The factors shaping the composition of the tree mycobiome are still under investigation. We tested the effects of host genotype, site, host phenotypic traits, and air fungal spore communities on the assembly of the fungi inhabiting Norway spruce needles. We used Norway spruce clones and spore traps within the collection sites and characterized both needle and air mycobiome communities by high‐throughput sequencing of the ITS2 region. The composition of the needle mycobiome differed between Norway spruce clones, and clones with high genetic similarity had a more similar mycobiome. The needle mycobiome also varied across sites and was associated with the composition of the local air mycobiome and climate. Phenotypic traits such as diameter at breast height or crown health influenced the needle mycobiome to a lesser extent than host genotype and air mycobiome. Altogether, our results suggest that the needle mycobiome is mainly driven by the host genotype in combination with the composition of the local air spore communities. Our work highlights the role of host intraspecific variation in shaping the mycobiome of trees and provides new insights on the ecological processes structuring fungal communities inhabiting woody plants. We hypothesized that (i) the needle mycobiome is genetically determined and that (ii) the effect of the host genotype on the needle mycobiome depends on the composition of the local air mycobiome.The aim of this study was to understand the ecological mechanisms contributing to the assembly of the tree mycobiome. For this, we determined the relative effect of host genotype, site, host phenotypic traits, and composition of the air mycobiome on the assembly of the needle mycobiome of Norway spruce trees  that belong to the Southern Swedish Norway spruce breeding programme  and tested its association with the needle mycobiome. The composition of both the needle Table  and air P = 0.66) in contrast to when all clones were considered, suggesting that differences in tree diameter at breast height (dbh) across sites was one factor underlying the interaction between clone and site. The same approach could not be taken in the North–South pair of sites because the tree diameters were not overlapping. In the subset of five clones with similar diameter across the East–West sites, both the clone (P = 0.008) and the site (P = 0.001) had a significant effect on the composition of the mycobiome. Moreover, the composition of the mycobiome was associated with the genetic similarity between the clones, i.e. clones that with higher genetic similarity also displayed more similar mycobiome communities, compared with clones with lower genetic similarity  and in the East–West (P = 0.037).We found an interactive effect between clone and site on the composition of the needle mycobiome and therefore the effect of the clone could not be interpreted alone. We explored whether differences in crown health or diameter were involved in the interaction by selecting sets of clones with the same diameter or crown health between sites and testing if the interaction ‘clone × site’ was still significant after controlling for phenotypic variation. When we selected the five clones with the same diameter in the East–West pair of sites, the interaction ‘clone × site’ was no longer significant  and location of the tree on the alpha and beta diversity of the needle mycobiome within each of the four sites. For both alpha and beta diversity, the marginal amount of variation explained by the host genotype within each individual site was consistently higher than the variation explained by phenotypic traits, which was always close to zero in most sites and indexes . RegardiThe aim of this study was to understand the ecological mechanisms contributing to the assembly of the needle mycobiome of Norway spruce. We showed that host genotype and site were the main factors shaping the composition of the needle mycobiome. The host genotypic effect was associated with the genetic similarity between the Norway spruce clones. The site effect was related to differences in the composition of local air mycobiome. Moreover, the effect of the host genotype on the composition of the mycobiome was site dependent, and it was mainly driven by differences of tree size for the same clone across sites. Altogether, our results suggest that Norway spruce trees recruit their needle mycobiome based on factors comprised of their genotype, the available air mycobiome and the tree size.et al., et al., et al., et al., The composition of the needle mycobiome differed between Norway spruce clones for both pairs of sites. This host genotype effect was consistently larger than that of the site, and that of the measured phenotypic traits, such as dbh and crown health. We found that the crown health did not influence the diversity of the needle mycobiome. This result is in line with a previous work showing that trees with contrasting physiological state as a result of the infection of a plant pathogen had similar needle mycobiome . The effect of the genotype reported here is similar to that reported for the phyllosphere microbiome of some perennial  and when separating the OTUs at different trophic modes and dominant guilds. In both analyses, the amount of variation explained by the genotype was between 8% and 10% for both pairs of sites  and gITS (primers spore traps) is that gITS have one more base pair degeneration and amplify some species that fITS do not, e.g. fungi belonging to the genera et al.  breeding programme  were established in different years with grafted clonal material. Söregärde was planted in 2009, Runesten and Larslund between 2011 and 2012, and Gåtebo in 2012. The establishment of the sites was also done in a pairwise design  shared between the two sites in the North–South pair (Larslund and Söregärde), and another 15 Norway spruce clones shared in the East–West pair of sites (Gåtebo and Runesten). From each of these clones, we sampled 10 vegetatively propagated Rosvall,  trees ‘. The Noret al., et al., et al., et al.  based on an identity‐by‐state matrix (IBS) using PLINK 1.9 , previously soaked in 4× TE buffer . Sequences with an average quality score < 10, base average quality < 2, lacking the primer sites  or containing false combinations of used primer tags, i.e. tag jumps  using a threshold value of 0.5 (plausible classification)  was tested on the alpha and beta diversity of the needle mycobiome across sites. The analyses were done separately for the two pairs of sites. To control for the fact that trees had different sizes between sites, as a result of being planted at different years, the diameter values were standardized for each clone within sites prior to the analyses. For all diversity analyses a non‐rarefied OTU table was used, and the square root of the total number of reads per sample as the first factor in all models was included. By doing this, we explicitly accounted for the potential bias resulting from different sequencing depth instead of rarefying , i.e. effect size, following the formula ω2 = (SSQeffect − dfeffect × MSerror)/ found on http://onlinestatbook.com, where SSQ is the sum of squares, df is the degrees of freedom and MS is the mean squares. The ω2 values were grouped by the effect size by trophic modes and most dominant guilds. To account for the fact that some OTUs belonged to more than one guild, the guilds and trophic modes were converted to dummy variables and assigned as 1 (presence) or 0 (absence) values of each guild and trophic mode to each OTU  of the East–West pair, the effect of ‘clone × site’ interaction was no longer significant. Only on those five clones in East–West pair of sites were further tested whether their genetic similarity was associated with the composition of their mycobiome. For this, the scores of each clone on the first and second axis of the PCA displaying their genetic similarity were obtained and fitted on the ordinations of the needle mycobiome using the envfit function in the vegan package.To test whether differences in crown health or dbh were causing the interaction clone × site, a set of clones with the same diameter or crown health status between sites were selected and tested if the interaction clone × site was still significant. We found that when considering clones with the same diameter between sites (www.smhi.se). These data were included as covariables in a global analysis of the composition of the needle mycobiome across all four sites. PERMANOVA analysis was performed for climate and weather separately.To test to which extent differences in climate and weather across sites influenced the composition of the needle mycobiome, we collected values of mean yearly air temperature and total yearly precipitation of the last 20 years  and also of the year before the sampling  from the closest active climatic station to each of the sites  using the variable ‘origin’, i.e., either needles or spore traps, as explanatory. This analysis was performed using both Bray–Curtis and Jaccard index.The air mycobiome was subdivided by month for May, June and July to identify which month had the highest similarity to the needle mycobiome. A principal coordinate analysis was performed for each site including needle samples and spore trap samples. The similarity between needle and spore trap communities was obtained for each month and site based on the et al., et al., Within each site, the contribution of host genotype, phenotype and relative location of the tree in the site  was tested based on alpha and beta diversity. The spatial descriptors, i.e. the distance‐based Moran eigenvector maps (dbMEM), were obtained using the adespatial package in the R environment (Dray Appendix S1: Supplementary InformationClick here for additional data file.Appendix S2: Table S2Click here for additional data file.


Lactobacilli sp. cell counts were higher than log 6 CFU/g at the first week of storage. In the case of sheep’s ice creams these values were maintained or increased until the 30th day, but decreased until the 60th day. Lactococci sp. counts surpassed log 7 CFU/g in all products, and these values were maintained until the end of storage, except in the case of G-Yoghurt and G-Kefir. Concerning the products containing probiotics, the sum of Lactococci sp. and Lactobacilli sp. counts was of the order log 8–9 CFU/g until the 60th day of storage, indicating that the probiotic characteristics of ice creams were maintained for at least 2 months. All products were well accepted by the consumer panel. Sheep’s SCW ice creams were better rated regarding aroma, taste and texture. However, only the ranking test was able to differentiate preferences among formulations.Second cheese whey (SCW) is the by-product resulting from the manufacture of whey cheeses. In the present work, sheep (S) and goat (G) SCW concentrated by ultrafiltration (UF) were used in the production of ice creams. Concentrated liquid SCW samples with inulin added as a prebiotic were fermented with yoghurt, kefir and probiotic commercial cultures before being frozen in a horizontal frozen yoghurt freezer. The physicochemical, microbiological and sensory properties of the products were evaluated over 120 days of frozen storage. The products presented significant differences regarding these properties, specifically the higher total solids and protein contents of sheep’s ice creams, which were higher compared to their goat ice cream counterparts. Sheep’s ice creams also presented higher hardness and complex viscosity, which increased with storage. These ice creams also presented higher overrun and lower meltdown rates. The color parameters of the ice creams showed significant differences between formulations resulting from storage time. In all cases,  Ricotta , Requesón (Spain) or Requeijão . Particularly in the case of Requesón or Requeijão, the whey results from sheep’s or goat’s cheeses. However, not all the whey resulting from the manufacture of small ruminant’s cheeses is transformed into whey cheeses, with a large proportion being used as animal feed or directly discarded [Cheese whey (CW) is the by-product resulting from the manufacturing of cheese. In some countries, CW is used to produce whey cheeses, such as iscarded ,2. Even iscarded ,5,6,7,8.iscarded ,10,11. Piscarded ,13. Otheiscarded ,15,16. Hiscarded .Fermented dairy products are commonly produced in different parts of the world. Furthermore, the use of specific microbial cultures such as probiotic bacteria offers opportunities to develop innovative foods with extended shelf lives. Frozen dairy products can have properties of both yoghurt and ice cream, and can be the carriers of lactic acid bacteria, prebiotics and probiotics. The production of frozen yoghurts or ice creams with low fat or lactose levels and/or with added prebiotics and probiotics created new opportunities for the development of dairy foods with health-promoting properties. An increasing number of papers describe the formulations and characteristics of such products ,29,30,31Hence, the objective of this research was to manufacture ice creams using goat and sheep liquid SCW concentrates (LSCWCs) produced by UF, and to study their physicochemical, microbiological and sensory properties in order to evaluate the feasibility of the use of this by-product by the dairy industry. No reports were found in the literature regarding the manufacture of ice creams with goat and sheep liquid SCW concentrates.2 and a 10 kDa cutoff, supplied by FipoBiotech, Spain. The process was carried out at 40–45 °C at a transmembrane pressure of 3 bar aiming at a volumetric concentration factor (VCF = Vol. feed/Vol. retentate) of 20, obtaining 25 L of concentrate. The concentrate was pasteurized  and then homogenized at 10 MPa using an APV Rannie™ homogenizer model Blue Top . Pasteurized and homogenized LSCWC were frozen at −25 °C until the moment they were used to produce ice creams.Sheep and goat second cheese whey (SCW), supplied by external dairy companies, was transported to the pilot plant of the Escola Superior Agrária de Coimbra , where it was processed. A total of 500 L of each type of SCW (ewe’s or goat’s) were subjected to ultrafiltration (UF) in a Proquiga Biotech SA pilot plant  equipped with a UF organic membrane (3838 PVDF/polysulfone) with an effective filtration area of 7 mw/v): sucrose (12%); inulin (5%), citric acid (0.4%) and xanthan gum (0.1%). The mix was allowed to mature for 12 h at 4 ± 2 °C. Finally, each mixture was inoculated with one of the following cultures:Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus) at a concentration of 0.005% (w/v);Yoghurt culture: Yoflex™  thermophilic yoghurt starter culture  mesophilic and thermophilic culture  at a concentration of 0.005% (w/v);Kefir culture: ExactBifidobacterium BB-12, Lactobacillus acidophilus LA-5) and Streptococcus thermophilus, at a concentration of 0.005% (w/v). ABT-3TM .A mixture of probiotic cultures: . Afterwards, they were heated to 85 °C and passed through the homogenizer valve again. The concentrates were then cooled to 44 °C and divided into 6 L portions, to which the remaining ingredients were added in the following proportions  at 43 °C, and the pH was monitored until it reached a value of 4.6. Fermentation was stopped by rapid cooling to 20 °C in less than 30 min. Afterwards, the fermented mixtures were placed in the refrigeration chamber at 4 ± 2 °C for 12 h.For each formulation, 6 L batches of ice cream were produced and placed in a horizontal frozen yoghurt freezer for 40 min. Immediately after freezing, the temperature of ice creams was −6 ± 1 °C. After this process, the ice creams were packaged in 500 mL polypropylene boxes and stored in a freezer at −21 ± 1 °C for 120 days. All experiments were performed in triplicate.Dry matter was determined by drying the samples in a Schutzart DIN 40050-IP20 Memmert™ oven  according to the AOAC (1997) procedure for frozen yoghurt . The ashThe pH was determined with a HI 9025 Hanna Instruments pH meter  in order to monitor its evolution over fermentation, immediately after the production of the fermented products and on the 1st, 30th, 60th and 120th days of storage of ice creams. The pH meter was previously calibrated with 7.01 (HI5007) and 4.01 (HI5004) Hanna buffer solutions. The titratable acidity, expressed in g of lactic acid/L, was determined by means of titration using a 0.1 N NaOH solution according to the technique described in NP 701:1982 for yoghurts  and AOACThe color of the ice creams was determined with a Minolta Chroma Meter, model CR-200B colorimeter  calibrated with a white standard . The following conditions were used: illuminant C, 1 cm diameter aperture, 10° standard observer. The color coordinates were measured in the CIEL*a*b* system.0, a*0, and b*0 and L*, a*, and b* were the values measured for the samples under comparison. A matrix of ΔEab* values between products was constructed. Five measurements were taken for each sample.Color difference (ΔEab*) was calculated as:The rheological properties of the different samples were determined in a Rheostress 1 rheometer  in oscillatory mode. The measurement system consisted of a cone and plate geometry, C60/Ti-0.052 mm (35 mm diameter and 1° angle). Stress sweep tests were conducted at 1 Hz to deter-mine the linear viscoelastic range of the yoghurts.The complex viscosity (η*) of the products was evaluated in the range of 0.3 to 6.5 rad/s at 3 Pa. Three measurements were taken for each sample.A Stable Micro Systems texture analyzer, model TA.XT Express Enhanced , was used to evaluate the hardness of the frozen samples one day after production, and the results were calculated using Specific Expression PC software.Overrun is the increase in the volume of the ice cream after freezing due to the incorporation of air. The method described by Skryplonek et al.  was folln = 3). The meltdown rate was expressed as the percentage of the melted ice cream weight divided by the initial ice cream weight. The determinations were done in triplicate.The meltdown rate was determined by the method described in Skryplonek et al.  by placiLactobacillus sp. and Lactococci sp. were determined after production and during the storage of frozen samples . Lactobacilli sp. and Lactococci sp. were enumerated on plates at 37 °C for 48 h on M17 agar (in aerobiosis) and on MRS agar (in anaerobiosis) , respectively, according to ISO 7889, IDF 117 (2003) [−5, 10−6 and 10−7 were inoculated in triplicate along with two controls for each medium.The microbial counts of lactic acid bacteria (LAB) of the genera 7 (2003) . In thisConsumer preference tests were conducted with an untrained panel at the 7th, 30th, 60th and 120th days of storage. The hedonic test was used to determine the degree of acceptability of the products . A categThe six formulations were presented to panelists, who were asked to evaluate the characteristics of aroma, taste, color and texture using a hedonic scale of 1 to 5. Thirty-one consumers participated in the panel. The members of the panel were also asked to rank the samples according to their preference, from 1 (most preferred) to 6 (less preferred) .p < 0.05 was used .Prior to statistical analysis, normal distribution was tested using the Kolmogorov–Smirnov test. The one way ANOVA and the Tukey’s post hoc test were used to test and compare, the statistical significance of differences among means, respectively. Results from the sensory ranking test were evaluated by the chi square test. For all mean evaluations, a significance level of The compositions of the ice creams are shown in The hardness results of sheep’s and goat’s SCW ice creams produced with different starter cultures are presented in The overrun values  are highFew works refer to the use of dairy by-products in the manufacture of ice cream. De Meneses et al.  evaluateLactobacilli sp. cell counts were higher than log 6 CFU/g at the first week of storage . Açu, et al. [Lactobacillus acidophilus), and synbiotic (L. acidophilus and inulin) ice creams and concluded that L. acidophilus counts were higher in synbiotic ice cream. Synbiotic ice cream received the highest overall acceptance scores. Sabet-Sarvestani et al. [L. acidophilus LA-5, Lacticaseibacillus paracasei L-26, and Lacticaseibacillus casei 431, respectively.Other manuscripts reported high probiotic counts in ice creams. Senaka Ranadheera et al.  observed, et al.  also obs, et al.  manufact, et al.  evaluate, et al.  evaluatei et al.  producedi et al.  observedi et al.  also obsWith regard to sensory quality, all products produced in the present work were well accepted by the consumer’s panel up to the 120th day of storage . Only thThe production of sheep’s or goat’s ice creams can represent an interesting opportunity for the valorization of SCW since it is infrequently reused. Whey and second cheese whey usually represent a problem to small and medium scale dairy processing units. These plants do not have adequate equipment to process SCW  and usually these by-products are offered for livestock feeding or are discarded into the public sewage system. The use of ultrafiltration, or even nanofiltration, to concentrate SCW originates liquid second cheese whey concentrates (LSCWCs) which can be directly used in the development of novel dairy products. This solution allows for the reduction in negative environmental effects resulting from the direct discharge of SCW. Furthermore, the addition of prebiotics and the production of fermented products containing probiotic bacteria increases the nutritional value of LSCWC’s and their market potential. The results of the present work have practical applications in small and medium scale dairy plants by reducing their environmental impact and improving their profitability via value-added products, which is crucial for sustainable rural development.


X-Ray Tomography (XRT) was used to visualize the microstructure of extruded meat replacers. The high moisture extrudates contained lamella, that became visible upon pulling the extrudate apart. In frozen state, these lamella could be visualized with XRT. The freezing increased the density difference between the water-rich and protein-rich layers, thus increasing the contrast obtained in the XRT. Differences in the physical structure were reflected in the measured structure. In non-frozen samples, no lamella were visible, indicating insufficient contrast.Because of the contrast obtained in frozen samples, we conclude that the XRT technique is a valuable addition to investigate extrudate structure, that can be used to quantify differences in extrudates obtained by for example variation in composition. Here we showed a higher lamella thickness for soy protein isolate (SPI) compared to more fibre-rich soy protein samples.  •XRT is a promising technique for non-invasive visualisation of high moisture extrudates.•In frozen state, lamella of high moisture extruded meat analogues could be visualized with XRT.•Structures measured in frozen state with XRT were comparable with lamella observed visually. It is tA key factor to better understand structure formation is the availability of good analytical techniques that can visualize resulting structure formed during the extrusion process. It enables the link between processing parameters and the resulting quality. However, visualisation of these structures is a challenge . An opti3 (3) is also sufficient to get an image of ice (X-ray tomography (XRT) is a non-invasive visualisation technique for which no staining or microtome slicing is required. XRT works based on density differences in the sample. It has been used in many applications, ranging from histology to soil research . The tec3 ,) and ore of ice . The maxe of ice .3  and soy protein concentrate  were obtained from Solae LLC . Insoluble soy fibre  was obtained from Vitablend and the soluble soy fibre  was supplied by Fuji Oil Co, Ltd., Japan.Soluble and insoluble fibre content of the soy protein concentrate, the Unifiber and the SoyaFibe S-CA100 were measured by Eurofins, using a modification of the protocol AOAC2011.25. The results of the fibre content is shown in 2.2The extrusion experiments were performed on a co-rotating twin-screw extruder, TwinLab-F 20/40 D , with a L/D ratio of 40 and a screw diameter of 20 mm. A 300 mm segmented cooling die was attached, consisting of three 100 mm cooling die pieces with a 25 × 7 mm geometry. The mounted screw is depicted in Solids were dosed via a gravimetric feeder  at the inlet located at 0D. The water was dosed via a peristaltic pump  at the inlet located at 10D.In these experiments the extrusion settings were kept constant. The screw speed was set at 800 rpm and the feed was set at 4.5 kg/h. The ratio of solid and liquid feed was adjusted on the determined moisture content of the blend to achieve an overall moisture content of 55% (w/w). The heating zones were set at 40 °C; 80 °C; 110 °C and 130 °C. The temperature of the cooling die was set at 50 °C. The extrudates were cut on a roller band and further analyzed by XRT.2.3A GE Phoenix v|tome|x m tomographer  was used for 3D non-invasive and non-destructive imaging Unless mentioned otherwise, a frozen sample was placed in a styrofoam shell and cooled using an air stream of −20 °C air to prevent the sample from defrosting. The system contains two X-ray sources. The 240 kV micro focus tube with tungsten target was employed. X-rays were produced with a voltage of 120 kV and a current of 120 μA. The measurements were done once for each sample composition.The images were recorded by a GE Dynamic 41|200 detector array with 2024 × 2024 pixels (pixel size 200 μm). The detector is located 815 mm from the X-ray source. The object was placed 78.1 mm from the X-ray source. This result in a spatial resolution of 20 μm. A full scan consist of 1500 projections over 360°. The 1st image was skipped. The saved projection is the average of 3 images where every image is obtained over 83 ms exposure time.GE reconstruction software  was used to calculate the 3D structure via back projection.Next to the seven extrudates, an extrudate with an added layer of pure ice was measured, to compare the grey value of the water-rich layer to that of pure water. The added ice showed the same grey value as the water-rich layers in the extrudate.2.4The 3D images, obtained using the v|tome|x XRT and the GE reconstruction software, were analyzed using Avizo imaging software Avizo3D 2021.2. A thickness map routine was performed to get a measure for lamella thickness. The thickness map is defined as the largest diameter that fit around the voxel in the selected volume. It represents the local size of the structure.3Seven samples, based on soy protein, were created with high moisture extrusion. Soy protein isolate (SPI) was mixed with soluble and insoluble soy fibre (respectively SF and IF), and compared with SPC and with SPC with added insoluble fibre. The fibre content ranged between 0% fibres based on dry matter (DM) (SPI), and 37% DM (soy protein concentrate (SPC) with extra added insoluble fibre (IF)), see Extruded samples were cut in a length wise direction, which revealed a homogeneous structure. Still, when pulled apart in the direction of the extrusion, a parabolic wedge formed . After fDespite the wedge being visible upon pulling the extrudate apart, in an XRT scan, a fresh sample showed a homogeneous density distribution of a structure without bubbles or density differences at the image resolution (20 μm) c. After The structure seen in the frozen sample was explained by the existence of multiple phases: ice and one or multiple biopolymer phases. The ice phase is consisting of pure water, as shown by the additional measurement of pure water that showed the same grey value as the water-rich phase. In this study, no distinction was made between the different polymer phases, and for ease of reference they are depicted as ‘the protein-rich phase’.After defrosting, the samples were pulled apart to show the wedge structure . These pTo quantify the structure, first the protein-rich, denser, lighter grey value part of the sample was selected, to estimate the size of the lamella. Subsequently, the water-rich, less dense and thus darker part of the image was selected, and the lamella size was studied as well. This yields a thickness distribution of the protein-rich and water-rich lamella. In 4Fresh samples show a homogeneous microstructure in the XRT image. Upon freezing, a layered microstructure becomes visible that disappears again upon defrosting. The frozen wedge structures observed with XRT qualitatively match the wedge structures obtained after pulling the defrosted extrudates apart. This reversible visibility of layers can be explained by the ice crystal formation during the freezing process. When the sample is cooled down, ice is formed first in the water-rich layers, and in the freezing process the ice crystallisation extracts water from the protein-rich phase. The ice phase consists of pure water, and solutes (including proteins) are excluded. Consequently, the protein-rich layers get more concentrated. The larger density difference created by water freezing that allows for visualisation in XRT stems both from the lower density of ice compared to water and from the difference in proteins and solutes in the protein-rich phase. The factors that are important in the formation of layers and that govern their thickness and the distribution over the phases are not fully elucidated. A number of hypothesis on the roles of different ingredients and the processes on formation of a fibrous phase are described in the recent review by 5The use of XRT is a promising tool to better understand the structure formed during extrusion. Here, we demonstrated that samples in frozen state can reveal differences in microstructure and XRT measurements allow quantification of the wedge length and lamella thickness. Samples have to be frozen to create contrast between low density ice crystals and a high density protein phase. In non-frozen samples, we did not find sufficient contrast. The wedge length found via XRT corresponded well with wedges observed when pulling apart non-frozen samples.The wedge structure formed upon extrusion depends on processing conditions and ingredient choice. In this article a number of ingredients were varied, and it was shown that with XRT differences in the structure could be quantified. Further research is necessary to understand the structural changes better.Maaike Nieuwland: Software, Validation, Formal analysis, Investigation, Writing – original draft, Writing – review & editing, Visualization. Walter Heijnis: Conceptualization, Investigation, Writing – review & editing. Atze-Jan van der Goot: Writing – review & editing. Remco Hamoen: Methodology, Software, Validation, Writing – review & editing, Visualization.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Fecal calprotectin in the non-IgE food allergy group was significantly higher compared to the control group (p < 0.05). Conclusion: This study provides a theoretical basis that Th2 cytokine expression in infants with a non-IgE mediated food allergy is significantly different than in healthy infants; this finding supports the use of early dietetic treatment with hydrolyzed formulas.Background: The aim of this study is to assess the serum values of IL-4, IL-5, IL-10, and IL-13 in a group of infants with non-IgE mediated food allergies treated with a hydrolyzed formula and compare them with a group of healthy peers. Methods: A total of 53 infants aged 1 to 4 months, of which 34 with non-IgE mediated food allergies and 19 healthy infants were enrolled in this study. Infants were eligible if they had gastrointestinal symptoms of food allergy and needed to switch from their initial formula to hydrolyzed formulas with an improvement of symptoms. Controls were fed with either breastmilk or standard formula. Blood samples were taken within one week of a special diet for cases. Interleukinsin in peripheral blood was detected and analyzed using the real-time PCR MAMA method. Fecal calprotectin was evaluated using a quantitative assay. Results: Values of IL-4 and IL-13 were significantly higher in the non-IgE food allergy group compared to the control group ( Non-IgE-mediated food allergies are immunologic reactions to food that present without serum-specific IgE antibodies and may occur in early life .The new guidelines of WHO for cow’s milk allergy diagnosis and treatment include the entity of non-IgE mediated food allergy in the most recent update, thus recognizing the relevance of this condition both in adults and particularly in children .From the immunological point of view, in the most frequent forms of CMPA, an abnormal proliferation of the Th2 subclass of T-lymphocytes occur and determines the production of lymphokines—such as IL-4—which induce an abnormal production of IgE by B-lymphocytes and amplify the inflammatory response and the production of Th2-associated cytokines , involved in atopic reactions [Atopy, in general, is a predisposition to respond immunologically to allergens leading to Th2 differentiation and induction of immunoregulatory mechanisms that occur via some cytokines production, such as IL-4, IL-5, IL-10, and IL-13 [Interleukins are small signaling proteins that have central roles in inflammation in subjects with cow’s milk allergy and may lead to a deeper knowledge of the pathogenesis of food allergy .In fact, despite advances in research, the pathophysiology of non-IgE-mediated food allergy has not been fully elucidated yet and it seems that the complex pathways leading to Th2 immune responses against food antigens cannot be attributed to a single driving force .While the immediate symptoms of immunoglobulin E (IgE) mediated CMA are readily recognized, the diagnosis of non-IgE mediated CMA can pose a challenge due to the delayed onset of symptoms and overlap with other common pediatric manifestations of functional gastrointestinal disorders such as infantile colic, gastroesophageal reflux (disease) and even infections, particularly in the first months of life ,2,6.Non-IgE-mediated gastrointestinal food allergies are a heterogeneous group of food allergies in which there is an immune reaction against food, but the primary pathogenesis is not a production of IgE and activation of mast cells: therefore, the pathway of interleukins response needs to be clarified .The major reason for the limited understanding of non-IgE food allergy is a delayed and less severe reaction to food ingestion and the fact that patients’ symptoms often improve with empiric food avoidance. Therefore, endoscopy and biopsy are not performed .As a consequence, if clinical symptoms indicate a non-IgE-mediated CMA, it is recommended to propose a diet with the elimination of cow’s milk proteins and subsequent reintroduction: extensively hydrolyzed formula (eHF) whey or casein-based are considered the first-line management of formula-fed infants with CMPA and suggested as therapy ,7,8,9.Subjects with non-IgE-mediated food allergy were proposed to be fed with an extensively hydrolyzed formula, leading to improvement of symptoms compared to what happens in atopic infants; however, laboratory data on their cytokines response are scanty .The importance to feed these subjects a diet similar to that of atopic has recently been suggested in the literature, observing an overall clinical improvement that is still not supported by laboratory evidence .The aim of this study was to evaluate the Th2 cytokine response  in a group of infants with non-IgE-mediated food allergic symptoms and compare it to the response in healthy peer control.Fecal calprotectin, which is a marker of gut inflammation whose use is emerging also in gastrointestinal allergic disorders ,11, has We screened for enrollment of all infants who were admitted to the Outpatient Clinic of the Special Care Unit of the Department of Pediatrics of the University of Turin, Regina Margherita Children’s Hospital, between October 2018 and October 2020. After discharge, the infants underwent blood tests during routine outpatient examinations. The study protocol was approved by the local Ethical Committee at Ospedale Mauriziano—Ospedale Infantile Regina Margherita—S. Anna Torino and written consent was obtained from infants’ parents.Group 1: patients with mild to moderate non-IgE-mediated CMA. Patients within 4 months of life, fed with adapted formula milk, with gastrointestinal  and/or cutaneous symptoms , and negative serum IgE to cow milk.Group 2: healthy infants of comparable age and not following any special diet, enrolled during outpatient routine health checks.Criteria for enrollment were as follows:We considered eligible for the study patients with mild to moderate non-IgE- mediated CMA that needed to switch from their initial formula to special formulas (extensively casein hydrolyzed formulas or amino acid-based formulas) with an improvement of symptoms. Controls were fed with either breastmilk or standard formula. In the group of cases, blood samples were taken within one week from the special diet introduction. Venous blood sampling was taken from 7.30 to 8.30 a.m. during routine clinical.Food-specific IgE for casein was determined by processing patients’ serums from venous blood samples obtained at the time of enrollment with the automated Pharmacia CAP system RAST , and the cut-off point for positivity was set at 0.35 kUA/L.Feces samples (about 5–10 g) were collected from each infant directly from the diaper and instantly analyzed for calprotectin values.® Calprotectin High Range . The analysis mechanism is based on a sandwich immunoassay with a measurement range between 100 and 1800 µg/g; it is for a quantitative assessment of fecal calprotectin value.For the determination of fecal calprotectin value, a rapid test was employed, the BÜHLMANN Quantum BlueThe automated extractor Maxwell  following the RNA Blood Kit protocol without modification was used to extract total RNA from whole blood samples. This equipment works with treatment with DNase during the RNA extraction process. Traditional Ultraviolet (UV) spectroscopy with absorbance at 260 and 280 nm was used to determine both purity and concentration of RNA. The Beer-Lambert law, which can describe a linear change in absorbance with concentration, allowed us to calculate the nucleic acid concentration. The RNA concentration range was in accordance with the manufacturer terms for the NanoDrop . UV absorbance measurements were acquired using 1 µL of RNA sample in an ND-1000 spectrophotometer under the RNA-40 settings at room temperature (RT). Applying this equation, an A260 reading of 1.0 is equivalent to ~40 µg/mL of single-stranded RNA. RNA purity was defined as the A260/A280 ratio, considering an A260/A280 ratio of 1.8/2.1 indicative of an RNA highly purified. RNA extracts were directly amplified without reverse transcription to verify the genomic DNA contamination. The RNAs were put in storage at −80° until testing.An amount of 400 nanograms of total RNA was reverse-transcribed with 2 μL of buffer 10×, 4.8 μL of MgCl2 25 mM, 2 μL ImpromII (Promega), 1 μL of RNase inhibitor 20U/l, 0.4 μL random hexamers 250 μM (Promega), 2 μL mix dNTPs 100 mM (Promega), and dd-water to obtain a final volume of 20 μL. The reaction mix was analyzed in a GeneAmp PCR system 9700 Thermal Cycle  under the following conditions: 5 min at 25 °C, 60 min at 42 °C, and 15 min at 70 °C for the inactivation of the enzyme. The cDNAs were kept at −20° until testing.We chose Glyceraldhyde triphosphate dehydrogenase (GAPDH) as the reference gene in all determinations, because it is known to be the most stable among reference genes and has already been validated in our previous study . The ABIWe assessed the expression values of IFN-1, IFN-2, and IFN-3 using 40 ng of cDNA in a 20 μL of total volume reaction containing 2.5 U goTaQ MaterMix (Promega), 1.25 mmol/L MgCl2, 500 nmol of specific primers and 200 nmol of specific probes as reported in ®. The quantitative variables were described in terms of medians (Me) and variation ranges. The qualitative variables were described through absolute frequencies and percentages. Depending on their distribution, comparisons of quantitative data samples were analyzed with Fisher’s test or the Mann-Whitney test. All tests were two-tailed and considered significant at values of p < 0.05.The data were analyzed with GraphPad Prism 7.01p < 0.05 was taken to be statistically significant.Mann-Whitney test was used to compare the transcriptional levels of each IL between each group of children with each other. Statistical analyses were done using the Prism software . In all analyses, We enrolled a total of 53 subjects. Of these, 34 infants had symptoms of non-IgE-mediated food allergy and 19 were healthy controls.IgE determinations in order to identify sensitization were performed and were found to be negative for both groups of infants.We defined “non-IgE-mediated food allergic patients” as those who had gastrointestinal symptoms , and/or cutaneous (eczema) and negative serum IgE to casein and whey protein and were then treated with hydrolyzed cow’s milk formula .The median postnatal age at enrollment was 10 weeks in patients with mild to moderate non- IgE-mediated CMA and 8 weeks in healthy controls. In the group with mild to moderate non-IgE-mediated CMA 18 (53%) subjects were male, in the healthy group 9 (47.4%).The median birth weight was 3180 g in the patients with mild to moderate non-IgE-mediated CMA and 3070 g in the healthy subjects. The median gestational age was 36 weeks in the group with mild to moderate non-IgE-mediated CMA and 37 in healthy infants, making the two groups reliably comparable.The patients with mild to moderate non-IgE-mediated CMA25 were Italian (73%), and 14 (74%) were in the healthy group.Feeding type: 29 patients (85%) with mild to moderate non-IgE-mediated CMA were fed with hydrolyzed casein formula and 5 (15%) with amino acid-based formula. Twelve healthy infants were exclusively breastfed (63%) and 7 formula-fed (37%).As detailed in p = 0.006; the IL5 values were lower: 0.70 +/− 0.48 inpatients with mild to moderate non-IgE-mediated CMA vs. 1.11 +/− 0.99 in healthy controls p = 0.0294; the IL10 values were lower: 0.73 +/− 1.12 in patients with mild to moderate non-IgE-mediated CMA atopic patients vs. 1.05 +/− 1.54 in healthy control, p = 0.0198, and the values of IL13 were higher1.95+/− 0.77 in patients with mild to moderate non-IgE-mediated CMA vs. 1.01+/− 1.2 in healthy controls, p = 0.0113.In particular, the IL4 values (mean +/− SD) were higher: 1.33 +/− 0.97 in patients with mild to moderate non-IgE-mediated CMA topic patients vs. 0.80 +/− 1.11 in healthy controls, We investigated the expression of some biomarkers of Th2 inflammation cytokines in infants with mild to moderate non-IgE-mediated CMA that needed to be fed with a hydrolyzed casein formula and showed also increased faecal calprotectin values.In newborns, gastrointestinal symptoms are very common and suggest the presence of disorders such as food intolerance, infections, metabolic disorders, congenital disorders of the gastrointestinal tract, and, more rarely, allergies ,13.In pediatric clinical practice, it occurs frequently to assess infants who develop symptoms suggestive of allergy after being introduced to standard cow’s milk formula. If these symptoms appear after the intake of cow’s formula milk, food intolerance or allergy to cow’s milk proteins (CMPA) must be suspected and maybe IgE or non-IgE-mediated .This clinical presentation is empirically treated by eliminating cow’s milk protein from the diet and special formula alternatives include hydrolyzed cow’s milk formula and amino acid-based formula that are all nutritionally adequate replacements of cow’s milk formula ,8,9,14.Nevertheless, the non-IgE-mediated CMA presents in the first period of life with a variety of symptoms, and in newborns, the most frequent at onset are the gastrointestinal ones: vomiting, diarrhea, constipation, severe colic, abdominal distension, and hematochezia. However, these manifestations are not specific to making the differential diagnosis, delaying the identification of these infants ,13.Moreover, in the field of neonatal medicine, an oral provocation  challenge test is not feasible because it can induce severe reactions and there are also no standardized diagnostic criteria. In common clinical practice, the specific IgE dosage and the execution of the Prik test in the newborn are also rarely performed, due to the poor reliability of the first examination and difficulty in carrying out the second, since newborns have intrinsic skin reactivity linked to age .Thus, it follows that the diagnostic process described by the DRACMA guidelines   is diffiInterestingly, in our study in the group of patients with mild to moderate non-IgE-mediated CMA when compared to peer healthy controls, increased mean values of IL-4 and IL-13 and decreased mean values of IL-5 and IL-10 were found.The Th2 polarization is characterized by IL-4/IL-13 dependent pathway activation in atopic eczema, allergic asthma, and food allergies ,16 and cInterleukin-5 (IL-5) is predominantly regarded as a Th2–cytokine, which contributes to an allergic response by activating eosinophils . Our finRegulatory T cells are able to secret IL-10, a cytokine with inhibitory properties; in fact, it inhibits Th1 and Th2 cells’ activity as well as the function of the innate immune system. When compared with adult T cells, the producing capacity and receptor expression of neonatal T cells is known to be reduced . TherefoWe have to consider that little is known about the immunologic maturation and cytokines pathways in the early stages of life in atopic subjects ,17 and nActivated cytokines, including also TNF-α, IFN-γ, IL-5, IL-8, and IL-10, play an important role in allergic disorders and may also affect immunologic development because cytokines levels change remarkably during the first year of life and are also influenced by some birth-related factors ,18.Our results showed significantly lower values of IL-5 and IL-10 in non-IgE-mediated food allergy subjects treated with an hydrolyzed milk formula: in this field, it is interesting to notice that it has been recently reported in an animal model that a probiotic may be useful to enhance the intestinal barrier and to reduce inflammation increasing IL-10 values .On the other hand, recent studies found that fecal markers of intestinal inflammation, such as fecal calprotectin, are possibly markers of intestinal barrier permeability, intestinal inflammation, and barrier disfunction; in fact, this is confirmed in our study, as infants with non-IgE-mediated food allergy also showed increased values of fecal calprotectin compared to those of healthy subjects .This is the first study that aims to detect the expression of Th2 cytokines  in infants with non-IgE food allergy and to compare it to peer healthy subjects. Interestingly we found that there were higher values of expression of IL-4 and IL-13, whereas values of IL-5 and IL-10 were lower. The study has some limitations. The most important one is the relatively small sample size in the non-IgE-mediated food allergy group, which may make it difficult to determine if our findings are conclusive. However, our study could lead to further investigations with larger cohorts of infants.Secondly, we did not investigate the expression of Th2 cytokines  before switching the formula to a hydrolyzed milk formula.Considering the very young age of the enrolled infants, blood sampling should be limited as much as possible: however, in order to confirm the observed data in peripheral blood, further longitudinal studies should be specifically designed to assess cytokines expression over time before and after switching the formula.Our data revealed that values of IL-4 and IL-13 are higher in infants with non-IgE-mediated food allergies, whereas IL-5 and IL-10 are lower, as observed in IgE-mediated food allergies . The praNevertheless, our cytokines findings showed that an inflammatory response is still present after a few days from a special diet introduction.This opens the way for future research with the aim of quantifying the cytokines response after a prolonged diet and better characterizing the causes of the onset of allergic symptoms in non-IgE-mediated food allergies in order to find some specific factors that could improve these conditions .


Cytoplasmic mislocalization and aggregation of TAR DNA-binding protein-43 (TDP-43) is a hallmark of the amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) disease spectrum, causing both nuclear loss-of-function and cytoplasmic toxic gain-of-function phenotypes. While TDP-43 proteinopathy has been associated with defects in nucleocytoplasmic transport, this process is still poorly understood. Here we study the role of karyopherin-β1 (KPNB1) and other nuclear import receptors in regulating TDP-43 pathology.We used immunostaining, immunoprecipitation, biochemical and toxicity assays in cell lines, primary neuron and organotypic mouse brain slice cultures, to determine the impact of KPNB1 on the solubility, localization, and toxicity of pathological TDP-43 constructs. Postmortem patient brain and spinal cord tissue was stained to assess KPNB1 colocalization with TDP-43 inclusions. Turbidity assays were employed to study the dissolution and prevention of aggregation of recombinant TDP-43 fibrils in vitro. Fly models of TDP-43 proteinopathy were used to determine the effect of KPNB1 on their neurodegenerative phenotype in vivo.Drosophila models of TDP-43 proteinopathy. Our results show that KPNB1 can rescue all hallmarks of TDP-43 pathology, by restoring its solubility and nuclear localization, and reducing neurodegeneration in cellular and animal models of ALS/FTD.We discovered that several members of the nuclear import receptor protein family can reduce the formation of pathological TDP-43 aggregates. Using KPNB1 as a model, we found that its activity depends on the prion-like C-terminal region of TDP-43, which mediates the co-aggregation with phenylalanine and glycine-rich nucleoporins (FG-Nups) such as Nup62. KPNB1 is recruited into these co-aggregates where it acts as a molecular chaperone that reverses aberrant phase transition of Nup62 and TDP-43. These findings are supported by the discovery that Nup62 and KPNB1 are also sequestered into pathological TDP-43 aggregates in ALS/FTD postmortem CNS tissue, and by the identification of the fly ortholog of KPNB1 as a strong protective modifier in Our findings suggest a novel NLS-independent mechanism where, analogous to its canonical role in dissolving the diffusion barrier formed by FG-Nups in the nuclear pore, KPNB1 is recruited into TDP-43/FG-Nup co-aggregates present in TDP-43 proteinopathies and therapeutically reverses their deleterious phase transition and mislocalization, mitigating neurodegeneration.The online version contains supplementary material available at 10.1186/s13024-022-00585-1. STMN2 (stathmin-2)  was inserted between fluorescent fusion proteins to ensure proper protein folding.The expression plasmids used in this study were obtained from multiple sources or generated as summarized in Supplementary Table HEK293T cells were cultured in DMEM media (Life Technologies) containing 10% FBS. Human SH-SY5Y neuroblastoma cells were cultured in DMEM/F12 media (Life Technologies) containing 10% FBS. Cells were transfected with NeuroMag Neo (OZ Biosciences) according to the manufacturer’s protocol. HEK293T and SH-SY5Y cells were purchased from ATCC . HEK293T cells were selected for certain experiments due to their robust protein aggregation and resistance to TDP-43 toxicity compared to SH-SY5Y neuroblastoma cells and primary neurons.100, cells were fixed 48 h after transfection. Cells were permeabilized with 0.2% Triton X-100 in PBS for 10 min, blocked with 5% bovine serum albumin (BSA) for 45 min and incubated with the following primary antibodies: anti-FLAG (1:500), anti-TDP-43 (1:500), anti-phospho-TDP-43S409/410 (1:2000), anti-Nup62 (1:250), anti-Nup98 (1:250), anti-Ran (1:200) and anti-KPNB1 (1:100) overnight at 4 °C, followed by incubation with fluorophore-coupled secondary antibodies for 1 h at RT.Cells were plated on coverslips in 12-well plates. Twenty-four hours after transfection, cells were fixed with 4% paraformaldehyde (PFA) in PBS for 15 min at room temperature (RT). For experiments with GFP-(GR)For high-resolution fluorescence microscopy, single and multiple z-plane images were acquired using an epifluorescence microscope (Nikon Eclipse Ti2) equipped with a Zyla camera . Within each experiment, all groups were imaged with the same acquisition settings. Image stacks were deconvolved using a 3D blind constrained iterative algorithm .To quantitate the spatial overlap between Nup62-mCherry and GFP-tagged TDP-43 constructs, Mander’s colocalization coefficient was calculated using the JACoP plugin on ImageJ . Values As a reporter for nucleocytoplasmic transport of proteins, NES-tdTomato-NLS was co-transfected with GFP or GFP-tagged KPNB1 constructs in SH-SY5Y cells. The mean pixel intensity of NES-tdTomato-NLS in the nucleus and cytoplasm was measured to calculate the nuclear-to-cytoplasmic (N-to-C) ratio.mNLS, HEK293T cells were co-transfected with GFP-TDP-43mNLS and different mCherry-KPNB1 constructs, fixed, DNA-stained with Hoechst and imaged. Z-stacks were acquired as described above. Images were blinded across conditions and TDP-43mNLS fluorescent signal was characterized as nuclear/nucleocytoplasmic/cytoplasmic for at least 20 cells per condition, in triplicate.To semi-quantitatively determine nuclear-cytoplasmic localization of GFP-tagged TDP-43Cells were seeded in 12-well plates. Forty-eight hours after transfection, cells were lysed in RIPA Lysis and Extraction buffer (Thermo Fisher Scientific) supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific) and centrifuged at 15000 rpm for 20 min at 4 °C. The supernatant was collected as the detergent-soluble fraction and the insoluble pellet was recovered in urea buffer . For experiments with total protein lysate, cells were collected in urea buffer. The samples were left at RT for 20 min then briefly sonicated and centrifuged at 15000 rpm for 20 min. The supernatant was collected as the detergent-insoluble fraction. For immunoblotting, the samples were boiled in Laemmli sample buffer for 5 min at 98 °C and separated in Bolt™ 4 to 12% Bis-Tris gels (Thermo Fisher Scientific). Proteins were blotted onto nitrocellulose membranes using an iBlot™ 2 Gel Transfer device (Thermo Fisher Scientific). Membranes were blocked with Odyssey blocking buffer (LI-COR) for 1 h followed by incubation with the following primary antibodies: anti-mCherry (1:1000), anti-GFP (1:2000), anti-β-tubulin (1:1000), anti-β-actin (1:1000) and anti-KPNB1 (1:1000) overnight at 4 °C and incubation with secondary antibodies  in PBS blocking buffer with 0.05% Tween 20 for 1 h at RT. Blots were imaged using an Odyssey scanner (LI-COR) and Image Studio software , quantification of bands intensity was performed using Image Studio Lite .HEK293T cells were seeded in 6-well plates. Forty-eight hours after transfection, cells were lysed in IP Lysis buffer (Thermo Fisher Scientific) supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific) and centrifuged at 15000 rpm for 20 min at 4 °C. A small aliquot of the recovered supernatant was collected as input extract, and the rest was used for IP. Pull-down was performed using GFP- and RFP-Trap magnetic beads (Chromotek) according to the manufacturer’s protocol. Briefly, beads were washed twice in wash buffer . The lysate was then incubated with the equilibrated beads overnight at 4 °C with end-over-end rotation. Beads were magnetically separated from the supernatant (flow-through) and washed 3 times with wash buffer. Bound proteins were released from the beads in an elution buffer (200 mM glycine pH 2.5). Samples were boiled in Laemmli buffer for 10 min at 95 °C and used for immunoblotting. The following primary antibodies were used: anti-mCherry (1:1000), anti-GFP (1:2000), anti-mAb414 (1:1000), anti-Nup62 (1:1000), anti-Nup50 (1:1000), anti-importin-α1 (1:1000) and anti-Ran (1:2000).EGFP  and human GAPDH  on QuantStudio 7 Flex Real-Time PCR system (Applied Biosystems) with QuantStudio Real-Time PCR software . Data was analyzed using the Design and Analysis software .RNA from cultured HEK293T cells was isolated using RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s protocol. Isolated RNA was treated with RQ1 DNase (Promega) and used for the cDNA synthesis with SuperScript First-Strand Synthesis System (Invitrogen) using oligo-dT and random hexamer primers. Real-time RT-PCR was performed using TaqMan Universal PCR Master Mix (Applied Biosystems) and TaqMan gene expression assays for Cell death of SH-SY5Y cells was assessed by the uptake of the membrane-impermeant Live-or-Dye™ Fixable Dead Cell Dye 640/662 . The dye penetrates dying cells that have compromised membrane integrity and labels amines on intracellular proteins. Cells were transfected with mCherry/mCherry-TDP-CTF and GFP/GFP-tagged KPNB1 constructs using FuGene6 (Promega). Forty-eight hours after transfection, cells were incubated with the dye for 30 min at 37 °C protected from light, then fixed in 4% PFA and imaged using an epifluorescence microscope (Nikon Eclipse Ti2). Cell death was assessed by scoring the proportion of dying cells that incorporated the dye.6)-tagged proteins (TDP-CTF and Nup62FG) were purified as described previously [E. coli cells (BL21-CodonPlus(DE3)-RIL, Agilent Technologies) harbouring the expression vector for His6-tagged IDRs were cultured in Luria–Bertani medium. Protein expression was induced by adding 1 mM IPTG, and the cultures were further incubated at 18 °C overnight. Cells were harvested by centrifugation , subjected to two rounds of freeze-thaw cycles and finally dissolved in urea-containing buffer  at 4 °C overnight for 2 days. The lysate was centrifuged  and the supernatant was collected and mixed with Ni-NTA agarose beads (Qiagen). The beads were gently agitated at 4 °C for 1 h, washed with wash buffer , and His6-tagged proteins were eluted with elution buffer . Eluted proteins were sequentially dialyzed at 4 °C in dialysis buffer 1 (0.1% (v/v) trifluoroacetic acid (TFA), 2 mM 2-mercaptoethanol) for 3 h, dialysis buffer 2 (0.05% (v/v) TFA, 2 mM 2-mercaptoethanol) for 3 h or overnight, and dialysis buffer 3 (0.05% (v/v) TFA) for 3 h. The purified proteins were lyophilized  and stored at 4 °C.Hexahistidine (Hiseviously . E. coliE. coli cells (BL21-CodonPlus (DE3)-RIL, Agilent Technologies) harbouring the expression vector for GST-tagged KPNB1 (full-length and HEAT1–9) were cultured in LB medium. Protein expression was induced by adding 0.5 mM IPTG, and the cultures were further incubated at 18 °C overnight. The cells were harvested by centrifugation  and stored at − 80 °C. The cell pellet was dissolved in lysis buffer  and subjected to three rounds of quick freeze-thaw cycles. The cell debris were removed by centrifugation  and the supernatant was mixed with Glutathione-Sepharose beads (Cytiva) at 4 °C for 1 h. The beads were then washed with wash buffer  three times. KPNB1 protein were separated from the beads by protease digestion. The protein-bound beads were incubated with PreScission Protease (Cytiva) in Digestion Buffer  at 4 °C for 14 h. Protein was concentrated using Amicon centrifugal filters (Millipore) and stored at − 80 °C.6-TDP-CTF and His6-Nup62FG) was dissolved in dissolving buffer  to a final concentration of 4 mM. Protein was incubated with 10 μM ATTO488/610-maleimide (ATTO-TEC) at RT for 1 h, and then with 5 mM dithiothreitol for 1 h. The labelled protein solution was diluted by a buffer  to a final concentration of 10 μM and incubated at RT for 30 min to allow aggregation. For dissolution assays, purified KPNB1 or BSA was added to pre-formed aggregates to a final concentration of 20 μM and incubated for 24 h. For aggregation prevention assays, TDP-CTF, Nup62FG and KPNB1 constructs were mixed at RT for 30 min. The mixture was then transferred to a 96-well clear-bottom plate (Greiner Bio-one) for microscopic observation . To measure turbidity, the mixture was transferred to a microcuvette and the optical density at 395 nm was measured using a V-630 spectrophotometer (JASCO).Lyophilized protein  and aged depending on the assay. For whole-mount immunofluorescence of pTDP-43, brains from 2-day old fly males were dissected in cold PBS, fixed in 4% formaldehyde, and stained with primary rabbit anti-human phospho-TDP-43S409/410 (1:300) and secondary goat anti-rabbit Alexa Fluor 594 (1:100) antibodies. In addition, fly heads were also used to quantify the levels of total TDP-43 protein by western blot as described [The stocks Gmr-Gal4 , Elav GeneSwitch (GS)-Gal4 , control UAS-LacZ BDSC:8529 and 8530), Ketel RNAi lines , and EP-Ketel (BDSC:15967) were obtained from the Bloomington Drosophila Stock Center. The Ketel RNAi line 107,622 was obtained from the Vienna Drosophila Resource Center. To generate flies expressing UAS-V5-Ketel, a synthetic DNA fragment carrying the Ketel coding sequence fused to the V5 tag at the N-terminal end was cloned into pJFRC-MUH via NotI and XbaI linkers, subsequently microinjected into fly embryos , and targeted to the attP2 landing site in the third chromosome. The UAS lines expressing human TDP-43escribed . For eyeescribed  and phenescribed , 27. Forescribed . For loc and 8530C9orf72, SOD1, or TARDBP. Post-mortem case demographics are provided in Supplementary Table 2O and antigen retrieval was performed by steaming tissue sections in citrate buffer, pH 6  for 30 min. Tissue sections were cooled to RT and rinsed with dH2O for 10 min. Sections were permeabilized and blocked with serum-free protein block containing 0.3% Triton-X 100 at RT for 1 h. Tissues were immunostained with antibodies against phospho-TDP-43S409/410 (1:200), Nup62 (1:100) or KPNB1 (1:2000), diluted in antibody diluent  and incubated overnight at 4 °C in a humidified chamber. Tissue sections were washed in tris buffered saline (TBS) containing 0.05% Tween-20 (TBS-T), three times for 10 min each. Alexa Fluor conjugated secondary antibodies anti-mouse 488 or anti-rat 555 or anti-rat 647, Thermo Fisher Scientific), were diluted in antibody diluent  and incubated for 2 h at RT. Tissue was washed with TBS-T and incubated with Hoechst 33342  for 20 min at RT before rinsing with TBS. To quench autofluorescence, tissues were stained with 1x True Black lipofuscin autofluorescence quencher  for 30 sec and rinsed with dH2O for 5 min. Tissue sections were mounted with glass slides using ProLong Glass Antifade Mountant (Invitrogen). High-resolution imaging was conducted, and optical sections were acquired, according to the depth of the tissue to capture the full contour of the pTDP-43 aggregate. Z-series were obtained using an Eclipse Ti2 fluorescence microscope (Nikon) equipped with a Spectra X multi-LED light engine (Lumencor), single bandpass filter cubes for DAPI, EGFP/FITC and TRITC (Chroma), and a Zyla 4.2 PLUS sCMOS camera (Andor), using NIS Elements HC V5.30 software (Nikon). Immunofluorescent staining of all cases from distinct CNS regions was conducted at the same time and all pathological subgroups were imaged with the same acquisition settings. Out of focus light from each z-series was eliminated using the Clarify.ai module of the NIS-Elements Advanced Research (V5.30) deconvolution package. Clarified z-series are shown as optical projections and 3D volume rendered views to demonstrate colocalization of KPNB1 and Nup62 with pTDP-43 inclusions. Fluorescent tile images of the same tissue sections were captured using a 40x objective on an inverted fluorescent microscope (Keyence BZ-X800) and high-resolution image stitching was completed using the Keyence analyzer package (BZ-X series).Well-characterized human post-mortem tissue cases were provided by the Mayo Clinic Florida brain bank. Tissue derived from spinal cord and hippocampus were obtained from patients with ALS or FTLD, respectively, in addition to primary motor cortex, and tissue from all three regions from neuropathologically normal control tissue blocks. The neuropathological curation of cases consists of sporadic and familial disease, where select cases were available for patients who developed ALS or FTLD due to genetic mutations in 2 in sterile-filtered culture medium containing Basal Medium Eagle (Thermo Fisher Scientific), 26.6 mM HEPES , 511 μM ascorbic acid, 1% (v/v) GlutaMAX (Thermo Fisher Scientific), 0.033% (v/v) insulin (Sigma Aldrich), 1% (v/v) penicillin/streptomycin (Thermo Fisher Scientific) and 25% (v/v) heat-inactivated horse serum (Sigma Aldrich). Culture medium was changed every 3–4 days.All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Mayo Clinic. Brain slice cultures (BSCs) were prepared from postnatal day 8–9 (P8–9) C57BL/6 mice as previously reported . Pups weFor AAV transduction of BSCs, AAV plasmids were first transfected in HEK293T using Polyethylenimine Hydrochloride (Polysciences). 3–4 days after transfection, cell media was collected and centrifuged at 800 rcf for 5 min to recover the supernatant containing secreted AAVs. AAVs were applied directly into the culture medium on the first day of the BSC (DIV0).For immunostaining of BSCs, slices were fixed 12–15 days after transduction (DIV12–15) with 4% PFA for 1 h at RT, permeabilized with 0.5% Triton X-100 overnight at 4 °C and blocked with 20% BSA for 4 h. BSCs were incubated with the following primary antibodies: anti-FLAG (1:500), anti-GFP (1:500) and anti-TDP-43 (1:500) in 5% BSA for 48 h at 4 °C, followed by incubation with fluorophore-coupled secondary antibodies for 4 h at RT. To stain the nuclei, slices were incubated with the Hoechst dye (1:5000) for 30 min at RT, then coverslipped with ProLong Glass Antifade Mountant (Thermo Fisher Scientific). Nuclear-cytoplasmic localization of TDP-43 was semi-quantitatively determined as described above for HEK293T cells.t-test or analysis of variance (one- or two-way ANOVA followed by Bonferroni’s post hoc test) in GraphPad Prism software (version 9.2.0). Differences were considered statistically significant when p < 0.05. Data is shown as mean ± SEM.Statistical comparisons between experimental groups were performed using either two-sided Student’s S409/410 (pTDP-43), p62/SQSTM1, and ubiquitin, and induces high levels of toxicity in neuronal cell lines and primary neurons [mNLS (full-length TDP-43 with mutant NLS) form pathological cytoplasmic aggregates that are positive for pTDP-43 but show diffuse localization with no pathological hyperphosphorylation in the presence of mCherry-KPNB1  in the cytoplasm, KPNB1 expression appeared to reduce its aggregation . TDP-43  neurons , 33. ExpMammalian cells express several members of the α- and β-karyopherin families of nuclear transport receptors that share a similar curved α-solenoid structure composed of stacks of related armadillo or HEAT repeats, respectively . To deteWe focused on KPNB1, the best characterized member of the NIR family, to gain a better mechanistic understanding of how NIRs reduce TDP-CTF aggregation and determine the region that is required and sufficient for this activity. Since KPNB1 is comprised of 19 highly flexible arrays of short amphiphilic tandem α-helical HEAT repeats, we generated a series of constructs based on these repeats. First, we tested the N-terminal  and the C-terminal half of KPNB1 (H10–19). Much like the full-length protein, KPNB1 H1–9 showed diffuse localization with enrichment in the nuclear envelope, whereas KPNB1 H10–19 was mainly present in the nucleoplasm Fig. a. ExpresTo further delineate the active region in the KPNB1 H1–9 fragment, we generated a series of constructs with C-terminal HEAT repeat deletions, ranging from H1–8 down to H1. Only KPNB1 H1–8 was strongly enriched on the nuclear envelope, and was identified as the smallest fragment with full activity towards reducing TDP-CTF aggregation, rendering TDP-CTF diffuse and significantly decreasing insoluble TDP-CTF protein levels by ~ 77% Fig. d,e. KPNBGTP, importin-α, and FG-Nups which can bind KPNB1 at two distinct Nup-interacting sites (NIS1 and 2) [Numerous studies have mapped binding sites for KPNB1 interactors that regulate its nuclear import activity, including Ran1 and 2) , 36  , our resmNIS) to reduce FG-Nup binding [WT expression caused diffuse nucleocytoplasmic distribution of TDP-CTF, reduced the formation of TDP-CTF cytoplasmic inclusions, and decreased insoluble TDP-CTF, KPNB1 H1–8mNIS had lost most of its activity  into the NIS1 of KPNB1 H1–8  dipeptide repeat protein, which has been shown to induce endogenous TDP-43 mislocalization in mice [100 forms both nuclear and cytoplasmic aggregates when expressed in HEK293T cells, but only cytoplasmic aggregates were positive for endogenous TDP-43 and Nup62 , and to a lesser degree its PrLD (aa275–414)  that interacts significantly weaker with KPNB1 in comparison to TDP-CTFWT , an alternative splicing isoform of TDP-43 where the PrLD is replaced by a nuclear export signal (NES) [mNLS aggregates, but also increased the nuclear localization of TDP-43mNLS  and TDP-PrLD are soluble when co-expressed with mCherry, only TDP-PrLD co-aggregates with Nup62-mCherry. Strikingly, sTDP aggregates that lack the PrLD do not colocalize with Nup62 but accumulate around Nup62 inclusions  with Nup62-mCherry and found it to still form aggregates surrounding Nup62 foci  that mediates liquid-liquid phase separation (LLPS) to form transient biomolecular condensates under normal physiological conditions, and adopts a unique double spiral-shaped fold to form pathological amyloid-like filament structures under disease conditions . The TDPain Fig. c, suggesrry Fig. . While tons Fig. . SimilarWe next tested whether expression of Nup62 can increase the association between TDP-CTF aggregates and KPNB1 in HEK293T cells. KPNB1 strongly reduces the prevalence of TDP-CTF aggregates; the rare remaining TDP-CTF aggregates weakly overlap with KPNB1, and co-expression of Nup62 dramatically increased the recruitment of KPNB1 into TDP-CTF aggregates Fig. e. As expWT strongly dissolved pre-formed aggregates and prevented the formation of these co-aggregates . For disaggregation assays, TDP-CTF and Nup62FG were incubated together for 30 min to allow the formation of aggregates, followed by KPNB1 addition for 24 h, whereas for prevention experiments all three proteins were mixed and incubated for 30 min. TDP-CTF and Nup62FG strongly co-aggregated in vitro, and KPNB1 WT and H1–9VLIM-F more aggregation-prone, but also abrogated its colocalization with Nup62 Ket (Ketel), the fly ortholog of human KPNB1, as one of the most robust enhancers of TDP-43 toxicity, thus confirming our findings from mammalian cells (full details of the screen will be published elsewhere). Expression of human TDP-43WT alone causes a very modest degenerative phenotype, whereas mutant TDP-43M337V shows partial cytoplasmic mislocalization and pathological hyperphosphorylation, leading to severe neurodegeneration [WT and TDP-43M337V Ket gene also rescued pathological and behavioral phenotypes induced by TDP-43M337V   in mammatel Fig. e. Similatel Fig. f. In summNLS , whereas KPNB1 constructs carrying NIS mutations to prevent FG-Nup-binding had no effect  tissue, we employed AAV-transduction of organotypic mouse brain slice cultures (BSCs) Fig. e 30, 4848. BSCs ion Fig. e. Expresect Fig. e,f. We aect Fig. a, arrowsCTF Fig. . Taken tDrosophila model of traumatic brain injury and human autopsy brain tissue from patients with chronic traumatic encephalopathy (CTE) and ALS/FTLD [Previous studies have reported cytoplasmic mislocalization of KPNB1 in ALS cases –53 and NALS/FTLD , 55. To ALS/FTLD , 57. AltALS/FTLD . Thus, sThe loss of nuclear TDP-43 together with its cytoplasmic accumulation and phase transition into insoluble aggregates plays a central role in the pathogenesis of the ALS/FTD disease spectrum and other TDP-43 proteinopathies , 58, 59.WT, but not TDP-43mNLS or TDP-CTF in vitro [mNLS aggregation, thus identifying the relevant KPNB1 interaction domain  aggregates in an FG-Nup-dependent fashion  can promote TDP-43 phase separation and aggregate formation , 70. TheDrosophila models  Immunofluorescence (IF) of SH-SY5Y cells co-expressing mCherry-TDP-CTF with GFP or GFP-KPNB1. mCherry-TDP-CTF forms cytoplasmic aggregates but shows diffuse localization in the presence of GFP-KPNB1. Hoechst staining was used to outline nuclei. Scale bar: 5 μm. (Bottom) Western blot analysis and quantification of insoluble mCherry-TDP-CTF and endogenous TDP-43 protein levels in SH-SY5Y cells expressing GFP or GFP-KPNB1. KPNB1 significantly reduced insoluble TDP-CTF levels, while endogenous insoluble TDP-43 levels were unaffected. β-tubulin was used as a loading control. Statistical analysis was performed using two-sided Student’s t-test . b, Western blot analysis of soluble and insoluble GFP-TDP-CTF co-expressed with mCherry, mCherry-KPNB1 or untagged KPNB1 in HEK293T cells. KPNB1 only reduces insoluble TDP-CTF levels. β-tubulin was used as a loading control. c, IF of HEK293T cells co-expressing GFP-TDP-CTF or GFP-TDP-43mNLS with mCherry or mCherry-KPNB1. TDP-CTF and TDP-43mNLS form cytoplasmic aggregates positive for phospho-TDP-43S409/410 but show diffuse localization with no pathological hyperphosphorylation in the presence of mCherry-KPNB1. Arrowheads point to colocalization. Scale bar: 5 μm. d, Western blot analysis and quantification of insoluble GFP-tagged TDP-CTFWT, TDP-CTFQ331K, TDP-CTFM337V and TDP-CTFA382T in HEK293T cells expressing mCherry or mCherry-KPNB1. KPNB1 similarly reduced insoluble wild-type and ALS-derived mutant TDP-CTF protein levels. β-tubulin was used as a loading control. Statistical analysis was performed using two-sided Student’s t-test . e, Western blot analysis of total protein levels of GFP-tagged TDP-CTF or TDP-43 in HEK293T cells expressing mCherry, mCherry-KPNB1 or untagged KPNB1. Cells were lysed in 7 M urea buffer to collect total protein. KPNB1 reduces total protein levels of TDP-CTF, but not full-length TDP-43. β-tubulin was used as a loading control. Statistical analysis was performed using one-way ANOVA and Bonferroni’s post hoc test . f, TDP-CTF and TDP-43 transcript levels were quantified in HEK293T cells co-expressing GFP-tagged TDP-CTF or TDP-43 with mCherry, mCherry-KPNB1, or untagged KPNB1. GAPDH was used as a reference to normalize the transcript levels. Statistical analysis was performed using one-way ANOVA and Bonferroni’s post hoc test (n = 3). Supplementary Fig. 2. Expression of full-length KPNB1 and its N-terminal half do not reduce endogenous insoluble TDP-43 levels. Quantification of endogenous insoluble TDP-43 protein levels in SH-SY5Y cells co-expressing mCherry-TDP-CTF with GFP, GFP-KPNB1 full-length (FL), H1–9 or H10–19. β-tubulin was used as a loading control. Statistical analysis was performed using one-way ANOVA and Bonferroni’s post hoc test (n = 4). Supplementary Fig. 3. Cytoplasmic GFP-(GR)100 aggregates sequester endogenous TDP-43 and Nup62. a, Immunofluorescence of HEK293T cells expressing GFP-(GR)100 and stained for endogenous TDP-43 and Nup62. Only cytoplasmic GFP-(GR)100 aggregates are immunopositive for TDP-43 and Nup62. White arrowheads point to cytoplasmic poly(GR) aggregates. Nuclear GFP-(GR)100 aggregates render TDP-43 staining diffuse (purple arrowhead), while Nup62 accumulates as puncta in the cytoplasm (red arrowhead). Hoechst staining was used to outline nuclei. Scale bar: 5 μm. b, Quantification of the percentage of cytoplasmic GFP-(GR)100 aggregates positive for TDP-43 or Nup62 in HEK293T cells. Almost all cells are positive for both proteins. Statistical analysis was performed using two-sided Student’s t-test . Supplementary Fig. 4. Untagged KPNB1 interacts with both the RRM2 and PrLD of TDP-CTF. a, (Left) Lysates from HEK293T cells expressing GFP alone, or untagged KPNB1 with GFP, GFP-TDP-CTF or GFP-TDP-43 were subjected to immunoprecipitation with GFP-Trap magnetic beads. Whole cell lysates (input) and immunoprecipitates (IP) were subjected to western blot analysis using indicated antibodies. (Right) Quantification of relative KPNB1 levels in IP normalized by KPNB1 levels in input. Statistical analysis was performed using one-way ANOVA and Bonferroni’s post hoc test . b, Schematic domain structure of TDP-CTF full-length (208–414). TDP-CTF contains part of the RRM2  and the C-terminal PrLD (275–414). c, (Left) Lysates from HEK293T cells expressing GFP alone, or untagged KPNB1 with GFP, GFP-TDP-CTF, GFP-TDP-CTFnon-PrLD or GFP-TDP-PrLD were subjected to immunoprecipitation with GFP-Trap magnetic beads. Whole cell lysates (input) and immunoprecipitates (IP) were subjected to western blot analysis using indicated antibodies. (Right) Quantification of relative KPNB1 levels in IP normalized by KPNB1 levels in input. Statistical analysis was performed using one-way ANOVA and Bonferroni’s post hoc test . *cleavage products. Supplementary Fig. 5. KPNB1 reduces TDP-CTF aggregation independent of its interaction with the RRM2 region. a, Schematic domain structures of full-length TDP-CTF (208–414), and its C-terminal and N-terminal deletion constructs used for co-IP experiments. b, Lysates from HEK293T cells co-expressing mCherry-KPNB1 with GFP, GFP-TDP-CTF or GFP-TDP-CTF C-terminal deletion constructs were subjected to immunoprecipitation with RFP-Trap magnetic beads. Whole cell lysates (input) and immunoprecipitates (IP) were subjected to western blot analysis using indicated antibodies. Deletion of amino-acids 230–240 in TDP-CTF reduced its binding to KPNB1. c, Lysates from HEK293T cells co-expressing mCherry-KPNB1 with GFP, GFP-TDP-CTF or GFP-TDP-CTF N-terminal deletion constructs were subjected to immunoprecipitation with RFP-Trap magnetic beads. Whole cell lysates (input) and immunoprecipitates (IP) were subjected to western blot analysis using indicated antibodies. Deletion of amino-acids 230–240 in TDP-CTF reduces its binding to KPNB1. TDP-PrLD weakly interacts with KPNB1. d, (Left) Lysates from HEK293T cells co-expressing mCherry-KPNB1 with GFP, GFP-TDP-CTFWT or GFP-TDP-CTFΔ230–240 were subjected to immunoprecipitation with RFP-Trap magnetic beads. Whole cell lysates (input) and immunoprecipitates (IP) were subjected to western blot analysis using indicated antibodies. KPNB1 interacts less with TDP-CTFΔ230–240. (Right) Quantification of relative GFP levels in IP normalized by GFP levels in input confirm that GFP-TDP-CTFΔ230–240 interacts less with mCherry-KPNB1. Statistical analysis was performed using two-sided Student’s t-test . e, Immunofluorescence of HEK293T cells co-expressing GFP-tagged TDP-CTFWT or TDP-CTFΔ230–240 with mCherry or mCherry-KPNB1. TDP-CTFΔ230–240 forms small nuclear and big cytoplasmic aggregates. KPNB1 reduces both TDP-CTFWT and TDP-CTFΔ230–240 aggregates and makes TDP-CTFΔ230–240 more nuclear. Hoechst staining was used to outline nuclei. Scale bar: 5 μm. f, Western blot analysis and quantification of insoluble GFP-tagged TDP-CTFWT or TDP-CTFΔ230–240 in HEK293T cells expressing mCherry or mCherry-KPNB1. KPNB1 equally reduced insoluble TDP-CTFWT and TDP-CTFΔ230–240 protein levels. β-tubulin was used as a loading control. Statistical analysis was performed using two-way ANOVA and Bonferroni’s post hoc test . Supplementary Fig. 6. The PrLD of TDP-43 interacts with FG-Nups and KPNB1. a, Immunofluorescence (IF) of HEK293T cells expressing GFP or GFP-TDP-PrLD and stained for endogenous Nup98 and Nup62. TDP-PrLD forms small cytoplasmic foci positive for Nup62. Hoechst staining was used to outline nuclei. Scale bar: 5 μm. b, IF of HEK293T cells expressing GFP or GFP-TDP-PrLD and stained for endogenous KPNB1. KPNB1 colocalizes with TDP-PrLD foci in the cytoplasm. Hoechst staining was used to outline nuclei. Scale bar: 5 μm. c, Lysates from HEK293T cells expressing GFP or GFP-TDP-PrLD were subjected to immunoprecipitation with GFP-Trap magnetic beads. Whole cell lysates (input) and immunoprecipitates (IP) were subjected to western blot analysis using indicated antibodies. The PrLD of TDP-43 interacts with Nup62, Nup98 and KPNB1, but not with Ran. Supplementary Fig. 7. Colocalization of Nup62 with TDP-43 constructs depends on the PrLD. Colocalization between Nup62-mCherry and GFP or GFP-tagged TDP-43 constructs was measured using the Mander’s overlap coefficient. Values close to 0 and 1 indicate a weak and strong colocalization, respectively. Statistical analysis was performed using one-way ANOVA and Bonferroni’s post hoc test . Supplementary Fig. 8. The NES of sTDP does not prevent it from binding to Nup62 aggregates. Immunofluorescence of HEK293T cells co-expressing Nup62-mCherry with either sTDP or sTDPmNLS ΔNES. Nup62 aggregates do not colocalize with sTDP even after mutating its NLS and NES. Hoechst staining was used to outline nuclei. Scale bar: 5 μm. Supplementary Fig. 9. TDP-CTF PrLD mutations that reduce its association with Nup62 abrogate its interaction and reduced aggregation by KPNB1. a, Immunofluorescence (IF) of HEK293T cells co-expressing Nup62-mCherry with either GFP-tagged TDP-CTFWT or TDP-CTF constructs harboring different mutations in the PrLD. All TDP-CTF mutant variants colocalize with Nup62 aggregates, except for TDP-CTFVLIM-F (green arrowhead) which accumulates in close proximity to Nup62 (red arrowhead). Scale bar: 5 μm. b, Colocalization between Nup62-mCherry and GFP-tagged TDP-CTFWT or TDP-CTFVLIM-F was measured using the Mander’s overlap coefficient. Values close to 0 and 1 indicate a weak and strong colocalization, respectively. Statistical analysis was performed using two-sided Student’s t-test . c, IF of HEK293T cells co-expressing Nup62-mCherry with either GFP-tagged wild-type or mutant TDP-43mNLS constructs. RNA-binding deficient TDP-43mNLS (TDP-43mNLS 5F-L) can still colocalize with Nup62. Only TDP-43mNLS VLIM-F aggregates (green arrowhead) do not contain Nup62 (red arrowhead). Scale bar: 5 μm. d, IF of HEK293T cells co-expressing Nup62-mCherry with GFP-FUSΔ14. Cytoplasmic FUS aggregates (green arrowhead) do not colocalize with Nup62 (red arrowhead). Scale bar: 5 μm. e, Protein sequences of TDP-CTFWT and TDP-CTFVLIM-F showing FG repeats (underlined) and VLIM-F mutations (in red) in the PrLD. f, (Left) Lysates from HEK293T cells co-expressing mCherry-KPNB1 with GFP, GFP-tagged TDP-CTFWT or TDP-CTFVLIM-F were subjected to immunoprecipitation with RFP-Trap magnetic beads. Whole cell lysates (input) and immunoprecipitates (IP) were subjected to western blot analysis using indicated antibodies. Introducing VLIM-F mutations in TDP-CTF significantly reduces its binding to KPNB1. (Right) Relative GFP levels in IP normalized by GFP levels in input confirm that GFP-TDP-CTFVLIM-F interacts less with mCherry-KPNB1. Statistical analysis was performed using two-sided Student’s t-test . g, IF of HEK293T cells co-expressing GFP-tagged TDP-CTFWT or TDP-CTFVLIM-F with mCherry or mCherry-KPNB1. KPNB1 only reduces the size of TDP-CTFVLIM-F aggregates. Hoechst staining was used to outline nuclei. Scale bar: 5 μm. h, Western blot analysis and quantification of insoluble GFP-tagged TDP-CTFWT or TDP-CTFVLIM-F in HEK293T cells expressing mCherry or mCherry-KPNB1. KPNB1 strongly decreased protein levels of insoluble TDP-CTFWT but not TDP-CTFVLIM-F. β-tubulin was used as a loading control. Statistical analysis was performed using two-way ANOVA and Bonferroni’s post hoc test . Supplementary Fig. 10. Nup98 strongly associates with TDP-CTFWT, but not TDP-CTFVLIM-F and sTDP. Immunofluorescence of HEK293T cells co-expressing GFP-Nup98 with mCherry or mCherry-tagged TDP-43mNLS, TDP-43mNLS 1–265, TDP-PrLD, sTDP, TDP-CTFWT or TDP-CTFVLIM-F. Arrowheads point to co-aggregation. Hoechst staining was used to outline nuclei. Scale bar: 5 μm. Supplementary Fig. 11. KPNB1 H1–8 reduces cytoplasmic Nup62 aggregates. Immunofluorescence of HEK293T cells co-expressing Nup62-GFP with mCherry, mCherry-tagged KPNB1 H1–8WT or H1–8mNIS. KPNB1 H1–8WT suppresses Nup62 aggregates, whereas NIS mutations abolished this activity. Hoechst staining was used to outline nuclei. Scale bar: 5 μm. Supplementary Fig. 12. Multiple Ketel RNAi lines enhance hTDP-43M337V toxicity in the fly eye when expressed under the control of the Gmr-Gal4 driver. Supplementary Fig. 13. Ketel overexpression does not change total levels of human TDP-43 protein in flies. Western blot analysis of hTDP-43 expression in fly brains. hTDP-43 is expressed upon administration of RU486, and Ketel overexpression had no effect on total protein levels of TDP-43. β-actin was used as a loading control. Statistical analysis was performed using one-way ANOVA and Sidak’s post hoc test (n = 3). Supplementary Fig. 14. Survival Kaplan-Meier curves of Elav-GS flies carrying the indicated transgenes in the absence of RU486. Note that all the negative controls yield similar results. Supplementary Fig. 15. The EP-Ketel insertion P{EPgy2}Fs(2)KetEY06666 rescues mutant hTDP-43 toxicity in flies. a, EP-Ketel suppresses TDP-43M337V toxicity in the Drosophila eye. b, EP-Ketel reduces phospho-TDP-43 staining in the adult brain when co-expressed using the Elav-GS driver and exposed to RU486. c, RU486-treated flies carrying the Elav-GS driver, mutant TDP-43 and EP-Ketel (green line) live longer compared to flies carrying the control LacZ transgene (red line). The blue line corresponds to LacZ; Elav-GS Gal4 flies without drug (solvent only) as a negative control. Survival analysis was performed using the OASIS online tool, and p-values were calculated using Fisher’s exact test . Supplementary Fig. 16. Downregulation of Ketel exacerbates toxicity of hTDP-43ΔNLS in the fly eye, but Ketel overexpression alleviates it. a, Eye phenotypes from flies expressing the indicated transgenes via the eye-specific Gmr-Gal4 driver. Arrow points to necrotic areas. b, Quantitative analysis of eye severity in flies co-expressing hTDP-43ΔNLS and the indicated transgenes via the eye-specific Gmr-Gal4 driver. Statistical analysis was performed using one-way ANOVA and Bonferroni’s post hoc test . Supplementary Fig. 17. The NIR cdm/IPO13 rescues severe eye degeneration in hTDP-43ΔNLS flies. a, Eye phenotypes from flies expressing the indicated transgenes via the eye-specific Gmr-Gal4 driver. Cdm overexpression (OE) rescues hTDP-43-induced neurodegeneration. b, Quantitative analysis of eye severity in flies co-expressing hTDP-43ΔNLS and the indicated transgenes via the eye-specific Gmr-Gal4 driver. Statistical analysis was performed using one-way ANOVA and Bonferroni’s post hoc test . Supplementary Fig. 18. KPNB1 increases the N-to-C ratio of different TDP-43 constructs in rodent primary neurons. Quantitative analysis of the N-to-C ratio of GFP or GFP-tagged TDP-43WT, TDP-43mNLS or TDP-CTF in primary neurons expressing mScarlet or mScarlet-KPNB1 24 h (a) or 48 h (b) post-transfection. Statistical analysis was performed using one-way ANOVA and Bonferroni’s post hoc test . Supplementary Fig. 19. KPNB1 reduces cytoplasmic aggregation of TDP-CTF and promotes nuclear localization in BSCs. a, Immunofluorescence of mouse BSCs (DIV12) co-expressing AAV-mScarlet-TDP-CTF with AAV-GFP, AAV-GFP-KPNB1 H1–9WT, AAV-FLAG-KPNB1 H1–9WT, H1–9mNIS, H1–8WT, H1–8mNIS or H10–19. KPNB1 H1–9WT and H1–8WT reduce TDP-CTF cytoplasmic aggregates (arrows) and render it very nuclear (arrowheads), whereas NIS mutations strongly reduce this function. Hoechst staining was used to outline nuclei. Scale bar: 50 μm. b, Quantification of the percentage of neurons exhibiting nuclear (N), nucleocytoplasmic (NC) or cytoplasmic (C) mScarlet-TDP-CTF distribution upon expression of different AAV-KPNB1 constructs. Statistical analysis was performed using two-way ANOVA and Bonferroni’s post hoc test . Supplementary Fig. 20. Nup62 colocalizes with pTDP-43 aggregates in the spinal cord of TARDBP ALS case. High resolution co-immunofluorescence staining of pTDP-43 (magenta) and Nup62 (green) with Hoechst (blue) was conducted in fixed spinal cord of a neuropathologically diagnosed TARDBP ALS case. pTDP-43 was stained with a 647/Cy5 secondary antibody and Nup62 with a 488/FITC antibody to ensure there is no bleed through between channels. The image is shown as a panel of projected optical sections from each z-series for magenta and green channels and merged with Hoechst DNA staining. Volume rendered z-series of all channels are shown as the 3D inset. Scale bar: 5 μm. Supplementary Fig. 21. KPNB1 expression does not dysregulate nucleocytoplasmic transport. a, Immunofluorescence (IF) of SH-SY5Y cells co-expressing the transport reporter NES-tdTomato-NLS with GFP or GFP-tagged KPNB1 full-length (FL), H1–9 or H1–8. Hoechst staining was used to outline nuclei. Scale bar: 5 μm. b, Quantification of the N-to-C ratio of the NES-tdTomato-NLS reporter. Statistical analysis was performed using one-way ANOVA and Bonferroni’s post hoc test . c, IF of SH-SY5Y cells expressing GFP or GFP-tagged full-length KPNB1 (FL), H1–9 or H1–8 and stained for endogenous Ran and Nup98. Both proteins remain mostly nuclear in presence of KPNB1. Hoechst staining was used to outline nuclei. Scale bar: 5 μm. Supplementary Fig. 22. Expression of the N-terminal half of KPNB1 does not affect the nuclear localization of endogenous TDP-43 in cells and BSCs. a, Immunofluorescence (IF) of HEK293T cells co-expressing GFP with an empty plasmid (ctrl), FLAG-tagged KPNB1 H1–8WT or H1–8mNIS and stained for endogenous TDP-43. TDP-43 remains nuclear in presence of KPNB1 H1–8. Scale bar: 5 μm. b, IF of mouse BSCs (DIV15) expressing AAV-FLAG-KPNB1 H1–9 and stained for endogenous TDP-43. TDP-43 remains nuclear in presence of KPNB1 H1–9. Scale bar: 25 μm. Supplementary Fig. 23. Nup62 and TDP-CTF aggregates are positive for amyloid staining and their co-expression promotes TDP-CTF aggregation. a, Immunofluorescence of HEK293T cells expressing mCherry-TDP-CTF or Nup62-mCherry which form Amylo-Glo-positive aggregates. DRAQ5 staining was used to outline nuclei. Scale bar: 5 μm. b, Western blot analysis of insoluble mCherry-TDP-CTF in HEK293T cells expressing GFP or Nup62-GFP. Nup62 strongly increases insoluble levels of TDP-CTF. Supplementary Table 1. Demographic information of human post-mortem cases used for Nup62 and KPNB1 co-immunostaining with pTDP-43S409/410. Supplementary Table 2. Summary of statistical analyses. Supplementary Table 3. Summary of DNA constructs and sources. Supplementary Table 4. Summary of Drosophila strains and sources. Supplementary Table 5. Summary of reagents and sources.


Allograft bone screws are rarely described for the fixation of the scaphoid. When fresh fractures are treated, metal screws are mainly used; when pseudarthrosis is the indication, plates in combination with vascularized or non-vascularized bone grafts are mainly used. The necessity of metallic screw removal is under debate, but it is mandatory for plates because of movement restrictions due to the plate. The use of biomaterials in scaphoid fracture fixation was described as leading to union rates of between 64 and 100%. Brcic showed the incorporation of an allogeneic cortical bone screw at 10 weeks postoperative, along with revascularization and stable osteosynthesis with primary bone healing, without any signs of immunological rejection. The purpose of this retrospective study was to explore the results obtained using an allogenic cortical bone screw (Shark Screw®) in patients with fresh scaphoid fracture fixation and pseudarthroses with respect to union rates and time to union.We retrospectively analyzed 75 patients: 31 with fresh fractures and 44 pseudarthrosis patients. The Shark Screw® was used for the fixation of the scaphoid in the fresh-fracture and pseudarthrosis patients. We evaluated the union rate, complication rate and time to union.Using the human allogeneic cortical bone screw for scaphoid fracture fixation led to a high union rate (94–96%). There were two nonunions in the fresh fracture group and two nonunions in the pseudarthrosis group. The complication rate was 1.3% (1 patient). Median time to union was 16, 18 and 29 weeks for the fresh-fracture, pseudarthrosis and delayed-union patients, respectively. The treatment of fresh scaphoid fractures and pseudarthroses showed similar union rates to those described in the literature, uses a shorter and less invasive surgical method with no need for hardware removal, and has a low complication rate.Using the human allogenic cortical bone screw (Shark Screw®) led to similar union rates in fresh fractures—but better union rates in pseudarthrosis patients—compared to those presented in the literature for other scaphoid fracture fixation techniques, and it enabled a short and low-invasive procedure without any donor site morbidity and without the necessity to remove the hardware in a second surgery. The pseudarthrosis patient group showed a particularly strong benefit from this new procedure. The physiological bone metabolism remodels the cortical bone screw without scars.Level of evidence: III: retrospective cohort study, therapeutic investigation of a treatment. Allograft use for scaphoid reconstruction was described for the first time by Carter et al.  using HeScaphoid fractures account for 50–90% of all carpal fractures in young active patients –15. ApprThe purpose of this retrospective study was to examine the first results of using the human allogenic cortical bone screw for fresh scaphoid fracture fixation and pseudarthroses with respect to union rates and time to union. Complication rates will be a second outcome. The hypothesis is that union rates when using the Shark Screw® are at least similar to union rates described in the literature for other scaphoid fracture fixation techniques.We received approval from the institutional review boards  before the beginning of the study. Due to ethical concerns, we did not use a control group who received a metal screw instead of the bone screw. Because of the retrospective nature of the study, the need for an informed consent was waved. No involvement of patients in the data collection and data treatment was necessary. All post-surgery visits were finished before the start of the study. The study was performed in accordance with the principles of the Declaration of Helsinki.Inclusion criteria were: patients aged 18–99 years; the use of the human allogeneic cortical bone screw , and the screw is put in place.A short (approx. 0.5 cm) skin incision was made above the scaphotrapezial joint at the radial border of the tendon of the flexor carpi radialis muscle. After the skin incision, a blunt probe was made with scissors and spread. Under X-ray image monitoring, the K-wire was inserted from the palmar-radial aspect of the scaphoid via the scaphoid tubercle towards the proximal pole of the scaphoid. The intraoperative X-ray image monitoring included at least three projection planes  and fluoroscopy during forearm rotation. After attaining the correct positioning, the core hole was drilled over the 1.2-mm K-wire. The thickness of the human allogeneic cortical bone screw was determined based on the size of the bone. After drilling the core hole, the thread for the cortical bone screw was cut over the 1.2-mm K-wire. The desired drilling and thread depth were made visible with lateral laser markings. The finished tunnel was rinsed with physiological saline solution to clean out fine bone chips. The cortical bone screw cut was then screwed in, without much resistance, with a hexagonal screwdriver, also under intraoperative X-ray monitoring  and fluoroscopy during forearm rotation. Then, as described above, after removing protruding material, the screw was ground back to below the cartilage surface with a small ball milling machine.As a rule, for postoperative care, immobilization in a cast was required for at least 2 weeks. In most cases, the resumption of extensive physical exertion should only be permitted after 12 weeks.p value of < 0.05 was defined as representing a significant difference.Data are presented as median (range). Due to the non-Gaussian distribution of the data, non-parametric Kruskal–Wallis ANOVA was used for calculating significant differences in quantitative values. Ordinal values were evaluated with contingency tables using Fisher’s exact test for significance. Kaplan–Meier curves were used to show differences in time to union between the groups. A Age and gender were not different between the groups Table . Time toThe size of the human allogeneic cortical bone screw used was mainly 3.5 and 4.0 mm Table . All patMean follow-up was 13 months , especially in pseudarthrosis patients (96%), when using the human allogeneic cortical bone screw for fixation. The two nonunions (4%) in the pseudarthrosis patients were due to non-compliance and a fracture gap bridging that was too short Fig. . Median Merrell et al.  describeIn the presented study, we recorded only one complication in 75 patients  and arthrosis at the last follow-up in 2 patients where union could not be achieved. This favorable outcome is related to the use of the human allogeneic cortical bone screw, which avoids donor site morbidity and problems related to metal hardware. Complication rates were reported to be between 21 and 26%, depending on the graft used , 37, 39.The union rate in the fresh fracture group (94%) was similar to values described by Dias et al. , even thBrcic et al.  describeTime between injury and surgery was reported as 7–50 (mean 14.8) weeks , similarDonor site morbidity, as described in the literature , 19, 41,Hardware removal is another problem which is avoided by using the human allogeneic cortical bone screw. In 17%  to 67%  of the pLangegger  describeThere are several limitations of this study. First, the retrospective nature of the study. Second, due to the explorative nature of the study and ethical concerns, no control group, using a metal screw, instead of the cortical bone screw was available for comparison. Third, no scores were available to interpret the results. Further prospective studies are needed to confirm these preliminary findings.In conclusion, using the human allogeneic cortical bone screw (Shark Screw®) led to similar union rates to those presented in the literature for other scaphoid fracture fixation techniques and enabled a short and low-invasive procedure without any donor site morbidity and without the necessity to remove the hardware in a second surgery. The pseudarthrosis patient group received a particularly strong benefit from this new procedure. The physiological bone metabolism remodels the cortical bone screw without scars.