Viruses are among the most infectious pathogens, responsible for the highest death toll around the world. Lack of effective clinical drugs for most viral diseases emphasizes the need for speedy and accurate diagnosis at early stages of infection to prevent rapid spread of the pathogens. Glycans are important molecules which are involved in different biological recognition processes, especially in the spread of infection by mediating virus interaction with endothelial cells. Thus, novel strategies based on nanotechnology have been developed for identifying and inhibiting viruses in a fast, selective, and precise way. The nanosized nature of nanomaterials and their exclusive optical, electronic, magnetic, and mechanical features can improve patient care through using sensors with minimal invasiveness and extreme sensitivity. This review provides an overview of the latest advances of functionalized glyconanomaterials, for rapid and selective biosensing detection of molecules as biomarkers or specific glycoproteins and as novel promising antiviral agents for different kinds of serious viruses, such as the Dengue virus, Ebola virus, influenza virus, human immunodeficiency virus (HIV), influenza virus, Zika virus, or coronavirus SARS-CoV-2 (COVID-19). Viruses are among the most infectious pathogens, responsible for the highest number of deaths worldwide. Although the pathogenic mechanisms of viruses are diverse, all existing viruses need a host to maintain their existence . The comViral infections result in millions of deaths and huge economic losses annually. In recent years, important examples are the viruses Ebola, Zika, SARS, MERS or recently the actual pandemic SARS-CoV-2 coronavirus (which have infected more than 140 million and killed more than 3 million people so far (data from 21 April 2021) .This means that novel systems for rapid and efficient detection of viruses are critical to control the infection spread. From the last decade, nanotechnology has signified an important advance in the development of nanomaterials for detection devices ,10,11,12Glycan molecules have been demonstrated to have a very important role in many biological recognition processes . Indeed,Therefore, in this review we summarized the recent advances in the fabrication of novel functionalized glyconanomaterials, evaluating the effect of different glycomolecules as biomarkers in the recognition of human viruses and the important role in the detection of viral glycoproteins in relevant cases such as the Dengue virus, Ebola virus, human immunodeficiency virus (HIV), influenza virus, Zika virus, and coronavirus SARS-CoV-2 as well as their novel application as potential antiviral agents. Different sections will be focused on proteins, oligosaccharide-functionalized nanomaterials, and glyconanoparticles as biosensors for virus detection and glyconanoconjugates for virus inhibition . AnotherOne of the most promising strategies for biosensor devices to detect viruses is based on protein-based biosensors ,18,19,20In such cases, some of the main examples using nanomaterials involve the functionalization with lectins, proteins which specifically interact with glycans from glycoproteins. In particular, Concanavalin A (ConA) is a well-known lectin, which binds specifically α-D-mannosyl and α-D-glucosyl residues , which aThus, Oliveira and coworkers  developeDifferences in viral envelope are an essential feature of immune response differentiation after arbovirus infection. The structural components of the viral envelope can be used to identify and differentiate arboviruses . TherefoThe degree of affinity of the sensor to the virus decreased from ZIKV > DENV2 > CHIKV > YFV. The sensor shows the highest response to ZIKV due to the interaction of ConA with specific carbohydrate residues present in the structural glycoprotein E of ZIKV b. Also, Another approach in the design of protein-based biosensors focuses on the use of proteins mimicking the carbohydrate-binding receptor of the virus.Fetuin-A  is a 64-kDa glycoprotein that is found in relatively high concentrations in human serum and recently has been used for the new selective nanobiosensor developed for influenza virus . The strTherefore, this could be a potential diagnostic sensor for clinical applications, because of the advantages of speed, the use of very small sample volumes, and its suitability for the selective determination of the virus from complex real samples.Another strategy for detection of viral infection involves the development of an imm unosensor capable of monitoring cytokines . CytokinA graphene-nitrocellulose ink was printed on a flexible polyimide film (Kapton) in an IDE pattern by aerosolizing graphene and depositing the aerosol mist in highly focused lines. The electrode was chemically modified to generate carboxylic groups as functional groups on the surface for covalent immobilization of anti-bovine antibody (IFN-γ or IL-10) which was then used for detecting the corresponding IFN-γ and IL-10 cytokines a. The syMost viruses recognize glycans on glycoproteins and glycolipids of the mammalian cells as receptors , especiaThus, Yates and coworkers  developeCarbon nanotubes were functionalized with pyrenemethylamine by π–π stacking interaction of the pyrene moiety and the SWNT. Then chemical modification was performed to incorporate heparin to the surface. This oligosaccharide is highly sulfated, having the highest negative charge density of any known biological macromolecule, whereas DENV envelope protein has positively charged β-strands located within a well-conserved binding pocket for electrostatic interaction with receptor sulfate groups. Therefore, interaction between the glycan and virus is detected by the resistance on p-type SWNTs. This method was highly selective since a functionalized sensor responded to the presence of DENV but not to influenza H1N1 . A limitHowever, actually the most important developments in glycan-sensors for virus detection have been focused on the coronavirus SARS-CoV-2, responsible for the pandemic respiratory disease COVID-19 (starting in December 2019) , which tIn this context, sialic acids were determined as a receptor (or recognition motif) for the glycoproteins from the coronavirus, especially the spike protein . In SARSThus, recently Gibson and coworkers  describeN-hydroxyethyl acrylamide), PHEA, was chosen as the polymer to give colloidally stable particles , by the displacement of an ω-terminal pentafluorophenyl (PFP) group, and immobilization onto gold particles via an α-terminal thiol. In the next reaction step, poly  to ensure glycosylation. The assays revealed that in this system sialic acid gave the strongest response and consequently was taken forward. With the successful identification of sialic acid as a target ligand, its application as the capture unit in lateral flow was examined. It is important to highlight that the performance does not only depend on the affinity of the capture ligand but also on the flow of the particles. For this reason, “half” lateral flow assays were established to optimize the particles. The negative test line was  2,3-silyllactose-BSA, which the nanoparticle should not bind to . The positive control was immobilized SARS-CoV-2 and the sialic acid-AuNPs 35 nm nanoparticles flowed over them. The lateral flow devices could clearly detect the virus-like particles at a concentration of 5 µg/mL protein. Sialic acid particle system had a clear preference for SARS-CoV-2, demonstrating selectivity in this glyconanoparticle system. This data supports the notion that the terminal sialic acid is the key binding motif. Therefore, this system can detect the spike glycoprotein from SARS-CoV-2 virus under 30 min, thus being interesting for the creation of rapid point-of-care diagnostics in a format which requires no infrastructure and limited training.Other glycans with a relevant role in cell communication, signaling and adhesion, also acting as immuno-modulators during infectious diseases are phosphoglycans such as glycosylphosphatidylinositol (GPI) . This moThus, recently a glycobiosensor based on synthetic GPI was developed by Orozco and coworkers  Figure . The sysT. gondii) was attached to screen-printed gold electrodes (SPAuEs) through a linear alkane thiol phosphodiester and the interaction with antibodies was detected and quantified by electrochemical impedance spectroscopy (EIS). No significant increase in the Rct (charge transfer resistance) value was observed when the GPI-electrode was incubated with the non-reactive serum whereas an increase was clearly observed with the reactive serum.This phosphoglycan -3-trimethyl chitosan (HTCC) cationic nano/microspheres were prepared by crosslinking of chitosan with genipin, followed by reaction with glycidyltrimethyl-ammonium chloride (GTMAC). After cytopathic and PCR analyses, these glycoparticles adsorbed in a strong and selective way human coronavirus NL63 (HCoV-NL63).Therefore, another strategy associated with detection of immunoresponses involves the fabrication of oligosaccharide nanoparticles in antitumor therapy ,41. DextGlycomolecules conjugated with nanostructures, such as nanoparticles have been also investigated for multiple applications, in particular for novel antiviral therapy.In this sense, sulfated glycopolymers such as heparin and carrageenin have been reported to interact with several types of viruses. However, inherent heterogeneity and toxicity of these naturally occurring glycopolymers may hamper clinical applications . To overThe recognition between cell surface glycans and carbohydrate binding proteins in pathogens and host cells is often the first stage of many infectious processes. Blocking these interactions provides a strategy to inhibit pathogen adhesion and subsequent infection. Importantly, this approach can prevent the virus from mutating and developing resistance . DC-SIGNIn this sense, recently Martin and coworkers  synthesi50 values as low as 0.37 μg/mL.The results showed that both the nanocarbon platforms  and the multivalent glycan display (glycodendrimers or glycofullerens) strongly influenced the glyconanomaterials’ ability to interact with DC-SIGN and prevent viral infection: glycoconjugates based on MWCNTs functionalized with glycofullerenes were found to be the most potent inhibitors, with ICGold nanoparticles (GNPs) displaying glycans have been extensively studied as multivalent glycomaterials for sensing and biomedical applications . Zhou anThis review emphasized the most recent advances in the design and application of different glyconanomaterials for the detection and inhibition of human viruses. This nanotechnological approach, in which it combines the power of nanomaterials, for example nanoparticles—which have been demonstrated in the last decade to be an important tool for biomedical applications—with glycan molecules—which have a key role in biological recognition systems—opens up timely a future way forward in biomedicine. Advantages of this methodology are for example high sensitivity and efficiency, whereas a potential disadvantage in the case of therapy application is the possible cytotoxicity of these nanomaterials.Novel systems using tailor-made oligosaccharides or particular proteins were emphasized with their highly sensitive, rapid, and economic features for potential industrial diagnosis kits, in particular of high relevance in the case of the actual pandemic (SARS-CoV-2 virus detection).


Individuals with bulimia nervosa (BN) experience persistent episodes of binge eating and inappropriate compensatory behavior associated with impaired physical and mental health. Despite the existence of effective treatments, many individuals with BN remain untreated, leading to a high burden and an increased risk of chronicity. Web-based interventions may help facilitate access to evidence-based treatments for BN by reducing barriers to the health care system.N = 152) will be randomly assigned to either (1) an intervention group receiving a 12-week web-based intervention or (2) a waitlist control group with delayed access to the intervention. Further assessments will be scheduled 6 (mid-treatment) and 12 (post-treatment) weeks after baseline. Changes in the number of binge eating episodes and compensatory behaviors will be examined as primary outcomes. Secondary outcomes include global eating pathology, functional impairments, well-being, comorbid psychopathology, self-esteem, and emotion regulation abilities.The present study will investigate the effectiveness of a web-based self-help intervention for BN in a two-armed randomized controlled trial. Individuals diagnosed with BN . •Although bulimia nervosa is associated with marked impairments, there is a treatment gap for patients in routine care.•Integrating web-based interventions into routine care might facilitate access to treatment for patients with bulimia nervosa.•A web-based self-help intervention will be evaluated by examining changes in core symptoms of bulimia nervosa.•Secondary outcomes include measures of psychopthology and psychosocial functioning as well as potential mechanisms of change.•Weekly questionnaires and ecological momentary assessment will increase the external validity and temporal resolution of results. Core syPrevious research provides evidence for the effectiveness of cognitive-behavioral therapy (CBT) for BN , and CBTConcerning the causes of this treatment gap, previous research indicates that a combination of patient-associated and healthcare-related barriers hinders the help-seeking process . On the Web-based self-help interventions might help facilitate access to mental health care by allowing higher temporal flexibility, being location-independent and cost-efficient while still providing the possibility of individual treatment adaptations . BesidesRegarding their effectiveness, first studies suggest that web-based self-help interventions can successfully reduce BN symptoms . Still, The present randomized controlled trial (RCT) addresses this research gap by evaluating whether applying a 12-week web-based self-help intervention in routine care will lead to a decline in eating disorder symptoms and improvements in the quality of life in patients with BN compared to a waitlist control group. Consequently, the primary goal of the current trial is to expand previous findings to a more representative sample. As such, we aim at overcoming the limitations of earlier studies by investigating the effectiveness of a web-based self-help program in a sample of full-threshold BN patients that would typically receive unspecialized, non-evidence-based care or no treatment at all .Our second goal is to explore how web-based interventions can affect symptom changes and whether patient characteristics influence treatment outcomes. So far, knowledge on moderators, mediators, and predictors of treatment success for internet-based CBT is still limited . Thus, wFinally, our trial will incorporate weekly symptom measurements and ecological momentary assessment (EMA) to increase the validity of results and obtain a more nuanced understanding of treatment effects .22.1Selfapy for BN, which is based on methods of CBT and was designed to directly target BN symptoms as well as topics related to BN such as emotion regulation, stress management, and self-esteem  and social media. Further study information will be shared via mailing lists, and a waitlist of subjects interested in the intervention will be contacted. Moreover, information material on paper will be distributed in Germany.The recruitment will include online and offline advertisements to reach a high number of German-speaking individuals potentially eligible for our study. Online advertisements will include postings on the intervention provider's website . The patient characteristics based on the PICO framework  can be f2.2.1Eligible participants will need to (1) be aged from 18 to 65, (2) have adequate German-language skills (C1), (3) have a smartphone with permanent internet access during the study period, and (4) be diagnosed with BN according to the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) criteria during the clinical interview . Exclusi2.3Selfapy for BN. A CONSORT flow diagram , six weeks after baseline (mid-treatment), and 12 weeks after baseline (post-treatment). Moreover, eating behavior, binge eating episodes, and compensatory behavior will be monitored weekly to assess changes in symptoms with a higher resolution. EMA will be scheduled pre- and post-treatment to measure eating disorder symptoms in daily life. All measures will be collected online using the assessment platform i Survey  and madeidelines  is provi2.4.4•Changes in the frequency of bulimic behaviors within the last 28 days: Based on previous meta-analyses and studies with a similar study design, the changes in the frequency of binge-eating episodes and compensatory behavior over the preceding four weeks were selected as the primary outcomes . The questionnaire's 28 items are answered on 5-point scales, and the internal consistencies for the subscales range from α = .78 to α = .86 : Smartphone-based EMA will be implemented to capture eating disorder symptoms, everyday affect, and emotion regulation with a high ecological validity in daily life. EMA will be conducted for five days at pre- and post-treatment and will consist of five daily signal-contingent and additional event-based assessments after bulimic behaviors. Binge eating episodes and compensatory behaviors will be assessed based on the DSM-5 criteria for BN and previous EMA studies  descriptive analyses, (2) confirmatory analyses of our primary outcomes, including sensitivity analyses, (3) analyses of secondary outcomes, including sensitivity analyses, (4) moderator and mediator as well as analyses of the EMA data. All statistical analyses will be conducted using R Statistics ; see the2.5.1d by For the statistical analyses of the effectiveness of the web-based intervention for patients with BN, growth models within a mixed linear models (MLM) framework will be performed. These have the advantage of considering the multi-level data structure of three repeated assessments (level 1) as nested within patients (level 2) and treating the outcome change as a continuous process. Additionally, MLMs have more power when handling missing data than traditional approaches . To eval2.5.2Both completer analyses and intent-to-treat analyses will be conducted, as previous studies on web-based interventions report notable dropout rates , and com2.5.3t-tests and chi-square tests. In case of significant group differences, these variables will be added as moderators of the treatment × time interaction effects to test the robustness of the findings. Further covariates possibly affecting the primary outcomes such as patient outcome expectancies, patient adherence, satisfaction, attitudes towards psychological online interventions, or emotion regulation will be included in our analyses.For a more detailed evaluation, additional statistical tests will include the analyses of potential moderators or confounding variables. Thus, we will check for possible pre-treatment differences between IG and CG in healthcare service utilization, demographic variables, and eating disorder symptom severity by conducting independent Moreover, we will analyze the weekly assessments of eating disorder symptoms and the data from the EMA. This will allow us to examine changes in core eating disorder symptoms with a higher ecological validity and temporal resolution. As patient safety indicators, the percentage of participants in the IG who experienced adverse intervention effects caused by the intervention will be quantified, and the amount of impairment due to these adverse effects will be calculated. Further, changes in emotion regulation difficulties and strategy use in patients both on a habitual level and in daily life will be evaluated as potential moderators and mediators of symptom reduction in BN.2.6powerlmm , strengthening our conclusion that our sample size will be sufficiently large to detect a medium effect under different assumptions.Sample size estimations were based on meta-analytic evidence for the effectiveness of psychological interventions for BN . Due to powerlmm . A powern of .40 , a power3Despite the effectiveness of psychotherapy for BN, only a limited number of patients with BN in routine care receive adequate evidence-based treatments . This trOur study aims to address this research gap by examining the effects of using a web-based self-help program for BN in routine care settings. Considering previous promising results concerning the effectiveness of such interventions for BN , we aim 3.1Based on our goal to evaluate the effectiveness of a web-based intervention, our study was designed as a randomized controlled trial with a waitlist control group maximizing the internal validity of results. To further decrease systematic biases, our randomization will follow the principles of sequence allocation concealment, and systematic pre-treatment differences will be minimized by excluding individuals receiving psychotherapy or pharmacotherapy for BN at baseline . FurtherMoreover, we aim at contributing to the research on treatments for BN by investigating changes in both cognitive and behavioral eating disorder symptoms. Network analyses indicate that it is possible to differentiate between various types of BN symptoms , includiApart from investigating changes in eating disorder symptoms, we will analyze whether and how the web-based intervention can help improve participants' daily life. Eating disorders are associated with a broad range of adverse outcomes, and thus examining changes in psychosocial functioning, well-being, and work capacity will help evaluate the web-based intervention from a broader perspective . FinallyNevertheless, these strengths will probably be associated with some limitations. First of all, to conduct our analyses with adequate power, a high number of participants needs to be included in our study. The recruitment of such a large-scaled sample might be challenging because the prevalence of BN is relatively low, and barriers in the help-seeking process might limit the number of eligible and interested patients . To maxiAdditionally, the current study does not employ follow-up assessments to investigate the long-term effectiveness of the web-based BN intervention. In general, BN is associated with a high risk for relapses, stressing the need for data on long-term symptom courses following treatment . HoweverAnother challenge that might occur is a high dropout from the study, which has been frequently reported in previous trials on interventions for eating disorders . Low adh3.2In sum, the contributions of the current study to the growing field of web-based interventions for BN are threefold. First of all, our trial is designed to evaluate whether applying web-based self-help interventions in routine care of BN can help reduce eating disorder symptoms, improve the overall quality of life, and illness-related topics such as psychosocial functioning. Secondly, our study will add knowledge on potential predictors of treatment outcomes and mechanisms of change. This will help us better understand how web-based self-interventions may reduce the burden for patients with BN. Third, by including a broad range of assessment methods and different outcomes instead of focusing only on changes in eating disorder symptoms, we will be able to further investigate whether and how web-based interventions impact the daily life of participants. Based on these innovations, the long-term goal of our research on web-based interventions is to implement evidence-based treatments into routine care, reduce social costs, and enhance the quality of life for individuals with eating disorders.APAAmerican Psychiatric AssociationAPOIAttitudes Towards Online InterventionsAZAktenzeichen [file number]BMIBody Mass IndexBNbulimia nervosaBPtKBundespsychotherapeutenkammer [German national association of psychotherapists]CBTCognitive-Behavioral TherapyCGcontrol groupCIAClinical Impairment AssessmentCONSORTConsolidated Standards of Reporting TrialsCSSRIClient Sociodemographic Service Receipt InventoryDERSDifficulties in Emotion Regulation ScaleDSMDiagnostic and Statistical Manual of Mental DisordersDIPS-OADiagnostic Interview for Psychological Disorders-Open AccessEDEEating Disorder Examination InterviewEDE-QEating Disorder Examination QuestionnaireEMAecological momentary assessmentEUEuropean UnionGAD-7Generalized Anxiety Disorder ScaleHFERSTHeidelberg Form for Emotion Regulation StrategiesICCintraclass correlation coefficientIGintervention groupiPCQProductivity Cost QuestionnaireLOCFlast observation carried forwardMICEmultiple imputations by chained equationsMLMmixed linear modelingiMTAInstitute for Medical Technology AssessmentNEQNegative Effects QuestionnairePATHEVPatients' Therapy Expectation and Evaluation ScalePHQPatient Health QuestionnairePICOPopulation Intervention Compared OutcomeRSESRosenberg Self-Esteem ScaleSPIRITStandard Protocol Items: Recommendations for Interventional TrialsWBQWeekly Binges QuestionnaireWHO-5World Health Organization-5 Well-Being IndexRecruitment started in January 2021 and is still ongoing. The first patient was enrolled in the study on January 25th, 2021. Assessments are expected to be completed by May 2022.10.13039/501100008530European Regional Development Fund awarded to Selfapy. Publication fees will be financially supported by the German Research Foundation (Deutsche Forschungsgemeinschaft) Open Access Publishing Fund at Heidelberg University. The funders have no authority over the study design, collection, management, analysis, and interpretation of data, writing of the report, and the decision to submit the findings for publication.The study will be funded by a Ethics approval has been obtained from the institutional review board at Heidelberg University (AZ Tim 2020 1/1). Informed consent will be obtained from all participants, and the trial will be conducted in compliance with the Declaration of Helsinki and good clinical practice. International data privacy regulations and EU legislation will be considered.Open Science Framework (https://osf.io/zdnea/?view_only=b204c63d3d6c4d569e5a140927708eb6).The de-identified and anonymized data and the R analysis script of the current trial will be made available on the LP and CT designed the study. SH and LP performed the sample size calculations and drafted the statistical design of the trial. SH and LP wrote the first draft of the manuscript. All co-authors  contributed to critical revisions of the paper and approved the final manuscript.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


COVID-19 caused by a highly pathogenic etiological agent, SARS-CoV-2, has imposed massive burdens on overall healthcare and public health systems worldwide, especially in low- and low-middle-income countries, including Bangladesh ,4.in vitro data demonstrated that the variant exerted approximately eight-fold reduced sensitivity to Oxford-AstraZeneca and Pfizer-BioNTech vaccine-generated immunity compared with the alpha variant [The sudden surge of cases caused by the newly identified SARS-CoV-2 double mutant Delta variant B.1.617.2) in the neighboring country India, has collapsed its healthcare and public health system; however, a mass vaccination program has proven effective, with new daily cases and deaths drastically reducing during recent days . All sig17.2 in t variant .As expected, the COVID-19 death toll has been rising since the start of July 2021, and there has been more than 200 associated deaths in a single day for the first time ever in Bangladesh . In addiAedes mosquitoes, an estimated 3.9 billion people in over 129 countries are at risk of contracting dengue, which is associated with an estimated 40,000 deaths every year [Dengue is the most prevalent vector-borne viral disease causing a critical flu-like sickness and sometimes precipitating a likely lethal complexity designated as severe dengue. Commonly transmitted by ery year . Approxiery year ,15. The ery year . Along wery year . In Bangery year . During ery year . RepeateThe combined burden of newly increasing dengue cases and the increase in COVID-19 infections caused by the Delta variant have the potential to exacerbate an already stressed healthcare system in Bangladesh. Earlier, a multicenter assessment study warned that dengue and natural disasters could worsen the COVID-19 situation in Bangladesh . In addiCo-infection with both SARS-CoV-2 and dengue virus in a patient poses a serious challenge for accurate diagnosis and the proper treatment of both viral diseases . The iniHigh population density is the primary reason for the rapid transmission of both dengue and COVID-19. The capital city of Bangladesh is Dhaka, one of the most populated cities in the world. Dhaka has experienced high numbers of dengue cases between 2012 and 2019. Since 2016, many dengue endemic countries approved the use of the first-licensed dengue vaccine (CYD-TDV); however, reports of more severe disease manifestations in seronegative individuals interrupt the implementation plan of dengue vaccine in Bangladesh , and mosIt is evident that an alarming upward trend has been observed in dengue infections during the last couple of months in Bangladesh. The bidirectional attacks mediated by the dengue epidemic and the massive third wave of COVID-19 have aggravated Bangladesh’s already vulnerable healthcare system . HoweverThe gravity of this co-epidemic should be underlined and the situation must not be handled with laxity by the authorities, especially as we did not observe countrywide vigorous campaigns against dengue fever, targeting both the common people and healthcare providers. Furthermore, if the upsurge of dengue cases in Bangladesh is equivalent to COVID-19, it would be unrealistic to provide health services and treatment to patients suffering from both infections.The concurrence of COVID-19 and dengue outbreaks requires urgent attention to avoid further disaster. Both public health and socioeconomic conditions will be highly disrupted if cases of both viral infections continue to increase in the near future. Efforts should be taken to prevent and control vector-borne viral spread. In the present circumstances, the first recommended step is the immediate implementation of mosquito-control measures, as the start of the monsoon season has already placed a significant burden on COVID-19 and dengue management. During this season, community participation and public awareness of mosquito-control activities should be strengthened. In addition to these measures, increased awareness at the diagnostic level must be promoted to improve the identification of both COVID-19 and dengue co-infection and ensure that physicians are aware of the possibility of co-infections. Furthermore, the major strategy for limiting dengue is the control of the mosquito vector, and the death toll can be minimized by ensuring the early diagnosis of both diseases. Overall, the mass campaigns and practical approaches that strive for immediate and steady identification and supportive remedial control of dengue and COVID-19 cases are influential factors that urgently need to be executed.


This is the first British multicentre study observing the impact of the COVID-19 pandemic on orthopaedic trauma with respect to referrals, operative caseload and mortality during its peak.A longitudinal, multicentre, retrospective, observational, cohort study was conducted during the peak 6 weeks of the first wave from 17 March 2020 compared with the same period in 2019.Hospitals from six major urban cities were recruited around the UK, including London.A total of 4840 clinical encounters were initially recorded. 4668 clinical encounters were analysed post-exclusion.Primary outcomes included the number of acute trauma referrals and those undergoing operative intervention, mortality rates and the proportion of patients contracting COVID-19. Secondary outcomes consisted of the mechanism of injury, type of operative intervention and proportion of aerosolising-generating anaesthesia used.During the COVID-19 period, there was a 34% reduction in acute orthopaedic trauma referrals compared with 2019 , and a 29.5% reduction in surgical interventions (993 down to 700 operations). The mortality rate was more than doubled for both risk and odds ratios during the COVID-19 period for all referrals  and for those undergoing operative intervention . Moreover, mortality due to COVID-19-related complications (vs non-COVID-19 causes) had greater odds by a factor of at least 20 times. For the operative cohort during COVID-19, there was an increase in odds of aerosolising-generating anaesthesia  by three-quarters, as well as doubled odds of a consultant acting as the primary surgeon.Although there was a reduction of acute trauma referrals and those undergoing operative intervention, the mortality rate still more than doubled in odds during the peak of the pandemic compared with the same time interval 1 year ago. This was the first representative observational study of the UK looking into the impact of COVID-19 pandemic on general trauma and orthopaedic surgical specialty.There is a valid comparison between two timeframes, exactly 1 year apart to represent pre-COVID-19 and during COVID-19.Other studies thus far have only shed light on local scales or cross-speciality within a shorter timeframe than this study and not necessarily commenting on mortality rates like this study.Weaknesses included loss of data points that have been accounted for in the tables  which did not affect the final analysis of data points.Operations conducted outside the specific study periods will not account for all those operations required such as for hip fractures.The COVID-19 pandemic will be remembered as one of the most unprecedented global health crises in modern history. With over 5 million deaths and over 263 million recorded cases globally, the pandemic has had a permanent impact on healthcare at the time of article submission.The English government responded by implementing social distancing measures on 17 March 2020 in an attempt to reduce the rate of transmission and therefore the demands on the National Health Service (NHS).To observe the impact of COVID-19 on trauma and orthopaedic acute referrals, operative casemix and mortality rates during the peak 6 weeks of the first wave of the pandemic compared with the same time interval in 2019.When comparing both years, there would be a difference in the prevalence of acute orthopaedic referrals, orthopaedic trauma casemix and aerosol-generating anaesthetic procedures due to social distancing/lockdown. Mortality rates and survival probabilities were also hypothesised to differ due to the first COVID-19 outbreak.This is the first multicentre longitudinal observational study observing patients who were acutely referred to the trauma and orthopaedic departments as well as those operated on within the same 6-week interval comparing 2019–2020 for general orthopaedic trauma.Seven principal NHS hospital trusts contributed data from six major urban cities including London, Gateshead, Middlesbrough, Dartford, Newport and Reading.All acute referrals, operative notes, inpatient medical records and discharge summaries were accessed using electronic patient databases at each contributing hospital trust.The 6-week study period was from the start of social distancing on 17 March 2020 to 28 April 2020 which encompassed the national lockdown measures instigated on 23 March 2020. This period was considered the peak 6 weeks of the epidemic in the UK as outlined by the recorded mortality rates and R-values published by the Office of National Statistics.Primary outcomes included the number of acute trauma referrals and those undergoing operative intervention, postoperative complications, mortality rates and the proportion of patients contracting COVID-19. Secondary outcomes consisted of the mechanism of injury, type of operative intervention and proportion of aerosolising-generating anaesthesia used.All acute orthopaedic trauma referrals presenting to the emergency department during the intervals 1 year apart were included. All orthopaedic trauma cases that required an operation, including those from acute orthopaedic trauma referrals, within the intervals 1 year apart. Those patients listed for an operation due to orthopaedic trauma prior to time period of data collection were included in the final analysis. We adhered to Strengthening the Reporting of Observational Studies in Epidemiology guidelines for observational studies.Any cases being referred internally from other specialties for trauma and orthopaedic advice and input, as well as referrals from any external centre asking for tertiary advice were excluded from further analysis. Any patients with postoperative complications arising from the period prior to the data collection were excluded. For operative trauma cases, those undergoing spinal procedures were excluded as these are jointly treated by neurosurgery in most hospitals. All non-urgent semi-elective procedures were excluded from analysis as well, as they would inaccurately assess the impact of any social distancing measures on the trauma workload. Routine elective orthopaedic cases were excluded.Demographics including age, sex and American Society of Anesthesiologists (ASA) grades were recorded for all patients. Injury characteristics were recorded, including the anatomical location and if the injury was open or closed. The mechanism of injury was categorised and whether the patient was referred as a trauma call. The nature of the operative procedures and the anaesthetic techniques were recorded. Patients undergoing multiple procedures were recorded for every episode where they were taken to theatre. Six-week mortality rate was recorded as well as the COVID-19 status of any symptomatic patients or suspected cases. Data points were divided into acute referrals and operative casemix as seen in Anaesthetic techniques: This was divided into anaesthetic aerosolising-generating procedures (AGPs) which consisted of any intubation  for a general anaesthetic. All other anaesthetic techniques including regional and local anaesthetics were deemed as non-AGPs.COVID-19 status: At the time, COVID-19 was being diagnosed with PCR from nasal and oropharyngeal swabs with a duration of 1–4 days where the sample was tested both locally in the hospital lab and corroborated with national lab testing to reduce risk of unequivocacy. Groups of patients were divided into either not swabbed (due to being asymptomatic) or swabbed due to presence of documented symptoms which yielded either negative or positive results.All the data were recorded, anonymised and verified by four members of the study group for their accuracy. The data were processed using Microsoft Excel . Shapiro-Wilk test indicated a normal distribution for age and days to discharge from hospital; hence, the mean  were calculated for both. ASA did not follow normality and was analysed using median (±median absolute deviation (MAD)) and IQR. Both prevalence ratio (PR) or risk ratio (RR) and odds ratio (OR) were calculated as well as a Fisher’s exact test for statistical significance for categorical data, defined as p≤0.05. Percentages and CIs were rounded off to one decimal place.The COVid Emergency-Related Trauma and orthopaedics (COVERT) Collaborative was founded at Imperial College Healthcare NHS Trust. It is currently a member of the COVID-19 Research Group and it has been endorsed by the Royal College of Surgeons of England and Imperial College Healthcare NHS Trust.Patients and the public were not involved in the study design, recruitment or conduct.A total of 4840 clinical encounters took place between the study periods. A total of 172 spinal operations and presentations were excluded from the final analysis. COVID-19 status for both acute referrals and operative casemix including results in all mortalities was demonstrated in A subgroup analysis of hip fractures was conducted due to its recognised risk of mortality within orthopaedic trauma. Those who were operated on in 2020, 20.2% tested positive for COVID-19, 47.3% tested negative and the remaining 32.4% were not tested due to being asymptomatic. Furthermore, 82.3% of all mortalities in 2020 sustained a neck of femur fracture in which only 35.7% of this cohort had a positive swab result, 21.4% with negative swab results and the remaining 42.9% were not swabbed due to being asymptomatic. There was no statistical difference in the OR and RR between both years for mortality rate in NOF fractures . The abs2 test was insignificant for both number of comorbidities and individual comorbidities between both years, except for cardiovascular and cerebrovascular disease in acute referral cohort.Taking into account that COVID-19 was a perioperative complication since patients may have been symptomatic with COVID-19 manifestations preoperatively but only had the swab results return with a positive finding either preoperatively or postoperatively; the most common postoperative complication in the COVID-19 period was a hospital-acquired pneumonia but with negative COVID-19 swab results or the decision not to test at all. The second most common postoperative complication in the year 2020 was extra-pulmonary sepsis . The pro10.1136/bmjopen-2021-054919.supp1Supplementary data10.1136/bmjopen-2021-054919.supp2Supplementary data10.1136/bmjopen-2021-054919.supp3Supplementary dataSix-week Kaplan-Meier survival probability analysis for mortalities between both years is plotted in There was a significant difference between pre-COVID-19 and post-COVID-19 periods at its peak. The alternative hypothesis was not rejected with respect to prevalence of (1) acute orthopaedic trauma referrals (reduced by 34%), (2) surgical interventions (reduced by 29.5%), (3) anaesthetic AGPs, (4) 6-week mortality rates (more than doubled in the COVID-19 period) and (5) survival probability between pre-COVID-19 and post-COVID-19 eras.et alet alThe 34% reduction in acute trauma referrals is in keeping with previous single-centre studies performed in the UK with results ranging between 26% and 59%.et al.With respect to the operative caseload , patientAlthough the expectation was to minimise the use of aerosolising-generating anaesthetic procedures, there was in fact an increased prevalence of using general anaesthesia±block up to an odds of 75%, in order to create a ‘closed circuit’ for the airways. As the anaesthetic methods was not well documented in the pre-COVID-19 era in a fifth (21.3%) of cases, this skewed the data as it may have been difficult to extract that data from 2019. The odds of a consultant-led operation doubled (OR=2.08) during the COVID-19 period as a consequence of all elective operations being suspended, hence more consultants were relocated to trauma theatre and increased pressure within the theatre environments led to consultant-delivered, rather than consultant-led care. With respect to surgical procedures, there was a significant reduction in PR of open reduction and internal fixation by a fifth (PR=0.81) and removal of metalwork and foreign bodies by three-quarters (PR=0.24), while there was a doubling (PR=2.02) in dynamic hip screw fixation in the COVID-19 era.Comparatively, the COVIDSurg Collaborative observed a mortality rate of 28.8% (p<0.0001) of orthopaedic patients who underwent surgery (both elective and trauma) within the first quarter of the year.Results from As expected, reduced survival probability reflected the most vulnerable patient profiles, usually with multiple preoperative and postoperative comorbidities . A reasoet alAs lockdown measures in the UK and globally eases and the incidence of trauma returns to pre-lockdown trends, it is imperative that we understand the true increased risk of mortality in acute trauma during the COVID-19 era. A recent publication by Kader Furthermore, the Corona Hands CollaborativeAlthough the trends in mechanisms of injury in our study were reflective of those within a US multicentre study, there was an opposing trend in the number medical/surgical procedures.With an overall mortality risk in 2020 doubled that of 2019, clinicians need to counsel patients presenting with acute orthopaedic trauma of the increased risk in the COVID-19 era, especially for those identified as increased risk stratification with multiple underlying comorbidities, elderly and frailty. With the ongoing risk of a subsequent wave and resurgence of COVID-19 cases on top of the inevitable winter pressures, this data is of critical importance in the risk management, decision-making and policymaking of trauma patients both in the UK and across the globe.Since the aetiology of neck of femur fracture is often low energy falls in the home environment, it is not unexpected to observe a consistency of neck of femur fractures in the elderly and the vulnerable during lockdown as seen in The increased mortality reflect the increased proportion of patients with NOF fractures that have a higher baseline mortality which has been echoed by the Scottish IMPACT-Restart study.If these ‘at risk’ patients were symptomatic with the virus, then aggressive preoperative optimisation would occur. Since 91% (n=10) of COVID-19 positive patients had sustained a NOF fracture, the National Hip Fracture Database (NHFD) best practice tariff of operating within an ideal 36-hour window set by the Royal College of Physicians was suspended until the patient was stabilised. All hip fracture patients in this cohort were operated on and had dedicated orthogeriatric input commencing from hospital admission. Hence the early perioperative period and surgery encompassed within the 10-day period post-admission. Moreover, NOF fractures are recognised as a pre-terminal illness and are known to carry a high risk of mortality in the first month which is trebled in the first year after the injury.et alThis was the first representative observational multicentre study of the UK looking into the impact of COVID-19 pandemic on general trauma and orthopaedic surgical practice. Studies thus far have only shed light on local scales, cross-speciality, reflecting a fraction of our study population or contain 30-day mortality at most.It is vital to continue exploring the impact of the pandemic on a larger scale. Ideally, more secondary care providers consisting of district general hospitals and major trauma centres will submit data. The diagnosis of COVID-19 was dependent on positive PCR swabs for this study rather than non-specific changes seen on chest CT or plain radiographs. This does not account for false negatives with clinical respiratory symptomatology or true positives in those asymptomatic. Nevertheless, this issue with data has been speculated on in another national study.This was the first, longest and largest British multicentre representation of the impact of COVID-19 pandemic on acute orthopaedic trauma referrals and mortality between mid-March and end-April 2020, representing the peak of the first wave during the lockdown. The mortality rate for acute referrals, as well as those undergoing operative intervention, more than doubled in odds when compared with the same time interval 1 year ago. The majority of mortalities consisted of the elderly with neck of femur fractures and cardiovascular and/or cerebrovascular diseases. This study will aid clinicians in counselling trauma patients of the increased risk of mortality during the era of COVID-19 and also aid in both healthcare infrastructure, resource allocation, decision-making and policymaking as we continue to battle with the pandemic.


The long-standing hypothesis that emotions rely on bodily states is back in the spotlight. This has led some researchers to suggest that alexithymia, a personality construct characterized by altered emotional awareness, reflects a general deficit in interoception. However, tests of this hypothesis have relied on heterogeneous assessment methods, leading to inconsistent results. To shed some light on this issue, we administered a battery of self-report questionnaires of interoception and alexithymia to three samples from Italy, the U.S., and Singapore (N = 814). Correlation and machine learning analyses showed that alexithymia was associated with deficits in both subjective interoceptive accuracy and attention. Alexithymics’ interoceptive deficits were primarily related to difficulty identifying and describing feelings. Interoception showed a weaker association with externally-oriented thinking as operationalized by the Toronto Alexithymia Scale (TAS-20) and no association with the affective dimension of alexithymia later introduced by the Bermond-Vorst Alexithymia Questionnaire (BVAQ). We discuss our results with reference to the theoretical and psychometric differences between these two measures of alexithymia and their shortcomings. Overall, our results support the view that interoceptive deficits are a core component of alexithymia, although the latter cannot be reduced to the former. A long standing philosophical and psychological tradition has argued that somatic sensations play a role in the experience of emotions. Suffice it to mention the famous subtraction argument proposed by James and Lange , 2, who A subclinical condition called alexithymia gives us an opportunity to investigate this claim more closely. The term “alexithymia”  was intThis dimensional vision of alexithymia is not shared by all researchers. A more recent, but less widely used instrument to assess alexithymia, the Bermond-Vorst Alexithymia Questionnaire BVAQ, , 15), wa, wa15]),In practice, the BVAQ-Cognitive and TAS-20 can be considered highly comparable measures : they caIn spite of their differences, the TAS-20 and BVAQ presuppose the existence of a strong association between affective and somatic experience. A lack of interoceptive awareness is certainly reflected in the first factors of both scales , which aim to measure difficulty distinguishing between bodily and emotional feelings. Moreover, a deficit in interoceptive awareness could be hypothesized to underly the characteristics that define the “emotionalizing” factor of BVAQ, if this factor reveals differences in physiological arousal. It is, therefore, not surprising that, in the last ten years, researchers have started to more deeply explore the extent to which interoception contributes to the etiology of alexithymia.The most radical proponents of this stream of research have formulated the “interoceptive hypothesis” of alexithymia. They argue that alexithymia is a general deficit in interoceptive ability , rather Although the association between alexithymia and interoception is sound, this strong version of the “interoceptive hypothesis” faces numerous theoretical and methodological issues. On the one hand, we can question whether interoceptive ability is related to all features of alexithymia – independently of whether we embrace the dimensional view subtended by the TAS-20 or argue that there are different types of this condition as suggested by the BVAQ. This point has not been adequately explored, since the majority of studies that examine the relationship between alexithymia and interoception have relied on the TAS-20 to assess alexithymia, and have only rarely adopted other measures such as the BVAQ. On the other hand, we need to resolve some outstanding issues concerning the operationalization of interoception, as detailed in the next sections.objective interoceptive accuracy (IAcc), (2) interoceptive sensibility (IS), and (3) interoceptive awareness (IA) [Khalsa and colleagues  have notess (IA) . IAcc (1ess (IA) . IS (2) ess (IA) , 32.Distinguishing between the different components of interoception is also useful for investigating the relationship between interoception and alexithymia since it helps to make sense of the apparently contradictory findings from different empirical studies. This was clearly revealed by a recent meta-analysis by Trevisan and colleagues  that conFirst of all, this meta-analysis shows that the association between Objective IAcc and alexithymia is non-significant. The explanation the authors give for this result is that the majority of the examined studies employed the heart-beat counting task , 34: altSecondly, Trevisan’s meta-analysis  examinesThis study aims to explore the extent of the association between subjective interoceptive ability and alexithymia. It places special focus on the still unresolved issues mentioned above.Specifically, our purpose is to clarify:(1a) Whether subjective interoceptive ability is associated only with alexithymia as it is operationalized by the TAS-20 and BVAQ-Cognitive or whether it also associated with the so-called Affective-type of alexithymia .(1b) Whether all or only some facets of alexithymia included in both the TAS-20 and BVAQ-Cognitive and Affective  are associated with subjective interoceptive ability.Which dimensions of subjective interoception provide better estimates of alexithymia levels; i.e., is alexithymia best predicted by the Subjective IAcc  or by the Subjective IAtt .Moreover, to assess whether our results are broadly replicable, we collected data from three large samples, which differed in terms of cultural and socio-demographic variables, and analysed them using two different statistical methods. Correlation analyses were used to compare our results to previous findings based on this same statistical method, while Machine Learning (ML) algorithms were applied to provide further data-driven confirmation of the results obtained from traditional inferential statistics .https://osf.io/5x9sg/) in the Open Science Framework (OSF).Data were collected online from three separate sources: an Italian opportunity sample, a Singaporean University sample, and a U.S. sample recruited from Amazon’s Mechanical Turk (MTurk) platform. Data collections were approved by the IRBs of the University of Trento (Protocol n. 2020-029) and the Nanyang University of Singapore . Prior to data analysis, this study was preregistered .Participants were students recruited from the Universities of Trento, Perugia, and Bari, or via social media platforms. No monetary compensation was provided but participants from the University of Trento could request university credits for time spent completing the tasks. Inclusion criteria comprised age (>18) and language . The final sample entered in the analyses included 325 subjects .Participants recruited through Amazon’s Mechanical Turk were English‐speaking USA residents aged over 18. An additional inclusion criterion was at least a bachelor level education, although this last criterion was only assessed by self-report. Respondents received $6 for completing the survey. This data collection was approved by NTU’s 2020-10-016 IRB. The final sample entered in the analyses comprised 250 subjects .Participants were recruited from undergraduate students at NTU and received university credits for their participation. The final sample entered in the analyses comprised 239 subjects . The TAS-20 comprises 20 items rated on a 5-point Likert scale ranging from 1 (strongly disagree) to 5 (strongly agree). The items load on three factors: (i) Difficulty Identifying Feelings (DIF), e.g., “I am often confused about what emotion I am feeling”, (ii) Difficulty Describing Feelings (DDF), e.g., “It is difficult for me to find the right words for my feelings” and (iii) Externally Oriented Thinking (EOT), e.g., “I prefer talking to people about their daily activities rather than their feelings”. Total scores range from 20 to 100 points, where higher scores are indicative of higher levels of alexithymia.Bermond-Vorst Alexithymia Questionnaire . The BVAQ consists of 40 items, rated on a 5-point Likert scale, where higher scores indicate higher levels of alexithymia. The items load on five subscales: (i) Identifying  (ii) Verbalizing , (iii) Analyzing , (iv) Fantasizing , (v) Emotionalizing . In addition, Vorst and Bermond [ Bermond  revealed Bermond  suggesteInteroceptive Confusion Questionnaire . The ICQ assesses the degree to which individuals experience difficulty interpreting their own non-affective interoceptive states, such as hunger, temperature and arousal . It consists of 20 items rated on a 5-point Likert scale, ranging from 1 (Does not describe me) to 5 (Describes me very well). The overall interoceptive confusion index ranges from 20 to 100, where higher scores are representative of more difficulty with interoception. The questionnaire has no internal subdivisions.Interoceptive Accuracy Scale . The IAS consists of 21 items rated on a 5-point Likert scale, ranging from 1 (strongly disagree) to 5 (strongly agree). Each item assesses self-reported accuracy  in the perception of a specific body signal . Total scores range from 21 to 105, where higher scores indicate greater self-reported interoceptive accuracy. The questionnaire has no internal subdivisions, although the initial factor analysis performed by Murphy et al. [y et al.  suggesteMultidimensional Assessment of Interoceptive Awareness - Version 2 . MAIA-2 consists of 37 items grouped in eight subscales: 1) Noticing ; 2) Not-Distracting  Not-Worrying ; 4) Attention Regulation ; 5) Emotional Awareness ; 6) Self-Regulation ; 7) Body Listening ; 8) Trusting . Responders assess how often each statement applies to their experience in daily life on a scale ranging from 0 (never) to 5 . Scores for each subscale are derived by computing the individual mean of each subject on the items composing the specific subscale. A total index of interoceptive awareness is derived by summing the individual scores at each of the 8 subscales.Body Perception Questionnaire . The BPQ is a self-report instrument used to assess body perception and interoceptive awareness. For this study, we used the BPQ Short Form, consisting of 46 items. Each item is rated on a five-point Likert scale ranging from 1 (never) to 5 . Items are divided into two main domains: Body Awareness and Autonomic Reactivity. Items in the first domain measure subjective perceived sensitivity to internal bodily functions. Values at the high end of the scale reflect perceived hypersensitivity and values at the low scale reflect hyposensitivity. Items in the second domain measure the respondent’s perception of the reactivity of his/her autonomic system.“interoceptive hypothesis” of alexithymia. Therefore, these measures represent crucial evidence for evaluating this hypothesis. This group specifically suggested that IAS and ICQ are measures of Subjective IAcc and should not be confused with measures of Subjective IAtt [There were two main reasons why we selected these four interoceptive measures from among many others available in the literature. The first is that both the IAS and ICQ were developed by the research group that formulated the ive IAtt . So, we All the described measures were administered to each of the three samples. The English version was given to the Singaporean and the U.S. samples, while Italian translations were provided for the Italian sample. Validated translations in the Italian language were available for TAS-20 , BVAQ 1, MAIA-2 Data analysis was conducted using RStudio (version 1.3.1093) and Python (version 3.8) scikit-learn library . PrelimIn this study, ML models were used to estimate the three alexithymia scores  based on interoceptive scores. Specifically, Support Vector Machine (SVM) regressors with a linear kernel were used. SVMs are supervised learning methods used for either classification or regression. SVMs offer advantages over other ML methods because they are (i) effective in high dimensional spaces and (ii) not computationally demanding since they use a subset of training points in the decision function .The same protocol was used to calibrate the three ML models, with each model estimating one of the three target variables. Firstly, the Italian dataset was randomly split into ‘train’ (75% of the subjects) and ‘test’ partitions (25% of the subjects). The ‘train’ partition from the Italian dataset was used to calibrate the models. The calibrated model was then evaluated on the remaining subsets: the Italian Test partition, the U.S. partition, and the Singaporean partition.The comparison of Mean Absolute Error (MAE) in the three ML models led to an unbiased data-driven quantification of how interoceptive scales and subscales were associated with the different measures of alexithymia. By considering the replicability of the results across the three samples , we could also assess how the findings generalized across different demographic groups. Finally, we used the rankings of the interoceptive scores computed by the ML models to obtain an indication of how different dimensions of Subjective IAcc  were linked to the alexithymia scores.A detailed description of all analytical steps including data pre-processing and further details on the ML models are available in italy <0.68; αU.S. <0.77; αSingapore <0.66). All the other measures revealed alpha values above the acceptance level.2 < = 0.04). Post-hoc analysis revealed higher differences between the Italian sample and the other two samples, while they failed to find a significant difference between the U.S. and the Singaporean samples in any psychological measure, with the exception of MAIA total scores (p<0.01). Results from one-way permutation ANOVAs were also significant for age, which was the only variable to show a large effect size (ηp2 = 0.37). Age was also found to significantly differ in all post-hoc comparisons (p = 0.001). Results from the chi-square test also revealed significant differences in the proportion of male and females between the three datasets ((p<0.001).Descriptive statistics of the socio-demographic and psychological data performed separately for the Italian, U.S. and Singaporean are summarized in Zero-order Spearman’s correlations for each sample are displayed in In all three samples there was a large correlation between the TAS-20 and BVAQ-Cognitive. The correlation between the BVAQ-Cognitive and BVAQ-Affective was not significant in either the Italian or the Singapore dataset and small in the U.S. sample. Similarly, there was no significant correlation between the TAS-20 and BVAQ-Affective in any dataset.Regarding the relationship between different self-report interoceptive measures, a large (negative) correlation was found between IAS and ICQ across the three samples. Medium correlations were found between MAIA total scores and IAS or between MAIA total scores and ICQ in all samples.The two dimensions of the BPQ showed a significant if moderate correlation in two out of the three datasets. However, correlating the two dimensions of the BPQ with the other scales of interoception, we obtained a different pattern of results. Indeed, BPQ-Reactivity always showed a significant negative correlation of medium magnitude with IAS and a significant positive correlation of medium-large magnitude with ICQ, but was not significantly correlated with MAIA total scores. By contrast, BPQ-Awareness showed an unstable pattern of results across the three samples and the direction of correlation with IAS, ICQ and MAIA was opposite to that between the same interoceptive measures and BPQ-Reactivity. For instance, in two out of three datasets, BPQ-Awareness showed a significant positive correlation of small magnitude with IAS and MAIA, while correlation with ICQ was never significant.Overall, the correlations between IAS, BPQ-Awareness, and MAIA total scores all went in the same direction but in the opposite direction to ICQ and BPQ-Reactivity.The relationship between measures of interoception and alexithymia as measured by the TAS-20 and BVAQ-Cognitive revealed a similar pattern of results. ICQ was the measure of interoception that showed the highest positive correlation with both the TAS-20 or BVAQ-Cognitive across the three samples. By contrast, BPQ-Awareness was the only measure of interoception that failed to show any overall significant correlation with either the TAS-20 or BVAQ-Cognitive. As for the correlation between the other measures of interoception and the TAS-20 or BVAQ-Cognitive, a consistent pattern of results emerged across the three samples, with a negative correlation of medium magnitude for both MAIA total scores and IAS and a positive correlation of medium magnitude for BPQ-Reactivity. Among the eight MAIA subscales , were of small magnitude and went in the opposite direction to correlations between these same interoceptive scales and the TAS-20 or BVAQ-Cognitive. The only exception was BPQ-Awareness in the Italian sample, which showed a small negative correlation with both the BVAQ-Cognitive and the BVAQ-Affective.BVAQ-Cognitive was the target variable best estimated (lowest MAE) within the training dataset and all the test datasets. Contrarily, BVAQ-Affective was the target variable that was most poorly estimated and did not differ substantially from the performance of the baseline “chance” model. Regarding the TAS-20, the MAE was lower than the baseline in the train set and in all the test datasets, but there was partial overlap with the baseline model.It is worth noticing that there were no remarkable differences in the performances between the Italian train and test datasets, indicating that the calibration process was performed without biases or overfitting. Furthermore, the performance on the three test datasets was comparable on the TAS-20 and BVAQ-Cognitive, suggesting that our results were independent of the socio-cultural differences between the three samples.By contrast, BVAQ-Affective has a performance close to baseline in the train dataset and in the Italian test dataset, and falls below the baseline in the U.S. and Singapore datasets. This result suggests a poor association between the BVAQ-Affective score and interoceptive measures.s Italy = - 0.31***) and BVAQ-Cognitive , its importance in ML models was far lower than that of ICQ, which is considered to highlight the same construct, Subjective IAcc.The main goal of this study was to understand if and to what extent the ability to recognize internal affective states depends on the ability to recognize internal bodily states. Therefore, we explored if, in the general population, a deficit in interoceptive ability co-occurs with higher levels of alexithymia. Our attempt to clarify the relationship between alexithymia and interoception went in two directions. Firstly, we examined whether self-reported deficits in interoception were associated with both the dimensional and categorical conceptualizations of alexithymia, respectively operationalized by the TAS-20 and the BVAQ. More specifically, we considered whether self-reported interoception levels can only estimate scores on the TAS-20 and BVAQ-Cognitive, or whether they are also an accurate estimator of BVAQ-Affective. Secondly, we aimed to clarify which components of subjective interoception are mostly closely related to alexithymia. This last objective aimed to clarify the mixed results in the literature regarding the association between subjective interoception and alexithymia, and the possibility that they derived from the different self-report instruments used to measure interoceptive ability.Regarding the first point, our results showed that interoceptive deficits are associated with the alexithymic characteristics captured by both the TAS-20 and BVAQ-Cognitive, but not with those operationalized by the BVAQ-Affective. From our analysis of the different components that make up alexithymia construct  and interpret (see ICQ) internal bodily signals, a deficit in the ability to direct top-down attention to regulating one’s bodily states (see MAIA), and increased symptomatology reflecting difficulties and problems of the autonomic system (see BPQ-Reactivity). The highest predictor of alexithymia levels across the three samples was the Interoceptive Confusion Questionnaire, which captures subjectively experienced difficulty in recognizing specific bodily sensations such as hunger, thirst, satiety, muscle tension, or nausea.More specific considerations are necessary to interpret the data on the Body Perception Questionnaire (BPQ). Employing this questionnaire or misleading interpretations of its results are probably the main causes for the inconsistencies found in the literature describing the relationship between alexithymia and interoception. Indeed, while studies employing other self-reported interoceptive scales suggest that alexithymia is associated with decreased interoceptive awareness, studies using BPQ often lead to the opposite result and suggest that high levels of alexithymia are associated with hypersensitivity to bodily sensations , or highTo account for these conflicting results, recent studies offer a more specific conceptualization of interoceptive awareness , 32. TheSome clarification is offered by Trevisan and colleagues’ meta-analysis , which hIn conclusion, we suggest that a weaker version of the “interoceptive hypothesis” of alexithymia would be appropriate. Our results show that interoceptive levels are good predictors of alexithymia levels. This suggests that deficits in interoception can reveal higher order impairment in the recognition of affective states. However, not all aspects of the construct of alexithymia clearly reflect an interoceptive deficit. We therefore disagree with the conclusion drawn by Brewer and colleagues  that “alS1 FileDetailed information on data pre-processing and ML analysis. Correlation matrices between all scales and subscales are also displayed.(DOCX)Click here for additional data file.


It is unknown how frequently damage control (DC) laparotomy is used across trauma centers in different countries. We conducted a cross-sectional survey of trauma centers in the United States, Canada, and Australasia to study variations in use of the procedure and predictors of more frequent use of DC laparotomy.A self-administered, electronic, cross-sectional survey of trauma centers in the United States, Canada, and Australasia was conducted. The survey collected information about trauma center and program characteristics. It also asked how often the trauma program director estimated DC laparotomy was performed on injured patients at that center on average over the last year. Multivariable logistic regression was used to identify predictors of a higher reported frequency of use of DC laparotomy.p = 0.02). Nearly half (48.4%) of high-volume volume level-1 trauma centers reported using the procedure at least once weekly. Significant adjusted predictors of more frequent use of DC laparotomy included country of origin , level-1 verification status , and the assessment of a higher number of severely injured  patients  and patients with penetrating injuries  in the last year.Of the 366 potentially eligible trauma centers sent the survey, 199 (51.8%) trauma program directors or leaders responded . The reported frequency of use of DC laparotomy was highly variable across trauma centers. DC laparotomy was used more frequently in level-1 than level-2 or -3 trauma centers. Further, high-volume level-1 centers used DC laparotomy significantly more often than lower volume level-1 centers (The reported frequency of use of DC laparotomy was highly variable across trauma centers. Those centers that most need to evaluate the benefit-to-risk ratio of using DC laparotomy in different scenarios may include high-volume, level-1 trauma centers, particularly those that often manage penetrating injuries.The online version contains supplementary material available at 10.1186/s13017-021-00396-7. Damage control (DC) laparotomy was developed to quickly control exsanguinating hemorrhage and gross contamination in injured patients with severe physiologic derangements . It was The above suggests that there is likely insufficient evidence to support the high DC laparotomy utilization rates reported by some trauma centers . LimitedReasons for variation in use of DC laparotomy between level-1 trauma centers in the United States are unknown. It is also unknown whether variation in use of DC laparotomy exists across trauma centers outside of the United States and how often the procedure is used across level-2 and -3 trauma centers . To address these knowledge gaps, a cross-sectional survey of trauma centers located in the United States, Canada, and Australasia  was conducted . This suA self-administered, electronic, cross-sectional survey of trauma program medical directors or leaders located in 4 high-income countries with similar emergency medical services was conducted , 20. StuThe population of interest included level-1, -2, and -3 trauma centers that treat adult or adult and pediatric trauma patients in the United States, Canada, and Australasia. The sampling frame of American, Canadian, and Australian trauma centers was identified using lists of those verified by the American College of Surgeons in 2013 , that coThe survey questionnaire was developed by modifying a previously developed questionnaire administered to trauma program directors or leaders in the above countries . The modAll trauma centers in the sampling frame were purposively sampled by sending an e-mail to their trauma program director or leader in September, 2014 . E-mailsCategorical survey responses were summarized using counts (percentages) and continuous survey responses using medians [with interquartile ranges (IQRs)]. Survey responses were summarized unstratified and stratified by country, reported frequency of use of DC laparotomy, and by volume and level of trauma center care. A high-volume trauma center was defined as per Nathens et al. as one that assessed > 650 major trauma [Injury Severity Scale (ISS) score > 15] patients in the last year . SummaryMultivariable logistic regression with robust standard errors was used to identify independent predictors of a higher reported frequency of use of DC laparotomy across trauma centers. As there have been no studies to guide selection of evidence-informed predictors for inclusion in the model, all variables felt to be potentially predictive that lacked evidence of multicollinearity were included in the model. We also tested whether the volume of severely injured patients modified the relationship between reported frequency of use of DC laparotomy and level-1 verification status.p values indicate that the model fits the data). Finally, overall classification performance of the logistic regression model was assessed by generating a receiver operating characteristic (ROC) curve that plotted sensitivity against false-positive rate (1-specificity) across a range of diagnostic thresholds.The degree of multicollinearity was estimated by calculating associations or correlations between variables and by using the Stata  command package “collin”. Model fit was tested using the Hosmer and Lemeshow’s goodness-of-fit test  responded and provided data on the frequency of use of DC laparotomy in their center.Of the 199 participating trauma centers, 156 (78.4%) were located in the United States, 26 (13.1%) in Canada, and 17 (8.5%) in Australasia. The 156 participating American trauma centers were located in 37 different states, with most in California (17.3%), Texas (9.6%), and Michigan (8.3%)  and pediatric (p = 0.008) trauma patients than those in Canada. Trauma centers in the United States also assessed a higher percentage of patients with penetrating injuries than those in Canada or Australasia (p = 0.008). However, the number of severely injured (defined as an ISS score > 15) patients assessed was similar across trauma centers in the three regions.Characteristics of the 199 participating trauma centers are outlined in Table The reported frequency of use of DC laparotomy for trauma by the 199 participating trauma centers was highly variable  . Variables describing teaching status and trauma center research activities were excluded from logistic regression models because there was evidence of multicollinearity between these variables and level-1 trauma center status. Significant adjusted predictors for more than once monthly reported use of DC laparotomy included country of origin (centers in the United States reported using DC laparotomy significantly more often than those in Canada), level-1 verification status, and the assessment of a higher number of severely injured patients and patients with penetrating injuries in the last year . The area under the ROC curve for the model was 0.88, indicating excellent performance for correctly classifying higher instead of lower reported use of DC laparotomy across trauma centers . Nearly half of high-volume, level-1 trauma centers reported using DC laparotomy at least once weekly. Trauma center and program characteristics that independently predicted higher reported use of DC laparotomy included country of origin (with centers in the United States reporting using DC laparotomy significantly more often than those in Canada), level-1 trauma verification status, and the assessment of a higher number of severely injured (ISS score > 15) patients and patients with penetrating injuries in the last year.Studying variation in use of DC laparotomy is important because surgeons are at risk of confirmation bias when only those with whom they work reflect their practice . HoweverTrauma centers in the United States reported using DC laparotomy for trauma more often than those in Canada despite adjustment for level-1 verification status and the volume of severely injured patients and patients with penetrating injuries. This could be because of differences in institutional cultures regarding use of DC laparotomy between countries. It could also be because of unmeasured differences in patient mix between countries aside from injury mechanism or ISS scores  or beliefs regarding appropriate indications for use of the procedure . Of the In addition to country of origin, other independent predictors of an increased reported use of trauma DC laparotomy included level-1 trauma verification status and the assessment of a higher number of severely injured patients and patients with penetrating injuries in the last year. In the post-hoc analysis of the PROPPR study, the ISS score  of the patients assessed at level-1 trauma centers also predicted an increased odds of use of DC laparotomy . This isThe study findings should be considered in the context of its strengths and limitations. First, the opinions of trauma program directors could be argued to be only estimates of the frequency of use of DC laparotomy. Surveys are a practical strategy to capture practice variation across hundreds of level-1, -2, and -3 trauma centers in the United States, Canada, and Australasia. However, because of the limitations of using cross-sectional survey data, they should be confirmed by future observational studies. Second, although we used several techniques shown to increase response rates to surveys (and the response rate is above what has been reported by many surveys reported in the trauma or surgery literature), it is possible that respondents’ answers on the reported frequency of use of DC laparotomy differ systematically from those who did not respond to the survey . Third, This study has important implications for future research, trauma surgery practice, and quality improvement efforts. First, a systematic review of 36 cohort studies found very little evidence to support that use of DC instead of definitive laparotomy in trauma patients was associated with an improvement in mortality or other patient-important outcomes . HoweverIn this large, international, cross-sectional survey of trauma program directors or leaders, the reported frequency of use of DC laparotomy was highly variable across level-1, -2, and -3 trauma centers. The reported frequency of use of DC laparotomy also varied significantly across level-1 trauma centers, especially between those that were high- versus lower volume. The procedure was used most often in level-1 trauma centers in the United States that assessed a large percentage of patients with penetrating injuries. Nearly half of high-volume, level-1 trauma centers reported using DC laparotomy at least once a week. Those trauma centers that most need to evaluate the benefit-to-risk ratio of using DC laparotomy in different scenarios may include high-volume, level-1 trauma centers, particularly those that often manage patients with penetrating injuries.Additional file 1. Location of the 156 participating American trauma centers.Additional file 2. Reported frequency of use of damage control laparotomy for trauma by trauma centers in the United States, Canada, and Australasia .Additional file 3. Receiver operating characteristic curve for the multivariable logistic regression prediction model.


By increasing the electron-withdrawing properties of the p-halogenated phenyl right-hand side fragment and maintaininga similar lipophilicity and size, an increased potency was achieved,indicating that the antibacterial activities of this series of novelbacterial topoisomerase inhibitors against all target enzymes aredetermined by halogen-bonding rather than van der Waals interactions.They show nanomolar enzyme inhibitory and whole-cell antibacterialactivities against S. aureus and methicillin-resistant S. aureus (MRSA) strains. However, due to the relativelyhigh substrate specificity for the bacterial efflux pumps, they tendto be less potent against E. coli and other Gram-negativepathogens.We designed and synthesizedan optimized library of novel bacterialtopoisomerase inhibitors with  Thesemolecular nanomachines are involved in maintaining the correct topologicalstate of the DNA in bacteria and consequently in regulating vitalbacterial processes such as cell replication, gene transcription,and genetic recombination.3 Despitetheir long-standing value in clinical practice, they now show a decreasedpotency, mainly as a consequence of the increased quinolone-based“acquired resistance” in bacteria.5 Still, some quinolones continue to be approved for clinicaluse. In 2017, the U.S. Food and Drug Administration (FDA) approveddelafloxacin for acute bacterial skin infections.6 Delafloxacin has a chemically distinct structure comparedto the currently marketed fluoroquinolones; the absence of a protonatablesubstituent confers a weakly acidic character to the molecule thatenhances its antibacterial activity in acidic environments such asone that occurs in Staphylococcus aureus infections.8 Recently, a new class of promising antibacterials known as “novelbacterial topoisomerase inhibitors” (NBTIs) has been discovered.12 They differ from 6-fluoroquinolones not only in their structure,but also in their binding mode and consequently their mechanism ofaction. Compared to 6-fluoroquinolones, NBTIs bind to a different,but vicinal and nonoverlapping binding site in bacterial topoisomerases,thus avoiding cross-resistance with 6-fluoroquinolones. Furthermore,while the mechanism of the bacterial topoisomerase inhibition of 6-fluoroquinolonesis based on the stabilization of double-strand DNA breaks, NBTIs stabilizesingle-strand breaks.14 The most advanced NBTI is gepotidacin, which is currently in thethird phase of clinical trials for the treatment of uncomplicatedurogenital gonorrhea.15 It is notably effectivefor infections caused by Gram-negative bacteria. However, it showsweaker antibacterial activity against Gram-positive bacteria , mainly as a consequence of some point mutations(GyrA D83N and ParC V67A). In particular, the lack of activity ofgepotidacin against S. aureus topoIV might be a pivotalreason for the emergence of resistant strains.16From the plethora of antibacterialsthat target bacterial topoisomerases,6-fluoroquinolones can be regarded as one most widely used intercalatingagents in antibacterial chemotherapy.17 DNA gyrase is comprisedof two GyrA subunits and two GyrB subunits , while topoIVis comprised of two ParC subunits and two ParE subunits .This structural similarity allows NBTIs to effectively inhibit bothenzymes. Thus, dual targeting should lead to a higher antibacterialefficiency and a lower effective bacterial mutation frequency , with the consequent lower risk for thedevelopment of bacterial resistance compared to that of single-targetinhibition.20Structurally, DNA gyrase and topoIV are heterotetramericenzymes,and their subunits share a high structural similarity in the bindingsite of NBTIs.21 A basic nitrogen on the linker is required to establish an ionicinteraction with Asp83 of the Staphylococcus aureus GyrA, which has been shown to be particularly important for NBTIsbinding and affinity.22Novel bacterial topoisomerase inhibitors havethree key parts:a heteroaromatic “left-hand side”, which can intercalatebetween the central DNA base pairs; a bicyclic or monocyclic heteroaromatic“right-hand side” (RHS), which can bind into the deepnoncatalytic hydrophobic binding pockets formed by GyrA of DNA gyraseand ParC of topoIV; and their connecting linker moiety.25 To achieve effective dual targetingthat can lead to synergistic antibacterial effects, NBTIs need tobe optimized toward the balanced inhibition of both of these enzymes.9 Based on this observation,we recently designed a series of NBTIs with p-halogenatedphenyl RHS fragments.13 We demonstratedthat the halogen atom at the para-position on thephenyl RHS fragment  can indeed establish strongsymmetrical bifurcated halogen bonds with the backbone carbonyl oxygensof these Ala68 residues, which are thus responsible for the high antibacterialpotencies. This was verified in our recently solved crystal structureof S. aureus DNA gyrase in complex with an NBTI (1) that included p-chloro phenyl on the RHSfragment .13The crystal structure of 1, the inhibitorypotency of which differs between S. aureus DNA gyraseand topoIV by almost three orders of magnitude 13 27 (see the Supporting Information). Moreover, to better define the dual targeting,the binding mode of this optimized NBTI series was also predictedthrough flexible molecular docking calculations using our in-house-constructed S. aureus and E. coli topoIV protein homologymodels.As the arbonyls2, a morese NBTIs 1.26 In tragments 1. The biS. aureus and E.coli). These allowed the identification of the crucial variationsin the amino acids that delineated the NBTI binding pockets for DNAgyrase and topoIV from both of the bacteria. The key amino acid differencesfor DNA gyrase were Met75 in S. aureus versus Ile74in E. coli; for topoIV, they were Ile71 in S. aureus versus Leu71 in E. coli .27Comparisons were then made amongthe available experimental structuraldata and our homology models for these four target enzymes , we opted to intensifythe propensity and strength by introducing the fluorine substituentson the aromatic ring to increase the size of the σ-hole.13 The synthesis of these NBTIs is given in detailin the Supporting Information, resultingin the focused library of an optimized series of NBTIs that is presentedin S. aureus DNAgyrase and E. coli topoIV that were in the low nanomolarrange, although they were relatively less potent for the inhibitionof the E. coli DNA gyrase and S. aureus topoIV.On this basis,and with the aim of boosting the strength of thehalogen bonds (as previously proposed),p-halogenated phenyl RHS fragments increase according to the increasingsize and polarizability of the halogen atoms at the para-position on the phenyl ring31 as well as their σ-holesize.34 As demonstrated by the data in 4–11 compared to the previously testedcompounds 1–331 for all targeted enzymes, except for E. coli topoIV. With the exceptions of 4 and 5, where the inhibitory potencies were similar, there was a particularimprovement in the inhibitory potency of 6 against allfour enzymes. Moreover, upon the enrichment of the p-halogenated phenyl RHS fragments by the incorporation of an additionalfluorine, the σ-hole size of the RHS fragment p-halogen atom increased even further,34 which additionallyenhanced the strength of the halogen bonds. Monofluorinated analogs 4–6 are generally more potent comparedto the previously tested nonfluorinated 1–3.31 Accordingly, the difluorinatedcompounds 8 to 10 showed even higher inhibitorypotencies, where 8 contained the symmetrical m-difluoro substitution and was the most potent.These data are in agreement with our previous findingsthat thepotencies of this series of NBTIs that include the 8–10 show that the increased lipophilicity was not the maindriving force for potency here; instead, it was the bifurcated halogen-bonding.Namely, 8–10 had very similar logPvalues and would thus be expected to be similarly potent if the lipophilicitywas the pivotal physicochemical property governing their potency  the symmetry of the binding site,(ii) the larger RHS fragment volume and consequently the additionalvan der Waals interactions of the fluorinated analogs, and (iii) theprotonation of the secondary amine of the linker relative to the positionof the electron-withdrawing fluorine. Put differently, the symmetryof the binding site provides an appropriate accommodation for thesymmetric RHS fragments. Analogously, the symmetrically difluorinatedphenyl RHS fragments appear to be better accommodated within the GyrA/ParCbinding sites relative to the monosubstituted RHS fragments.As well as this apparent increase in the strength of the halogenbonds, there are three further potential reasons why the highest potencyis visible for S. aureus and E. coli DNA gyrase and topoIV potencies of 8  indicate that it has an equilibrated activity onall selected targets that could lead to a synergy of its antibacterialactivities  did not markedly affect the antibacterial activityof NBTI 5463 against E. coli. Accordingly, it canbe concluded that even if a synergistic effect is not achieved, dualtargeting is still a plausible way to avoid resistance due to single-targetmutations.Comparisons of the tivities 2. HoweveKa of the secondary amine on thelinker of these NBTIs influences theirionization and their propensity to form an ionic interaction withthe aspartic acid residue  are 6.31 and 6.32, respectively, indicating that theamine is mainly in an unprotonated form at a physiological pH andis thus preventing ionic interactions with the GyrA Asp83 of DNA gyraseand the ParC Asp79 of topoIV. The pKa ofthe secondary amine in 8 is 9.30, whereby the majorityof 8 is in the protonated form at a physiological pH.The protonated form interacts with one GyrA Asp83 or one ParC Asp79through an ionic interaction that is crucial for the antibacterialactivity.17The effects of basicity andthe p residue 1. Thus, 12 and 13 show only about one tenth the inhibitorypotency of 1, as was similarly observed by Li et al.24 and Singh et al.37 The derivative with the nitrogen in the ortho-position(13) showed slightly better results compared to thatwith the nitrogen in the meta-position (12). However, the drop in potency compared to that of phenyl analog 1 shows that the mesomeric effect of the ring nitrogen doesnot influence the σ-hole size of the p-halogen,unlike the inductive effect for the fluorine substituent at the sameposition.To show that the nitrogenin the pyridine does not increase theσ-hole size (unlike the substitution with fluorine), we introducedpyridine instead of phenyl as the RHS core aromatic substituent. Both compounds with pyridine RHS S. aureus and E. coli (S. aureus and MRSAstrains) than against E. coli as a representativeGram-negative bacterium . The potency was improvedfor the AcrA knockout strain of E. coli N43 (the strain lacking the cell membrane efflux pump) and alsoto a lesser extent for the strain with a mutation in the lpxC gene (which increases the membrane permeability) of E. coli D22  compared to that of wild-type E. coli. This shows that our compounds have two shortcomingsrelated to their inhibition of the growth of Gram-negative bacteria.First, their membrane permeability is suboptimal for crossing themembranes of Gram-negative pathogens and, even more importantly, theyare efficiently pumped-out by the E. coli effluxpumps. This results in reduced overall cellular activities against E. coli and most probably against other Gram-negative bacteria, assummarized in Despite the very balanced on-target inhibitory activitiesagainstboth DNA gyrase and topoIV from  E. coli 1, these 6–8 are significantlymore potent against E. coli topoIV than the othercompounds here . While this is already a notable result, their on-targetpotencies indicate the conclusion that their whole-cell potency mightbe further improved by the co-application of efflux pump inhibitors.We plan to evaluate this possibility in future studies.Compounds nds here 1, and th50 values, compounds 4–11 showed weaker effects by up to three orders of magnitude on humancells compared to those for the Gram-positive bacteria .Allthese NBTIs show good selectivities for bacterial topoisomerasescompared to the orthologous human topoisomerase IIα enzyme 1. Their 12 and 13 are presented as the residualmetabolic activities (%) of cells treated with 50 μM NBTIs fromtwo independent experiments, each of which was performed in triplicate.As demonstrated, 12 and 13 show only partialor little cytotoxicity at 50 μM, suggesting that the potenciesagainst S. aureus and E. coli donot overlap with the cytotoxicities. Therefore, the more potent compoundsagainst these bacteria should have better safety profiles in termsof effects on human cells. Of note, an important NBTI class-relatedeffect is a tendency to inhibit hERG potassium channels.38 Accordingly, to further assess the toxicityprofiles of these compounds, it is necessary to evaluate their hERGcardiotoxic potential, which we plan to do in the future.The data in p-halogenated phenyl RHS fragments for the dualtargeting of DNA gyrase and topoIV activities. By introducing variousfluoro substitutions to the RHS fragments, a good enhancement of thebifurcated halogen-bonding propensity and strength was achieved betweenthe p-halogenated phenyl RHS fragments and the Ala68and Ala64 carbonyl oxygens in the GyrA and ParC hydrophobic bindingpockets. These optimized NBTIs showed highly improved enzyme inhibitorypotencies that exceed those of previously published NBTIs.Insummary, we have reported here on the design, synthesis, andbiological evaluation of an optimized series of highly potent NBTIswith 10 they offer additional evidence for the existence and importanceof this halogen-bonding. Namely, by increasing the size of the halogen,the van der Waals contact area can be increased such that just thesize and lipophilicity can guide the NBTI potency. However, with thisnew series we show that their potencies are amplified by increasingthe electron-withdrawing properties of the phenyl substituents whileretaining the same lipophilicity and approximately the same size.This indicates that halogen-bonding rather than van der Waals bondingguides the observed potencies. Moreover, these compounds showed relativelywell-balanced inhibitory activities against both S. aureus and E. coli due to the appropriate steric positioningof the optimized RHS fragments in the enzyme binding sites. For Gram-positivebacteria , it is evident that our NBTIs have satisfied the aims ofthis study, as exemplified by their high antibacterial potencies.However, they show relatively weaker potencies against E.coli and other Gram-negative bacteria. This appears to bebecause they are substrates of the bacterial efflux pumps, as demonstratedby the significant improvements in MICs for the AcrA (efflux pump) knockout of E. coli N43. Among theseNBTIs, 8 shows the highest enzyme inhibition and whole-cellpotency, making it a promising hit for further hit-to-lead optimizationtoward achieving optimized NBTIs with the desired potencies and safetyprofiles.Animportant conclusion from this series of NBTIs is that, alongwith the structural data from the crystal structure,