Advanced Practice Nurse (APN) is a specialist who has acquired clinical skills to make complex decisions for a better professional practice. In the United States, this figure has been developed in different ways, but in some European countries, it is not yet fully developed, although it may imply a significant advance in terms of continuity and quality of care in patients with chronic or multiple pathologies, including cardiac ones and, more specifically, heart failure (HF). The follow‐up of HF patients in many countries has focused on the medical management of the process, neglecting all the other comprehensive health aspects that contribute to decompensation of HF, worsening quality indicators or patient satisfaction, and there are not updated reviews to clarify the relevance of APN in HF, comparing the results of APN interventions with doctors clinical practice, since the complexity of care that HF patients need makes it difficult to control the disease through regular treatment. For this reason, this systematic review was proposed in order to update the available knowledge on the effectiveness of APN interventions in HF patients, analysing four PICO questions : whether APN implies a reduction in the number of hospital readmissions, if it reduces mortality, if it has a positive cost‐benefit relationship and if it implies any improvement in the quality of life of HF patients.A systematic review was performed based on the PRISMA statement, searching at four databases: PubMed, CINAHL, Scopus and Cuiden. Articles were selected based on the following criteria: English/Spanish language, up to 6 years since publication, and original quantitative studies of experimental, quasi‐experimental or observational character. Papers were excluded if they do not comply with CONSORT or STROBE checklists, and if they had not been published in journals indexed in JCR and/or SJR. For the analysis, two separate researchers used the Cochrane Handbook form for systematic reviews of intervention, collecting authorship variables, study methods, risks of bias, intervention and comparison groups, results obtained, PICO question or questions answered, and the main conclusions.A total of 43,754 patients participated in the 11 included studies for the development of this review, mostly from United States and non‐European countries, with a clearly visible lack of European publications. Regarding the results related to first PICO question, researches reviewed proved that APN implied a reduction in the number of hospital readmissions in patients with heart failure (up to 33%). Regarding the second question, mortality was always lower in groups assisted by APN versus in control groups (up to 7.8% vs. 17.7%). Regarding the third question, APN was cost‐effective in this type of patient as the cost reduction was eventually calculated in 1.9 million euros. Regarding the last question, quality of life of patients who have been cared for by an APN had notoriously improved, although one of the papers concluded that no significant differences were found. All the questions addressed obtained a positive answer; therefore, APN is a practice that reduced hospital readmissions and mortality in HF patients. The cost‐effectiveness is much better with APN than with usual care, and although the quality of life of HF patients seems to improve with APN, more studies are needed to support this focused on this. Advanced practised nursing addressed at patients with heart failure has proven reduce hospital readmissions and also mortality.Advanced practised nursing also has positive effects on cost‐benefit and on the improvement of quality of life of heart failure patients.The advanced clinical competences are needed for the enhanced professional practice to improve heart failure patients’ care.This review is the first comprehensive analysis of clinical trials, quasi‐experimental and observational studies with the greatest rigour and quality bringing together the best evidence that confirms the ability of advanced practical nursing to reduce hospital readmissions and mortality of HF patients.The verification made by this review about the impact of advanced practice nursing to improve cost‐effectiveness of care for patients with HF and their quality of life makes the development of this care system as a highly recommended task to improve the efficiency of and effectiveness of health services.Confirming the positive impact that advanced practice nursing can have on the results of care of patients with HF allows us to extend this model of advanced care to other chronic, multiple and/or complex pathologies.1Advanced Practice Nursing was defined in 2002 by the International Council of Nurses as “a specialist nurse who has acquired the expert knowledge base, the abilities to adopt complex decisions and the clinical competences needed for an enhanced professional practice, whose characteristics are given by the context or country in which the nurse is accredited to work in”  includes different specific and well‐differentiated Nursing roles such as: Nurse Practitioners (NPs), Certified Nurse‐Midwives (CNMs), Certified Registered Nurse Anaesthetists (CRNAs) and Clinical Nurse Specialists (CNSs), always in the scope of the United States  sometimes causes great confusion, leading to forgetfulness and mistakes in taking medications, for example  model for the formulation of clinical questions in the practice based on evidence , CINAHL , Scopus  and Cuiden , since they are the most relevant information sources with the most likelihood of finding the best quality scientific papers on the theme and the objectives of the review. Although only 4 databases were consulted, in accordance with recommendations for conducting systematic reviews in the clinical setting . Later, to complement the search, the descriptor "Clinical Nurse Specialist" was added. In the case of the Cuiden database, as it is in Spanish, the following descriptors were used: “Enfermería de práctica avanzada” and “Insuficiencia cardiaca,” both included in DeCS. The search strategy applied systematically combining the Boolean operators with the aforementioned descriptors was (“Nurse Practitioner” OR “APN” OR “Advanced Practice Nursing” OR “Nurse‐led”) AND “Heart failure” NOT “Review,” with the exception of Cuiden, in which the following strategy was applied: “Enfermería de práctica avanzada” AND “Insuficiencia cardiaca.” The search was repeated incorporating the aforementioned descriptor "Clinical Nurse Specialist" with the boolean "OR" together with the rest of the terms mentioned linked to the descriptor "APN" into the search strategy.The subsequent process for the selection of the articles was exactly the same for all the databases used, using the following descriptors: “Nurse Practitioner,” “APN,” “Advanced Practice Nursing,” “Nurse‐led” and “Heart failure,” all included in MeSH  and in DeCS , the methodological design employed, the sample of participants, the objectives of the study and the main results and its conclusions, classifying the research based on the PICO question(s) it definitely answered and excluding from the review those articles which, after analysing their full text, were found to not answer correctly at least one of the PICO questions.Finally, a search was performed to check if the journals in which the chosen articles were published were indexed in the JCR  and/or in the SJR , excluding those articles whose journal did not appear in at least one of the lists, verifying the impact factor of each of them and the corresponding quartile occupied by the journal in the year of their publication.The review was approval by Research Ethic Committee of Red Cross Nursing School, University of Sevilla.4In the preliminary search performed by the reviewers in the mentioned databases, 365 articles were obtained, 6 records being deleted due to duplicity. Subsequently, 336 records were deleted by applying the inclusion and/or exclusion criteria after analysing the abstracts, and another 11 records were also deleted after applying these same criteria but in the analysis of the full texts, thus obtaining a total of 11 articles for the qualitative synthesis of the systematic review. The flow diagram of the information through the different phases of the review, based on the PRISMA statement  share their geo‐location between the U.S. and Australia, while the rest are distributed among countries as diverse as Sweden, China or Brazil. With regard to the research designs employed, most are experimental papers (n = 8), while two are observational (n = 2), with only one paper left of a quasi‐experimental character (n = 1). The sampling size varied considerably in the different articles included in the review, ranging from 78 patients to 38,618 patients. The articles were published between 2012 and 2017.All the selected articles are written in English, and more than half  that produce a very positive effect on the patient. When the “head” health professional, or the one who really manages the patient, is the nurse, health promotion acquires a really privileged position in the map of actions performed for the benefit of the patient. Studies have been analysed which, although not including the concept of “Advanced Practice Nursing” in their content, do deal with nursing leadership in terms of managing the patient, a concept which has been commented on only a few lines above, and which implies higher control over all the interventions performed on the patient.5.5A search was not performed on grey literature pages or secondary sources. This can lead to increased publication bias.Consensus was reached by the authors not to use meta‐analysis in this review. A meta‐analysis was not considered feasible owing to the diversity of the primary studies included; a factor that can render meta‐analysis meaningless. The external validity was not measured. The setting of the trials differed; selection of participants; characteristic of participants; hence undermining external validity (Rothwell, 6If we understand the effectiveness of Advanced Practice Nursing as the sum of all the concepts analysed in this review, it could be said that this branch of the nursing profession produces very positive effects not only for the patient but also for society as a whole since, in addition to generating positive results in health such as a reduction in the number of hospital readmissions, an improvement in quality of life or the reduction in mortality, it reduces the costs of the health services, enabling economic and social savings within the health system.It could be considered that all the PICO questions addressed by this review have a positive answer, always with the presence of differences among the individual papers included; however, for this reason, it is to be concluded that a much higher volume of studies would be needed to verify, by means of more evidence, that Advanced Practice Nursing is a branch of the profession capable of facing the new care needs of an ever‐ageing and increasing population, pluripathological and, consequently, polymedicated. It is necessary to further investigate and advance in this field, not only because of the aforementioned but also because of the changes in the health system and its funding, a crucial and motivating aspect which confers and modulates competences to the health professions.7This paper represents a descriptive contribution to the usefulness of Advanced Practice Nursing as a developmental strategy in the clinical practice in the management of patients with heart failure. Verifying the impact that this care clinical model can have, largely implemented in the United States and in many other countries but still in development in others, either on mortality, on quality of life or on the reduction of the number of hospital readmissions, and even on the health expenses, can be extremely useful to justify the setting in motion of advanced practice procedures in countries and contexts in which this has not yet been implemented. The demographical and epidemiological changes and the challenge imposed by the economic crises are some of the factors which are facing us with the need to transform the health systems so as to be able to respond to these demands, and the response to heart failure is a concrete example which is requiring us to transform the traditional health care response nurses are providing to these patients into a care model in which nurses show the abilities to adopt complex decisions and the clinical competences needed to offer an enhanced professional practice to this type of complex patient.In view of the results obtained in this review, advanced practice nurses can contribute to reduce the risk of readmission and death in these patients, improving their quality of life and reducing the associated health costs, reason for which there is a substantial body of international evidence on the positive impact of the Advanced Practice Nursing roles to improve the health results of the patient, the quality of care and the efficiency of the health system, which must lead us to recommend the implementation of this approach in all the health systems aspiring to a universal coverage with the maximum care quality. It has been established that nurses have an important role in comprehensive HF management. Nurse‐led education supports patients in gaining knowledge and through that, empowers patients to become an active participant in their health matters.The authors declare that they have no conflict of interests.JOP and JAPB contributed to research project elaboration, review of articles, searching of the databases and manuscript writing. JMRR, JGS and NJP contributed to review of articles and manuscript writing. MRM contributed to research project elaboration, manuscript writing and study orientation.


Moreover the correlation between all the variables here analysed enlightens that the same origin can cause the temporal behavior of all these variables. We interpret the seismological, geochemical and geodetic observable in terms of the injection of magmatic fluids into the hydrothermal system or its pressurization.The knowledge of the dynamic of the Campi Flegrei calderic system is a primary goal to mitigate the volcanic risk in one of the most densely populated volcanic areas in the world. From 1950 to 1990 Campi Flegrei suffered three bradyseismic crises with a total uplift of 4.3 m. After 20 years of subsidence, the uplift started again in 2005 accompained by a low increment of the seismicity rate. In 2012 an increment in the seismic energy release and a variation in the gas composition of the fumaroles of Solfatara  were recorded. Since then, a slow and progressive increase in phenomena continued until today. We analyze the INGV - Osservatorio Vesuviano seismic catalogue of Campi Flegrei from 2000 to 2020 in order to look for any variation in the seismic parameters and compare them with geochemical monitored ones. A remarkable correlation between independent variables of earthquake cumulative number, CO/CO A slow regular and persistent subsidence occurred for most of the period since the last eruption. Uplift episodes are documented in 1950–1952, 1969–1972 and 1982–19843. The last one was associated with a total uplift of 1.8 m in the central area of the caldera with the occurrence of more than 16,000 earthquakes4 without culminating in an eruption.Within the bradyseismic phenomenon which characterize the Campi Flegrei volcanic field, an uplift of the central part of the caldera has been recorded since 2005, reaching a value of 65 cm at the beginning of 2020. The last eruption of Campi Flegrei occurred in 1538 on the western side of a raised marine terrace, and, on the basis of historical and geological reconstructions, the seismicity and uplift had been increasing for about 100 years before the eruption. Some days before the eruption occurred a rapid and localized increase of the ground uplift5 together with an increment of the seismicity rate6 and of the degassing activity7. An increment in the seismic energy released occurred in 2012 . The duration-magnitude empirical relation can be found inMc. For magnitudes larger than Mc the network is able to detect any earthquake occurring in the area, conversely, not all the events with magnitude smaller than Mc are detected. As a consequence, the number of earthquakes with a magnitude smaller than Mc reported in the catalogues is smaller than that expected by the Gutenberg–Richter relationship.The detection threshold of any seismic network can be characterized by the completeness magnitude Mc is usually estimated with different techniques: the maximum curvature method11, Goodness-of-Fit Test11, b-value stability approach12, Entire Magnitude Range14, and others. To take into account the improvement of the Campi Flegrei seismic network with time we used a theoretical method that allows to define the detection and location sensibility in space of a seismic network16 and apply it for different time windows. The method consists in simulating earthquakes with different magnitudes distributed on a grid at a certain depth. More precisely, a grid of synthetic earthquakes, located in the area plotted in Fig. 17 source model), average rock density of 2000 kg/m318 mean quality factor Q=10019. The seismic noise at each station has been evaluated as the mean daily noise. The magnitude threshold for location is defined as the minimum magnitude of an earthquake recordable at least by 4 seismic stations. The same method is used for the detection threshold but the number of stations recording the earthquakes has to be larger than 1.Analyzing a seismic catalogue Mc. For the analysis we considered the time periods indicated in Fig. As we are considering the seismic catalogue of the located earthquakes we assume that the location sensitivity of the network in the epicentral area represents a theoretical evaluation of Mc range between 0 and 0.5 in the inner part of the caldera for the whole analysed period.Figure b parameter. The analysis has been performed dividing the catalogue in moving time windows. In order to analyse the influence of the window on the b value estimation we used, as suggested by20, different window lengths, going from 100 to 350 earthquakes. As can be seen in Fig. b value tends to assume smaller values for larger window size and shows higher variations for smaller window sizes. Our main interest is in these variations to be compared to the trend of other variables. As a consequence we decide to use a window of 150 events representing a good compromise. Moreover the simulation, described in the Supplementary Materials, reveals that using 150 events it is possible to obtain a reliable estimation of the b value. Due to the small occurrence rate of earthquake in the first part of the analyzed catalogue, taking a window length of 100 earthquakes the first sample point with magnitude above 0.2 occurs in 2010. With a window length of 350 earthquakes the first sample occurs in 2016. Using 150 samples the analysis starts from 2014.As a first step we estimate the temporal variations of the Gutenberg–Richter Mc and the b value minimizing the residuals of the Gutenberg–Richter fit12. More precisely we analyse the residuals associated with the b value in respect to the Mc variations. The obtained Mc values span from − 0.2 to 0.4, with some points at 0.8. However the Mc variation does not exhibit any trend enlightening that there is no evidence of a visible improvement of the Mc estimates in the epicentral area since 2012. From the sensibility analysis we notice that after 2011 the sensibility location threshold of the central part of the caldera remain almost constant, confirming the empirical data. Moreover we observe that, if we use two different values of the completeness magnitude, namely Mc = 0.2 and Mc = 0.4, the b parameter variation trend does not exhibit any substantial difference. Even if the b value assumes smaller values for Mc = 0.2 in respect of Mc = 0.4, the trend and the variation points are the same .The obtained seismological time series are finally compared with independent compositional data of the main fumarolic emission of Campi Flegrei  where the elevation reached 65 cm at the beginning of 2020. The uplift started slowly, 1–2 cm per year, until 2012 when the uplift rate increased to 6–7 cm per year. The temporal evolution of these phenomena, together with the variations in the gas composition of the monitored fumaroles, lead the Civil Defence of Italy to move the alert state of the volcano from green to yellow in 2012.23. Its increasing rate and energy released, together with surface deformation, and degassing, is used to define caldera unrests24. Seismicity is usually described using occurrence rate, magnitude and the Gutenberg–Richter b value. In volcanic areas the magnitude is usually small to moderate and the b value is higher than 1, due to the presence of fluids within a highly fractured rock. Siniscalchi et al.25 estimated the b value for the Campi Flegrei area during the period 2005–2016. They found a value of 0.92 whereas9 found a value of 1.03 for the period 2005–2019. A significantly smaller value (0.72) was found, for the period 1983–1984, by6. Such a small value can be related to high stress regimes26. It is interesting to notice that, during the 1982–1984 bradiseismic crises the seismicity was spread over the caldera inner and the b value was very small 29. Again it occurred after an increment in the caldera uplift  were recorded in the area of Solfatara/Pisciarelli and an increment in the b value is detectable. At the same time the interdistance and the mean depth are decreasing. The CO/CO2 showed a marked change in its trend . In other cases the relationship cannot be so easily established . However, we would like to remark that the functional form of the dependence of each variable on the other is less relevant in this study than the existence of the dependence itself.We would like to enlighten the common features of the analysed variables plotting each one versus the others. The plots reveal a clear dependence of each variable on the others. Figure 2 increase in time. This quantity is remarkably correlated with the cumulative number of events and the ground vertical deformation (see Figs. All the temporal variation of our observable can be possibly led back to a common cause, namely the fluid heating or pressurization clearly revealed by the CO/COVs velocity, compatible both with fluid presence and temperature increment, was evidenced by30 using the cross correlation of the seismic noise recorded in the caldera.Taking into account all the variations we suggest that the common cause can be identifiable with a fluid injection or pressurization. Injection of magmatic fluids within the hydrothermal system which feeds the Solfatara/Pisciarelli fumaroles would heat up the system increasing the pore pressure and facilitating the earthquakes occurrence being the medium less prone to bear stress and being the faults lubricated. Considering the vertical uplift the surficial manifestation of a deeper crustal bending, it could plays both as cause and consequence in this process being induced by the heating of the hydrothermal system and inducing fracture generation which recalls fluids towards the surface. A slightly decrement in the 23, but starting from middle 2018 the behavior of the system showed a clear variation. The available tomographies [e.g.32] evidence that a seismic layer, characterized by low Vp/Vs and low attenuation, was located at a depth of ~ 2.5 km, where most of the seismicity occurred. Its rheological properties could have suffered little variations due to vertical movement and heating. Geochemical analysis show that the gases involved in the hydrothermal system and sampled at the Solfatara/Pisciarelli fumarolic vents originate from a higher pressured system.The seismological and geochemical anomalies recorded in Campi Flegrei can be interpreted in terms of magmatic fluid circulation and permeability increment within the hydrothermal system. A magmatic intrusion or the injection of magmatic fluids were claimed as cause of the variations recorded from the beginning of the uplift until the 201233. Successions of brittle structures such as faults in contrast with levels of water saturated loose sands have been observed within the Campi Flegrei caldera. An increment in the temperature could recall hot fluids from the lower system increasing the pore pressures and lubricating the faults facilitating earthquake occurrence. The increment of the b value observed in the last decade as well as more dense and shallow swarms are compatible with this hypothesis. These observables temporal change appears to be correlated among themselves and with the ground deformations evidencing a variation in the hydrothermal system supported also by the increment in the CO/CO2 fumarolic ratio. In particular, the last quantity signs an increase of the temperature of the hydrothermal system since the end of 2017 that could be interpreted as due to fluid injection into the system or their pressurization. The CO/CO2 fumarolic ratio is remarkably correlated with the earthquakes cumulative number since 2000 (Fig. The rheological conditions of the central part of the caldera seems to be changing in time. Different rheological behaviors at Campi Flegrei have already been observed several times during the unrest and volcanic episodes occurred over the last 15 kyr000 Fig.  indicatiSupplementary Information 1.Supplementary Information 2.Supplementary Information 3.


The radial forearm free flap (RFFF) is the most commonly used flap for defects of the oral cavity. The facial artery musculomucosal (FAMM) is a safe and effective method to reconstruct medium sized defects of the oral cavity. No comparison exists between the FAMM flap and RFFF.1) Retrospective chart review from 2007 to 2016. 2) Cost difference analysis.p = 0.065) as well as lower rates of complications not requiring return to the OR with 1 complication in 1 patient as opposed to 10 patients with 15 complications (p = 0.008). Also, FAMM flap had shorter operative times compared to the RFFF group . The average operative room related costs for a FAMM flap were 6510 CAD vs 10,703 CAD for RFFF (p < 0.0005). Speech and swallowing outcomes were similar (p > 0.05).Thirteen FAMM flap cases and 18 RFFF met inclusion criteria. The FAMM flap showed a tendency to lower rates of return to the operating room (The FAMM flap can be used for reconstruction of medium-size defects of the oral cavity with functional outcomes similar to the RFFF while decreasing the associated costs and morbidity. The modern reconstructive paradigm has evolved around the use of free tissue transfer preferentially for reconstruction of the oral cavity. The radial forearm free flap (RFFF) emerged as the workhorse of free flap soft tissue oral cavity reconstructions due to its versatility and capacity to reconstruct medium and large size defect in all subsites of the oral cavity . The altIn the last decade, an expansion of the indications and the development of the surgical techniques for these locoregional flaps has led to a slow but important paradigm shift in oral cavity reconstruction making way for these locoregional flaps as an alternative reconstructive method in selected cases. Indeed, accumulating evidence points towards a decrease in health care costs and resource utilization, as well as decreased operating times and reduced donor site morbidity with the use of some locoregional alternatives, with no compromise in surgical or functional outcomes –5, 7.A particularly fitting option for the oral cavity is the FAMM flap. It has been successfully used for the reconstruction of complex oral cavity defects demonstrating an excellent safety profile . MoreoveFollowing institutional board review approval, we conducted a retrospective review of all patients who had undergone an oral cavity resection for which a FAMM flap or RFFF was selected for the reconstruction in one academic referral center from January 2007 to June 2016 inclusively. Operative notes were reviewed in detail to identify patients with oral cavity cancers who had a defect spanning at least three distinct subsites of the oral cavity or oropharynx for inclusion in the study . The continuous variables describing the mean total costs, costs of surgery and costs of hospitalization were compared between groups using the Man Whitney U test.p = 0.312). The available data from 7/13 FAMM flap patients and 15/18 RFFF patient regarding flaps surface area show a higher surface area for RFFF . The FAMM flaps covered defects created by the removal of specimens measuring from 3x3x2 cm to 6x4x2 cm in pathology after fixation. For the RFFF, the range spanned from 4 × 3,7 × 2 cm to o7,4 × 5,4 × 3,5 cm. All dimensions are detailed in Table Demographic data and data pertaining to the size of the primary tumor and the surgical defects to be reconstructed are shown in Table p = 0.008). The distribution of these complications is detailed in Table p = 0.065). It is worth noting however, that in the 11 instances of return to the OR, 9 were from the RFFF group while only 2 were from the FAMM group. The details and distribution of these complications are showed in Table As far as surgical complications are concerned, there was a statistically significant difference in the proportion of patients with postoperative complications not requiring return to the OR with only 1 patient in the FAMM group with 1 complication as opposed to 10 in the RFFF group with 15 complications (p = 0.002). For the remainder of the RFFF cases, a split-thickness skin graft was required to complete the closure while a secondary closure of a segment of the donor site incision was favored for FAMM cases.With regards to closure at the donor site, primary closure at the donor site was achieved in 33% of RFFF cases while 92% of FAMM flap donor sites were closed primarily  and anesthesia time was 8.5 h for FAMM and 10.7 h for RFFF (p = 0.000).The average surgical time for FAMM cases was 7.2 h compared to 8.9 h for RFFF (p = 0.717), time to decannulation (p = 1.00), duration of tube feeding (p = 0.449), speech (p = 0.314) or long-term feeding (p = 0.139) outcomes. The detailed distribution of speech and feeding outcomes is detailed in Fig. No difference was noted between the groups for length of hospitalization (p = 109). When broken down into its component, the average difference in the price of hospitalization was 6866 CAD (p = 0.482) while that of the cost of surgeries was 4193 CAD (p = 0.000). The details of the cost difference analysis are shown in Table As far as the cost difference analysis is concerned, we have calculated an overall cost difference in the total average cost between the two reconstructive options at 11,059 CAD  section the pedicle to allow for oral rehabilitation and 2) to revise a check scar for trismus. We have chosen to discuss these as part of the “complications” as they are not necessary in every case, and they must be accounted for in the cost involved in doing a FAMM flap. There was no flap failure in any group but 2 patients in the RFFF group required revision of the anastomosis, which was successful. While no flap was lost in our series, these cases highlight the fact that free flap reconstruction, although associated with a generally excellent rate at success in excess of 90%, is still associated with a 3–25% rate of re-exploration as a consequence of micro-anastomosis –18. We hIn well selected patients, we have demonstrated that functional results are similar between RFFF and FAMM flaps. The present study demonstrates comparable groups on the basis of the demographic and tumor specific data Table  as well Patients in the RFFF group had an average flap surface area that was larger than those in the FAMM flap group. However, a difference in flap size does not imply a difference in defect size in our group of patients. In the FAMM flap group, we tended to close the tongue primarily or by secondary intention while the FAMM flap was used to reconstruct the remaining subsites involved Fig. . In the Due to the retrospective nature of our study, we could not measure specifically the defect sites. But it is safe to infer that defects were similar in size based on the similar T classification distribution between the groups. As well, by excluding all FAMM and RFFF cases that did not meet the criteria of a “3 subsites defect”, we have created an extra condition to ensure the indications for a FAMM or RFFF would be overlapping as much as possible.From a health economics perspective, the cost difference analysis presented in this study shows a significant OR cost saving associated to the use of the FAMM flap. Free flap surgery is performed by a dual surgical team approach at our institution. While this approach has the merit of providing a setting for consultants to share ideas and expertise on a same case, it results in an obvious increase in the cost of the initial surgery. Moreover, the longer operative times and the presence of more nursing and paramedical staff for setting up the case and assisting two teams all contributed to increasing the price of the initial surgery. Of note, in the cases of FAMM flap, there was no specific code that could be billed for the reconstructive part of the surgery. Indeed, in the absence of a free flap performed by a second surgeon specifically, the price of the ablative and reconstructive portions of the surgery are bundled and determined based on the duration of the surgery. Hence, the absence of a specific code for the FAMM flap can further contribute to creating a price gap for the cost of surgery. This price gap is further compounded by the higher tendency to return to the OR in the RFFF group. Furthermore, while the number of nights of hospitalization did not vary significantly between the two groups, the average price of hospitalization was markedly elevated in the RFFF group. This is due to a subgroup of patients in the RFFF group requiring transfer to the intensive care unit (ICU) while no patient in the FAMM flap group was transferred to the ICU.specifically for the flap monitoring as opposed to other activities on the ward. This results in an underestimation of the cost of hospitalization of the RFFF group. Moreover, an in-depth analysis of preoperative work up costs, and costs of postoperative follow up visits, cost of care for recurrences, and cost of follow up with allied health professionals for each type of reconstructive method was not provided. Also, due to some difference in local practice habits, the use of certain OR personnel (for e.g. respiratory therapist) or the number of personnel involved in each case may vary and limits the applicability of this cost difference analysis outside of the setting in which it was conducted. For instance, in this study an average hourly rate of 31.63 CAD was considered for the presence of respiratory therapist. This represents on average an added 281.5 CAD per RFFF case and 227.7 CAD per FAMM case. However, the 53.8 CAD difference in the average cost difference between RFFF and FAMM due to the respiratory therapist’s salary does not invalidate our findings even in a different setting where respiratory therapists are not routinely involved in the OR. Indeed, the main driver of cost difference was not in the cost of surgery itself rather than in the cost of hospitalization.Our cost difference analysis was carefully designed to include as many direct and indirect costs related to the choice of the reconstructive method, a few caveats must be pointed out. Our institution has developed a post-operative monitoring protocol which allows patients with free flap reconstructions to be sent directly to a regular post-operative head and neck oncology ward. This has a major impact on the cost of hospitalization as it decreases the numbers of nights spent in the ICU hence making the cost difference less important at our institution compared to institutions where free flaps are routinely sent to the ICU at least for the first 24 h. Also, when calculating the price of the hospitalization for the RFFF group, the added nursing time dedicated to the monitoring of the flap following our protocol could not be accounted for precisely due to a lack of quantification by our institution’s accounting office of the extra personnel requested The FAMM flap is a useful versatile and safe option for the reconstruction of oral cavity defects but it has some technical limitations that should be considered when choosing a reconstructive option. Notably, in fully dentate patients it may require the extraction of a molar tooth for inset. While most of our patients in this series were edentulous at baseline, the dental status of the patient should be taken into account when considering a FAMM flap. The decision to extract a tooth should be weighed against the morbidity of the RFFF and discussed in conjunction with the patient in cases were both flap are acceptable options. Moreover, it may require pedicle sectioning for proper dental rehabilitation in up to one third of cases . HoweverA review of the literature demonstrates that locoregional flaps in head and neck reconstruction are undergoing a revival with multiple studies in the 5 years demonstrating the safety, functional, oncological and financial efficiency of locoregional flaps when compared to free flaps , 12, 21.The use of the FAMM flap for reconstructing medium-size defects in selected patients is associated with lower costs, shorter OR time, similar functional outcomes and a tendency to lower complication rates. A nuance should therefore be introduced in the framework of thought of the reconstructive surgeon to include, in selected medium size defects, this locoregional flap as an alternative to the RFFF.


Seasonal influenza viruses repeatedly infect humans in part because they rapidly change their antigenic properties and evade host immune responses, necessitating frequent updates of the vaccine composition. Accurate predictions of strains circulating in the future could therefore improve the vaccine match. Here, we studied the predictability of frequency dynamics and fixation of amino acid substitutions. Current frequency was the strongest predictor of eventual fixation, as expected in neutral evolution. Other properties, such as occurrence in previously characterized epitopes or high Local Branching Index (LBI) had little predictive power. Parallel evolution was found to be moderately predictive of fixation. Although the LBI had little power to predict frequency dynamics, it was still successful at picking strains representative of future populations. The latter is due to a tendency of the LBI to be high for consensus-like sequences that are closer to the future than the average sequence. Simulations of models of adapting populations, in contrast, show clear signals of predictability. This indicates that the evolution of influenza HA and NA, while driven by strong selection pressure to change, is poorly described by common models of directional selection such as traveling fitness waves. Seasonal influenza A viruses (IAV) infect about 10% of the global population every year, resulting in hundreds of thousands of deaths . VaccinaThe number of available high-quality HA and NA sequences has increased rapidly over the last 20 years  and viruThe underlying assumption of all these methods is that 1) differences in growth rate between strains can be estimated from sequence or antigenic data and 2) that these growth rate differences persist for long enough to be predictive of future success. Specific positions in surface proteins are of particular interest in this context. The surface proteins are under a strong positive selection and change their amino acid sequence much more rapidly than other IAV proteins or than expected under neutral evolution . EpitopeA mutation with a fitness benefit should have a tendency to fix, whereas detrimental mutations fix less often. Fixation and loss of a mutation is, however, not only determined by its intrinsic fitness benefit, but also by the fitness of the background it arose on and the fitness of competing variants . The degX in the genome of influenza that we observe at a frequency f in the population at a given date, what can we say about the future of X? The trajectory of a mutation will depend on its own effect on fitness, the contribution of the genetic background on the same segment, and the effect of the remaining seven segments. Here, we investigate properties of broad categories of mutations effectively averaging over different genetic backgrounds to isolate the effects intrinsic to novel mutations in this category.The main underlying question asked in this work is the following: given a mutation X at future times f(t). In other words, having observed a mutation at frequencies X will fix in the population, will disappear, or whether the site will stay polymorphic.First, we ask whether we can quantitatively predict the frequency of We use amino-acid sequences of the HA and NA genes of A/H3N2 (since the year 2000) and A/H1N1pdm (since the year 2009) influenza available in GISAID   for two time bins in a row, in which case amino acid iX is considered as “fixed” (resp. absent) in the population. Otherwise, the trajectory is considered “active.” Examples of trajectories can be seen in The central quantities that we derived from this data are “frequency trajectories” of amino acids at each position in the sequences. If an amino acid Since genomic segments of influenza do not recombine, concurrent trajectories of mutations on the same segment will be correlated. In the extreme cases where the two mutations always appear in the same strains, their two trajectories will be identical. This “nesting” of frequency trajectories may introduce biases when performing statistical analysis. In f(t) of a mutation until a given date t0, how much can we say about the future values of f after t0? We consider the idealized case sketched in f0 at time t0, what is the probability f at time Having observed the frequency trajectory posed by  to quantf0. The number of trajectories is limited and the frequency estimates themselves are based on a finite sample and are hence imprecise. Therefore, we consider trajectories in an interval To answer this question retrospectively, we use all frequency trajectories extracted from HA and NA sequences that satisfy these conditions for a given f0 while decreasing, even though they crossed that bin at an earlier time. This is due to the fact that some trajectories “skipped” the interval f0 in question on their initial rise due to sparse sampling. These trajectories are nevertheless rising in the sense that they start at frequency 0 for For f0, at least for some time. A fraction of those would then sweep through the population and fix.Since rapid sequence evolution of influenza HA and NA mediates immune evasion, one could expect that a significant fraction of new amino acid mutations on rising trajectories in f after a time f0 at time 0. The results for different f0. If a large fraction of the trajectories keep increasing after this time, we should see the “mass” of To quantify the extent to which this preconception of sweeping adaptive mutations is true, we estimated the probability distribution t = 0 and is essentially flat for t > 0 in the case of A/H3N2, and slightly increasing for A/H1N1pdm , and the initial peak at f0 is not visible anymore. The only information remaining from the initial frequency is the fraction that fixed or was lost (see below). This behavior is expected in neutral models of evolution  differs between D) of We conducted the same analysis for A/H1N1pdm, with results shown in f. f, mutations found in many regions or those that are older (in the sense that they have taken more time to reach frequency f) tend to fix more often than geographically localized mutations or more recent ones, but the effect is small. These two scores are in fact correlated, with older trajectories representing mutations that are more geographically spread, as can be seen in Since influenza is seasonal in temperate regions, geographic spread and persistence might be predictive of the success of mutations. We quantify geographic spread of a mutation by the entropy of its frequency distribution across regions  and its persistence by the age of the trajectory by the time it reaches frequency The results shown in L = 200 evolving in a fitness landscape that changes through time. A similar setting was used in . Each time bin is considered as a snapshot of the A/H3N2 influenza population and we will refer to sequences in time bin t for To assess the ability of the LBI to pick a close representative of the future, we compute the LBI of each node of one time bin in the tree using only the leaves that belong to that time bin. The top panel in The strain with the highest LBI is consistently closer to the future than the average strain by about one to two amino acids, whereas the overall distance increases linearly due to the continuous evolution of the population. We hence reproduce previous results showing that the LBI picks closer than average representatives . To inveThis near equivalence of the consensus and the strain with the highest LBI can be explained as follows: The LBI tends to be high for nodes in a tree that are close to the root of a dense and large clade. A typical sample of influenza HA sequences fall into a small number of recognizable clades, and the strains with maximal LBI will often be close to the root of the largest of those clades. This root of the largest clade will often be close to the consensus of the whole population, explaining the similar distance patterns. To test that hypothesis, we measure the hamming distance from the sequence of the top LBI strain to the consensus sequence for populations of all time bins. Predicting the trajectory of a mutation requires 1) significant fitness differences between genomes carrying different variants at the site and 2) a selection pressure that changes slowly over time. Under such conditions, it is expected that frequency trajectories will show a persistent behavior which would make them predictable for some time. However, we could find little evidence for such persistent behavior in the past 19 years of A/H3N2 evolution. This leads us to conclude that 1) A/H3N2 influenza virus evolution is qualitatively different from models of rapidly adapting populations (despite clear evidence for frequent positive selection), and 2) many methods to predict influenza evolution are unable to predict future frequency dynamics even though they work well for predicting the composition of the future population. This is illustrated by the case of LBI, which works primarily because it picks strains that represent the future well and not because it is predictive of future dynamics. A/H1N1pdm, in contrast, shows stronger signatures of predictability.t < 0 to t > 0, instead of showing “inertia.” This suggests that selective sweeps are not representative of typical trajectories  is not necessarily inconsistent with a lack of predictable dynamics. In fact, At the same time, influenza virus surface proteins show strong signatures of selection . Their pA recent study proposed that influenza virus evolution is primarily limited by an asynchrony between population level selection and generation of new variants within infected hosts . Along thttps://github.com/PierreBarrat/FluPredictibility/tree/master/src/config were removed.The sequences used are obtained from the GISAID database . Acknowlhttps://github.com/PierreBarrat/FluPredictibility.The code used to generate the figures presented here is available at For a set of sequences in a given time bin, we compute frequencies of amino acids at each position by simple counting. We make the choice of not applying any smoothing method in an attempt to be as close to the data and “model-free” as possible. This is especially important for the short-term prediction of frequency trajectories, as estimations of the “persistence time” of a trajectory might be biased by a smoothing method.t if an amino acid is seen under the lower frequency threshold of 5% (resp. above the higher threshold of 95%) for the two time bins preceding t, and above this lower threshold (resp. below the higher threshold) for time bin t. It ends in the reciprocal situation, that is when the frequency is measured below the lower threshold (resp. above the higher threshold) for two time bins in a row.We compute frequency trajectories based on the frequencies of amino acids. A trajectory begins at time In order to avoid estimates of frequencies that are too noisy, we only keep trajectories that are based on a population of at least ten sequences for “each” time bin. As said in the Results section, we also restrict the analysis to trajectories that begin at a 0 frequency, in part to avoid double counting. We find a total of 460 such trajectories. However, only 106 reach a frequency of 20%, on which Note that the fact that we use samples of relatively small sizes—at least for some time bins—can lead to biases in the estimation of frequencies. We show in i is the integral of all of the tree’s branch length around i, with an exponentially decreasing weight t being the branch length. When considering a time binned population, the LBI is computed once for each time bin by considering only the leaves of the tree that belong to the time bin. This means that only branches that ultimately lead to a leaf that belongs to the time bin are considered in the integration.LBI was introduced in  as an apτ parameterizes the distance over which the tree is informative of the fitness of a particular node. Here, we use a value of τ equal to a tenth of τ has little effect on the pick of the top LBI strain. By retrospectively optimizing its value, it is possible to reduce the average distance to the population 2 years ahead by The time scale X, we define its regional distribution using the numbers r that carry X. Regional weights are then defined as: For a mutation G(X) of X by using the Shannon entropy of the probability distribution We can then measure the geographical spread G(X) is a positive quantity that is larger when X is equally present in many regions, and equal to zero when X is concentrated in only one region.Region used are the ones defined in Nextstrain/flu . Those aN sequences i is a position in the sequence, i in sequence nσ, a a character of the alphabet and δ the Kronecker delta. The profile a at position i.Given a set of σcons such that: We then simply define the consensus sequence In other words, the consensus sequence is the one that has the dominant character of the initial set of sequences at each position.nx and my are sequences, and np and mq are the frequencies at which these sequences are found in their respective populations. For convenience, we also define In order to measure the distance of several predictor sequences to the future population, we rely on the Earth Mover’s Distance (EMD), a metric commonly applied in machine learning to compare collections of pixels or words . Here, wF under the conditions: We now introduce the following functional: nmw tells us how much of sequence nx is “moved” to sequence my. The functional F sums all of these moves and attributes them a cost equal to the Hamming distance nmd. The conditions on weights in w ensure that all the weight np of nx is “moved” to elements in Intuitively, the weight The minimization is easily performed by standard linear optimization libraries. Here, we use the Julia library JuMP .Molecular Biology and Evolution online.msab065_Supplementary_DataClick here for additional data file.


Essentially, we trap a single vortex along the length of a nanobeam and observe the transitions as a vortex is either trapped or released, detected through the shift in the beam resonant frequency. By exciting a tuning fork, we control the ambient vortex density and follow its influence on the vortex capture and release rates demonstrating that these devices are capable of probing turbulence on the micron scale.Since we still lack a theory of classical turbulence, attention has focused on the conceptually simpler turbulence in quantum fluids. Reaching a better understanding of the quantum case may provide additional insight into the classical counterpart. That said, we have hitherto lacked detectors capable of the real-time, non-invasive probing of the wide range of length scales involved in quantum turbulence. Here we demonstrate the real-time detection of quantum vortices by a nanoscale resonant beam in superfluid  4He with millisecond and micron resolution at temperatures of 10 millikelvin.Previous work has shown the detection of quantum turbulence with mechanical resonators but with limited spatial and temporal resolution. Here, the authors demonstrate real-time detection of single quantum vortices in superfluid  Quantum turbulence, the turbulence which occurs in quantum fluids, is much simpler than its classical counterpart, comprising single-quantised identical vortices. It can be thought of as an “atomic theory” of turbulence. This simplicity has led to the hope that understanding quantum turbulence can lead to insights into understanding the classical equivalent. Unfortunately, quantum turbulence is a microscopic phenomenon with significant length scales in the nanometre to micrometre range and we largely lack the tools to study liquid motions on these scales. Since quantum turbulence appears to behave as an ensemble of independent quantum vortices on short length scales but behaves very similarly to classical turbulence on larger scales, a critical scale length here seems to be the typical vortex separation. Thus, to advance our understanding, we need to be able to detect turbulence at the single-vortex level and on the scale of the vortex separation. Systems for trapping single vortices have been used previously4, but with much larger dimensions. Using another approach, the SHREK project5 has compensated for relatively large detector sizes by expanding the experimental space thereby making the scale of the flow patterns much larger.Despite the long history of classical turbulence, and its very significant impact on human affairs we still lack a theory, since the governing Navier-Stokes equations are only soluble in quite specific simple situations. The importance of a general solution to these equations is underlined by its inclusion in the Clay Institute, Millennium Problems4He in real time. Vortices can be trapped on nanobeam resonators, signalled by an increase in the beam resonant frequency. This allows us to follow the capture of a single vortex, its presence in the trapped state and its subsequent release via reconnection with a nearby vortex in the surrounding vortex tangle, controlled by the excitation of a nearby quartz tuning fork transducer, advancing our capability to probe vortex tangles on much smaller length scales and faster timescales than has hitherto been possible.Here we demonstrate the detection of single quantum vortices in superfluid 7 and ballistic 4He 9.The nanobeams we use for detecting single vortex events in real time have characteristic dimension <1 μm and response times faster than 1 ms. Such devices have recently emerged as highly sensitive probes of hydrodynamicQ ~ 2.8 × 103. The beam is driven at a velocity of only a few millimetres per second. This is orders of magnitude below the expected velocity for the onset of turbulence production10. Therefore, in all our measurements the beam response is linear. The frequency width arises almost entirely from intrinsic losses in the beam material. Although while operating the beam in the liquid, we do see a vanishingly small increase in damping above the vacuum value arising from a very small level of acoustic emission into the liquid, but the contribution from the superfluid is essentially negligible.The beam used here has a vacuum frequency of 2.166 MHz, and at the measurement field of 5T has a 3N4 nanobeam with a 130 × 200-nm cross-section provides the vortex detector.Figure 12. The 2-ms time-analysis interval represents an optimal compromise between fast detection and the frequency resolution of the high-Q resonator.Figure α (see figure) it gradually increases and stabilises in the region β to γ before abruptly resetting to the initial low-frequency state at time δ. We can identify and associate each change with the successive stages of the nanobeam’s interaction with the vortex tangle.The pattern of the events in the figure, with the frequency intermittently jumping from a low to a higher value and back again, is maintained over the many hundreds of such interactions we have recorded. Initially the beam frequency is low and stable. At time δiαi+1). This is the only beam response in turbulence-free superfluid, and we identify it with the vortex-free beam. In this state, the beam resonance frequency is reduced by 50 kHz from its vacuum value, consistent with the added effective mass contributed by the volume of superfluid displaced.Referring to Fig. 8.The damping of the beam in this state, inferred from the resonance width, is identical with that in vacuum. Therefore there is no significant added dissipation mechanisms in the presence of the superfluid, as expected from the low phonon and roton damping at a temperatures of ~10 mKβiγi, some ~3 kHz higher than the low state, represents the case where the nanobeam has trapped a singly quantised vortex along its entire length. The state is metastable, but will last for several days in the absence of local turbulence, and survives even if the beam motion is ceased, restarted or driven quite hard. However, upon restarting the turbulence source, the beam relaxes to the default state with the lower frequency described previously.We believe that the plateau state F = ρvs × κ, with ρ the fluid density, vs the superflow created by the image at the position of the beam’s vortex and κ the circulation2, which displaces the beam towards the substrate, thereby increasing its tension and thus yielding an increased beam resonant frequency when a vortex is trapped along it. For a fuller treatment see the The identification of the capture of a singly quantised vortex by the beam is confirmed by several observations. First, the captive vortex generates an additional restoring force increasing the beam’s resonance frequency. This arises from the attractive interaction between the trapped vortex and its image in the nearby substrate. The interaction of the vortex with the parallel image vortex gives rise to a static force Q ~ 3000) and since the beam has a very non-symmetrical vertical/horizontal cross-section (130 × 200 nm), and also very different terminal geometries in the vertical and horizontal planes, the horizontal and vertical resonances are likely to be very widely separated with a vanishing probability of any overlap. With our multi-frequency methods we can readily probe for other resonances in the vicinity of the beam’s horizontal frequency and have not seen anything. We can thus justify discounting this source of frequency change. It would be beneficial if we could indeed specifically excite such a Magnus mode as it might help to reduce the influence of the substrate.At this point we should note that because the trapped vortex generates a circulation around the beam, there is a possibility that a Magnus force also comes into play, applying a vertical force as the beam moves transversely. If this force can distort the beam further, an additional frequency shift should occur. Any vertical Magnus force would necessarily be applied at the frequency of the resonant horizontal motion. Thus, for a Magnus force component to come into play, the vertical and horizontal resonant lines would have to overlap to excite a vertical motion. Bearing in mind that these are very narrow resonances , supporting the idea of the capture of a singly quantised vortex. While double or even higher-order quantisation is not energetically unfavourable, it is hard to imagine any creation mechanism. Trapped multiply quantised vortices would yield discrete higher-frequency plateaus which have not been observed.Secondly, the damping of the beam hardly differs from that of the vortex-free or vacuum state, as expected, since the capture of a single vortex should not significantly change the acoustic emissionαiβi and γiδi between the default and metastable states to the capture and the release of a vortex by the beam. The latter process is always instantaneous on the scale of our detection time and is governed in some way by reconnection of the trapped vortex with a crossing vortex in the surrounding superfluid.We now can attribute the transitions 3, and as shown in the cartoon of Fig. We have better understanding of the trapping process. A length of nearby vortex, lying more or less parallel to the beam, interacting with its negative image in the beam, will experience both an attractive force towards the beam and a perpendicular transverse force parallel to the beam surface. Therefore, the vortex will gradually spiral into the beam, finally being trapped, as discussed by GriffithsThe observed process of vortex capture by the beam is indeed a much more gradual process than the release. In addition to the “completed” events shown in Fig. We should emphasise here that the behaviour of the capture and release processes is completely different. We can show that by looking at the effect of the local vortex density on these two processes.13. At our temperatures, with vanishing normal fluid density, and thus slow vortex decay, the action of the fork fills the entire experimental volume with essentially uniform vorticity. In panel a of the figure, we show the tuning fork velocity as a function of the driving force. The clear jump in the slope of the tuning fork response (marking greatly increased dissipation) indicates the onset of turbulence production, see for example references15. As discussed below, the results suggest that the trapping process is governed by the ambient vortex density in the immediate surrounding of the beam, say within a distance of a few micrometres.To discuss the results, we begin with the effect on the capture process shown in Fig. a we also plot a summary of the single-frequency measurements of the detection event rate, τ−1, defined as the inverse mean waiting time τ for an event to occur. The event rate increases with the fork’s velocity confirming that the nanobeam probes the surrounding tangle density. We only detect vortices at tangle densities corresponding to fork velocities above 73 mm s−1. At this velocity, the rate of detection is very low with the shortest waiting time between the events being ~40 s and the longest ~1000 s.  emitted by the local tangle. However, whatever the detailed process, it is worth emphasising again that the capture process is governed by the surrounding vortex tangle density.Panel tαγ of vortices on the nanobeam at five tuning fork velocities. First, the typical lifetime of a captured vortex state is three orders of magnitude shorter than the wait time between events. Secondly, the data display no discernible dependence on the tuning fork velocity, showing that the release is insensitive to the overall vortex tangle density. This is surprising since we know that the captured state can exist essentially indefinitely if the vorticity is turned off (carefully to avoid dislodging the vortex in the process). Thus, although we understand the “on” and “off” states of the beam, we do not yet fully understand the processes leading to jumps between them.Once the vortex is captured, its lifetime follows a very different dependence. The release must depend on the proximity of another vortex for annihilation and thus should also carry information on the surrounding tangle. Figure 19 and simulations of quantum vortex behaviour20 show that the timescale, t, for vortex–vortex interactions displays a square root relationship with the vortex spacing δ as κ is the circulation quantum and A a constant of order 1, depending on the geometry of the approaching vortices19. This expression and our range of lifetimes of 3–100 ms  composite nanobeam. The beam’s dimensions are defined lithographically, with length l = 70 μm and width w = 200 nm. The 100-nm-thick Si3N4 layer determines the mechanical properties of the beam, while the Al layer allows the magnetomotive excitation and measurement of the beam motion. The combined thickness of the aluminium and silicon nitride layers is t = 130 nm, with a combined density of 3062 kg m−3. The vacuum frequency of the fundamental mode is determined experimentally to be f0 = 2.166 MHz. The nanobeam is suspended approximately d ~ 1 μm above the silicon substrate. The experiment is housed in a brass experimental cell containing superfluid 4He at a temperature of 10 mK, mounted on the mixing chamber of a cryogen-free dilution refrigerator.The nano-electromechanical device system (NEMS) consists of a doubly-clamped aluminium-on-silicon nitride  lock-in amplifier. With the driving frequency fixed on resonance, any change in the nanobeam resonance frequency is detected as a drop in the measured signal.11 is used in place of the signal generator and high-frequency lock-in. The MLA instrument employs a frequency comb comprising integer multiples ni of a base tone fb such that all measurement frequencies fi satisfy fi = nifb. For distinguishing between tones, the measurement time tm must be larger then the inverse separation between frequencies tm > 1/fb. This constrains the time resolution and frequency spacing of the instrument, and faster measurements require the frequencies to be spaced further apart. It is also pertinent to note that the non-linearities of the resonator will cause mixing between the frequency tones although the use of low-excitation drives avoids this problem12.For simultaneous detection at multiple frequencies, a multi-frequency lock-in amplifier (MLA)For both resonance tracking techniques, an oscilloscope is used in conjunction with the lock-in demodulation in order to record vortex capture events. The lock-in demodulated signal at the vortex-free resonant frequency of the beam is monitored by the oscilloscope, which triggers the lock-in amplifier to record data when the signal strength falls sufficiently from the resonance frequency shift. For single-frequency measurements the fall and subsequent rise in the signal would then yield the event lifetime. In the multi-frequency measurements the recorded data is fitted with a Lorentzian peak to obtain the beam’s resonate frequency as a function of time, and the lifetime is then found from this data.22 (trans-impedance amplifier) to recover the signal which can then be used to find the fork velocity24. The driving force on the fork can be found from the drive signal using well-established techniques24.Similarly, the tuning fork is measured with a vector network analyser, using an I–V converterSupplementary informationPeer Review File


Following the publication of the original article , we wereThe corrected Fig. Errors have subsequently been identified in the original publication, and the following correction should be noted:While analyzing the results of “nanoparticles in the concentration and size of exosomes” in the research, the result analysis of the original Fig. 1b is inaccurate. The corrected Fig.


Hearing is one of the earliest senses to develop and is quite mature by birth. Contemporary theories assume that regularities in sound are exploited by the brain to create internal models of the environment. Through statistical learning, internal models extrapolate from patterns to predictions about subsequent experience. In adults, altered brain responses to sound enable us to infer the existence and properties of these models. In this study, brain potentials were used to determine whether newborns exhibit context-dependent modulations of a brain response that can be used to infer the existence and properties of internal models. Results are indicative of significant context-dependence in the responsivity to sound in newborns. When common and rare sounds continue in stable probabilities over a very long period, neonates respond to all sounds equivalently (no differentiation). However, when the same common and rare sounds at the same probabilities alternate over time, the neonate responses show clear differentiations. The context-dependence is consistent with the possibility that the neonate brain produces more precise internal models that discriminate between contexts when there is an emergent structure to be discovered but appears to adopt broader models when discrimination delivers little or no additional information about the environment. By the time a new life exits the mother, it has quite a lot of experience with sound information2. We know newborns can recognize familiar voices from birth or earlier4, localize sound sources5, and even separate concurrent sound sources6. However, less is known about how sound is processed in relation to its context in the early stages of brain development. This study was designed to further our knowledge on how the neonate brain utilizes sound information within structured sequences.Hearing is one of the earliest senses to mature with the developing fetus being capable of registering sound from as early as 18 weeks gestation7. Electrodes are placed over the scalp surface while a structured sequence of sound is presented. This methodology has provided insight into statistical learning in the neonates that enables extraction of the kinds of abstract pattern learning required for music and language learning10. For sound sequences of predictable structure, with increased exposure, the brain becomes less responsive to predictable events and more responsive to events that mismatch one’s predictions11. This is reflected in the event-related potential (ERP) responses. The ability to discriminate between responses to predictable and prediction-violating sounds can therefore be used to infer the existence of internal models of the environment representing a prediction or inference about the sensory input most likely to be encountered next13. This methodology is ideal for use in neonates because the process occurs automatically and does not require a task or explicit attention21.The recording of auditory event-related brain potentials in this study utilizes a methodology that can be applied in neonates in the same way as in adults23). A significant literature places the precision-weighting in the gain on output from specific cell populations in responsive cortical areas  acknowledging that this reflects a hierarchical network interaction both within and between brain regions. In other words, a seemingly simple deviance-detection indicator is subserved by a sophisticated iterative inferential process that operates over many different timescales27. Within scalp-recorded responses, this process has been linked to change over time in several components of the auditory ERP28. In adults, the key difference between pattern-matching and mismatching sound emerges within 100–250 ms after the point of deviance and is maximally recorded over fronto-central electrode sites when a nose or mastoid reference is used. This “mismatch” sensitivity (mismatch negativity or MMN) is captured in a difference waveform but in the tone responses it often overlaps early components known as the N1 and P2 and is considered part of the N2 complex31. The precision-weighting can be seen both as changes in the response to pattern matching sounds that become less negative with repetition and pattern stability over time and in the mismatch response which becomes more negative over this period.In adults, the amplitude of the response to sounds that violate established patterns is proposed to reflect a precision-weighted signal influenced by the goodness of evidence upon which the internal model is built  is typically dominated by a positive waveform appearing 200–400 ms from the onset of the deviation , which may be surrounded by an early and a late negative difference, depending on the acoustic properties of the stimuli, the amount and type of separation between the regular and irregular sounds, etc34. Although initially understood to be an indicator of probability30, the mismatch process shows sensitivity to violations of established sound transitions36 requiring a more contextualized form of learning. This type of statistical learning involves extraction of regularities in how features and objects co-occur in the environment over space and time. Importantly, previous studies showed that properties of the infantile electric brain response to auditory deviance suggest that it reflects a prediction error37, similarly to the analogue response in adults38. The infantile MMR response is elicited both by violating regularities based on individual sounds and on short sound patterns39, bringing up the possibility that, again similarly to adults40, they may also reflect the large-scale structure of the auditory context.While the morphology of ERPs is rapidly changing within the first year of life and the stable componentry of adulthood only emerges during childhood, ERP signs of detecting auditory deviance are present right from birth44 might also be observed in neonates. Here we provide evidence that the brain of newborn infants can indeed form internal models that differentiate sounds based on probability, and weight that response based on precision, but that the propensity to do this depends on contextual factors that may be related to a determination of the inferred value of tracking information over time. Differential responses to sound may only occur when a sound provides information about longer-term structures in the environment. Thus, the same acoustic differentiation may or may not be automatically made, depending on its relevance for revealing important information about the context.The study was designed to determine whether the differential precision-weighting based on large scale structure seen in adults11 such that the two sounds were delivered with largely different probabilities . Experiment 1 is referred to hereafter as the context 1 control. A different group of newborns heard similar sequences but with the sounds in reverse probabilities (Experiment 2), which is hereafter referred to as the context 2 control. When adults hear sequences of this kind, the responses to the common and rare tones differentiate such that the response becomes more “suppressed” or more positive at fronto-central scalp sites with increased exposure, and “larger” or more negative to rare deviations11. Stable sequences of this kind are also considered ideal for formation of precise internal models and were designed based on sequences for which probability-dependent response were observed previously in neonates16. However, neither group of infants produced responses to rare tones that were significantly different to the common tones, and indeed the responses from each group were remarkably similar, both in amplitude and morphology.The study featured three experiments with three separate groups of neonates with the sound sequence design depicted pictorially in Fig. 45. In newborn infants, the latency of responses elicited by auditory sensory deviance have been found to depend on both the specific acoustic feature and the amount of separation32. In previous neonate studies of tone duration sensitivity, responses to common and rare sounds tended to differentiate at about 150 ms through to 300 ms16. To capture this window, responses were quantified using the mean amplitude over 150–300 ms at the Cz scalp electrode. An additional period of analysis was included to capture earlier components using a mean amplitude over 50–150 ms. The component amplitudes are presented in Fig. context  and a within-subject factor for probability .In auditory ERPs, the early period of response is considered to reflect relatively more sensory-based processes with the later components progressively more cognitive in nature48. Certainly, the brain responses of our two groups of infants show no significant evidence of a discrimination being made between the rare and common sounds in either control sequence. Thus, by these data alone, one would infer that these neonates were not able to discriminate the duration difference between 100 and 250 ms long tones. However, contexts 1 and 2 were quite clearly distinguished when the same tone sequences were concatenated alternatingly in a continuous sequence. Further, there was also indication of the rare properties being differentiated over time.In adults and neonates, the absence of a differentiation in response to a rare and common sound with different physical properties is typically considered evidence that the two tones could not be discriminated50 , another group of newborns were presented with the same tones as those used in the control contexts, but organized into an alternating sequence, switching back and forth between periods of context 1 and 2 modelled on sequences used in adultscontext  and probability  revealed a significantly larger positive polarity response over 50–150 ms in context 1  = 6.269, p < 0.019, η2 = 0.188) reflecting this earlier positive response to tones in context 1 than 2. A mixed model ANOVA comparing context 1 early responses between the corresponding control and alternating experiments also indicated significantly more positive responses over 50–150 ms in the alternating sequence group than in the control group  = 16.968, p < 0.001, η2 = 0.210). There were no significant main effects or interactions for the equivalent comparisons of response amplitudes for context 2. These differences are clear in Fig. A repeated measures ANOVA of amplitudes obtained in the alternating sequence with within-subjects factors of 50, the data was further explored for evidence of probability differentiation by separately examining responses obtained from the first 2.1 min and second 2.1 min periods . The component amplitudes for these divisions of the data are presented in Fig. probability  × context ] was conducted to test the responses for effects of probability difference, separately for the first and the second 2.1 min periods and for the early and late measurement intervals. Examination of the responses in the first 2.1 min period revealed a significant effect of context over the 50–150 ms time window  = 11.393, p < 0.002, η2 = 0.297), where responses were significantly more positive in the first than the second context. The effect was further modified by tone probability  5.464, p < 0.027, η2 = 0.168) with the differences being larger between rare than between common tones, of which only the former reached significance (t(27) = 4.034, pholm < 0.001). There were no significant main effects or interactions for amplitudes measured in the 150–300 ms window over this first 2.1 min. However, importantly, no significant main effect was found for tone probability over this first 2.1 min period for either context.Given evidence that the infants could differentiate the contexts, and evidence in adults that the precision-weighting on auditory responses change dramatically over time within alternating sequencesF = 7.284, p < 0.012, η2 = 0.212). As can be seen in Fig. m = 0.325 µV vs. m = 0.944 µV, respectively). In summary, the differentiation in the event-related brain responses within the first 2.1 min primarily reflected the context that the tones were presented in, but by the second 2.1 min the tone probabilities were differentiated with the rare ones eliciting more negative response over the 150–300 ms; the same window as previously observed to capture a negative mismatch response16.In contrast, examination of responses in the second 2.1 min period revealed no differences over 50–150 ms. However, there was a main effect of probability for the 150–300 ms period (25). The estimate of volatility/precision then weights the brain response leading to changes in response amplitude over time. Where a model has been formed based on highly reliable information, the response to a model-matching sound should be very suppressed and responses to model-mismatches highly salient51. The classic oddball sequences used for control contexts 1 and 2 are considered ideal learning environments for building precise internal models provided that the brain can distinguish between the common and rare elements. Earlier studies indeed suggest that the infant brain can distinguish the durations used here16, and the present data from the alternating sequences indicates that our infants did also discriminate the two durations. The key question is why the neonates did not do so in the classic oddball control sequences. The main findings of the present study are discussed below in relation to a thesis that the infant brain is sensitive and responsive to the information carried by sound, and that brain responses are adjusted to this determination accordingly. This sensitivity to the information carried by sound would be reliant upon the extraction of regularities in how features and objects co-occur in the environment over longer timescales which expands the view of statistical learning to the large scale structure.The context-dependence of neonate responses to sounds imply that the infant brain should be considered an intelligent consumer of information rather than as a passive processor of sound sequence material. Internal models of the environment are proposed to contain an estimate of how volatile the environment is . The responses elicited to the common and rare instances of these two sounds were remarkably similar in the control experiments. In contrast, the juxtaposition of the two contexts in the alternating sequence revealed an ability to discern the boundaries within the sequence where the probabilities of the two sounds changed, moving from a stronger tendency towards encountering the shorter tone after a period of hearing a stronger tendency towards encountering the longer tone and vice versa. The key indicator that the babies detected these boundaries lies in the point at which the positive shift onsets in the response morphology, which is much earlier for context 1 than 2, and much earlier in context 1 for the alternating experiment than for the control experiment. There was therefore a strong dependence of the morphology of the infant response based on the very long-term structure of the listening environment.53). Stimulus specific adaptation is an important mechanism through which the auditory system tunes responsivity to reflect redundancy and sharpen discrimination ability54 and distinguish information carrying signals from background noise55. The processes underlying how sound statistics affect adaptation are complex and sophisticated53, and while they are argued to be insufficient to fully account for deviance sensitivity24, they could contribute to the general context effect in the alternating sequence. Each of these is discussed in turn with respect to how well they fit the current data. Firstly, sounds of different duration carry different levels of sound energy56, and as such, it is possible that context 1 and 2 could have been differentiated based on a general shift in sound intensity over time. This sensory property-based distinction could be considered consistent with an effect appearing early within the event-related response45, possibly even earlier than the point of difference in tone length at 100 ms ) but only 250 ms in context 1 (250 ms common tones with a 500 ms SOA). In adults, this difference in silent interval changes the degree of adaptation. However, once again in adults this is evident mostly approximately 200 ms after tone onset57. Finally, the silent intervals are slightly longer in the control conditions (550 ms for context 1 and 700 ms for context 2 which) which also could be associated with different levels of adaptation and could again affect the amplitude of the positive component that occurs at about 200 ms after tone onset. However, a difference in adaptation is not evident between the two control conditions. Further, this effect would create a more positive response in the control context 1 than in the alternating context 157. In contrast the opposite of this has been found here. In sum, the current data does not support the notion that adaptation alone is likely to explain the effects in neonates distinguishing the different contexts or the alternating and the control sequences. Therefore, instead it is proposed that the distinction in the responses only emerges where the statistical properties of sound carry information about a longer-term structure within the sound sequence. In other words, we propose that the babies can distinguish between the two sound contexts, but they do so only when differentiation is meaningful to isolate information needed to encode larger scale structure.There are a number of properties that could influence the responses to tone changes at the boundaries through a process called adaptation . Interestingly, the data also suggest this was particular to the first context heard. The first context, regardless of whether long or short tones are common, is also associated with higher precision models in adults44. The alternating sequence responses therefore provide evidence that newborns can not only determine the sequence structure60, but can also form internal models that build up in precision over time enabling an outlier to be detected. A progressive refinement of cortical response in neonates over time has also been seen in statistical learning of language8 which is also compatible with the concept of precision-weighting but in the present case it is demonstrated with respect to relative probability. These observations add to a body of work showing how such learning is shaped by many longer timescale characteristics including hierarchy, order, entropy, and uncertainty9.It was also only when the sounds were embedded within a longer structure that evidence of an ability to discern one sound as a local rare outlier emerged. When responses from the first and second half of the contexts were separately averaged, it became evident that responses appearing initially within a “new” context tended to reflect the differentiation of the context only . Later on, when the context had been stable for a while, a significant longer-latency negative shift was seen in response to the rare tone in context 1, consistent with prior observations of deviance detection16 the infants always heard sequences of less than 7 min duration with breaks between successive presentations. Thus, a potential explanation for the absence of tone differentiation in the current control sequences is that rare changes to the duration of sound within a long constant sequence carried insufficient information to warrant considering a different model, as there was no alternative offered by the sound input. Once again, the babies can distinguish between the two sounds, but perhaps cease to do so when it offers no new information about structure.So why were infants not sensitive to the outliers when presented within a listening context that should have been ideal to observe differential responses? One possible reason for the longer-term structure of the sound sequence influencing sound differentiation may relate to a trade-off between the information value of maintaining precise internal models and the associated investment of effort. In the control sequences, the babies heard 3000 tones in a constant uninterrupted probability arrangement over a 40-min period. In the alternating sequence, the exposure to the two contexts occurred in blocks of 500 stimuli: thus, the maximum period of exposure to one sound probability arrangement was shorter than 7 min. Similarly, in prior studies showing the ability of young infants to distinguish between sounds of two different durations61. Under this broader more inclusive model, all sounds heard may be captured as predicted such that the absence of differentiation in the responses does not reflect an inability to distinguish them, but rather a determination that distinguishing between them carries insufficient benefit to warrant the effort involved. In contrast, when these same two sounds occur within the alternating sequences, two different acoustic states are present in the environment and modelling these two different states may become energetically more beneficial. This suggests that the optimization of resources, a potential driver of cognitive development62, is already evident in the first few days after birth. The above account is fully compatible with a predictive coding theory of brain function suggesting that a fundamental operating principle is to seek to reduce variational free energy, that is to reduce surprise by utilizing statistical information to anticipate upcoming states63. Observations enable accumulation of a probability density over their hidden causes, and through a process of active inference, an internal model of the most likely next state can be inferred and refined over time based on the adequacy of the fit to new evidence. The optimization of a model to reduce free energy can be governed by weighting whether to aim for accuracy of predictions over the shorter or longer term by the energy required to achieve that level of accuracy. Developing or maintaining sensitivity to the two sound durations is beneficial to extracting the predictable structure in the alternating sequence, but not in the control sequence. This kind of contextualized processing is another example of how expectations can shape responses to sound64, with the present observations suggesting that some such modulations are so fundamental to learning that evidence can be seen in the first few days of life, even in sleeping neonates.When exposed to oddball sequences over a long period of time, a broader and more inclusive model that includes both sound durations would avoid attention capture by the unimportant deviations, potentially conserving energy for modelling other aspects of the environment32) which complicates the study the computational principles underlying them (see for discussion32). There are a number of studies, for example, that refer to a larger central positivity as being indicative of deviance detection for some features in infant data33 but this was not observed here and may not be how duration deviance is reflected in neonate ERPs where previously, a more negative response has been observed66. Notwithstanding, we suggest the context-sensitivity and changes over time in responsiveness present in these neonate data are indicative of the way perception is shaped by modelling of complex patterning in the environment.A limitation of the present study, as noted in the introduction, is that the stable componentry of adulthood only emerges during childhood. The responses observed in neonates are incomparable to those in adults  and the emergence of differentiation of probable features in a precision-weighted manner. These feats are important for infants to learn from the environment, finding regular sequences of events within constantly changing stimulation of typically several sources of information/stimulation present concurrently. This ability would seem pivotal to explain the readiness with which infants learn critical interaction skills such as understanding speech and entering into communication exchange and dialogues16 and g*power estimates suggest the power of observing a significant mismatch response if present exceeded 0.99. All participants had an Apgar score of 9/10. Infants were predominantly asleep during the recordings, in quiet sleep 78% of the time, and in active sleep 16% of the time. Informed consent was obtained from one (mother) or both parents. The experiment was carried out in a dedicated experimental room. Experiments 1 & 2 were carried out concurrently with babies allocated to experiments in counterbalanced order. Experiment 3 was carried out separately with all babies hearing the same sequences. Importantly, all recordings were carried out in the same manner, in the same setting. The study was conducted in full accordance with the Declaration of Helsinki and all applicable national laws and was approved by the relevant ethics committees: Medical Research Council-Committee of Scientific and Research Ethics (ETT-TUKEB), Hungary.Electroencephalographic (EEG) data useful for the current analyses was recorded from 110  healthy, full-term infants 0–6 days postpartum. Data collected from another 14 infants had to be discarded: three due to recording errors (no trigger was recorded) and eleven due to failing to meet criteria for data quality  with ER-1 headphones  connected via sound tubes to self-adhesive ear cups  placed over the infants’ ears.11 with one sound being common (p = 0.85) and the other rare (p = 0.15), and the sounds occurring pseudo randomly constrained such that a minimum of three common sounds occurred between rare sounds. In context 1, the long sound was common, and the short sound was rare, and in context 2, the probabilities were reversed.The sequence presented in the control conditions were based on sounds used previously in similar infant studies yielding significantly different responses to common and rare sounds . The two harmonic complex tones of different duration , comprised of three pure sine frequency components . All tones were delivered with a uniform 70 dB intensity. The tones were presented in a typical oddball design41 and SOAs of 300–500 ms have been used successfully in other neonates studies69. Each group of 500 sounds began with a minimum of four occurrences of the new common sound. The sequence was presented three times with a 1 min silent period between them and always started with a context 1 block.A diagram depicting the structure of the sequences presented to each group is presented in Fig. 70 plugin to EEGLAB71 within MATLAB. The EEG signals were passed through a digital bandpass filter between 1 and 25 Hz and epochs of − 100 to 500 ms with respect to the sound onset were extracted for each stimulus. All amplitude measurements and illustrations are referred to the 100-ms pre-stimulus interval, the baseline.EEG was recorded with Ag/AgCl electrodes attached to the Fp1, Fp2, Fz, F3, F4, F7, F8, T3, T4, Cz, C3, C4, Pz, P3, P4 locations according to the International 10–20 system. The reference electrode was placed on the tip of the nose and the ground electrode on the forehead. Data was recorded using a direct-coupled amplifier  at 24-bit resolution and a sampling rate of 1000 Hz. Data processing was conducted using the ERPLab72.Epochs were subjected to an artifact rejection process where epochs with a voltage change exceeding 150 μV were rejected from analysis. The data quality was considered acceptable if a minimum of 50% of the epochs were retained for every condition tested, for electrodes F3, Fz, F4, C3, Cz, C4, P3, Pz, P4. Responses were averaged separately according to sound duration and probability . For testing the long vs. short discrimination, the responses in the alternating condition were further divided into averages that separately captured the first and second halves . The first was the mean amplitude between 50 and 150 ms after sound onset to capture early components of the response. The second was the mean amplitude between 150 and 300 ms after sound onset which captures the period over which a significant difference was present in the response to common and rare tones in prior work (12) indicating that a mismatch to the prevailing internal model was detected. Separate ANOVA analyses were conducted on the 50–150 ms and 150–300 ms amplitude measurements. These are described in the results section with some being within-subject and others between-subject as required. Post-hoc Holm-Bonferroni corrected tests were employed to explore significant interactions. Effect sizes are reported as partial eta squared and the significance criterion level was α < 0.05. All statistics were computed using JASP (version 0.13.173).Two time windows were used to quantify the amplitudes within the central (Cz) responses, the electrode selected as it typically best captures deviance-related effects in neonates (e.g., Supplementary Information.