigma company (USA), respectively. Coomassie Brilliant Blue was obtained from Fluka (Switzerland). The Micro BCA assay kit was purchased from Pierce  and all other chemicals used were of analytical grade. Synthesis and characteristics of TMC polymeret al, 1998 3]/[H] × 1/9 ] × 100DQ (%)=[[(CH (1)3)3] is the integral of the trimethyl amino group at 3.3 ppm and [H] is the integral of the 1 H peaks of TMC polymer between 4.7 and 5.7 ppm  technique. All samples were loaded on a 10% acrylamide gel and the silver nitrate staining was used to visualize the bands of protein.Loading efficiency of TT antigen in TMC nanoparticlesThe TT-loaded TMC was identified from the difference between the free and total amounts of TT in the supernatant followed by centrifugation at 15,000 g for 20 min at 10 °C. The BCA assay was applied to obtain the concentration of non-entrapped TT antigen in TMC NPs and the suspension of empty TMC NPs was utilized as a blank. The loading efficiency (LE) was determined by the following equation:  (2) Additionally, the yield of TMC NPs and also TT loading content (LC) were calculated as follows:(3) (4) Release assay The nasal release of TT antigen from TMC NPs was performed using the vertical Franz diffusion cell, to simulate the humidity of the nasal cavity. The mentioned diffusion cell is comprised of two compartments with a membrane fixed between the donor and acceptor chambers. The acceptor chamber was filled with 20 ml of fresh PBS solution at pH 7.4, which stirred by a magnetic stirring bar. The nanospheres (25 mg) were poured evenly across the hydrated filter paper mounted in the diffusion cell, which was in contact with the buffer of the acceptor compartment. The release experiments were carried out at 37 °C for 4 hr. At regular time intervals, 400 µl of each sample was withdrawn from the acceptor chamber and replaced with a fresh PBS. The concentration of released TT antigen from TMC nanospheres was measured using the Micro BCA protein assay method. All experiments were conducted in triplicates. Immunization studiesin vivo study. All animal experiments were performed according to the Mashhad University of Medical Sciences Ethical Committee Acts, which complied with the ARRIVE guidelines and also conducted under the National Institutes of Health (NIH) guide for the care and use of laboratory animals. Rabbits were immunized in groups of four, three times at days 0, 14, and 28  via nasal administration as the following formulations: 40 Lf TT antigen solution (TT); 40 Lf TT antigen in TMC + CDM (TMC (TT) + CDM); and 40 Lf TT antigen in TMC + lactose (TMC (TT) + Lac). Additionally, intramuscular administration of the alum-adsorbed TT vaccine (Alum TT) was performed as a positive control to compare the efficacy of vaccine formulations. White albino rabbits weighing between 2 to 2.5 kg were supplied by Pasteur Institute (Iran) to accomplish the Rabbits were sedated with ketamine hydrochloride) 40 mg/kg  or TT antigen solution (100 µl) by the intranasal route to assess the immune responses against the TT antigen. For the nasal delivery of dry powder formulations, powders were poured in polyethylene tubes (2.0 mm diameter) and linked to a syringe. The mentioned tubes were placed in an animal nose (0.5 cm) and 10 ml of air was injected into the tube. The inoculated animals were bled two weeks after the last booster to collect sera for antibody assays. Subsequently, rabbits were sacrificed and the nasal cavity was rinsed with PBS . All samples were kept at -70 °C until immunological assays.Determination of antibody responsesThe serum sample of each rabbit was collected to evaluate the total IgG antibody. Additionally, the nasal lavage samples from the nasal cavity of animals were used for the sIgA antibody assay. TT-specific antibody responses in the sera and nasal lavages were determined by end-point titration using an enzyme-linked immunosorbent assay (ELISA) as described previously . AccordiStatistical analysispost hoc Tukey-Kramer test. The results are presented as the mean (n = 4) ± standard deviation (SD). The P-values of less than 0.05 were considered significant.Immunization data were performed by one-way ANOVA using GraphPad Prism (version 7.0) to assess the significance of the differences between formulations followed by the Characteristics of synthesized TMC by NMR spectroscopyThe analysis of NMR spectra revealed that a reproducible degree of quaternization was about 24%, followed by synthesizing TMC polymer using the two-step methylation procedure (data not shown). Preparation and characteristics of TMC nanospheresThe TMC NPs were produced using ionotropic gelation of TMC polymer with the TPP solution. According to this method, the negatively charged TPP solution can interact with the positive charges of CHT (amino groups) by electrostatic interactions. The mentioned method has several advantages including the absence of heat and organic solvent during the preparation of TMC NPs. The mean intensity diameters of TMC NPs (n=5) without or with TT antigen was 378.13±50.9 nm and 447.3±58.07 nm, respectively. Additionally, the zeta potential of TMC NPs in the absence or presence of TT antigen was 3.32±0.05 mV and 2.28±0.07 (n= 5), respectively.Determination of the protein stabilityThe SDS-PAGE analysis was utilized to assess the effect of the TMC preparation procedure on TT antigen integrity. As shown in Loading efficiency of TT antigen in TMC nanospheresThe loading efficiency of TT in TMC NPs was calculated to be 43.25%±3.56 (n=5). The yield of TMC NPs and TT loading content was determined as 86%± 23.4 and 90%±27.1, respectively.The release study of TT from TMC nanoparticlesThe release assay of the TT antigen was accomplished in a diffusion chamber to mimic the humid environment of the nasal mucosa . The nasAnti-TT IgG titersP<0.05) was observed in the rabbits received Alum TT and TMC (TT) + CDM formulations through IM or nasal administration, respectively. Moreover, no significant difference (P>0.05) was determined between vaccinated animals by TMC (TT) + CDM and Alum TT vaccines. The serum IgG titers of immunized rabbits with various vaccine formulations were obtained via the end-point titration using an ELISA method . The titAnti-TT sIgA titersP< 0.01) higher for nasal administration of TMC (TT) + Lac vaccine compared to TMC (TT) + CDM or TT solution. The lowest sIgA titer was obtained in the group received the TT solution by the nasal route. The sIgA titers were identified from nasal lavage of immunized rabbits using the end-point titration via an ELISA method. As shown in Dry powder forms of particulate delivery systems with muco-adhesive properties including CHT and its derivatives were developed as appropriate candidates for mucosal immunization. The purpose of our investigation was to evaluate the immune efficacy of synthesized TMC NPs as an antigen delivery system for nasal delivery of TT antigen in the presence of CDM or lactose excipient. The co-administration of lactose and TT-loaded TMC NPs were accomplished to promote the inhalation efficacy of the vaccine formulation. Additionally, the CDM adjuvant was physically mixed with TMC NPs loaded with TT to elevate the immunogenicity and efficiency of vaccine formulations. The dry powder formulations were delivered via the nasal route in a rabbit model to determine the titers of sIgA and IgG antibodies against the TT antigen. The dry powder form of vaccine formulations has many advantages including more microbiological and chemical stability, easier storage without cold chain, mass vaccination, and better distribution as well as more affordable compared to liquid formulations , 40, 41.The mean size of synthesized TMC NPs without or with TT antigen was about 378 nm and 447 nm, respectively that is expected to interact efficiently with DCs. Similar to the viruses, the NP formulations with a size less than 500 nm can be quickly recognized by antigen-presenting cells (APCs) during processing and presentation of antigen followed by strongly uptake by DCs to evoke the immune response against pathogens. In contrast, the particle formulations with a size range of 0.5 to 5 µm are effectively uptaken by macrophages -45. AddiThe nasal release study of TT-loaded TMC NPs was performed in a diffusion chamber to simulate the temperature and humid environment of the nasal cavity. Therefore, to assess the release profile of the TT antigen, the nanospheres were in contact with a wet and warm membrane similar to the nasal mucosa. Based on the release profile, the TT antigen demonstrated a burst release within 1 hr, suggesting that most of the TT was released from TMC NPs followed by a plateau release up to 4 hr. Previous studies revealed that the half-life of clearance is approximately 15 to 20 min in a nasal cavity of humans , 49, 50.According to the antibody assays, the incorporation of TT into TMC NPs could significantly enhance the titers of sIgA and IgG antibodies in rabbits vaccinated through the nasal route compared to the antigen alone. The sIgA antibody indicates mucosal immunity against pathogens, whereas the systemic humoral immune response is mediated by IgG antibody . Our resFollowing the intranasal delivery of TMC formulations, the higher titer of mucosal IgA antibody was observed for TMC (TT) + CDM and TMC (TT) + Lac vaccines than the TT solution. The TT-loaded TMC NPs in the presence of lactose excipient could considerably enhance the sIgA titer indicating a potent mucosal immune response. Based on our results, TMC formulations could pass through the NALT in the respiratory tract to interact with mucosal inductive tissues such as the Peyer’s patches. Subsequently, the TT-loaded NPs could be efficiently uptaken by the M-cells located in the mucosal inductive tissues to secrete the mucosal sIgA antibody and induced robust mucosal immunity , 54, 55.et al. reported that the addition of CDM to TT antigen could induce the lower mucosal sIgA titer in comparison with the TT solution, while the high mucosal immunity was stimulated by the TT solution. This study demonstrated that the lower titers of the sIgA antibody elicited with CDM + TT formulation might be due to the size of microspheres and their inability to interact with the M-cells of NALT (Although the TMC (TT) + CDM vaccine induced the higher titer of the sIgA antibody compared to the TT solution, no significant difference was found between the mentioned formulations. Some of our previous studies illustrated that the mixture of CDM with PLGA NPs or alginate microspheres loaded with TT could enhance the titers of the sIgA antibody followed by nasal administration , 41. How of NALT . Additio of NALT . Accordi of NALT . et al. demonstrated that the co-administration of insulin and diethylaminoethyl-dextran (DEAE-Dextran) promoted the nasal absorption of insulin formulation in a mouse model (Although the co-administration of CDM or lactose excipient with TMC (TT) formulation could induce robust systemic immunity against TT, the intranasal delivery of TMC (TT) + CDM formulation was more effective to elicit the high titer of IgG antibody compared to TMC (TT) + Lac vaccine. Additionally, no significant difference was found between the nasal delivery of TMC (TT) + CDM and IM injection of the Alum TT vaccine. Accordingly, the addition of CDM to TT-loaded NPs has an effective role to produce a high level of systemic IgG antibody response. The CDM has been reported as a safe absorption enhancer with adjuvant effect to improve the epithelial absorption and permeation of various antigens due to opening the intercellular tight junctions , 57-59. se model , 41, 58.se model . The co-se model , 58. Taken together, the co-administration of CDM with antigen delivery systems such as TMC nanospheres can induce potent systemic immune responses, whereas CDM has no significant effect to elicit mucosal sIgA response. The utilization of particulate delivery systems was developed to overcome the enzymatic degradation, rapid mucociliary clearance, and low absorption of antigens and drugs following nasal administration. The dry powder form of TT-loaded TMC NPs with or without CDM adjuvant was evaluated after intranasal immunization in a rabbit model. The encapsulated TT in TMC NPs induced high sIgA and IgG titers indicating the robust mucosal and systemic responses against TT antigen, respectively. The addition of CDM powder to TMC vaccine formulation could significantly promote the systemic IgG responses compared to TMC (TT) in the presence of lactose. Although, the TMC formulation with CDM increased the mucosal sIgA titer, no considerable difference was observed between the mentioned vaccine formulation and TT solution. According to our study, CDM excipient can be used as an efficient adjuvant to induce potent systemic responses.


Xenopus tropicalis frogs. The adult male offspring of pesticide-exposed fathers (F1) showed reduced body size, decreased fertility, and signs of endocrine system disruption. Impacts were further propagated to the grand-offspring (F2), providing evidence of transgenerational effects in amphibians. The adult F2 males demonstrated increased weight and fat body palmitoleic-to-palmitic acid ratio, and decreased plasma glucose levels. The study provides important cross-species evidence of paternal epigenetic inheritance and pollutant-induced transgenerational toxicity, supporting a causal and complex role of environmental contamination in the ongoing species extinctions, particularly of amphibians.Underlying drivers of species extinctions need to be better understood for effective conservation of biodiversity. Nearly half of all amphibian species are at risk of extinction, and pollution may be a significant threat as seasonal high-level agrochemical use overlaps with critical windows of larval development. The potential of environmental chemicals to reduce the fitness of future generations may have profound ecological and evolutionary implications. This study characterized effects of male developmental exposure to environmentally relevant concentrations of the anti-androgenic pesticide linuron over two generations of offspring in   Interesechanism . The ecoXenopus tropicalis frogs at environmentally relevant concentrations. Linuron is used in many parts of the world, and measured environmental concentrations are around 10 μg/L  using a semistatic system (50% water change three times per week) to maintain good water quality  and housed at the Evolutionary Biology Center, Uppsala University. Adult frogs were bred to obtain F0 tadpoles according to  quality . Three r quality . Water q2.2F0 males and 11 linuron-exposed F0 males with naive females as described in F1 =12; linuronF1 = 32) were mated with naive females to generate the grand-offspring (F2) and enable studies of transgenerational effects  were mated with naive females to analyze fertility.The overall study design is presented in  effects . When th2.3Male and female frogs were placed in pairs in breeding tanks and observed every 45th minute to determine the time to enter amplexus (mating position), time to spawning and total time in amplexus. The mating experiment was discontinued if the frogs did not enter amplexus within six hours. To study male fertility, a random subsample of the spawned eggs was collected using three glass Petri dishes on the bottom of the breeding tanks and transferred to holding tanks as described in 2.4After breeding, the frogs were anaesthetized in tricaine , weighed, and photographed on top of a millimeter-scale paper , and after measurements of snout-vent length (SVL), fore limb width (secondary sex characteristic) and hind limb length (endpoint for disruption of the thyroid axis), killed by decapitation. The photographs of the nuptial pads were analyzed using ImageJ . The body mass index (BMI) was calculated using the body weight and SVL. The testes were collected and weighed to calculate the gonadosomatic index (GSI). Fat bodies (fat depots associated with the gonads) and liver samples were collected and stored at −80 °C until analysis. The left testis was fixed in neutral buffered formaldehyde . The examiner was unaware of to which experimental group the frogs belonged.2.5The left testes were embedded in hydroxyethyl methacrylate for high-resolution morphological observation as previously described . Transve2.6After decapitation, trunk blood was collected at the decapitation site in heparinized tubes before centrifugation at 1000 ×g for 10 min. The plasma was collected and stored at −80 °C until analysis. Glucose levels were analyzed with enzymatic colorimetric methods using an Architect c4000 automated chemistry analyzer .2.7Lipids were extracted from 50 to 100 mg of liver and fat body tissue using a modified Folch procedure . An inte2.8U test or Fisher's exact test for categorical variables. Differences were considered statistically significant at p values <0.05.The results are presented as the mean ± SEM, and the data were analyzed using the nonparametric Mann-Whitney 33.1F1) demonstrated significantly altered body measures compared with the corresponding controlF1 males  and the controlF1 frogs (1.90 ± 0.15 mmol/L).The analyses of fatty acids and the desaturation index based on the 16:1/16:0 and 18:1/18:0 fatty acid ratios  in the f3.3F1 frogs was significantly higher relative to their body weight than that in the controlF1 group, resulting in a 35% increase in the GSI. The area of the male breeding glands, the nuptial pads, was decreased by 23% compared to that in controlF1 frogs  than in the controlF1 group (0/12). The average fertility rates in the linuronF1 and controlF1 groups were 30.8% and 44.9%, respectively (p = 0.0519).The weight of the testes in the linuronF1 frogs . The breF1 males . Notably3.4F1 frogs. These testes lacked distinct germ cell nests, and both spermatocytes and spermatogonia were freely dispersed in the seminiferous tubule lumen. In addition, there was a higher abundance of interstitial connective tissue surrounding the seminiferous tubules  demonstrated an increased body weight and BMI (by 11%) compared with those in the controlF2 group  lower in the linuronF2 males (1.93 ± 0.14 mmol/L) than in the controlF2 males (2.79 ± 0.22 mmol/L).There was a significant increase in the oleic acid (18:1) levels in the fat bodies, but not in the liver, of linuronF2 frogs . The fatF2 group . A similF2 group . The 18:F2 frogs . The pla3.7F2 males compared with the controlsF2. The breeding experiments revealed no significant differences in breeding behavior or fertility for linuronF2 males compared to those in the controlF2 group , but relatively little attention has been given to assess how this affects biodiversity and ecosystem function  provides further evidence of disturbed androgen regulation in the linuronF1 males pyrene from the tadpole stage to adult age in Xenopus tropicalis resulted in a smaller F1 generation , an enzyme that converts saturated fatty acids into monounsaturated fatty acids  released into the environment  and thatAppendix A. Supplementary datahttps://doi.org/10.1016/j.scitotenv.2021.145771.Supplementary data to this article can be found online at Supplementary material


Acute hydrocephalus is a common complication of spontaneous or traumatic intracranial bleeding with extensive subarachnoid hemorrhage (SAH) or ventricular extension. However, it has never been reported to be secondary to pneumocephalus.A 32-year-old man was admitted following a motorcycle accident. Head computed tomography (CT) performed right after the accident revealed a skull base fracture and mild perimesencephalic SAH. Three days later, repeated CT revealed delayed perimesencephalic pneumocephalus and an evident enlargement of the ventricular system.The patient was diagnosed with acute obstructive hydrocephalus, which was secondary to pneumocephalus and traumatic SAH.The patient was treated with temporary external ventricular drainage (EVD).The patient experienced an unremarkable recovery process. At follow-up 3 months later, he showed no recurrence of the hydrocephalus and the score of Glasgow Outcome Scale was 5.Transient mechanical obstruction of CSF circulation and disturbance of CSF physiology might conjointly lead to the acute obstructive hydrocephalus. A gradual accumulation of a large amount of air inside the cranium may lead to increased intracranial pressure and mass effect . In addition to headache, pneumocephalus may cause confusion, lethargy, hemiparesis, and hemiplegia. Hardly could we ever imagine that acute hydrocephalus occurs after traumatic pneumocephalus. In this report, we present a rare case of acute hydrocephalus secondary to traumatic mild SAH and delayed perimesencephalic pneumocephalus. To the best of our knowledge, no previous similar case was reported.Acute hydrocephalus is a common complication of spontaneous or traumatic intracranial bleeding with extensive subarachnoid hemorrhage (SAH) or ventricular extension.2A 32-year-old man was admitted to the local hospital following a motorcycle accident. Head computed tomography (CT) performed right after the accident revealed a skull base fracture and mild perimesencephalic SAH Fig. . He was 3Being a Case Report with written consent, our institution does not require formal Ethical Approval. Written informed consent was obtained from the patient for publication of this case report.4 However, it has never been reported in patients with traumatic pneumocephalus. In a report by Lee et al, a case of delayed shunt-dependent communicating hydrocephalus was reported 2 months after surgery for tension pneumocephalus. However, the patient is different from ours in that our patient developed delayed hydrocephalus concurrently with the development of pneumocephalus. In addition, the hydrocephalus of this case was transient and no permanent shunting was needed. Therefore, they might have different pathophysiological mechanisms. The possible explanations that involved in the development of acute hydrocephalus in this patient might be as follows.Acute hydrocephalus refers to rapid ventricular enlargement and is commonly encountered after spontaneous or traumatic intracranial bleeding, CSF infection, or intracranial occupying lesions. In contrast, the patient did not develop hydrocephalus at the acute stage of head injury but 3 days later with the concurrence of perimesencephalic pneumocephalus. The hydrocephalus did not recur after resolution of the pneumocephalus and withdrawal of the EVD. CSF upward circulation to the supratentorial basal cisterns might be blocked by entrapped air at the perimesencephalic cistern.First, obstruction of CSF circulation caused by perimesencephalic pneumocephalus. Our deduction was mainly based on the following facts. In contrast, acute hydrocephalus was previously reported following pneumoencephalography, which was thought to be obstruction of CSF circulation.,8 According to this hypothesis, cerebral interstitial fluid (ISF) as well as CSF, is formed by water (constituting 99% of CSF) filtration across arterial capillary walls throughout the entire central nervous system (CNS). A continuous turnover of water takes place in microvessels between the plasma and ISF/CSF. The exchange of water inside the CNS is influenced by osmotic and hydrostatic forces. The water exchange between the entire ventricular system and the ISF depends on changes in fluid osmotic and hydrostatic pressures in different CNS compartments. That is to say, the volume of CSF is regulated by the osmotic and hydrostatic difference between the CSF/ISF and microvessels. In this patient, perimesencephalic SAH and its degradation products might cause local inflammatory responses and relatively hyperosmotic environment and lead to water movement from the ISF and microvessels to the ventricular system. Although this theory of CSF formation has not been widely accepted and could not overturn the traditional theory at present, it could at least shed some light on the formation of hydrocephalus in this case.Secondly, relatively hyperosmotic environment caused by SAH. From the traditional and widely accepted viewpoint, CSF is actively secreted by the choroid plexuses in the ventricles and circulates unidirectional along the ventricular system toward the cortical subarachnoid space and is ultimately absorbed into the venous sinuses through arachnoid villi. However, with some new findings in experimental investigations on animals and humans, a new hypothesis of CSF physiology and hydrocephalus development was proposed which was named Bulat-Klarica—Orešković hypothesis.5We report a rare case of acute hydrocephalus secondary to traumatic SAH and delayed perimesencephalic pneumocephalus. The underlying mechanism might be transient mechanical obstruction of CSF circulation and disturbance of CSF physiology.Conceptualization: Guangming Wang, Kun Hou, Jinlu Yu.Data curation: Guichen Li.Project administration: Jinlu Yu.Writing – original draft: Guichen Li, Tengfei Luan.Writing – review & editing: Kun Hou, Jinlu Yu.


A total of 559 adult patients were hospitalized in the Sheba Medical Center with a diagnosis of COVID-19, 320 (57.24%) of whom were tested for troponin levels within 24-hours of admission, and 91 (28.44%) had elevated levels. Predictors for elevated troponin levels were age , female sex , low systolic blood pressure  and increased creatinine level . The risk for death  and a composite outcome of invasive ventilation support and death  was significantly higher among patients who had elevated troponin levels. In conclusion, in hospitalized patients with COVID-19, elevated troponin levels are associated with poor prognosis. Hence, troponin levels may be used as an additional tool for risk stratification and a decision guide in patients hospitalized with COVID-19.Myocardial injury in hospitalized patients is associated with poor prognosis. This study aimed to evaluate risk factors for myocardial injury in hospitalized patients with coronavirus disease 2019 (COVID-19) and its prognostic value. We retrieved all consecutive patients who were hospitalized in internal medicine departments in a tertiary medical center from February 9 The coronavirus disease 2019 (COVID‐19) pandemic is a global health concern. Its main manifestation is pulmonary inflammation with potentially life-threatening complications, such as pneumonia and acute respiratory distress syndrome .Myocardial injury, as heralded by elevated troponin levels, was previously observed in various medical conditions, including pneumonia –6, and wTo date, several studies have described myocardial injury in patients diagnosed with COVID-19 . AccordiOur study aimed to further evaluate the incidence of myocardial injury in COVID-19 hospitalized patients, to characterize risk factors for myocardial injury among these patients, to determine its prognostic significance.th, 2020, to August 28th, 2020, with a diagnosis of COVID-19 and a reverse-transcription polymerase chain reaction confirmed SARS-CoV-2 infection. All patients included were 18 years old or older at the time of diagnosis and had at least one high sensitivity troponin (hs-TnT) measurement within 24 hours after admission. If there was more than one hs-TnT measurement into the first 24-hours, the highest level was chosen.This is a retrospective cohort study. Data were obtained from the electronic medical records of the Sheba Medical Center, the largest tertiary medical center operating in Israel. We retrieved all consecutive patients who were hospitalized from February 9th Revision codes and included heart failure, ischemic heart disease, chronic kidney disease, malignancy, chronic obstructive pulmonary disease, diabetes mellitus, hypertension, cerebrovascular accident, and dyslipidemia. When death occurred outside the hospital, mortality dates were obtained from governmental mortality records.For each patient, baseline demographic characteristics and clinical information were extracted from the medical records. Clinical data included medical comorbidities, home medications, vital signs on admission , and laboratory tests that were taken within 24-hours from admission , defined according to the Kidney Disease: Improving Global Outcomes (KDIGO) definition as a ratio of more than 1.5 between the maximal creatinine level to its minimal value in less than seven days . MyocardTo better characterize COVID-19 patients with increased troponin, we reviewed their medical records, and examined their electrocardiogram (ECG) at presentation and compared them to previous ECG exams, coronary catheterization during hospitalization, and diagnostic imaging for pulmonary embolism (including chest computed tomography angiography (CTA) and lower limb venous Doppler ultrasound (US)).The study was conducted and reported in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guidelines .Baseline clinical data were compared between patients with at least one troponin measurement within 24-hours from admission and patients without troponin measurement and between patients with elevated troponin and non-elevated troponin. Continuous variables were compared using the Mann–Whitney–Wilcoxon test, and categorical variables were compared using the Chi-square test.Logistic regression was applied to identify variables that are best associated with an elevated troponin level following COVID-19 diagnosis. Covariates for the multivariable models were pre-specified based on clinical relevance.Outcomes of mortality, invasive ventilatory support, length of hospital stay, and AKI were analyzed in the following manner. The crude association between troponin levels and mortality, and between troponin levels and the composite outcome of invasive ventilation and mortality was modeled using Kaplan Meier curves and compared using the Log Rank test. A Cox proportional hazard regression analysis was performed to adjust for age, sex, background comorbidities , chronic medications , low systolic blood pressure at admission (defined as < 90 millimeters of mercury [mmHg]), and maximal creatinine level as measured up to 24-hours from admission.Data analyses were performed using the R programming language .The study was approved by the institutional review board (IRB) of Sheba Medical Center. Informed consent was waived by the IRB committee.th, 2020 to August 28th, 2020, a total of 559 patients over the age of eighteen were hospitalized with a diagnosis of COVID-19 at the Sheba Medical Center. Among these patients, 320 (57.24%) were tested for troponin levels in the emergency room and up to 24 hours from admission. Among patients tested for troponin, 91 (28.44%) were found to have an elevated level. A visualized presentation of troponin levels is available in From February 9Patients who were tested for troponin were more likely to be male, were older, and had more underlying diseases than those who were not tested .Characteristics of patients who had troponin levels measured are presented in In a multivariable analysis, age , low systolic blood pressure at admission , and elevated creatinine level at admission  were found to be significantly associated with an elevated troponin. Male sex was significantly negatively associated with elevated troponin . Other background comorbidities and chronic medications were not found to be significantly associated with elevated troponin levels. The complete data regarding predictors for elevated troponin in COVID-19 patients are presented in Prior to adjustment, patients with elevated troponin level were more likely to develop acute kidney injury , hypoxemia during hospitalization , to be intubated within 30-days of admission  and to die within 60 days of admission  than patients with normal troponin level . The lenA Cox proportional hazards regression analysis for the same association, adjusted for sex, age, elevated troponin, comorbidities , chronic medications , low admission systolic blood pressure, admission creatinine level is presented in Among 91 patients diagnosed with COVID-19 and elevated troponin, eight patients (8.79%) had newly significant ECG changes. Three of them had a low voltage of QRS amplitude, two had new-onset atrial fibrillation, and three had extensive ST elevation (STE). Of the three patients with STE, one was diagnosed with STE myocardial infarction (STEMI) a week before the COVID-19 diagnosis, and underwent percutaneous coronary intervention (PCI)- Therefore the ECG findings were regarded as reminder findings with no special treatment. The two other patients presented with STE on the ECG were immediately taken to coronary catheterization with a suspected STEMI. Of these, one had significant coronary stenosis requiring the placements of two drug-eluting stents, while the other was found to have normal coronaries. A detailed specification of ECG findings of the sub-group of deceased patients hospitalized with COVID-19 and elevated troponin can be found in Two patients with COVID-19 and elevated troponin were diagnosed with pulmonary embolism based on a computed tomography angiography and doppler ultrasound of the lower extremities demonstrating deep vein thrombosis.To further analyze the cause of death of the patients who were deceased and had elevated troponin, we reviewed their medical records for the cause of death . Out of In this retrospective cohort study, we demonstrated factors that are associated with elevated troponin in patients with COVID-19, and showed that elevated troponin is associated with a poor prognosis in patients hospitalized with COVID-19. Moreover, its prognostic significance remains after adjustment for other variables. We showed that elevated troponin levels were prevalent among hospitalized COVID-19 patients (28.44% of tested patients) and were found to be associated with reduced survival as well as with an increased risk of a composite outcome of invasive ventilatory support and death. Elevated troponin was also associated with a higher risk for other in-hospital adverse events, such as acute kidney injury and low oxygen saturation. These findings strengthen the prognostic value of the myocardial injury, as measured by serum troponin, in patients hospitalized with COVID-19, as was shown in prior studies. A retrospective cohort conducted by Shi et al. , as wellAs described in prior studies, patients with elevated troponin levels were older and had higher rates of underlying comorbidities , 13–15. Low systolic blood pressure (<90mmHg) and increased admission creatinine levels were found to be associated with the development of cardiac injury in patients hospitalized with COVID-19. Female sex was shown to be a predictor for myocardial injury, but it may be explained by selection bias. Male patients were more likely to be tested for troponin levels, and possibly female patients were tested when being older or exhibiting more severe symptoms. When stratified by sex, there was a significant age difference between female patients with elevated troponin levels and males, with female patients being significantly older.Several possible explanations were suggested for the mechanism by which COVID-19 causes myocardial injury , 22. IncWe assume that as in other medical conditions, the elevated troponin levels reflect myocardial injury as a part of the inflammatory state of the COVID-19 infection. As most patients in our study died from respiratory complications, we believe that the presence of myocardial injury signifies a more severe infection and inflammatory state, leading to a worse phenotype of its most common presentation, COVID-19 pneumonia. This assumption is supported by a recently published study by Metkus et al.  which deCardiac arrests and arrythmias were described in COVID-19 hospitalized patients in several studies and reports , 27. TheTo-date, this is the first study to evaluate ECG findings in a large population with COVID-19 infection and elevated troponin levels, comparing them to previous individual exams, and further tracking their coronary catheterization results. This in-depth analysis enabled us to demonstrate that troponin levels may be of prognostic significance even without any evidence for myocardial ischemia on ECG, with two patients demonstrated to have normal coronaries on coronary catheterization.The small sample size in this study limits the interpretation of the results. Furthermore, this research was conducted in a single tertiary medical center. troponin levels were measured according to the discretion of the attending physician and, therefore, selection bias might have occurred. Indeed, it is evident that patients who had troponin measured were older and tended to suffer from more background diseases, including ischemic heart disease.Acute kidney injury was defined according to the KDIGO definition of a ratio of more than 1.5 between the maximal and minimal creatinine levels at a seven day period. Urine output and 48-hours elevation were not considered, as well as changes in creatinine values that were non-minimal or non-maximal. Thus, there is an underestimation of AKI events.Body mass index was not calculated routinely thus was not retrieved, as well as di-dimer and Creatine kinase, which were not routinely measured. Furthermore, echocardiography test, which may have added aetiologic and prognostic information was not performed during hospitalization to the studied population.This study demonstrates that myocardial injury, as indicated by elevated troponin levels, is an important prognostic factor for people diagnosed and hospitalized with COVID-19. Myocardial injury represents a poor prognosis and a higher risk for kidney function deterioration, oxygen support, invasive ventilation support, and mortality. This research emphasizes the importance of measuring troponin levels in all COVID-19 hospitalized patients, even without clinical evidence of myocardial injury. This will enable risk stratification, directing clinical decisions and intensity of care in the emergency room, and throughout hospitalization.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S4 Fig(DOCX)Click here for additional data file.S5 Fig(DOCX)Click here for additional data file.S6 Fig(DOCX)Click here for additional data file.


Corynebacterium striatum (C. striatum) have been described sporadically in the literature over the last two decades. However, C. striatum meningitis combined with multiple abscesses has not been published before.Intracranial infections with C. striatum and combined with suspected brain and lung abscesses. This patient who underwent multiple fractures and a recent cut presented with headache and paraphasia. C. striatum was isolated in cerebrospinal fluid and supposedly transmitted from the skin purulent wound through blood. The patient was treated with intravenous vancomycin and had a transient improvement, but died finally. Multiple abscesses, especially in the brain, could be a reason to explain her conditions were deteriorating rapidly.In this report, we describe the clinical and imaging findings in a 54-year-old woman with meningitis caused by C. striatum can cause life-threatening infections. Early identification and diagnosis, early administration of antibiotics to which the bacterium is susceptible, and treatment of complications will be beneficial in patients with C. striatum-related infection.Note that  Corynebacterium species are part of the resident skin flora. Among these, Corynebacterium striatum (C. striatum) is a kind of causative bacteria to induce various types of infections mainly including lower respiratory tract infection, isolated bacteremia and central line infections [C. striatum over the last two decades [C. striatum meningitis combined with multiple abscesses has not been published before. In this report, we describe one patient with meningitis caused by C. striatum and combined with suspected brain and lung abscesses.fections . However decades –5. FurthA 54-year-old woman presented to the neurology department of our hospital on 29 October 2019 complaining of headache, blurred vision, vomiting and paraphasia. This patient had had rheumatoid arthritis and osteoporosis, and taken the drugs irregularly over the past decade. She broke her right femur 5 years ago and right leg 2 years ago. She underwent fracture repair surgeries successively and sustained the plate internal fixation in her right leg. The surgical wounds had healed without obvious infection. Accidentally, she cut her left leg over 40 days ago and smeared Chinese herbal ointment to promote healing. However, the treatment effect was poor and she had not paid enough attention to protect against infections. Ultimately, she left an unhealed and purulent wound visibly. Family and psycho-social history including relevant genetic information was normal.At the time of admission, the patient’s vital signs were normal without fever. She was confused and had simple language reaction to painful stimuli. She did not cooperate to complete neurological examination which showed a stiff neck and positive right pathological sign.9/L with 84.5% neutrophils. High-sensitivity C-reactive protein was 256.59 mg/L and procalcitonin was 4.89 ng/mL, both far above normal values. Lumbar puncture was then performed after excluding contraindications, in especial brain shift on computed tomography (CT) plain scanning. A lumbar puncture yielded cloudy cerebrospinal fluid (CSF) with initial pressure over 400mmH2O. CSF analysis revealed an elevated total nucleated cell count of 1210 × 106/L (90% multiple nuclear cells), an increased protein concentration of 1.58 g/L  and a decreased glucose concentration of 0.39 mmol/L (blood glucose 5.44 mmol/L). Considering the available information, we empirically made a diagnosis of purulent meningitis and immediately treated with intravenous vancomycin (1 g every 12 h) and meropenem (1 g every 8 h) to cover all common causative microorganisms until culture results were known.Laboratory tests of blood taken at admission showed a white blood cell (WBC) count of 14.61 × 106/L, and CSF culture yielded no bacterial growth. Five days later, the first bacterial culture was subsequently identified as Corynebacterium striatum (C. striatum). Susceptibility testing was not performed by our laboratory department due to a lack of drug sensitivity guidelines for this kind of bacteria in our country. Intravenous vancomycin and meropenem therapy was continued. Blood culture taken before the start of antibiotic therapy remained negative. The CSF and blood levels of vancomycin were not measured.After a 24-h incubation period, gram staining of the cloudy CSF demonstrated the presence of gram-positive bacteria Fig. . After 72O, an increased total nucleated cell count of 536 × 106/L and protein concentration of 1.40 g/L. In the next day, this patient had respiratory and circulatory failure gradually. In spite of giving cardio-pulmonary resuscitation and mechanical ventilation, this patient eventually died. As far as available information was concerned, we speculated that the intracranial abscess ruptured and formed cerebral hernia. In the course of the hospitalization, the patient’s family firmly refused any surgical operations because of the costs and risks.However, head and chest CT with contrast enhancement examinations  showed intracranial and pulmonary abnormal lesions, which were considered as abscesses Fig. . After 9C. striatum has been established as an emerging multidrug-resistant and opportunistic pathogen, which was traditionally known as a common component of the normal microbiota of skin and mucosal surfaces [C. striatum was commonly isolated from wounds, respiratory specimens, tissue specimens, bone specimens and blood cultures. Therefore, most cases of C. striatum infection manifested as pneumonia, bacteremia/septicemia, endocarditis and peritonitis. And the scope of these infections has been broadening.surfaces . C. striC. striatum was isolated from CSF and identified as the causative pathogen. To our knowledge, only a few previous reports have implicated C. striatum in central nervous system infection. The first proven case of meningitis caused by C. striatum was published in 1996, which was a 23-year-old Canadian man with a direct communication between the skin and the CSF through a catheter left after an extensive nerve reconstructive surgery [C. striatum from three children at the ages of 13-month, 20-month and 6-year, respectively [C. striatum. Two Turkish cases of meningitis due to C. striatum both underwent an external ventricular drainage after neurosurgery operations [C. striatum meningitis [C. striatum meningitis combined with multiple abscesses has not been published before. Bacteria can enter the brain to induce intracranial infection through contiguous spread, hematogenous dissemination or with other unknown mechanisms [C. striatum invades to CSF and causes meningitis. In the present case, it seemed that there was no direct communication between the skin and the CSF, in spite of those extracranial bone fractures. Hence, it suggested hematogenous spread of bacteria from the skin purulent wound was the primary cause of meningitis and forming abscess.In the present case, erations . Additioningitis . HoweverC. striatum, in the present case was identified from CSF culture. However, the association between the C. striatum and abnormal lesion in brain was not definitely clear without evidence from biopsy or drainage culture. Additionally, pulmonary cavitary lesions on CT were detected in the present patient and abscesses could not be ruled out as well. The most common causes of lung abscess are aspiration pneumonia, transdiaphragmatic spread, and hematogenous seeding commonly associated with right-sided endocarditis or septic thrombophlebitis [Cranial imaging should be performed in patients with intracranial infection, especially suspected brain abscess. CT scanning with contrast enhancement provides a rapid means to detect abscesses, but lacks sensitivity and specificity. Head CT scanning with contrast enhancement of the present patient showed the lesion characterized by a hypodense center and a hyperdense ring that was considered to be a suspected brain abscess. Comparatively, MRI combined with diffusion-weighted and apparent-diffusion coefficient images is a more valuable diagnostic tool . Unfortuhlebitis . These cC. striatum is relatively resistant to many of the commonly used antibiotics with Gram-positive activity. It was demonstrated that vancomycin, linezolid and daptomycin were generally classified as active antibiotics [C. striatum meningitis published in the above-mentioned studies and the present case report. Note that the different treatment effects and clinical outcomes were also attributed to severity of infection and patients’ clinical manifestations. The fact that the present patient had coma, anisocoria, respiratory and circulatory failure associated with the intracranial infection could explain why she died. Multiple abscesses, especially in the brain, could be a reason that her conditions were deteriorating rapidly.ibiotics . Howeveribiotics . VancomyC. striatum has the potential to cause meningitis especially in patients with direct communication between the skin and the CSF. No denying that hematogenous spread of C. striatum is also worthy of attention. The frequency of C. striatum infections appears to be increasing along with the improvement of feasibility and accuracy of the identification from clinical cultures. Note that C. striatum can cause life-threatening infections. Early identification and diagnosis, early administration of antibiotics to which the bacterium is susceptible, and treatment of complications will be beneficial in patients with C. striatum-related infection.As a conclusion, these published cases confirm that


A cardiologist-only approach to procedural sedation with midazolam in the setting of elective cardioversion (DCC) for AF has already been proven as safe as sedation with propofol and anaesthesiologist assistance. No data exist regarding the safety of such a strategy during emergency procedures. The aim of this study is to compare the feasibility of sedation with midazolam, administered by a cardiologist, to an anaesthesiologist-assisted protocol with propofol in emergency DCC.Single centre, prospective, open blinded, randomized study including all consecutive patients admitted to the Emergency Department requiring urgent or emergency DCC. Patients were randomized in a 1:1 fashion to either propofol or midazolam treatment arm. Patients in the midazolam group were managed by the cardiologist only, while patients treated with propofol group underwent DCC with anaesthesiologist assistance.Sixty-nine patients were enrolled and split into two groups. Eighteen patients (26.1%) experienced peri-procedural adverse events , which were similar between the two groups and all successfully managed by the cardiologist. No deaths, stroke or need for invasive ventilation were registered. Patients treated with propofol experienced a greater decrease in systolic and diastolic blood pressure when compared with those treated with midazolam.p < .001).As the procedure was shorter when midazolam was used, the median cost of urgent/emergency DCC with midazolam was estimated to be 129.0 € (1st-3rd quartiles 114.6–151.6) and 195.6 € (1st-3rd quartiles 147.3–726.7) with propofol (Procedural sedation with midazolam given by the cardiologist alone was feasible, well-tolerated and cost-effective in emergency DCC. Direct current cardioversion (DCC) represents the most widely used and effective method to restore sinus rhythm in patients with persistent atrial fibrillation (AF) –3. DCC iTo this day, no specific guidelines or recommendations with regards to the most appropriate drug that should be used for procedural sedation is available and different sedation strategies have been described in literature but no consensus on the most efficient strategy has been reached. One of the most commonly used agent for sedation in DCC, is propofol  an intraThe aim of this prospective, open-blinded, randomized study is to compare the feasibility of sedation with midazolam, administered exclusively by a cardiologist, to an anaesthesiologist-assisted protocol with propofol in urgent or emergency DCC.The “Intravenous beNzodiazepine Safety and Tolerability in Emergency Atrial fibrillation Direct-current cardioversion” (INSTEAD) is a single centre, prospective, open blinded, randomized study included consecutive patients admitted to the Emergency Department or the University Hospital “Ospedali Riuniti Umberto I - Lancisi - Salesi” requiring urgent or emergency DCC. The present study follows a pilot trial  that hasEmergency DCC was defined as DCC performed in haemodynamically compromised patients with new-onset AF, according to European guidelines . Urgent The inclusion criteria consisted in age > 18 years old and admission for high rate AF or atrial flutter requiring urgent/emergency cardioversion. A documented or suspected allergy or intolerance to midazolam or propofol represented the only exclusion criterion. No patient was excluded based on comorbidities or concomitant diseases in order to reflect “real-life” population data.The study conforms to the Declaration of Helsinki and was performed in accordance with the Consolidated Standards of Reporting Trials (CONSORT) standards and national recommendations by competent authorities  . The carIn the midazolam group, both procedural sedation and DCC were carried out by the cardiologist who administered a starting dose of midazolam 3 mg, followed by 2 mg every 2 min until satisfactory response  . The anaA maximum of three shocks were delivered, following a step up published protocol : a firstOne-hundred percent oxygen supplementation and saline infusion were provided to each patient during the procedure.At the end of the procedure, 1 mg of flumazenil was administered to the patients in the midazolam group in order to revert the effects of the benzodiazepine.When awake, all patients were asked to rate the pain and distress on a scale going from no pain to the worst possible pain and distress according to a visual analogue scale (VAS).A 12-lead ECG was performed at the beginning and at the end of the procedure in order to assess the restoration of sinus rhythm and all vital status parameters  as well as delay, length of procedure, length of monitoring and hospitalization were registered.The primary safety endpoint was a composite of death and the following peri-procedural adverse events requiring medical intervention: bradycardia (defined as heart rate < 50 bpm for at least 30 s), severe hypotension (defined as systolic blood pressure < 80 mmHg), severe hypoxia (defined as oxygen saturation < 85%), stroke, transient ischaemic attack and need for orotracheal intubation.Other safety endpoints included the variation of blood pressure, heart rate and oxygen saturation registered before induction, before delivery of the shock, after the shock and after recovery. Tolerability of the procedure was assessed using the VAS.Efficacy was assessed based on the number of very early recurrences (within 2 h) and early recurrences (within 24 h). All patients discharged before 24 h from the Emergency Department were called by phone the day after the procedure in order to confirm stable sinus rhythm. Time-related issues  were also considered.Finally, we analysed the cost-effectiveness of the procedure carried out by the cardiologist alone with midazolam, compared to procedural sedation with propofol and anaesthesiologist assistance. Direct total costs were defined as the total of personnel costs, material costs and hospitalization costs. For a more detailed definition of costs calculation please refer to our previous study . IndirecQuantitative variables were checked for normality by the Kolmogorov–Smirnov test, and described as mean and standard deviation  or median and first to third quartile . ANOVA adjusted by age and sex was used to compare normally distributed quantitative variables. Kruskal– Wallis ANOVA adjusted by age and sex was used to compare non-normally distributed quantitative variables. Categorical variables were described by absolute number and assessed by using χ2 analysis. General linear model for repeated measures was used to assess time-dependent changes of blood pressure, heart rate and oxygen saturation. As one patient per group ended up having both interventional drugs administered Fig. , an inteBeing a feasibility study, a formal, a-priori, sample size calculation was not performed. However, considering an expected composite endpoint rate of 18% from our pilot study , a populp < 0.05 were taken as statistically significant.SPSS 25.0 for Windows  was used for statistical analysis. Values of Sixty-nine patients were consecutively enrolled in our study, 61 of which (84.4%) had atrial fibrillation and eight (11.6%) were admitted with atrial flutter. General characteristics of the population, including risk factors, comorbidities, underlying heart disease, echocardiographic parameters and previous arrhythmic episodes are described in Table Eighteen patients 26.1%) met the composite safety endpoint (Table 6.1% met p = .336) and diastolic (p = .347) blood pressure than those treated with midazolam .Ten recurrences were registered in the first 24 h. Of those, six were registered in the propofol group  and four were registered in the midazolam group (two very early and two early), with no significant difference between groups (p = .013) and were burdened by a 30 min higher delay which was needed to get more personnel ready , nor did median hospitalization length  and was mainly driven by an increased delay and lack of coordination between the cardiologist and the anaesthesiologist. In terms of material costs, the median cost in the midazolam group was higher than the propofol one as the former implied the use of flumazenil . Hospitalization costs in the midazolam group added up to a median of 28.1 € and 48.7 for the propofol group (p = .022). The total median cost of urgent/emergency DCC with midazolam was estimated to be 129.0 € (1st-3rd quartiles 114.6–151.6) and 195.6 € (1st-3rd quartiles 147.3–726.7) with propofol (p < .001).The median medical cost was of 16.4 € for the midazolam group and 47.8 € for the propofol group (Procedural sedation plays a key role in many procedures carried out for arrhythmia related issues such as device implant, electrophysiological studies, catheter ablations  and DCC The results of the present study confirm that procedural sedation with midazolam could be a viable alternative to the propofol-based protocol, even in an emergency/urgency setting. Both drugs showed a similar safety profile as no patient experienced death, neurological events or needed orotracheal intubation. The number of adverse events in the midazolam group were fewer than those experienced in the propofol group and more importantly all of them were easily managed by the cardiologist alone with no need of intervention or assistance by the anaesthesiologist. On a note, the number of adverse events and the recurrence rates during emergency/urgent DCC were higher when compared to events in elective procedures . This reRegarding recurrences, the difference was not statistically significant between the two groups. However, it is important to underline that the rate of recurrence could be different in patients who undergo urgent or emergency DCC compared to elective procedures. In fact, arrhythmia recurred in 14% of our patients, more than what usually expected in elective DCC . PotentiThe procedure carried out with the benzodiazepine was well tolerated as demonstrated by the fact that all patients scored a zero on the VAS scale. We feel that such a result is not only secondary to the sedation obtained with midazolam, but also partly related to the anterograde amnesia caused by the drug. Moreover, as described above, while oxygen saturation and heart rate had a similar trend in both study groups, patients who received propofol experienced a more important drop in systolic blood pressure than in those in the midazolam group. This data confirm the effect of propofol as a peripheral vasodilator, therefore causing a greater impact on haemodynamic parameters .In addition to this, the results clearly show how the length of the procedure and the associated delays can be shortened by using midazolam instead of propofol. We believe that the use of flumazenil as an antagonist to midazolam is the key factor contributing to a reduced procedural time. Flumazenil has been described to be a safe and effective reversal agent  and alloOur findings show that the use of midazolam can reduce costs by decreasing the time spent for the procedure itself and by having fewer operators needed to perform the procedure, as the cardiologist can safely manage both sedation and shock delivery. Standardizing a protocol which does not imply the need for anaesthesiologist support appears cost-effective.Some limitations of the present trial deserve to be discussed. Firstly, double-blinding was deemed technically unfeasible due to the different number of physicians involved, the different intravenous formulations and the different titration protocols. Second, differences in experience and expertise of the cardiologists with midazolam could have provided an unintentional bias, albeit very difficult to measure. Third, the lower-than-expected difference in the composite endpoint between the two groups leaves open the chance of a type II error, even if of minor magnitude from a clinical point of view. However, it must be stressed out that the rate of severe complications was too low to allow for any firm conclusion regarding safety, being this a feasibility study with a relatively small sample size. Fourth, the present results could be difficult to extrapolate in other clinical settings, especially in those countries in which cardiologists are trained in advanced airways management or in which DCC are not primarily managed by the cardiologist .Finally, we found that one patient who regularly took benzodiazepines for insomnia had built a tolerance which limited our possibility of using midazolam and had to be crossed over to propofol. Nonetheless, we also had one patient who probably metabolized propofol too quickly and as a consequence midazolam had to be used for sedation. However, we feel that this examples strongly confirm the need of finding easily manageable, safe and well tolerated options for procedural sedation.A cardiologist-only approach to procedural sedation with midazolam could represent a feasible and well tolerated option for DCC even in the emergency/urgency setting. Adverse events were managed by the cardiologist in both groups with no need for intervention by the anaesthesiology team. The length of procedure was shortened when midazolam was used and fewer delays were registered, thus potentially leading to reduced costs.Additional file 1: Supplementary Table 1. CONSORT 2010 checklist of information to include when reporting a randomised trial.


Clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing techniques find applications in many fields, such as molecular biology, cancer biology, and disease modeling. In contrast to the knock-out procedure, a key step of CRISPR knock-in experiments is the homology-directed repair process that requires donor constructs as repair templates. Therefore, it is desirable to generate a series of donor templates efficiently and cost-effectively. In this study, we developed a new strategy that combines (i) Gibson assembly reaction, (ii) a linker pair composed of eight in silico screened restriction enzyme sites, and (iii) a hierarchical framework, to remarkably improve the efficiency of producing donor constructs for common genes as well as for the genes containing unbalanced guanine-cytosine content and requiring a selectable marker. Furthermore, the approach provides the ability of inserting additional elements into the donor templates, such as single guide RNA recognition sites that have been reported to enhance the efficiency of homology-directed repair. Conclusively, our modularized process is simple, fast, and cost-effective for making donor constructs and benefits the application of CRISPR knock-in methods. In a cell, there are multiple signaling pathways that respond to intracellular and extracellular signals, thereby regulating gene expression. To investigate mechanisms of signal transduction, it is desirable to generate a series of knock-in mutants fused with fluorescence proteins (FPs) for tracking protein dynamics , 2, and A typical donor construct has four major components: the vector backbone, the IF, such as an FP, and the two homologous fragments (5’ and 3’ arms) localized at the two sides of the insertion point. Mainly, there are two categories of methods for assembling these components into a complete donor construct: restriction enzyme (RE)-based approaches and sequence-independent overlapping techniques. Examples for the first category are BioBricks , BglBricGiven the need of generating donor constructs more efficiently and cost-effectively, we established a new strategy that combines Gibson assembly, the Modular Overlap-Directed Assembly with Linkers (MODAL) standard , and theOur method incorporates the advantages of the RE-based approaches and sequence-independent overlapping techniques. On one hand, the assembly step is based on Gibson reaction, for which we introduced the linkers to cope with the constraints and to increase the efficiency up to eight-fold. On the other hand, the RESs employed here can be easily used for subsequent experiments. We demonstrated that, the HDR efficiency was improved to approximately two-fold by inserting the sgRNA recognition sites, which comprise the sgRNA target sequences and the protospacer adjacent motif (PAM) sequences (5'-NGG-3'), to our IF vector through a simple digestion–ligation step. In our group, we already created the donor vector bank where the donor vectors with various reporter proteins (RPs), FPs, and/or removable selectable markers (SMs), flanked by the linkers on standby. Depending on the different experimental purposes, the corresponding IF vector is chosen to perform one digestion plus one assembly reaction, and then the donor template is ready. Overall, the new approach and our donor vector bank facilitate the production of donor constructs required for CRISPR knock-in method, hence expanding the application of CRISPR-based techniques to a broader range of genes, discovering the unknown functions and mechanisms in cells.AgeI site at the 5’ end of linker a, XhoI site at the 3’ end of linker b, and three other RESs on each linker  into Gibson assembly reaction. The linkers were composed of specific RESs, with nker See . The linnker See . Throughnker See  and seleThe FP vector was purchased from Addgene  and the linker a and b were inserted, therefore becoming the IF vector used in The digested DNA fragments and vectors were purified and then mixed with NEBuilder HiFi DNA Assembly Master Mix (NEB#E2621), incubating for 1–3 hours. The DNA assembly mixtures were transformed into DH5α cells. The next day, 60 colonies were picked for PCR verification to check the IFs. The efficiency was calculated as the number of the colonies containing the desirable constructs divided by the number of tested colonies.GRCh38/hg38) was downloaded from the National Center for Biotechnology Information database (GCF_000001405.34_GRCh38.p8_genomic.gbff), and split into different GenBank files for every chromosome as well as one for mitochondrial DNA. Analysis was performed on each chromosome individually. Alternate assemblies and unassembled reads were not included in the analysis. A combination of homebrewed scripts and Biopython .The human reference genome . Afterward, the cells were incubated for 48 hours and subcultured in 10 cm dishes for 5–7 days before subjected to image-taking or knock-in efficiency assay. The sgRNA sequence of CDH1 is 5′-AAGCTGGCTGACATGTACGG-3′, which was designed by the program named “CRISPR gRNA Design tool – ATUM” (https://www.atum.bio/eCommerce/cas9/input). For the sequences of the sgRNA recognition site, the PAM sequences were added to the 3’ end, marked in bold: 5′-AAGCTGGCTGACATGTACGGAGG-3′. After annealing the two single-stranded oligonucleotides, the sgRNA-PAM fragments were inserted into the donor construct. Through flow cytometry, the knock-in efficiency was calculated as the number of EGFP-positive cells divided by the total number of the cells.T47D cells (ATCC#HTB-133) were cultured in Dulbecco’s Modified Eagle’s Medium (Gibco) supplemented with 10% fetal bovine serum (Biochrom), 100 units/ml penicillin and 100 µg/ml streptomycin (Invitrogen) in 5% COFor generating CRISPR knock-in donor constructs, Gibson assembly is the method of choice to join the vector backbone, the IF, and the two homologous fragments (5’ and 3’ arms) up. DNA synthesis is a straightforward and fast way to obtain the 5’ and 3’ homologous arms of target genes. However, this approach is only suitable for low complexity fragments with few repeat sequences and homopolymeric regions, and without unbalanced GC content . AlternaOur idea came from the MODAL strategy that applied linkers to conduct Gibson assembly . HoweverIn order to verify our approach in the real-world CRISPR knock-in practice, we chose genes involved in the epithelial-to-mesenchymal transition (EMT)  as a modCDH1 (coding E-cadherin), CTNNB1 (coding β-catenin), and SNAI1 (coding Snail1), were picked as examples to test our method for producing donor constructs. Here, we aimed at inserting EGFP to the 3’ end of CDH1, the 5’ end of CTNNB1, and the 5’ end of SNAI1. To do so, we designed primers to amplify the homologous arms from human genomic DNA and attach the linker sequences at the same time by PCR. Consequently, the results showed that all the 5’ and 3’ arms of the three genes, except the 3’ arm of CTNNB1 with MODAL linkers (red arrow), were successfully amplified by the designed primers  than CDH1 and CTNNB1 , and SM-EGFP were the IFs used in the experiments to also test whether the length of IFs would affect the outcome. The size of EGFP, SM, and SM-EGFP was around 0.75 kb, 1.3 kb, and 2.2 kb, respectively. As shown in the left part of SNAI1 or VIM as examples to confirm our approach. First, the IF vectors carrying the linker pair 1, removable SM, and the FP were proceeded for RE, AgeI, digestion. Next, the 5’ homologous arm was incorporated into the vectors through Gibson assembly. Then, the previous two steps were repeated but with XhoI digestion and 3’ homologous arm integration. To produce a donor construct with a removable SM and an FP for SNAI1, we successfully achieved an assembly efficiency at approximately 70% for both the 5’ and 3’ homologous arm incorporation steps .Conflict of interest statement. None declared. bpaa006_Supplementary_DataClick here for additional data file.