Uncontrolled bleeding after cardiac surgery can be life-threatening. Factor eight inhibitor bypassing activity (FEIBA) is a prothrombin complex concentrate empirically used as rescue therapy for correction of refractory bleeding diathesis post-cardiopulmonary bypass (CPB). FEIBA used as rescue therapy for bleeding diathesis after CPB has been associated with a low incidence of complications and a reduction in transfusion requirement and re-exploration. The feasibility and efficacy of early administration of FEIBA after the termination of CPB have not been studied in a prospective randomized trial.We designed a small randomized, double-blinded, placebo-controlled pilot trial to determine the feasibility of a larger trial testing the hypothesis that FEIBA decreases transfusion requirements after CPB. The study was designed to evaluate the feasibility of a larger pivotal trial to determine the effectiveness of FEIBA in reducing the total volume of blood products transfused perioperatively, and its safety profile. Study participants were adult patients undergoing elective major aortic cardiovascular surgery at a tertiary referral hospital, who were equally randomized to receive a single dose of either FEIBA or matched placebo intraoperatively at the end of CPB.Twenty patients were screened and 12 were randomized and included in the analysis. Protocol adherence was high, and all patients received the study drug per intention-to-treat except one patient. There were no protocol deviations or events of unblinding, and adverse events were not different between groups. Patients in the FEIBA group were older and more likely to be female and had higher BMI, lower hematocrit, and longer hypothermic circulatory arrest. There were no differences in post-randomization blood product transfusions  or in the administration of open-label FEIBA.This pilot trial confirmed the adequacy of the trial design that involved the early, blinded administration of FEIBA, by demonstrating excellent protocol adherence. We conclude that a larger trial establishing the effectiveness of early prothrombin complex concentrate administration to reduce the use of blood products in the setting of high-risk cardiac surgery is feasible.NCT02577614. Registered 16 October 2015ClinicalTrials.gov,  What uncertainties existed regarding the feasibility?What are the key feasibility findings?FEIBA used as rescue therapy for bleeding diathesis after CPB has been associated with a low incidence of complications and a reduction in transfusion requirement and re-exploration. However, FEIBA has not been prospectively investigated as an early treatment for the reduction of transfusion requirements.What are the implications of the feasibility findings for the design of the main study?There were no protocol deviations or events of unblinding, and adverse events were not different between groups. There were no differences in post-randomization blood product transfusions or in the administration of open-label FEIBA.A larger trial establishing the effectiveness of early prothrombin complex concentrate administration to reduce the use of blood products in the setting of a high-risk cardiac surgery is feasible.It is recognized that blood product transfusion has been associated with adverse outcomes in cardiac surgery, including increased risk of infection, hospital length of stay, and mortality. While cardiac surgeons and anesthesiologists appreciate the risk of morbidity and mortality with blood product transfusion –7, it isIn cardiac surgical procedures at high risk for bleeding after separation from CPB such as those involving the aorta with long CPB and aortic cross-clamp times, refractory bleeding diathesis is commonly managed with the initiation of rescue therapy with factor concentrates. The evidence for prothrombin complex concentrate products, albeit limited, suggests decreased intraoperative transfusion requirements , 9. HoweFactor eight inhibitor bypassing activity (FEIBA) is principally composed of the clotting factors of the prothrombin complex, chiefly factors II, VII, IX, and X. Whereas in licensed prothrombin complex concentrates (PCCs), the coagulation factors are present as zymogens, FEIBA does contain small amounts of activated coagulation factors, in addition to the zymogens , 12. FurOur institution has experience with the use of FEIBA for the rescue treatment of CPB-associated coagulopathy. In a retrospective study of 25 high-risk patients that received FEIBA as rescue therapy for post-CPB bleeding diathesis using a conservative dose of 10–25 IU/kg , we found that the use of fresh-frozen plasma and platelet transfusion after FEIBA administration was significantly lower compared to the amount of blood products transfused prior to FEIBA. No patients were returned to the operating room for re-exploration for bleeding. One patient developed an upper extremity deep vein thrombosis in the setting of central venous catheterization .We conducted a pilot study to evaluate the feasibility of the prophylactic administration of FEIBA after the termination of CPB and a reversal dose of IV protamine sulfate. The study was designed to demonstrate the feasibility of a trial investigating the potential role of FEIBA administration in reducing the need for allogeneic transfusion to treat refractory bleeding diathesis in patients undergoing a high-risk cardiovascular surgery.We conducted a single-center, double-blind, placebo-controlled, randomized pilot trial to assess the feasibility and safety of prophylactic FEIBA administration in patients undergoing elective major aortic cardiovascular surgery requiring CPB. The study was conducted at the Oregon Health and Science University adult cardiac operating rooms between August 1, 2016, and August 31, 2017. The study protocol was approved by the Institutional Review Board. Patients were enrolled in the trial after providing written informed consent preoperatively. The study adheres to CONSORT guidelines.Patients were eligible if they were adults 18 years or older, scheduled for elective aortic procedures, including ascending, arch, and descending repair or reconstruction, with cardiopulmonary bypass, aortic valve repair or replacement, coronary re-implantation , and/or deep hypothermic circulatory arrest. Patients were excluded if they were unable to receive the study drug based on contraindications stated by the manufacturer, such as known anaphylactic or severe hypersensitivity reactions to FEIBA or any of its components or received a blood transfusion within 28 days. Patients were also excluded if their scheduled procedure included coronary artery bypass grafting; had a history of myocardial infarction, thrombosis, or embolism; disseminated intravascular coagulation; or were pregnant women, decisionally impaired, prisoners, or unwilling to provide informed consent.A computer-generated random list using a uniform distribution to equally (fair-coin randomization) assign patients to either FEIBA or placebo was created and maintained by the investigational pharmacy. The investigational pharmacy prepared a study drug that was delivered to the providers in the operating room by an anesthesia technician. Patients, surgeons, anesthesiologists, and nurses were blinded to treatment assignment. The research pharmacy prepared the FEIBA (at the concentration of 40 IU/mL) or placebo  in an opaque syringe. To maintain blinding, the volume of FEIBA and the matched placebo were prepared based on a milliliter per kilogram of actual body weight. Blinded assessors collected data on primary and secondary endpoints, including transfusion requirements, chest tube drainage, safety endpoints, and protocol adherence auditing.After anesthesia induction, patients were equally randomized by the research coordinator to receive either a single dose of FEIBA or a matched volume of saline administered after separation from CPB. The active study drug was prepared in the dose of 20 IU per kilogram in a concentration of 40 IU/mL, at a rate of 0.5 mL/kg via infusion pump over 10 min. The placebo consisted of a matched volume of 0.9% sodium chloride supplied in an identical syringe and tubing at a rate of 0.5 mL/kg via infusion pump over 10 min. Patients were otherwise managed per usual care according to a standardized protocol.After separation from CPB, a reversal dose of intravenous protamine sulfate, calculated according to the heparin dose-response curve, was given with a target goal of a heparin concentration of zero or a return to baseline activated clotting time (ACT). As part of our standard care, routine post-CPB labs, a complete blood count, and coagulopathy panel including the hemoglobin, hematocrit, platelet count, INR, aPTT, and fibrinogen were sent after the administration of IV protamine immediately following separation from CPB, with a post-protamine ACT and arterial blood gas. In addition, samples were collected for thromboelastogram (TEG) by the research lab. After ACT normalized and labs were drawn, the study drug was administered, and the field was subsequently inspected for ongoing microvascular bleeding. In the presence of refractory bleeding diathesis, the anesthesiologist was permitted to administer a dose of FEIBA in an open-label manner as a rescue measure based on the algorithm described below and illustrated in Fig. We planned to enroll 12 patients for the initial feasibility and safety study. As this study was designed to evaluate the feasibility and safety of FEIBA administration in the setting of high-risk cardiovascular surgery with CPB, it did not have adequate power for efficacy endpoints.The primary goal of this pilot trial was to document the feasibility of the prophylactic blinded administration of FEIBA in the context of cardiovascular procedures at high risk of coagulopathy and requirement for blood product transfusion. Indicators of feasibility were protocol violations, maintenance of blinding, use of open-label FEIBA, and occurrence of serious adverse events. The primary endpoints for a larger pivotal trial would be a cumulative volume of blood products transfused including the packed red blood cells (PRBCs), fresh-frozen plasma (FFP), platelets, and use of cryoprecipitate, after the administration of the study drug until the end of anesthesia, and the evaluation of the safety profile.The study was analyzed using a modified intention-to-treat approach. Descriptive summaries are presented using means and standard deviations (SD) for quantitative characteristics and frequencies (%) for categorical characteristics. Data were tested for normality and summaries reported accordingly. Since the study design was randomized, we tested for treatment differences using Welch’s t-tests for mean comparisons of quantitative characteristics and chi-square tests of associations for binary or categorical characteristics. Welch’s t-test was used to test for a treatment effect for the primary endpoint, volume in mL/kg of actual body weight of any blood products transfused after randomization, and for the secondary endpoints. There were no plans for interim analysis; however, safety data was monitored on an ongoing basis during the study. All hypothesis tests evaluated were two-sided, and all analyses were conducted using the Stata (version 15.1) statistical package. The same statistical package was used to create the random sequence for the fair-coin randomization assignment. A two-sided alpha value of 0.05 was required for statistical significance.Twenty patients were screened for eligibility and 13 patients were randomized. Six were allocated to the placebo group and 7 were allocated to the FEIBA group Fig. . One subIntraoperative characteristics are shown in Table During the study implementation, protocol adherence was high, without protocol violations, with the exception of one patient randomized in error. There were no reports of drug administration events requiring troubleshooting that would have unmasked providers as to which study drug was delivered. Likewise, there were no events requiring unblinding.The administration of open-label FEIBA was not different between the two groups. Additional study endpoints to estimate variability for the planning of a larger trial are shown in Table There were a total of five serious adverse events in two patients resulting in death. One patient experienced postoperative complications including acute kidney failure requiring renal replacement therapy and cerebrovascular accident. The other patient had refractory bleeding diathesis and bleeding after separation from CPB requiring the administration of open-label FEIBA. Postoperatively, the patient developed a cerebrovascular accident Table . These eIn this small pilot trial evaluating the feasibility of prophylactic administration of FEIBA in a blinded fashion, we found that it was possible to comply with study procedures and adhere to the study protocol, with patients assigned to the FEIBA group receiving the active study drug in the appropriate dose, while the open-label use of rescue FEIBA was limited to refractory bleeding diathesis, according to the study algorithm.This pilot study has several limitations including small sample size, imbalances between study groups, high variability in the requirements for blood product transfusion, and inadequate study power for efficacy endpoints. Furthermore, three patients required the use of open-label FEIBA, which could potentially negate the differences between groups in the effects of the intervention, especially if the open-label administration was provided to patients in the placebo group. However, the concern for contamination between groups was attenuated because the study was specifically designed to determine the role of the early administration of FEIBA on subsequent needs of blood product transfusion. On the other hand, it would not have been ethical to disallow the use of open-label FEIBA in the context of ongoing bleeding. To that effect, the study had equipoise for early empiric FEIBA administration in high-risk patients given with the intent of reducing potential bleeding, but not given with the intent of treating ongoing, refractory bleeding.Due to the small sample size, the randomization did not produce equally balanced groups, with the FEIBA group resulting in an older population with more comorbidities and a longer duration of deep hypothermic circulatory arrest. Although not different between the two groups, more serious adverse events were reported in the FEIBA group. In addition, the study was not powered to detect differences in the efficacy endpoint of the total amount of blood product transfused after randomization. However, the study provides estimates of variability that will allow the calculation of the sample size for the pivotal efficacy study. We failed to find differences in the cumulative amount of blood product transfused or in the amount transfused intraoperatively or in the ICU. This is in contrast with prior findings from observational studies suggesting a relevant reduction in blood component transfusion compared to no treatment . SeveralThis trial provides important insights to inform the planning of the pivotal trial including the population selection to include patients who are more likely to require transfusion and the determination of the appropriate dose of FEIBA. Because we did not detect differences in coagulation profile between the groups after the administration of the study drug, the dose of FEIBA administered for the study may have not been adequate. It is possible that a higher dose might have resulted in more detectable changes in the coagulation profile. It is also possible that correction of the coagulation profile in the placebo group occurred by replenishing factor deficiency derived from other sources such as blood components. Due to the unbalances in baseline and intraoperative characteristics, it is challenging to further interpret differences between groups with regard to the blood product transfused. It should be emphasized that bleeding complications typically have a multifactorial origin. To what extent FEIBA treatment may reduce bleeding and transfusion requirements would require a larger clinical trial.While this trial was not powered to detect differences between groups, future trials will need to be powered to assess comparative outcomes including cumulative volume of blood products transfused. If alternatives to packed red blood cells, fresh-frozen plasma, and platelet transfusion were available, complications could potentially be reduced. Prothrombin complex concentrates may turn out to be a lower-risk alternative to the blood products from which they are derived.We demonstrated the feasibility of a double-blind, placebo-controlled trial to test whether administration of prophylactic FEIBA decreases transfusion of blood products in patients at high risk for bleeding diathesis in cardiac surgery. Although the trial was not powered for the evaluation of efficacy, the study provides estimates of variability for the planning of a pivotal trial. We conclude that a larger trial of FEIBA administration to prophylactically replenish coagulation factors and correct post-CPB coagulopathy is feasible; however, further refinements of the study design are needed specifically with regard to the study population selection, the choice of study drug dosing, and the use of a standardized transfusion algorithm to minimize the effect of practice variability among centers. Whether a prophylactic approach might prevent refractory bleeding diathesis and reduce the need for high volumes of blood product transfusion needs to be determined in a future, larger trial.


Despite the implementation of effective paediatric vaccination programmes, pertussis remains a global health problem. Disease epidemiology has changed over time, shifting towards the adolescent and adult populations. In adults, the true burden of pertussis is greatly underestimated and pertussis vaccine coverage rates are suboptimal, including individuals with chronic conditions. Here, we report the outcomes of a virtual international scientific workshop to assess the evidence on the burden of pertussis in older adults and identify potential solutions to improve uptake of pertussis vaccines. In adults, pertussis is underdiagnosed in part due to atypical or milder clinical presentation and the lack of testing and case confirmation. However, contemporary epidemiological data denoted an increase in the burden of pertussis among adolescents and adults. This might be related to a variety of reasons including the waning of immunity over time, the lack of booster vaccination, and the improved diagnostic methods that led to increased recognition of the disease in adults. Pertussis sequelae can be severe in older adults, particularly those with existing chronic medical conditions, and the vulnerability of these groups is further enhanced by low pertussis vaccine coverage. Possible measures to increase vaccine uptake include strengthening and harmonisation of immunisation guidelines, healthcare professionals taking a more active role in recommending pertussis vaccination, involvement of vaccination centres and pharmacies in the vaccination process, and improving knowledge of pertussis burden and vaccine efficacy among the general population. Bordetella pertussis [Pertussis is an infectious disease of the respiratory tract caused by the bacterium ertussis . Universertussis , 3. A reertussis . The Worertussis . In its ertussis . Availabertussis , 8.B. pertussis exposure and infection and can also transmit the disease to susceptible patients [Adolescents and adults can be a source of transmission to infants who are too young to be fully vaccinated against pertussis and who are most susceptible to develop severe disease when infected. Previous studies have shown that household members, especially mothers, are the most common source of pertussis infection in infants less than 6 months old , 10. Matpatients , 14.Adult vaccination strategies against pertussis might be the most effective measure to not only protect the individuals from the disease and its complications, but also mount an indirect protection of new-born infants through immunisation of parents, older siblings, and healthcare workers , 15.Combination vaccines containing reduced-antigen-content tetanus toxoid, diphtheria toxoid, and acellular pertussis (Tdap) are currently widely available for vaccination of children, adolescents and adults . The safThe coronavirus disease (COVID-19) pandemic has highlighted the value of vaccination of vulnerable adult populations as a preventive measure against infectious diseases. The International Council on Adult Immunization stressed the urgent need for global adult immunisation strategy that covers available vaccines against a variety of pathogens other than the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and called upon global community and various stakeholders to implement evidence-based adult immunisation programmes and policies .The authors, stakeholders, and experts in the field of adult vaccination from multiple countries  gathered during a virtual scientific workshop organised by GSK on the 30th of June 2020. The objectives were to 1) assess the evidence on the burden of pertussis in older adults and those with respiratory conditions and (2) identify solutions to improve uptake of Tdap vaccines. This manuscript summarises those discussions. A lay language graphical summary is also available in Fig.  assess tAdults > 18 years of age are now proportionally more affected by pertussis than 10–20 years earlier , 8, 25. Immune responses against pertussis, whether natural or acquired through vaccination, decline over time . BoosterToday, the demographic dispersion of pertussis includes adolescents and adults, in addition to infants. Furthermore, the true burden of pertussis in adolescents and adults is greatly underestimated , 35.One factor of underestimation is related to the atypical clinical characteristics of cases compared to the classical child symptoms, making the diagnosis difficult . After tBesides atypical presentation of pertussis in older adults, late presentation to HCPs (leading to no or late testing) contribute to under-diagnosis of the disease in this age group. Given the limited sensitivity of laboratory diagnostic methods, there is a need for more and earlier testing of patients presenting with a cough. Awareness of the symptoms of pertussis  in adults and of the time-sensitivity of laboratory testing needs to increase.Another cause of underestimation is the challenge associated with laboratory case confirmation. Culture of nasopharyngeal secretions and polymerase chain reaction are traditionally used for diagnosis but are effective only at the early phase of the disease. Patients, however, may present late to HCPs and it typically takes several visits to reach a diagnosis. Serologic testing targets pertussis toxin, but results can be affected by pre-existing immunity (induced either by previous vaccination or infection). Moreover, serologic methods for the diagnosis of pertussis are not standardised .B. pertussis with dementia. This is based on the well-established correlation existing between systemic inflammation and Alzheimer disease (AD) and accounts one intriguing epidemiologic observation [B. pertussis colonisation, in close proximity to the central nervous system and olfactory pathway, could explain how B. pertussis and its toxin account for the activation of the microglia, inflammation, atrophy, and accumulation of the amyloid plaques and tau tangles in the brain [B. pertussis in the aetiology of AD is still the topic of ongoing debates, in a life-course perspective, the important role of infant vaccinations and boosters later in life is well-established.An additional concern of geriatricians is the possible links of ervation . The subhe brain . In addihe brain . While tDuring the workshop, extensive discussions were conducted on the long-term consequences of pertussis and the increased impact from comorbidities. The clinical course and the potential complications and sequelae of the disease may also be severe among older adults, not just in infants. Studies suggest that older adults have an increased risk for pertussis-related mortality than children or adolescents, though it is likely to be underestimated . ComplicThe COVID-19 pandemic has highlighted the vulnerability of older adults to respiratory infectious diseases and their complications. It has been revealed that individuals with comorbidities are more vulnerable to infection and are more prone to develop severe symptoms. Some of the most common comorbidities in hospitalised COVID-19 patients were diabetes and obesity , 55, conB. pertussis antigens play a fundamental role in the protection against pertussis. There is, however, no correlate of protection, meaning that antibody levels cannot be precisely correlated with clinical protection [Vaccination is one of the most effective strategies to prevent infection and reduce severity of pertussis in adolescents and adults . Antibodotection . Tdap vaotection . Althougotection , 30, 58.Since 2005, the US Advisory Committee on Immunization Practices recommended vaccination with one dose of Tdap for previously unvaccinated adolescents aged 11–18 years and adults aged 19–64 years. This recommendation was expanded to adults aged > 65 years in 2012 . The 201In contrast with the fairly high vaccination coverage achieved in children, that is currently hampered by the COVID-19 pandemic , optimisDuring the COVID-19 pandemic, most national and international agencies, such as the WHO, the Centers for Disease Control (CDC), the Robert Koch Institute, and the Italian public health authority have all reinforced their recommendations on adult vaccination, including Tdap, to help protect HCPs and the residents of care facilities, and to reduce the risk of double infection, the number of possible clinical visits, and the difficulties related to differential diagnosis of COVID-19 and other respiratory infections with similar clinical features –70. ThisThere are multiple barriers found at the patient, HCP, infrastructure, and funding level that prevent reaching a high vaccine uptake among adults. The belief by some HCPs and patients that childhood vaccination provides lifelong protection, the low awareness of pertussis as an adult disease, as well as the low awareness of safety and benefits of adult pertussis vaccines might lead to lack of recommendation among HCPs and to vaccine hesitancy or refusal among patients , 71. ExtImplementation of vaccination programmes for high-risk adults is even more troublesome, likely due to difficulties in identifying and reaching out to these people. Disparities of recommendations in different countries may impede efforts to improve adult vaccination. International guidelines would create a harmonised recommendation for adult vaccination.The vaccination process involves many stakeholders: general practitioners, nurses, pharmacists, geriatricians, specialists (e.g. pulmonologists for asthma and COPD), and funding bodies. Cooperation across disciplines and societies could improve pertussis awareness and adult vaccination compliance. Vaccination centres other than primary care providers, such as community pharmacies can be an attractive channel and be an efficient way to improve vaccine access and coverage among adults , 74. PeoWaning of immunity, immunosenescence, and burden of pertussis in adults provide a strong rational for booster vaccination, considering immunogenicity and safety of Tdap. It might have better acceptance among older adults than the concomitant administration of Td and monovalent pertussis vaccines. Implementation of Tdap for the decennial booster might also be cost-effective . RandomiTdap vaccination in the COVID-19 era was also discussed during the workshop. On one hand, despite the value of adult vaccination being reinforced both by the current pandemic situation and by several international and national recommendations, less funding is likely to be directed towards the diagnosis of pertussis and Tdap vaccination in the near future. On the other hand, disease severity and the high morbidity and mortality observed among older adults and at-risk populations during the COVID-19 pandemic may redound to adult vaccination efforts and direct more attention towards protection of adults and at-risk groups. As pertussis is difficult to diagnose and it could increase the risk of hospitalisation in adults , vaccinaPertussis is still a frequent infectious disease, with high burden among adolescents and adults, particularly for those with underlying health conditions. This adult population represents a significant source of transmission for unvaccinated or partially vaccinated infants. Atypical clinical presentation of pertussis in adolescents and adults makes clinical diagnosis difficult. Despite recent improvements, a more accurate epidemiological surveillance is desirable to assess the real burden of disease and the impact of vaccination. Pertussis control in older adults could be improved by engaging scientific leaders for a multidisciplinary approach of pertussis and its complications, obtaining a political commitment for the need of research and long-term vaccination policies, and by creating a dialogue on the value of vaccination to increase public engagement.


Genetically identical populations of mammalian cells can demonstrate inherent heterogeneity in gene expression and consequent functional behavior. This non-genetic heterogeneity can emerge due to stochasticity in gene expression, plasticity emerging due to gene regulatory networks, and the epigenetic state of cells. The non-genetic heterogeneity of cells can determine cell fate decision and differential response and adaptation to varying environmental conditions. Non-genetic heterogeneity of stromal and tissue resident stem cells are also known to play an important role in development, cancer, and infectious diseases. Thus, non-genetic heterogeneity is emerging as a major player in mediating resistance to existing therapies. In this Research Topic, we aimed to assemble a collection of manuscripts that address the following important questions: What is the major source of non-genetic heterogeneity, and what is its role in homeostasis and pathobiology? Why and how does non-genetic resistance occur?Capp and Laforge propose to consider an alternative model of development named ontophylogenesis where the generation of a differentiated state is considered as a constrained random process. The chance-selection principle governing cell differentiation would be based on the randomness of biochemical reactions at lower scales on which the multiscale constraints produced by the dynamical organization of the biological system retroact, thus driving the system toward a stabilized state of equilibrium. Mitchell highlights the experimental and computational systems biology studies that have been instrumental in decoding how B-cells achieve distinct fates and the implications of various mutations. The article focuses on mechanisms leading to cell-to-cell variability in B-cell terminal differentiation, and consequences on population heterogeneity in terms of decision-making timings and population distribution proportions. Next, Sha et al. demonstrate using single-cell transcriptomic data on epithelial-mesenchymal transition (EMT) that intermediate cell-states along the EMT spectrum can play a crucial role in TGFb-induced EMT. Analyzing the trajectory of cell-state transitions induced by various growth factors, they highlight how intermediate states can be instrumental for cell-cell communication, highlighting a role of non-cell-autonomous factors in decision-making. Tonn et al. discuss the “metabolic” phenotypic heterogeneity at single cell resolution. They propose a mixture model for systematic prediction of the impact of biochemical parameters on the metabolite distribution at single cell level. This study opens the avenue for uncharted territory of single cell metabolic heterogeneity.After exploring the literature revealing the stochastic nature of cell differentiation and the role of stochastic gene expression  in this process, Gessain et al. discuss the non-genetic heterogeneity in the immune system particularly in macrophages. They specifically discuss the heterogeneity of macrophages in human diseases in the context of infection, inflammation, metabolism, aging and cancer. Finally they summarize how non-genetic heterogeneity in macrophages may impact the pathophysiology in humans and its implication in therapeutic targeting. Specifically on cancer, Guinn et al. propose in a stimulating article that the role of gene expression noise in metastasis should be investigated by two complementary approaches. On the one hand, the authors discuss the monitoring and cataloging of naturally occurring gene expression variability to establish associations with cancer progression and metastasis, and suggest that three different types of noise-modulated threshold crossing  should be more particularly studied in the context. On the other hand, they propose to experimentally modulate protein noise independently of the mean through synthetic biological gene circuits to confirm the role of non-genetic heterogeneity in disease development, stress survival, and metastasis.Biswas discusses the phenotypic heterogeneity in treatment response, specifically the heterogeneity in cellular response and downstream signaling and its impact on treatment response. Interestingly, he discusses a similar mechanism of action during heterogeneous cellular responses. Farquhar et al. discuss how a combination of computational and experimental approaches helps decoding the design principles of fractional killing and non-genetic heterogeneity implicated in antimicrobial resistance (AMR). They also expound the implications of these ideas in cancer drug resistance and underscore the importance of synthetic biology attempts based on the design principles of regulatory networks, which can help discover effective strategies against AMR.From a therapeutic viewpoint, Deshmukh and Saini adopt a broader perspective by considering the evolutionary implications of phenotypic heterogeneity at all levels of life, from viruses to mammals. On the one side, the authors particularly emphasize the role of non-genetic variability during organismal development (with Caenorhabditis elegans as an example) or within a specialized organ system . On the other side, its potential initiating and promoting role in the onset of cancer is largely discussed, with detailed examples over apoptosis, signaling, metabolism, as well as drug resistance. Finally, moving beyond investigating phenotypic plasticity and heterogeneity, Clairambault takes the focus on cancer progression from an evolutionary perspective, presenting a breakdown of multicellularity as one of the hallmarks of cancer. The article asks poignant questions about the connection between emergence of multicellularity and that of cancer, and argues that investigating how multicellularity originated can have important insights into understanding how it breaks down during multiple stages of cancer progression. This series of stimulating articles highlight that non-genetic heterogeneity should be considered as a central component in development and disease, and reveal that innovative theories and experiments in modern biology can be elaborated and conducted by considering non-genetic heterogeneity as a driving force in physiological and pathological systems.J-PC, MJ, and AS conceived, wrote, and edited the final version of this editorial. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Chi-square tests and logistic regression models revealed important temporal patterns in practicing different hygienic and mobility-related prevention measures and the respondents’ risk perceptions during the three timelines. The factors predicting the level of prevention practices vary across the three timelines, based on the specific type of prevention, and within the changing public health contexts. This study contributes to the literature on COVID-19 by incorporating a temporal perspective in conceptualizing prevention predictors. It provides crucial insights for developing timely public health strategies to improve infectious disease prevention at different stages and for individuals with varying backgrounds. The effectiveness of public health measures in containing an infectious disease largely depends on how the general public is taking the prevention practices in daily lives. Previous studies have shown that different risk perceptions and sociodemographic characteristics may lead to vastly different prevention behaviors. This paper applies a temporal perspective in examining the changing patterns of prevention practices over time and their dynamic relationships with the perceived risk towards COVID-19 and its individual characteristics. Three key timelines  were identified to represent the early, lockdown, and reopening stages of the first wave. Data were drawn from an online survey conducted in the Greater Toronto Area (GTA) of Canada ( Reacting to an infectious disease outbreak in the absence of a vaccination, traditional public health measures  have been rigorously implemented to contain the spread of the disease ,2,3. TheResearchers from different disciplines are interested in factors shaping prevention practices . A groupStill, much of the conceptual frameworks on the relationship between prevention practices and risk perception are characterized by “interpretational ambiguity” , p26). I6. I2]. HThis paper aims to understand the complex relationships among prevention behaviors, risk perception towards COVID-19, and individual characteristics from a temporal perspective, based on an online survey conducted in the Greater Toronto Area (GTA) of Canada. It seeks to address the following research questions: (1) How did individual prevention behaviors and risk perceptions change over time during the first wave of COVID-19? (2) How do different prevention practices relate to risk perception and individual characteristics across the timelines? The GTA is the most populous metropolitan area and the largest COVID-19 hotspot in Canada. Three key timelines during the first wave of COVID-19 in 2020 were selected to capture individual psycho-behavioral changes over time and the factors associated with such changes. They are February , April , and June . The results of the study provided important insights and timely evidence for the further analysis and modeling of infectious disease, as well as developing and implementing public health interventions in a timely manner. The data used for this study were collected from an online survey conducted from June to November in 2020. The survey was administered among adult residents in the GTA using Qualtrics. It is part of a larger research project examining various aspects of the community response to COVID-19 in Toronto. All aspects of the survey and data collection received ethical approval from the Ryerson University Research Ethics Board (REB) and Queen’s University General Research Ethics Board (GREB). The online survey was deemed appropriate, as a traditional paper-based survey was impossible to conduct during a pandemic. Additionally, a timely and efficient survey administration through an online survey platform would help to collect more accurate responses regarding participants’ recent experiences. Recruitment was primarily done via key informants, community collaborators, and online advertisements on social media and in local newspapers and newsletters. To accommodate those interested individuals with limited Internet access, assistance was provided through the telephone to help someone to complete the survey on their behalf. A nonrandom and purposeful sampling strategy was adopted primary via referral from our community collaborators and connections. Recruiting residents of the Greater Toronto Area (GTA), we aim for balanced representations of women and men, urban and suburban residents, different age groups, etc. Due to the challenges in conducting an online survey during the pandemic, sampling bias is acknowledged as a limitation. In total, over 770 responses were collected, in which 300 invalid cases were removed for reasons such as an abnormally large number of autogenerated survey completions within a short time period , those from outside of the study area, and those that did not complete the questions with key variables. In the end, 470 valid cases were included in the final sample. The survey included questions on prevention behaviors, the perceived risk associated with COVID-19, and the socioeconomic and demographic backgrounds of the survey respondents. The survey incorporated a temporal perspective in collecting information on prevention practices and risk perception across the three key timelines . The survey was launched in June, and participants were asked to recall their experiences in February, April, and June respectively. There were two broad types of prevention practices examined: hygienic-related  and mobility-related prevention practices . Questions on prevention and risk perceptions were repeated for the months of February, April, and June, and the remaining questions, such as those on socioeconomic background, did not have such a temporal dimension. The overall temporal changes in prevention practices and risk perceptions within the sample were tabulated to provide a general understanding of the trends and patterns. Chi-square tests were then conducted to explore the bivariate relationships between prevention and risk perception and the individual characteristics, which were all measured categorically. The initial chi-square tests on individual characteristics and prevention practices included a wider range of variables . Only those that showed significance in at least one prevention practice at one timeline were further included in the logistic regression models. Separate logistic regressions were conducted for the three timelines in order to examine the temporal differences in the effects of the explanatory variables on the dependent variable (prevention practices). For each timeline, five logistic regressions were conducted for the five prevention variables, respectively. The different regression models provided critical insights into the dynamic relationships of prevention behaviors, risk perceptions, and individual characteristics. The final explanatory variables included risk perception towards COVID-19, age, household income, gender, and attitude towards public health interventions. The multicollinearity was manually tested among selected explanatory variables on their bivariate relationships, and no noticeable degree of correlation among the explanatory variables was found. p < 0.001). In April and June, some of these associations continued to be significant, such as in the case of hand washing and mask wearing , beiFinally, this study indicates that public health interventions played a powerful role in shaping the practices of individual disease prevention. From February to April, the percentages of participants who always practiced various prevention measures increased fairly rapidly. Further, about 36% of the survey participants held a conservative attitude towards public health interventions. This variable was found significant in April and June in explaining several prevention practices. Before public health intervention started, risk perception was an important determinant of prevention practices instead. Prevention practices that were clearly recommended in the local public health guidelines at different timelines  were practiced more often by participants compared to self-imposed measures . Further, the percentage of those always wearing a mask increased consistently from February to June, reflecting the effect of revised public health recommendations at that time. This paper provides crucial insights for developing timely public health strategies to improve prevention practices for individuals with different backgrounds. Recent studies of COVID-19 suggest that early-stage preventions, either self-imposed or government-imposed, are crucial in containing transmission, before a surge in cases overwhelms the tracing and testing capacities ,46. ThisA number of limitations exist in this study. Firstly, conducting a questionnaire survey during a global pandemic has demonstrated many unique challenges in terms of sampling, sample size, recruitment, and representativeness . For exaThe limitations lead to a number of paths for future research. Firstly, qualitative research methods can be a valuable addition to the study by identifying personal contexts and reasons for the changing psycho-behavioral responses towards COVID-19 at different stages of the pandemic. Future primary data collection through interviews and/or focus groups will be particularly helpful in providing in-depth insights to improve our understanding of the findings from the statistical analyses; reveal more nuanced patterns in the temporal changes of prevention and its predictors; and contextualize them in the wider socioeconomic, cultural, and regulatory frameworks. Secondly, future research could be conducted to expand the scope of the survey to improve the sample size and representativeness, which would also allow for incorporating a more comprehensive set of variables. For example, different types of risk perceptions (e.g., using the CoWoRP scale of probability, severity, worry, and unsafeness developed by Reference  to measu


Stress and burnout are serious and growing threats to the mental health of medical trainees. Recent estimates of burnout in medical students and residents are quite high, with more than half displaying signs of stress, anxiety and depression. The COVID-19 pandemic has only heightened the state of poor mental health in these student populations. It is the position of LSU Health Shreveport Office of Institutional Wellness that a critical need exists for academic institutions to evaluate challenges to self-care and wellbeing in medical trainees. Such evaluations may pave the way for the development of effective institutional wellness initiatives and strategies, with the goal of reducing barriers to self-care to promote better mental and physical health, and facilitate improved quality of life in medical students and residents. The COVID-19 pandemic created widespread stress and anxiety in the US population. Social distancing, increasing joblessness and limited access to mental health services, created an increased mental health burden during and beyond the pandemic . StudentStudents of color (SOC) have been disproportionately impacted during the pandemic, displaying poorer outcomes with COVID-19 as well as increased rates of anxiety and depression , 14. PooOne tactic to promote wellness and reduce stress during the rigors of medical training is to engage in self-care activities , 16. TheAyala et al., in a 2017 study on self-care, found “a robust inverse relationship between perceived stress and medical students' quality of life” , and provide a 24/7 crises management hotline to assist with emergent mental health issues.Further, it is important to remember that the COVID-19 pandemic has not affected trainees equally, and has exacerbated the distinct mental health issues faced by SOC, lesbian, gay, bisexual, transgender, queer (LGBTQ) students, and students that are economically disadvantaged , 14. AcaPhysical and emotional wellbeing are key to academic success in medical education. However, medical students and residents face poor mental health due to the rigors of their training and decreased ability to practice self-care. Research studies report medical trainees and healthcare professionals are at increased risk for poor nutrition, reduced sleep health, depression, and suicide. This is especially true in underrepresented students and residents. These conditions have become even more prevalent after COVID-19. Self-care has been shown to attenuate poor mental health and stress, however due to time constraints during medical training, few seek out or practice wellness activities. Increasing opportunities for medical trainees to participate in self-care activities during their training, can assist them in developing strong values for self-care, and become better wellness advocates to future patients. A fundamental shift recognizing the essential role of self-care is needed in order to address the epidemic of stress and burnout among medical trainees. Academic institutions must also understand and address the barriers students and residents face in regards to practicing self-care and wellbeing, and emphasize initiatives that address burnout-related issues and provide beneficial, culturally competent resources. Helping students and residents recognize the importance self-care and wellness during their medical training assists them in developing resilience, which will help them deal with stress more effectively during their matriculation and post-graduation, and improve their quality of life.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Public concern regarding environmental contaminants (ECs)-related reproductive disorders has increased due to increasing global rates of infertility. All kinds of ECs are on rise rapidly in developing and industrializing low- and middle-income countries. The aquatic environments throughout the world are repositories for enormous amounts of ECs. As the biology of the reproductive system is highly conserved in vertebrates, wildlife or laboratory studies on fish provide significant information to establish a detailed risk assessment, and to identify novel or more sensitive endpoints for ECs-related reproductive disorders. The adverse effects of ECs on endocrine regulation of reproduction in male fishes have been extensively studied and reviewed; however, our knowledge on the effects and mechanisms of action of ECs on determinants of male fertility is limited. The present study is a state-of-the-art comprehensive review on the ECs-related fertility threat in male fishes with emphasis on the ECs effects on sperm production, morphology, genome, and motility kinetics. After a brief introduction to reproductive biology, fertility indicators, and determinants of fertility in male fishes, wildlife evidences for reproductive disorders were reviewed in fishes from the polluted aquatic environment. The laboratory studies show that ECs detected in aquatic environment are capable of causing fertility threat at environmentally relevant concentrations associated with a decrease in fertility determinant(s). This study suggests an urgent need to better elucidate mechanisms through which ECs affect sperm functions to cause fertility threat.Increasing global rates of diminished fertility in males has been suggested to be associated with exposure to environmental contaminants (ECs). The aquatic environments are the final repository of ECs. As the reproductive system is conserved in vertebrates, studies on the effects of ECs on fertility endpoints in fishes provide us with valuable information to establish biomarkers in risk assessment of ECs, and to understand the ECs-related fertility threat. The aim of the present review was to evaluate associations between ECs and fertility determinants to better understand ECs-related male fertility threat in male fishes. Wildlife studies show that the reproductive system has been affected in fishes sampled from the polluted aquatic environment. The laboratory studies show the potency of ECs including natural and synthetic hormones, alkylphenols, bisphenols, plasticizers, pesticides, pharmaceutical, alkylating, and organotin agents to affect fertility determinants, resulting in diminished fertility at environmentally relevant concentrations. Both wildlife and laboratory studies reveal that ECs adverse effects on male fertility are associated with a decrease in sperm production, damage to sperm morphology, alternations in sperm genome, and decrease in sperm motility kinetics. The efficiency of ECs to affect sperm quality and male fertility highly depends on the concentration of the contaminants and the duration of exposure. Our review highlights that the number of contaminants examined over fertility tests are much lower than the number of contaminants detected in our environment. The ECs effects on fertility are largely unknown when fishes are exposed to the contaminants at early developmental stages. The review suggests the urgent need to examine ECs effects on male fertility when a fish is exposed at different developmental stages in a single or combination protocol. The ECs effects on the sperm genome are largely unknown to understand ECs-related inheritance of reproductive disorders transmitted to the progeny. To elucidate modes of action of ECs on sperm motility, it is needed to study functional morphology of the motility apparatus and to investigate ECs-disrupted motility signaling. Global rates of environmental contaminants (ECs)-related reproductive disorders have been increasing over the past 50 years. In human beings, the incidences of testicular dysgenesis syndrome, including hypospadias (urethra opens on the underside of the penis instead of the tip), cryptorchidism (one or both testes not descended into the scrotum), testicular cancer, low semen quality, and infertile men, show global increases associated with ECs ,7,8,9. LThe aquatic environment is at greatest risk from pollutants since all chemicals will eventually find themselves in the rivers, lakes, and oceans as the final repository . As biolWe have recently reviewed the toxicity of ECs on sperm morphology and motility in fishes, in vitro . The revThe present study is a state-of-the-art comprehensive review on the ECs-related fertility threat in male fishes with emphasis on the adverse effects of ECs on determinants of fertility, including sperm production, morphology, genome, and motility kinetics. After a brief introduction to reproductive biology, fertility indicators, and determinants of fertility in male fishes, wildlife evidences for reproductive disorders are reviewed in fishes from the polluted aquatic environment. To understand whether reproductive disorders in wildlife is associated with ECs, we review laboratory studies in which the adverse effects of particular ECs are studied on determinants of fertility, in vivo. Finally, we discuss urgent needs to better elucidate mechanisms through which ECs affect sperm functions to cause fertility threat. The present review provides us with valuable information to understand ecotoxicological impacts of ECs on fish fertility, which can be useful to establish biomarkers in ECs risk assessment.It is essential to review reproductive biology, fertility indicators, and determinants of fertility in male fishes before delving into the ECs-related fertility threat. These provide the basic information to better understand multiplicity of sites through which ECs interfere with fertility. To clarify the terminology, “semen” refers to seminal plasma and sperm and “sperm” refers to sperm cells in the present review.In general, the male reproductive organ consists of a paired testes, the testicular duct, and the sperm duct in fishes ,29 Figu. In someThe testes are divided into the “tubular type” and the “lobular type” according to the distributions of spermatogonia in the seminiferous region ,31,32,33Danio rerio) and guppy (Poecilia reticulate).In the tubular type, as spermatogonia divide and enter in meiosis, the cysts migrate towards the region of the spermatic ducts located in the central region of the testis, where the cysts open to release sperm E. This tOryzias latipes), common carp (Cyprinus carpio), goldfish (Carassius auratus), and rainbow trout (Oncorhynchus mykiss).In the lobular type, the testis is composed of numerous lobules that are separated from each other by a thin layer of fibrous connective tissue, and spermatogonia are spread along the germinal compartment throughout the testis. The cysts do not migrate or become displaced during their development, and sperm is released into the lobular lumen F. This tThe testicular compartment contains Sertoli cells, Leydig cells, blood/lymphatic vessels, macrophages and mast cells, and neural and connective tissue cells G. The LeThe testicular ducts are located adjacent to the testes, which continue into the sperm ducts on the ventral sides. Testicular and sperm ducts possess very similar structural and enzyme-histochemical characteristics, and play key roles in nutrition of sperm, storage of sperm, synthesis of steroids, secretion of proteins and enzymes, and formation of the seminal plasma ,35. MatuSperm is produced from spermatogonia following divisions ,38,39. DSperm is differentiated into a head, midpiece, and flagellum in fishes ,41,42 F. The heaThe seminal plasma is a product of Sertoli cells, testicular ducts, and sperm ducts, and its composition is different among fishes that may reflect species variations. The main role of seminal plasma is to create an optimal environment for the storage of sperm during maturation in the sperm ducts. Seminal plasma maintains sperm viability, motility, and fertilizing ability, and protects sperm against damage caused by proteolytic or oxidative attacks ,51.2+) ions in both freshwater and marine fishes, while cyclic adenosine monophosphate (cAMP) remains unchanged. However, studies show that demembranated sperm of salmonid and sturgeon fishes require cAMP for the axonemal beating 2 [194], 2 ,164.2 or BPA  to ECs causes damage to sperm and decreases sperm motility and velocity to decline sperm fertilizing ability; however, the effective concentrations are far exceeding the WHO recommended limits . The pre2, EE2, 17α-MT, 4-NP, BPA, BPS, and DEHP and its metabolite (MEHP) cause diminished fertility at environmentally relevant concentration (The present review shows that: (a) Unlike the source of contaminants from industrial, municipal, treatment plant, or agricultural activities, abnormalities in sperm morphology ,139, damntration . (c) Thentration . It is wntration . HoweverThe present review highlights the following aspects that should be taken into account in further studies to better elucidate the ECs-related fertility threat in male fishes.(a) One can easily distinguish that the number of contaminants that have been used in fertility tests are comparable to the number of contaminants in our environment. Future studies should examine the effects of other chemicals with major public concerns. Among them, there is an immediate need to examine the effects of pharmaceuticals  that are highly consumed in global medicine use, agriculture, and animal husbandry ,241. Fer(b) Examination of the adverse effects of ECs at environmentally relevant concentrations that cover the lowest and highest detected concentrations is highly needed. Most studies have failed to include the lowest environmental concentration among treatments .(c) The ontogeny-dependent effects of the ECs on male fertility are largely unknown. Basically, developmental stages are the main parameters to be carefully defined based on the objective. For instance, if the study is aiming at investigating the adverse effect of ECs on sperm production, it is a prerequisite to expose the fish from the early developmental stage of the testis. However, the present review shows that a number of studies that exposed fishes to ECs at early life stages is considerably lower than those that examined the adverse effects of ECs in adult-exposed individuals . When em(d) Assessment various types of determinants of fertility to better elucidate site of action of an EC that cause diminished fertility. There are a few studies that have evaluated the ECs effects on sperm production and motility .(e) The present study shows that ECs may cause phenotypic changes in the reproductive system and affect determinants of fertility . It has To better elucidate ECs effects on sperm morphology, future studies should investigate functional structure of sperm including nucleus, mitochondria, centrioles, and axoneme, with particular consideration to the organization of the microtubules and motor protein called “dynein” in the latter case ,246,247.Regarding the ECs effects on sperm genome, it is needed to examine effects of ECs on incidence of aneuploidy , to stud2+, and cAMP) that regulate sperm motility signaling are fully unknown and should be considered in future studies [2+ through activation of a Ca2+ channel to interfere sperm motility in human. The laboratory studies suggest that ECs may affect sperm swimming pattern as sperm velocity parameters are decreased  was exposed to 0.1 µg/L EE2, which was associated with decrease in VSL. This suggests that the adverse effects of EE2 on sperm velocity could be related to amount of ATP available to generate flagellar beating force. Further studies are needed to investigate sperm energetics along with sperm motility kinetics to better understand the mechanisms through which ECs cause fertility threat.(f) Sperm requires energy for motility and flagellar beating force ,68,106. However, assessment of the adenylate energy charge (AEC = ATP + 0.5 ADP/ATP + ADP + AMP) is suggested to better understand the adverse effects of ECs on sperm energetics and metabolism of energy ,66,249. (g) In an aquatic environment, there are mixtures of ECs that may exhibit pharmacodynamic interactions. These interactions can be antagonistic or synergistic, which may cause an increase in the toxicity of a particular chemical through the cocktail effect ,142. For(h) Our current knowledge on the ECs-related male fertility threat is mainly from freshwater fishes inhabiting lakes or rivers . Future (i) Recently, epigenetic inheritance of the adverse effects of ECs received high scientific and public considerations. As ECs have been shown to affect the sperm genome, further studies need to characterize transgenerational inheritance of the adverse effects of ECs to the offspring. There are studies that show the adverse effects of bisphenols and 4-NP that were inherited to the next generations ,167; how(j) The threat of ECs to fish populations is still an open question. There are historical reports that show populations of fishes have declined ,251,252;We also would like to offer the following suggestions to normalize the studies for better understanding of the ECs-related fertility threat in fishes.(a) The large variations among results of studies that have examined a particular contaminant suggest the need to optimize a protocol for harmonization of studies, which should encompass both biological and technical issues. These may include a protocol for optimizing the design of exposure tests, methods for evaluating male fertility, and laboratory tools to assess determinants of fertility. For example, (a-i) CASA has become very popular for the analyses of sperm motility kinetics. Technical conditions for recording of sperm motility and setting of CASA affect the results , thus ne(b) The present study suggests species-specific adverse effects of an EC on male fertility and its determinants. To better understand sensitivity of a species to an EC, the effects of ECs could be examined on small laboratory fishes including zebrafish, guppy and medaka, which serve as aquatic model organisms. Application of species with different biology of reproduction will provide us with valuable information to assess ecological risk assessment of ECs. Among species, zebrafish and Medaka, in contrast to guppy, are external fertilizers, and the testes are of the tubular type in zebrafish and guppy compared to those of medaka that are of the lobular type.(c) Although all studies have considered this note, it is worth reminding that one should take into account renewal of exposure medium based on the half-life of the EC.


The objective of this study was to assess nursing homes’ COVID-19 preparedness in southern Portugal, including staff’s work experiences during the pandemic. (2) Methods: We used a COVID-19 preparedness checklist to be completed by management teams, followed by follow-up calls to nursing homes. Thereafter, a survey of staff was applied. Data analysis included descriptive statistics, exploratory factor analysis, and thematic analysis of open-end questions. (3) Results: In total, 71% (138/195) of eligible nursing homes returned the preparedness checklist. We conducted 83 follow-up calls and received 720 replies to the staff survey. On average, 25% of nursing homes did not have an adequate decision-making structure to respond to the pandemic. Outbreak capacity and training were areas for improvement among nursing homes’ contingency plans. We identified teamwork as an area of strength for safety culture, whereas compliance with procedures and nonpunitive response to mistakes need improvement. (4) Conclusions: To strengthen how nursing homes cope with upcoming phases of the COVID-19 pandemic or future public health emergencies, nursing homes’ preparedness and safety culture should be fostered and closely monitored. It has been more than a year since the World Health Organization (WHO) declared the coronavirus outbreak a pandemic. Currently, as the pandemic unfolds and national vaccination campaigns intensify, countries are still struggling to contain the spread of different virus strains and their effects on the population’s health. Each country has been facing its specific challenges, depicted by the large variability in countries’ set of responses to this global health crisis ,2,3. A cThe effects of the pandemic in nursing homes worldwide have taken a toll on all of us and in our humanity, as these facilities have been affected in an unprecedented manner . The iniWhilst caring for residents, nursing home staff are also managing concerns about their health and well-being and the safety of their family members. In the first stages of the pandemic, the focus on the health and well-being of nursing home staff was not immediately prioritized. Only later, balancing safety, well-being, and quality of life among staff became a topic of discussion, leveraging from the focus attributed to these topics by international organizations, such as the Organization for Economic Co-operation and Development (OECD)  and WHO In Portugal, the first death from COVID-19 occurred on 16 March 2020. During the initial phases of the pandemic, 40% (450/1126) of the deaths from COVID-19 were among residents in nursing homes [The main goal of this study is to assess the COVID-19 preparedness of nursing homes in southern Portugal and explore its effects on nursing home staff safety culture and well-being in the early phases of the pandemic (from March to July 2020). Thus, the objectives of this study are: (1) to assess COVID-19 preparedness of nursing homes in two regions of southern Portugal (Algarve and Alentejo) in the early phases of the pandemic; (2) to better understand safety concerns and well-being of nursing home staff; and (3) to understand nursing home staff work experiences during the pandemic, including resident safety culture. Findings will be used to inform current ongoing responses targeting nursing homes, optimize dealing with upcoming phases of the SARS-CoV-2 pandemic, and strengthen nursing home preparedness to other public health emergencies.We conducted a cross-sectional survey-based study in nursing homes of 2 regions in southern Portugal (Algarve and Alentejo) in the early phases of the COVID-19 pandemic (from March to July 2020). Nursing homes were identified by the regional offices of the Social Security Institute. Nursing homes participated in our study voluntarily and independently of parallel ongoing COVID-19 testing initiatives. Participating nursing homes self-assessed COVID-19 preparedness using a checklist filled by managers; thereafter, a web-based self-administered survey was conducted among staff. For this observational study, a formal ethics approval was waived because of the non-intrusive and non-interventional characteristics of the study; the anonymization of all personal data prior to analysis and reporting; the existing protocol between the Social Security Institute and the ABC research institute; and the urgency of gathering key information about the problems that nursing homes had been facing in a timely fashion. Nursing home staff responding to our survey granted consent at two moments: before answering the survey and when submitting their answers.The checklist  was deveSafety concerns and well-being of nursing home personnel survey  , which rWe used descriptive statistics to characterize attributes of participating nursing homes and survey respondents and to examine missing data patterns. We summarized the share of facilities that had fully implemented each set of items in the preparedness checklist by computing the geometric mean across items. Composite response frequencies of the resident safety culture from the perspective of staff were computed by averaging positive responses. We used a spreadsheet to group data from open-ended questions from participating nursing homes and survey respondents into meaningful emerging categories.We used Goodman and Kruskal’s Gamma test to measure the correlation between ordinal variables in the survey. We performed exploratory principal axis factor analysis via oblimin rotation with Kaiser normalization on the 10-item questions about perceived fear/anxiety and absenteeism amongst staff. Missing values were excluded listwise. The number of factors was determined based on parallel analysis, Kaiser criterion of eigenvalues , and a visual inspection of the scree plot. We assessed the final factor loading structure for internal consistency with Cronbach’s alpha. Thereafter, we conducted a K-means cluster analysis with the regression method.We used IMB SPSS Statistics version 26  for all analyses. The confidence level was set at 95%.n = 138) of nursing homes returned the COVID-19 preparedness checklist: return rate was of 53% in Algarve (51/96) and 88% in Alentejo (87/99). In parallel, we conducted 83 follow-up calls with nursing homes, of which 65 (78%) were with a facility that had previously returned its COVID-19 preparedness checklist. In total, 720 nursing home staff replied to the safety and well-being survey.Between March and July 2020, 96 nursing homes in Algarve  and 99 licensed nursing homes in Alentejo were engaged in the COVID-19 initiatives set forth by the ABC . A totalOn average, a quarter of nursing homes did not fully observe an adequate structure for planning and decision-making in response to the pandemic, and 17% did not have a fully implemented and disseminated contingency plan, with designated key people to operationalize thereof . The keypoor communication channels, both internal and external, often failing to disseminate the contingency plan among key stakeholders  and engaging with health and other competent authorities;inexistent or poor planning to isolate or transfer residents if need be;poor surveillance systems to monitor for symptoms among residents and staff;insufficient planning to overcome hindrances related to staff shortages and absenteeism, and infrastructure constraints ;the inexistent monitoring system of the effectiveness of the measures aiming at addressing behavioral factors, both at the institutional and individual level;misuse of personal protective equipment (PPE) attributed to poor training and a generalized shortage of specific equipment .Based on the returned checklists and follow-up calls with nursing homes, we identified key items that were most frequently overlooked:continuous revision of the contingency plan to reflect any updates to the guidelines set forth by the Directorate—General of Health and other relevant competent authorities;emergency protocol with the nearest primary health care centers for a quick response in case of an outbreak;systematically maintaining an inventory of PPE in close collaboration with governmental authorities;using social media and other platforms to update families and carers on residents’ well-being and on the public health measures that the nursing home is developing.We also highlight good practices found at some facilities:A detailed list is available in Most of the 720 survey respondents were female (93%), and the average age was 45 years old . The higp < 0.001) between the risk perception of becoming infected with coronavirus and that of becoming severely ill with COVID-19 (p < 0.001). Our results did not suggest a strong or significant association between the importance of being tested for COVID-19 and the respondents’ risk perception of becoming infected or severely ill with COVID-19.Our data suggested a strong positive association  and regional offices of the Social Security Institute (MLSSS), which resulted in increased administrative burden and increased waiting time in receiving support. Some respondents also perceived that the competent authorities had little knowledge of the real context and circumstances of most nursing homes, and thus, were not aware of the structural hindrances these institutions were facing to follow many of the guidelines and orientations set forth by health authorities.A second category focused on the psychological support available to residents and staff throughout the pandemic crisis, or the lack thereof. The availability of psychological support to staff was highlighted as an important tool for managing stress levels and the emotions of colleagues, residents, and those of residents’ families and carers. Some respondents also highlighted increased levels of stress and anxiety amongst nursing home managers, which made them more reactive and less supportive towards staff. Notwithstanding, some respondents found in other colleagues the much-needed support to cope with fear and other daily challenges.The third category refers to a generalized perception of misinformation in regard to the contingency plan, where respondents identified not knowing its content or how to best proceed to activate and comply with the foreseen planning. Related to this is the common misuse of PPE, which could signal shortages in PPE, as well as inadequate and insufficient training about its appropriate use.p = 0.035) and support with dealing with fear/anxiety   .We computed composite percent positive scores related to the constructs of teamwork, staffing, compliance with procedures, training and skills, and nonpunitive response to mistakes . The lowWe examined nursing home COVID-19 preparedness and the staff’s perceived well-being and safety, including nursing home resident safety culture, in the early phases of the pandemic, in two regions of southern Portugal. Our findings suggest that COVID-19 preparedness in nursing homes was poor. Often, nursing homes overlooked the importance of planning a thorough contingency plan and of having an adequate structure for planning and decision-making to respond to the COVID-19 pandemic. In general, nursing home staff showed concerning well-being scores. Our data support that staff perceived fear and absenteeism among peers differently, the latter with greater variability. We identified teamwork as a strength for resident safety culture, whereas compliance with procedures, nonpunitive response to mistakes, training and skills, and staffing were signaled as areas for improvement.Some nursing homes did not use the preparedness checklist as a tool to inform the preparation of a contingency plan. We explain non-response by considering three key factors. First, the use of the preparedness checklist by nursing homes occurred on a voluntary basis and was independent of getting access to COVID-19 testing. Second, nursing homes had to quickly react to threats posed by COVID-19 and adapt their operations; hence, non-responders may have felt overconfident with the actions implemented in their facility and did not assign substantial importance to other initiatives wherein no additional value is perceived. And third, by the time when news in the Portuguese media started linking fragilities in nursing homes’ contingency plans and COVID-19 outbreaks, these facilities were overwhelmed with daily mandatory and uncoordinated data requests from different agencies; contrary to governmental data-driven initiatives targeting hospitals, this was not the case for social care. Contributing to this context could be the current levels of integration of health and social care information, which is uncoordinated and poor regarding data interoperability . In the Our findings suggest that many nursing homes overlooked the importance of thorough and exhaustive planning and the role of the contingency plan in that. For example, in Portugal, one of the most serious events in a nursing home which resulted in the death of 18 people seems to have been strongly related to the absence of a proper contingency plan; a few months later, little had improved . This isMany of the vulnerabilities in the COVID-19 preparedness of nursing homes occur atop of long-lasting structural barriers  ,38, whicpsychological PPE [Nursing home staff perceived their working realities on fear and absenteeism very differently. Several features may influence how staff are affected by psychological distress  aical PPE , nurturiSafety culture studies in social care settings such as nursing homes are widely lacking, and much of the available literature refers to the USA context . To the Our study strength relies on the large sample of nursing homes involved, the engagement of staff with our survey, and the use of safety culture indicators. Also, we featured in our survey international standardized measures in hopes of future possible international comparisons. However, this study should be interpreted considering some limitations. First, our sample is not representative of the nursing home population and social and health care workers in these facilities in Portugal, and thus, hampering the generalizability of our findings outside the context of the study. Notwithstanding, these data, in parallel to the COVID-19 testing, were crucial to inform and support health and social authorities in better supporting nursing homes; also, these findings are well aligned with those of other international studies. Second, we only used one section of the resident safety culture questionnaire to reduce the length of our survey. By doing so, we are aware that other areas that may be significantly affecting the safety culture of a nursing home may not have been captured by our data. Finally, we understand that the data collection method chosen for our survey may have limited the ability of some people responding to the survey, particularly people without access to a computer or smartphone and with lower literacy on information and communications technology. Also, we are aware that to increase the response rate, some nursing homes had a computer available for staff interested in answering the survey. The extent to which this commodity influenced their answers was not considered in data analysis.The objective of this study was to assess the COVID-19 preparedness of nursing homes in southern Portugal in the early phases of the pandemic, including the work experiences and safety concerns of staff. In Portugal, COVID-19 seems to have exacerbated the longstanding and systemic fragility of social care and the underinvestment in high-quality long-term care, including nursing homes. That disinvestment is partly exposed by the generalized unpreparedness across nursing homes and their difficulties with keeping residents and staff safe during the pandemic. Hence, rethinking the positioning of nursing homes in the social and health care systems could potentially strengthen nursing home preparedness. In addition, future response actions to the pandemic should involve nursing home representatives in decision-making processes to the extent where it is feasible and leverage from available evidence at the national and international level.Communication between competent authorities and nursing homes should be clearer in scope, actions, time, and responsibilities, increasing transparency and accountability in responding to the challenges of the COVID-19 pandemic. Improved communication channels are also key inside nursing homes to better discuss the effects of the pandemic within the facility and as a mechanism to bolster staff resilience. Additionally, assessing safety culture amongst nursing home staff can be a meaningful approach to understanding and dealing with staff’s experiences of fear and anxiety. These could be addressed by involving staff in decision-making, recognizing their efforts and merits, enhancing collective learning and sharing, learning the current priorities of staff, and normalizing the sharing of feelings of fear, anxiety, and psychological frailty. Dealing with a sudden threat like the first phase of the COVID-19 pandemic posed challenges to the nursing home community as a whole, involving residents, relatives, social and health care workers, and management alike. Lessons learned as discussed in this study on preparedness and perceived safety should result in a more resilient nursing home sector in Portugal for the challenges still to come.


Frequency-based measures of heart rate variability have been shown to be a useful physiological marker in both clinical and research settings providing insight into the functioning of the autonomic nervous system. Ongoing interactions between the autonomic nervous system control of the heart and lung occurs during each ventilation cycle because of their anatomical position within the closed thoracic cavity. Mechanical ventilation and subsequent removal change the normal ventilator mechanics producing alterations in the tidal volume, intrathoracic pressure, and oxygen delivery. A noninvasive method called heart rate variability (HRV) can be used to evaluate this interaction during ventilation and can be quantified by applying frequency-based measures of the variability between heartbeats. Although HRV is a reliable method to measure alteration of the autonomic nervous system (ANS) function and cardiopulmonary interaction, there have been limited reports concerning the changes in the frequency-based measure of HRV during both spontaneous and mechanical ventilation. The purpose of this methodological study is therefore to describe the physiological influence of both spontaneous and mechanical ventilation on frequency-based measures of HRV. Heart rate variability (HRV) is a noninvasive method to evaluate the alteration of autonomic nervous system (ANS) function and cardiopulmonary interaction . HRV anaHRV analysis is used as a measure of cardiac ANS regulation . HRV canThe benefit of frequency-based method or spectral analysis of HRV is that it can identify different arrhythmias during different types of ventilation  on adults, children, and high-risk infants . MoreoveRSA is a marker for ANS control  and contributes to improved gas exchange during mechanical ventilation (MV) , 10, 11.The influence of MV on HRV has been studied in nonhuman subjects such as canine models and human subjects, including newborn babies, children with brain death, and healthy young adults placed on sedation and paralysis –16. The It is important to know that breathing is a form of exercise, and normal respiratory frequency is the main factor maintaining the balance of the inspiration/expiration ratio (I : E ratio) . FurtherDespite the recognition that different types of MV are associated with alteration in the ANS, there have been limited reports concerning the changes in the frequency-based measure of HRV, which is a reliable method to detect ANS activity, particularly during MV . TherefoThe purpose of this research study is to describe the physiological influence of spontaneous and MV on frequency-based measures of HRV. In this study, the difference between spontaneous respiration and MV was described. Additionally, a deep physiological review of autonomic tone, cardiovascular effects during spontaneous respiration, and HRV changes during spontaneous respiration, MV, and weaning from MV was described and illustrated by using figures.Both spontaneous and positive-pressure MV (which is currently the most commonly used MV in ICUs) induce alterations in ITP and lung volume above an end-expiratory baseline . These aSeveral hemodynamic effects of all forms of MV are similar despite variances in the mode of MV , 22. ITP2 requirements of cells and organs while excreting the CO2 byproduct of the metabolism [2 delivery to the body along with expected tissue ischemia, progressive organ dysfunction, and if untreated, death. Therefore, reestablishment and maintenance of controlled cardiopulmonary function is an essential goal in the management of critically ill patients in ICUs [The heart and lungs are closely coupled by their anatomical locations within the thorax and their responsibility to deliver the Otabolism . In crit in ICUs . Critica in ICUs . Ventila in ICUs , 28, 29.Autonomic tone neural processes probably play a primary role in all the long-term effects of ventilation on the cardiovascular system. Most of the immediate effects of MV on the cardiac function are secondary to changes in autonomic tone . The lunDuring spontaneous respiration, negative ITP induces a decrease in right atrial pressure (RAP) and an increase in right ventricular (RV) transmural pressure  . The decAs a result, LV stroke volume decrease, and a fall (<10 mmHg) in systolic blood pressure during inspiration is observed. The decrease in systolic blood pressure stimulates the baroreceptor reflex, which consequently increases sympathetic outflow to the heart, and a slight increase in HR occurs .Unlike spontaneous respiration, positive-pressure MV results in an increase in ITP during inspiration and throughout the respiratory cycle if positive end-expiratory pressure (PEEP) or CPAP is used . During The result of the increases of these neurohormones includes tachycardia, vasoconstriction, oliguria, retention of sodium and water, and subsequently increases in blood pressure. An increase in HR occurs as an initial response to lower cardiac outcomes. This can be seen in the beat-to-beat variations in HR that compensate for cyclical differences in RV and LV outputs , 37. In Transition from MV to spontaneous ventilation during the weaning process also produces hemodynamic alterations and ANS dysfunction (reflected by decreased HRV), especially in patients with underlying cardiovascular dysfunction . ANS altDuring weaning, ITP decreases, and the degree of ITP reduction depends on the mode of MV used. The decrease in ITP may result in rapid increases in stoke volume  . AdditioHRV is the analysis of beat-to-beat variation in HR, which results, in large part, from ongoing changes in sympathetic and parasympathetic inputs to the sinoatrial node. It is related to the influences of the ANS during invasive and noninvasive MV . The ANSFrequency-based measure analysis, or spectral analysis of HRV, is a technique that provides discrete measures of the vagal as well as the sympathetic and parasympathetic modulation of the heart . FrequenThere are some factors that possibly contribute to most of the measurement of frequency-based measures of HRV: the basal firing rate of nucleus ambiguous motor neurons in the brain , respiratory patterns, and central respiratory drive . NucleusI/E ratio (1 : 2 in rest or sleep state and 1 : 1 in exertion), there will be hemodynamic equilibrium and ANS control. When there is a normal respiratory pattern, the frequency-based measures of HRV are less affected and result in the higher parasympathetic nervous system (HF) and lower LF and LF/HF ratio [By reviewing the cardiovascular effect of spontaneous respiration, frequency-based measures of the HRV effect during spontaneous breathing can be evaluated. During spontaneous inspiration, there is a decrease in ITP and an increase in HR and stroke volume. While during spontaneous expiration, there is an increase in ITP and a decrease in HR and stroke volume. During normal HF ratio , 57 , decreasing LF (sympathetic nervous system), and decreasing LF/HF.This review described the physiological influence of spontaneous and MV on HRV (particularly frequency-based measure). Despite the recognition that different types of MV are associated with alteration in ANS, there have been limited reports concerning the changes in the frequency-based measure of HRV , which is a reliable method to detect ANS activity, particularly during the MV and weaning process . MV is cTransition from MV to spontaneous ventilation (the weaning process) produces hemodynamic alterations and ANS dysfunction (reflected by decreased HRV), especially in patients with underlying cardiovascular dysfunction. This will lead to a great change in HRV (RSA or HF) and may cause cardiac dysrhythmias. Understanding the physiological changes of frequency-based measure of heart rate variability, specific physiological changes of frequency-based measure of heart rate variability have been reviewed primarily during spontaneous respiration, mechanical ventilation, and weaning from mechanical ventilation based on the physiological analysis or review of the cardiovascular effect of spontaneous respiration, mechanical ventilation, and weaning process.Of great scientific interest is the effect of spontaneous respiration, mechanical ventilation, and weaning from mechanical ventilation on frequency-based measure  of HRV by using data-based analysis for critically ill patients. In conclusion, ventilation is a ubiquitous phenomenon and its effects on HRV are a mandatory result. By understanding the effect of different types of ventilation  on the cardiovascular system, the simple determinants of lung-heart interactions will be accurate, provide the most efficient treatment to patients in critical setting, and consider respiratory support as a confounding variable that might affect the high-frequency measure of HRV in respiratory distress patients with MV and during the weaning process.


Ovarian cancer (OC) is one of the most frequent malignancies of the female genital tract, and is still the leading cause of death from gynecological tumors . AlthougOne of the most significant findings regarding high-grade serous ovarian cancers (HGSOC), being one of the most frequent and aggressive OC histotypes , is thatHGSOCs were also among tissue and cellular OC models in the study of Batool et al., focused on the biological role of the CD83 molecule. The role of this glycoprotein, which exists in both membrane-bound and soluble forms and controls the reactivity of numerous immune cells , has nevLAG3, HAVCR2, TIGIT, and CTLA4, could be taken into account as promising targets. Immunologic status of a tumor microenvironment is critical for both OC progression and its therapy . AccordiThe importance of immune cells, specifically neutrophils, in the OC microenvironment was also tested by Muqaku and colleagues . The fouAnother element of the tumor microenvironment, apart from immune cells, are cancer stem cells. Importantly, they are usually therapy-resistant and responsible, at least to some extent, for OC relapse. In their study, Terraneo et al. show that the L1 cell adhesion molecule (L1CAM) is a new OC stem cell marker, responsible for tumor insensitivity to radiation and the high tumor-initiating capacity of the stem cells. These observations direct our attention to a specific population of OC-associated cells, namely L1CAM+/CD133+ cells, which may determine some tumor-promoting phenotypes and represent a target for novel therapeutic approaches in OC patients .Some summary of the threads related to the importance of the tumor microenvironment in OC development is the review paper by Hassan et al., in which the role of selectins as mediators of OC cell adhesion to normal peritoneal cells is widely presented and discussed .As per potential therapeutic solutions, van den Brand and colleagues show that epithelial cell adhesion molecule (EpCAM)-directed designed ankyrin repeat proteins (DARPins) combined with the photosensitizer IRDye 700DX may be attractive targets for photodynamic therapy in OC patients. Notably, employment of this modality resulted in a significant reduction of vitality within OC cell lines , 3D tumor spheroids, and mice xenografts. The conjugates also expressed specific binding in primary patient samples . BrückneTaken together, in this Special Issue, we were able to gather an exciting mix of articles dealing with either already opened or completely new avenues in OC research. As co-editor of this Special Issue, I am convinced that the publications contained herein will be of great interest to basic scientists and clinicians, translating into further development of the research threads described above. Personally, I note with great satisfaction the apparent increase in the proportion of research conducted using primary material from OC patients instead of established, permanent cell lines. This desirable trend should translate into a greater value of findings, from those obtained in the laboratory under in vitro conditions to the complex and individual-specific situation observed in human organisms in vivo.