The results were discussed as regards analogous systems where cholesterol (Chol) was the third component. Moreover, the phosphatidylcholine–lauryl gallate (PC–LG) interactions were monitored by the attenuated total reflectance Fourier transform infrared spectroscopy and time-of-flight secondary ion mass spectrometry. Besides lipid composition, the addition of LG was found to be a significant factor to modulate the model membrane properties. The LG molecules adjust themselves to the PC monolayer structure. The hydrophobic fragment is dipped into the membrane interior while the hydroxyl groups of phenolic gallate moiety associate with the polar groups of PC mainly through hydrogen bonding inducing the compacting effect. LG is found to be deeply submerged within DOPC, closer to the double bonds, and its insertion practically does not affect the DPPC/DOPC membrane fluidity. This is crucial for getting more profound insight into the role of LG in stabilizing the non-raft domains, mostly exposed to oxidation in which LG can co-localize and serve its antioxidant function.Lauryl gallate (LG) is an antioxidant agent. However, it exhibits poor solubility in water. Its interactions with the membrane result in structure evolution thus affecting the membrane functionality. In this paper the Brewster angle microscope coupled with the Langmuir trough was applied to determine the morphology, phase behaviour, thickness and miscibility of ternary Langmuir monolayers with equal mole fractions of 1,2-dipalmitoyl- Domain formation in the model membranes has been intensively studied since the hypothesis of lipid rafts was put forward  enriched in unsaturated PL susceptible to oxidative attack. Oxidation of unsaturated fatty acids in biological membranes lowers the membrane fluidity, damages its structure and functionality and finally causes undesirable lesions and diseases Yagi . To reduThe aim of this work was to investigate the phase behaviour, morphology and stability of model phospholipid membranes containing cholesterol or antioxidant LG. From the wide spectrum of gallates only LG seemed to optimize high antioxidant activity with sufficient hydrophobicity for the Langmuir monolayer formation. The effect of LG on the binary DPPC/DOPC monolayers was compared to that of cholesterol, which unlike LG is a structural component of biological membranes. The ordering of PL induced by Chol is driven by steric interactions  isotherms . Moreover, the PC–LG interactions in the hydrated multibilayers were monitored by the attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. The studies were supplemented with the time-of-flight secondary ion mass spectrometry (TOF-SIMS) analysis which identified chemically the lateral distribution of components in the PC/LG monolayers. Formation of intermolecular hydrogen bonds between the galloyl moiety and the head groups of PC was examined based on the signal intensity of characteristic ions.For these purposes, the ternary DPPC/DOPC/Chol (or LG) Langmuir films on the water subphase were investigated using the Brewster angle microscope (BAM) coupled with the Langmuir trough. The kind and magnitude of interactions between molecules in the ternary systems were estimated based on the experimentally determined surface pressure–area per molecule  and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol (Chol) and LG of purity > 99% were provided by Sigma and used without purification. Chloroform (p.a.) and ethanol   were solvents for compound dissolution. Redistilled water was purified by the Milli-Q system to obtain resistivity of 18.2 MΩcm. Such water was used as a subphase for the Langmuir monolayer formation.PL: 1,2-dipalmitoyl-x = 0.5) and the ternary mixtures of the constant DPPC and DOPC ratio (x = 0.5) with varying Chol or LG mole fraction  were prepared from the respective stock solutions. The droplets of the solution were placed on the subphase surface top by means of a Hamilton microsyringe. After chloroform evaporation, the surface pressure–area per molecule (π–A) isotherms were recorded during symmetrical monolayer compression with the rate of 10 mm/min. The solubility of LG in water was estimated for 0.035 mg/mL, i.e. 1.034 × 10−4 M (http://www.hmdb.ca/metabolites/HMDB38720). It was sufficiently low to form the Langmuir monolayers on water in the wide range of compression rates (from 5 to 100 mm/min) and its monolayers were stable in time even at a high surface pressure. The temperature of 20 °C was maintained owing to the water system circulation . The surface pressure was measured by the Wilhelmy plate method. Simultaneously, the morphology of the floating monolayers was monitored by the BAM coupled with the trough. The Langmuir trough and BAM were placed in the laser safety cabinet and isolated from vibrations coming from the surroundings by the active vibration isolation system.The one-component , binary (DPPC/DOPC 1:1) and ternary (DPPC/DOPC/Chol and DPPC/DOPC/LG) Langmuir monolayers were formed on the water subphase using a computer-controlled KSV standard trough . First the compounds were dissolved in chloroform to obtain the total concentration of 1 mg/mL. Then the binary mixture of DPPC/DOPC  to 10−8. As p-polarized light illuminated the pure water surface at the Brewster angle of about 53.2°, practically no intensity reflection was observed (dark background without contrast). When the monolayer with the refractive index different from that of water (i.e. 1.33) was spread onto the subphase, reflection occurred. The reflected light passed through the objective lens into the analyzer, and finally to the CCD camera . These elements were positioned in the reflected beam path to acquire high quality contrast images of the lateral morphology. Imaging of 720 µm × 400 µm areas was performed with the lateral resolution of 2 µm. The black glass plate was placed under the water subphase on the bottom of the trough to prevent diffraction of the laser beam and to minimize light scattering.A computer-controlled nanofilm_ultrabam  was used for direct visualization of substructures with a long range orientational order and for evaluation of layer thickness at the air–water interface. The light source of the BAM was a solid-state 50 mW laser-emitting 658 nm wavelength light. The laser beam accounts for purely R into the film thickness d  and the mean molecular areas of components in the monolayers (A12), as determined from the π–A isotherm at 35 mN/m, first the quantity of compounds needed to cover the support with one statistical monolayer was calculated. Then the concentration and volume of the chloroform solutions were chosen so that after evaporation of the solvent, the 40 statistical bilayers were formed in each case. Such number of bilayers was found to be sufficient for obtaining good quality spectra. Before the solution pouring, the ZnSe crystal plate was cleaned with ultra pure ethanol. After the solvent evaporation the supported layers were transferred to a vacuum (117 mbar) for 30 min to remove the possible chloroform residuals, and then placed into the closed chamber with a relative humidity 80% for 30 min in order to hydrate the dried multibilayers.The multibilayers built of DPPC or DOPC and LG at different mole fractions (0.25 and 0.5) were prepared on the ZnSe crystal by spreading of the chloroform solutions. Knowing the geometric surface area of the ZnSe crystal .Infrared absorption spectra were obtained with a Nicolet 8700A Thermo Scientific spectrometer. The attenuated total reflection (ATR) configuration was used with the Varimax HATR ZnSe crystal plate (45° cut). Typically 256 Fourier-transformed and averaged scans were collected for each measurement. Absorption spectra at a resolution of 4 cmxLG = 0.25) were transferred at 35 mN/m with the rate of 5 mm/min from the water subphase onto the mica plates using the Langmuir–Blodgett trough . The samples were dried under vacuum (117 mbar) overnight. Mass spectrometric measurements were made using a TOF-SIMS 5 device . The primary ion source of Bi3+ was used at 25 keV. The scanning area of secondary ions was 100 µm × 100 µm with 256 × 256 pixels. All measurements were performed in the static mode (dose no larger than 1013 ions/cm2). Two different places of each sample were analysed. No statistical difference was found between them. The post processing data analysis was conducted using the SurfaceLab 6.7 software (ION-TOF). For all samples charge effects were compensated using a flood gun adjusted individually for each sample area. Only positive spectra were recorded and calibrated using the positions of CH3+, C4H7+, C5H9+ peaks. The mass peaks with m/z of 27, 29, 39 and 58 were removed and then the mass spectra were compared after normalization of the intensity, proportionally to the total intensity recorded for each spectrum.The Langmuir monolayers of DPPC, DOPC, LG and mixed PC/LG  mixtures by monitoring differences in the shape of the compression isotherms at the air–water interface. Figure s Gaines . Therefos Gaines b.π–A isotherms which would suggest LG leaving the monolayer for the bulk subphase, or forming a multilayered film. These findings allow to confirm that the LG molecules remain in the mixed monolayer and associate with PC in such a way that changes the polar head configuration yielding smaller areas per molecule.To prove that LG is not ‘squeezed out’ of the PC monolayer, the isotherm of pure DOPC monolayer was recorded and then taking the same amount of DOPC the mixed DOPC/LG film containing 10 mol% LG was obtained. The measurements were repeated three times. The same experiment was conducted for the systems with DPPC instead of DOPC. The results are presented in Fig. Moreover, as reported previously for the DOPC/LG, POPC/LG and DPPC/LG binary systems in the full range of the LG mole fraction, no LG squeezing out of the PC monolayers was found  there is observed the liquid–liquid phase coexistence (region of coexisting liquid phases) with one phase rich in the unsaturated DOPC and the other in the saturated DPPC and cholesterol which can be seen as dark and bright regions, respectively. Hence the bright regions seem to be equivalent to the liquid-ordered state (Veatch and Keller xChol = 0.5 two liquid phases still coexisting as micron-sized liquid domains are observed and their heterogeneity of size is larger than that at xChol = 0.25. At a high mole fraction (xChol = 0.75) one uniform liquid phase is observed at first as a result of coalescence of domains into larger ones, and finally at ca. 30 mN/m into one homogeneous phase. Domains coalesce close to a critical point due to a small difference in the dipole density between phases (Keller and McConnell Both DPPC and DOPC form homogeneous monolayers at 35 mN/m but that of DPPC is tightly packed (condensed) while that of DOPC is loosely packed (expanded) Fig. . In the x = 0.25–21 mN/m; at x = 0.5–30 mN/m; at x = 0.75–38 mN/m. At low LG concentration xLG = 0.25 and surface pressure, the area fraction of bright oval domains is small but the average size of the condensed domains increases with the increasing surface pressure up to 35 mN/m. Contrary to DPPC/DOPC, further compression does not lead to the change of domain sizes or shapes but it increases only their number per unity area. Hence the domains can be identified individually. At xLG = 0.5 the domains are much smaller and rather sparsely spread. With the LG mole fraction increase up to x = 0.75 the ternary monolayer is homogeneous at 35 mN/m  in DPPC/DOPC the difference in x = 0.75 it is as large as 337 mN/m. The drastic increase in the degree of the monolayer condensation, i.e. packing of the lipid molecules, makes it more ordered.The s (Israelachvili a is estimated to be 87 Å2 (http://www.hmdb.ca/metabolites/HMDB38720), the volume of hydrocarbon chain V = 350.2 Å3, the length of hydrocarbon chain lC = 16.68 Å, the critical packing parameter s was calculated according to Eq. Additionally, the geometric packing (arrangement) of molecules was described in terms of the molecular shape of the constituents. It was predicted based on the critical packing parameter s parameter for LG is 0.24 which is lower than 1/3 suggesting a cone shape of molecule  for which coexistence of condensed DPPC-rich domains and a fluid DOPC-rich matrix can be seen . The thickness values of DPPC/DOPC/LG membranes depend insignificantly on the composition and they are located between those of DPPC/DOPC and LG. The thickness of pure LG monolayer is as low as 0.93 nm. The difference in thickness of both types of membranes becomes bigger as the mole fraction of the third component increases. As reported earlier Chol exerts a condensing effect on DPPC and DOPC  at different values of the surface pressure were determined directly from the isotherms, and then compared with the values obtained for the ideal miscibility or complete immiscibility of molecules based on Eq. A1, A2, A3 denote the mean molecular areas in the one-component monolayers of DPPC, DOPC and Chol (or LG), respectively at a given surface pressure; and x1, x2, x3 are the molar ratios of components 1, 2 and 3 in the mixed monolayer.Miscibility and interactions between molecules in the mixed monolayers were qualitatively analysed in accordance with the additivity rule Gaines . TherefoA123 is the mean molecular area in the ternary films. If Aexc is equal to 0, the dependence of A123 on the composition is linear, and the mixture behaves ideally indicating that the components are completely miscible or immiscible. When Aexc ≠ 0, i.e. any deviation from linearity of the A123 = f (x) function occurs, this indicates miscibility of the compounds due to the mutual interactions. The non-ideality of these mixed systems was expressed in the excess area (Aexc)-composition plots shown in Fig. Then the effect of Chol or LG on the DPPC/DOPC phospholipid films was estimated from the excess area per molecule values according to Eq. Aexc are lower  was determined at 35 mN/m from Eq. N denotes the Avogadro’s number, Aexc is the excess area, and π is the surface pressure which changes in the range of integration from 0 to 35 mN/m.To explore the magnitude of these interactions and to find out how different amounts of Chol or LG modulate their interactions with the DPPC and DOPC model membranes, the excess Gibbs energy of mixing . Although not corresponding to the more densely packed monolayers, at which xChol = 0.25 (∆Gexc = − 1660.9 J/mol) (Jurak xLG = 0.5 (∆Gexc = − 1424.2 J/mol)  Jurak  whereas R is the gas constant and T is the temperature.Furthermore, in order to determine the thermodynamic stability of the mixed systems the total Gibbs energy of mixing was determined Eq. :7\documeGmix are negative regardless of the monolayer composition  of infrared spectra of PC membranes is represented by the bands of antisymmetric and symmetric stretching vibrations of CH2 groups ) of lipid hydrocarbon chains that form a hydrophobic core of the lipid bilayer. These bands are sensitive to the lateral packing of hydrocarbon chains. Spectral parameters (wavenumber of absorption maximum and bandwidth) of the νas,s(CH2) bands monitor the alterations of the ratio of trans to gauche conformers of lipid CH2 moieties  bands of DOPC/LG to a higher-frequency region and increase in the bandwidths of these two vibrational modes. This indicates that the DOPC/LG bilayers adopt a conformationally disordered gauche-rich fluid state while the DPPC/LG bilayers are enriched in the trans conformer population in the hydrocarbon part of the membrane. The absorption maxima of both νas,s(CH2) bands for pure LG are between those of DPPC and DOPC, and simultaneously the peaks become broader/tighter and shifted to higher/lower frequencies than those of DPPC or DOPC, respectively. Such observation implies the intermediate ordering of hydrocarbon chains.In Fig. +–(CH3)3 stretching vibrations of choline, symmetric and antisymmetric stretching vibrations of PO2− group, stretching vibrations of the C–O–P–O–C fragment, stretching vibrations of the ester carbonyl group C=O which represents the polar–apolar interface in the PC membrane  and its maximum shifts to the higher-frequency region compared to the pure PC films, and at xLG = 0.5 finally splits into two bands. This is due to contribution of the C=O stretching coming from the C=O ester groups of LG. In the spectrum of pure LG the band ascribed to the stretching vibrations of ester C=O group appears at 1683.28 cm−1. Its shift suggests participation of carbonyl groups in the interaction process. The other characteristics for the LG bands at 1245.81, 1196.76 and 1034.07 cm−1 correspond to the C(=O)–O stretching vibrations of ester groups, in-plane bending vibrations of the phenolic group and O–C–C stretching vibrations of the ester group, respectively  are very sensitive to hydrogen bonds formation  and 1220 cm−1 (in the fully hydrated PC state)  + ion. Other characteristic ions produced by PC head appear at m/z 104.12 ((C5H14NO)+) and m/z 166.07 ((C5H13PO3N)+) +) Fig. a intensi)+) Fig. b signifi)+) Fig. . During .38 Fig. b, c. Hig.38 Fig. .3+ beam and different packing of LG within the DOPC monolayer.To illustrate how insertion of LG into the DPPC or DOPC monolayer affects the molecular organization, simple models are presented in Fig. Interestingly, intensity of molecular ion of DOPC after the addition of LG is 38.3 times higher than that of pure DOPC. This is due to weak binding of LG into DOPC through the hydrogen bond that is favourable for desorption of molecular species. In the presence of LG the yield of DPPC molecular ion increases only eight times indicating that molecules are more strongly bound within the DPPC/LG monolayer due to the additional interactions by hydrogen transfer between the hydroxyl group of LG and the phosphate group of DPPC. As a result, in the molecular desorption region (far away from the impact region)  phase at the experimental temperature. Consequently, because of the presence of C18 chains with one double bond which induces a kink, the DOPC monolayer is less condensed  and ∆Gexc (1277.8 J/mol)  Fig.  and weak ∆Gexc 127.8 J/molLo phase is stronger than for the DOPC-rich liquid-disordered Ld phase , the border of ordered domains blurs which is associated with the fluidizing effect of LG due to disturbing the ordered structure of DPPC-enriched domains. This strongly supports the increased mutual miscibility between DPPC, DOPC and LG in the monolayer state. Hence similarity in the phase behaviour of LG and DPPC/DOPC maintain the membrane fluidity.LG analogously to Chol can modulate the fluidity and ordering of the membrane leaflet, thus influencing its function. LG forms the disordered monolayer at the air–water interface similarly to DOPC Fig. . Althougx = 0.25, is lower in the DPPC/DOPC/LG system as the LG area is lower than that of Chol at that surface pressure. However, in this system the thickness and packing of monolayers change only little with the increasing LG due to the similar phase state/condensation degree of the LG and DPPC/DOPC monolayer . Chol is primarily a structural component of biological membranes. LG can interact with the lipid components of membrane and simultaneously being an antioxidant molecule, it is able to protect the unsaturated lipids from oxidation by free radical scavenging. Chol in the DPPC/DOPC monolayers causes their condensation leading to an increase in density of lipid molecules and their ordering, thus improving the film stability. LG practically retains the DPPC/DOPC membrane fluidity and minimizes the unfavourable interactions between DPPC and DOPC also increasing the phospholipid miscibility and stability. As regards the distribution between the coexisting phases, LG preferentially localizes in more fluid areas, which is imposed by antioxidant activity, whereas Chol in more ordered ones. This results from differentiated affinity of both molecules for unsaturated or saturated PL. The favourable interactions contribute to the formation of heterogeneities within the membrane. They seem to reflect the specialization of various regions in the membrane processes.


Field cancerisation was originally described as a basis for multiple head and neck squamous cell carcinoma (HNSCC) and is a pre-malignant phenomenon that is frequently attributable to oncogenic human papillomavirus (HPV) infection. Our work on β-HPV-induced cutaneous squamous cell carcinomas identified a novel Lrig1+ hair follicle junctional zone keratinocyte stem cell population as the basis for field cancerisation. Herein, we describe the ability for HPV to infect adult tissue stem cells in order to establish persistent infection and induce their proliferation and displacement resulting in field cancerisation. By review of the HPV literature, we reveal how this mechanism is conserved as the basis of field cancerisation across many tissues. New insights have identified the capacity for HPV early region genes to dysregulate adult tissue stem cell self-renewal pathways ensuring that the expanded population preserve its stem cell characteristics beyond the stem cell niche. HPV-infected cells acquire additional transforming mutations that can give rise to intraepithelial neoplasia (IEN), from environmental factors such as sunlight or tobacco induced mutations in skin and oral cavity, respectively. With establishment of IEN, HPV viral replication is sacrificed with loss of the episome, and the tissue is predisposed to multiple cancer stem cell-driven carcinomas. Human papillomavirus (HPV) infection is associated with oropharyngeal and anogenital cancers in both men and women. Approximately 90% of all cervical cancers are attributed to high-risk alpha-genus HPV (α-HPV) infections, also ∼60% of squamous cell carcinomas (SCC) of the vulva, vagina, anus and penis are due to infection of α-HPV . HPV infSequential genetic and epigenetic changes occur over several years and provide the transformational basis for both intraepithelial neoplasia (IEN) and ensuing epithelial cancers (carcinoma). The proportion of transformed cells within IEN can be graded and used to define the risk of invasive disease . Progres1), hence the American Association for Cancer Research Task Force on the treatment and prevention of IEN recognizes the importance of early treatment to prevent invasive disease    . Howeverigure 1) . Within igure 2) . TherefoFigure 2). Clonal selection and expansion may result in a single clone in continuous epithelia  or multiple clones in discontinuous epithelia (breast and lung)  . Within nd lung) . Hence, Figure 2)  . Howeverigure 2) . As a coigure 2) . Hence, Human papillomavirus (HPV) binds epithelial cell heparan sulfate proteoglycans and cell specific receptors to gain entry by both clathrin-dependent and -independent endocytosis . InfectiMost HPV infections are spontaneously cleared. For example, the risk of α-HPV female genital infection over a lifetime is up to 80% , but witIn cervical lesions caused by the α-HPVs, the viral oncogenes E6 and E7 increase proliferation of suprabasal epithelial cells. Along with E1 and E2, viral replication requires E6 and E7 for entry into S-phase. Upon leaving the basal layer, keratinocytes enter into a program of terminal differentiation in order to produce a protective barrier. However, in HPV infection, suprabasal cells continue to proliferate and are prevented from entering terminal differentiation . OncogenFluorescent labeling studies in mice using lineage tracing have concluded that stem cell division is prominently (∼90%) asymmetric; in which there is renewal of the stem cell and a daughter cell that is committed to terminal differentiation . StochasAdult tissue stem cell expansion, as proposed for the mechanism of HPV-induced field cancerisation, is dependent on symmetrical division of existing stem cells. As discussed, HPV viral oncogenes will drive proliferation of infected adult tissue stem cells by targeting p53 or pRb. Importantly, the binding of E7 to pRb releases repression of both sex determining region Y-box 2 (Sox2) and octamer-binding transcription factor 4 (Oct4) . Similarin vitro of increased colony forming efficiency  or cutaneous warts. Similarly, mucosal HPV lesions include condyloma or leukoplakia within the genitalia and oral mucosa . In addiThe transition from benign to premalignant lesion has been characterized by TP53 immunostaining, resulting from mutation acquisition, and manifesting as a small micro-clonal expansion comprising of 60–3000 cells presenting clinically as an actinic (solar) keratosis or leukoplakia . In the Figure 2)  . For exaigure 2)  Similarligure 2) . Hence, igure 2) .Field cancerisation emerging from HPV induced amplification of adult tissue stem cells results from additional environmental induced mutations. The area of IEN can be large, in the oral cavity it can be over 7 cm in diameter and is predisposed to multiple primary HNSCC and therefore poor prognosis . IntriguMany cancers exhibit hierarchical growth with evidence of differentiation consistent with the cancer stem cell model . Whereinin vitro and in vivo assays (Head and neck squamous cell carcinoma (HNSCC) identification and characterisation of cancer stem cells has been supported by o assays . Similaro assays . The enso assays . Overallo assays . High nuo assays . Hence, This review has focused on HPV infection, notably oncogenic genotypes from both the alpha and beta genus. Within the tropic tissue that was breached to allow viral entry, persistent infection requires that resident adult tissue stem cells are infected. HPV-infected adult tissue stem cells, similar to other HPV-infected cells are forced to proliferate, leading to their expansion as adult tissue stem cells beyond their native niche. This expansion renders them susceptible to environmental carcinogens. In the case of skin, β-HPV genotypes induce hair follicle junctional zone keratinocyte stem cells to proliferate and expand into the overlying epidermis, whereupon they are susceptible to UV-induced mutations. Transformational mutations result in field cancerisation, with additional driver mutations, causing clonal selection as IEN progresses from mild to severe. Additional mutations then can give rise to multiple cancers. Hence, HPV-induced stem cell expansion predisposes to and, through viral oncogene expression, induces the generation of cancer stem cells, which in turn can define the fate of tumor and patient prognosis. Hence, we propose that the ability of oncogenic HPV infection to manipulate adult tissue stem cells underpin its ability to drive cancer growth through promotion of cancer stem cells.CO, SL, and GP conceived the idea. CO, SL, CB, MG, and GP drafted the manuscript with inputs from all authors. All authors have made final approval for the final version to be submitted.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Stem cells and cellular plasticity are likely important components of tissue response to infection. There is emerging evidence that stem cells harbor receptors for common pathogen motifs and that they are receptive to local inflammatory signals in ways suggesting that they are critical responders that determine the balance between health and disease. In the field of papillomaviruses stem cells have been speculated to play roles during the viral life cycle, particularly during maintenance, and virus-promoted carcinogenesis but little has been conclusively determined. I summarize here evidence that gives clues to the potential role of stem cells and cellular plasticity in the lifecycle papillomavirus and linked carcinogenesis. I also discuss outstanding questions which need to be resolved. Human papillomavirus (HPV) infection occurs via micro wounds which allow the virus access to the basal layer of stratified epithelia. This target site of infection has been the subject of intense scrutiny, leading to significant progress regarding the molecular mechanisms governing the attachment and eventual entry of the virus into initially infected cells ,2. HowevPapillomaviruses can productively infect both mucosal and cutaneous stratified epithelia. Infection and the ensuing life cycle of the virus are intimately linked to the differentiation program of the tissue ,9 thus oStudies using HPV virus like particles (VLPs), or infectious virions for attachment and viral entry argue against the exclusive targeting of a small subpopulation such as the tissue stem cells. Rather, the virus associates with the exposed basement membrane at a site of wounding, and furin cleavage leads to conformational changes on the capsid which allow it to attach to nearby basal cells via a receptor which recognizes L1 capsid protein . The higIf one takes for granted the more likely scenario that both stem and committed cells can be the targets of infection, then a vital question concerns the fate of infection in a stem as opposed to a committed cell. A popular hypothesis has been that long-term maintenance of the viral DNA can take place within infected tissue stem cells ,5. This Epidermodysplasia verruciformis (EV) patients, transplant recipients etc.) [Hair follicle stem cells have also been proposed as the reservoirs for human cutaneous HPV infections. DNA of β-papillomaviruses is frequently isolated from plucked eyebrow hairs ,18 suggets etc.) .The inability to define the concrete characteristics of the human cervical stem cells has complicated studies aiming to understand the maintenance of the human mucosotropic viruses. It is thought that the transformation zone which is characterized by a transition from columnar to squamous epithelium is the site of the so-called reserve cells which may act as the cervical stem cells. Some markers have been proposed for reserve cells , p63, Keratin 7 (K7), etc.) ,22 but tEscherichia coli [Infected tissue stem cells are of interest due to their potential links to carcinogenesis. However, more recently stem cells have also been implicated in the tissue response to infection. There is an emerging understanding that tissue stem cells have evolved to respond directly both to commensal and pathogenic microbes as evidenced by the expression of pattern recognition receptors (PRRs) in tissue stem cells ,24,25. Ihia coli  mobilizeWhile the effects of infection on tissue stem cell dynamics are less well understood in cutaneous and mucosal epithelia compared to the gut, studies investigating the expression of viral gene products on skin stem cell populations suggest that important changes occur. Compelling evidence regarding the changes in stem cell dynamics during papillomavirus infection comes from studies using transgenic animals for both mucosotropic ,29 and cInitial focus has been on uncovering how the behavior of stem cells may change upon papillomavirus infection. There is however also evidence which supports an alternate, not mutually exclusive scenario: that during infection it is possible that reprogramming events can contribute to the emergence of stem-cell like cells.The increased stem cell mobilization seen in transgenic animals is also concurrent with the expression of stem cell markers such as Keratin15 (K15) outside the typical stem cell niche ,29. The In light of technologies describing cellular reprogramming developed after Yamanaka’s seminal discoveries in 2006 , the re-Mycobacterium leprae [M. leprae infection leads to such events are incompletely understood, initial evidence suggests that they involve in part the innate immune response and inflammation which precede such reprogramming events [One of the most intriguing and compelling reported examples of reprogramming in vivo implicates the intracellular m leprae . The bacm leprae . While Sg events .Inflammation is naturally of relevance to the HPV lifecycle as well, particularly if one takes into account the mode of infection. Both infection-associated and sterile inflammation are understood to contribute to a regenerative response and inflammation is emerging as an evolutionarily conserved mechanism of tissue regeneration . RegenerOther than leading intracellular lifestyles HPVs do not have many commonalities with mycobacteria at a first glance. However, upon closer inspection of the available evidence there is good reason to suspect that the virus may be contributing to similar events. The viral oncogenes have prominent targets which are implicated in stem cell biology and may contribute to epigenetic reprogramming via their inactivation.INK4A/ARF locus, some of which are implicated in papillomavirus pathogenesis are also important during reprogramming [Work aimed at illuminating the critical steps during the process of reprogramming cells to pluripotency, clearly implicated prominent tumor suppressors in stem cell biology. p53, retinoblastoma protein (pRb) and other key players in tumor suppression have been shown to control checkpoints during cellular reprogramming ,52,53,54Hox genes. Critically this expression has been linked to the high p16 expression characteristic of HPV positive cancers and may represent a way of targeting HPV-positive cancer cells since they are dependent on its continued expression [In addition to targeting tumor suppressors which have been linked to cellular reprogramming there is also evidence to suggest that the viral oncogenes may contribute to cellular reprogramming in ways which are independent of their ability to target p53 or pRb. High-risk HPVs  have recently been shown to upregulate Kruppel-like factor 4 (Klf4) and contribute to its hypoSUMOylation . This uppression . The E7 pression . High-ripression ,66. Of cThere is a dearth of studies which would allow definitive conclusions about the upregulation of stem cell related proteins in the viral life cycle. However, there is an accumulating body of evidence suggesting they may be linked to disease. The upregulation of genes related to stem cells may be a way in which the virus contributes to the formation and maintenance of cancer stem cell populations . RecentlINK4A as a useful biomarker it remains to be seen whether the use of these are of added prognostic benefit. Nevertheless, the expression of these proteins in cancer biopsies and cervical cancer cell lines may provide insights into potential roles in the carcinogenic process. Critically, the links of early gene products, particularly the viral oncogenes of human papillomaviruses to the upregulation of such markers lends credibility to this notion.The expression of stem cell related markers is now increasingly reported in HPV-related cancers. Proteins such as Sox2, Nanog, and Oct4 are now thought to serve not only as biomarkers tumor stage and therapy response in cancer but also may be directly implicated in the process of carcinogenesis. In HPV-associated cancers stem cell markers Oct4, Sox2  , and LriA source of skepticism for studies supporting a changing cellular state during infection, derives from the refractoriness of keratinocytes to cellular reprogramming and perceived lack of plasticity. It is clear that no condition has been described thus far where a viral gene product or set of products have been shown to independently lead to an isolatable stem cell. However, it is critical to bear in mind that infection occurs in the context of a wound. It is an aspect of infection which has been poorly accounted for particularly since many experimental models used in papillomavirus research do not take it into consideration. In virion-infected monolayer keratinocytes, organotypic cultures of stably transfected keratinocytes or transgenic animals expressing viral gene products, the wound environment is not routinely modelled. However, during real-life infection, in addition to the viral invasion which triggers an inflammatory response via the presence of pathogen associated molecular patterns (PAMPs) there are also damage associated molecular patterns (DAMPs), and reactive oxygen species (ROS) released as a result of the wounding. Thus, both infection-associated, as well as sterile inflammation should be kept in mind as additional important factors which likely remove constraints towards increased cellular plasticity .The role of stem cells and cellular stemness has been proposed to be important in the lifecycle and disease promotion by HPVs. Viral genomes have been detected in anatomical locations which are consistent with those of stem cells yet conclusive evidence as to whether infected stem cells are the sites of viral maintenance of all HPVs or the cancer initiating cells of HPV-related cancers is to this day tenuous. Early gene products, and particularly the viral oncogenes have been shown to modify stem cell dynamics and cellular stemness, however the extent to which this is critical for the viral lifecycle or for ensuing disease remains elusive. As HPV-related cancers account for about 5% of human cancers  and pot. Critica


Fcgr2b knockout or antagonistic FcγRIIb antibody inhibited Aβ1-42-induced tau hyperphosphorylation and rescued memory impairments in AD mouse models. FcγRIIb phosphorylation at Tyr273 was found in AD brains, in neuronal cells exposed to Aβ1-42, and recruited SHIP2 to form a protein complex. Consequently, treatment with Aβ1-42 increased PtdInsP2 levels from PtdInsP3 to mediate tau hyperphosphorylation. Further, we found that targeting SHIP2 expression by lentiviral siRNA in 3xTg-AD mice or pharmacological inhibition of SHIP2 potently rescued tau hyperphosphorylation and memory impairments. Thus, we concluded that the FcγRIIb-SHIP2 axis links Aβ neurotoxicity to tau pathology by dysregulating PtdInsP2 metabolism, providing insight into therapeutic potential against AD.Amyloid-β (Aβ)-containing extracellular plaques and hyperphosphorylated tau-loaded intracellular neurofibrillary tangles are neuropathological hallmarks of Alzheimer's disease (AD). Although Aβ exerts neuropathogenic activity through tau, the mechanistic link between Aβ and tau pathology remains unknown. Here, we showed that the FcγRIIb-SHIP2 axis is critical in AβDOI:http://dx.doi.org/10.7554/eLife.18691.001 In Alzheimer’s disease, damage to neurons in the brain gradually causes memory loss and difficulties with thinking. The main hallmarks of this damage are seen in the accumulation of proteins in and around neurons. First, a protein called amyloid beta forms aggregates outside the cell. This appears to lead to the build up of an abnormal form of a protein called tau inside the cell. These abnormal tau proteins have excessive numbers of phosphate groups attached to them, and so are known as “hyperphosphorylated”. The molecular mechanism underlying amyloid beta’s role in the hyperphosphorylation of tau proteins was not known.Amyloid beta binds to many different receptor proteins – including one called Fc gamma receptor IIb – on the surface of neurons. Kam, Park et al. investigated whether interactions between amyloid beta and Fc gamma receptor IIb might regulate the phosphorylation of tau within neurons. Adding amyloid beta to mouse neurons caused tau proteins to become hyperphosphorylated. However, removing Fc gamma receptor IIb from the neurons, or stopping it from binding to amyloid beta, abolished this effect.When amyloid beta was bound to Fc gamma receptor IIb, the receptor became phosphorylated. This in turn triggered a series of further phosphorylation events, culminating in an increase in the level of a molecule that relays signals from cell receptors – called SHIP2 – inside the neurons. This molecule increases tau phosphorylation when added to neurons. Reducing the activity or amount of SHIP2 in mice that present the symptoms of Alzheimer’s disease reduced the hyperphosphorylation of the tau protein in their neurons and restored their memory to normal levels.Kam, Park et al. also looked at samples taken from the brains of human Alzheimer's disease patients. Unlike samples taken from people without Alzheimer’s disease, neurons in these samples contain both phosphorylated Fc gamma receptor IIb and hyperphosphorylated tau proteins.By uncovering the molecules that link amyloid beta with tau hyperphosphorylation, Kam, Park et al.’s results suggest new targets for therapies to treat the symptoms of Alzheimer’s disease. More research is now needed to investigate whether this could lead to the design of new drugs.DOI:http://dx.doi.org/10.7554/eLife.18691.002 Alzheimer's disease (AD) is characterized by the progressive loss of memory and the neuronal degeneration . The patAPP transgenic mice prevented Aβ pathologies, including learning and memory impairment P2, and PtdInsP2, are known to play a major role in signal transduction upon cellular stimulation P2 have been relatively well characterized in cell survival, proliferation, and synaptic function via their binding proteins P2, PtdInsP2 and PtdInsP3 are formed when cells respond to signals P2 , such as PtdInsPmulation . Among tproteins , but the signals . SH2 domsP2 . Increasmeric Aβ , and rece models . HoweverUntil now, Aβ was reported to bind to many receptors, including alpha7 nicotinic acetylcholine receptors (α7 nAChR), NMDA receptor, receptors for advanced glycation end- products (RAGE), Aβ-binding alcohol dehydrogenase (ABAD), the Ephrin-type B2 receptor (EphB2), cellular prion protein (PrPc), and paired immunoglobulin-like receptor B (PirB) . Althoug1-42 to mediate Aβ neurotoxicity, synaptic dysfunction, and memory impairment in AD pathogenesis  to increase PtdInsP2 levels for tau hyperphosphorylation. Further, Fcgr2b or Inppl1 deficiency in 3xTg-AD mice or pharmacological inhibition of either protein abrogates all of these observations, highlighting the importance of the FcγRIIb-SHIP2 axis in the Aβ-induced tau pathology.Recently, we showed that Fc gamma receptor IIb (FcγRIIb) is also expressed in neurons and directly interacts with Aβogenesis . Here, w1-42 and a mediator of memory impairment in hAPP-J20 mice expressing only familial mutant APP , Thr231/Ser235 (AT180), and Ser202 (CP13), but these phosphorylations were abrogated in Fcgr2b knockout (KO) neurons (1-42 in the neuron-specific enolase (NSE)-positive cortical neurons, but not in the Fcgr2b KO neurons . Each group of mice was tested for spatial working memory in a Y-maze task. Although the total number of arm entries was not significantly different between groups, Fcgr2b deficiency improved the spatial working memory of 3xTg-AD mice  region of wild-type (WT) mice  mice , the micWT) mice . Interesbehavior , object behavior , and pasbehavior . On the behavior . From weted mice . In contted mice . Thus, w1-42  in the cytosolic region of FcγRIIb in SH-SY5Y cells  or cytoplasmic region (Δcyto), or substituting the tyrosine residue with phenylalanine (Y273F), and examined their effects on Aβ22 cells . Moreove22 cells , blockediated it , indicat neurons . On the ns , but not in normal and mild cognitive impairment (MCI) patients (stage III) . In addi1-42  is known to bind to the phosphorylated ITIM region of FcγRIIb and inhibit downstream responses triggered by immune receptors . Given t1-42 , suggest1-42. Treatment of SH-SY5Y cells with Aβ1-42 enhanced subcellular localization of SHIP2 to the plasma membrane and thus colocalization of SHIP2 with FcγRIIb P2 P2 with a protein lipid overlay (PLO) assay using the purified His-tagged pleckstrin homology (PH) domain of tandem PH domain-containing protein-1 (TAPP1) as a probe PsP2 , we exam a probe . From tho Aβ1-42 . In contwn cells . Converscells P3 was decreased by 17% P2 and PtdInsP2 levels. On the other hand, the levels of PtdInsP2 were lowered by Aβ1-42 in WT neurons, consistent with a previous report P2 and PtdInsP3 levels in neurons via FcγRIIb.When we directly measured the levels of phosphoinositides with an ELISA assay, we observed that the levels of PtdInsPd by 17% . Howevers report , and als neurons . We furtmembrane . In contwn cells . As repown cells , hydrogeficiency . These o2 by Aβ1-42 can influence tau phosphorylation, we directly delivered phosphoinositide into living neurons using a carrier  in primary cortical neurons in a dose-dependent manner P2, PtdInsP2, and PtdInsP3, failed to do so. Consistent with the activation of GSK3β by Aβ1-42, PtdInsP2 treatment also reduced the inhibitory phosphorylation of GSK3β at Ser9 in neurons , as well P carrier . Comparet manner . Interes neurons . Moreove as well . We furtectively . We confl1 siRNA . Combinel1 siRNA , these r1-42 in control cells, knockdown of SHIP2 expression in SH-SY5Y cells abrogated tau phosphorylation by Aβ1-42 . We confirmed that infection with lenti-siInppl1 reduced SHIP2 levels in both HT22 cells and primary cortical neurons  in primary cortical neurons . These results indicate that SHIP2 is critical to memory impairment and tau hyperphosphorylation in 3xTg-AD mice.Then, we examined the role of SHIP2 (INPPL1) in memory impairment in vivo using  neurons . Consist neurons . When wenjection . When we-AD mice . The ame1-42 in mice, we directly delivered AS1949490 into mouse brains because of its poor bioavailability   . Taken t1-42, with FcγRIIb accounts for such neuropathogenic defects of AD as an initiation step, the selective interaction of oligomeric Aβ1-42 with FcγRIIb may hint at why Aβ1-42, but not Aβ1-40, is important in tau pathology . Thus, it is likely that Lyn phosphorylates the ITIM of FcγRIIb in neuronal cells in response to Aβ1-42. Moreover, we also observed this phosphorylation of FcγRIIb in AD brains (stage V and VI) in which tau was highly phosphorylated. Then, how is FcγRIIb different from other Aβ receptors? It is reasonable to propose that different mechanisms or even the same mechanism exerts multiple effects at different stages of disease progression (Unlike other FcγRs that have an immunoreceptor tyrosine-based activating motif (ITAM), FcγRIIb has a unique ITIM in its cytoplasmic tail and thus acts as an inhibitory receptor in B cells . Excitingression . For insgression  and ABADgression . In casegression . Furthergression . Thus, wgression .1-42 to tau by binding to the phosphorylated FcγRIIb. Many studies on SHIP2 have focused on its inhibitory effect on insulin signaling P2 is scarce under normal conditions and increases through signaling P3 are known to overlap to some degree in their functions, they apparently have distinct roles in neurodegeneration. Recently, it was shown that PtdInsP2, but not PtdInsP3 and PtdInsP2, potentiates glutamate-induced cell death in neurons P2 and leads to neurodegeneration in brains  also dephosphorylates PtdInsP3 at position 3 and generates PtdInsP2 P2 deficiency P2, and leads to AD pathogenesis.Dysregulation of phosphoinositide metabolism is increasingly recognized as important in various diseases, such as cancer, diabetes, and myopathy . The phoignaling . While P neurons . In addin brains . We alsosP2 . The redsP2 . More reficiency . Becausels in AD , as we a1-42 induces FcγRIIb phosphorylation to recruit SHIP2, leading to disruption of phosphoinositide metabolism for tau hyperphosphorylation and memory impairment in neurons and AD model mice. Together with the Aβ-lowering strategy, our results provide new ways for AD therapeutics to rescue Aβ and tau pathology: i) selective inhibition of the interaction between FcγRIIb and Aβ1-42, ii) inhibition of a kinase  which phosphorylates FcγRIIb upon Aβ1-42 stimulation, and iii) inhibition of SHIP2 which disrupts phosphoinositide metabolism.In conclusion, the FcγRIIb-SHIP2 signaling axis provides the missing link between Aβ and tau pathologies. Notably, AβFcgr2b KO C57BL/6 .WT (C57BL/6),  C57BL/6 , 3xTg-ADg for 20 min at 4°C, aliquoted and stored at –80°C until use. The supernatants were subjected to SDS-PAGE.Hippocampal tissues from AD (Braak V-VI) , MCI (Braak III)  and non-AD patients  were kindly provided by the Harvard Brain Tissue Resource Center . Hippocampal tissues of AD patients were homogenized in ice-cold Tris-buffered saline (TBS) buffer . The homogenates were clarified by centrifugation at 14,000 Inppl1 (5′-GAA GGG AGG GCA CGT TAA TTT-3′) (Sigma-Aldrich) was used for injection and pLKO.1-Neo-CMV-tGFP non-target virus was used as a control. The lentivirus  was stereotaxically injected bilaterally into the dentate gyrus (2 μl per hemisphere at 0.4 μl/min) with the following coordinates: anteroposterior = 2.1 mm from bregma, mediolateral = ± 1.8 mm, dorsoventral = 2.0 mm. After the injection, the cannula was maintained for an additional 5 min for a complete absorption of the virus. Behavior tests were performed 20 and 30 days after the injection. For western blot analysis, brains were removed 25 days after viral injection, hippocampal region was dissected and its protein samples were prepared as described above. Intracerebroventricular injection of PBS or Aβ1-42  was performed as described previously  and xylazine (10 mg/kg). Lentivirus expressing shRNA against mouse eviously . In the Behavior tests for double transgenic or injected mice were performed as described previously . All app1-42 oligomers were prepared from lyophilized monomers . The hydroxyfluroisopropanol (HFIP)-treated Aβ1-42 peptide was dissolved in dimethylsulfoxide (DMSO) and then diluted in phosphate-buffered saline (PBS). The stock solution was incubated at 4°C for 24 hr and stored at –80°C until use. Before use, the solution was centrifuged at 12,000 g for 10 min and the supernatant was used as an oligomeric Aβ (ADDLs). The oligomeric status of Aβ1-42 was evaluated by western blot analysis and atomic force microscopy  kit  following the manufacturer’s instructions. Briefly, the mice were anesthetized and the brains were microdissected. The hippocampus was carefully isolated and homogenized in 10 volumes of ice-cold guanidine buffer  and then mixed for 3 hr at room temperature. The brain homogenates were further diluted 1:10 with cold reaction buffer  and then centrifuged at 16,000 g for 20 min at 4°C. The diluents were mixed 1:1 with standard dilution buffer in the assay kit.Aβ levels in the 3xTg-AD and 3xTg-AD/2. Primary neurons were transfected using Lipofectamine 2000 reagent (Invitrogen), whereas other cells were transfected using Polyfect reagent  according to the manufacturer’s instructions. If required, cells were treated with SB-415286, roscovitine (Sigma-Aldrich) or AS1949490  as indicated.Primary cortical and hippocampal neurons were cultured from embryonic day 17 (E17) mice. The neurons were plated on poly-L-lysine -coated glass coverslips and maintained in neurobasal medium containing 2% B-27 supplement (Invitrogen) and 0.5 mM L-glutamine (Invitrogen). Half of the medium was exchanged every 3 days. Primary neurons were not authenticated, and were not tested for mycoplasma. SH-SY5Y, HEK293T and CHO cells , which were authenticated by ATCC and were negative for mycoplasma, were cultured in DMEM  supplemented with 10% fetal bovine serum (FBS) (HyClone), penicillin and streptomycin (Invitrogen). Cells were grown at 37°C under an atmosphere of 5% COFCGR2B and human INPPL1 shRNAs were synthesized, annealed and cloned into the pSUPER-neo vector. Human tau (0N4R) cDNAs were subcloned into pcDNA3-HA and pEGFP-C1 vector as described previously (All primers used in this study are listed in eviously . His-tagFCGR2B shRNAs or pINPPL1 shRNAs for 36 hr and then cultivated in the selection medium containing 1 mg/ml G418 (Invitrogen) for at least two weeks. A single cell was further cultivated to form stable cell colony and the expression of each cell lines was analyzed by western blotting and reverse transcriptase (RT) PCR.SH-SY5Y cell were transfected with pcDNA3-HA, pFCGR2B-HA, pSuper-neo, p3VO4, 1 mM NaF, 1 μg/ml each of aprotinin, leupeptin and pepstatin A). The lysates were centrifuged at 14,000 g for 10 min at 4°C and the supernatant was separated by SDS-PAGE and blotted onto PVDF membrane. The blots were blocked for 1 hr at room temperature and incubated with following antibodies: anti-phospho-FcγRIIb, anti-FcγRIIb , anti-Aβ , anti-phospho-tau , anti-NSE , anti-phospho-GSK3β, anti-p35/25 , anti-GSK3β , anti-SHIP2, anti-mFcγRIIb, anti-GFP, anti-His , anti-α-tubulin and anti-β-actin (Sigma-Aldrich), Nu-1 , PHF1, CP13 and TG5 , and 12E8 . Membranes were rinsed three times with TBS-T , further incubated for 1 hr with peroxidase-conjugated secondary antibodies and visualized using ECL detection system.Cells were lyzed in lysis buffer  (Sigma-Aldrich) for 10 min, rinsed three times with PBS and then permeabilized with 0.1% Triton X-100 in PBS. After blocking with 5% BSA in PBS, neurons were incubated overnight at 4°C with the following antibodies: AT180 (1:200), AT8 (1:200), anti-NSE (1:200), anti-FcγRIIb (1:500) and anti-SHIP2 (1:250). After rinsing three times with PBS, cells were incubated with FITC- or TRITC-conjugated secondary antibodies (Jackson Laboratory Inc.) at room temperature for 1 hr. The coverslips were placed with mounting solution (Sigma-Aldrich) and observed on a confocal laser scanning microscope .1-42-treated SH-SY5Y cell extracts were incubated with anti-FcγRIIb or anti-SHIP2 antibodies in IP buffer  for 12 hr at 4°C, and then pulled-down by protein G-Sepharose beads . For co-immunoprecipitation assay, HEK293T cells which transiently overexpressed His-tagged SHIP2 with either GFP-tagged FcγRIIb or FcγRIIb Y273F mutant were lyzed and incubated with anti-GFP or anti-His antibodies for 12 hr at 4°C, and then pulled-down by protein G-Sepharose beads. After a short centrifugation, the beads were washed three times with IP buffer and subjected to western blotting.For endogenous immunoprecipitation (IP) assay, Aβ1-42 were harvested with the buffer  and then mechanically disrupted using a 26-gauge needle with passing it 20 times. Cell lysates were centrifuged at 1000 g for 10 min at 4°C to remove the nuclei or unbroken cells. The supernatant was collected into new tube and again centrifuged at 100,000 g for 1 hr at 4°C with Beckman SW41 rotor. The pellet was resuspended in lysis buffer and used as a crude membrane fraction, whereas the supernatant used as a cytosolic fraction. The separated fractions were confirmed with western blot analysis using anti-FcγRIIb and anti-α-tubulin antibodies for the membrane and cytosolic markers, respectively.SH-SY5Y cells treated with AβThe lipid extraction from primary cortical neurons or SH-SY5Y cells was performed as described . After sE.coli BL21 cells were transformed with the plasmids and cultured to reach an OD600 of 0.6, before induction with 1 mM IPTG. After incubation for 18 hr at 16°C, cells were harvested and lyzed by sonication. His-fused TAPP1-PH and GRP1-PH were purified from cell lysates using Ni-NTA chelating agarose CL-6B . PLO assays were performed as described previously with minor modification P2 and PtdInsP3 diC16  were reconstituted in a 1:2:0.8 solution of chloroform, methanol and water, and used as positive controls. The lipid extracts from the cells were serially diluted and spotted on PVDF membranes which were pre-wetted in methanol, washed in TBS-T buffer and then air-dried. The membranes were dried completely and blocked with 3% BSA in TBS-T buffer for 1 hr. The blots were incubated overnight at 4°C with gentle rocking in the fresh blocking buffer containing purified 10 μM His-TAPP1-PH or His-GRP1-PH. The membranes were washed 5 times over 50 min in TBS-T buffer and incubated with anti-His antibody for 1 hr at room temperature. The membranes were further incubated for 1 hr with peroxidase-conjugated secondary antibodies and bound proteins were visualized using ECL detection system. PtdInsP2, PtdInsP2 or PtdInsP3 levels were quantified by ELISA kit (Echelon) following the manufacturer’s instructions.For the purification of recombinant proteins, the PH domain of TAPP1 and GRP1 was inserted into pET-28a vector. fication . LyophilThe synthetic phosphoinositides diC16 were incubated with histone carriers (Echelon) with a 0.5–3:1 molar ratio for 15 min with a vigorous vortexing. The histone-phosphoinositides complex was diluted 1:10 with neurobasal media and added to primary cortical neurons. The phosphoinositides without carriers and the only carriers without phosphoinositides were used as negative controls.t-test. Differences among multiple means were assessed by one-way ANOVA, followed by Tukey’s post-hoc test. Error bars represent s.d. or s.e.m. as indicated.Statistical analyses were performed with GraphPad Prism software. Differences between two means were assessed by paired or unpaired  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor.Thank you for submitting your article "FcγRIIb-SHIP2 axis links Aβ to tau pathology by disrupting phosphoinositide metabolism in Alzheimer's disease model" for consideration by The work describes how FcγRIIb deficiency protects neurons against Aβ-induced toxicity. Especially the identification of SHIP2 linking Aβ to Tau pathology is novel and potentially of high interest. Overall the referees were in support of publication, but before reaching consensus a few critical questions should be addressed. The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Essential revisions:eLife is not misled in the sense that this paper resolves the controversies in the field. Therefore, critical literature should be included in the Introduction and Discussion, and some balancing of the conclusions in the current manuscript versus other work should be provided. Why do the authors think that their receptor is the major driver of pathology in AD and why are other receptors not considered? Does this need any qualifiers?1) Overall the paper should take more into account other publications in both Introduction and Discussion sections. There are in the main time at least ten other receptors for Aβ oligomers described. Also some critical perspective on what is meant with Aβ oligomers is indicated. For instance, the 7PA2 cell line also produces 'zeta' peptides and the relation between these in vitro generated mixtures of Aβ peptides and the in vivo situation is far from clear. It is important that the broader non-specialized reader of 2) There are two major gaps in the proposed cascade and this should be mentioned at least in the Discussion to clarify this for the reader. First there are no mechanistic data in this work to link Aβ to FcγRIIb phosphorylation. The authors mention lyn kinase briefly  but don't show any data. The second gap is a mechanistic link between increased PtdIns and tau pathology. GSK-3-β kinase that catalyzes tau hyperphosphorylation, uses ATP as a donor of phosphate groups, so phosphoinositides are not a substrate. Therefore a possible link could be PtdIns-mediated regulation of GSK-3β.3) Two referees have concerns with the idea that the action of FcγRIIb is cell autonomous. First there are issues with the expression of FcγRIIb in neurons: Figure.1——figure supplement 1 A shows in fact that a large part of FcγRIIb mRNA is in glia and not in neurons. Additional evidence to proof that FcγRIIb is expressed in neurons is needed, it could be that the weak signal in the experiments in neurons is background. The authors could consider in situ hybridisation to check their claim. In addition, FcγRIIb is highly expressed in microglial cells and may play a role in microglial activation. Therefore, the impact of FcγRIIb KO on tau pathology in 3X TG mice can be also mediated by reduced microglial activation (cell autonomous effects vs non-autonomous effects). Did the authors check microglial activation and cytokine levels in these mice (WT 3XTG vs. KO 3Xtg)? Moreover, have the authors looked at whether FcγRIIb plays a role in sensing Aβ in microglial cells?4) The authors should provide in the next version the pictures of 5) In the last paragraph of the subsection “FcγRIIb is essential for tau hyperphosphorylation and memory deficit in 3xTg-AD mice”, it is said that 9months old 3xTgAD mouse are "plaque-free". What happens in the cortex of these 3xTg mice where Aβ plaques should be present, is tau phosphorylation more pronounced there? And is that protected by FcγRIIb deficiency? Does the age of mice affect tau hyperphoshorylation, i.e. is tau pathology still absent in 3xTg FcγRIIb KO mouse even at a very advanced age when there are loads of Aβ?6) In Figure.3—figure supplement.2A the authors analyze the effects of the receptor mutations on Aβ-induced cell death. "Cell death was determined after 24h under fluorescence microscope". What method/dye/criterion was used? How many spots were analyzed per sample per condition to produce statistics on panel B? […] Essential revisions:1) Overall the paper should take more into account other publications in both Introduction and Discussion sections. There are in the main time at least ten other receptors for Aβ oligomers described. Also some critical perspective on what is meant with Aβ oligomers is indicated. For instance, the 7PA2 cell line also produces 'zeta' peptides and the relation between these in vitro generated mixtures of Aβ peptides and the in vivo situation is far from clear. It is important that the broader non specialized reader of eLife is not misled in the sense that this paper resolves the controversies in the field. Therefore, critical literature should be included in the Introduction and Discussion, and some balancing of the conclusions in the current manuscript versus other work should be provided. Why do the authors think that their receptor is the major driver of pathology in AD and why are other receptors not considered? Does this need any qualifiers?Until now, Aβ was reported to bind to many receptors, including alpha7 nicotinic acetylcholine receptors (α7 nAChR), NMDA receptor, receptors for advanced glycation end-products (RAGE), Aβ-binding alcohol dehydrogenase (ABAD), the Ephrin-type B2 receptor (EphB2), cellular prion protein (PrPc) and paired immunoglobulin-like receptor B (PirB) . Although these receptors were shown to be responsible for Aβ neurotoxicity, especially memory impairment in AD mice, their role as neuronal receptors in Aβ-induced tau pathologies was limitedly shown in α7 nAChR and NMDA receptor . Of particular note, while α7 nAChR was reported to mediate Aβ-induced tau phosphorylation, the finding was based on in vitroand ex vivosystem . It thus remains to be confirmed in detail using animal models of AD. Furthermore, evidence showing a correlation of the proposed molecular mechanism with pathologic evidence was not much provided. In particular, the CAMKK2-AMPK at down-stream of NMDA receptor was recently proposed to mediate the synaptotoxic effects of Aβ oligomers through tau phosphorylation and this event is very likely caused by NMDA receptor-induced increase of intracellular calcium, not by direct interaction of NMDA receptor with Aβ . Therefore, a neuronal receptor that is important in Aβ- induced tau pathology has not been identified and needs to be elucidated for controlling overall AD pathology. We added this information in ‘Introduction’ (fourth paragraph).Then, how is FcγRIIb different from other Aβ receptors? It is reasonable to propose that different mechanisms or even the same mechanism exerts multiple effects at different stages of disease progression . For instance, RAGE is now believed to mainly function to transport Aβ in the blood brain barrier  and ABAD acts for mitochondrial toxicity as an intracellular binding partner of Aβ . In case of PrPc, it's debatable whether it is involved in Aβ-induced memory impairments and thus needs to be further characterized . Further, compared to those receptors, our observations that the phosphorylation of FcγRIIb at tyrosine 273 is found in the brain of AD patients and is required for both oligomeric Aβ neurotoxicity and tau hyperphosphorylation can make it distinct from other Aβ- binding receptors. In the case of PirB that shares structural similarity with FcγRIIb and also acts as an Aβ receptor for synaptic plasticity, the phosphorylation of PirB is not associated with Aβ signaling . Thus, we believe that FcγRIIb facilitates tau phosphorylation and neuronal loss in AD brains, consistent with the proposed role of tau in AD pathogenesis, such as severe memory impairment and neuronal loss . Following the reviewer’s suggestion, we added the specificity of FcγRIIb in ‘Discussion’ (fourth paragraph).n oligomers, and the proposals on which species of Aβ oligomers are responsible for the pathogenesis are a little in debate . We have here used 3 different sources of Aβ oligomers; synthetic Aβ oligomers, naturally secreted Aβ oligomers (7PA2 cells), and Aβ of 3xTg-AD model mice. Although synthetic Aβ oligomers are well-characterized and have been used widely for neurotoxicity, the acting concentration of synthetic Aβ (μM range) is relatively higher than that in AD brains. Compared to synthetic Aβ oligomers, conditioned medium from 7PA2 cells mainly contains not only Aβ dimers and trimers but also the different pools of Aβ oligomers, including zeta peptide, and show more potent neurotoxic properties (nM range) . Moreover, the oligomers generated in 3xTg-AD mouse brain may be more complicated and needs to be identified. Nonetheless, we propose here that FcγRIIb plays a crucial role in tau phosphorylation and neurotoxicity in vitroand in vivoin response to these species of Aβ, probably a certain common species among the different sources of Aβ oligomers. We added above information in ‘Discussion’ (second paragraph).In general, Aβ oligomers play a key role in AD pathogenesis, while soluble Aβ oligomers are still heterogeneous, including low or high 2) There are two major gaps in the proposed cascade and this should be mentioned at least in the Discussion to clarify this for the reader. First there are no mechanistic data in this work to link Aβ to FcγRIIb phosphorylation. The authors mention lyn kinase briefly  but don't show any data. The second gap is a mechanistic link between increased PtdIns and tau pathology. GSK-3-β kinase that catalyzes tau hyperphosphorylation, uses ATP as a donor of phosphate groups, so phosphoinositides are not a substrate. Therefore a possible link could be PtdIns-mediated regulation of GSK-3β.1-42 treatment in primary cortical neurons and SH-SY5Y neuronal cells . Thus, we initially hypothesized that and tested whether FcγRIIb was phosphorylated by Lyn after treatment with Aβ. We found that Lyn was activated (phosphorylation at Tyr 397) by Aβal cells . In addial cells . To furtal cells  and Aβ-ial cells . Moreoveal cells . Taken tThe second gap; the previous study showed that GSK3β, a well-known tau kinase, is stimulated by ER stress . We previously demonstrated that ER stress is elicited by the interaction between Aβ1-42 and FcγRIIb, and is responsible for Aβ neurotoxicity . Thus, we decided to examine the gap among SHIP2, ER stress, and GSK3β. To determine the relationship between PtdInsP2 (a SHIP2 product) and ER stress, we first analyzed the alteration of GRP78, a typical marker of unfolded protein response (UPR), following the delivery of various phosphoinositides into primary cortical neurons. Among the phosphoinositides, we found that PtdInsP2 only increased the level of GRP78. We also found that PtdInsP2 abolished the inhibitory phosphorylation of GSK3β at Ser9 (GSK3β activation) and increased tau hyperphosphorylation  Two referees have concerns with the idea that the action of FcγRIIb is cell autonomous. First there are issues with the expression of FcγRIIb in neurons: To evaluate neuronal expression of FcγRIIb, we have really paid great attention and by performing many experiments.First, as already shown in our previous manuscript , FcγRIIb mRNA (RT-PCR) and proteins  were found in the neurons of mouse and AD patient brains . MoreoveFcgr2b2 mRNA that is selectively increased by oligomeric Aβ1-42 treatment in primary cortical neurons   mice of FcγRIIb is the best. However, as you known well, generation of neuron-specific knockout (nsKO) mice of FcγRIIb and of FcγRIIb nsKO/3x AD double transgenic mice will take too long time (3 more years). Thus, as an alternative approach, we generated a dominant negative mutant of FcγRIIb  lacking its cytoplasmic region (a.a. 232-300) and thus unable to transmit the signal into cells, and confirmed its protective activity in Aβ-induced neuronal cell death after its transient expression in neuronal cells (data not shown). We then generated a transgenic mouse expressing FcγRIIb- ΔCyto-HA under ., 1996) . We conf., 1996) . To addr., 1996) . The beh., 1996) . These rhttp://www.brain-map.org/). Although fcgR2b expression is quite low, fcgr2b-positive ISH signals were detected in the hippocampus of P56 male mouse  results provided from Allen Brain Atlas  Cahoy et al. [J. Neurosci. (2008)] created a transcriptome database of the expression levels of >20,000 genes by gene profiling from astrocytes, neurons and oligodendrocytes. Based on their list, FcγRIIb is significantly expressed in postnatal day 16 (P16) neurons and P17 forebrain. (2) Suemitsu et al. [Neuroscience (2010)] detected FcγRIIb protein in parvalbumin neurons, whereas FcγRIII and FcγRI proteins were detected in microglial cells.In summary, we confirmed that, though neuronal expression is very low compared to non-neuronal cells, FcγRIIb is evidently expressed in neuronal cells as well. Again, please note that its expression is increased at least several folds in primary neurons by Aβ treatment and in AD brains compared to age-matched non- AD brains. Moreover, presence and distinct function of a neuron-enriched FcγRIIb2 provides more compelling evidence for the pathogenic role of FcγRIIb in AD.Fcgr2b KO in the cortex .While we have mainly focused on the role of neuronal FcγRIIb in tau pathology of AD model, we were also attempted to examine the role of FcγRIIb in the microglial activation in 3x Tg-AD mice. From immunohistochemical analysis, we identified that, compared to that of age-matched control mice, expression of Iba1, a microglia marker, was significantly reduced by e cortex  and slige cortex  of 3x Tg-AD mice . Though 4) The authors should provide in the next version the pictures of We are sorry for missing the pictures. We added the figures and separated those into 5) In the last paragraph of the subsection “FcJRIIb is essential for tau hyperphosphorylation and memory deficit in 3xTg-AD mice”, it is said that 9months old 3xTgAD mouse are "plaque-free". What happens in the cortex of these 3xTg mice where Aβ plaques should be present, is tau phosphorylation more pronounced there? And is that protected by FcγRIIb deficiency? Does the age of mice affect tau hyperphoshorylation, i.e. is tau pathology still absent in 3xTg FcγRIIb KO mouse even at a very advanced age when there are loads of Aβ?Fcgr2b KO in the same age of 3x Tg-AD mice . Since we mostly analyzed tau phosphorylation at the age of 8-9 months, we performed additional experiments to identify the effect of FcγRIIb KO in more aged mice following the reviewer’s suggestion. While transgenic expression of human tau (detected by HT7 antibody) in 3x Tg-AD mice was not altered during aging, tau phosphorylation in 20 month-old 3x Tg-AD mice was heavily detected and occurred more than that of 6 month-old 3x Tg-AD mice . Compare-AD mice . These r-AD mice  (subsect6) In Apoptotic cell death was determined by morphology-based death assay as described in the previous literatures . After transfection with the DNAs, GFP-positive cells showing condensed and fragmented nuclei after staining with Hoechst 33258 were counted under a fluorescence microscope. We counted at least 900 cells in 3~4 random spots per sample per condition. We added this information into the figure legend of the revised manuscript  legend.


Optimizing economic welfare in environmental governance has been criticized for delivering short-term gains at the expense of long-term environmental degradation. Different from economic optimization, the concepts of sustainability and the more recent safe operating space have been used to derive policies in environmental governance. However, a formal comparison between these three policy paradigms is still missing, leaving policy makers uncertain which paradigm to apply. Here, we develop a better understanding of their interrelationships, using a stylized model of human-environment tipping elements. We find that no paradigm guarantees fulfilling requirements imposed by another paradigm and derive simple heuristics for the conditions under which these trade-offs occur. We show that the absence of such a master paradigm is of special relevance for governing real-world tipping systems such as climate, fisheries, and farming, which may reside in a parameter regime where economic optimization is neither sustainable nor safe. Economic optimization in environmental governance was criticized for delivering short-term gains at the expense of long-term environmental degradation. Here, the authors use a stylized model of human-environment tipping elements to show no paradigm guarantees fulfilling another paradigm. Yet, it remains challenging to translate these aims into concrete policy implementations, accounting for non-linearities, such as tipping elements3, regime shifts5, and multi-stabilities6, as well as multiple kinds of uncertainties9, and extreme events10.The Sustainable Development Goals11 and how these findings relate to the three real-world systems of climate, fisheries and farming.To support the decision making processes in these contexts, we ask the question how the three prominent decision making paradigms of economic welfare optimization, sustainability and safe operating space compare with each other. Specifically, we investigate the parameter regimes for synergies and trade-offs when applying these paradigms to the management of tipping elements13. Most often, the present value of macroeconomic social welfare, i.e., the sum of discounted future benefits minus costs, is the target to be optimized. Such optimization approaches have been criticized regarding the discount rates used, delivering short term gains at the expense of long-term environmental degradation15. Further criticism targets the lack of a systems perspective required to understand the structural landscape of model dynamics, as well as the assumptions made due to imperfect information10. This critique is partly dealt with in optimization variants, such as robust16 or viable19 control, which are dealing with multiple types of uncertainty20. Naturally, other or multiple objectives21 and criteria23 with possible constraints24 can be optimized as well. In this work, we use the term solely in the narrow economic sense of maximizing the present value as defined in Eq. Optimization approaches have emerged as the primary guiding principle to derive a policy strategy for environmental governance25 the policy paradigm of sustainability has emerged in the scientific and political discourse27. The economics of sustainability has brought up many definitions of sustainability alone31. In these analyses sustainability is usually imposed as a constraint within an economic welfare optimization paradigm. Trade-offs to economic welfare optimization are well known32. However, these classic social welfare optimization approaches are challenged through the increasing recognition of non-linearities, such as tipping points, regime shifts, uncertainties and the risk of catastrophic outcomes9. Taking up these challenges, e.g., non-convexities33 and climate tipping elements35 have been studied within an economic framework. Here, we derive our formal definition of sustainability from the Brundtland report26. Its design is deliberately simple and targeted to the mathematical framework we use (see below). We do not intend our definition to be applicable to a general model of a welfare economy27.In recognition of increasing environmental and social threats36 or safe operating spaces38 as a policy paradigm to derive concrete actions from39. These concepts originate from resilience thinking40 and a precautionary principle41 to deal with potential dangerous tipping elements in the environmental governance system. Trade-offs but also synergies with optimization thinking have been therefore discussed42. Also formal analyses studying relations between resilience as a system property and sustainability were conducted44.Recent advances in sustainability science have brought forth tolerable windowsHowever, the reciprocal relationships between these three paradigms of economic optimization, sustainability and safe operating space is still insufficiently explored. Such an understanding is important in order to judge, for example, when economic optimization is, or is not, an appropriate policy goal. Also, guidance is required when a sustainability paradigm may conflict with a safe operating space paradigm and vice versa.Here, we report progress towards a better understanding of the mutual relationships between these three paradigms of economic optimization, sustainability and safe operating space by applying them to a stylized model of a human-environment tipping element. We do so because of the increasing importance of tipping points and regime shifts in environmental governance. Our model is deliberately stylized, thereby applicable across multiple cases and scales, to gain a deeper understanding more complex models might miss. The formal definitions of the three paradigms are designed to fit our mathematical framework (see below). Since we do not focus on intragenerational justice in this article, one agent suffices as a decision making subject, in contrast to a multiagent setting. We find that there exists no master paradigm between the three examined, i.e., a policy can be any combination of optimal or not, sustainable or not and safe or not. This is of special relevance to the climate system which may reside at the edge in the parameter regime where economic welfare optimization becomes neither sustainable nor safe. This suggests the use of more advanced paradigms to support decision making in climate policy.46, in which an agent makes decisions about how to interact with its environment , but risks triggering a collapse of the environment to the degraded system state with non-zero probability δ and no immediate reward at all. From there, only the low pressure action opens the option to recover to the prosperous state with non-zero probability ρ.We use the mathematical framework of Markov Decision Processesent Fig. . Our pard r Fig. . At the For example, the high pressure action could correspond to emitting a business-as-usual amount of carbon to the atmosphere yielding a reward of high, short-term economic output as long as the system has not tipped. The low pressure action resembles emitting a reduced amount of carbon, assuming a lower short-term economic output for the guarantee to not trigger climate tipping elements into a disastrous state.π is a function that specifies what action a to apply at a system state s. The agent receives reward rt at time step t. The value vπ(s) of a state s under a given policy π is given by the expected value of the normalized accumulated discounted rewards r with discount factor 0 ≤ γ ≤ 1 when starting in state S0 = s and following policy π:A policy γ = 1 corresponds to no discounting , whereas γ = 0 corresponds to completely myopic, fully discounting agents.Note that the discount factor actually denotes the farsightedness of the agent. Thus, πr(p) = h, πr(d) = l), applying the high pressure action at the prosperous state and the low pressure one at the degraded state and the cautious policy (πc(p) = l, πc(d) = l), applying the low pressure action at the prosperous, as well as the degraded state.We classify policies according to whether they are economic welfare optimal or not, sustainable or not, and safe or not. For the sake of simplicity we focus on two deterministic policies, distinguishing whether the agent should apply the low or the high pressure action at the prosperous state Fig. : the risπ is defined as optimal (in the economic welfare sense) if its value vπ(s) (Eq. 46.A policy π(s) Eq.  for ever26 a sustainable policy should fulfill two requirements: First, meet the needs of the present. We translate this formally into the agent evaluating the present state s as acceptable , if its value  has much similarity with the notion of weak sustainability48.Note that this reduction of sustainability to the one-dimensional value 37 is typically defined as a subset of the whole state space 49. In practice, the position of these potential tipping thresholds is always uncertain and the boundaries are placed at the lower end of the uncertainty zone. In that way the definition of the safe operating space states constitutes a normative judgment about the risk the decision maker is willing to tolerate. In the subsequent analyses we take the extreme position of no risk tolerance and identify the SOS with only the (more favorable) prosperous state, independent of the collapse probability δ.The Safe Operating Space (SOS)π as safe if every state the agents eventually visits under policy π lies within the SOS.We define a policy In contrast to acceptable and unacceptable states, safe states are independent of the policy used.δ, ρ, γ, rl/rh, rmin/rh: the probability of a collapse from the prosperous to the degraded state under the high pressure action δ, the probability of recovery from the degraded to the prosperous state under the low pressure action ρ, the agent’s discount factor γ, the high reward receivable from the high pressure action when staying at the prosperous state rh, the low reward receivable by taking the low pressure action at the prosperous state rl, and the normatively chosen minimum acceptable reward rmin a state value must have to be perceived as acceptable under a certain policy. Since all three rewards come in arbitrary units, the policy classification only depends on their ratios.In summary, our stylized model of a human-environment tipping element depends on the five parameters δ, ρ, γ, rl/rh, rmin/rh) .Further, we find a decreasing critical collapse probability with increasing farsightedness rl ≥ rmin, then the cautious policy is sustainable for all parameter combinations δ, ρ, γ, rl/rh . This is because in this parameter region the risky policy is acceptable also at the degraded state (Methods).Provided the low pressure reward exceeds the normative minimum acceptable value threshold, δ, ρ, γ, rl/rh, rmin/rh . This yields a classification of policies into eight different categories Fig. .Fig. 3Parmin is too large. The cautious policy does not return enough value to be sustainable  and the risky policy at the degraded state produces too little future reward to be sustainable, due to the low chance of recovery and lack of farsightedness.In particular, optimal policies are not necessarily sustainable  which additionally accounts for social indicators51, such as the number of people living in extreme poverty. Thus, SAJOS policies can be interpreted as the overlap of safe with sustainable policies. Within our model, we can give a definite criterion for when this form of SAJOS exists: as long as the reward from a low pressure action rl exceeds the normative threshold value rmin (rl > rmin), the cautious policy is both safe and sustainable . Additionally, we added a parameter sensitivity analysis by visualizing the likelihood of ending up in a certain parameter regime by color gradients between regimes , the climate system is likely to reside in a parameter sweet spot (gray area), where applying an optimization, sustainability or SOS paradigm results in the cautious policy as the advisable way of governing the climate system. However, if the collapse probability per time step is smaller  the situation is different. Here, an SOS and a sustainable paradigm would still yield the cautious policy . At the collapsed and degraded state the conditions for the fishers are not acceptable. Therefore they have to leave the system and cannot wait for the fish’s recovery. But further investigation is needed to reduce the uncertainty with respect to the other parameters.For fishery systems, both transition probabilities certainly depend on a variety of factors, e.g., fisher’s technical and cultural traits or the dominant fish species in the system, as well as external factors such as climate change influencing habitat condition63. Nevertheless, land degradation by farming has been identified as a tipping element by Kinzig and others64, where the authors discuss the case of the western Australian wheatbelt with a typical collapse timescale of about 100 years. Soil recovery is estimated to take place within 20 to 1000 years65, which is roughly consistent to Kitzing et al., where the duration to reach equilibrium again is estimated with up to 300 years. We therefore assume a typical recovery timescale of about 300 years. In contrast to climate and fisheries, the transition probabilities we associated with the process of land degradation by farming suggest, that here an optimality paradigm is very likely to yield the risky policy which is neither sustainable nor safe despite considerate parameter uncertainty  = h, πr(d) = l, (2) πc(p) = l, πc(d) = l, (3) π3(p) = h, π3(d) = h, (4) π4(p) = l, π4(d) = h. We concentrate on deterministic policies only to simplify the calculation without loss of generality, because if an optimal policy exits there exits also a deterministic optimal policy46. We further focus here only on the first two policies, named the risky and the cautious policy, since the remaining two apply a high pressure action at the degraded state. This will trap the agent at this position for eternity without receiving any reward. The math on these policies is left to the interested reader.There are four deterministic policies in our Markov decision process model: (1) δ, ρ, γ, rl, rh). From Eq. γ < 1 one can derive the recursive relationship between state values, known as the Bellman Equation69:p) being the probability to enter state s′ given the agent has started in state s and applied action π(s).In the following we derive the analytical expressions of the state values of these policies as functions of the parameters  = h, πr(d) = l) we solve the system of equationsTo obtain the explicit state values for the risky policy (πc(p) = l, πc(d) = l) we solve the system of equationsTo obtain the explicit state values for the cautious policy  by multiplying the stationary state of the effective Markov chain with the reward vector For Pπ of the effective Markov chain readThe components of the transition matrix σπ is the normalized eigenvector of the transition matrix with eigenvalue one. Hence,The stationary state γ = 1 the value vπ can be obtained by simply inserting γ = 1 into Eqs. Performing this calculation for risky and cautious policy explicitly yields consistent results with the calculation for 0 ≤ γ < 1 from above. For To derive the analytical expression of the hypersurface in parameter space that separates the regions where either the risky or the cautious policy is optimal we set π we apply the definition from Eq. To obtain the hypersurface that separates state s being acceptable from being not acceptable under policy For the risky policy at the degraded state we set For the cautious policy at the prosperous state we set For the cautious policy at the degraded state we set To get from acceptability to sustainability for the risky policy one has to logically combine Eqs. The division of the parameter space according the safe operating space paradigm is obvious from its definition. Only the cautious policy is a safe policy since it will eventually end up and remain in the prosperous, safe operating space state. The risky policy switches recurrently between the prosperous and the degraded which makes it, by definition, not safe.p be the probability per time step that a system state will transition into another state. The average number of time steps the system will be in that state is given by 〈N〉 = (1 − p)/p. Inverting yields p = 1/(〈N〉 + 1). We map a model time step to a year. Thus, a collapse time scale of e.g., 50 years corresponds to a collapse probability of δ ≈ 0.02. Supplementary Tab. Let by 〈N〉 =  − p/p. Ihttps://github.com/wbarfuss/Paradigms.Python code for the reproduction of the reported results plus interactive versions of the figures can be downloaded from Data sharing not applicable to this article as no datasets were stored on disk during the production of the figures (see Code availability).Supplementary InformationPeer review file


We have read with great interest the article by P. Faverio et al. published in this journal . We thanWhile the authors have tested the methodology of NIV interruption well, the number of days at each stage of weaning, NIV during afternoon and night or nocturnal NIV only, was at physician discretion. This is an exciting aspect and confusing as well because, eventually, the criteria may be influenced by wide individual variability, which may make it impossible to extrapolate the results. Good numbers (39%) of the patients in the chronic domiciliary NIV group were not able to be weaned by the new method and continued to be in the domiciliary NIV settings. As there are no clear criteria for such patients, it will be worth knowing the settings, whether these patients were finally stabilized, and if yes, “how.” These aspects will help readers in better application or exclusion of the new interruption-based weaning in this subgroup of patients in the future. Pneumothorax due to severe bullous emphysema is considered by the authors as a risk factor for not weaning. While we agree that such patients are prone to not weaning, pneumothorax it is more related to the selected positive inspiratory and end-expiratory pressure levels selected . HoweverWe again thank the authors for another interesting observation of no recurrence of AHRF during hospitalization after weaning by the new method. This is interesting and raises speculation whether this can be considered as a protective aspect of the NIV. A prospective randomized study will give us more evidence on these aspects, especially which subgroup of patients will be benefited by this new technique.