This study aimed to jointly model HIV disease progression patterns based on viral load (VL) among adult ART patients adjusting for the time-varying “incremental transients states” variable, and the CD4 cell counts orthogonal variable in a single 5-stage time-homogenous multistate Markov model. We further jointly mapped the relative risks of HIV disease progression outcomes (detectable VL (VL ≥ 50copies/uL) and immune deterioration (CD4 < 350cells/uL) at the last observed visit) conditional not to have died or become loss to follow-up (LTFU).Secondary data analysis of individual-level patients on ART was performed. Adjusted transition intensities, hazard ratios (HR) and regression coefficients were estimated from the joint multistate model of VL and CD4 cell counts. The mortality and LTFU transition rates defined the extent of patients’ retention in care. Joint mapping of HIV disease progression outcomes after ART initiation was done using the Bayesian intrinsic Multivariate Conditional Autoregressive prior model.λ23): HR = 1.83 and (λ34): HR = 1.42 respectively). Regarding the time-varying effects of CD4 cell counts on the VL transition rates, as the VL increased, (λ12 and λ23) the transition rates increased with a decrease in the CD4 cell counts over time. Regardless of the individual’s VL, the transition rates to become LTFU decreased with a decrease in CD4 cell counts. We observed a strong shared geographical pattern of 66% spatial correlation between the relative risks of detectable VL and immune deterioration after ART initiation, mainly in Matabeleland North.The viral rebound from the undetectable state was 1.78times more likely compared to viral suppression among patients with VL ranging from 50-1000copies/uL. Patients with CD4 cell counts lower than expected had a higher risk of viral increase above 1000copies/uL and death if their VL was above 1000copies/uL (state 2 to 3 (With high rates of viral rebound, interventions which encourage ART adherence and continual educational support on the barriers to ART uptake are crucial to achieve and sustain viral suppression to undetectable levels. Area-specific interventions which focus on early ART screening through self-testing, behavioural change campaigns and social support strategies should be strengthened in heavily burdened regions to sustain the undetectable VL. Sustaining undetectable VL lowers HIV transmission in the general population and this is a step towards achieving zero HIV incidences by 2030.The online version contains supplementary material available at 10.1186/s12976-021-00145-y. Antiretroviral therapy (ART) has since been the backbone of HIV prevention and control as it reduces viral load (VL) replication in the human host by blocking the virus life cycle . Once thMultistate Markov models are mathematical models which have been used to evaluate HIV disease processes; however, the number of states, state cuts-off points and the number of transitions vary across studies , 4. ThisThe low-middle-income countries (LMIC) have traditionally relied on the use of CD4 cell counts in HIV disease progression monitoring as this has been the readily available laboratory marker; however, if both VL and CD4 cell counts are available, it is imperative to model these prognostic markers jointly to understand better the HIV disease progression patterns. Joint modelling of these two prognostic markers helps to explain those effects which one marker cannot explain in the absence of the other marker.In HIV disease monitoring programmes, Bayesian spatial modelling is an emerging tool to analyze spatially related multidimensional data with an underlying spatial process to guide policy , 10; howThe main objective of our study was to jointly model HIV disease progression using two 5-stage time-homogenous multistate Markov models based on CD4 cell counts and VL. We firstly fitted two time-homogeneous multistate Markov models with states defined by CD4 cell counts and VL. In each multistate model, we jointly model these prognostic markers with one marker defining the finite multistate model states and the other marker forming the covariate matrix components of the regression model. The first covariate was an orthogonal variable generated using the principal component analysis (PCA) . The secThis study was a secondary analysis of data from patients’ records compiled for monitoring and guiding programme planning. The data used in this study came from the Zimbabwe National ART programme collected through the electronic patients’ management system (ePMS) database described elsewhere , 13. TheWe considered individuals aged 15 years and above, with at least two repeated measurements of both CD4 cell counts and VL who initiated ART between 2004 and 2017. However, since this was programme data, the follow-up visits of the patients were intermittent, and there may have been clinical reasons for requesting CD4 count and VL measurements from patients. Each individual had an average follow-up period of 2 years, and the majority of the patients (79.2%) in the final sample considered for analysis were enrolled on ART between 2009 and 2015.X(t), t ∈  for both the CD4 cell counts and VL multistate models.Table λij, VL(t) = λij(0) exp(βij × CD4state)and λij, CD4(t) = λij(0) exp(βij × VLstate)] (model 3) were better fit compared to the no covariate models (model 1). Simultaneously adjusting for the orthogonal variable and the time-varying effects on both the CD4 cell count model and VL model (model 4) further improve the model, P-value< 0.05; hence, the interpretation of results was based on these models (model 4).Both the CD4 cell count and the VL multistate models showed that the model with the orthogonal variable (model 2) was a better fit compared to the no variate model (model 1). Similarly, the multistate models which adjusted for the time-varying effects of the other prognostic marker, [i to state j for i > j defines an increase in CD4 cell counts which indicates immune recovery process while i < j defines a decrease in CD4 cell counts, which means immune deterioration process.The orthogonal and the time-varying VL effects were regressed in a single model. In this model, the movement between state λ23 = 0.0404) were 11.22 times higher than the rates of increase in CD4 cell counts (immune recovery) among patients in state 3 . Patients in state 2 (CD4 cell count range of 350–500 cells/uL) were more likely to become LTFU (λ25 = 0.0551). The positive VL log-linear effects showed an increased risk of immune deterioration for an individual in state 2 (CD4 cell count range of 350–500 cells/uL) to state 3 (CD4 < 350cells/uL) λ23 transition .In Table λ23), the transition rates increased with an increase in VL levels over time. Similarly, individuals with CD4 ≥ 500cells/uL (λ15) and those with CD4 < 350cell/uL (λ35) showed an increased risk of becoming LTFU as their VL increased over time. A comparable trend was observed for individuals with CD4 < 350cell/uL (λ34) who exhibited an increased risk of death with an increase in VL over time.The time-varying VL values and the negative VL  had an increasing effect on the risk of death in this cohort for individuals with CD4 < 350cell/uL (hazard ratio (HR) =1.75 and HR = 1.67, respectively). Regarding the time-varying effects of VL on the CD4 cell counts transition rates, as the CD4 cell counts increased for an individual with a CD4 cell count range of 350–500 cells/uL  were 1.78 times higher than the rates of VL suppression among patients with a VL range of 50-1000copies/uL (λ21 = 0.0161). However, patients with VL ≥ 1000copies/uL were 7.77 times more likely to die (λ32 = 0.0035 vs λ34 = 0.0272) and 9.8 times more likely to become LTFU (λ32 = 0.0035 vs λ35 = 0.0343). The time-varying CD4 cell counts had an increasing effect on the risk of viral increase from a VL range of 50-1000copies/uL to VL ≥ 1000copies/uL in this cohort, HR =1.23.In Table . Regarding the time-varying effects of CD4 cell counts on the VL transition rates, as the VL increased (λ12 and λ23) the transition rates increased with a decrease in the CD4 cell counts over time while as the VL decreased  the transition rates decreased with a decrease in the CD4 cell counts over time. The mortality rates of individuals with VL < 50copies/uL and those with VL range between 50-1000copies/uL increased over time as CD4 cell count decreases (λ14 and λ24). Regardless of the individual’s VL state, the transition rates to become LTFU decreased with a decrease in CD4 cell counts. Individuals with high CD4 cell counts were more likely to become LTFU in this cohort ; however, patients with CD4 ≥ 500cells/uL and a VL range of 50-1000copies/uL had an increased risk of becoming LTFU.Similarly, the negative CD4 cell count residual was associated with an increased risk of viral increase from a VL range of 50-1000copies/uL to VL ≥ 1000copies/uL We performed a post-estimation test to assess which of the two multistate models fit better on this data in describing the HIV disease progression, mortality and becoming LTFU. We used the prevalence plot to compare the observed and the expected percentage prevalence for CD4 cell counts and VL multistate models. The VL multistate model showed a perfect fit for state 3(VL ≥ 1000copies/uL) and fair (moderate) fit for state 1 (VL < 50copies/uL) and state 4 (died). State 3 (VL ≥ 1000copies/uL) and state 5 (LTFU) of this model were not perfectly fitted by this model Fig. .Fig. 2TRegarding the CD4 cell counts multistate model, state 1 (CD4 ≥ 500cell/uL) was perfectly fitted while state 2 (350 ≤ CD4 < 500) and state 4 (died) were fairly fitted. State 3 (CD4 < 350cell/uL) was perfectly fitted between time 0 and time 5 years only while state 5 (LTFU) was not perfectly fitted  on the VL and CD4 cell count multistate models to identify any association between the observed transition processes and the region. We estimated the HR summarised in Table Becoming LTFU was observed to be highest among patients from Mashonaland East (P4) regardless of their CD4 cell count. We found that the risk of viral rebound to above 50copiels/uL was high among patients from Matabeleland North (P7) (state 1 to 2), Mashonaland East (P4) (state 1 to 3) regions. In contrast, viral suppression to undetectable levels (VL < 50copies/uL) was evident among patients from Masvingo (P6) (state 2 to 1) and Matabeleland North (P7) (state 3 to 1). The risk of becoming LTFU was high among patients from Mashonaland East (P4) if their VL < 50copiels/uL, Masvingo (P6) if their VL range was between 50-1000copies/uL and Mashonaland West (P5) if their VL ≥ 1000copies/uL.To get a pictorial view of the spatial patterns and correlation between the CD4 cell counts marker and the VL measurements, we fitted the multivariate intrinsic CAR prior model with the province as the spatial unit. We jointly modelled those patients who had a VL ≥ 50copies /uL (VL state 2 and 3 combined) at the end of the follow-up to define that group that might not have attained undetectable VL or have a VL rebound to detectable levels, and those patients who had a CD4 < 350cells/uL (CD4 state 3) to define that group that is still in the immune-deterioration phase at the end of the follow-up period.Table The joint mapping of the posterior relative risk of the HIV disease progression defined by the VL (RR1) and CD4 cell counts (RR2) under the MCAR model is shown in Fig. The main objective of our study was to jointly model HIV disease progression using two 5-stage time-homogenous multistate Markov models based on CD4 cell counts and VL. The multistate models accounted for the orthogonal and the time-varying covariates simultaneously which has been a gap in previous studies. The spatial overlap of the HIV disease progression outcomes (detectable VL (VL ≥ 50copies/uL) and immune deterioration (CD4 < 350cells/uL) at the last observed visit conditional not to have died or become loss to follow-up (LTFU) was also described.We fitted different time-homogeneous multistate Markov models and observed that multistate models with both the orthogonal and the time-varying variables were the best fitting models to describe the HIV disease progression. The VL multistate model was superior in predicting the HIV disease progression patterns in this cohort compared to the CD4 cell count multistate model. These findings support what earlier studies have reported on the superiority of VL in monitoring HIV disease progression patterns compared to CD4 cell counts , 18. Ourλ12 and λ23) the transition rates increased with a decrease in the CD4 cell counts over time while as the VL decreased  the transition rates decreased with a decrease in the CD4 cell counts decreased over time. This finding aligns with the negative correlation which exists between these two prognostic markers that as CD4 cell count levels decreases, VL increases. These finding could be explained by poor ART adherence which remains a challenge among ART patients [The VL multistate model with CD4 cell counts in the covariate matrix showed that VL rebound and VL increase transition rates  were higher than the VL suppression transition rates . This means viral rebound was more likely compared to viral suppression in this cohort. We also found that as the VL increased . Patients with CD4 cell counts lower than expected  were associated with an increased VL and mortality if VL > 50copies/uL. This means as patients with high VL immune deteriorate, the risk of death becomes higher. These findings support other earlier studies that once the immune system deteriorates, the chances of immune recovery become slim, and thus when most deaths occur –22. The Adjusting for the spatial covariate in the VL multistate model, as expected, the regions with a high risk of viral rebound were also more likely to have a high risk of low CD4 cell counts. The intrinsic MCAR prior model further confirmed a strong overlapping geographical correlation between individuals with a detectable VL (VL ≥ 50copies/uL) and immune deterioration (CD4 < 350cells/uL) of 66%; hence, a shared geographical pattern of relative risks between these two outcomes exists. Patients staying in provinces that border with nearby countries had high relative risks of immune deterioration and detectable VL, particularly, those from Matabeleland North province in the northern part of Zimbabwe. Matabeleland North province has a busy truck route to neighbouring countries and high mobility of local and tourists. Earlier studies looking at spatial heterogeneity of viral suppression in this province reported similar results , 25. PeoThe reported results should be inferred in light of some limitations. Firstly, the dataset used in this study is very small and might not be an accurate representation of the Zimbabwean HIV population. We included only health facility data liked to the ePMS with both VL and CD4 cell counts repeated measurements. In this regard, the MCAR assumption might not have been fully satisfied. The spatial effects were observed at a higher level which might not precisely pinpoint the marginalized areas to guide policy. This was as a result of our small sample size in this study; however, future studies should consider lower administrative level spatial units. Moreover, the MCAR model only describes the spatial interaction across the error terms to explain spatial autocorrelation; however, this model falls short when a more direct presentation of spatial interaction is desired. The VL and CD4 cell counts measurements were not randomly done rather differential monitoring was implemented due to resource constraints. The joint model could not account for the biological order of the association between VL and CD4 cell count, that is, VL change may precede CD4 change. We could not adjust for ART adherence, comorbidities ; and demographic characteristics (age and gender) due to model convergence issues. Multistate Markov models can estimate multiple transition rates and outcomes simultaneously compared to the Cox proportional hazard models. However, the assumption of constant hazard function does not reflect reality and also the Markov process has a memory loss property which may be a limitation in HIV studies . DespiteIn conclusion, the findings from this study provide a foundation on the HIV disease progression patterns in Zimbabwe to guide policy going forward and motivate future statistical modelling to consider such modelling approaches in infectious disease to guide policy and programme management. We found that VL models were much more superior compared to CD4 cell count models in HIV monitoring. We also observed that after joint modelling of CD4 cell counts and VL in a single model, the rates of VL rebound risks are still higher than viral suppression. We observed that having a high VL (VL ≥ 1000copies/uL) increased the risk of becoming LTFU and death. The spatial overlap between VL and CD4 cell counts indicates the inter-linkages between these two markers in HIV monitoring and the shared geographical correlation of HIV disease progression immunological outcomes was high. With the global efforts to achieve zero HIV incidence, interventions which encourage ART adherence like support groups and continual educational support on the barriers for ART uptake is crucial. Region-specific interventions which focus on behavioural change, social support, increased uptake of HIV preventions like PrEP should be strengthened among high-risk groups all in the quest to achieve zero HIV incidences by 2030.Additional file 1.


PD) aggregates can act as proteinaceous nuclei (“seeds”) able of self-templated propagation. Since this is strikingly reminiscent to properties of proteinaceous infectious particles (prions), lessons learned from prion diseases suggest to test whether transferred αSynPD can propagate and induce neurological impairments or disease in a new host. Two studies that addressed this question provided divergent results. Intracerebral (i.c.) injection of Lewy body extracts from PD patients caused cerebral αSyn pathology, as well as nigrostriatal neurodegeneration, of wild-type mice and macaques, with the mice also showing motor impairments . In contrast, i.c. transmission of homogenates from PD brains did not stimulate, after “> 360” days post-injection (dpi), pathological αSyn conversion or clinical symptoms in transgenic TgM83+/− mice hemizygously expressing mutated (A53T) human αSyn . To advance the assessment of possible αSynPD hazards by providing further data, we examined neuropathological and clinical effects upon i.c. transmission of brain, stomach wall and muscle tissue as well as blood from PD patients in TgM83+/− mice up to 612 dpi. This revealed a subtle, yet distinctive stimulation of localized αSyn aggregation in the somatodendritic compartment and dystrophic neurites of individual or focally clustered cerebral neurons after challenge with brain and stomach wall homogenates. No such effect was observed with transmitted blood or homogenized muscle tissue. The detected stimulation of αSyn aggregation was not accompanied by apparent motor impairments or overt neurological disease in TgM83+/− mice. Our study substantiated that transmitted αSynPD seeds, including those from the stomach wall, are able to propagate in new mammalian hosts. The consequences of such propagation and potential safeguards need to be further investigated.Cerebral deposition of abnormally aggregated α-synuclein (αSyn) is a neuropathological hallmark of Parkinson’s disease (PD). PD-associated αSyn (αSynThe online version contains supplementary material available at 10.1007/s00401-021-02312-4. Numerous findings indicate that pathologically aggregated species of amyloid-β (Aβ) and tau protein in Alzheimer’s disease (AD), and of α-synuclein (αSyn) in Parkinson’s disease (PD) can act as proteinaceous nuclei (‘seeds’) which recruit endogenous precursor proteins and convert them into their own misfolded oligomeric or polymeric aggregate structures. Such self-templated propagation of pathological protein states in AD and PD shows obvious parallels to the replication of proteinaceous infectious particles (prions) in transmissible spongiform encephalopathies that essentially consist of pathologically misfolded and aggregated prion protein Recent evidence suggests that possibly harmful transmissions of Aβ seeds of AD or cerebral amyloid angiopathy (CAA) did occur in the past between humans via contaminated human cadaveric pituitary growth hormone or dura mater transplants , 21, 30.PD in Lewy body (LB) extracts or homogenized brain tissue from PD patients were examined in two animal studies this produced contrasting findings. Recasens et al.  casein in PBS) two times for 10 min., sections were incubated with the primary antibody  diluted 1:2500 in blocking solution containing 0.2% (w/v) Triton X-100 for 60 min. at 37 °C. After washing three times for 5 min. in PBS (pH 7.4), sections were incubated for 40 min. at RT in blocking solution containing biotinylated goat anti-rabbit IgG  at a dilution of 1:1000. Antibody labeling of pSer129 αSyn was prepared by applying the Vectastain ABC peroxidase kit  for 30 min. at RT according to the instructions of the manufacturer, and visualization of immunolabelling was done by exposure to 3,3′-diaminobenzidine for 3–5 min. Subsequently sections were counterstained with haematoxylin for 20 s (sec.), followed by a differentiation with 1% (w/v) HCl in 70% (v/v) EtOH and blue-staining in tap water. Slides were light-microscopically examined and photographed using an ICC50 HD  or Axioscope 40  microscope.After rehydration, paraffin-embedded tissue sections (6 µm) were incubated for 10 min. in ready-to-use haematoxylin solution  and differentiated in 1% (w/v) HCl in 70% (v/v) ethanol for 2 s. Staining was completed by incubation in tap water for 10 min., followed by counterstaining with 1% (w/v) eosin solution.PD aggregates in human tissue samples, we applied a Western blot assay the principle of which has been outlined by Kramer et al. [To detect pathological insoluble high molecular weight (hMW) αSynr et al.  and Thomr et al. . This We+/− mouse tissue in Fig. +/− mouse tissue in Fig. In brief: 10% (w/v) tissue homogenates were prepared as described. 5 µl aliquots of 10% (w/v) tissue homogenates were mixed with 5 µl sample loading buffer , incubated for 5 min. at 100 °C, and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) using 12.5% (w/v) SDS–polyacrylamide gels. After transfer of proteins from the stacking as well as the separating gel to polyvinylidene difluoride membranes  using a semi-dry blotting apparatus  as previously described , membranrec) into oligo- or polymeric aggregates rich in β-strand secondary structure. Vigorous periodic shaking (or “quaking”) is employed to stimulate this conversion reaction in various formats of well plates. Thioflavin T, a benzothiazole dye added to the reaction mixture, exhibits enhanced fluorescence and a characteristic red shift of its emission spectrum after binding to the amplified αSynrec aggregates. The resulting fluorescence signals indicate the occurrence and progress of aggregate amplification, i.e., the effect of αSyn seeding activity present in the test sample, and can be recorded in real time [The rationale of the RT-QuIC seeding activity assay is that PD- and other disease-associated αSyn aggregates are able to induce by seeded polymerization in vitro the conversion of monomeric recombinant αSyn  or from a human donor without neurodegenerative protein aggregation disease (n = 40). Any possible findings in test mice that should be attributed to the effect of injected exogenous αSynPD seeds had to differ significantly from the unspecific αSyn- or clinical background in our sham-challenged negative controls. As a second prerequisite for our bioassay study, we validated that our TgM83+/− mice behaved after exposure to reference αSyn seeds in brain homogenate from clinically ill TgM83+/+-mice as previously reported [+/− reporter mice. In addition, all bioassayed PD tissue homogenates were characterized by Western blotting for the presence of aggregated αSynPD. Furthermore, caudate nucleus as well as stomach wall tissue samples had been previously or were in this study, respectively, also scrutinized for αSynPD seeding activity after a suitable RT-QuIC assay had become available to us for this purpose.To determine whether, and if so, which seeding effects i.c. injected PD tissue samples had in the TgM83reported . Based oPD aggregates could be detected by Western blotting in caudate nucleus homogenate from control donor 1  and the stomach wall of PD patient A was homogenized and also examined by Western blotting for hMW αSynPD seeding activity after our TgM83+/− bioassay had provided positive results for PStH (see below) despite the negative Western blot findings obtained with this tissue. Since the donor of negative control NStH had Covid-19, his tissue sample was heated to 65 °C for 45 min to inactivate coronavirus SARS-CoV-2 for biosafety reasons. To match these tissue-processing conditions required for our negative control sample, a subset of PStH specimens was also subjected to the same heat treatment prior to RT-QuIC analysis.The stomach wall homogenate was additionally analyzed retrospectively, in a pilot experiment, by RT-QuIC assay for in vitro detection of αSynRT-QuIC analysis of unheated 10% (w/v) PStH that had been spiked with PBH revealed an inhibition of the detection of the exogenously added seeding activity in this sample material  PStH, which is consistent with the high resistance of αSynPD seeds to heat inactivation [PD seeds in PStH from PD patient A.Since the heated PStH and NStH samples had been treated equally prior to RT-QuIC analysis, the positive RT-QuIC readout for heated PStH is unlikely to have resulted from an unspecific non-αSyntivation . Thus, o+/− mice injected with human NBH, 5 did not reach the minimum incubation period for our bioassay of 400 dpi so that the readout of their IHC examination was unusable . This reduced the number of NBH sham-challenged mice available for IHC analysis to n = 35. In contrast, all of the animals sham-challenged with murine NBH (n = 5) were eligible to IHC analysis.Of 40 TgM83+/− mice injected with murine or human NBH almost ubiquitously showed a background staining of spots of spheroid-like αSyn immunolabelling in varying numbers and intensities  along with pronounced widespread cerebral inclusions of pathologically aggregated αSyn in the somatodendritic compartment (SDC) and dystrophic neurites (DNs) either congenitally at ≥ 660 days of age (see homepage from The Jackson Laboratory: https://www.jax.org/strain/004479) [extensive (or phenotype “E'”) somatodendritic/dystrophic neurite (SDC/DN) pathology. As expected, we did neither observe this pathological phenotype nor motor impairments in the five relatively young sham-challenged TgM83+/− mice exposed to murine NBH which were examined at incubation times of 180–219 dpi and an age of 231–272 days. However, when we examined the 35 TgM83+/− mice that had been injected with human NBH and killed after 456–612 dpi within an age-range of between 511 and 654 days, we found that 2 (6%) of these negative control animals showed extensive SDC/DN pathology  , or due /004479) , 39. Tha+/− mice of this negative control group, 3 (9%) developed severe neurological disease with motor impairments at an age of 513, 526 and 566 days . While only one of these animals  also exhibited extensive SDC/DN pathology, another mouse of this group  had presumably suffered an ischemic stroke, as indicated by a lens-shaped blood clot in the brain stem.Of the 35 sham-challenged TgM83+/+ mouse (euthanized at an age of 288 days) in five 7- to 9-week-old TgM83+/− mice resulted in an accelerated onset of pronounced neurological symptoms typical for this experimental paradigm [+/+ mice or MSA patients, respectively [I.c. injection of brain homogenate from a clinically ill TgM83paradigm  in the rectively .+/+ brain homogenate  than in age- and incubation time-matched mice injected with murine NBH . Thus, our validation experiment recapitulated consistently the previously reported observations, and the conclusion that the stimulation of extensive SDC/DN pathology as well as of disease symptoms could be attributed to the αSyn seeding activity present in the tested TgM83+/+ brain homogenate.Extensive SDC/DN pathology as well as pronounced neurological symptoms occurred with statistical significance more frequently in the mice challenged with TgM83+/− mice challenged with PBH did not reach the minimum incubation period for our bioassay of 400 dpi . Since the readout of their IHC examination was, therefore, unusable, this reduced the number of PBH-challenged mice available for IHC analysis to n = 58.Two out of 60 TgM83+/− mice that had been i.c. inoculated with PBH almost ubiquitously showed a background of spheroid-like αSyn immunostaining . This resulted in group sizes of PBH- and NBH-challenged TgM83+/− mice available for this purpose of n = 56 and 33, respectively  SDC/DN pathology. Immunostaining of this pathological phenotype was either confined to isolated individual nerve cells or DNs in the brain slices . These findings strongly suggest that the injected PBH from both patients consistently stimulated a subtle cerebral aggregation and deposition of human αSyn in male and female TgM83+/− mice, thereby leading to the development of the localized phenotype of SDC/DN pathology.The statistical analysis of immunohistochemical findings summarized in Table +/− mice, we failed to detect neurological or other disease symptoms that could be associated with the observed seeding effects of αSynPD. Similarly to the negative control group only three cases of neurological disease with movement impairment were observed in test mice at an age of 511, 566 and 602 days . Again, only one of these animals  showed extensive SDC/DN pathology. Thus, the clinical monitoring of our study animals over long incubation times up to 612 dpi did not provide evidence for a higher incidence or faster onset of neurological impairments in TgM83+/− test mice than in negative control animals.Despite the obvious stimulation of aggregation and deposition of endogenously expressed human αSyn by i.c. injected PBH in the brain of TgM83+/− mice which had been injected with homogenates of skeletal muscle  or whole blood  from PD patient A did not show pathological αSyn aggregation and deposition in their brains  or in combination (p = 0.0001). Subject to the considerations outlined in the discussion this indicated that the injected homogenate from the stomach wall of PD patient A had stimulated the aggregation and deposition of human αSyn and the development of localized SDC/DN pathology in 50% of TgM83+/− recipient mice.The group of TgM83 L+ Fig. c. In add L+ Fig. d. Thus, BH Table . That wa+/− test mice exposed to PStH, PMuH or PlBd than in the negative control animals sham-challenged with human NBH.Neurological disease with movement impairment was observed only in one PStH-challenged animal  not showing SDC/DN pathology, and in no animals that had been injected with PMuH or PBld. Thus, no higher incidence or faster onset of neurological symptoms could be detected in TgM83+/− mice. 54 out of 56 test mice challenged with PBH and eligible to IHC and statistical data analysis showed this pathological phenotype, but none of the 33 corresponding control mice injected with NBH.In our study, we found that i.c. injected brain homogenates from two PD patients resulted in the consistent detection of a subtle phenotype of cerebral αSyn deposition, referred to by us as localized SDC/DN pathology, in TgM83PD of the inoculated PBH but pathologically phosphorylated and aggregated endogenous αSyn. First, Recasens et al. [PD could be detected by 24 h after i.c. injection in the brain of wild-type mice, but not at any later time point between 4 weeks and 17 months post-injection.It can be concluded for three reasons that the localized SDC/DN pathology detected specifically in our test mice did not represent residual exogenous αSyns et al.  have sho+/− mice. As outlined above, this homogenate contained predominantly presynaptic αSynPD micro-aggregates instead of LBs, which are only phosphorylated at Ser129 to a limited extent. Our caudate nucleus inoculum showed only a weak immunolabelling of αSynPD aggregates by anti-pSer129 αSyn antibody ab51253 in the Western blot . This strongly argues against residual exogenous αSynPD as the molecular species constituting the localized SDN/DN pathology observed in younger and older mice.Third, localized SDC/DN pathology could not be detected by us in 4 out of 7 369- to 531-day-old test mice challenged with PBH that were neuropathologically examined at 314–488 dpi, while it was consistently present in 51 test mice with incubation times of > 488 dpi and aged > 531 days. If the localized SDC/DN phenotype would have been based on equally present residual αSyn7 SD50 per animal), the earliest occurrence of localized SDC/DN pathology was detected in one animal at 404 dpi (aged 455 days). The incubation period between 404 and 488 dpi (455–531 days of age) then apparently represented a transitional phase in which three animals were found positive for this pathological phenotype, while four other animals were still negative. After this transition phase, i.e., when the incubation period exceeded 488 days (age > 531 days), all eligible test animals had developed localized SDC/DN pathology.Rather, the occurrence of this pathology correlated plausibly with the incubation time and age of our test mice. With the seeding doses injected  showed the extensive phenotype of SDC/DN pathology.+/− mice. However, further studies are necessary to definitely resolve this question.Thus, the available body of data is rather suggestive of the localized SDC/DN pathology not being a merely accelerated stage of congenital pathogenesis but a novel phenotype of cerebral αSyn deposition that is not intrinsically expressed in TGM83PD aggregates as inoculum in their transmission studies, while we injected non-concentrated brain tissue homogenate into our test animal which reflects a more realistic worst-case scenario for iatrogenic αSynPD transmission.The results of our study reconcile the apparently contradictory findings reported by Recasens et al.  and Prus+/− model as we used. The tissue homogenates tested in these experiments originated from the frontal cortex or the substantia nigra. These specimens were not biochemically enriched or concentrated for αSynPD which may provide one explanation for the apparent discrepancy to the findings by Recasens et al. [+/− mice incubation times of at least about 400 dpi are required to detect stimulation of αSyn deposition in the brain upon an i.c. challenge with caudate nucleus homogenates.While our study supported the positive test results by Recasens et al.  in this s et al. . More im+/− mice challenged with tissue homogenates from the substantia nigra or frontal cortex were kept for “> 360” dpi until neuropathological examination. Thus, there was a high likelihood of negative test results such as the reported ones if the incubation time (the upper limit of which was not specified in the report) had not substantially exceeded 400 dpi in this study.In the study by Prusiner et al. , TgM83+/PD micro-aggregates and αSynPD seeding activity, the TgM83+/− mouse model exhibited a clear stimulation of the cerebral aggregation and deposition of endogenous αSyn by injected αSynPD seeds, and thus provided results consistent with those reported by Recasens et al. [However, our study demonstrated that with sufficiently long incubation times, and inoculums containing high levels of presynaptic αSyns et al. .+/− mice after “> 360” dpi, which is in agreement with our failure to do so up to 612 dpi.Finally, Prusiner et al.  could no+/− test mice, a challenge with homogenized stomach wall from this donor did not so and resulted, with statistical significance (p = 0.0001), in the occurrence of localized SDC/DN pathology in 50% of the recipient mice. To the best of our knowledge, this is the first report on a stimulation of pathological αSyn aggregation and deposition in the brain of bioassay animals by transmitted non-CNS tissue from a PD patient.While i.c. injection of whole blood or homogenized muscle tissue (biceps) from PD patient A failed to stimulate cerebral αSyn aggregation and deposition in our TgM83+/− mice. Apparently, a component of NSpH other than αSyn seeds did unspecifically, yet fulminantly accelerate the congenital pathogenesis of the animals in these experiments.However, special care must be taken when interpreting this finding, as Sacino et al.  reported+/− bioassay, because NBH was considered as sufficient a negative control material by us and the authority for animal protection before the report by Sacino et al. [Unfortunately, no negative control of non-PD stomach wall tissue was included in our TgM83o et al.  appearedo et al. :First, the authors concluded that the NSpH component responsible for the observed unspecific effect originated likely from the white matter, since they did not find an induction of αSyn deposition by injected cortical brain homogenate from normal donor mice. The stomach wall homogenate used in our study, though, did not contain high proportions of white matter material.+/− mice injected with NSpH developed an extensive SDC/DN pathology and severe motor disease after very short incubation periods of 112–154 dpi. Our test mice, in contrast, displayed a much milder localized SDC/DN pathology, and no onset of clinical disease, even only after much longer incubation times of 524–566 dpi. Thus, the neuropathological and clinical effects of injected NSpH and PStH differed profoundly from each other.Second, TgM83Third, only 50% of our test mice showed localized SDC/DN pathology whereas all of the NSpH-challenged animals detailed in the study of Sacino et al.  developePD seeding than an injection of PBH.Fourth, the only localized SDC/DN pathology and negative clinical readout of our PStH-injected test mice corresponded exactly to the findings after a challenge with PBH, with the only difference that the administration of PStH resulted in a lower attack rate of αSynPD seeds via the injected PStH, since our RT-QuIC pilot analysis indicated that this tissue contained seeding-active αSynPD, though apparently at a significantly lower concentration than PBH as demonstrated by the negative Western blot finding for PStH.Fifth, this set of observations can be explained offhandedly by the transmission of αSynPD seeding activity in this tissue. Such alternative hypothesis would require, in a rather implausible way, two premises: (i) that the transferred αSynPD seeds in the injected PStH did not stimulate the localized SDC/DN pathology, and (ii) that instead an unspecific non-αSyn component in PStH mimicked the pathogenic effect exactly to be expected for the detected αSynPD seeding activity.In contrast, the alternative hypothesis, according to which the observed seeding effect may be due to an unspecific non-αSyn component in the injected PStH, seems very unlikely in the light of the detection of αSynPD.Notwithstanding this, we cannot rule out with absolute certainty that the observed stimulation of localized SDC/DN pathology was caused by an unspecific non-αSyn component of the stomach wall analogously to that reported by Sacino et al.  in norma+/− mice. In contrast to this clear neuropathological effect of the seeding-active PBH and PStH inocula, these did not induce detectable health impairments in the used animal model. That is particularly noticeable since TgM83+/− mice develop a severe neurological phenotype upon challenge with αSyn seeds from diseased TgM83+/+ mice or patients with MSA [PD aggregates. This has been also recently reviewed by Asher et al. [I.c. injection of homogenized human PD brain or stomach wall tissue, but not of blood or homogenized muscle (biceps) tissue, from PD patients stimulated cerebral deposition of endogenous pathologically aggregated and phosphorylated αSyn in form of localized SDC/DN pathology in our TgM83with MSA , 39. At r et al.  who concPD seeds could possibly have harmful effects below full-blown PD transmission [PD seeds between humans should be carefully considered and further investigated. The same holds true with respect to precautionary measures for patient safety, which in the light of our findings should pay special attention to tissues from the brain as well as from the alimentary tract, the enteric nervous system of which is known to often contain pathological αSyn aggregates in PD . WePD seeds should be removed or inactivated as far as possible when reprocessing these and other medical devices. To achieve this requirement as optimally as possible, the efficacy of physical and chemical reprocessing procedures against αSynPD seeds needs to be quantitatively assessed on a broader scale. A systematic expansion of the data basis on both transfer risks and precautionary strategies can significantly contribute to adequately counter in the clinical practice the phenomenon of transmissible αSynPD seeding activity.Currently, there is no experimental or epidemiological justification for new special recommendations modifying existing guidelines on the reprocessing of medical devices such as gastroscopes, colonoscopes or surgical instruments, but potential contaminations with αSynSupplementary file1 (DOCX 46 KB)Supplementary file2 (TIF 9201 KB)Supplementary file3 (DOCX 13 KB)Below is the link to the electronic supplementary material.


This data article refers to the paper “Assessing Nearly zero energy buildings (NZEBs) development in Europe” Data allow an overview of the status of most commonly implemented efficiency measures and renewables in NZEBs. In particular, data are available in relation technologies, such as heating, domestic hot water, lighting, renewable sources, ventilation, cooling. Heat recovery efficiency data are also collected. U-values are detailed for roofs, walls, floors, windows. Further data can be visualized in relation to technologies costs, cost of construction and maintenance. It is composed of different sheets:•Building/project name and category •Member State location (country in which the building is located)•Year of construction or refurbishment (year in which the building was constructed or renovated)In Sheet 1 (named “NZEBs best practices”), the following information is available for each collected best practice building:•2y) Primary Energy Demand (PED) (kWh/m•Renewables (RES) (%) (percentage of renewable energy in the building)•2) (area of the building)Floor area (m•2y) (amount of heating required to heat the building)Space heating demand Implemented technologies •Building/project name•Member State location•Year of construction and refurbishment•Building type•Implemented technologies In Sheet 2 (named “Technologies”):•Implemented technology•Member State location•Installation •Grid connection (in relation to photovoltaic systems)•Product cost (cost to have the technology)•Initial design cost •Operational energy use revenues (from electricity sold to the grid)•Trends/objectives In Sheet 3 (named “Technology cost”):•Building/project name•Member State location•2) (cost to build the building)Construction cost (€/m•Average construction cost per Member State (€)•2) (cost to maintain the building over its lifecycle)Lifecycle maintenance cost (€/m•Average lifecycle maintenance cost per Member State•Data source (where the information was taken)In Sheet 4 (named “Cost”):•Building name/project•Member State location;•Renewable (%)•2y)Primary Energy Demand (kWh/m•2y)Space heating Demand (kWh/m•Average RES share in best practice example buildings by MS (average of the renewable energy present in the buildings)•Average primary energy demand in best practice example buildings by MS (average of the primary energy demand)•Average space heating demand in best practice example buildings by MS (average of the space heating demand)•Data sourceIn Sheet 5 (named “Energy consumption-Renewables”):•Building/project name•Member State location;•Heat Recovery Efficiency •Average heat recovery efficiency per MS (average of the above)•Data sourceIn Sheet 6 (named “Heat recovery”):•Implemented technologies•Number of best practice buildings where a technology was installed (buildings having a certain technology)•Relative percentage (derived percentage of the building having a certain technology)In Sheet 7 (named “Systems”):•Building/project name•Member State location•Data source•U-value for walls, roofs, floors, windows •Average U-Value for NZEB exemplary buildings per MS In Sheet 8 :2The 51 buildings included in the data are located in 20 Member States . Italy and Austria represent the largest part of NZEBs of the sample (12% each), followed by France and Germany (10% each). Various types of buildings are included in the sample, like educational, commercial, office and residential buildings from 19 Member States. The 43% of the buildings are educational buildings . Some of them are pilot projects. The 33% are residential buildings . The 12% are office/administration buildings. Finally, there are also hotels and mixed used urban projects. The building/projects included have been constructed during the period from 2002 to 2019. However, for some of them the construction year is not available.Data allow visualize the average primary energy demand values (including renewables) of NZEBs best practices per country, as shown in École François Mitterand in France (−7.5 kWh/m2y) to the Plus Energy Settlement in Kleehäuser, Freiburg in Germany (152 kWh/m2y) (2006) 2y) In the collected NZEBs best practices, the projects with available on energy demand data are 28, ranging from the Primary School in Bielawa in Poland. An example of NZEB using ambient air heat pumps for heating is the Technical University Sofia in Bulgaria. Finally, an example of a building using water heat pumps is the Faculty of Agriculture, University of Osijek in Croatia Data allow also visualise the most diffused NZEBs technologies. As example, a NZEB best practice building using geothermal heat pumps for heating is the Main costs associated with technologies are available. As example, École François Mitterand in and the Hill Primary School in the United Kingdom. For biomass boilers, Elementary School and Kinder-garten Albrechtsberg in Austria is a best practice example of a pilot education building using biomass boiler for space heating and domestic hot water.Data are also collected for specific technologies. For boilers, there are NZEBs pilot education buildings in Europe using this technology for space heating Elementary School and Kinder-garten Albrechtsberg in Austria F3 Brdo, Ljubljana, uses external shading, manually operated roller blinds Green Lighthouse in Denmark uses both heat pumps a well as the night ventilation for cooling Leaf House in Loccioni, Italy uses heat pumps and radiant floors for cooling Technical University Sofia, Bulgaria uses a VRF system Data linked to this paper allow to visualise NZEBs that use exclusively passive space cooling methods. École François Mitterand in France Multifunctional School Houthaven in the Netherlands uses heat recovery units for ventilation ENERPOS University complex in Saint Pierre, France has ceiling fans as well as a VRV system The majority of the collected buildings with available information about ventilation includes heat recovery systems . Heat reBeddington Zero Energy Development in London heat recovery system presents an efficiency of 70% Bushbury Hill Primary School in the United Kingdom has a mechanical ventilation system with heat recovery efficiency by 80% during winter, while it is based on windows during summer Green Home Nanterre in France has a decentralised ventilation system of 96% heat recovery In relation to heat recovery efficiency in collected NZEBs best practices , the BedData are also collected for efficient insulation, an important passive method to reduce energy demand and achieve high energy savings 2K (NZEB After School day care center in Belgium) to 0.4 W/m2K  2K (Primary School in Budzów in Poland) to 0.26 W/m2K  2K (Beddington Zero Energy Development in London and in Plus Energy Settlement in Weiz-Gleisdorf in Austria) to 0.41 W/m2K  2K (in Maardu Day Care Center in Estonia 2K (in École François Mitterand in France) U-values range from 0.08 W/mPrimary School Mariagrün in Austria and the Bushbury Hill Primary School in the United Kingdom Plus Energy School Hohen Neuendorf in Germany uses biomass, CHP and PV to cover the 21% of its energy needs while the École François Mitterand in France includes 406 m2 of PV to cover an equal share Väla Gård in Sweden  Lehtomäki day care center in Finland NZEB multi-family house F3 Brdo in Ljubljana, Slovenia Plus Energy School Hohen Neuendorf in Germany has 400 m2 of photovoltaics Among examples that do not impose the RES requirement in their national NZEB standards, there are the Multifunctional School Houthaven in the Netherlands uses presence detectors while Sustainable Building Energy Information Centre Debrecen in Hungary includes both presence and daylight detectors in its lighting. Best practice examples are the Luthaa Nursery in Finland (LED lamps) and the Childcare Centre Cologno Monzese in Italy (energy saving lamps) In relation to lighting, Bushbury Hill Primary School in the UK, a building based on passive solution for cooling and ventilation in summer Luthaa nursery in Finland and in Kleehäuser, Freiburg in Germany the additional costs are only 10% 2) in Green Lighthouse in Denmark, a building that uses geothermal, solar thermal and PV as renewables, district heating, heat pumps and LED lights Väla Gård is calculated to be around 61% . The building includes ventilation with heat recovery, ground source heat pumps and PV Regarding the costs due to additional NZEB technologies, the lowest ones are identified in 2 in A-32 building, Salburua in Spain  to 2624 €/m2 in Moretti More in Italy 2 in Brussels Rings to 1106 €/m2 in Isola nel Verde in Italy 2. A broad scale shift towards NZEBs requires an effort in the current market. Cost-effective integration of efficient solutions and renewables remain a major challenge.The data from 24 NZEBs show that the project costs range from 682.3 €/mNo ethics fields involved.Delia D'Agostino: Conceptualization, Methodology, Investigation, Writing – original draft, Writing – review & editing. Sofia Tsemekidi Tzeiranaki: Data curation, Software, Investigation, Writing – review & editing. Paolo Zangheri: Data curation, Investigation. Paolo Bertoldi: Supervision.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


The complex chemicalstructure and the fact that many areas inpulping and lignin chemistry still remain unresolved are challengesassociated with exploiting lignin. In this study, we address questionsregarding the formation and chemical nature of the insoluble residuallignin, the presence of fatty acids in kraft lignin, and the originof secoisolariciresinol structures. A mild thermal treatment of ligninat maximum kraft-cooking temperatures (∼170 °C) with talloil fatty acids (TOFA) or in an inert solvent (decane) produced highlyinsoluble products. However, acetylation of these samples enableddetailed chemical characterization by nuclear magnetic resonance (NMR)spectroscopy. The results show that the secoisolariciresinol (β–β)structure in kraft lignin is formed by rearrangement of the β-arylether structure. Furthermore, fatty acids bind covalently to kraftlignin by reacting with the stilbene structures present. It is highlyprobable that these reactions also occur during kraft pulping, andthis phenomenon has an impact on controlling the present kraft pulpingprocess along with the development of new products from kraft lignin. However, part of the β-O-4 linkages,3–7% according to quantitative NMR experiments,4 remain intact during the process. The main side product formed fromthe kraft process, that is, sulfur-containing kraft lignin, is stillusually burned as energy but could potentially be used to replacemany applications based on scarce oil sources.Materialproduction from sustainable resources is an ongoing globalchallenge. Kraft pulping is currently the main processing techniqueto produce renewable cellulosic fibers used in various products.1 After the fast bulk delignification stage, about 90–95%of the original lignin has been dissolved, depending on the processingconditions.5 The rate of delignification is much slowerat the final residual delignification stage. Compared to the conventionalkraft pulping process, processing parameters have been optimized,for example, by lowering the processing temperatures from 170 °C,and by tuning the concentrations of hydroxyl and hydrogen sulfideions for more efficient delignification .5Kraft pulpingdelignification can be divided into three stages,namely initial, bulk, and residual delignification.8 According tothe present view, the aromatic condensed structures are accumulated,rather than formed by chemical reactions during the process.8 Secoisolariciresinol is also one of the structuresfound in residual kraft lignin (2%), in addition to resinol  and phenyl coumaran .9 Processing conditions  affect the structure of residual lignin, in termsof the amount of phenolic groups, β-O-4 linkages, and condensedlignin units.10 Lignin–carbohydratecomplexes have also been suggested to play some role in the more difficultdelignification of residual lignin.12 Earlier studies by Gellerstedt et al. suggested that residual lignin contains also material resulting fromthe formation of aliphatic C–C bonds because correspondingsignals appear in the NMR spectra of kraft lignins and insoluble materialthat remains in kraft lignin cooked with 2,6-xylenol after thioacidolysis.13 Furthermore, the involvement of radical reactions during the formationof residual lignin has been suggested, as fatty acids are incorporatedinto lignin during kraft pulping.9Although the kraft lignin process is currentlyhighly efficient,all aspects are not totally understood. Examples include the residuallignin in kraft pulping, which is highly insoluble and difficult toremove, and the occurrence of fatty acids in kraft lignin. From earlierstudies, residual lignin is known to contain β-O-4 structures(3–7%) and “condensed aromatic structures”.15 The amount of secoisolariciresinolstructure in kraft lignin is 3%.4 Secoisolariciresinolis also one of the structures found in residual kraft lignin (2%).9 However, comparison of lignins in the same studyshowed that the amount of secoisolariciresinol structure, along withfatty acids, is higher in kraft and residual lignins as compared tomilled wood lignin.8The resinol structure is the major type β–βstructure of lignin, whereas the secoisolariciresinol structure presentsanother, a minor type β–β structure. The secoisolariciresinolstructure is probably not, as initially believed, formed through theresinol structure, that is, the more typical β–βstructure in lignin.16 For example, kraft lignin has been suggestedto be suitable for preparing pyrolysis oils or as a starting materialfor carbon fibers.18 Torrefaction is a milder thermal pretreatment for biomass takingplace at 200–300, °C, which aims for improved fuel productionor consumption.19 In all of these processes,lignin is thermally treated and information on the reactions involvedis highly beneficial for their efficient development.In additionto the reactions occurring during kraft pulping, understandingthe thermal reactions of lignins is important in the development ofnew processes based on pyrolysis and milder heat treatments.2) as the acid.20 TOFAs,obtained by distillation, were used in the reaction as a model compoundrepresenting the fatty acids released during the kraft pulping process.TOFA was also used as a reaction medium for heat transfer. The useof alkaline water solution containing hydrogen sulfide as the reactionmedium was omitted, because the aim was to investigate radical reactionsinstead of ionic reactions. The material formed as a result of theheat treatment of lignin and fatty acids was hard, but very brittle,and furthermore dissolved very poorly in all of the tested organicsolvents. According to initial chemical analysis, the material wascomposed of lignin and fatty acids, suggesting that the starting materialswere attached through covalent bonding (it was impossible to “extract”any of the components with any solvents or their combinations). Tounderstand the compositions of the materials and chemical reactionsinvolved, a set of further experiments and analyses was performed.To define lignin–lignin reactions, a similar mild heat treatmentwas performed using the chemically inert decane instead of TOFA asthe solvent. Chemical and thermal properties of the formed productswere characterized thoroughly using various complementary techniques.In orderto investigate mild thermal heat treatments of lignin,the reactions were performed in tall oil fatty acids (TOFA) or inan inert solvent decane. Our initial aim was to study the reactionsof kraft lignin and TOFA during mild thermal treatment at moderatetemperatures (∼170 °C) to assess the incorporation offatty acids into kraft lignin. The kraft lignin used was from theLignoboost process, where lignin is precipitated using carbon dioxide was purchased from Eurisotop .Kraft softwood lignin , received in the powdered form, was dried furtherin an air-circulating oven at 40 °C before use. Distilled TOFA(product name: FOR2) was received from Forchem , andtypically comprises of certain rosin acids , and the followingfatty acids  as the main components: linoleic acid , oleic acid , and pinolenic acid . Decanewas purchased from Sigma-Aldrich . The solventused for NMR (d =0.91 g mL–1) or decane  were placed in an inner PTFE cupof a 450 mL stainless-steel Parr reactor  equipped with a heating mantle, temperature control,and a pressure monitor (controller model 4843). The reactor was flushed,to remove air, by filling and emptying it three times with N2 . After flushing, N2 pressure wasset at 70 psi (0.48 MPa), which was maintained during heating by releasingN2 through a valve. The reaction mixture was heated to170 °C and maintained at that set temperature for 2 h. The productstarted to form already after 15 min, but to confirm that all ligninreacted, and for the purpose of comparison, a reaction time of 2 hwas used. The monitored heating temperature was approximately 170–180°C in decane and 170–195 °C in TOFA. After cooling,the floating reaction product (lignin–decane or lignin–TOFA)was collected from the surface of the liquid, washed with acetone,and dried in an air-circulation oven at 40 °C. Part of the productcould not be easily removed from the temperature sensor of the Parrreactor, and therefore reliable mass balance calculations could notbe performed.To obtain solutionswith 5 wt % of lignin with a similar level of volume, lignin (5.0g in TOFA and 4.0 g in decane) and TOFA  was placed ina round bottom flask. Pyridine (2 mL) and acetic anhydride (2 mL)were added. The mixture was allowed to react at room temperature withoutstirring for 20 h, which resulted in dissolution of the starting lignin.The polymerized samples were further refluxed at 80 °C with stirringfor 7 h, resulting in mainly soluble products. The acetic anhydridewas quenched by adding and evaporating ethanol twice (20 mL), thetrace of pyridine was evaporated with toluene ,and finally the trace of toluene was evaporated with chloroform .–1.TheFT-IR spectra of the starting lignin, TOFA, lignin–decane,and lignin–TOFA were recorded with a Bruker ALPHA  attenuated total reflectance (ATR)-FT-IR spectrometerin a spectral range of 375–4000 cm–1 and an N2 flowrate of 50 mL min–1 were used.Thermal decomposition wasanalyzed by thermogravimetric analysis (TGA) using a Mettler ToledoTGA/SDTA 851e . The samples (5–10mg) were analyzed in alumina crucibles with pinhole lids. A heatingrate of 10 °C min–1 anda cooling rate of 5 °C min–1, and the measurementswere performed under nitrogen. The maximum temperature used was belowthe degradation temperature of the material to be analyzed. A secondheating stage was used to analyze the data of samples containing lignin.Differential scanningcalorimetry (DSC) measurements were performed using a DSC Q200 . The samples (5–10 mg) were analyzedin aluminum pans with lids. A heat–cool–heat cycle wasused with a heating rate of 10 °C mind6 as the solvent. The thermallytreated acetylated samples were mainly soluble in DMSO-d6 (1.6 mL). After filtration through a cotton wool plugusing a Pasteur pipette, the sample was concentrated under vacuumto a volume of 0.7 mL.The starting lignin, lignin–decane, and lignin–TOFAwere characterized using NMR spectroscopy. All samples were analyzedin DMSO-1H–13C heteronuclear single-quantum coherence (HSQC) NMR spectrum of thestarting lignin was acquired using a Bruker 600 MHz spectrometer,and the 2D HSQC NMR spectra of lignin–decane and lignin–TOFAusing a Varian Unity Inova 600 MHz spectrometer. The temperature usedduring measurement was 25 °C for the starting lignin and 40 °Cfor heat-treated lignins. The pulse sequence used was hsqcetgpsisp2.2(Bruker) or gHSQC (Varian). The 90° pulse width was determinedseparately for all samples. The number of increments used in the measurementswas 256 (Varian) or 300 (Bruker), and the number of scans was 64 (Varian)or 512 (Bruker).The 2D 1H NMRbefore and after heat treatment. Spectra are presented in the Supporting Information. The fatty acid contentsof the starting lignin and lignin–TOFA were evaluated fromthe 1H NMR spectra (Supporting Information) by comparing the integrals of fatty acid Fω  and lignin Ar–OCH3 .The TOFA used for the reaction was analyzedby –1. Injector temperaturewas 250 °C and a 1:20 split ratio was used. Pyrolysis productswere separated in an Agilent DB-5MS UI  capillary column.Column oven temperature was kept at 50 °C for 2 min after whichthe temperature was increased at a rate of 10 °C min–1 to 280 °C and kept at 280 °C for 5 min resulting in a30 min overall run time. Ion source temperature was 250 °C withan electron ionization of 70 eV. The MS scan range was 40–400 m/z. Compounds were identified by comparingthem to the National Institute of Standards and Technology (NIST)database.Measurementswere performed using a Pyrolab2000 pyrolyzer connected to a BrukerScion SQ 456 GC–MS. The pyrolysis chamber temperature was keptat 150 °C and the samples were pyrolyzed by heating the platinumfilament in 8 ms to 200 °C and keeping the temperature elevatedfor 2 s before rapid cooling of the filament. The second heating to580 °C was also 2 s long. Helium was used as a carrier gas ata flow rate of 1 mL min–1 in tetrahydrofuran (THF). Sampleswere filtered through a 0.2 μm syringe filter  before injection (50 μL).Total run time was 12 min, with a flow rate of 0.8 mL min–1. Molar masses were evaluated using data from the UV range (254 nm),and the following polystyrene standards were used for calibration:474, 840, 2500, 5010, 13,300, 32,300, 43,400, 76,000, and 139,400Da along with 321,000 and 526,000 Da (Fluka).Molar masses of acetylatedlignin–TOFA and starting lignin acetylated in the presenceof TOFA (roughly 50 wt %) were analyzed using SEC. The Waters AcquityAPC system was used for the analysis , equipped with Acquity APC XT columns 45, 200, and 450 Å,and using 30 °C as the column oven temperature. Sample concentrationwas 1 mg mL1 The heat treatment in decaneprovided information on thermal reactions of kraft lignin alone inthe selected temperature, whereas the participation of fatty acidsin reactions was evaluated in TOFA. The first difference between treatmentswas observed when monitoring the reaction temperature: the temperatureset at 170 °C remained fairly constant in decane (170–180°C), whereas it increased spontaneously (170–195 °C)in TOFA, indicating that exothermic reactions were occurring in thereaction vessel.The starting kraft lignin was heat-treated in decane or TOFA todiscriminate the internal reactions of lignin from cross-reactionsof lignin with TOFA. The treatments were performed at ca. 175 °C,which is the maximum temperature during the conventional kraft pulpingprocess.d6), and it therefore should bekept in mind that the minor insoluble fraction may have some structuraldifferences compared to the observed results.To gain insights into the reactions occurringunder thermal treatments, the chemical structures of the startingand heat-treated lignins were analyzed using nuclear magnetic resonance(NMR) spectroscopy. The samples were acetylated because the very lowsolubility of nonacetylated samples prevented analysis. The underivatizedproducts were not soluble in many of the typical organic solventstested, and only slightly soluble in aqueous NaOH-solutions. The mainpart of the acetylated products was soluble in the NMR solvent used. According to visualinspection, decane did not contain lignin-based materials.In practice, allof the lignin was floating on the surface of the liquid used for reactionmedia after the reaction in TOFA or decane. However, part of the materialwas tightly attached to the temperature sensor of the reaction vesseljeopardizing determination of accurate mass fractions. To confirmthat lignin was not dissolved during reaction, the used TOFA was analyzedbefore and after the reaction by 22 structures of acetylated secoisolariciresinol and non-acetylateddihydrocinnamyl alcohol,23 stilbene structuresformed from phenylcoumaran (β-5) and spirodienone structures(β-1),24 and fatty acids of variouschain lengths and degrees of unsaturation.25 It is worth noting that some of the published data are for nonacetylatedsamples and, therefore, some of the values may differ compared toacetylated samples used here. Furthermore, there might be some variationas a result of different solvents used for NMR analysis. Lists ofidentified cross peaks are presented in 24 As a rough estimation based on the 1H NMR spectrum, thefatty acid content of starting lignin was around 4 mol % (Supporting Information).The2D HSQC NMR spectrum of the starting lignin and the structuresidentified in NMR spectra are presented in 1H NMR spectrum, the fatty acid content oflignin–TOFA was around 7 mol %, almost twice as much comparedto starting lignin (Supporting Information).Based on the NMR spectraof both lignin–decane 2 and lig14 Our findings support the earlier conclusions,but instead of a separate pathway in lignin biosynthesis, secoisolairciresinol(C) may form directly from β-aryl ether (A).According to the results from the NMR analysis presentedabove,two sites seem reactive during heat treatment, also depending on thereaction media. In decane, β-aryl ether structures (A) are reactingbut stilbenes (S1 and S5) are not, whereas both the β-aryl etherstructures (A) and stilbenes (S1 and S5) react in TOFA. The formationof secoisolariciresinol (C) seems to be associated with reactionsof β-aryl ether (A) because secoisolariciresinol (C) is formedand β-aryl ether (A) is reacting in both heat treatments, thatis, decane and TOFA. The study by Zhang et al. 2003 concluded thatsecoisolariciresinol (C) is not formed from resinol (B) but insteadis somehow connected to the β-aryl ether structure.24 Apparently, another experimentalsetup with some simpler model compounds is required for the identificationof the formed products, which would be beyond the scope of the experimentspresented in this paper.Stilbenes(S1 and S5) also react during heat treatment in TOFA,whereas these structures are stable in decane. Stilbenes and fattyacids, therefore, possibly form certain adducts or condensation products,although the site of connection could not be identified from the NMRspectrum of lignin–TOFA. The reaction pathway must be clearlymore complex as more reacting components are present. The very obvioushypothetical product between lignin and TOFA, formation of an esterin a reaction with fatty acid and lignin could not be observed neither,in alignment with previous NMR work with kraft lignin, which containedfatty acids.Supporting Information. The FT-IR assignmentsfor lignin are based on the previously published results for lignin.26 The FT-IR assignments for TOFA, a mixture offatty acids produced as side products of the forest industry, arebased on earlier studies on vegetable oils.28To further support the NMR analysis findings and to evaluate thecompositions of the materials, we analyzed starting lignin, TOFA,and lignin heated in TOFA (lignin–TOFA) or decane (lignin–decane)using FT-IR 4. A list30 and the values measured in thiswork, using FT-IR with ATR accessory with no need for separate samplepreparation, may therefore vary slightly. For example, the spectrumof starting lignin has a weak absorption band at 1361 cm–1, which can be assigned to the aliphatic C–H stretch in methyl(not in −OMe) and to the phenolic hydroxyl group. Althoughthe value slightly differs from the literature (1365–1370 cm–1) the position and shape of the absorption band arecomparable.26 Similarly, the absorptionband of lignin at a value of 1208 cm–1 may be assignedarising from C–C plus C–O plus C=O stretching(G condensed > G etherified),althoughthe literature value is 1221–1230 cm–1).26The lignin samples were measuredin earlier studies with KBr,–1). Similarly,all the typical absorption bands found in the starting lignin, fromabsorption band 1703 cm–1 (C=O group) to816 cm–1 , are also found in the heat-treatedsamples. These results also confirm that the material after heat treatmentcomprises of lignin, rather than totally charred components.As seen in –1) has a similar strength compared to starting lignin,whereas the strength of the C=O absorption band (1708 cm–1) for lignin–TOFA is similar compared to TOFA.Furthermore, the absorption band for C–H stretching in methine(3006 cm–1) is present in the spectrum of the lignin–TOFAsample, providing further proof that lignin–TOFA contains anotable amount of additional fatty acids.In –1) were more intense comparedto starting lignin, which would also be compatible with the formationof the secoisolariciresinol (C) structure with a relatively increasingamount of −CH2–.For both heat-treatedsamples, the absorption bands at the areacorresponding to either methyl or methylene C–H stretching of the starting lignin was 156 °C, whichis in the same range with previously reported values for kraft lignin(120–174 °C).32 For TOFA, transitionsfor crystallization and melting were observed at temperatures of −23°C (Tc) and −19 °C (Tm), respectively. These values are reasonablecompared to the DSC analysis of fatty acids in the literature, consideringthat the aim of the method used in this study was to compare the materialscontaining lignin and TOFA.33 The observed Tg was 142 °C for lignin–TOFA (Tm) for lignin–TOFA was observedat −8 °C, which was in a similar range compared to transitionsobserved for TOFA.Thermal properties of startinglignin, TOFA, and heat-treated lignin–TOFAwere also determined using DSC, presented for lignin–TOFA in nin–TOFA 6, which 1H NMR integralsof lignin Ar–OCH3 and fatty acid Fω). Furthermore,the glass transition temperature of lignin–TOFA was slightlydecreased compared to starting lignin and an additional melting transitionwas also observed.The results from the thermal analyses also suggest that ligninand TOFA were connected by a covalent bond, because no separate evaporationof fatty acid was observed in the TGA of heat-treated lignin–TOFA,while this material contains some fatty acids according to chemicalanalyses (∼7 mol % by comparing Supporting Information.Pyrolysis-gaschromatography/mass spectrometry (Pyr-GC/MS) was used to identifyvolatiles in order to understand the underlying mechanisms occurringduring heat treatments. The Pyr-GC/MS analyses were performed by firstheating the samples to 200 °C, mimicking the heat treatment performedearlier, and then bringing the temperature up to 580 °C. Theresults of the main peaks, each representing more than 3% peak areaof the total amount of all peaks (100%), except for silylated compoundsmost probably originating from other sources, are shown in Pyrolysis at higher temperature(580 °C) produced a typicalfragmentation pattern of kraft softwood lignin, and no significantdifference was observed between the samples, lignin, and lignin–TOFAmixture. However, only two products, that is, dimethyl disulfide andguaiacol, were formed from the starting lignin at lower pyrolysistemperature (200 °C). In the lignin–TOFA mixture, vanillinwas the main lignin-based compound released followed by guaiacol anddimethyl disulfide, and fatty acid-based 2,4-decadienal, 2-decen-1-ol,and hexanal were concurrently released.35 The significant release of guaiacolat lower pyrolysis temperatures (200–300 °C) has alsobeen observed earlier.35 Based on the structural analysisby the NMR presented above, the release of guaiacol also supportsthe hypothesis that secoisolariciresinol (C) is formed from β-arylether (A). Secoisolariciresinol (C) has previously been suggestedto form during the biosynthesis of lignin from two coniferyl alcoholradicals and this structure would then be attached to the β-arylether structure (A).14 Previously, (C)has also been observed in DFRC (derivatization followed by reductivecleavage) analysis of lignin dimers, resulting from the DFRC procedure,in which β-aryl ether bonds are cleaved.15 Our results suggest that the Cβ–O bond ofβ-aryl ether structure is homolytically cleaved at mildly elevatedtemperatures (ca. 170–200 °C) or in other suitable conditionsfor prompting radical reactions, and the resulting Cβ-radicalscouple to form the β–β bond (14 The formation of thesecoisolariciresinol (C) structure then takes place after releaseof H2O2, which can react with either ligninor TOFA. The mechanism for the release H2O2 isnot clear at this point, but the required reducing component couldbe present in a reaction media containing lignin, fatty acids, andsulfur compounds. Therefore, (C) formation does probably not occurduring lignin biosynthesis from coniferyl alcohol, as suggested previously,36 but instead, the rearrangement of β-aryl ether, prompted byradical formation, may create a more chemically resistant, and possiblymore insoluble structure.The products formedfrom the pyrolysis of starting lignin wereconsistent compared to previous studies in both applied temperatures.β–β bond 1. The foThe presence of TOFA clearly affected pyrolysis at 200 °C,but evaluation of plausible reaction pathways involved is more complexbecause of the higher number of reacting components. The similar formationof secoisolariciresinol (C), as suggested above, would be reasonable,because guaiacol was also released in the presence of TOFA. However,vanillin was the major lignin-based component released, suggestingcleavage of the Cα–Cβ bond. Results from the NMRanalysis showed that stilbenes were concurrently consumed (disappearing).The formation of aldehydes from stilbenes is a known reaction undercertain conditions, but based on the results presented here, it seemsto require the presence of fatty acids, which also attach to lignin.The reaction pathway leading to the covalent bonding of TOFA underheat treatment is thus not evident from the present results, and requiresfurther investigation.Supporting Information). By visual inspection, the acetylated samples seemed to be solublein the used solvent (THF). According to the results, presented in Supporting Information), the molar masses beforeand after heat treatment were very similar, and in fact, the molarmass of lignin–TOFA was slightly lower. Based on the low solubilityof the product, this initially seemed surprising. However, this iswell in line with the reaction pathway suggested for the formationof a secoisolariciresinol structure (C). This result is also consistentwith the results of Balakshin et al. (2003), in that aromatic condensationproducts, that is, formed via coupling to the 5-position, are notproduced during kraft pulping.8 Instead,cleavage of the C–O bond and rearrangement of β-arylether structure and formation of a new aliphatic C–C bond wouldresult in a more hydrophobic and chemically resistant structure (10H12O4H = C20H24O8H2) and the secoisolariciresinoldimer (C20H22O6H2) showthat the relative reduction (−9%) in molar masses (from 394to 360) is very similar compared to the molar masses acquired fromSEC.The molar masses of acetylated startinglignin and heat-treatedlignin–TOFA were evaluated by SEC. The lignin sample was acetylatedin the presence of TOFA for improved comparison of the samples (theSEC chromatograms can be found in tructure 1. Notabl9 However, the structural patterns of lignin taking part in theseradical reactions is novel information, in addition to the reactionmechanism for the formation of secoisolariciresinol structure.Cleavage of the β-O-4linkage by the action of hydroxyl and hydrogen sulfide ions is themajor ionic reaction pathway during kraft delignification. In addition,according to the results presented here, minor radical reaction pathwaysare involved in the kraft process. The existence of fatty acids andsecoisolariciresinol structures in the kraft and residual ligninsis known from earlier studies, and a radical pathway leading to theseproducts has been proposed previously.In this study, we have shown that temperature of 170–175 °Cis one of the essential reaction conditions that induce the formationof these products through a radical pathway. Considering this temperaturewith the fact that delignification improves by lowering the temperatureof conventional kraft pulping process, the formation of these productscould be one of the reasons for the slow residual delignificationphase. Unless derivatized by acetylation using harsh conditions, theproducts are highly insoluble in any common solvents, and could, therefore,form a protective layer, which is not easily accessible by the pulpingchemicals anymore. Furthermore, the formed C–C bonds are morestable, less reactive, and more hydrophobic compared to the functionalstructures reacting in the starting materials. Understanding all underlyingmechanisms of the kraft delignification process, even the minor ones,is useful for improved control of the whole process.In additionto kraft pulping process, it seems that radical reactionsof lignin are also most probably taking place during all heat treatmentprocesses of lignin, and knowledge of the possible reaction pathwaysis useful in developing possible applications for lignin. However,further studies, in addition to the kraft lignin, are required toevaluate if the rearrangement of β-O-4 linkage to secoisolarisiresinolis taking place with other types of lignins as well.Concludingthe results presented here, β-aryl ether structuresof lignin can rearrange to form secoisolariciresinol structures duringmild thermal heat treatment. In addition, the formed structure withnew C–C bonds and less hydroxyl groups is chemically more stableand at least partially responsible for the observed lower solubilityof the formed product. On the other hand, if fatty acids are present,the stilbene structures of lignin react and form covalent bonds withfatty acids. Whereas the rearrangement of β-aryl ethers to secoisolariciresinolis likely to occur through radical reactions, the reaction pathwaywith fatty acids remains unclear. Finally, the knowledge of the reactingsites under mild thermal treatment of kraft lignin is valuable inthe development of new applications based on heat treatment, alongwith controlling and optimizing the existing kraft pulping process.


Pes) and nasal (ΔPnos) as a potential measure of inspiratory effort in spontaneously breathing patients with de novo ARF.Excessive inspiratory effort could translate into self-inflicted lung injury, thus worsening clinical outcomes of spontaneously breathing patients with acute respiratory failure (ARF). Although esophageal manometry is a reliable method to estimate the magnitude of inspiratory effort, procedural issues significantly limit its use in daily clinical practice. The aim of this study is to describe the correlation between esophageal pressure swings  admitted to the Respiratory Intensive Care Unit (RICU) of the University Hospital of Modena  and candidate to escalation of non-invasive respiratory support (NRS) were enrolled. Clinical features and tidal changes in esophageal and nasal pressure were recorded on admission and 24 h after starting NRS. Correlation between ΔPes and ΔPnos were strongly correlated at admission  and 24 h apart . The nasal plug insertion and the mouth closure required for ΔPnos measurement did not result in significant change of respiratory rate and ΔPes. The correlation between measures at 24 h remained significant even after splitting the study population according to the type of NRS .ΔPnos might warrant further investigation as a measure of inspiratory effort in patients with ARF.In a cohort of patients with ARF, nasal pressure swings did not alter respiratory mechanics in the short term and were highly correlated with esophageal pressure swings during spontaneous tidal breathing. ΔTrial registration: NCT03826797. Registered October 2016.The online version contains supplementary material available at 10.1186/s13054-022-03938-w. L) plays a key role in the progression of lung damage during acute respiratory failure (ARF) of different etiology , if not stated otherwise. Correlations were sought using Pearson’s R, between-groups differences with the Fisher’s and Mann–Whitney tests, as appropriate. Changes in RR and ΔPes before and after the insertion of the nasal probe and mouth closing were sought for using the Wilcoxon signed ranks test. A sample size of at least 37 patients would have provided 90% power (1-β) to detect a correlation with R > 0.5 at an α level of 0.05. In a sensitivity analysis, we compared the agreement between ΔPes measured at T2 and ΔPes, estimated based on ΔPnos using the Bland–Altman method, to assess whether ΔPnos could serve as a surrogate of ΔPes. We further performed the analysis according to the type of NRS. In another sensitivity analysis, the differences of ΔPes and ΔPnos in patients that required endotracheal intubation versus those who were still under NIV or HFNC at 3 days from inclusion were assessed. Statistics were performed using R . Statistical significance was assumed with two-tailed p < 0.05.The correlation between ΔN = 62), unavailability of research staff (N = 18), refusal to receive esophageal manometry (N = 19). The clinical and physiological characteristics of the study population at the study time points are shown in Table Sixty-one out of 160 consecutive patients admitted to the RICU in the considered time-lapse with de novo ARF and candidates to receive HFNC were enrolled. Of these 51 (83.6%) were diagnosed with COVID-19-related pneumonia while 10 had ARF of different etiology. Reasons for exclusion were: presence of chronic respiratory disease , while the ΔPes was 12 [10–17] cmH2O and 12 [10–18] cmH2O (p = 0.29).The insertion of the nasal plug did not change the pattern of breathing. The RR before and after insertion of the nasal plug was 25 [24–29] minPes and ΔPnos were 12 [10–17] cmH2O and 5.6 [4.2–8.0] cmH2O, while at T2 were 7 [5–11] cmH2O and 3.0 [2.1–4.7] cmH2O, respectively. Patients with COVID-19 presented lower values of ΔPes and ΔPnos at T1 but not at T2 , with all patients receiving HFNC , and at T2 , with 16 (26.2%) and 45 (73.8%) under HFNC and NIV, respectively. The correlation at T2 remained significant when analyzing separately patients receiving HFNC  or NIV  . The ΔPes/ΔPnos ratio at T2 was similar in patients receiving HFNC versus NIV . Moreover, the ΔPes/ΔPnos ratio at T1 was similar in patients with low versus high respiratory drive, defined as ΔPes > 12 cmH2O, .The distribution of the ΔPes/ΔPnos ratio at T1 was 2.27 (standard deviation 0.44), and this value was used as multiplication factor to compute ΔPes, estimated from ΔPnos. Bland–Altman method at T2 showed a bias of 0.1 cmH2O and 95% limits of agreement, LoA, from -2.0 to 2.1 cmH2O , while ΔPnos was 6.5 [4.3–8.4] and 5.6 [4.3–7.5] cmH2O (p = 0.76), respectively. At 24 h, patients who were further intubated had both higher ΔPes  and ΔPnos  as compared with those still under NRS.Characteristics of patients who were intubated versus those still on NRS at day 3 are displayed in Additional file Pnos measured with closed mouth was highly correlated with ΔPes during non-assisted and assisted spontaneous breathing. The correlation between these two physiological variables showed persistence over time and low inter-patient variability regardless the application of HFNC or NIV. The assessment of ΔPnos did not affect inspiratory effort and respiratory rate during non-assisted spontaneous breathing. Moreover, ΔPnos and ΔPes after 24 h from NRS start resulted significantly higher in those patients that were subsequently intubated.The main findings of this study are that, in a population of patients with prevalently COVID-19-induced ARF, ΔPes could be estimated by nasal pressure during a sniff test [Pes/Pnos of 1.05 during maximal sniff and of 1.09 during submaximal sniff, being Pnos always less than Pes. During tidal spontaneous breathing, instead, the posterior nasal valve remains open. When Pnos and Pes are measured during spontaneous breathing without the collapse of the posterior nasal valve, simultaneous pressure waveforms did not show phase difference, though the pressure ratio increased [Pnos swing is likely to mirror variation of Pes during spontaneous breathing. Indeed, our study confirmed that ΔPnos was highly correlated with ΔPes with a narrow range of ratio between the two values, irrespective of the strength of the inspiratory effort.To our knowledge, this is the first study assessing the correlation between tidal changes of esophageal and nasal pressure in patients with ARF during spontaneous breathing. Previous studies already reported that iff test . Howeveriff test . Moreovencreased . Based oPes/ΔPnos ratio across our patient cohort was relatively consistent showing low inter-patient variability over time and under different type of support  test showed that the transmission of pressure changes from the alveoli to the upper airways is altered in case of airflow limitation [Pes on average by 14% in patients with acute asthma and by 19% in patients with stable COPD [Pnos and SNIP exhibit different physiological behaviors, dynamic hyperinflation may affect ΔPes/ΔPnos ratio also during spontaneous breathing. As we have excluded patients with hypercapnic respiratory failure and chronic obstructive respiratory disease, the present results should not be automatically extended to patients affected by significant dynamic intrinsic positive end expiratory pressure. Second, the measurement of ΔPnos during spontaneous breathing may be affected by the collapse of the posterior nasal valve induced by exaggerated respiratory drive [Pnos. Third, all the measurements were performed with patients asked to keep the mouth closed for the entire evaluation time. This task, however, might be difficult to accomplish in some clinical conditions , and during severe nasal congestion or anatomical alterations of the nostrils. Moreover, although we did not measure the flow change induced by the ΔPnos equipment, it cannot be excluded that the insertion of the nasal plug would have modified the flow (and thus the positive pressure) delivered through HFNC. In this line, there is evidence that closing a nostril can modify the final flow delivered through HFNC, thus reducing the degree of positive pressure generated [Pes) after placing the Pnos equipment, we feel that the plug insertion did not significantly affect the impact provided by HFNC on patients’ respiratory drive. Also, our study population mainly included patients with COVID-19-related ARF, much reducing the generalizability of results. Finally, the clinical evidence provided by our investigation should be taken with caution as the study was not sufficiently empowered. In this line we believe that a different study design with a pre-calculated sample size would be needed to find a reliable threshold of ∆Pes and ∆Pnos able to predict the risk for intubation.Although our study reported high correlation between Δmitation . Moreoveble COPD . Despitery drive , 21. In enerated . Howeverenerated  demonstrPnos could be extended in different settings of care, thus implementing the respiratory effort monitoring of patients with ARF at impending risk of deterioration.With this proof-of-concept physiological study we have showed that nasal pressure swing during spontaneous tidal breathing was highly correlated with esophageal pressure swing in patients with ARF. The ratio between these variables showed persistence over time and low inter-patient variability regardless the application of NRS. Should data be confirmed on larger studies and in heterogeneous populations with ARF, noninvasive and easy-to-use measurement of ΔAdditional file 1: eTable 1. Esophageal and nasal pressure swings according to acute respiratory failure etiology. Data are presented as median and interquartile ranges (IQR). eFigure 1. Pearson’s R showing correlations between ΔPes and ΔPnos at 24 hours after splitting the study population according to the NRS received. eFigure 2. Bland-Altman analysis assessing the agreement between ΔPes measured with esophageal manometry and estimated based on ΔPnos  and computed as k·ΔPnos, where k is the average ratio of ΔPes to ΔPnos measured at baseline. At T2 Bland-Altman methods showed a bias of 0.1 cmH2O and 95% limits of agreement, LoA, from −2.0 to 2.1 cmH2O (95.1% of measurements within LoA). Among patients receiving HFNC bias was 0.1 cmH2O and 95% LoA were from −2.1 to 2.3 cmH2O, while in patients receiving NIV bias was 0.0 and 95% LoA from −2.0 to 2.0 cmH2O. eTable 2. General and clinical characteristics of the study population according to respiratory support at day 3. Data are presented as number (n) and percentage for dichotomous values or median and interquartile ranges (IQR) for continuous values.


Establishing a quantitative link between magmatic processes occurring at depth and volcanic eruption dynamics is essential to forecast the future behaviour of volcanoes, and to correctly interpret monitoring signals at active centres. Chemical zoning in minerals, which captures successive events or states within a magmatic system, can be exploited for such a purpose. However, to develop a quantitative understanding of magmatic systems requires an unbiased, reproducible method for characterising zoned crystals. We use image segmentation on thin section scale chemical maps to segment textural zones in plagioclase phenocrysts. These zones are then correlated throughout a stratigraphic sequence from Saint Kitts (Lesser Antilles), composed of a basal pyroclastic flow deposit and a series of fall deposits. Both segmented phenocrysts and unsegmented matrix plagioclase are chemically decoupled from whole rock geochemical trends, with the latter showing a systematic temporal progression towards less chemically evolved magma (more anorthitic plagioclase). By working on a stratigraphic sequence, it is possible to track the chemical and textural complexity of segmented plagioclase in time, in this case on the order of millennia. In doing so, we find a relationship between the number of crystal populations, deposit thickness and time. Thicker deposits contain a larger number of crystal populations, alongside an overall reduction in this number towards the top of the deposit. Our approach provides quantitative textural parameters for volcanic and plutonic rocks, including the ability to measure the amount of crystal fracturing. In combination with mineral chemistry, these parameters can strengthen the link between petrology and volcanology, paving the way towards a deeper understanding of the magmatic processes controlling eruptive dynamics.The online version contains supplementary material available at 10.1007/s00445-021-01476-x. The interplay between the chemical and physical processes experienced by magma within the crust is intimately linked to the style and frequency of eruptions observed at the surface  that represents a millennial timescale of eruptive history. Given its ubiquity throughout the stratigraphy, we use plagioclase to provide us with a “crystals-eye view” of the magmatic plumbing system, using a series of quantified chemical maps to interrogate mineral chemistry. Our results show that the systematic investigation of plagioclase chemistry can reveal temporal trends not evident in whole rock geochemistry alone. Using the method of Sheldrake and Higgins , we quanth December 2020–present) but also noted on Saint Kitts, Montserrat, Dominica and Martinique , microvesicular pumices with sparse phenocrysts (e.g. SK386B), and mafic scoria with abundant phenocrysts of plagioclase and crystal clots (SK392). Scans of thin sections in plane polarised light can be found in Online Resource 2, Fig. 2 (wt%) from XRF analysis used as the internal standard. Major element whole rock measurements and selected trace elements for the samples used in this study are found in Online Resource 3, Table Selected samples were cleaned and dried in an oven at 60 °C for 24 h. The dried samples were then crushed, sieved to < 500 µm and milled to a fine, homogeneous powder using an agate mill. Glass beads were made from the resulting powder using a PANalytical EAGON-2 fusion machine on a pre-set silicate programme at the University of Geneva. Major element analysis was performed on the glass beads by X-ray fluorescence (XRF) using a PANalytical AXIOS MAX with a rhodium anode tube at 4 W at the University of Lausanne. DA-12, NIM-N and NIM-G standards were used for quality control. A suite of trace elements was measured on the glass beads by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) at the University of Lausanne using a Quadrupole Agilent 7700 spectrometer interfaced to a GeoLas 200 M 193-nm excimer ablation system. The NIST SRM 612 external standard was measured at the beginning and end of every four unknowns. Data were reduced using the SILLS data reduction software  with an analysis time of ~ 21 h per sample . Analysis conditions were 15 keV, 100 nA, 150 ms dwell time and a 5-µm beam. All plagioclase EPMA data are presented in Online Resource 4, Table In situ mineral analyses of plagioclase were performed using a JEOL 8200 Superprobe at the University of Geneva and a JEOL JXA-8530F at the University of Lausanne. Both microprobes were equipped with a five-channel wavelength-dispersive spectroscope system (WDS) and were operated at an accelerating voltage of 15 keV, a beam current of 15 nA and a beam diameter of 5 µm. Quantitative analyses were made using a variety of internal standards . Mineral analyses were targeted to span the full variability of plagioclase zoning in a given thin section, typically using transects. WDS maps were measured on a representative part of 9 selected thin sections using a 20 µm pixel size to map a total area of ≥ 100 mm2+ / Ca2+  + Na+  + K+] × 100; Online Resource 6, Fig. Phase separation was performed using a finite mixture model on the EPMA count maps of all 10 measured elements  to a minimum at 500 cm (50.6 wt%), followed by a resurgence to andesitic values. The silica minimum is followed by a 70-cm-thick palaeosoil layer. This sequence is punctuated by non-systematic oscillations between ~ 56 and ~ 62 wt% SiO2. SiO2 and CaO variations on Saint Kitts are remarkably well correlated, not only for this sequence but all lava and pyroclastic samples on the island. FeO has a negative correlation with SiO2 accordant with Saint Kitts’ classification as tholeiitic–calc-alkaline transitional , typically associated with orthopyroxene as clots. Orthopyroxene is easily identified by its pale green–pale brown colour in plane polarised light and the coprecipitation of Fe oxides growing as inclusions in the rim and, less commonly, in the core. It is typically unzoned but may exhibit weak oscillatory or reverse zoning (SK408). Where both orthopyroxene and amphibole are present, a higher fraction of orthopyroxene may result in a lower fraction of amphibole  can be seen in Fig. ole Fig. . Amphiboole Fig. . Phenocr2 (81 pixels) and (ii) matrix plagioclase, with crystal areas < 32400 µm2 (81 pixels). Importantly, by using the method of Sheldrake and Higgins (Plagioclase varies in its chemistry (expressed as An#) and texture both within and between erupted samples. The area fraction of An# for each sample is plotted in Fig.  Higgins , we ensu2  broadly increases vertically through the section (55–87), with units in the upper half displaying more pronounced bimodality in An# distributions Fig. . Whole rSK385 61. wt%; FigThe complex, bimodal phenocryst distributions Fig.  reflect Complex textures and zoning features that reappear frequently throughout the sequence suggest that plagioclase zoning may be correlated between samples. Figure Crystal populations can be defined as groups of crystals that contain the same combination of zoning group(s) Fig. . Each zo2O pathways , where measured glass chemistry may span a significantly wider chemical spectrum than the whole rock chemistry  scoria layer. In order to map a total area of > 100 mm2, three separate scoria pieces from the deposit were individually scanned, effectively representing three random samples of the same eruption. Segmentation identified the same five zoning groups in all three scoriae independently , as well as several commonalities in crystal populations , as well as the contrasting populations observed in a subsequent event (SK394A), suggests that the crystal populations present, and their proportions, provide a unique fingerprint for samples of a given volcanic eruption Fig. . Hence, Following the verification of the method for a single sample, we can now examine how crystal populations change through time between eruptions. Figure Considering the number of populations present in a given sample Fig.  as a proSuperimposed on this overall reduction in complexity is a dependence between the bed thickness and the textural complexity, such that thicker beds  contain the highest number of crystal populations. Additionally, the thickest units  contain the highest number of populations composed of three and four zoning groups per crystal .where In all samples FI and the rim An# interquartile range are positively correlated, suggesting that fracturing exposes the inner portions of the phenocrysts, effectively increasing the compositional range of the outer rims Fig. . There iThe quantified chemical mapping of plagioclase has allowed us to interrogate magmatic processes with a greater statistical significance compared to whole rock chemistry and EPMA spot analyses alone. Further subdividing crystals on the basis of their size and textural relationships Supplementary file2 (PDF 25401 KB)Supplementary file3 (DOCX 23 KB)Supplementary file4 (XLSX 48 KB)Supplementary file5 (PDF 1453 KB)Supplementary file6 (PDF 821 KB)Supplementary file7 (PDF 2478 KB)Supplementary file8 (PDF 3950 KB)Below is the link to the electronic supplementary material.