Chronic kidney disease (CKD) affects over 10% of the global population and poses significant challenges for societies and health care systems worldwide. To illustrate these challenges and inform cost-effectiveness analyses, we undertook a comprehensive systematic scoping review that explored costs, health-related quality of life (HRQoL) and life expectancy (LE) amongst individuals with CKD. Costs were examined from a health system and societal perspective, and HRQoL was assessed from a societal and patient perspective. Papers published in English from 2015 onward found through a systematic search strategy formed the basis of the review. All costs were adjusted for inflation and expressed in US$ after correcting for purchasing power parity. From the health system perspective, progression from CKD stages 1–2 to CKD stages 3a-3b was associated with a 1.1–1.7 fold increase in per patient mean annual health care cost. The progression from CKD stage 3 to CKD stages 4–5 was associated with a 1.3–4.2 fold increase in costs, with the highest costs associated with end-stage renal disease at $20,110 to $100,593 per patient. Mean EuroQol-5D index scores ranged from 0.80 to 0.86 for CKD stages 1–3, and decreased to 0.73–0.79 for CKD stages 4–5. For treatment with renal replacement therapy, transplant recipients incurred lower costs and demonstrated higher HRQoL scores with longer LE compared to dialysis patients. The study has provided a comprehensive updated overview of the burden associated with different CKD stages and renal replacement therapy modalities across developed countries. These data will be useful for the assessment of new renal services/therapies in terms of cost-effectiveness. These studies used different HRQoL assessment tools, which comprised the EuroQol-5D (EQ-5D) [eight studies], the EuroQol visual analogue scale (EQ-VAS) [three studies], the Kidney Disease Quality of Life Short Form , which can be converted into a single index score reflective of societal preferences . The EQ-Similarly, CKD stages 4–5 were associated with lower VAS scores compared to CKD stages 1–3 . For RRTThe KDQOL-SF is a self-report measure consisting of generic and kidney-specific parts . The genvs 0.52± SD: 0.11, respectively).Patients with CKD stages 4–5 had the lowest mean PCS and MCS at 34.8–49.2 and 50.3–60.1 respectively ,61–67 S. For RRTvs 0.52 ± 0.11, respectively). Regarding place of dialysis delivery, Nagaraja et al. [vs 0.52 ± 0.11, respectively).Peritoneal dialysis was associated with better scores than HD for both generic and kidney specific domains ,85. Nagaa et al.  reportedvs 0.61 ± 0.13, respectively).Finally, Shah et al. ,87 indicvs 0.53 ± 0.38, respectively).The HUI system is a generic, preference-based measure used to produce utility scores providing a summary index of HRQoL on a (0.0–1.0) scale . A crossIn total, seven studies addressing LE and survival among CKD patients were included.2) to 2.0 years at an eGFR < 6 ml/min/1.73 m2.Bowling et al.  conducteSteenkamp et al.  analyzedTwo reports by the US Renal Data System (USRDS) ,94 compaFinally, a cohort study by Verberne et al.  comparedAll nephrologists concurred with the review findings in relation to the increase in costs and decrease in HRQoL as CKD stage advanced. The nephrologists suggested that costs at different stages could vary considerably depending on what is bundled into the episode of care , aetiology of CKD and associated comorbidities. These factors may help explain the heterogeneity with respect to costs found in this study. The nephrologists agreed that societal costs might be inaccurately estimated and poorly captured in the current reports. Examples of societal costs include productivity losses  amongst CKD patients, donors and/or carers due to either absenteeism (work absences) or presenteeism (reduced productivity among those with CKD in work). Societal costs also include transportation costs, and avoidable co-morbidity such as depression occasioned while on HD and awaiting transplantation.The nephrologists attributed the higher costs in the US, relative to Europe, to systems that carefully itemize more costs. In the US, in contrast to Europe, there may be more comprehensive attribution of health care expenditure resulting in higher unit costs (i.e. prices for drugs and procedures), higher rates of comorbidities in CKD/ESRD patients (those presenting may have been sicker), and a lower threshold for investigation in some clinical settings . They suggested that HRQoL data should be interpreted cautiously due to potential confounders .Concerning different RRT modalities, all nephrologists confirmed that KTx is accepted to be consistently associated with lower costs, better HRQoL and longer survival compared to dialysis. They also supported the review findings regarding the substantial decline in costs in subsequent years following the first year after KTx, and attributed that to reduced burden of procedures, hospitalizations and outpatient appointments following stabilization of post-transplant follow-up and to the availability of relatively cheap generic immunosuppressive drugs. The nephrologists suggested that HRQoL and LE data for KTx might be subject to a degree of selection bias, as healthier patients are more likely to be transplanted.All nephrologists acknowledged that the review findings would be useful for commissioners considering the cost-effectiveness of new therapies. They also advised that the study would inform future research, resource allocation, training of health personnel, preventative health care, and opportunities for reducing costs .This scoping review has explored the reported burden associated with CKD in order to inform provision of convenient renal services and economic evaluations of innovative therapies. While broadly consistent with the assumptions and experiences of practicing nephrologists, the findings of this review provide a deeper quantitative understanding of the influence of CKD on costs, HRQoL and LE in developed countries during the last five years.The results showed great variations in costs across different CKD stages, where costs substantially increased with each advance in CKD stage. Most studies (86%) estimated total health care costs among CKD patients. From the health system perspective, the progression from CKD stages 1–2 to CKD stages 3a-3b was associated with a 1.1–1.7-fold increase in per patient mean annual costs, with CKD stages 1–2 demonstrating the lowest costs , with Europe and the US at the lower and the higher limits of the range, respectively. The transition from CKD stage 3 to CKD stages 4–5 was associated with a 1.3–4.2-fold rise in costs, with ESRD showing the highest costs ranging from $20,110 to $100,593, with Europe and the US at the lower and the higher limits of the range, respectively. Although health care costs of CKD show some variation, they are consistent in the sense that the US and Europe have the highest and lowest costs for all stages, respectively. The substantial variation between Europe and the US may be linked to differences in health care accountancy, difference in clinical practices  and/or differences in the cost of care between countries . While cThe review produced limited data addressing costs from a societal perspective in Europe and none in the US. These costs were also highest amongst patients with CKD stages 4–5 . Productivity loss was highest in the Nordic countries, accounting for 2/3 of the total societal costs among patients with CKD stages 4–5 . DespiteDifferent HRQoL assessment tools showed variations in HRQoL scores across CKD stages, where scores showed a notable decrease with each progressive CKD stage. The transition from CKD stages 1–3 to CKD stages 4–5 was associated with a decrease of 8–11% in mean EQ-5D index scores, with CKD stages 4–5 showing the lowest scores ranging from 0.74 to 0.79. A systematic literature review of 21 studies from developed countries showed that mean EQ-5D scores among patients with type 2 diabetes (without complications) were in the range of 0.71–0.94 . A systeThis study produced limited LE data among CKD patients, where available this suggested that LE substantially decreased with each age group and with worse kidney function. Similar findings have also been reported by Turin et al. in a large abridged life table analysis study in Canada . The lowOur study explored costs and outcomes associated with different CKD treatment modalities. From a health system perspective, the costs of HD were 1.1–1.8-fold those of PD. This has also previously been reported in a cohort study by Berger et al. . RegardiThis review adopted a comprehensive systematic search strategy for scoping literature to address its objectives. A broad range of study designs/methodologies from both recent peer- reviewed and non-peer reviewed literature were included. Furthermore, costs in this study were adjusted for inflation and purchasing power parity to improve comparability. This study also considered a consultation exercise with nephrologists. The review nevertheless still had some limitations. The authors only assessed and included English language articles, which posed a possibility of missing potential papers from continental Europe. The quality of the included studies was not assessed in order to include an extensive range of evidence from both peer-reviewed and grey literature and provide a better understanding of the burden associated with CKD. This review included only studies from North America, Europe, and Australia, which may not make the study findings transferable to developing countries, where the delivery of health care services is different and RRT is not available for many ESRD patients. Since, most studies examined total healthcare costs, irrespective of whether it is related or not related to CKD, the itemisation of all services and costs was infeasible.Costs and outcomes associated with CKD varied considerably across different stages. From a health system perspective, the transition from CKD stage 3 to CKD stages 4–5 was associated with a 1.3–4.2-fold increase in costs, with ESRD showing the highest costs . The progression from CKD stages 1–3 to CKD stages 4–5 was associated with a decline of 8–11% in mean EQ-5D index scores, with CKD stages 4–5 demonstrating the lowest scores (0.74–0.79). Recent studies of LE associated with different CKD stages in different countries have been limited in number and scope. Amongst treatment modalities, KTx showed the lowest costs , highest EQ-5D scores (0.82–0.83), and longest LE among both males and females across all age groups.In addition to identifying gaps in knowledge, the current study contributes to a better understanding of the economic costs likely to attend increases in CKD prevalence, informing health care planning and resourcing in developed countries. This study can also inform the development of cost-effectiveness models of novel therapies that prevent or slow the progression of CKD. Based on the study findings, the following areas are recommended for future research;Investigation of CKD aetiology-specific costs and HRQoL as they could substantially vary depending on aetiology of CKD.A more comprehensive and consistent estimation of societal costs associated with different CKD stages and RRT modalities with particular emphasis on productivity losses and costs related to induced conditions such as depression.A careful itemization of healthcare costs aspects and examining whether they are paid for by a third part or out of pocket.Conducting abridged life table analyses for estimation of LE across different CKD stages in different countries.S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.S3 Appendix(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


To estimate the economic burden of cholera in Africa.Cholera affected 44 countries in Africa.The analysis used data from public sources in Africa published until September 2019.Based on existing data from field-based cost-of-illness studies, estimated cholera incidence rates, and reported cholera cases to WHO, this research estimates the economic burden of cholera in Africa from a societal perspective with 2015 as the base year. The estimate included out-of-pocket costs, public health system costs, productivity loss related to illness and an optional productivity loss related to premature deaths valued by the human capital approach. As various input data such as cholera incidence, hospitalisation rates and the number of workdays lost were not well defined, a series of scenario analyses and uncertainty analyses, accounting for unknowns and data variability, was conducted. Similarly, the value of time lost due to illness and deaths using the human capital approach was explored through scenario analyses.In 2015, an estimated 1 008 642 cases in 44 African countries resulted in an economic burden of US$130 million from cholera-related illness and its treatment. When the estimated 38 104 cholera deaths were included in the analysis, the economic burden increased to US$1 billion or international $2.4 billion for the same year. At the same time, when only the 71 126 cases and 937 deaths reported to the WHO are considered, the economic burden was only US$68 million for the year 2015. The estimates of economic burden are thus heavily dependent on the cholera incidence rate, how time lost due to illness and deaths are calculated, hospitalisation rates and hospitalisation costs.The findings can be used as an economic justification for cholera control in Africa and for generating value-for-money evidence to underpin Ending Cholera—A Global Roadmap to 2030 with considerations to study limitations. Model-based estimation of the economic burden of cholera in 44 African countries was conducted using various secondary data sources.The average inpatient health system costs, out-of-pocket expenses and the number of days of productivity lost related to cholera treatment from five African papers that presented data was directly applied to countries that did not have data.The estimated number of cholera cases was derived from the global burden of disease study and WHO report.The productivity loss related to cholera illness and premature deaths were valued by the human capital approach using gross domestic product per capita.Cholera incidence rates, cholera hospitalisation rates and cholera hospitalisation costs are the most uncertain parameters that drove the results.Vibrio cholerae is linked to poor water, sanitation and hygiene (WASH) conditions.3Cholera, an acute, diarrhoeal illness caused by infection of the intestine with the toxigenic bacterium When measured as an economic burden, cholera has a significant economic impact on health systems and individuals. The societal cost per cholera case is believed to be more than US$1000 when the wider socioeconomic costs such as cost to the health system, the patient’s family and income loss due to death, are taken into account.A recent economic burden estimate in Asia used the actual estimated cost of illness collected from countries to project the economic burden while using a variety of scenario and sensitivity analyses to demonstrate the uncertainties around the estimated and reported cholera cases to WHO.The research involved the selection of countries, identification of cost-of-illness studies to define out-of-pocket (OOP) expenses, productivity loss and health system costs to estimate and extrapolate the economic burden, and finally, conduct sensitivity analysis and scenario analyses. Patients or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.The primary input needed for estimating the economic burden is the cost-of-illness studies conducted around cholera cases at health facilities in African countries. Such studies provide three sets of key information.The cost-of-illness data are typically not available from most African countries. In such situations, cost-of-illness was extrapolated to African countries using available data from field studies in the region  and WHO estimates (outpatient costs) such that each country has a unit cost of illness (cost of illness per cholera case). When the total number of cholera cases is multiplied by the unit cost-of-illness, the economic burden for that country is derived which is summated at the regional level. However, all the input costs and the number of cases are not constant numbers. There may be several biases and reported variations such as under-reporting or overestimations because of which we conduct a series of scenario analyses and uncertainty analyses as described below so that the limitations and boundaries are well understood.African countries as defined by the United Nations,Base case: The Global Burden of Cholera study2Liberal case: The incidence rate from Beira, Mozambique was extrapolated to the rest of Africa after applying an incidence correction assumption by African WHO mortality strata D (AFR-D).2Conservative case: As other sensitivity analyses used in the 2015 global burden of disease estimate produced a similar number of cholera cases in Africa, a conservative estimate was derived by applying half of the incidence rate as the base case.WHO report case: A separate scenario analysis was conducted based on the number of cholera cases and deaths reported to WHO in 2015.The 2015 global burden of disease studyAs described previously in the conceptual framework, there are three types of costs: OOP costs, productivity loss and health system costs. These costs were extracted from literature based on actual studies conducted in healthcare settings. A previous systematic literature reviewThe OOP costs for hospitalised cases were reported for all five papers used in the study, while only Malawi reported the OOP costs for outpatient cases. There are two types of OOP costs, direct medical and direct non-medical costs. Direct medical costs include expenditure accrued by patients for diagnosis, medicines and other costs directly related to the treatment of cholera. Direct non-medical costs include expenditures on travel to the healthcare facility, room and boarding, food, and other costs not directly related to treatment. All the costs reported in the different years were converted to 2015 US dollars by adjusting them based on annual percent changes in world consumer prices as reported by the International Monetary Fund (IMF).Loss of productivity due to illness was estimated based on the average number of lost workdays for patients and caregivers throughout the illness and the recuperation period documented separately for patients and caregivers. Lost workdays for Ghana, Malawi and Mozambique were extracted directly from their respective sources.Service delivery costs were also extracted from the papers for each hospitalised and outpatient cases. Health facility costs for service delivery included personnel, medicines, diagnostic tools, medical equipment, infrastructure, beds and utilities that patients are not responsible for paying. Of the five publications used in this study, Malawi, Tanzania and Mozambique reported health service delivery costs for hospitalised cases. For countries without reported costs, the mean hospitalisation health facility costs for these three African countries were applied. Only Malawi reported outpatient service delivery costs. The country-specific average outpatient costs for health centres with and without beds from the WHO-CHOICETo estimate the productivity loss due to premature death from cholera, the mean age of cholera death was derived from a multicountry cholera surveillance project that reported 873 deaths in six African surveillance zones.Only the study conducted in Malawi reported a hospitalisation rate, at 90% hospitalised and 10% receiving care for less than 12 hours, which is analogous to outpatient care. Other studies included in the analysis were unclear whether all cases were hospitalised, or only hospitalised cases were accounted for. Since 90%–100% hospitalisation is considered high, the analysis was conducted based on three scenarios: (1) under the assumption of 90% hospitalisation, (2) average of 75% hospitalisation and (3) average of 55% hospitalisation. Assumption (2) was determined based on a previous investment case studyA probabilistic multivariate sensitivity analysis was conducted using Ersatz.Among all input parameters, disease burden (the number of cases) and hospitalisation rates had limited information that could be captured in one single number with uncertainty ranges. Therefore, scenario analyses were conducted for both disease burden and hospitalisation rates. Disease burden scenarios included three estimates as explained earlier and is based on a previous publicationAll the costs were converted to their equivalent value in 2015 US dollars (US$). For the conversion, the annual percent changes in world consumer prices as established by the IMF were applied.18The previous cholera economic burden study estimated the costs in 2002 prices for WHO reported cholera cases in 2005, 2006 and 2007.610.1136/bmjopen-2020-044615.supp1Supplementary dataBased on a literature review, average workday loss of 5.76 (range 2.96–9.60) and 3.86 (range 3.09–4.64) was estimated for cholera cases and caregivers, respectively, irrespective of their hospitalisation status . The OOPIn the base-case analysis, 1 008 642 cases of all ages were included from 44 African countries, while 71 176 cholera cases from 16 countries were reported to WHO . The numThe scenario analyses showed that when assumptions on the number of cholera cases changed, the economic burden excluding death ranged from US$64.6 million to US$227.2 million. The estimated/reported number of cases influences the economic burden considerably and drops to US$8.6 million when WHO reported cholera cases are considered in the analysis .The scenario analysis that varied hospitalisation rates had a relatively small impact on the overall economic burden. When workdays lost are valued at 75%, 50% and 25% of GDP per capita, a large reduction of economic burden was observed .Probabilistic multivariate sensitivity analysis showed OOP costs during hospitalisation, service delivery costs at hospitalisation, and the number of workdays lost among cholera patients and caregivers, have the largest impact on the results .The total economic burden of cholera in Africa was estimated at US$1 billion (I$2.5 billion) which was around US$1.1 per capita for the year 2015, of which only US$0.06 is borne by the public health system. Lost productivity due to premature death due to cholera was estimated to be US$900 (I$2.2 billion) for the year 2015. Productivity loss due to premature death accounted for 87.7% of the total economic burden of cholera in Africa. When costs linked to productivity loss are excluded, 45.6% of the costs were borne by the health system, with the rest borne by affected families including 23.6% as OOP costs and 30.8% as productivity loss due to inability to work. Probabilistic multivariate sensitivity analysis demonstrated that the costs around hospitalisation and loss of workdays are driving the overall costs, which the scenario analysis showed that the economic burden is heavily dependent on how we value loss of workdays.The Global Task Force for Cholera Control has proposed an ambitious plan to reduce 90% of cholera cases by 2030 as a part of the Ending Cholera—a Global Roadmap to 2030.A previous study on the economic burden of cholera in Africa showed costs of US$39 million to US$156 million, depending on the assumptions on life expectancy  and year of reported cholera cases .The economic burden of cholera estimation study in AsiaThis estimate of the economic burden of cholera used the latest available field-based information in its analysis, but not without limitations.First, the incidence data on cholera were estimated previouslySecond, the entire cost-of-illness field data inputs came from five African papers, four of which had only costs for hospitalised cases which means that the cost-of-illness data are heavily dependent on either extrapolation or assumptions. It is known that in many African countries cholera treatments are provided through specific centres known as ‘cholera treatment centres’ often set up adjacent to existing health facilities, close to cholera-affected communities. Most severe cases are hospitalised in these centres, while others are kept under observation along with the provision of rehydration and treatment. This situation creates a challenge in differentiating the typical categorisation of cholera cases as hospitalised and outpatient cases, with most studies resulting in reporting everyone as hospitalised. The health facility costs of such specialised treatment centres could be different from typical facility-based service provision. In the absence of outpatient data, we have used WHO-CHOICE data that may not represent typical cholera treatment situations. Because the base-case assumes 90% of cholera cases are hospitalised, this bias is minimised. In addition, a scenario analyses with a range of hospitalisation rates was presented.Third, OOP costs and the number of workdays lost are collected from patient and family surveys which are subject to many biases. The OOP costs are different in different countries, and range depending on the patient’s location, type of health facility, travel time and distance, and the healthcare system. It is also subject to the limitations of surveys such as how and when the data was collected. This data was extrapolated to all African countries from five study sites. The extrapolation methods may have resulted in underestimation or overestimation of OOP costs, the direction of which is difficult to ascertain. Extensive sensitivity analyses are conducted to understand these limitations.Fourth, the overall economic burden is heavily dependent on how the lost workdays and years lost due to death are valued. Cholera is known to affect economically marginalised people more often, and a GDP per capita estimation of valuing their lost time could be an overestimation. Also, some of the people may not be in the workforce, may not earn in the future, but still, their time is valued. If an ill worker is replaced, it may affect family income but may not affect the overall economy. Thus, the potential income loss presented in this analysis need not represent the true income loss in African countries. To understand these limitations a scenario analysis where lost workdays and death years are valued at 75%, 50% and 25% of GDP per capita.Lastly, cholera is known to affect the tourism and export industries which are not accounted for here, resulting in an underestimation of the economic burden. Similarly, the costs of outbreak control are not included here. Also, health facility costs do not include diagnostic costs.0, with additional information such as vaccine delivery costsThis study estimates the economic burden of cholera in 44 African countries, which can be used as evidence for making the case for cholera control measures such as investments in improving WASH, a better surveillance system, the use of oral cholera vaccination, and other measures described in the Ending Cholera—a Global Roadmap to 2030.


These substitutions were hypothesized to influence the pH optimum of fusion. The pH optimum of fusion activity was measured by a luciferase assay and biological properties of viruses were monitored. The in vitro and in vivo replication ability and pathogenicity of mutants were comparable (64) or lower  than those of the wild-type virus. However, the HA2 mutation V662H and double mutation T642H, V662H shifted the fusion pH maximum to lower values (ranging from 5.1 to 5.3) compared to pH from 5.4 to 5.6 for the wild-type and 64 mutant. The decreased replication ability and pathogenicity of 66 and D mutants was accompanied by higher titres in late intervals post-infection in lungs, and viral RNA in brains compared to wild-type virus-infected mice. These results have implications for understanding the pathogenicity of influenza viruses.Influenza A viruses (IAVs) enter into cells by receptor-dependent endocytosis. Subsequently, conformational changes of haemagglutinin are triggered by low environmental pH and the N terminus of HA2 glycoprotein (gp) is inserted into the endosomal membrane, resulting in fusion pore formation and genomic vRNA release into the cytoplasm. However, the pH optimum of membrane fusion is host- and virus-specific and can have an impact on virus pathogenicity. We prepared mutants of neurotropic IAV A/WSN/33 (H1N1) with aa substitutions in HA2 gp at the site of HA1/HA2 interaction, namely T64 Influenza A viruses (IAVs) are common respiratory pathogens, which under certain circumstances cause a serious disease with a fatal outcome. The broad host range and high variability of IAVs are linked to influenza epidemics and pandemics. To better understand the virulence potential of new influenza viruses in humans, the factors influencing the virulence of IAVs need to be studied . The surThe fusion process mediated by IAV is activated by the low pH environment in endosomes, where HA undergoes large irreversible structural changes . The pH http://www.rcsb.org) we designed and introduced amino acid substitutions at the interface of HA2 gp and HA1 gp of A/WSN/33 (H1N1) virus. By reverse genetics were prepared three viruses with mutations in HA2 gp: T642H , V662H  and a double mutant with both amino acid substitutions in HA2 (T642H and V662H). Molecular interactions at the HA1 and HA2 interface determine the pH required for fusion activity of IAV, and thus amino acid substitutions observed or introduced in this region may have further implications for virus virulence and pathogenicity. We examined the impact of the introduced mutations on the pH stability of mutant viruses and their replication ability in vitro and in vivo as well as their pathogenicity in a mouse model.The fusion of viral and cell membranes is essential for the infectivity of IAV and its replication in the host . Underst2H substitution in the HA2 subunit; H1 numbering – position 407 in HA); 66  and D (T642H and V662H substitutions in the HA2 subunit). Viruses were propagated in the allantoic fluid of 9-day-old fertilized chicken eggs.Recombinant wild-type (wt) and mutant influenza A/WSN/33 (H1N1) viruses were generated by reverse genetics using the pHW2000 eight-plasmid system . Mutatio−1 gentamicin (Sandoz) and 2 mM glutamine (Lonza). BSRT-7/5 (BHK-21 – Baby Hamster Kidney cell line expressing T7 RNA polymerase) cells were propagated in the same medium supplemented with geneticin (1 mg ml−1) instead of gentamicin.Madin-Darby Canine Kidney (MDCK) cells, Madin-Darby Bovine Kidney (MDBK) epithelial cells, adenocarcinoma alveolar basal epithelial cells A549, human embryonic kidney 293T (HEK 293T) cells, and VERO African green monkey kidney cells were propagated in Dulbecco’s modified Eagle’s medium  supplemented with 10 % FBS (Lonza), 40 μg mlThe wild-type A/WSN/33 (H1N1) virus, as well as its HA2 mutants, were prepared by transfection of HEK 293T cells with eight reverse genetics plasmids bearing the genetic information corresponding to individual gene segments encoding all IAV proteins using Lipofectamine 2000 (Invitrogen) as the transfection reagent . Transfeet al.  was added. Plates were incubated at 37 °C in a humidified atmosphere containing 5 % CO2. After 72 h, infected cell monolayers were fixed with 20 % trichloroacetic acid, overlay medium was removed, and cells were washed with PBS and subsequently permeabilized by fixation with cold methanol at 4 °C for 20 min. After this interval mAb specific for IAV nucleoprotein 107L [−1 in PBS containing 1 % bovine serum albumin was added. The bound mAb was detected with goat anti-mouse IgG conjugated with horseradish peroxidase  and the reaction was visualized by the addition of a substrate solution containing 3-amino-9-ethylcarbazole (Sigma) in 0.03 % H2O2. The visible immune-stained viral plaques were evaluated to determine infectious virus titre [The virus inactivation test at low pH was performed using the method described by Zaraket et al.  with modein 107L , at a cous titre . The expet al. [6 cells per well), constitutively expressing the bacteriophage T7 RNA polymerase and incubated for 1 h at 37 °C in DMEM without FBS. After this interval, medium was removed and infected cells were exposed to 0.1 M MES buffer (1 ml per well) adjusted to the appropriate pH ranging from pH 4.6 to 6 for 10 min at 37 °C. MES buffer was then replaced with the complete growth medium DMEM for 6 h to allow the IAV HA to mediate the fusion of infected VERO cells with target BSRT-7/5 cells, enabling the transfer of T7-luciferase plasmid into target cells and expression of firefly luciferase. Cells were lysed with 0.5 ml Reporter Lysis Buffer (Promega) and cell debris was removed by centrifugation at 15 000 g at 4 °C. Luciferase activity was measured in the supernatants using luciferase substrate oxyluciferin (Promega) and measured on a BioTek Synergy 2 Luminometer. Cell–cell fusion luciferase reporter assays were independently repeated at least three times in triplicate. Mock infected controls and no-transfection controls were done for boundary pH values (4.6 and 6.0) to determine the background signal. Data were normalized and analysed by non-linear regression fitting the bell-shaped curve using the least squares fit.A cell–cell fusion luciferase reporter assay was used to quantify the fusion activity of viruses at different pH. The assay was performed according to Galloway et al.  modifiedMultistep growth curves in MDBK, A549 and VERO cells were determined for each recombinant virus. Confluent monolayers in six-well plates in triplicate were infected with an m.o.i. of 0.001 of each virus for 1 h at 37 °C. After this interval monolayers were washed and incubated at 37 °C for 12, 24, 36, 48, 60 and 72 h in MEM with 0.5 % FBS. The infectious virus titres in the supernatant of infected cells at each time-point were measured by a plaque assay .50, groups of mice (n=5 per group) were intranasally (i.n.) infected with four different doses of examined virus diluted in PBS in a volume of 40 µl per mouse. Mice were anaesthetized with 2.5 % isoflurane in oxygen before virus inoculation. The survival of mice was monitored for 15 days. The infectious dose 1LD50 was determined by the method of Reed and Muench [Six-week-old female BALB/c mice provided by the Faculty of Medicine, Masaryk University, Brno, Czech Republic, were used. In all experiments presented in this study, the animals were treated according to the approval and standards of the European Union and State Veterinary and Food Administration of the Slovak Republic. The State Veterinary and Food Administration of the Slovak Republic specifically approved this study (permission number 1301/14–221). Fundamental ethical principles and animal welfare requirements were respected. For determination of 1LDd Muench .4 cells per well. After removing the culture medium, two-fold dilutions of virus sample in PBS or serum-free DMEM (starting from 50×) were applied onto the cell monolayer for 1 h at 37 °C. Medium with un-adsorbed virus was then removed and Ultra-MDCK medium with 0.5 % FBS was added. After 18 h of incubation under the same conditions, cell monolayers were washed, fixed with cold methanol and the virus was detected using monoclonal antibody (mAb) 107L specific to IAV nucleoprotein as described before [−1 in PBS with 5 % non-fat dry milk). After 1.5 h of incubation with HRP-conjugated goat anti-mouse IgG , a positive reaction was visualized by addition of substrate solution containing 3-amino 9-ethyl-carbazole with 0.03 % H2O2. Stained red coloured cells identified by light microscopy were considered as positive. The virus titre was expressed as the reciprocal value of the highest sample dilution positive for replicating virus in cell culture [To measure infectious virus in the lungs of infected mice, the lungs from three mice per group were excised at 2-day intervals. Lung homogenate suspensions in PBS  were prepared and cell debris was pelleted. The supernatants were examined for infectious virus as described . Brieflyd before   using Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher Scientific) according to the manufacturer’s instructions. To detect vRNA, a volume of 150 µl of supernatant from brain cell homogenates was used. Total RNA was extracted by the phenol–chloroform method and examined for vRNA . Total R2H, referred to as mutant 64) and at position 66 instead of valine . The third mutant we prepared contained both substitutions, i.e. T642H and V662H (this double mutant is referred to as ‘D’).To assess the influence of amino acids at critical positions of the HA trimer on the optimal pH value of IAV fusion, we prepared mutant viruses with amino acid substitutions at the interface between the globular and stem parts of the HA trimer. The positions for introduced mutations were selected on the basis of the known 3D structure of the HA trimer . The intAmino acid substitutions were introduced by site-directed mutagenesis into the HA gene of IAV of H1N1 subtype A/WSN/33 (H1N1) and mutant viruses were generated using reverse genetics. The presence of the HA mutations was confirmed in the rescued viruses after their propagation in cells and fertilized chicken eggs by Sanger sequencing of the HA gene. Haemagglutinin and neuraminidase (NA) sequences of the rescued recombinant viruses were confirmed to have no other mutations for three consecutive passages in embryonated chicken eggs (data not shown).in vitro and in vivo. To monitor viral and endosomal membrane fusion we used a modified luciferase fusion assay described by Galloway et al. [The pH optimum of fusion and biological properties of the IAV mutants were compared with the parental wt virus y et al. , which epH) for each virus (pH=6.2) than other tested mutants (IR50pH=6.00) and the wt virus (IR50pH=5.96).It is known that pre-exposure of virus to the critical low pH before infection causes irreversible conformational change of HA, which results in the loss of virus infectivity. The activation pH triggering such refolding of HA is characteristic to the particular virus strain. We evaluated the acid stability of the HA2 mutant viruses after their exposure to different pH, ranging from 4.8 to 6.8 (Δ 0.2) for 10 min at 37 °C. The infectivity of all viruses was determined by a modified plaque assay on MDCK cells . Comparech virus . We obse9 p.f.u. ml−1. Maximal viral titres of all viruses were approximately 2–4 logs lower in A549 cells and 2–3 logs lower in VERO cells. The differences were observed in the exponential phase of virus growth in each cell line, namely 66 and D viruses reached lower titres than the wt and 64 mutant. However, at later time-points, the titre of examined viruses in a given cell line reached a plateau, the height of which was dependent on the cell type: it was reached after 48 h in A549 cells, where the maximal titre of all viruses was lower (5.1–5.35 log10) than in the other two cell lines. The maximal titre in VERO cells after 60 h was about 1 log10 higher than in A549 cells. In MDBK cells, a plateau was reached after the 72 h for wt, 64 and 66 viruses (titre 8.5 log10), where the log10 of the maximal titre was 8.4–8.5 and was higher than that of the double mutant virus (D) . However, the in vitro replication profile of mutant 64 was close to that of the wt virus in all three cell lines (P<0.01) than wt virus.To determine the replication properties of the viruses with mutations in the HA2 gp, we measured the multicycle replication of recombinant viruses (m.o.i. 0.001) in MDBK, A549 and VERO cells . All recll lines . No sign50. Mice (n=5 per dose per virus) were infected with five different doses of a given virus and the course of the infection as well as survival of mice was monitored (results not shown). The in vivo titration of viruses showed that a similar infectious dose of mutant virus 64 (1.3×103 p.f.u. 40 µl–1 per mouse) and wt virus (1.4×103 p.f.u. 40 µl–1 per mouse) was required to achieve 50 % lethality for mice (1 LD50). The infectious dose required for the 50 % lethality of mice was 30 times higher for mutant 66, i.e. 4.0×104 p.f.u. 40 µl–1 and seven times higher for the double mutant, i.e. 1.0×104 p.f.u. 40 µl–1 in comparison to the wt virus. Thus mutant 66 and double mutant D were less virulent (pathogenic) than wt and 64 viruses.To compare the pathogenicity of each virus mutant with wt virus, we determined the amount of pfu causing lethal infection of mice, corresponding to 1 LDn=15 per virus) and the virus replication in mouse lungs . To normalize the impact of infection, we used an infectious dose of 1 LD50 of each virus. The virus titre in lungs of mice infected with 1 LD50 was determined by rapid culture assay (RCA). All viruses reached maximal replication on day 2 or 4 post-infection, with peak viral titres ranging from 13.31 (log2) for virus 64 to 14.56 (log2) for wt virus. Interestingly, mutant viruses 66 and D remained in mouse lungs longer, at late points post-infection (8 days p.i.), and reached higher titres than wt and 64  infected with wt and 64 viruses were comparable, being most severe on days 8–10 post-infection. The damage to lungs of mice infected with mutant virus 66 was not as severe and lungs were not oedematous, suggesting a milder course of infection caused by the 66 mutant. Although the lung damage of virus D-infected mice was milder than that of the wt and 64-infected mice, it was still higher than in 66-infected mice. Moreover, we were able to detect vRNA in lungs of mice infected with mutant D for a longer period of time, even on day 15 p.i. (two of three mice) (data not shown).Simultaneously we examined the lung damage in mice infected with a dose 1 LDnfection . PatholoInfluenza A/WSN/1933 (H1N1) virus is considered to be a neurotropic virus and therefore we tested the presence of the virus also in the brains of mice. The aim was to determine whether the neurotropism of this virus could be influenced by the introduction of described HA2 mutations. Although we did not detect the infectious virus in mouse brain in any of the experimental groups, the presence of vRNA was confirmed . We therThe replication efficiency, virulence, pathogenicity and transmissibility of influenza viruses depend on a number of viral and host factors . HA, the2H and V662H, was prepared.To determine the impact of changes in the fusion pH optimum on the biological properties of IAV, we prepared mutants with amino acid substitutions in the area of interaction between the HA1 and HA2 subunits. The basic amino acid histidine, the charge of which is extremely pH-sensitive under physiological conditions, was chosen in our study as the most suitable substituent to generate an HA trimer mutant with altered pH stability. Histidine was introduced into the HA2 gp of wt virus A/WSN/33(H1N1), namely at position 64 instead of threonine (mutant virus 64) and at position 66 (mutant virus 66) instead of valine. Moreover, a double HA2 mutant comprising both substitutions, T64in vitro as well as in vivo properties of A/WSN/33 (H1N1) influenza viruses. First, a luciferase method was used to monitor the pH optimum of viral and cell membrane fusion mediated by particular mutants. The maximum fusion activity of mutant 64 was at pH 5.4-5.6, which was comparable with that of the wt virus. Mutant viruses 66 and D had a lower pH maximum of fusion, estimated at pH 5.1-5.3. By monitoring IAV fusion activity in the range from neutral pH towards low pH, we showed that a pH of 6.0–5.8 was a threshold pH at which fusion activity became detectable. These results are consistent with published data [We examined how these substitutions influence the pH optimum of fusion and the hed data .in vitro analysis of replication of mutant viruses we chose three different cell lines – MDBK, A549 and VERO. It is generally known that MDBK cells promote effective virus replication, which we confirmed experimentally. In contrast, the lowest virus titres were obtained by propagation of viruses in human lung cell line A549. The in vitro replication profile of the mutant 64 was the most similar to the wt virus. In all cell lines, lower replication of 66 and D mutants in comparison to wt and 64 viruses was observed, but only in the A549 cell line were differences in growth rates of 66 and D mutants vs. wt and 64 viruses significant (P<0.01).For in vitro  is determined by many factors. One of them is the stability of virus infectivity after exposure to low pH. The influence of HA pH stability on virus infectivity was analysed, statistically evaluated and the 50 % reduction of virus infectivity was determined. This analysis showed that the D mutant was most sensitive to pH decrease, where 50 % of infectivity was observed between pH 6.2– and 6.0, while other viruses dropped to 50 % at lower values, between pH 6 and 5.8. The highest low pH sensitivity of the D mutant could be attributed to two histidine residues in this area, present at positions 64 and 66 of HA2 gp instead of theronin and valin. At lower pH (from 6 to 5) the infectivity of all viruses rapidly decreased. However, the low pH pre-exposure influences not only the conformation of HA. The impact of pre-exposure of virus to low pH is more complex and can have many consequences, as its impact on the NA or M2 ion channel activity, which in cooperation with HA and host factors also influences ribonucleoprotein release from the viral particle, resulting in the character of virus infectivity [The difference in the replication ability of virus mutants ectivity .in vitro replication activity, the in vivo replication of mutants 66 and D was reduced in comparison with the wt and mutant 64. A 30-fold higher amount of the 66 mutant virus and a seven-fold higher amount of the D mutant than of wt and 64 viruses was required to achieve the 1 LD50 infection dose, i.e. 50 % lethality in mice. In accordance, inspection of the lung damage of mice infected with 1 LD50 of the examined virus unambiguously pointed to a milder course of infection in mice infected with mutant virus 66 than with wt or 64 viruses. In mutant virus 66, however, we were able to detect vRNA in mouse lungs also at later points and, surprisingly, we detected RNA even on day 15 p.i. . Nevertheless, this mutant as well as mutant D, which showed lower replication activity in vitro and required several times higher dose of virus to achieve 50 % lethality in mice, reached the comparable virus titres in mouse lungs as did the wt and mutant virus 64. However, 66 and D mutants were eliminated from mouse lungs more slowly than the wt and 64 virus. Namely mutant D was detected in mouse lungs even on day 10 p.i.Similar to In vivo, the T64H substitution in mutant 64 caused greater lung damage in infected mice (70–90 %), which was comparable to the wt virus, while the V66H substitution confer to virus 66 a milder pathogenicity, resulting in 10–20 % lung damage. The increased manifestation of lung damage in D mutant-infected mice compared to 66 virus could be attributed to the second, T64H substitution introduced into the D mutant.50, the longer time for its elimination is needed. Similar results were described by Fislová et al. [We hypothesized that the higher amount of virus is needed for the lethal dose 1 LDá et al. , where tNervus olfactorius), as previously experimentally demonstrated in mice [Bulbus olfactorius) [As the influenza A/WSN/33 (H1N1) virus strain used in this study is considered to be neurotropic, we attempted to detect infectious virus not only in the lungs but also in the brains of infected mice. There are several theories explaining how the neurotropic IAV enters the brain. One possibility is that the virus enters the central nervous system via the olfactory nerve ( in mice . The olfctorius) . Here thctorius) . In our ctorius) . Howeverctorius) . On the ctorius) .The mutation introduced into HA2 gp had an impact on the pH optimum of fusion in the case of mutant virus 66 or D, where the broad pH maxima were shifted to lower values. There was recorded a difference in sensitivity of mutant 66 and D infectivity to low pH when compared to the wt. The changes are very subtle, so it is hard to say if this is in accordance with previous observations, namely that increased stability of IAV at low pH corresponds to decreased fusion pH, while a decreased HA stability corresponds to higher fusion pH . Howeverin vitro virus replication and virus pathogenicity were detectable also in the 64 mutant virus that showed no shift in the pH optimum of fusion, none of the introduced mutations resulted in increased virulence and the worsening of the course of infection in vivo. The differences in in vitro and in vivo replication of mutant viruses in comparison to the wt were accompanied also by other changes such as lung damage, the persistence of infectious virus in lungs and vRNA in brains of infected mice. After infection of mice with an equal lethal dose of virus, we observed that the more virulent was the virus used for infection, the earlier its titre in lungs decreased.However, it should be stressed that although changes in in vitro and in vivo characteristics as the wt, was present in brains of mice in a higher amount in comparison to wt-infected mice and therefore could be more neurotropic than the wt. Previous data show that fusion pH optimum of highly pathogenic avian IAV, in particular isolated from poultry, is higher than that of human IAV. This parameter, linked to the pH at which virus HA is stable, can influence IAV tropism, which plays an important role during interspecies transmission of IAVs and in IAV ecology [In summary, the constructed IAV mutants 66 and D had lowered virulence in comparison to the wt. Both of them (mutant 66 and D) had broad maximum of fusion activity shifted to lower pH in comparison to the pH of fusion maximum of wt or 64 mutant virus. Mutants 66 and D were eliminated from lungs of infected mice later than the wt. However, vRNA of mutant 64 with fusion optimum in the same pH range as the wt, and with similar  ecology .In general, HA activation pH values for IAV typically range from 4.8 to 6.2 . HoweverIt cannot be excluded that some other, undescribed HA2 fusion mutants could emerge in nature, namely of avian or swine IAV, with replication ability in a new mammalian/human host, but with a more virulent and more pathogenic phenotype, and thus representing a threat to humans .


To investigate the impact of biological subtypes in locoregional recurrence in Chinese breast cancer patients receiving postmastectomy radiotherapy (PMRT).About 583 patients who received postmastectomy radiation between 2010 and 2012 were retrospectively analyzed. According to immunohistochemical staining profile, patients were classified into: Luminal A‐like, Luminal B‐like, HER2‐positive, and triple‐negative breast cancer (TNBC). Local and regional recurrence (LRR) cumulative incidences were calculated by competing risks methodology and the power of prognostic factors was examined by Gray's test and the test of Fine and Gray.P < .05), TNBC , and pN3 . In the multivariate model, HER2‐positive subtype , TNBC , and pN3  were independent predictors of LRR. LRR without trastuzumab was similar to that of TNBC  in HER2‐positive subtype patients, while LRR with trastuzumab was approximate to Luminal A . Additionally, endocrine therapy also significantly reduced LRR incidence in the luminal subtype cohort .The median follow‐up was 70.9 months. About 34 LRR events occurred. For Luminal A, Luminal B, HER2‐positive, and TNBC patients, the 5‐year LRR cumulative incidence rates were 1.57%, 4.09%, 10.74%, and 10.28%. Compared with Luminal A, HER2‐positive subtype and TNBC had a significant increased risk of LRR . In univariate analysis, predictive factors for higher LRR were HER2‐positive subtype  Expert Panel serves as a reliable prognostic predictor for Chinese breast cancer patients receiving PMRT. Treatment with trastuzumab and endocrine therapy dramatically decreased the risk of LRR in HER2‐positive and hormonal receptor‐positive patients. Wright et al analyzed 582 consecutively treated patients receiving PMRT and demonstrated that biological subtype could predict the prognosis of different biological subtypes of breast cancer.Whether biological subtype can serve as a prognostic biomarker for patients who received PMRT has been seldom examined in a Chinese cohort. Therefore, we conducted the present analysis on the relationship between LRR and biological subtypes of breast cancer patients undergoing PMRT in Fudan University Shanghai Cancer Center in order to provide more insights into clinical decision.22.1From January 2010 to July 2012, 688 patients who treated with mastectomy and PMRT at the Department of Radiation Oncology, Fudan University Shanghai Cancer Center were retrospectively reviewed. Their medical records were consecutively collected. Review of data for this investigation was approved by the Institutional Review Board of our center. Before treatment, all patients were required to have a complete physical examination, routine blood test, biochemical test, mammography and/or breast ultrasonography, chest X‐ray, an abdominopelvic computed tomography scan and/or ultrasonography, and a bone scan if indicated. The 2018 eighth edition of the AJCC Cancer Staging Manual was used for restaging.The inclusion criteria were as follows2.2All patients received mastectomy and axillary dissection. Radiotherapy and/or chemotherapy was performed according to the National Comprehensive Cancer Network practice guideline. PMRT was compulsory for patients with T3‐T4 and/or N2‐N3 breast cancer, while elective for the T1‐T2N1 cohort. With a 6‐MV linear accelerator, radiotherapy was delivered to the ipsilateral chest wall, supraclavicular region of lymph nodes, and/or intramammary lymph nodes. The total dose was 50 Gy /25 fractions. Nine patients received an additional 10 Gy to the tumor bed.2.3ER, PR, and Ki‐67 were assessed by IHC staining. IHC staining of ER, PR, and Ki‐67, more than 1%, 20%, and 20%, respectively, was considered positive or high expression.Tumors were subtyped according to the St Gallen International Breast Cancer Conference (2013) Expert Panel as Luminal A‐like , Luminal B‐like , HER2‐positive (HER2 overexpressed or amplified‐ER and PR absent), and TNBC .2.4Follow‐up was determined from the date of diagnosis. Outpatient department records and regular telephone calls were conducted to perform follow‐up. Information from personal contact with the patients was also collected. LRR diagnosis was defined by pathology or image information . The date of LRR was the date of the first relapse that occurred in the ipsilateral chest wall, supraclavicular lymph nodes, axillary nodes, or the intramammary chain.2.5LRR rate was analyzed as the first site of failure. Cumulative incidence of LRR was calculated by competing risks data analysis, as described by Gray,33.1Clinicopathological factors of all patients and the four subgroups are shown in Table 3.2With a median follow‐up time of 70.9 months, a total of 34 patients developed LRR. For the entire cohort, the 5‐year cumulative incidence of LRR (± synchronous distant metastasis) was 5.54%. Twenty‐eight patients were diagnosed of LRR concurrently with distant failure and six patients were diagnosed with isolated LRR. Among the 34 patients who developed LRR, there were 39 sites of recurrence, including supraclavicular area n = 16), chest wall (n = 15), internal mammary area (n = 4), and axillary nodes (n = 4) Figures  and S2. 6, chest 3.3P < .05), TNBC , and pN3 . Grade, pT, and LVI statuses were not related to LRR (Table P < .05), TNBC , and pN3  were independent predictors of LRR. HER2‐positive and TNBC had increased risk of LRR, while Luminal B had a nonsignificant higher risk than Luminal A.Factors predictive of LRR were analyzed by univariate analysis and multivariate analysis. In univariate analysis, factors predictive of higher LRR were HER2‐positive subtype . This was not consistent with the report of the Danish Breast Cancer Cooperative Group and the report from Wang and his colleaguesP < .05). Interestingly, the LRR incidence for HER2‐positive with trastuzumab was similar to that of Luminal A . In addition, the LRR incidence for HER2‐positive without trastuzumab was similar to that of TNBC . In addition to trastuzumab, endocrine therapy also significantly reduced LRR incidence .In the present study, the LRR incidence for HER2‐positive and ‐negative was at a similarly low level .We also observed remarkably different patterns of relapse among the subtypes. Despite the highest risk of LRR in TNBC, the LRR curve for TNBC demonstrated the steepest incline before the peak, indicating earlier local relapse compared with HER2‐positive or luminal subtypes. In the TNBC cohort, all local recurrence occurred within the first 3 years, which was 1 to 3 years earlier than HER2‐positive and luminal subtypes. A similar relapse pattern was also observed in other studies. Dent et al examined a cohort of 1601 breast cancer patients and analyzed timing of relapse among different biological subtypes. TNBC patients had an earlier local recurrence than other subtypes  Expert Panel serves as a reliable prognostic predictor for Chinese breast cancer patients receiving PMRT. Treatment with trastuzumab and endocrine therapy dramatically decreased the risk of LRR in HER2‐positive and hormonal receptor‐positive patients. Click here for additional data file. Click here for additional data file.


Virusmentha network, an online virus–virus or virus–host interaction tool. Furthermore, we summarize the role of the 14-3-3 family in RNA and DNA viruses. The participation of 14-3-3 in viral infections underlines its significance as a key regulator for the expression of host and viral proteins.The 14-3-3 proteins are a family of ubiquitous and exclusively eukaryotic proteins with an astoundingly significant number of binding partners. Their binding alters the activity, stability, localization, and phosphorylation state of a target protein. The association of 14-3-3 proteins with the regulation of a wide range of general and specific signaling pathways suggests their crucial role in health and disease. Recent studies have linked 14-3-3 to several RNA and DNA viruses that may contribute to the pathogenesis and progression of infections. Therefore, comprehensive knowledge of host–virus interactions is vital for understanding the viral life cycle and developing effective therapeutic strategies. Moreover, pharmaceutical research is already moving towards targeting host proteins in the control of virus pathogenesis. As such, targeting the right host protein to interrupt host–virus interactions could be an effective therapeutic strategy. In this review, we generated a 14-3-3 protein interactions roadmap in viruses, using the freely available  Viruses are a leading source of death and have a significant impact on global health. They are unique pathogens which rely on living organisms to complete their life cycle . TherefoRecently, the 14-3-3 family of proteins has received much attention in the field of disease progression and drug development ,7,8,9. THistorically, 14-3-3 proteins were named based on their elution and the migration pattern of their two-dimensional DEAE-cellulose chromatography and starch gel electrophoresis .The 14-3-3 proteins are essential regulators of intracellular signaling pathways and upon interacting with their target protein, they modify its function and intracellular localization . FurtherThe crystal structure of 14-3-3 proteins comprises of highly helical and cup-shaped dimers . The cupThe general function of 14-3-3 proteins can be classified based on the following characteristics : (A) conFurthermore, various post-translational modifications, such as phosphorylation, acetylation, acylation, polyglycylation and oxidative stress, are documented for 14-3-3 proteins ,24. EvenViruses are a major contributor to the global burden of diseases because they cause acute and chronic infectious diseases ,27. ManyProtein–protein interaction (PPI) is one of the primary components of system biology and a key mediator for host–virus interactions. PPI can be detected by in-vitro, in-vivo, and in-silico studies. The in-vitro methods include tandem affinity purification (TAP), co-immunoprecipitation (co-IP), protein fragment complementation, protein arrays, phage displays, X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy ,30,31,32The interaction between proteins is one of the most critical processes in the execution of cellular processes. In system biology research, protein–protein interaction networking (PPIN) is an essential tool for the identification of interactions between protein pairs. Therefore, the importance of understanding this PPI has led to the development of experimental and computational interaction network tools. As discussed above, a wide variety of methods have been developed to study these interactions.Virusmentha network  such as the influenza A virus (IAV), measles virus, human respiratory syncytial virus, human immunodeficiency virus (HIV), La Crosse virus, and double-stranded DNA (dsDNA) viruses like herpes simplex virus type I, human herpes 4, hepatitis B virus (HBV), Nipah virus, Hendra virus and Murid herpesvirus . InvestiThe 14-3-3 proteins are associated with many cellular processes, which could probably implicate them in many human illnesses. Therefore, the characterization of their functions at the molecular level is of the utmost importance. Given the multitude of its binding partners and its essential roles in numerous biological processes, the 14-3-3 family of proteins should be considered pathfinders for further exploration of closely related viruses.Viruses have been shown to arrest host cells by hijacking and manipulating host protein complexes to promote the development of infection. Generally, viruses are classified based on the Baltimore Classification System invented by David Baltimore, which is primarily based on their genome composition and replication strategy . AlthougRNA viruses have ssRNA or dsRNA as their genetic material . In addiIn 2000, Aoki et al. demonstrated that 14-3-3 proteins associate with the hepatitis C virus (HCV) core protein to activate RAF-1 kinase and contribute to hepatocyte growth regulation . FurtherThe 14-3-3 proteins have a crucial role to play in the regulation of cell cycle progression ,68. TheySimilarly, in the case of adeno-associated virus type 2 (AAV-2) infection, 14-3-3 ε and 14-3-3 γ interact with the Rep68 protein of AAV-2 by phosphorylation at serine 535 . InteresMembers of the14-3-3 family regulate numerous intracellular processes, one of which is immunity . ImmunitPrevious studies from our laboratory have shown that the 14-3-3 protein also binds to the severe acute respiratory syndrome (SARS) coronavirus nucleocapsid (N) protein . This biIn addition, a proteomics report showed that the influenza A virus (IAV) NS1 protein interacts with several 14-3-3 isoforms, although the functional implication of this interaction remains unclear .DNA viruses have DNA as their genetic material and replicate either by host or by virally encoded DNA polymerases . IsoformIn the case of HSV-I, ICP27 is an essential protein for viral replication because it is involved in the nuclear export of viral mRNA and the suppression of host protein synthesis by inhibiting cellular mRNA splicing ,83. ICP2Gupta et al. identified several members of the 14-3-3 protein family  as interacting partners of EBV-encoded large tegument protein deneddylase (BPLF1) using co-IP and mass spectrometry approaches . InteracIn addition, we also demonstrate and summarize all the roles of 14-3-3 proteins in different viruses . The apoWe also elaborate on all the signaling pathways that are manipulated by 14-3-3–virus interactions . It is iVirusmentha, an online tool. Based on this database, 14-3-3 ζ, ε, and θ are well known in the context of viral infections.Viruses exploit the molecular machinery of the infected host to support their replication. To achieve this, viruses establish virus-specific protein interactions to perturb several cellular processes in the infected host ,91. ThusFurthermore, 14-3-3 family members display a significant role in interacting with several RNA and DNA viruses through multiple pathways, including apoptosis, cell signaling, cell cycle, and ubiquitination. These viral–14-3-3 interactions may change the typical distribution and disrupt the original functions of 14-3-3 proteins. For example, HBV, HCV, and HSV viruses were directly associated with, or inhibited, 14-3-3 proteins from inducing apoptosis. Therefore, monitoring and manipulating 14-3-3 proteins may represent new diagnostic and therapeutic targets for virus infections. The development of therapeutics, including drugs and vaccines, is highly dependent on the knowledge gained from investigating host–virus interactions. As such, interrupting host–virus interactions by targeting the right host factor might be a highly effective strategy for treating viral infections.


Metaplastes ornatus and examined the biomechanics of male and female genital structures, involved in their SRB as a model case. During an initial phase of mating, males of this species thrust their subgenital plate with hook-like spurs and many microscopic spines into the female genital chamber. By moving the subgenital plate back-and-forth, males stimulate females, and this stimulation induces the ejection of sperm previously stored in females. We aimed to uncover the mechanics of the interaction between the subgenital plate and genital chamber during SRB. The genital morphology and its material composition were investigated using modern imaging and microscopy techniques. The obtained results showed a pronounced material heterogeneity in the subgenital plate and the genital chamber. The material heterogeneity was completely absent in that of a second bushcricket species, Poecilimon veluchianus, which does not exhibit SRB. Finite element simulations showed that the specific material heterogeneity can redistribute the stress in the subgenital plate of M. ornatus and, thereby, reduces stress concentration during SRB. This may explain why only a few examined males had a broken spur. We suggest that the observed structural features and material heterogeneity in M. ornatus are adaptations to their SRB.Sperm removal behaviour (SRB) is known in many animals, and male genital structures are often involved in the SRB, e.g. rubbing female genitalia vigorously. However, it remains unclear how those male genital structures function properly without severe genital damage during SRB. In the present study, we focused on the bushcricket The online version contains supplementary material available at 10.1007/s00114-020-01706-w. Sperm removal behaviour triggered by male stimulation (hereafter SRB) is an offensive male adaptation to achieve fertilisation success under sperm competition Parker . There aFrom a male perspective, the removal of rival sperm from female sperm storage organs is always likely to be beneficial due to increased fertilisation success. On the other hand, sperm removal could be counter to female interests since females actively invest in maintaining sperm in female sperm storage organs, e.g. providing nutrition to sperm and protecting sperm against pathogens  as a study species because it is highly suited to a biomechanical study of genital interactions during SRB. Males of this species thrust a part of the postabdomen—the subgenital plate—into the female genitalia prior to the transfer of their own ejaculate . In the current study, to unveil if there are any adaptive strategies to avoid genital damage in both sexes, we aimed to address the following questions: (1) how often the male and female genitalia get damage in field-caught bushcrickets and (2) are there any strategies to reduce genital damage that could be inflicted during SRB? For this purpose, first, we observed field-caught males and females to quantify the frequency and severity of genital injuries. We then used computed tomography and microscopy to investigate the morphology and material distributions of the relevant male and female genital structures, using the results from which to perform finite element simulations, to address how males avoid breakage of the delicate, paired hook-like spurs of the subgenital plate. As a comparison, we also analysed the material distribution from corresponding structures of Poecilimon veluchianus veluchianus Ramme, 1933 , a bushcricket species which does not exhibit SRB. Based on our results, we discuss how M. ornatus males reduce damage to their genitalia during SRB.M. ornatus and P. v. veluchianus were collected in Paleokastro, central Greece (Nomos Fthiothis), and further specimens of M. ornatus were collected a few kilometre north of the village of Vitoli, central Greece (Nomos Fthiothis), between 16 June 2019 and 26 June 2019. The specimens were first frozen in a conventional freezer and preserved in 70% ethanol for a few weeks before we conducted further experiments.Specimens of M. ornatus were scanned using a SkyScan 1172 micro-computed tomography (microCT) scanner . For this purpose, the samples were dehydrated with an ascending series of ethanol to absolute ethanol, which was then substituted with 1,1,1,3,3,3-hexamethyldisilazane , and were dried in a fume hood. HMDS was used for drying the samples as an alternative method of critical point drying, which was first applied to insect specimens for scanning electron microscopy . Outlines of the genital chambers and wounds were traced using the software Adobe Illustrator CS4 . Colours were assigned to the scabs (black), scratched areas (dark grey) and genital chamber (light grey). Based on the resulting images, the areas of the genital chambers and wounds were measured using the software Fiji  and embedded in glycerin on glass slides. Subsequently, we visualised autofluorescence using a Zeiss LSM 700 confocal laser scanning microscope (CLSM)  equipped with four stable solid-state lasers with wavelengths of 405 nm, 488 nm, 555 nm and 639 nm. To selectively detect emitted autofluorescence for each of the four laser wavelengths, we applied respectively either a bandpass emission filter transmitting light with wavelengths 420–480 nm or long-pass emission filters transmitting light with wavelengths ≥ 490 nm, ≥ 560 nm or ≥ 640 nm. Objective lenses with × 5  = 0.16) and × 10  magnification were used. To obtain overview micrographs of each structure, tile scans were performed. To each image produced by the abovementioned four sets of lasers and filters, we assigned blue, green, red (50% saturation) and red (50% saturation). The resulting overlaid micrographs were interpreted according to the colour code reported by Michels and Gorb  was used to develop a geometric model of the subgenital plate of nal Fig. . Based onal Fig. . The parnal Fig. . Taking nal Fig. .Fig. 2GTo address the question of how the male reduces risks of damage of the fine spurs during the SRB, we focused on and simulated movements that may cause spur breakage. Considering that the subgenital plate itself is a symmetric and relatively flat plate, we expect that 2D models provide sufficient data for our purpose. Therefore, to reduce the computational time, here we developed a 2D model of the subgenital plate. We cut the model in half about the central line, and a symmetric boundary condition was applied to this symmetric axis , which are general-purpose elements. The simulations were performed using a general ‘static step’ ABAQUS . Each siM. ornatus, we carefully checked the outline of the intact female genital chamber in the volume rendering of the micro-CT scanned female and aligned the subgenital plate on it with the same size-scale  but has sclerotised ridges (yellow)  Fig. . In the ow) Fig. .Fig. 3CM. ornatus (i.e. ‘reference model’). The results were given for the three steps of the spur hooking, pulling and pushing of the subgenital plate. As seen in Fig. Figure The results obtained from the ‘stiff model’ are shown in Fig. ESM 2(MP4 5048 kb)ESM 3(MP4 5586 kb)M. ornatus the biomechanics of a male genital structure involved in SRB and how they are adapted to reduce mechanical damage of the fine spur. Our experiment of genital coupling simulated using a micro-CT-based visualisation technique suggested that the depth of the subgenital plate is much greater than that of the female genital opening at rest in M. ornatus. Although females slightly open their genital chamber just before males start the SRB  presumably reduces the risk of severe mechanical damage and increases female tolerance. This is thus functionally equivalent to the resilin-rich composition of the spermalege in female bed bugs, Cimex lectularius, which likely functions in avoiding damage at the site of hypodermic traumatic insemination . Sperm removal ability is essential for M. ornatus subgenital plates that contrasted strongly with the homogeneously and weakly sclerotised subgenital plate of P. v. veluchianus, which do not show the SRB. It is reasonable to expect that males cannot even insert the P. v. veluchianus type of the subgenital plate since such a subgenital plate would collapse due to external forces generated by the narrow female genital chamber. Comparative finite element modelling comparing models with the realistic material heterogeneity versus a homogeneously stiff material demonstrated that in all simulated loadings, the subgenital plate with the material heterogeneity experiences lesser stresses than the homogeneous model. Considering that the number of thrusts  in a single mating can exceed 2000


Oligoarticular juvenile idiopathic arthritis (JIA) is a common disease in pediatric rheumatology. The management of oligoarticular JIA can result in a considerable economic burden. This study is a four-year, retrospective cost identification analysis performed to determine the annual direct cost of care for patients with oligoarticular JIA and possible predictive clinical factors. Direct healthcare costs were defined as those associated with office visits, laboratory studies, hospital admissions, joint injections, medications, infusions, radiology tests, and emergency room visits. Disease characteristics and patient information included ANA status, gender, age at diagnosis, duration from diagnosis to initial visit during the study period, and whether uveitis had been diagnosed. We identified 97 patients with oligoarticular JIA eligible for the study. The median age of diagnosis was 4.3 years. Positive ANA were noted in 75% of patients. 34% of patients received at least one intra-articular steroid injection. 32% of patients were prescribed a biologic during the study period, predominantly with other medications, while 23% of patients received only NSAIDs. 20% of patients were prescribed oral steroids. The average total direct medical cost in this study per year for an oligoarticular JIA patient was $3929 ± 6985. Medications accounted for 85% of annual direct medical costs. Clinic visits and laboratory testing accounted for 8% and 5%, respectively. Patient characteristics and demographics were tested for association with direct medical costs by the Wilcoxon rank sum test and Kruskal-Wallis test. Patients who were ANA positive had increased annual costs compared to patients who are ANA negative. ANA-positive patients were found to have statistically significant costs, particularly, in laboratory tests, procedural costs, radiology costs, and medication costs. The results reported here provide information when allocating healthcare resources and a better understanding of the economic impact oligoarticular JIA has on the United States healthcare system. Juvenile idiopathic arthritis (JIA) is characterized by inflammation and damage to the joints and affects approximately 1 in 1,000 children worldwide . Little Evolving health care reform requires managing direct costs of care incurred by different treatment modalities for children with arthritis and other chronic illnesses. The multidisciplinary management of oligoarticular JIA, including frequent hospital visits, laboratory evaluation, and medical treatments, can result in a considerable economic burden. Data on the cost of JIA are less available than for other illnesses . With thA four-year, retrospective cost identification analysis was performed to determine the annual direct cost of care for patients with oligoarticular JIA and possible predictive clinical factors.Patients meeting ILAR criteria for the diagnoses of persistent and extended oligoarticular juvenile idiopathic arthritis  between Health care costs were measured as a direct cost to the hospital for acquisition of materials and services. Direct healthcare costs were defined as those associated with office visits, laboratory studies, hospital admissions, procedures such as joint injections, medications, day hospital infusions, radiology tests, and emergency room visits. All costs were obtained by the hospital's business analytics department.Costs of outpatient medications were calculated using a combination of prescriptions sent, (extracted from the EMR) and formulary pricing. Costs from infusions received at Lurie Children's Hospital were obtained via the business analytics department; infusion center visits were considered hospital encounters. Our study did not include infusions done at other centers or through home health since those costs could not be obtained. All statistical analyses were performed using SAS statistical software . Percentages, median (with interquartile range), or mean ± SD were used where appropriate to express disease characteristics, patient demographics, and direct medical costs. The Wilcoxon rank sum test was used to determine correlations between patient characteristics and direct medical costs.We identified 97 patients  with oligoarticular JIA eligible for the study. The median age of diagnosis and median duration from diagnosis (in 2011) for patients in our study were 4.3 and 8.2 years, respectively. Positive anti-nuclear antibodies were noted in 75% of patients. 34% (33 patients) had received at least one intra-articular steroid injection. Of the patients who received intra-articular joint injections, 62% (21 patients) had joint injections performed by interventional radiology, whereas 30% (10 patients) had joint injections in the operating room, and 6% (2 patients) in our outpatient clinic, respectively. 27% (26 patients) had a diagnosis of uveitis documented in our EMR. 76% of patients were classified as having persistent oligoarticular JIA. A medication profile of the 97 patients is shown in p values for the associated variable. The ANA status and oligoarticular JIA subtype were statistically significant, with p values of 0.02 and 0.04, respectively. Patients who were ANA positive had increased annual costs compared to patients who are ANA negative. ANA-positive patients were found to have statistically significant costs, particularly, in laboratory tests (p = 0.08), procedural costs (p = 0.04), radiology costs (p = 0.06), and medication costs (p = 0.02). Extended oligoarticular JIA patients had higher costs than patients classified as persistent oligoarticular JIA subtype. Extended oligoarticular JIA patients particularly had statistically significant medication costs (p = 0.04) compared to persistent oligoarticular JIA patients, with medication costs of $3932 ± 3943 and $2613 ± 4725, respectively. When evaluating costs for the number of arthritic joints at the start and end of the study, an increase or decrease in joint count over four years detected no difference in annual total direct cost.Patient characteristics and demographics were tested for association with direct medical costs by the Wilcoxon rank sum test for all two group comparisons and the Kruskal-Wallis test for all comparisons with three or more groups. Variables that were evaluated for associations with direct costs included ANA, gender, diagnosis of uveitis, subtype of oligoarticular JIA, and intra-articular steroid injections. JIA is a complex chronic condition associated with a considerable economic burden to families affected as well as the health care system. We investigated costs associated with oligoarticular JIA. In our study, the mean annual total cost of oligoarticular JIA was estimated as $3929 ± 6985. As expected, the cost of medications, including infusions, (85%) was the largest contributor for costs. Extended oligoarticular JIA had a statistically significant higher mean annual cost of care than persistent oligoarticular JIA patients. ANA positivity was also found to be associated with higher annual costs of care. This result could be explained by the association of ANA positivity with uveitis, resulting in more aggressive treatment plans, often including biologic agents.Our study had cost component results similar to other cost studies of JIA patients. In previously published studies, the majority included and categorized costs by the various subtypes of JIA. Our findings are similar to studies finding that patients with JIA have high costs, particularly, those related to office visits, medication costs, and diagnostic testing . An asseResults of costing have been similar in studies for other rheumatologic conditions. In a South Korean cost study for systemic lupus erythematous patients, the largest component of direct medical costs were medications, with a mean annual cost amounted to 3305 US dollars in 2010 . In adulOur study was subject to several limitations. First, data was primarily extracted from the EMR and was dependent on proper diagnosis coding, physical examination documentation, procedure coding, and updated medication lists. Provider variability in medication treatment plans and laboratory evaluations could lead to variability in costs. Micro costing was performed on prescription drugs ordered in the EMR, which were assumed to be filled. Costs extracted were limited to services provided within our institution. Outside costs such as laboratory studies and infusions were not included, although most oligoarticular JIA patients do not receive infusions. This study only evaluated direct costs, not indirect costs such as time missed from school, transportation costs, or work absences for guardians, which could also pose a significant economic burden. In a United Kingdom study, it was found that JIA is likely to have a considerable economic impact, especially with regards to nonhealth care costs due to its societal impact . We alsoIn summary, we identified the annual direct medical costs for oligoarticular JIA. The results reported here provide information when allocating healthcare resources and a better understanding of the economic impact oligoarticular JIA has on the U.S. healthcare system. Even though oligoarticular JIA is considered one the more mild subtypes of JIA, it is still associated with high costs. Our study found that direct medical costs were higher in oligoarticular JIA patients who were ANA positive or had an extended course and that medications account for over 80% of direct medical costs in patients with oligoarticular JIA. The costs involved with the care of JIA patients are important for physicians and policy makers to consider when making informed decisions about patient care, especially as targeted drug therapies become more widely available for children.


The aim is to perform a pilot study evaluating the differences in healthcare service use and its associated costs by infant feeding mode in an infant’s first year of life. Data from a prospective cohort study and administrative databases were linked to examine healthcare use in healthy full term infants (N = 160). Exposure was categorized as exclusively breastfed, mixed fed and exclusively formula fed. Outcomes included hospitalizations, emergency room and physician visits. Descriptive statistics and generalized linear modelling were performed.Overall $315,235 was spent on healthcare service use for the sample of infants during their first year of life. When compared to exclusive breastfeeding, mixed feeding and exclusive formula feeding were found to be significant predictors of total healthcare service use costs (p < 0.05), driven by costs of hospital admissions. Due to the human and economic burden associated with not breastfeeding, policies and programs that support and encourage breastfeeding should be priority. Human breastmilk is universally acknowledged as the gold standard of infant feeding, as it offers a unique matrix of compounds for optimal growth, health and development in a newborn child . Studieseeding infants in Newfoundland and Labrador (FiNaL) study have been previously published [Methods from the Fublished –19. In bThe series of administrative data was created by the NL Centre for Health Information (NLCHI). Individual level record data were extracted from the Provincial Discharge Abstract Database (PDAD) for hospital admissions, Live Birth System (LBS) for demographics and infant characteristics, MCP Fee-for-Service Physician Claims (FFS) for family doctor and specialist visits, and the MediTech Module (MTER) for emergency room (ER) visits.IFM was defined using the WHO criteria. ‘Exclusive breastfeeding (EBF)’ defined infants receiving only breast milk  and nothing else, except for oral rehydration solution, medicines, vitamins and minerals when needed. ‘Mixed feeding’ (MF) defined infants receiving breastmilk and other food or liquid including water, non-human milk, and formula. ‘Exclusive formula feeding’ (EFF) defined infants fed only breastmilk substitutes. Due to the self-reporting of breastfeeding, EBF could only be valid for the first month of life, though information on IFM was collected throughout the first YOL.Primary outcomes were HSU and the associated costs for hospital admissions, ER and physician visits. Direct HSU costs reflect those to the payer over a 1 year time horizon.Deterministic linkage was used to link the FiNaL study and administrative data provided by the NLCHI. Descriptive statistics, either frequencies for categorical variables or means and standard deviations (SD) for continuous variables were conducted. Generalized linear modelling (GLM) was used to compare HSU costs, and maternal and child characteristics between IFM. Billable claims for each healthcare provider visit were summed using the claims provided by the NLCHI. Costs were converted to 2015 Canadian dollars, as all resource intensity weights (RIW) and cost per standard hospital stay (CSHS) used were for the 2015 fiscal year. The average cost of an ER visit for the EH regional health authority was calculated using the average of the 2014/2015 and 2015/2016 fiscal years. Due to the skewed HSU costs, both base case and robustness GLM analyses were performed. For the base case GLM a gamma distribution and a log link function were used. Following findings from the modified park and box cox tests, the robustness check followed an inverse gaussian distribution and a reciprocal function. A power calculation was conducted for the GLM at an alpha level of 0.05, by IFM; EBF (n = 107), MF (n = 32), EFF (n = 21). Statistical analysis was conducted using SPSS (IBM 25) software [There were 160 infant health insurance numbers provided for the data linkage. The recruitment process is illustrated in the Additional file The majority of infants were seen by a family doctor n = 151) within their first YOL, irrespective of feeding mode, and over half (n = 92) were seen by a specialist. MF and EFF infants had a higher percentage of specialist visits , while 55.8% of EBF infants visited a specialist during the first year. The highest frequency of billable claims was: no specific illness diagnosed at the visit (n = 591), signs and symptoms not otherwise diagnosed as an infection or disease (n = 225), upper respiratory tract infection (n = 92), otitis media (n = 61), common cold (n = 49), disorders of eyes and ears (n = 46), thrush (n = 46), and atopic dermatitis (n = 37). There were 12 infants hospitalized at least once during the first YOL. The length of stay (LOS) ranged from 1 to 7 days. Most commonly, admissions were related to respiratory complications . EBF infants had fewer hospital admissions (2.8%) than that of MF (15.2%) and EFF (19.0%), p < 0.05. Half of infants (n = 83) were brought to the ER at least once during the firstYOL. More MF infants had ER visits (56.3%) than that of EBF (53.3%) and EFF infants (38.1%). MF and EBF infants had significantly more ER visits when compared to EFF infants, p < 0.01. Additional information regarding hospital admissions, ER and physician visits can be found in the Additional file 51 withinThe direct HSU costs of 160 healthy full term infants during their first YOL amounted to $315,235.56  to examine the primary outcome of total HSU costs. The base case analysis for the GLM is presented in Table In the present study, 160 mother-infant dyads were enrolled in a data linkage pilot examining the impact of IFM on total HSU costs. Cumulative HSU cost in the first YOL for all healthy full term infants in one provincial region in Canada was $315, 235.56, including cost of birth. The highest percentage spent on HSU was for hospital admissions, followed by family doctor, ER and specialist visits. Higher HSU costs were associated with EFF infants when examining hospitalizations (birth and admissions), and significant differences were found between IFM when examining total HSU costs.Our findings are consistent with the literature, where studies in other countries have shown that infants who had early exposure to formula experienced higher volumes of visits to family doctors, infectious episodes and hospital admissions –25. SimiThere are a number of strengths of the current study. To our knowledge, this is the first estimate of HSU costs by IFM of a sample of full term healthy infants living in Canada. Our ability to link maternal and child data allowed us to examine the specific characteristics that are associated with higher HSU costs among infant’s , and control for these covariates in our analysis. The advantage of conducting a pilot study has allowed us to assess the feasibility of collecting data in a Canadian context and estimate variability in outcomes to help determine sample size for future larger provincial studies. The administrative database allowed for the calculation of individual level data and the direct costs associated with HSU through the claims of family doctors and specialists.In one region of Canada, EFF and MF were found to be significant predictors of the total HSU costs during the first YOL. Further research is needed in a Canadian context examining larger samples, with a necessity for more reliable and valid measures of exposure to capture longer durations of IFM. Due to the human and economic burden associated with no breastfeeding, policies and programs that support and encourage breastfeeding should be a priority for governments and regional health authorities.Our results are based on a relatively small sample size, where the subsample of the FiNaL Study had a selection bias of primiparous, Caucasian, higher education and household income mothers. We found differences among groups for the total HSU costs, driven mainly by hospitalizations, however significant differences were not observed when examining the costs associated with other HSU . This could be explained by either having no differences among groups, or that the study was underpowered to examine the differences of ER and physician visits. Due to challenges with collecting exposure data on feeding mode and its duration, our MF and EFF covered the first 6 months of life, but our EBF rate was considered valid and reliable for the first month only. The data on exposure were self-reported by mothers and therefore could result in misclassification. Based on the health insurance claims in NL, the administrative databases can only collect information on FFS physicians. Therefore there are a proportion of family doctors and pediatricians that are salaried that we would not have HSU on, therefore this an underestimate of the use and cost experienced in this region. In addition, although we used a health systems perspective to examine the costs, not all costs were included .Additional file 1: Figure S1. Flowchart of participant requirement process. Table S1. Healthcare Provider Visits by Infant Feeding Mode ). Table S2. Number of Unique Visits to Healthcare Providers and the Associated Costs by IFM (Counts). Table S3. Description of Hospitalizations During The First Year of Life. Table S4. Mean (SD), Median (IQR), Min & Max of the Total Costs Associated with each Healthcare Provider, By Infant Feeding Mode.