Samples were subjected to an ultrasound water bath treatment for 5 min, 15 min, and 30 min at 60 °C (±2 °C) and with an amplitude of 100% as an equivalent to the heat-treatment conditions combined with an ultrasound effect. Furthermore, the effect of temperature on the polymer barrier (water vapour and oxygen permeability) properties was tested at 20 °C, 40 °C, and 60 °C. Results showed that PPAcPVDC possessed better properties of water vapour permeability and oxygen permeability properties to LLDPE. Statistical analyses showed a significant (p < 0.001) impact of ultrasound treatment on the overall migration value, regardless of the food simulant used.In this study, we investigated the impact of ultrasound treatment on barrier properties of linear low-density polyethylene (LLDPE) and acrylic/poly(vinylidene chloride) polypropylene (PPAcPVDC)-coated pouches intended for food packaging before and after exposure to food simulants. Packaging pouches were filled with two food simulants, namely ethanol (10% ( The main role of packaging is to protect foods from unfavourable external factors, including gases and vapour, and to preserve product characteristics  during the shelf life ,2. PolyoPolypropylene (PP) is a polymer available at low cost that has excellent thermal stability, optical transparency, and barrier properties . Linear Ultrasound treatment (UST) is defined as treatment with acoustic waves with frequencies between 20 kHz and 100 kHz. UST is a non-ionizing, non-invasive, and non-polluting form of mechanical energy. It represents a novel method for use in food technology, with the ability to control and improve the preservation of treated food ,10. HoweVarious small compounds  could eventually migrate to packaged food, resulting in a dietary consumption of chemicals that may be harmful to human health . BecauseThe present study focuses on the determination of barrier performance (water vapour and oxygen permeability) of two commercially available food packaging films, namely linear low-density polyethylene (LLDPE) and polypropylene coated with acrylic/poly (vinylidene chloride) (PPAcPVDC), both treated with a UST. In addition, we determined the impact of the FS (ethanol (EtOH) and acetic acid (HAc)) on the packaging film barrier properties during UST, as well as the overall migration (OM) in FSs, as influenced by UST.v/v))  and acetic acid )  were used for preparation of FSs.In this study, we used linear low-density polyethylene-LLDPE  and polypropylene coated with acrylic/poly(vinylidene chloride)-PPAcPVDC , without any graphic treatment. Ethanol  and aqueous acetic acid at 3% (w/v), representing hydrophilic and acidic foodstuff, respectively. The samples were coded according to FS type and UST  operated at 35 kHz. The maximum ultrasonic power of the system was used (640 W adjusted to an amplitude of 100%). All samples were treated at 60 °C to simulate the in-package heat treatment for 5, 15, and 30 min . After tv/v) ethanol; Hac—3% (w/v) acetic acid. Mean values within a column that do not share a common superscript letter are significantly different (p ≤ 0.05).LLDPE—linear low-density polyethylene; PPAcPVDC—polypropylene coated with acrylic/poly(vinylidene chloride); control—treatment time equal to 0 min; EtOH—10% (v/v) ethanol; Hac—3% (w/v) acetic acid. Mean values within a column that do not share a common superscript letter are significantly different (p ≤ 0.05).PPAcPVDC—polypropylene coated with acrylic/poly ethanol; Hac—3% (w/v) acetic acid. Mean values within a column that do not share a common superscript letter are significantly different (p ≤ 0.05).LLDPE—linear low-density polyethylene; control—treatment time equal to 0 min; EtOH—10% (v/v) ethanol; Hac—3% (w/v) acetic acid; nd—not determined. Mean values within a column that do not share a common superscript letter are significantly different (p ≤ 0.05).LLDPE—linear low-density polyethylene; PPAcPVDC—polypropylene coated with acrylic/poly. The average value at five positions per type of sample was used in all calculations.WVP) of films was determined under controlled conditions according to the gravimetric standard method [WVP (g·m−1·s−1·Pa−1) was calculated from the change in the cell weight versus time at the steady state, using the following Equation (1):m/Δt is the weight loss per unit of time (g·s−1), A is the film area exposed to the moisture transfer (9.08 × 10−4·m2), x is the film thickness (m), and Δp is the water vapour pressure difference between the two sides of the film (Pa). All measurements were performed in triplicate.The water vapour permeability (d method  adapted d method . Samplesq: cm3 ∙ m−2 ∙ d−1 ∙ bar−1) was determined according to the International Organization for Standardization (ISO) [P: cm3·cm−1·s−1·bar−1), solubility (S: cm3·cm−3·bar−1), and diffusion (D: cm2∙s−1) coefficients, calculated from the time lag (tL: s) value at the provided sample thickness [DE), permeability (PE), and heat of sorption (SE) through untreated and UST-treated LLDPE and PPAcPVDC films.The gas permeance (on (ISO)  on a GDPhickness . All coev/v)) and aqueous acetic acid (3% (w/v)) were used as FSs for all measurements. Measurements were performed with a migration cell  and either following (1) the immersion method (for untreated and control samples with UST only) or (2) the article filling method (for pouches filled with FSs and UST-treated samples) according to the modified EN 1186-1 and EN 1186-5 standards [2) of the sample was measured prior to experiments. Migration cells (for method 1) or pouches (for method 2) were kept for 10 days at 40 °C. Afterwards, FS solution from the upper part of the migration cell (for method 1) and from the pouches (for method 2) was decanted in a previously weighed glass cell and evaporated at high temperatures (>300 °C). All measurements were performed in triplicate. The overall migration (OM) was calculated according to the following equation:m1 is the mass of the empty glass cell before evaporation (mg), m2 is the mass of the glass cell after evaporation (mg), and A is the surface of the test specimen intended to come into contact with the simulant (dm2). All measurements were performed in triplicate.The overall migration (OM) values were determined to check the maximal quantity of low-molecular-weight compounds that had migrated from the packaging materials to the FSs. Ethanol  were evaluated and reported with Origin software . All experiments were carried out at least in triplicate, and results were reported as the means and standard errors of differences of the means of these measurements.Statistical analysis was performed in Minitab using analysis of variance (ANOVA) , with packaging material (Pack), ultrasound treatment (UST), food simulant (FS), and temperature (Temp) as fixed factors. Tukey’s test was used as the post hoc test, and −13 g·m−1·s−1·Pa−1) compared to the LLDPE (control) (3.51 × 10−13 g·m−1·s−1·Pa−1) (p < 0.001). When the UST time was increased from 5 min to 30 min (both food simulants (FSs) used), the WVP of PPAcPVDC significantly decreased  was decreased compared to the control (only carboxymethyl cellulose added: WVP = 0.4525 × 1012 g·cm−1·s−1·Pa−1) [The PPAcPVDC (control)  showed b−1·Pa−1) . Ščetar −1·Pa−1) . Thus, t−1·Pa−1) ,29.p < 0.001)  for both tested polymers [P decreased by about 8% for LLDPE and 19% for PPcast film [Results of the oxygen permeability parameters for PPAcPVDC and LLDPE are given in < 0.001) . The excpolymers . Namely,ast film .P (8.6 × 10−3 cm·m−2·d−1·bar−1), which was responsible for the remarkably increased barrier properties of biaxially oriented PP films [As already mentioned, the better barrier characteristics of PPAcPVDC compared to LLDPE were mostly attributed to the presence of an AcPVDC coating. Similarly, Daniloski et al. (2019) showed that PPAcPVDC film was a better barrier to oxygen compared to biaxially oriented coextruded PP . MoreovePP films .S) (p < 0.001). A similar trend was observed for most of the treated (UST and FSs) PPAcPVDC samples, except for PPAcPVDC: HAc 15 min, and no specific behaviour could be seen for the treated LLDPE samples. Mrkić, Galić, Ivanković, Hamin, and Ciković (2006) and Daniloski et al. (2019) observed a significant increase in the S of gases at higher temperatures (above 50 °C) in PE and monoaxially and biaxially oriented PP films [S (as obtained for PPAcPVDC samples) was due to the larger volume fraction of the crystal region in contrast to the LLDPE samples [In both control samples, the oxygen solubility coefficient (S)  increasePP films ,38. Acco samples .D values slightly increased with increased temperature. In LLDPE samples treated with UST and EtOH ; the exception was LLDPE: HAc 30 min. In PPAcPVDC (control) samples  samples, and EtOH , there w samples , there w control .S, D, and P for both PPAcPVDC and LLDPE films are presented in S and D, with good correlation . Similarly, Daniloski et al., (2019) found a good correlation for D values of biaxially oriented PPAcPVDC  [P. Hong and Krochta (2006) showed a good fit of the Arrhenius plot for P of PP and PE films  [p < 0.001). S, D, and p values were not significantly affected (p > 0.05) by UST.The Arrhenius plots of < 0.001) . In the = 0.999) . AccordiEa) for P, D, and S, the rate and affinity of transferring molecules can be predicted under various conditions. Ea is defined as the energy required to start the diffusion of a particular gas through the packaging material [Ea values of all parameters in the LLDPE (control) samples were lower than in the PPAcPVDC (control) samples. PPAcPVDC was shown to be a better barrier against oxygen; the Ea of PPAcPVDC was higher than that of LLDPE. After UST in contact with FSs, all Ea values for P increased compared to the control samples. Ea (D) and Ea (S) were lower for UST at 5 min, with no particular behaviour in other samples. An ample knowledge of these parameters is demanded for estimating the efficiency of packaging material and the quality properties of the packed food during storage, including the distribution chain. Fluctuations in temperature might lead to changes in material permeability, affecting the transfer of gases through the package, resulting in limited shelf life of the packed food item [With the activation energy  and possess low wettability due to their hydrophobic properties. The passage of migrants through these materials in normal conditions  is very unlikely [p < 0.05). Nevertheless, the above-mentioned treatments might either decrease the hydrophobicity of the polymer or introduce C=O or –OH groups that are more prone to degradation. Once treated, it was observed that PP was more prone to degradation in comparison to PE [p < 0.05). This indicates that there is an impact of the packed simulant or foodstuff on the surface of the material during UST (p < 0.05). Several authors have explained that a number of factors could affect these adjustments [Overall migration (OM) tests are performed to determine the overall quantity of chemicals that may migrate from polymer films into food (or a simulant). Namely, migration could be affected by numerous factors, such as packaging materials, food nature, temperature, characteristics of migrating additives, and the number of potential migrants in packaging materials, to name a few . Polyoleunlikely . The OM on to PE . Similaron to PE . These iustments ,47,48. Np < 0.05). Hafttananian, Zabihzadeh Khajavi, Farhoodi, Jahanbin, and Ebrahimi Pure (2021) presented that LDPE possessed more affinity to HAc than distilled water, and as such, a greater penetration of HAc was observed in the polymer matrix [p < 0.05). A similar observation was made by Liu Jing-Min et al. (2020), who investigated the migration of HAc through PE wrap films. The authors observed a poor affinity of the PE wrap film for HAc, which caused a small swelling effect of the film and, consequently, less migration of that FS through the packaging material [Additionally, for both treatment methods, only UST on raw material and pouches filled with FS showed higher OM values for HAc in comparison with EtOH-exposed samples  and hydrophilic (OM results obtained for EtOH) foodstuff. Correspondingly, the OM values should be less than the legally permitted values of 10 mg·dmterature . Notablyp < 0.001). The diffusion process controlled oxygen permeation through both materials. In LLDPE, the lowest WVP was reached for the control sample, and the highest for the sample treated with UST and HAc for a period of 30 min. On the contrary, in PPAcPVDC, the treated samples (UST and FSs) were less permeable to water vapour than the control samples. The OM was shown to be significantly higher when samples were treated with UST and in contact with FSs.LLDPE and PPAcPVDC, commonly used commercial food packaging materials, were treated with an ultrasound while in contact with two FSs representing hydrophilic and acidic foodstuff. Treated (UST and FSs) LLDPE and PPAcPVDC showed different barrier properties for water vapour and oxygen under the tested conditions. PPAcPVDC was shown to be a better gas barrier in contrast to LLDPE. Temperature was presented as a statistically significant factor for permeability properties, leading to a remarkable increase in the permeability properties of the packaging material samples with temperature increase  are achieve commercial importance, it is envisaged to study the physicochemical and surface properties of the tested materials. Therefore, further studies are still needed to extend our results to real foodstuffs because different food products have specific packaging requirements based on their permeability characteristics and storage conditions.


Invariant chain  is a multifunctional protein expressed in antigen presenting cells. It assists the ER exit of various cargos and serves as a receptor for the macrophage migration inhibitory factor. The newly translated Ii chains trimerize, a structural feature that is not readily understood in the context of its MHCII chaperoning function. Two segments of Ii, the luminal C-terminal region (TRIM) and the transmembrane domain (TM), have been shown to participate in the trimerization process but their relative importance and impact on the assembly with MHCII molecules remains debated. Here, we addressed the requirement of these domains in the trimerization of human Ii as well as in the oligomerization with MHCII molecules. We used site-directed mutagenesis to generate series of Ii and DR mutants. These were transiently transfected in HEK293T cells to test their cell surface expression and analyse their interactions by co-immunoprecipitations.Our results showed that the TRIM domain is not essential for Ii trimerization nor for intracellular trafficking with MHCII molecules. We also gathered evidence that in the absence of TM, TRIM allows the formation of multi-subunit complexes with HLA-DR. Similarly, in the absence of TRIM, Ii can assemble into high-order structures with MHCII molecules.Altogether, our data show that trimerization of Ii through either TM or TRIM sustains nonameric complex formation with MHCII molecules.The online version contains supplementary material available at 10.1186/s12865-021-00444-6. Ii is a non-polymorphic type II transmembrane glycoprotein , 2. It iFour Ii isoforms have been described in humans , 11. IipHelicobacter pylori [Best characterized as a MHCII chaperone, recent studies have revealed that Ii is also engaged in a number of other immune functions –27. For r pylori , 31. In r pylori .Once translated and translocated into the ER, Ii rapidly trimerizes –35. The While there is ample experimental and computational evidence that these different regions of Ii can trimerize, their relative importance remains debated. For one, the cytoplasmic region is not believed to play a role in self-association of full-length Ii because of its antiparallel nature. Rather, trimerization of this domain was proposed to facilitate sorting and promote endosomal retention as well as the generation of large endosomes , 43. WhiHere, we have revisited these issues using a cellular system that allows assessing the capacity of Ii to trimerize and to associate into high-order structures with MHCIIs. Our data demonstrate that neither TM nor TRIM are essential for hIi trimerization. In addition, we show that any of these domains is sufficient to trigger the assembly into nonameric structures with MHCIIs, as long as the Ii moieties involved share the same domain. The importance of these trimerization regions for Ag presentation by MHCIIs and for Ii functions in general are discussed.LIML) or p35 lacking the TRIM motif and the sorting signals (p35LIMLTRIM). Mutation of the two leucine-based endosomal localization motifs favors the accumulation of Ii at the plasma membrane in the presence of MHCII molecules, thus providing a simple, indirect flow cytometry readout for ER egress [LIML and the IiLIML/MHCII complex are nevertheless internalized  and TRIM domain (Δ20TRIM)  bearing a stringent KKAA cytoplasmic ER retention motif (which cannot be overcome in any ways) , or even trimers (α1β1Δ20TRIM1). Thus, the use of control IiΔ20TRIM could not inform on the capacity of IiTRIM to assemble different MHCIIs into the same complex.First, we tested the control Ii Δ20try Fig. b–e. A frtry Fig. b, d. Wheane Fig. b–d. Alsoane Fig. e, f. In ane Fig. c, d and,KAA Fig. b trafficKAA Fig. e, f. Intane Fig. b–d. AlsoKAA Fig. e, f ThesLIMLTRIM in cells expressing DR. Our results show that DR was found at the plasma membrane together with Ii and CLIP . Interestingly, when αSCD, DRβKKAA and WT DRβ were all co-expressed, there was no CLIP, Ii or DR at the plasma membrane . The data are compatible with a model where the αSCD first trimerizes [KKAA. We conclude that the TM domain of Ii is not a prerequisite for the assembly of multimeric structures comprising multiple MHCII molecules. These data are in agreement with those of Cresswell and collaborators showing that the proteinase K digestion of Ii in MHCII/Ii complexes generates a C-terminal K3 fragment, which includes TRIM and by itself can retain the complex in its nonameric conformation [Experiments using truncated soluble molecules have demonstrated the rapid trimerization of Ii and the subsequent formation of complexes of variable stoichiometry with MHCIIs . We haveimerizes  and the ormation .Fig. 6DRNewly translated full-length Ii chains swiftly trimerize upon translocation into the ER , 36. The3 were first described in the early 1990s and are a direct consequence of Ii’s ability to form trimers [3 [TRIM. Indeed, we have shown in Fig. 2) can be visualized on Western blots after crosslinking. Interestingly, these dimers have been described almost 40 years ago by Koch and Hammerling [When considering the chaperone role of Ii, the need for trimerization in the context of MHCII transport is not readily apparent. Nonameric complexes (αβIi) trimers . Howeverimers [3 . While oimers [3 , 50. Indimers [3 , 70. Micimers [3 . This meimers [3 . It is immerling . They weBeyond the debate regarding the stoichiometry of the complexes leaving the ER, the need for multimerization of MHCII in Ag presentation remains nebulous. At one extreme, Ii was even shown to be dispensable for MHCII assembly/trafficking in some cell lines and knockout mice , 73, 74.3αβ or (Iiαβ)3 complexes. The difficulty resides in our capacity to generate structurally comparable complexes of defined stoichiometry. In our recent studies, we made use of the αSCD and the results suggested that the TM of Ii is not required in living cells for the formation of Ii/MHCII complexes of variable stoichiometry. Here, we have confirmed these results and extended the conclusions to the TRIM of hIi. Also, we have shown that no region other than the TM or TRIM (or even MHCIIs themselves) promote Ii self-association. By using SCDs devoid of TRIM, we were able to compare the trafficking of Ii1α1β1 with WT nonameric Ii3α3β3 complexes. While we have not monitored Ag presentation per se, the capacity of all these constructs to generate MHCII/CLIP complexes and to interact with DM suggest that they are structurally and functionally similar. Interestingly, the group of Hirano has recently provided evidence that HLA-DPβ allotypes bearing a glycine at position 84 (DP84Gly), such as DP4, do not bind Ii through CLIP [1(αβ)1 complex. While Ii chaperones DP4 to the endocytic pathway, more studies will be needed to determine if this peculiar stoichiometry intervenes in the association of these alleles with autoimmune diseases [No study has tackled the systematic comparison of Ag presentation efficiency using Iiαβ, Iiugh CLIP . They fudiseases .In conclusion, the purpose of the two distinct trimerization domains of Ii in the chaperoning of MHCIIs remains an open question. As mentioned above, it is possible that the luminal TRIM serves some MHCII-independent functions and that the structural features required for Ii to chaperone other cargos are dependent on TRIM. Future structure–function studies addressing the interaction of Ii with other molecules, such as CD70 and possibly CD1d, should shed light on this issue –81.myc and pcDNA3.1 DRβKKAA, pcDNA3 Ii, pcDNA3 p33, pcDNA3 p33LIML, pcDNA3 p35, pcDNA3 p35LIML and pBud Δ20 Ii have been described previously [3(Ser)1(Gly)3 linker was created has described for αSCD [LIML, Δ20 and βSCD, giving rise to the pBud p35LIMLTRIM, pBud Δ20TRIM and pBud βSCDTRIM, respectively.pBud DR, pBud DM, pcDNA3.1 DRα, pBud αSCD, pcDNA3.1 DRβeviously , 58, 82.for αSCD . MutantsThe following mouse mAbs were described previously , 82: BU4Alexa Fluor 488- or 633-coupled goat anti-mouse secondary Abs  were used for flow cytometry. For WB, Peroxidase-AffiniPure goat anti-mouse IgG (H + L) and Peroxidase-AffiniPure goat anti-mouse Fc specific  were used. For IPs, cells were lysed at 4 °C in 1% Triton-X100. Lysates were analyzed as controls and all samples were subjected to reducing SDS-PAGE. Proteins on immunoblots were detected by chemiluminescence . For crosslinking experiments, cells were lysed in 1% Triton and 400 µg/mL DSP (dithiobis (succinimidyl propionate)) . For Endo H resistance assays, total lysates were treated with Endo H (New England Biolabs), according to the manufacturer’s recommendations. Proteins were analyzed in non-reducing conditions by SDS-PAGE.For transient expression, HEK293T cells were transfected using polyethyleneimine  and stained after 48 h. To determine surface expression, live cells were stained on ice and analyzed by flow cytometry on a FACSCalibur or FACSCantoII. To determine total expression of MHCII and Ii, cells were fixed in 4% paraformaldehyde, permeabilized, and stained, as described previously . ForwardAdditional file 1. Internalisation of Ii mutants lacking the endosomal targeting motif. HEK293T cells were transiently transfected with DR+p33, DR+p33LIML or αSCD/β. After 48h, cells were stained on ice with BU45. Cells were shifted to 37°C and aliquots were stained after 0, 15 and 30 minutes using a goat anti-mouse IgG shifted to 37oC and aliquots were stained after 0, 15 and 30 minutes using a goat anti-mouse IgG experiments and error bars indicate the standard deviation of triplicates. Paired Student’s t-tests were performed; *: p ≤ 0.05 and **: p ≤ 0.01. Autoradiograms used to prepare figures in the paper. A and B, see figure 1. C, D and E, see Fig.


To protect these films from rapid oxidation, we add a thinALD coating of Al2O3, which increases the lifetimeof the films from 1 day to several months. Encapsulation of the filmsalso influences photochromic bleaching, changing the time dependencyfrom first-order kinetics. As well, the partial annealing which occursduring the ALD process results in a dramatically slower bleachingspeed, revealing the importance of defects for the reversibility (bleachingspeed) of photochromism.Thin films of rareearth metal oxyhydrides show a photochromiceffect, the precise mechanism of which is yet unknown. Here, we madethin films of NdH Ix3–2Ox) thin films receive attentiondue to their reversible photochromic effect,1 where the material reversibly changes color triggered by UV light.In the presence of this incident light, the films “darken”,absorbing light over a wide range of wavelengths (visible to near-IR).Yet, when the light is removed, the original transparency is returnedby “bleaching”. Such optical properties are attractivefor smart window applications, especially since the bleaching speed(time required to recover the transparent state) has recently beenreported as low as 9 min.2Rare earth metal oxyhydrides  are prepared by reactive magnetronsputtering of a metallic REH2 film, which oxidizes to asemiconducting photochromic oxyhydride when exposed to air.4 The extent of oxidation (O2–:H– ratio) is related to the deposition pressure during sputtering,where more oxidized films are achieved by sputtering the parent REH2 at a higher pressure which invokes a higher porosity of theas-deposited REH2. In this way, both the type of cation(RE) and the O2–:H– ratio of thesematerials can be tuned, impacting their photochromic properties.2Thin filmsof REH5 This is partly because some REHx3–2Ox powders have shown pure H– conductivity.7 In general, these large RE cations lead to tetragonal lattices,which has sometimes been associated with anion order,8 although this last aspect is debated.9 Smaller RE cations, instead, result in anion-disordered cubic lattices,thus behaving as ion insulators.7Although the mechanism of photochromismin these materials is notwell-defined, it has been proposed that ion mobility plays a rolein the process.4 it may be thatshort-range mobility, rather than long-range, influences the photochromiceffect. An NMR study of YHx3–2Ox, for example, showed the presence of a mobileH fraction which reversibly disappeared during photochromic darkening.5 However, it should be noted that other theoriesabout the photochromic mechanism have been proposed, and not all involvea diffusion-related step, namely the formation of hydroxides, colorcenters, and dihydrogen species.10Because most of the reported photochromic oxyhydrides fall in thecubic ion insulator range ,x3–2Ox thin films and theirphotochromic performance. While photochromism in Nd-based oxyhydrideswas reported earlier,11 a complete opticaland structural analysis has been lacking so far. Rare earth oxyhydridesbased on Nd are of particular interest because they show a high H– conductivity,7 have a largeRE cation, and have sometimes been described as anion-ordered witha tetragonal crystal structure.12 These structural properties of Nd oxyhydrides differ from the cubicoxhydrides we reported earlier,4 allowing for the unique opportunityto assess which structural aspects are relevant to the photochromiceffect.Here, we investigate the structural properties of NdHx3–2Ox thin films can be made by air oxidationof NdH1.9+δ films, where the O2–:H– ratio of the resultant film depends on thedeposition pressure (pdep). However, thesefilms are unstable in airand require a protective coating of Al2O3 depositedby ALD. Remarkably, the c/a ratioof our tetragonal NdHx3–2Ox films depends on the pdep (or O:H ratio). While all these films are photochromic,samples made at the same pdep showed verydifferent color changing kinetics during photochromism, despite beingequivalent in terms of crystal structure and optical properties. Thevariability in bleaching time is found to be due to (1) the encapsulationof the film by the protective layer and (2) the heating occurringduring ALD. The former changes the order of bleaching (no longer first-orderkinetics), while the latter may lead to a partial annealing of thefilms which eliminates some defects, slowing the bleaching time constant.This suggests that a certain “metastability” of an as-depositedREHx3–2Ox film and the associated structural defects are necessary ingredientsfor photochromic bleaching.We find that NdHII1.9+δ (∼300 nm) were depositedby DC reactive magnetron sputtering of a neodymium target  at 100 W in an Ar/H2 gas mixture at a ratioof 7:1. The vacuum system was operated at a base pressure of <10–6 Pa. The films were grown at various deposition pressures(pdep = 0.3–0.9 Pa) on 10 ×10 mm2 fused silica (f-SiO2) substrates at roomtemperature (∼21 °C). After deposition, the films wereoxidized in ambient air to form the oxyhydride (NdHx3–2Ox). For comparison,some GdHx3–2Ox thin films were made by the same methods and conditions (pdep = 0.7 Pa).Thin films of NdH–),which is seen as a widening of the optical band gap in the transmissionspectra . To protect the films from this completeoxidation, they were coated with a conformal Al2O3 layer by atomic layer deposition (ALD) . After taking the as-sputtered films out of vacuum, theywere brought to the ALD system, limiting the ambient air exposureduring sample transport (detailed in Table SI of the Supporting Information).The Nd oxyhydride filmsare not stable in ambient air over longperiods of time. Within a few days of removal from the vacuum chamber,the films fully oxidize  as the precursor and O2 as the reactant. The TMA pulse time was set to 0.06 s, followedby a waiting time of 4 s, and an O2 plasma for 6 s at 300W. The base pressure was (p ∼ 2 μbar),while the process pressure varied between 0.1 and 0.2 mbar. After300 cycles (1.8 h), the ALD layer was ∼47 nm thick, determinedby X-ray reflectometry (XRR) . ALD-coated NdHx3–2Ox films showed remarkably longer lifetimes, maintaininga stable composition  for at least 138 days or 5 months . A band gap shift is noticeable in the ALD-coated films comparedto the as-deposited uncoated films (day 0) , which may be due to slight oxidation from the combinationof O2 plasma and heating during ALD, an effect that likelydisappears as more monolayers are deposited.The Alry (XRR) S6. ALD-cFigure S3 and Figure S8).Optical transmissionspectra were acquired by a custom-built opticalfiber spectrometer containing a deuterium and a quartz tungsten halogenlamp  and a Si array wavelength-dispersivespectrometer . The transmission spectraof Nd-based thin films were measured for several days to monitor theextent of oxidation for both ALD-coated and uncoated films. The opticalband gap energies of the films were calculated via the Tauc method(x3–2Ox were measured by illuminatingthe films with a narrow wavelength LED (λ = 385 nm) for 1 hand measuring the average transmittance (λ = 450–1000nm) with respect to time. After 1 h the LED was turned off, and thebleaching process was measured for several hours until the originaltransparency was recovered. All optical measurements were taken atroom temperature (∼21 °C). The photochromic effect wasonly measured for ALD-coated films because uncoated NdHx3–2Ox films oxidize constantlyover time, preventing any reliable time-dependent measurements.The photochromicproperties of ALD-coated NdHkα1 and kα2. Theevaluation of the unit cell symmetry can be misinterpreted due tothe influence of thin film stress and texture on the observed XRDpattern. To investigate their presence, these properties were measuredin Bragg–Brentano (θ–2θ) geometry with varyingψ angles (ψ = 0–80°) to probe crystallitesof different orientation. The angle ψ describes the tilt ofthe sample perpendicular to the X-ray beam. Only ALD-coated NdHx3–2Ox filmswere analyzed by XRD since such measurements take several hours duringwhich the uncoated samples oxidize.The structural properties of the thin films were analyzed by X-raydiffraction  with a Cu source in grazing-incident(GI-XRD) geometry . Lattice constants were derived based on pseudo-Voigtfitting of each diffraction peak considering both IIIAx3–2Ox deposited between pdep = 0.3–0.9Pa result in various optical properties upon airoxidation, similar to our previous work using other rare-earth cations.4 Films deposited at low pressures(pdep = 0.3–0.5 Pa) are opaque, typical of RE dihydrides.19 The transmission spectrumfor the 0.5 Pa sample, however, shows a larger transparency window,extending toward longer wavelengths. This film could be very minimallyoxidized, yet still maintaining the metallic properties of as-depositedNdH1.9+δ.17The dihydride phase ofthe films deposited at 3 (pdep* ∼ 0.6 Pa) are more transparent(1.9+δ film in air and the appearance of semiconductingproperties that are characteristic of oxyhydrides (NdHx3–2Ox).3However, films depositedat and above a critical deposition pressurensparent2a,b and nsparent2c. This pdep ≥ 0.6 Pa. The relationship between the anioncomposition and the band gap is a phenomenon seen often in multianioncompounds such as oxyhydrides,4 oxyhalides,20 and oxynitrides.21 Because the oxyhydride valence band is composed of the oxide andhydride states, and O2– is more electronegativethan H–, a replacement of H– byO2– shifts the valence band down.22 This was further investigated experimentally by using acombination of RBS and ERDA to confirm that REHx3–2Ox  thinfilms deposited at higher pressures contain more O2– and have a larger band gap.4 Because the NdHx3–2Ox filmsdescribed here are produced by the same methods, we expect the sametrends to appear here, namely, that NdHx3–2Ox films deposited at 0.9 Pa havea larger optical band gap and O2– content than thosedeposited at 0.6 Pa.The optical band gap increases with the deposition pressure 2c for pdx3–2Ox films (Eg = 1.91–2.61 eV)span over a wider range thanwhat was found for other rare-earth metal oxyhydrides 4 fora similar set of pdep . Because the optical band gap and the O2–:H– ratio are related, this may indicate that stableNd-based oxyhydride thin films can be made in a larger compositionrange than for the other RE cations. A similar trend has been observed,for example, by Fukui et al.,9 where alarger stable composition range was found for La oxyhydride powderscompared to Y oxyhydrides. Another possibility is that a larger spreadin Eg can be generated for a similar O2–:H– range due to the higher polarizabilityof Nd compared to the smaller Sc, Y, and Gd cations. Cation-basedband gap engineering was shown, for example, in oxysulfides, wherethe conduction band was shifted by changing the RE cation graduallyfrom Gd to Ce.23Notably, the band gap energies observedhere for NdHBx3–2Ox, where large cations(La–Nd) often lead to tetragonal (P4/nmm) lattices24 with anion ordering8 and long-range anion mobility.7 Smaller RE cations (Sm–Er) should then result incubic (Fm3̅m), anion-disordered,and anion insulating materials.25 However, alternative structures were reported for RE = Y, La, Dy,Er, and Lu .27 Eventually, though, the crystal structure of the best H– conductor thus far (LaHx3–2Ox)6 was identifiedas tetragonal, but anion-disordered,9 challengingthe view that anion order is a necessity for long-range diffusionand a direct consequence of a tetragonal lattice.The cation sizeis an important determining factor for the structure of REHx3–2Ox, and often only instoichiometric compositions (REHO). For thin films, only Fm3̅m has been reported for RE = Sc, Y, Gd,Dy, and Er.28 The situation is lessobvious for thin films of NdHx3–2Ox, where some authors obtained a cubiccrystal structure by epitaxy29 and otherswere not able to assign a crystal structure from XRD. Specifically,many authors use the low-intensity (101) reflection to distinguishbetween Fm3̅m and P4/nmm since it only appears for the latterspace group.25 However,A∂̅alsteinsson et al.11 didnot find this reflection for their NdHx3–2Ox films and assigned no spacegroup. Therefore, it is unclear whether Nd oxyhydride thin films exhibita tetragonal crystal structure as their powdered counterparts do.12Importantly,all of the aforementioned studies dealt with powder REHx3–2Ox thin films, we did not observeany(101) reflection, even with careful measurement at low θ . This could be due to (1) the presenceof a cubic lattice, (2) the inherent low intensity of this reflection(at least 10× lower intensity than (220) and (002)8), (3) an absence of anion order,6 and(4) thin film texture . Toexclude the latter possibility, we performed XRD measurements tiltingthe film in the direction perpendicular to the X-ray beam by ψ= 0–80°. Since none of these measurements show a (101)peak, its absence is not caused by thin film texture. Because it isnot immediately apparent if our NdHx3–2Ox thin films are tetragonal orcubic, we assign the reflections observed from XRD by their expectednotation for a face-centered cubic lattice. Here, the (200) reflectionis used to calculate “a” (or the (220)in the case of 0.3 Pa), while (111) is used to calculate “c”. In case the unit cell is truly cubic, the twovalues should be equal (c/a = 1).Otherwise, if c/a ≠ 1, thereis a degree of tetragonal distortion.aAlso for our ALD-coated NdHpdep* are shown in Figure S9. The result for 0.3 Pa is in agreementwith the fcc NdH1.9+δ structure. The average latticeconstant .15 For 0.5 Pa, on the other hand, a200 and a compression of c111, meaning that this film is tetragonal (c/a = 0.985). Apparently, even a minimal addition of O2– is sufficient to induce a tetragonal structure.XRD patterns for films deposited below x3–2Ox thin films produced at and abovethe critical pdep . In general, as pdep increases, the (200) reflection shifts to larger 2θ,while the (111) peaks largely remain at the same position. Based onthe calculated a200 and c111 lattice constants . The 0.3 Pa sample is close to thecubic line, in accordance with the notion that it is NdH1.9+δ. Nd oxyhydride samples made at 0.8–0.9 Pa also tend towardthe cubic line, while all the others are clearly tetragonal (a > c). Therefore, by changing the depositionpressure, we can produce NdHx3–2Ox films of slightly different crystalstructures.This difference in a200 are in agreement with those ofstoichiometric NdHO, but our c111 is consistentlysmaller. Although substrate clamping of the thin film can preventcomplete expansion during air-oxidation, we found no residual stressin our films and no significant peak shifts during heating of the films for ∼30h at 87 °C , suggestingthat the tetragonal distortion c/a ≠ 1 we observe is an intrinsic material property.In c/a of 0.973 for 0.6 Pa, while Widerøe et al.12 and Ubukata et al.7 report 1.000 and 0.998–1.000, respectively. This could bedue to the composition, where films produced at pdep > 0.8 Pa tend toward a stoichiometric NdHO composition,while all the others are H-rich. This aligns with our previous work4 where we show that our photochromic REHx3–2Ox thin films produced by air oxidationof a sputtered dihydride generally encompass the H-rich regime ofthe REH3–RE2O3 compositionline.22 We therefore consider that the c/a ratio is a function of the O2–:H– ratio, where less tetragonal distortion ispresent for a composition close to the stoichiometric NdHO, perhapsdue to the decreased occupation of octahedral interstitial sites.30Wefurther note that some of our films are more tetragonally distortedthan the literature reports, with a minimum 22 are anion-disordered, especiallysince we did not find any superstructure reflections in the XRD indicativeof anion ordering. The disordered nature of the films may be due tothe methods by which we produce these materials and the apparent greaterstability of anion-disordered RE oxyhydrides away from the stoichiometricREHO composition.22At this point, it is not possible to determine if these filmsdifferin terms of anion ordering due to the lack of neutron diffractiondata. However, we assume for now that these films, similar to ourprevious studies,Cx3–2Ox thin films  have photochromic properties, where the films darkenduring UV-light exposure and bleach back to their original transparencywhen the light is removed. The relative photochromic contrast ((T – T0)/T0) over time for our NdHx3–2Ox films is shown in T).REHx3–2Ox films measured and presented in B)28 isno longer valid. This is visible for ALD-coated NdHx3–2Ox  when compared to a Gd-based filmwithout the coating. If the assumption of first-order kinetics iscorrect, a linear time dependency should be visible in uncoated GdHx3–2Ox film. Therefore, for this work, we definea new value, τB,50%, which is the time required tolose 50% of the darkened contrast (gray line in All of the NdHdH3–2xOx 4c and ALOx films S15 when Ox films S15 when x3–2Ox thinfilm depends not only on the RE cation but on the pdep (O2–:H– ratio)of the film.2 Briefly, films made at higher pdep resulted in a higher O content, lower ΔT, and faster τB. Compared to the photochromiccontrast of ALD-coated Nd-based films, this expected pdep-dependent trend is reproduced, since the largest contrastappears for pdep = 0.6 Pa and the lowestfor 0.9 Pa.We have shown earlier that the photochromic efficiency of a REHB,50%) does not follow any specific trend, and we find a widearray ofvalues . Without the ALD coating, the bleachingspeed of GdHx3–2Ox films made at the same pdep isfairly reproducible, which changes dramatically with the additionof the coating.On the other hand, the bleaching speed  bandgaps (O2–:H– ratios) .We can eliminate some reasons for why τ ratios) 2c, (2) c ratios) 3c, and (p ∼ 2 μbar),we made several NdHx3–2Ox films at 0.6 Pa and deposited the ALD coating ontothem. Some films were removed directly after the ALD procedure wascompleted (theating = 1.9 h), while theothers were left in the vacuum chamber at 87 °C for additionaltime.Instead, we studied the procedureused to deposit the ALD coatingwhich requires heating of the films to 87 °C for a minimum of1 h 48 min along with a few minutes of transfer time between the vacuumand air 5a.BecauB,50%) is stronglydependenton theating . However, we find that the latticeconstants (peak positions), texture (peak intensity ratios), microstrain(FWHM), and crystal structure (c/a) do not change significantly during heating, suggesting that long-rangeordering that is probed by XRD is not affected by ∼30 h ofheating at 87 °C, but local/short-range order may be altered.These latter aspects are then relevant to the photochromic effect.These can include, for example, reorganization of occupied and vacantinterstitial sites , partial removal of pointand/or line defects, growth of grains/removal of grain boundaries,and others.The bleaching speed . Whether or not encapsulationof REHx3–2Ox thin films influences the photochromic effect is debated33 but is outside the scope of this work. We suppose that the ALD-coatedfilms are better described by a series of processes with no singlerate constant or by kinetics of a different order. Precise conclusionsrequire more insight about the underlying mechanism of the photochromiceffect, which is still missing.The protective ALD coating changes the kineticsof bleaching fromthe first-order behavior we normally find.x3–2Ox films. During thisheating, local/short-range changes such as reorganization of the anionsublattice, removal of line defects, and a slight growth of grainsare possible. Although these changes are difficult to quantify, theycan play an important role during photochromism. Several theorieshave been put forth to explain this effect in REHx3–2Ox thin films10 without unanimous consensus. However, importantphenomena can be identified and assessed within the context of thiswork.Therefore, we focus primarilyon the heating in the ALD chamberand the effect of this on the photochromic properties of NdHFigure S17). Thus, althoughthe contrast and bleaching speed have often been considered related,this does not appear to be true for ALD-coated NdHx3–2Ox films. We suggestthat darkening and bleaching do not depend on the same factors. Darkeninglikely depends on the presence and concentration of H– and O2– ions in the material since neither REhydrides nor RE oxides are photochromic, and the photochromic contrasthere only depends on the pdep (O:H ratio). Bleaching, on the other hand,is more difficult in an annealed material, perhaps due to a greaterstability of the optically absorbing species in a material with fewermicrostructural defects.We note that while the bleaching speed was dramaticallyinfluencedby heating (becoming ∼6 times slower after 30 h of heating),the photochromic contrast did not show the same trend, barely changingwith heating .Importantly, these filmsare unstable in air without a protectivecoating of AlWe assume that the heatingwhich occurs during the deposition ofthe protective coating results in a reduced defect concentration.As the samples were left in the ALD chamber for longer periods oftime, the bleaching rate became slower, suggesting that the presenceof defects in the material ,and the overall imperfections of the as-deposited material are importantto the reversibility of the photochromic effect. The stability ofthe dark species in the oxyhydride matrix may determine the bleachingspeed, and annealing the oxyhydride acts to stabilize the darkenedstate, increasing the time needed for bleaching.


Scientific Reports 10.1038/s41598-018-32402-5, published online 21 September 2018Correction to: The original version of this Article contained errors in Table 2, where the “Irregular” and “Regular” “Median (IQR)” for “Total Sleep Time (minutes)” was incorrectly stated in 30-second epochs instead of minutes. Furthermore, the population medians for “Sleep in Clock Day (minutes)”, “Sleep in Clock Night (minutes)” and “Daily Light Exposure (minutes > 250 lux)” contained errors. The first 30-second epoch of each hour was not included when counting sleep and light exposure in the clock day and night.The original Table As a result, the minute values in the Results’ sub-section “Group differences in sleep timing and light exposure” have been corrected.“Specifically, on average, irregular sleepers slept for 65 minutes more during the clock day and 59 minutes less during the clock night compared to regular sleepers. In addition, irregular sleepers tend to be exposed to fewer minutes of light (>250 lux). On average, irregular sleepers were exposed to 48.2 fewer minutes of light per day than regular sleepers.”now reads:“Specifically, on average, irregular sleepers slept for 66.5 minutes more during the clock day and 60.4 minutes less during the clock night compared to regular sleepers. In addition, irregular sleepers tend to be exposed to fewer minutes of light (>250 lux). On average, irregular sleepers were exposed to 48.4 fewer minutes of light per day than regular sleepers.”The original Article has been corrected.


Sus scrofa) were domesticated very early in Neolithic China, but far less is known about the processes by which pig husbandry intensified so that pork became the most important animal protein for humans are less clear. Here, we explore pig feeding practices using the carbon and nitrogen isotope composition of bone collagen, focusing on developments in pig husbandry during the Yangshao period (7000–5000 BP) in the middle Yellow River region of China, and at the site of Xipo (5800–5000 BP) in particular. The results show that the diets of domestic pigs at Xipo were dominated by millet foods. Comparisons with other Yangshao sites in the region show a trend of increasing millet foddering for pigs throughout the Yangshao period. These results, and comparisons of the isotopic data for pigs against those for humans from the Xipo cemetery (5300–5000 BP), suggest that pigs were closely managed by humans. The evidence points to an intensification of Neolithic pig husbandry in the middle Yellow River region from this period.It is well-known that pigs ( Sus scrofa) came to provide the primary domestic animal protein source for humans [It is widely acknowledged that a combination of domesticated crops and animals provided the subsistence basis and fostered human population growth and the rise of social complexity in Neolithic farming societies –3. In Ner humans –6. Howevr humans , 8, and Although pig domestication was initiated c. 8500 BP in China , 9, 10, Intense pig husbandry practices would likely include control over movement, diet and reproduction , and herQiminyaoshu–suggest that a seasonal mixed mode of pig husbandry was practiced in the middle and lower Yellow River region during the mid-6th century AD, whereby pigs grazed in the spring and summer when plants are growing, during the autumn pigs grazed but were not foddered, and cereal bran was collected for fodder during the harsh winter and early spring [In turn, pig feeding strategies closely correlate with their mode of management. Pig foddering requires confinement under close supervision, typically penned or at least tethered near the homestead. The way in which pigs were penned at a household or farmstead level can be seen in the miniature pottery sculptures of pigpens, sometimes also connected to a toilet, as burial objects found in the tombs dated to the Han dynasty (202 BC–220 AD) –17. Histy spring . The rec13C/12C) and nitrogen (15N/14N) isotope values of Sus bone collagen, to provide a proxy for pig diets under human control. Carbon isotope values (expressed as δ13C) of terrestrial plants are principally determined by their photosynthetic pathway [3 and C4 pathways–which result in distinct, non-overlapping δ13C values in C3 (−37 to −22 ‰) and C4 plants (−16 to −9 ‰) globally  [3 plants [4 millets–broomcorn (Panicum miliaceum) and foxtail millet  [3 plants, while if domestic pigs were foddered with isotopically distinct millet foods, we should be able to detect the extent of such practices.To explore pig feeding practices in Neolithic northern China, we determined the stable carbon  , 21. The −37 to − ‰ and C43 plants , includiitalica) –31. Thus15N) values of plants are determined by those of their nitrogen source, uptake mechanisms and assimilation pathways [15N values than those that access nitrogen through mutualistic mycorrhizae (e.g. legumes) [15N of 3 to 6 ‰ between source (diet) and consumer [15N trophic step for humans falls at the higher end of these estimations (c. 5–6 ‰) and this is likely to be the case too for pigs as feeding studies suggest that pigs show a similar trophic enrichment pattern to humans [15N values directly, through provisioning with human food scraps, some of which are animal products [15N values than pigs foraging in the absence of human interference.Nitrogen isotope (δpathways . Plants legumes) . Such isconsumer –36. Accoconsumer  the δ15No humans , 38. Antproducts –41, or iproducts . Thus, p13C, n = 6; δ15N, n = 2) [Previous stable isotope studies have suggested that millet foddering of pigs was practiced on the Wei River Plain from the Early-Yangshao period (7000–6000 BP) , 44. How, n = 2) , 53, theIn this study, we first explore the diets of domestic pigs at Xipo based on the stable isotopic composition of bone collagen, and we then compare these results against those published for humans from the Xipo cemetery. Next we compare the Xipo results with those of pigs from other Yangshao sites in the middle Yellow River region, aiming to:Understand the pig feeding practices and management modes at Xipo;Disentangle the relationships between pig husbandry, human diets and local farming;Gain greater insights into pig husbandry regimes in the middle Yellow River region during the Yangshao period.The Yangshao Culture (7000–5000 BP) is characterised by distinct painted ceramics, the appearance of large, densely populated settlements with sophisticated layouts, and a mixed mode of human subsistence . These c2 in size in the central area (F105 over 500 m2 including surrounding corridor)  , construOryza sativa) and soybean (Glycine max) remains were also recovered. Many of the ceramics recovered from the burials in the Xipo cemetery are clearly functional and assumed to be associated with the cooking of millet (2) suggests that pigs were likely consumed during feasting events held in or near these buildings [According to the recovered plant remains, stable isotope values of human bones, and starch grain analyses of dental calculus , 59, foxf millet . Pigs douildings . ArticulSus and 17 other faunal species were selected for stable isotope analysis , cattle (Bos), sheep/goat (Ovis/Capra), deer (Cervus), bear (Ursus), and bird (Avis), which were selected to compare the diets of domestic animals and to establish an isotopic baseline of the local ‘wild’ ecosystem. Cattle and dog specimens came from contexts dated to the later Western Zhou period (1046–771 BC). Details of the contexts and phases of animal bone samples are given in In total, bone specimens of 70 analysis . The SusPermission to perform the analysis of the faunal materials from Xipo was issued by the National Cultural Heritage Administration, China . The specimens are stored in a repository of Henan Provincial Institute of Cultural Heritage and Archaeology in Zhengzhou, Henan, China, which are not publically accessible. For research purposes, any individuals or groups can seek access to the materials from this institution, with support from the excavation co-director (Dr. Xinwei Li). This described study complied with all relevant regulations.Bone collagen was extracted following a standardised protocol . Approxi13C/12C and 15N/14N ratios using a Sercon 20/22 continuous flow mass spectrometer coupled to a Callisto elemental analyser. Stable carbon and nitrogen isotope ratios are expressed using the delta (δ) notation as:13C/12C or 15N/14N. The δ13C and δ15N values are reported relative to the international Vienna PeeDee Belemnite (VPDB) and Ambient Inhalable Reservoir (AIR) standards, respectively. An internal standard, Alanine, and two further internal bone collagen reference materials, COW and SEAL, all previously calibrated to international reference materials were included in each run to correct for drift, to monitor measurement uncertainty and to calibrate the raw data. Precision of measurement was calculated as ±0.08 ‰ for δ13C and ±0.12 ‰ for δ15N based on multiple measurements of the collagen standards. Accuracy was determined to be ±0.10 for δ13C and ±0.16 for δ15N based on the difference between the observed and known δ values of the standard Alanine and its long-term standard deviation. The total analytical uncertainty was estimated to be ±0.13 for δ13C and ±0.2 for δ15N using the equations given by Szpak et al. [For stable isotope analysis, approximately 1 mg of bone collagen for each sample was weighed into tin capsules to determine the k et al. . AdditioThe state of collagen preservation was assessed by collagen yield, weight percentage of C and N and the atomic C:N ratio . The C:N13C and δ15N values for all faunal samples from Xipo are shown as a bivariate plot in The δCervus specimens show a mean δ13C value of −19.7±1.1 ‰ (n = 6), similar to δ13C values for other wild fauna, including Ovis/Capra (−19.6 ‰), Ursus (−20.0 ‰) and Avis (−18.0 ‰) and indicating that wild herbivores foraged in similar open C3 environments in the vicinity. Cervus δ15N values range from 3.3 to 5.6 ‰ (mean 4.2±0.9 ‰) and that of the single ovicaprid is 4.2 ‰. These values likely reflect the baseline range of δ15N values for herbivores feeding in an undisturbed wild environment. Pigs have the highest δ13C and δ15N values of all of the fauna sampled  range widely, from −13.0 to −6.2 ‰ (−7.8±1.3 ‰) and from 5.5 to 8.8 ‰ (7.2±0.6 ‰), respectively, indicating that most pigs had an omnivorous diet that included significant amounts of C4 resources. A few pigs exhibited δ13C values below −8 ‰, suggesting that they also consumed some C3 foods. We found significant differences between the wild herbivores and domesticated pigs for both δ13C  and δ15N values . Two other domestic animals, dogs (n = 2) and cattle (n = 6), are from the later Western Zhou period. The dogs with δ13C values of −7.7 ‰ and −8.9 ‰, and δ15N values of both 6.6 ‰ are very similar to the range of pigs, while the cattle show δ13C (−10.4±1.4 ‰) and δ15N values (5.3±0.6 ‰) lower than those of the pigs. sampled . Compare13C values clustered between −8.5 and −6 ‰ . Thea et al.  and Zhana et al. : millet o grains , 71. It 3 or even diets comprising >50% C3 resources , however, fall squarely into the middle of the range for all pigs , indicative of omnivorous diets with a significant contribution of C4 resources, i.e. millets. Referring to the modelled values of the pure millet consumer, the distribution of δ13C and δ15N data suggests that most pigs ate more millet foods than humans  and/or animals that consumed C3 plants, like wild fauna .On average, compared to pigs, humans are enriched in t in 15N . For simn values . These oWe further explored the relationship between human diets and social status by a re-analysis of the published collagen isotopic results for Xipo humans from the cemetery, against a hierarchy of four burial groups Figs  and 5. T15N and δ13C values are correlated with their burial class. The results show that human δ15N values are correlated with their assigned burial class with statistical significance (p = 0.01), but no clear differentiation in δ13C values between ‘high’ and ‘low’ status individuals (p = 0.20) is apparent  is larger than that between Class 2 and Class 3 (p = 0.01) and also between Class 3 and Class 4 (p = 0.02). Specifically, the high-status people with large graves and more valuable grave objects tend to have higher δ15N values than those with small graves and few grave goods. The most parsimonious explanation is that these high-status individuals consumed more animal protein and the animal source was most likely pig meat since wild animals have significantly lower δ15N values. Together these data suggest that high-status people had preferential access to protein sources enriched in 15N compared to lower-status individuals, but there is no significant difference in carbon isotope values. Therefore, this isotopic evidence suggests that millets were eaten by both high and low status individuals, rather than being reserved for individuals of a certain social status.In order to explore the relationship between human diets and social status, we use ordered logistic regression  and lowest δ13C value (−12.0 ‰) of all individuals published. It seems that in addition to millets and pigs, some C3 food sources led to his bone collagen exhibiting a more negative δ13C value and higher δ15N value. Eating hunted wild herbivores does not appear to be a plausible explanation, however, since those analysed in this study show significantly lower δ15N values than domestic pigs , having mean δ13C and δ15N values of −13.2±2.8 ‰ and 5.6±0.9 ‰ (n = 13) [3/C4 diets. This indicates that from the Early-Yangshao period pigs were partly fed with millet, and in varying proportions. Dongying (5900–5600 BP) has too few datapoints, but the available pig δ13C values from this site are also consistent with a significant contribution of millet foods in their diets [13C and δ15N values of Quanhucun pigs largely overlap with those of Xipo pigs, but show slightly more negative δ13C values and higher δ15N values  [13C and δ15N values of −7.5±2.5 ‰ and 8.5±0.9 ‰ (n = 5) [Notably, both the δ(n = 13) , 44, andir diets . The Qua n = 10) . In the  (n = 5) . Their d13C and δ15N values hint at the presence of some C3 foods with elevated δ15N values in their diets  would be penning. Some small walled features at Xipo have been interpreted as possible pigpens [The stable isotopic results suggest that Xipo pigs had a predominantly C pigpens , but no  pigpens , 76. HerPigs were also used in diverse ritual activities. The burial of pig remains, particularly mandibles, along with humans in graves, is a frequently observed phenomenon initiated from the Pre-Yangshao period c. 8500 BP onwards , 9, 77. in situ rather than brought to the site from a hinterland. We suggest that pig husbandry practices at Xipo were predicated on taking advantage of crop surplus production, and were tightly integrated into the millet farming system.In spite of the strong dependence in these Yangshao sites on pig husbandry, there is a paucity of strong evidence for built features, such as pens, indicative of local pig raising. Yet compared with other Yangshao sites in the middle Yellow River region, the relatively narrow range of isotopic data for Xipo pigs indicates that a relatively homogenous pig foddering regime was employed, suggesting that the pigs were raised Isotopic data from the site of Xipo show that pigs were intensively fed on millets, along with other minor sources such as household waste. This observation suggests that they were confined within or close to the Xipo settlement in close proximity to humans. The intensive foddering strategy for fattening pigs supported the growing human population, and at the same time demanded a high labour input. In spite of the evident economic importance of pig husbandry and close proximity of pigs and humans, the evidence for their use in ritual activities is, so far, surprisingly limited. Certainly, if they were used for such activities, the isotopic data lacks evidence for the presence of any specially fed pigs. Comparisons with other Yangshao sites elsewhere in the middle Yellow River region suggest a trend towards intensified pig husbandry practices, particularly millet foddering, during the Yangshao period. From the Middle-Yangshao phase, millets became the predominant pig fodder, which were likely derived from crop surpluses. Together, the importance of millet foods in pig foddering and pig consumption in human diets at Xipo indicates that pigs became an integral part of both the local farming regime and subsistence economy in the middle Yellow River region from this period.S1 TableDetails of instrument measurement, data calibration, uncertainty calculation, collagen yield and sample information.(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 TableOrdered Logistic Regression in 5.2 and 5.3.(XLSX)Click here for additional data file.


Recent evidence shows that more complex clusters of chronic conditions are associated with poorer health outcomes. Less clear is the extent to which these clusters are associated with different types of disability  and functional mobility (FM)) over time. This was a longitudinal analysis using the National Health and Aging Trends Study (NHATS) . Using latent class analysis, we determined the optimal clusters of chronic conditions, then assigned each person to a best-fit class. Next, we used mixed-effects models with repeated measures to examine the effects of group (best-fit class), time (years from baseline), and the group by time interaction on each of the outcomes in separate models over 4 years. We identified 5 chronic condition clusters: “multisystem morbidity” (13.9% of the sample), “diabetes” (39.5%), “osteoporosis” (24.9%), “cardio/stroke/cancer” (4.5%), and “minimal disease” (17.3%). Group by time interaction was not significant for any outcome. For ADL outcome, only time was significant . For IADL, both group  and time  were significant. For FM, both group  and time were significant . We did not find evidence that any cluster experienced greater increases in disability over time, but all clusters containing multiple chronic conditions had risk of IADL and FM disability. Increased screening for IADL and FM disability could identify early disability and prevent decline.


There are many pathological changes in the brains of Alzheimer’s disease (AD) patients. For many years, the mainstream view on the pathogenesis of AD believes that β-amyloid (Aβ) usually acts independently in addition to triggering functions. However, the evidence now accumulating indicates another case that these pathological types have synergies. The objective of this study was to investigate whether effects of Aβ pathology on cognition were mediated by AD pathologies, including tau-related pathology (p-tau), neurodegeneration , axonal injury (NFL), synaptic dysfunction (neurogranin), and neuroinflammation .Three hundred seventy normal controls (CN) and 623 MCI patients from the ADNI (Alzheimer’s Disease Neuroimaging Initiative) database were recruited in this research. Linear mixed-effects models were used to evaluate the associations of baseline Aβ with cognitive decline and biomarkers of several pathophysiological pathways. Causal mediation analyses with 10,000 bootstrapped iterations were conducted to explore the mediation effects of AD pathologies on cognition.APOE ε4 carrier status have a moderating influence on some of these relationships. There is a stronger association of Aβ with biomarkers and cognitive changes in the elderly and females. In CN group, Aβ pathology is directly related to poor cognition and has no mediating effect (p < 0.05). In mild cognitive impairment, tau-related pathology  and neurodegeneration  mediate the impact of Aβ on cognition.Tau-related pathology, neurodegeneration, neuroinflammation are correlated with the concentration of Aβ, even in CN participants. The results show that age, gender, and In conclusion, early Aβ accumulation has an independent effect on cognitive decline in CN and a tau, neurodegeneration-dependent effect in the subsequent cognitive decline in MCI patients.The online version contains supplementary material available at 10.1186/s13195-021-00943-z. An essential pathology of Alzheimer’s disease (AD) is the gradual aggregation of β-amyloid (Aβ) in the brain, a process that begins decades before cognitive symptoms appear. The detection of abnormal Aβ accumulation may support the clinical diagnosis of AD –3. For mThe objectives of our study were (1) to investigate whether Aβ pathology is related to downstream pathophysiological processes and cognitive levels; (2) to explore whether the major unchangeable AD risk factors such as age, gender, and APOE ε4 status regulate these associations; and (3) to investigate to what extent those associations represent particular downstream changes related to Aβ or to what extent they are driven by other relevant biomarkers. Advances in developing new cerebrospinal fluid (CSF) or blood biomarkers provide insights into tracking pathological processes , 9–12. RTo implement these objectives, we investigated biomarkers that reflect the pathophysiology of AD, including tau-related pathology , neurodegeneration , axonal injury , synaptic dysfunction (neurogranin), and neuroinflammation  . All thoThe data used in the study was acquired from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) database. We included 993 subjects with available basic clinical characteristics, cerebrospinal fluid data, imaging data, and cognitive assessment data from the ADNI database. The ADNI database classified subjects clinically as cognitively normal , mild cognitive impairment  or AD dementia following predefined criteria . Individ42, p-tau, t-tau, sTREM2, YKL-40, plasma NFL, and MRI information were included in the analysis. The methods and analyses for biomarkers and imaging have been previously described in detail [42, p-tau, and t-tau levels (unit: pg/mL) were completed at the University of Pennsylvania through the multiple xMAP Luminex platforms  and INNOBIA AlzBio3 kit  [APOE genotype was determined by genotyping two single-nucleotides  [APOE ε4 carrier if carrying at least one APOE ε4 allele.Those CN and MCI subjects with available baseline CSF Aβn detail . CSF Aβ4Belgium) . CSF sTRBelgium) . The CSFBelgium) . All CSFBelgium) . APOE ge rs7412) . Genotyphttp://adni.loni.usc.edu/) and in previous publications [The protocol of the ADNI FreeSurfer-based pipeline is available online (ications . The MRIWe downloaded the episodic memory (MEM) and executive function (EF) comprehensive scores, as well as the recently verified language (LAN) and visual-spatial functions (VS) from the website as tools to track the trajectory of cognitive measurement. These scores are extracted from ADNI neuropsychological tests, which are comprehensive scores after optimization of psychological measurement. These measurements have been verified before, proving robust and externally effective , 23.42 concentrations are considered as three standard deviations (SD) higher or lower than the overall population means. These subjects (n = 6) were excluded. Baseline characteristics were compared between diagnostic groups using Student’s t-tests or Wilcoxon rank-sum tests for continuous and χ2-tests for categorical measures. The first aim of this study was to measure the direct associations of CSF Aβ42 with cognitive measurements and various available biomarkers. To this end, we used each biomarker and cognitive assessment results as the dependent variable of interest and the CSF Aβ42 level as the independent variable in the multivariate linear regression model. To determine the association of CSF Aβ42 levels with biomarkers and cognitive ability changes, we utilized a pre-established method in which we fitted linear mixed-effects (LME) models with various measurements as the dependent variable and time as the independent variable, controlling for random slope and intercept. The LME model was also used to simulate the rate of changes of various measurements for subsequent analyses. We added a new variable resulting from the product of risk factors and CSF Aβ42 to the model to evaluate the interaction effects of CSF Aβ42 levels and main AD risk elements  on each indicator.All statistical analyses were performed in R v.3.6.3. Outliers of baseline CSF Aβ42 and multiple cognitive measures. The purpose of this analysis is to assess whether the previously discovered association between CSF Aβ42 and cognition is partially, completely, or not mediated by AD pathology. In each model, CSF Aβ42 values were included as an independent variable, cognitive measurements as the dependent variable. All mediational tests were performed with 10,000 bootstrap replications. The cut-off value of biomarker which used to indicate normal (negative) and abnormal (positive) might be considered as a characteristic for the existence of Aβ pathology [42 is 192 pg/ml. This cut-off value was used to divide the participants into A− and A+ groups for subsequent subgroup analyses.We used various biomarkers as mediators for our main hypothesis to analyze the mediation between CSF Aβathology . The cutAPOE ε4 carrying status, and educational level as covariates, and additionally adjusted intracranial volume during MRI measurements analyses. Since all outcome variables in the model were converted to standardized z-scores, the coefficient refers to the standardized effect.All analyses were adjusted age, sex, p < 0.001; EF, p < 0.001; LAN, p < 0.001; VS, p < 0.001). The cognitive decline rates of MCI participants are significantly higher than that of CN population . There is lower CSF Aβ42 (p < 0.001) and higher p-tau (p = 0.002), t-tau (p < 0.001), Neurogranin (p < 0.001), and plasma NFL (p < 0.001) in the MCI group compared with CN group. We did not find any difference between the two groups in terms of educational level, CSF sTREM2, and CSF YKL-40 concentration, which was closely resembled previous findings of this cohort [The demographics, CSF Aβ, mediators, and neurocognitive data at baseline and follow-up are shown in Table s cohort , 26.Tabl42 with all mediators and cognitive measures. Individuals with lower CSF Aβ42 levels had more significant tau pathology, neurodegeneration, and severe synaptic dysfunction, as indicated by CN participants’ CSF p-tau, t-tau, and neurogranin levels  and VS  in CN participants. No significant interactions with age and APOE ε4 status were detected using CSF Aβ42 as a marker of amyloid pathology , NFL (p = 0.017), MRI , and cognitive measurements . CSF Aβ42 interacted with sex on longitudinal entorhinal volume (p = 0.042), MEM (p = 0.003), and EF (p = 0.015). Regarding the interaction between APOE ε4 status and CSF Aβ42, it was only significant for hippocampus volume (p < 0.001) and mid temporal volume (p = 0.011) (Additional file We only found CSF Aβ p = 0.01. RegardiAPOE ε4 status. We tested whether the association between CSF Aβ42 and cognitive measurements was mediated by tau pathology and/or neurodegeneration, synaptic dysfunction, neuroinflammation. There is no evidence that CSF Aβ42 contributes to cognitive impairments via modulating other AD pathology in CN participants (Additional file Preliminary regression analyses of different cognitive groups showed the associations of the pathological index with cognitive measurements in the model controlling for age, gender, educational level, and The direct impact of tau pathology, neurodegeneration, and synaptic dysfunction on cognition was also observed in the MCI participants. Figures Although the results of this study show a particular association between neuroinflammation and cognitive decline, we have not observed a mediating effect (Additional file APOE ε4 carrier status had moderating effects on some of these correlations. The interaction analyses showed there were stronger associations of Aβ with cognitive changes and biomarkers in the elderly and females. In the mediation analysis, most p values with statistical significance were less than 0.001. This means that even the FDR correction is not likely to affect the final conclusion.In this study, we investigated the associations of Aβ pathology with cognitive changes and biomarkers which represent the downstream pathophysiological processes of Aβ in a cohort of non-demented participants. Our results showed that the concentration of Aβ was negatively correlated with tau-related pathology, neurodegeneration, and neuroinflammation, even in CN participants, suggesting that a reduction in Aβ could slow its downstream pathophysiological processes. Previous studies showed that anti-oligomeric Aβ antibodies effectively reduced plaque load, tau hyperphosphorylation, and microglial activation, and these antibodies improved the cognitive ability in mouse models. These may be the protective effects of anti-oligomeric Aβ antibodies before cognitive decline . We alsoWe conducted a series of mediation analyses to explore which factors mediate the impact of Aβ on cognition. In the CN group, Aβ, tau pathology, neurodegeneration, axonal injury, synaptic dysfunction, and neuroinflammation were directly related to cognitive decline without mediation effects. Several previous studies reported that people with decreased Aβ levels had a faster cognitive decline in memory and executive function –31. But In MCI participants, the effects of Aβ on cognitive measurements were partially mediated by tau pathology and neurodegeneration. Accumulating evidence has shown that upstream Aβ accumulation is related to abnormal changes in downstream pathological biomarkers, including abnormal tau, neurological dysfunction, glial activation, neuron loss, and brain atrophy, which is consistent with our result . HoweverPrevious mediation studies did not include all the pathological factors , 40. TwoIn addition, we only observed the direct effects of tau pathology on cognitive changes in A + individuals. Contrary to the opinion that there is no particular interaction between Aβ and tau except for the former’s triggering function, there is now evidence that functions of Aβ may be more complex . Some ev42 was expressed in a ratio to Aβ40 to assess the pathologic species while accounting for individual differences in amyloid production [42 as a marker for Aβ pathology. Future researches may consider using ratios to explore whether there are differences in research results.There were still some possible limitations in our research. Firstly, since this study is a single-center study, the results still need to be verified in larger longitudinal cohorts. Secondly, ADNI has a relatively pure AD population because it mainly includes MCI patients. Reproduction of findings from different AD phenotypes and participants from other cohorts will be helpful. Thirdly, the relationships between Aβ and cognitive decline still need to be further explored. In several previous studies, Aβoduction , 51. We In conclusion, our research found associations of Aβ pathology with cognition and several AD pathologies, including tau-related pathology, neurodegeneration, axonal injury, synaptic dysfunction, and neuroinflammation. Though the underlying mechanisms were not completely clear, these results still offer new evidence for the synergistic effect among pathological processes. Early Aβ accumulation has an independent impact on cognitive decline and a tau, neurodegeneration-dependent effect in the subsequent cognitive decline. Our results need to be repeated in large samples, and the underlying mechanism needs to be explored in further research.Additional file 1. Mediation analyses of Aβ and baseline cognitive measurements with biomarkers as mediators in CN participants.Additional file 2. Mediation analyses of Aβ and rates of cognitive measurements with biomarkers as mediators in CN participants.Additional file 3. Longitudinal clinical characteristics of participants in individual groups in the current study.Additional file 4. Main and interactions effects of Aβ on biomarkers and cognitive measures in CN participants.Additional file 5. Main and interactions effects of Aβ on biomarkers and cognitive measures in MCI participants.Additional file 6. Effects of biomarkers on cognitive composite measures in CN participants.Additional file 7. Effects of biomarkers on cognitive composite measures in MCI participants.Additional file 8. Mediation analyses of Aβ and cognitive measurements with biomarkers as mediators in A-CN participants.Additional file 9. Mediation analyses of Aβ and cognitive measurements with biomarkers as mediators in A+CN participants.Additional file 10. Mediation analyses of Aβ and cognitive measurements with biomarkers as mediators in A-MCI participants.Additional file 11. Mediation analyses of Aβ and cognitive measurements with biomarkers as mediators in A+MCI participants.


Purpose: Testicular germ cell tumors are the fourth most common neoplasm in adolescents, accounting for 8% of all tumors in the age group 15–19 years. On rare instances, the primary testicular lesion is not clinically or radiologically evident while nodal or visceral metastases represent the clinical manifestations of the disease. This phenomenon is described as “burned-out testicular tumor.” In this paper, the authors report a single-institution experience with burned-out testicular tumors in adolescents and discuss their clinical implications.Patients and Methods: All the patients diagnosed with metastatic testicular germ cell tumors at Bambino Gesù Children Hospital between January 1, 2010, and June 30, 2020, were included in the study. Patients were categorized into two groups: “primary testicular” and “burned out.” All the patients were staged and treated according to the AIEOP–TCGM 2004 protocol.Results: Eleven patients were classified as “primary testicular,” and five patients were classified as “burned out.” “Burned-out” tumors were associated with the presence of systemic symptoms compared to “primary testicular” tumors  and higher aFP, hCG, and LDH levels (p < 0.00001). The “burned-out” population had a statistically significant higher incidence of relevant toxicity than the “primary testicular” population  and a worse outcome in terms of both mean overall survival  and mean event-free survival .Conclusion: “Burned-out” testicular tumors seem to be a well-distinct clinical entity with a high treatment-related toxicity and poor prognosis. Further studies are needed to clarify the “burned-out phenomenon” and to identify more effective therapeutic strategies for these patients. Testicular germ cell tumors are the fourth most common neoplasm in adolescents, accounting for 8% of all tumors in the age group 15–19 years .The prognosis of germ cell tumors is generally excellent, even though a subset of patients, i.e., patients older than 11 years, with elevated alfa-fetoprotein levels at diagnosis, extragonadal primary tumors, non-germinomatous tumors, and stage III and IV disease, have a worse outcome and therefore need an intensified treatment –6.On rare instances, the primary testicular lesion is not clinically or radiologically evident while nodal or visceral metastases remain viable and represent the clinical manifestations of the disease . In thesIn the present paper, the authors report a single-institution experience with burned-out testicular tumors in adolescents and discuss their clinical implications.All the patients diagnosed with metastatic testicular germ cell tumors at Bambino Gesù Children Hospital between January 1, 2010, and June 30, 2020, were included in the study.2/day iv for 4 days, Etoposide 100 mg/m2/day iv for 4 days, Bleomycin 15 UI/m2 on day 2), three courses for stage III disease, and four courses for stage IV disease; indications for resection of secondary lesions after chemotherapy were considered for individual cases.The clinical notes of all the patients with stage III and IV tumors were retrospectively reviewed and categorized in two broad groups: “primary testicular” patients, i.e., patients who presented with testicular pain or testicular enlargement at initial diagnosis and sonographic evidence of testicular mass, and “burned-out” patients, i.e., patients who had no testicular symptoms at initial presentation and non-specific findings at ultrasound, such as microcalcification  and parep < 0.05 was considered statistically significant for each analysis.Seven variables have been examined: age at diagnosis, clinical presentation, serum markers level, stage, primary histology, toxicity, and outcome. Follow-up time was calculated from the time of diagnosis until the time of last follow-up visit with a cutoff in October 2020. Statistical analyses were performed using Prism 9.0.0.121 . A value of t-test.Mean age at diagnosis has been calculated separately in the two groups and data have been compared using Student's The presence at initial diagnosis of “mass effect” symptoms, defined as extra-testicular palpable mass or symptoms related to compression due to metastatic mass, or “systemic” symptoms, defined as fever > 38°C, weight loss, and deep venous thrombosis, was searched for in the clinical notes of each patient in both groups. Data were compared using Fisher's test.t-test.Alpha-fetoprotein (aFP), human chorionic gonadotropin (hCG), and lactate dehydrogenase (LDH) levels were measured at initial presentation in all the patients. Mean and standard deviation for each serum marker were calculated separately in the two groups and data were compared using Student's In each group, patients were divided into two subgroups according to the stage of the disease, i.e., stage III and IV. For stage IV subgroup, patients with extra-pulmonary visceral metastases were analyzed separately on the basis of the evidence of a distinct worse prognosis in adult population . Data weAll the patients had their diagnosis confirmed by histological examination of the affected testis or a specimen from the metastatic mass. Patients were classified according to the AIEOP–TCGM 2004 protocol as mature teratoma, immature teratoma, yolk sac tumor, choriocarcinoma, embryonal carcinoma, seminoma, or mixed histology . Data wePatients were defined as having experienced “relevant toxicity” during treatment if they had at least one episode of grade 3 or higher toxicity in at least one apparatus as defined in the “Common Terminology Criteria for Adverse Events (CTCAE) version 5.0” . Data wep < 0.05 was considered statistically significant.Patient's outcome has been registered at the time of last follow-up visit as “remission” if the patient had no clinical, radiological, or serological evidence of disease; “progression” if the patient had clinical, radiological, or serological signs of disease; or “death” if the patient had passed away. Event-free survival (EFS) was calculated from the date of initial treatment to the date of whichever came first among progression, death, and last follow-up visit. Overall survival (OS) was measured from the date of initial diagnosis to the date of death or last follow-up visit. Survival was analyzed with Kaplan–Meier plots and the log-rank test. A value of In the examined period, 16 patients were diagnosed with stage III and IV testicular germ cell tumors at our institution and all were included in the present study.Eleven patients were classified as “primary testicular:” four patients (36%) had right testicular tumor and seven (64%) had tumor in the left testis. Five patients were classified as “burned out:” two patients (40%) had right-side tumor, two had left-side tumor (40%), and in one patient (20%), the location of the primitive tumor could not be determined.None of the patients had pre-existing comorbidities.p < 0.00001).“Burned-out” patients were significantly older  than “primary testicular” patients .All the “burned-out” patients presented with symptoms related to mass effect due to metastases. Four “burned-out” patients (80%) presented with systemic symptoms . The association between “burned-out” tumors and systemic symptoms was statistically significant  vs. 530 mIU/ml , p < 0.00001; mean LDH 1,558 IU/L  vs. 424 IU/L (range 238–620 IU/L), p < 0.00001.aFP, hCG, and LDH levels were significantly higher in the “burned-out” population than in “primary testicular” patients: mean aFP 2,215 ng/ml  vs. 979 ng/ml , In the “primary testicular” population, six patients (55%) were stage III and five patients (45%) were stage IV; one patient (9%) had extrapulmonary visceral metastases in the central nervous system.In the “burned-out” population, one patient (20%) was stage III and four patients (80%) were stage IV; three patients (60%) had extrapulmonary visceral metastases, including liver (three patients), bones (two patients), and central nervous system (one patient) .p = 0.3077). A trend toward a higher incidence of extra-pulmonary visceral metastases was noted in the “burned-out” population (three patients vs. one patient in the “primary testicular” group), although such difference failed to prove statistically significant (p = 0.0632).There was no statistically significant difference in the stage distribution between the two groups , two patients had yolk sac tumor (18%), and one patient had immature teratoma (9%).in situ neoplasia associated with areas of fibrosis and interstitial lymphoplasmacytic infiltrates. One patient affected by choriocarcinoma with multiple pulmonary metastases progressively developed respiratory distress and was considered unfit to undergo surgery.In the “burned-out” group, diagnoses were made on biopsies from metastatic sites. Two patients had choriocarcinoma (40%), two patients had embryonal carcinoma (40%), and one patient had mixed histology (20%). Four patients underwent orchiectomy after the diagnosis of metastatic germ cell tumor had been established: in all four cases, histology revealed p = 0.0833).A trend toward a higher incidence of choriocarcinoma and embryonal carcinoma in the “burned-out” group was noted, although it was not statistically significant  experienced “relevant toxicity” during treatment; both had grade 3 anemia and thrombocytopenia (18%), one had infection (9%), and one had renal and hepatic grade 3 toxicity associated with grade 3 arterial hypertension (9%).In the “burned-out” population, four patients (80%) had “relevant toxicity;” all of them had grade 3 anemia and thrombocytopenia (80%), three had infectious complications (60%), two had simultaneous renal and pancreatic grade 3 toxicity (40%), two had neurological complications (20%), one had a severe hypertensive crisis (20%), one had chronic pulmonary toxicity (20%), one patient with massive pulmonary metastases had acute respiratory distress and pleural hemorrhage secondary to “choriocarcinoma syndrome” , 15 thatThere were no toxicity-related deaths in both groups.p = 0.0357)  .The 11 patients in the “primary testicular” group were followed up for a mean time of 43 months (range 13 to 119 months). Five patients underwent surgery on residual secondary lesions after chemotherapy. At the last follow-up visit, 10 patients (91%) were alive and in complete clinical, radiological, and serological remission while one patient (9%) had radiological evidence of progression of disease; this patient decided to refer to a different institution for further treatment and was subsequently lost at follow-up. A second patient experienced relapse 29 months after the initial diagnosis and was successfully treated by salvage therapy with surgical resection of residual masses followed by high-dose chemotherapy and autologous stem cell transplantation. No deaths were recorded. Mean OS time for the “primary testicular” group was 43 months (range 13–119 months) and mean EFS was 38 months (range 5 to 119 months).The five patients in the “burned-out” group were followed up for a mean time of 15 months (range 6 to 28 months). At the last follow-up, two patients (40%) affected by pure embryonal carcinoma were alive and in complete remission at 28 and 27 months, respectively; one of them underwent surgery on residual metastases after chemotherapy. One patient (20%) initially diagnosed with mixed tumor experienced clinical and radiological signs of progression of disease with negative serum markers at 3 months with histological evidence of mature teratoma, a condition described as “growing teratoma syndrome” ; this pap = 0.0299) and EFS (p = 0.0164) as shown by the Kaplan–Meier plots ; despite this difference, both populations are comprised in the range 13 to 19 years, which is reported to be the age range at highest risk of adverse events in testicular germ cell tumors . HistoloWe may therefore assume that the two groups are comparable and that the differences in terms of clinical presentation, treatment-related toxicity, and outcome are attributable to the “burned-out” vs. “primary testicular” status.p = 0.0027).All the patients in the “burned-out” population presented with a palpable metastatic mass, and four out of five patients also presented with systemic symptoms, i.e., weight loss, fever, and deep venous thrombosis, while none of the “primary testicular” patients in our series presented mass-related symptoms or systemic symptoms  vs. 530 mIU/ml , p < 0.00001; mean LDH 1,558 IU/L  vs. 424 IU/L (range 238–620 IU/L), p < 0.00001.Serum markers at diagnosis were significantly higher in the “burned-out” population than in the “primary testicular” group: mean aFP 2,215 ng/ml  vs. 979 ng/ml , The differences in clinical presentation and serum markers at diagnosis may be interpreted as a sign of higher tumor burden in the “burned-out” group; a different explanation could be a substantially different biological behavior of “burned-out” tumors compared to “primary testicular” tumors even in the face of similar disease stage. At the state of the art, there is poor evidence about the biological mechanism of the “burned-out” phenomenon and its clinical implications; further studies are needed to clarify this issue.p = 0.0357); such event is unexpected since both groups received the same therapeutic regimen and no patient had pre-existing comorbidities. One patient in the “burned-out” group experienced “choriocarcinoma syndrome,” a treatment-related complication that occurs in patients affected by choriocarcinoma with multiple pulmonary metastases at the beginning of chemotherapy, characterized by acute respiratory distress and pulmonary hemorrhage  and EFS (p = 0.0164). To our knowledge, this is the first study to compare outcome between “burned-out” and “primary testicular” patients. Several factors may contribute to such difference. In the present series, “burned-out” patients had significantly higher levels of serum markers  than “primary testicular” patients: elevated aFP, hCG, and LDH are known adverse prognostic factors in adults (p = 0.0632); the presence of such secondary lesions is a documented poor prognostic factor in adults (The present data show that “burned-out” patients have a worse outcome compared to “primary testicular” patients in terms of both OS (n adults , althougp = 0.016. To our n adults . “Burnedn adults  but not The different clinical behavior could be explained by a biological difference between “burned-out” and “primary testicular” tumors. Further studies are needed to clarify this issue.The present study has an obvious limitation in its retrospective nature. A second limitation is the small number of patients included in the study.On the other hand, the two groups compared in this study showed a similar distribution in terms of age, stage, and histology; moreover, all the patients were treated according to the same protocol . It is rIn conclusion, “burned-out” testicular tumors seem to be a well-distinct clinical entity with a higher treatment-related toxicity and poorer prognosis compared to metastatic “primary testicular” tumors. Further studies are needed to clarify the “burned-out phenomenon” from a biological point of view and to identify more effective therapeutic strategies for these patients.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.GP, AC, MD, RC, and AI contributed to conception and design of the study. FB and CM organized the database and revised the data. RA and FD revised the pathology specimens. PD revised radiology images. All authors contributed to manuscript revision and read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.