To compare the von Mises stresses of the pedicle screw system and the displacement of injured vertebrae using 3‐D finite element analysis, and to evaluate the curative effect of the pedicle screw system.Finite element methods were used for biomechanical comparison of four posterior short segment pedicle screw fixation techniques. The different pedicle screw models are traditional trajectory (TT), Universal Spine System (USS), cortical bone trajectory (CBT), and CBT at the cranial level and pedicle screw (PS) at the caudal level (UP‐CBT). The stress distribution of the screws and connecting rods under different working conditions and the displacement of the injured vertebrae were compared and analyzed.After the pedicle screw system was fixed, the stress under vertical compression was mainly concentrated at the proximal end of the screw, while the stress was mainly concentrated on the connecting rod during flexion, extension, lateral flexion, and rotation. The TT group had the greatest stress during the flexion, extension, and left and right rotation. The UP‐CBT group was most stressed when the left and right sides were flexed; the stress of the USS screw system was less than that of the other three models during flexion, lateral flexion, and rotation. The maximum von Mises stress values of pedicle screws in all exercise states were 556.2, 340.7, 458.1, and 533.4 MPa, respectively. In the USS group, the displacement of the injured vertebra was small in the flexion, and the left and right lateral flexion and the right rotation were higher than in the TT group and the CBT group. The maximum displacements of the injured vertebrae in all motion states were 1.679, 1.604, 1.752, and 1.777 mm, respectively.Universal Spine System pedicle screws are relatively less stressed under different working conditions, the risk of breakage is small, and the model is relatively stable; CBT screws do not exhibit better mechanical properties than conventional pedicle screws and USS pedicle screws. With the increase in the occurrence of high‐energy trauma injuries, such as from traffic accidents and high‐altitude falls, the incidence of thoracolumbar fractures is increasing. Twenty percent of thoracolumbar fractures are burst fractures, which are usually caused by high‐energy damage, and are due to axial pressure. The buckling rotation force causes the front column and the center column to be unable to supportAccording to a recent epidemiological survey, the incidence of spinal fractures is approximately 32.8/100 000The optimal treatment for thoracolumbar fractures remains controversial. Despite potential complications, surgical treatment is very effective, surgical treatment is still very effective when there are severe compression and neurological impairmentsThe purpose of treatment for spinal fractures is to restore the original sagittal and coronal planeset al.et al.et al.12–L2 to simulate the USS pedicle screw fixation model at different depths, and found that the insertion of screws with 60% or more depth can achieve sufficient strength and make the injured vertebrae more stable.The pedicle screw was first designed and manufactured by Kinget al.et al.These methods have their own advantages and disadvantages. With the wide application of the pedicle screw system, several years of follow up have revealed that many patients have experienced loose internal fixation, broken nails, kyphosis, and height loss of the anterior edge of the vertebral body. In 2009, Santoni 12–L2 spine vertebrae and implant; (ii) peak stress of the pedicle screw system; and (iii) smallest axial displacement of the injured vertebrae.The objective of the present study is to use the USS pedicle screw system to compare the traditional trajectory (TT), CBT, and CBT at the cranial level and pedicle screw (PS) at the caudal level (UP‐CBT), which are used to treat thoracolumbar fractures, based on the following criteria: (i) stress distribution on the TThe working hypothesis was that of the four implants, the USS had the best biomechanical performance when used for thoracolumbar fracture fixation.The software Mimics 20.0 , 3‐matic 12.0 , SolidWorks 2014 , and Abaqus 6.13  were used. A CT scanner  was used to collect raw data in DICOM format with a scan slice of 0.625 mm.12–L2 spine, which included three vertebrae and two discs, was reconstructed. Geometrical details of T12–L2 spine vertebrae were obtained from 64 spiral CT images of a 26‐year‐old healthy male (65 kg and 172 cm) with no history of spine injury or osteoporosis or radiographic evidence of degeneration. The CT images were scanned and imported into Mimics 20.0 . The surface model was then exported into 3‐matic 12.0  to generate and enhance the quality of the solid model. Eventually, it was imported to Abaqus 6.13  for meshing and analysis. Each vertebral body consisted of cortical bone and cancellous bone, and each vertebral disc was composed of nucleus pulposus, annulus fibrosus, and endplates. Posterior elements were built separately from the vertebral bodies. Based on Boolean algebra, approximately 60% of the L1 segment was resected, we established a 3‐D finite element model of injured vertebrae through the removal of the anterior and middle column of the L1 vertebrae.A finite element model of the TFour fixation models were established for unstable thoracolumbar fractures: the traditional trajectory (TT) model, the Universal Spine System (USS) model, the cortical bone trajectory (CBT) model, and the CBT at the cranial level and PS at the caudal level (UP‐CBT) model. The implants and the vertebral structure were precisely engaged. The diameter of the TT screw was 6.5 mm and the total length of the screw was 45 mm. The diameter of the USS screw was 6.2 mm and the total length of the screw was 45 mm. The diameter of the CBT screw was 5.0 mm and the total length of the screw was 35 mm. The fixation models are shown in Fig. A posterior pedicle screw system involving four nails and two rods was used Table .The entry point and direction of the TT screw are along the vertebral arch, with the root anatomical axis into the nail, parallel to the upper and lower endplates of the vertebral body.The USS consisted of four Schanz screws and two connecting rods. The Schanz screw can have a sliding support of up to 15° when resetting. The connecting rods of the USS are located directly on both sides of the spinous process and behind the lamina, and spread out to the adjacent vertebrae. Rear support can be improved when resetting, and resetting and fixation can occur without the need to fix the injured vertebra, reducing the dissection and injury of the diseased paravertebral muscle.In CBT, a relatively new technique for pedicle screw insertion in the lumbar spine, a screw follows a sagittal caudocephalad path and a laterally directed path in the transverse plane to maximally engage the cortical bone from the pedicle to the vertebral body. Given the more medial approach, the new technique is less invasive as it requires a smaller incision and less lateral tissue dissection.In the UP‐CBT group, the upper segment is fixed with CBT screws, and the lower segment is fixed with conventional pedicle screws. This UP‐CBT procedure requires an incision of 5–6 cm. In terms of incision length, the UP‐CBT screw is less invasive than the traditional trajectory screw.2 was constrained in all degrees of freedom. A pure moment of 10 Nm combined with a pre‐compressive load of 150 N was applied to the top surface of T12. Flexion, extension, left/right lateral bending, and left/right axial rotation were simulated.The inferior endplate of LTo validate the rationality of the models, including model simplification, material properties, boundary conditions, and loads, a moment of 10 Nm and a compressive load of 150 N were applied at the reference point. These loading conditions are adopted from the biomechanical experiments and published finite element analyses. The range of motion (ROM) among different models was compared. There was little difference between the models. Therefore, the models in the present study are effective for further analyses Table .12–L2, von Mises stress, and the stress nephogram of the pedicle screws and rods of the four fixation finite element models under six loading  conditions were analyzed. No statistical analysis was performed in the manuscript because only one subject was modeled.The ROM of TIn the four pedicle screw models, the minimum stress was observed in the posterior extension state, and the maximum stress was most significant in the rotating state. The TT group had the greatest stress during the flexion, extension, and left and right rotation. The UP‐CBT group was most stressed when the left and right sides were flexed; the stress of the USS screw system was less than that of the other three models during flexion, lateral flexion, and rotation. In the post‐extension state, the stress of the upper screw is significantly greater than that of the lower screw Fig. .In the TT group, the USS group, the CBT group, and the UP‐CBT group fixed model, the maximum von Mises stress values of pedicle screws in all exercise states were 556.2, 340.7, 458.1, and 533.4 MPa, respectively (Fig. During all the motion states, the stress of the rod in the CBT group was significantly greater than that of the other three groups in the flexion and rotation state of the rod. The UP‐CBT group showed relatively little stress on the left side and the left and right rotation. The maximum stress of the rod mostly occurs in the lateral flexion and rotation of the fixed model. In the TT group, the USS group, the CBT group, and the UP‐CBT group fixed model, the maximum von Mises stress values of the rods under all motion conditions were 188.6, 293.3, 379, and 181.2 MPa, respectively Fig. .In the four models, the displacement of the injured vertebra was the largest with flexion, and the displacement of the injured vertebra was the smallest when the extension was extended. In the USS group, the displacement of the injured vertebra was small in the flexion, and the left and right lateral flexion and the right rotation were higher than for the TT and CBT groups. The displacement of the injured vertebra was the largest when flexing forward and left and right. In the fixed models of the TT group, the USS group, the CBT group, and the UP‐CBT group, the maximum displacements of the injured vertebrae in all motion states were 1.679, 1.604, 1.752, and 1.777 mm, respectively (Fig. reduce kyphosis, reduce the indirect decompression of the spinal canal, and help to restore nerve function. Although lumbar pedicle screw fixation has the advantage of the biomechanical stability of pedicle screws, screw loosening and rupture still occur, especially in patients with osteoporosis, leading to failure of correction and nonunion. With the development of pedicle screw technology, a variety of pedicle screw systems have emerged in the clinic.The posterior pedicle screw technique has become the gold standard for spinal internal fixation. Thoracic and lumbar fractures can be treated with a variety of posterior surgical techniques. A posterior pedicle screw system involving four nails and two rods was used. Fixation can improve et al.et al.et al. also pointed out that the load of the USS pedicle screw system is not transmitted directly from the upper screw to the lower screw but from the upper screw to the connector to the rod to the connector to the lower screw. The load of the traditional pedicle screw is transmitted directly from the upper screw to the lower screw.Currently, the USS pedicle screw system is widely used in clinical practice. Some scholars have applied the USS pedicle screw system in clinical practice, and a lot of research has been conducted. Aono et al.et al.et al.et al.Because the rod of the USS is close to the sides of the spinous process and close to the rear of the lamina, it can have a better supporting effect, and can avoid the “parallelogram effect” of the conventional screw, thereby achieving a better resetting and fixing effect. The cortical nail screw internal fixation technique is currently a hot topic. The CBT screw internal fixation technique was first proposed by Santoni 12–L2 finite element model. The lower surface of L2 is constrained by all degrees of freedom. A pure torque of 10 Nm was applied in combination with a precompression load of 150 N to the top surface of T12. We simulated buckling, stretching, left/rightward bending and left/right axial rotation, consistent with other experimental models. The pedicle screw system simplifies the threading and binds the screw to the vertebral body to avoid failure of the mesh due to the thread and affects the calculation of the model.Cortical and cancellous bones were simulated using 3‐D homogeneous solid elements. The anterior/posterior longitudinal ligament, the transverse ligament, the ligamentum flavum, the interspinous ligament, and the supraspinous ligament were modeled using spring unit elements. We constructed a TPrevious researchers have used the Edit Masks function in MIMICS in the construction of intervertebral discs. This function needs to be depicted in the cross‐section of the intervertebral disc according to the shape of the intervertebral disc, and then smoothed. This method is more complicated and there will be errors. In this study, Anatomy Reconstruction, a new function of 3‐matic 12.0 software, was used to form the general shape of the intervertebral disc by constructing the upper and lower vertebral bodies of the upper vertebral body, and then constructing the annulus fibrosus, nucleus pulposus, and end plate.12–L2 segment was used, and the injured vertebra was modeled for L1. Partial resection of L1 or partial elastic modulus were conducted and Poisson's ratio for partial vertebral fractures was applied. The finite element method does not precisely model the actual injured vertebral body. To establish a model close to the clinical fracture reduction, a thoracolumbar fracture model of healthy normal people is used to perform the injured vertebrae osteotomy, and the vertebral screw system is used to restore the injured vertebrae. In this study, a V‐shaped osteotomy was performed on 60% of the vertebral bodies on the L1 vertebral endplate, and the injured vertebrae model of the anterior and posterior column injury was constructed.For the finite element study of thoracolumbar fractures, the TBy applying a vertical load of 500 N to the upper surface of the model and a torque of 15 Nm to simulate different motion states such as flexion, extension, lateral flexion, and rotation, it can be observed that in the four pedicle screw models, in the four pedicle screw models, it can be observed that the minimum stress is displayed in the extended state and the maximum stress is displayed in the rotated state. The minimum stress shows the maximum stress in the rotating state. The stress of the screw is the largest in the TT group during flexion, extension, and rotation. The stress in the UP‐CBT group is greatest when lateral flexion occurs. The USS screw system has less stress during forward flexion, lateral flexion and rotation than the other three models. The stress during bending and rotation is smaller than in the other three models. In the extended state, the stress peak of the internal fixator mainly appears in the lower screw, and in the post‐extension state, the stress of the upper screw is significantly greater than that of the lower screw.For the stress of the rod, the stress of the CBT group was significantly greater than that of the other three groups in the flexion, left flexion, and left and right rotation states. The UP‐CBT group showed relatively little stress on the left side flexion and the left and right rotation. The maximum stress of the rod mostly occurs in the lateral flexion and rotation of the fixed model. Through the force cloud diagram of the pedicle screw, it can be observed that the maximum stress of the pedicle screw system of the four models is concentrated on the proximal rod end of the pedicle screw, which is consistent with the clinically observed screw fracture site. Usually, the junction of the screw and the connecting rod is the most common site of clinical pedicle screw fracture. The fixation of the pedicle screw system stabilizes the spine and promotes healing of the injured vertebra. Early functional exercise is beneficial to patients, but inappropriate or exercise of excessive intensity is very likely to cause pedicle screw fracture. Under the four kinds of exercise state, the maximum stress of the pedicle screw is from large to small: for rotation, flexion, lateral flexion, and extension, respectively. The USS pedicle screw system has the lowest rotation, flexion, and lateral flexion among the four pedicle screw systems, and the risk of nail breaking is minimal. At the same time, bed rest and wearing a brace can reduce the force of the injured vertebrae and limit the range of lumbar motion, as well as reduce the risk of nail breakage.The displacement of the injured vertebra can reflect the stability of the pedicle screw system. Under the four kinds of motion states, the four pedicle screw system models have the same trend in change of the injured vertebrae displacement. The maximum displacement of the injured vertebra is from large to small. For flexion, rotation, lateral flexion, and extension, in the flexion state, that is, when the displacement of the four models is at a maximum, the maximum displacement of USS in the four models is the smallest, indicating that USS has better stability.At present, finite element analysis is more common in orthopaedics, and can explain clinical problems and provide a reference for clinical medical activities. However, due to the complexity of the human body structure, the finite element analysis method has certain limitations. The model is simplified and the material properties differ, which may lead to errors in the experimental results. However, because the finite element method is relatively simple, it can explain some practical problems and plays a very important role in the clinic.


In vitro, activated GRK2 phosphorylated WT IL3 and WT CT proteins but not Ala-substituted forms. TAS2R14s with mutations in IL3 (IL-5A), CT (CT-5A), and in both regions (IL/CT-10A) were expressed in human embryonic kidney 293T cells. IL/CT-10A and CT-5A failed to undergo desensitization of the intracellular calcium response compared with WT, indicating that functional desensitization by GRK phosphorylation is at residues in the CT. Desensitization of TAS2R14 was blocked by GRK2 knockdown in human airway smooth muscle cells. Receptor:β-arrestin binding was absent in IL/CT-10A and CT-5A and reduced in IL-5A, indicating a role for IL3 phosphorylation in the β-arrestin interaction for this function. Agonist-promoted internalization of IL-5A and CT-5A receptors was impaired, and they failed to colocalize with early endosomes. Thus, agonist-promoted functional desensitization of TAS2R14 occurs by GRK phosphorylation of CT residues and β-arrestin binding. However, β-arrestin function in the internalization and trafficking of the receptor also requires GRK phosphorylation of IL3 residues.For most G protein–coupled receptors, the third intracellular loop (IL3) and carboxy-terminal tail (CT) are sites for G protein–coupled receptor kinase (GRK)–mediated phosphorylation, leading to β-arrestin binding and agonist-specific desensitization. These regions of bitter taste receptors (TAS2Rs) are extremely short compared with the superfamily, and their function in desensitization is unknown. TAS2R14 expressed on human airway smooth muscle cells relax the cell, suggesting a novel target for bronchodilators. To assess IL3 and CT in agonist-promoted TAS2R14 desensitization (tachyphylaxis), we generated fusion proteins of both the WT sequence and Ala substituted for Ser/Thr in the IL3 and CT sequences.  These e protein , 11, 12.ocytosis . This incoupling , 15, 16.coupling , 18. Thu2+]i, causing hyperpolarization of the HASM cell membrane and marked HASM relaxation , a widely expressed receptor that may play a role in metabolic and inflammatory diseases, and has been considered a target for a new class of bronchodilators because of its expression on human airway smooth muscle (HASM) cells. In these cells, TAS2R14 agonists stimulate i response . On the other hand, the third loop mutant IL-5A displayed [Ca2+]i responses that were similar to R14-WT, with no statistical difference in the slopes . Taken together, these results suggested that despite both regions displaying GRK-mediated phosphorylation in vitro, functional desensitization of TAS2R14 requires only phosphorylation of one or more of the C-terminal Ser residues.To ascertain the functional effects of potential GRK phosphorylation of TAS2R14, three mutant receptor complementary DNAs were constructed substituting Ala in the encoded receptor for all Ser/Thr in the IL3 (IL-5A), the cytoplasmic tail (CT-5A), or both (IL/CT-10A). WT and mutant receptors were expressed in HEK-293 cells, and i responsiveness in siCtrl- and siGRK2-transfected cells (A). The [Ca2+]i response to agonist (B) was markedly altered by the GRK2 knockdown, revealing a pattern consistent with less desensitization, with mean slope of −0.03 ± 0.35 versus −1.74 ± 0.03 for siCtrl-transfected cells (p < 0.001).To further implicate GRK phosphorylation as a mechanism of rapid desensitization, and, to ascertain relevance in a cell type of interest, cultured HASM cells were utilized to ascertain i in microdomains acts to hyperpolarize the cell membrane and relax the cell  as well as a small cytoplasmic C terminus . Thus, TAS2R14 is compact, and prior to this study, it was not clear whether a GRK-based mechanism was in play with this receptor with such small intracellular domains. We found that using TAS2R14 peptides that purified activated GRK2 phosphorylates both the intracellular loop and cytoplasmic tail regions in vitro and failed to do so with the same peptides having Ala substituted at the Ser/Thr candidate sites. In the in cellulo studies, mutation of the five phosphoacceptor sites in the third loop along with the five in the C terminus (IL/CT-10A) ablated desensitization. And, knockdown of GRK2 resulted in a signaling phenotype of WT receptor that was quite similar to IL/CT-10A . Furthermore, β-arrestin recruitment, and binding, to TAS2R14 was absent with this mutated receptor. Since we have previously shown that agonist-initiated TAS2R desensitization is insensitive to PKC inhibitors  are the only direct bronchodilators available. The clinical need for a different class of direct bronchodilators to treat obstructive lung diseases such as asthma has been promoted for a number of years (2AR) prompted studies to assess their function in HASM, as potential therapeutic targets with an alternative mechanism of action  using site-directed mutagenesis methods as previously described  or Lipofectamine 2000 (Thermo Fisher Scientific) as described , was treated with isopropyl β-d-1-thiogalactopyranoside to induce protein expression. Protein purification was performed using glutathione Sepharose 4B  as previously described (GST fused TAS2R14 IL3 and cytoplasmic tail complementary DNA was generated using TAS2R14-WT and TAS2R14 to 10A as templates. GST-TAS2R14-IL3–specific primers were 5′cgcggatccatgatcttctccatgtgg3′ (forward) and 5′ccgctcgagtttaactcctctgtgggc3′ (reverse). GST-TAS2R14-CT–specific primers were 5′cgcggatccatgaacaagaagctgagac3′ (forward) and 5′ccgctcgagagatgattctctaaattct3′ (reverse). The PCR products were cloned into the prokaryotic expression vector pGEX-4T1  at the escribed .32P-ATP (PerkinElmer) and 10 μM of unlabeled ATP in 30 μl of buffer containing 20 mM Tris–Cl (pH 8.0), 4 mM MgCl2, active GRK2 recombinant protein , and the GST-tagged fusion proteins as substrates. After incubation at 30 °C for 30 min, the reactions were stopped by adding protein loading buffer and the proteins electrophoresed through 12% SDS-PAGE gels. The relative amounts of incorporated radioactivity were determined by autoradiography and quantitated by Image J . Negative controls consisted of the reaction without GRK and the reaction in the presence of activated protein kinase B (where the peptides have no predicted phosphorylation sites for this kinase).Assays were performed with 10 μCi of γ-2+]i signal after the peak response (which occurs at 10 s after agonist exposure) to 50 s, and the slope of the decrease was determined for each experiment and used for statistical comparisons between mutant receptors.Transfected cells were transferred onto eight-well chamber slides and 48 h later loaded with Fluo-4 followed by treatment with 200 μM of the agonist DPD for the indicated time. Images were acquired using the EVOS Cell Imaging System (Thermo Fisher Scientific) with excitation at 488 nm and a 515 to 540 nm emission filter. Desensitization was defined as the decrease in the i data from 10 to 50 s were fit by linear regression. For each individual experiment, the slopes were calculated based on the best fit regression and were compared by t tests. Unless otherwise stated, p values <0.05 were considered significant. Bar graphs are presented as mean ± SD along with the results from the individual experiments.Bands from Western blots were quantitated by ImageJ, and the pixel density was compared by paired or unpaired All data described in the article are present in the main text, figures, and the The authors declare that they have no conflicts of interest with the contents of this article.


We proved that Fyn tyrosine kinase elicits APP phosphorylation on Tyr682 residue, and we reported increased levels of APP Tyr682 and Fyn overactivation in AD neurons. Here, we want to contemplate the possibility of using fibroblasts as tools to assess APP Tyr682 phosphorylation in AD patients, thus making the changes in APP Tyr682 phosphorylation levels a potential diagnostic strategy to detect early pathological alterations present in the peripheral cells of AD patients’ AD brains.Alzheimer’s disease (AD) is a devastating neurodegenerative disorder with no cure and no effective diagnostic criteria. The greatest challenge in effectively treating AD is identifying biomarkers specific for each patient when neurodegenerative processes have not yet begun, an outcome that would allow the design of a personalised therapeutic approach for each patient and the monitoring of the therapeutic response during the treatment. We found that the excessive phosphorylation of the amyloid precursor protein (APP) Tyr Alzheimer’s disease (AD) is a prominent neurodegenerative disease which takes a major toll on the elderly and places an enormous burden on the healthcare system.The amyloid precursor protein (APP) is one of most extensively studied molecules in AD, and its cleavage, mediated by β-site APP-cleaving enzyme 1 (BACE1 or β-secretase) and by γ-secretase, leads to the dysregulated production of Aβ peptides, which mostly accumulate at central synapses [682 residue in the processes responsible for the generation of Aβ peptides in human neurons [682 residue is located in the highly conserved 682YENPTY687 motif, which binds specific adaptor proteins depending on its phosphorylation/dephosphorylation state [682 phosphorylation prevents the APP from binding to the Clathrin and AP2 endocytic proteins [682 residue in neurons differentiated from neuronal stem cells of AD patients [682 phosphorylation might be an early marker of neuronal degeneration [682 phosphorylation, that in turn triggers biochemical events downstream, finally resulting in Aβ production and neuronal death [682 phosphorylation and Aβ release in AD neurons [682 residue as a biomarker to identify patients with high levels of APP Tyr682 phosphorylation that may benefit from personalised treatments with Fyn TKI.Our group have previously underlined the crucial role of APP Tyr neurons . APP Tyron state . The APPon state ,6,7. In proteins , and affproteins . Consequproteins . Notablypatients ,10 preceneration ,6. Furth neurons . These fWe found elevated APP Tyr phosphorylation levels and Fyn overactivation both in skin fibroblasts and cortical tissues of minipigs carrying PSEN1 M146I mutation . The inchttps://catalog.coriell.org/). All fibroblast cultures derived from an ante mortem skin biopsy from the forearm, and had approximately the same biological age in culture (number of passages of cell lines and cell divisions). Fibroblast cell cultures grew very slow, but they all showed the same morphology and shape in culture. No differences were found in the extent of neuronal survival or proliferation. In addition, Aβ40 and Aβ42 were undetectable in media of fibroblast cultures, because the levels appeared below the sensitivity of a commercially available ELISA kit (data not shown).Human fibroblasts were purchased from the Coriell Institute  and cultured following the guidelines reported on the manufacturer’s web site , using the source number (#) reported in Some more information about patient clinical features and specific characteristics of the fibroblast cultures are available on the Coriell online catalogue , mouse anti-p-Tyr  and monoclonal anti-β-actin-peroxidase  antibodies.Fibroblasts were kept in culture until cell confluence was reached. Lysates were firstly processed for IP (pTyr) using mouse anti-p-Tyr magnetic beads, and subsequently analysed by WB. For pTyr protein enrichment, we used Phospho-Tyrosine Mouse mAb Magnetic Bead Conjugate (P-Tyr-100) . The samples were loaded on a 4–20% precast gel. Western blot analysis was performed using anti APP, clone Y188 , rabbit anti-pan Fyn , rabbit anti-Src pTyrData are expressed as mean ± standard error of the means. All the experiments were performed at least three times. The appropriate statistical test was selected using GraphPad Prism software (version 9.0c) ; details are reported in the legend for each figure.In the study, we included fibroblasts from ante mortem skin biopsies of 14 AD patients, six unaffected spouses, and four healthy donors with a familiar history of AD. In addition, four patients with a diagnosis of dementia, three associated to Parkinson’s disease (PD) and one to epilepsy were included. Post-mortem histopathology brain examinations were performed in all patients with mild cognitive impairment or dementia diagnosis to confirm AD.682 residue (APP has indeed other two Tyr(s) whose changes in the phosphorylation can also easily be detected by the same procedure), previous findings from our group proved that only APP Tyr682 residue phosphorylation is crucial in initiating APP amyloidogenic processes in AD neurons, whereas Tyr phosphorylation at different APP sites does not affect the extent of A𝛽 production or accumulation [420 phosphorylation was also assessed as an indirect measure of Fyn TK activity [The levels of phosphorylation of APP Tyr were assessed using the same procedure previously applied to neurons ,3 and comulation . The incactivity , and theWe found a significant increase in the level of APP Tyr phosphorylation in the majority of fibroblasts from AD patients when compared to healthy donors, as well as patients with other dementias . In part420 phosphorylation levels  and interacting directly with tau [18 [682 residue, which would then induce the amyloidogenic cleavage [682 phosphorylation levels are elevated, thus preventing off-target effects, and improving the outcome of the disease.It is well established that Fyn can play a dual role in both the Aβ and tau pathologies observed in AD , exacerbwith tau  by phosp tau [18 . We prevcleavage . This cacleavage . This ob682 phosphorylation in AD neurons opens the scenario to either test already commercially available Fyn TKIs or develop more specific compounds that can potentially control Fyn hyperactivity in AD patients and prevent Aβ42 production in patients in whom APP Tyr682 phosphorylation is increased.Indeed, the identification of Fyn as an actor in APP Tyr682 phosphorylation.Overall, the observations reported here prospect the possibility that events related to APP Tyr phosphorylation in fibroblasts may reflect Aβ related abnormalities in the brain, and that, if correlated with changes in cognitive performance and with other biomarkers including neuroimaging approaches or to the levels of Aβ and phosphorylated tau in biofluids, may allow the identification of patients who could benefit from a personalised pharmacologic approach, by using Fyn TKI. Indeed, these results underline the need to extend this evidence to a larger number of samples/fibroblasts, as well as to develop a procedure selective and quantitative for the detection of the levels of APP Tyr682 phosphorylation levels in peripheric cells, such as fibroblasts, as a potential diagnostic approach to design personalised therapy in patients in which APP Tyr682 phosphorylation is increased. Indeed, further studies will be necessary to extend this analysis to a larger number of patients.In conclusion, this study proposes to use changes in APP Tyr


The megalithic jar sites of Laos (often referred to as the Plain of Jars) remain one of Southeast Asia’s most mysterious and least understood archaeological cultures. The sites, recently inscribed as UNESCO World Heritage, host hollowed stone jars, up to three metres in height, which appear scattered across the landscape, alone or clustered in groups of up to more than 400. Until now, it has not been possible to estimate when the jars were first placed on the landscape or from where the stone was sourced. Geochronological analysis using the age of detrital zircons demonstrates a likely quarry source for one of the largest megalithic jar sites. Optically Stimulated Luminescence (OSL) dating suggests the jars were positioned at the sites potentially as early as the late second millennium BC. Radiocarbon dating of skeletal remains and charcoal samples places mortuary activity around the jars from the 9-13th century AD, suggesting the sites have maintained ritual significance from the period of their initial placement until historic times. Northern Laos is home to one of Southeast Asia’s most enigmatic archaeological cultures. The megalithic jar sites of Laos comprise one to three-metre-tall carved stone jars dotted across the landscape, appearing alone or in groups of up to several hundred. The majority of these sites are found in Xieng Khouang Province, and while collectively termed the ‘Plain of Jars’, the sites are mostly located on mountain ridges, saddles or hill slopes surrounding the central plain and upland valleys. While brought to the attention of Western scholars in the late 1800s ,3. Sincehttps://whc.unesco.org/en/list/1587/). This recent research identified varied mortuary practices at Site 1  and similar evidence for burial markers at the sites 2 and 52 although human bone was not recovered at these latter two sites, likely due to taphonomic processes. How the burials uncovered at Site 1 relate to the megaliths is unclear as the timing of the placement of the jars has, until now, proven difficult to estimate.In recent years archaeological research conducted by the authors has resulted in the excavation of selected areas at three jar sites and broader surveys of the region, increasing our knowledge of the sites and their enduring ritual significance –8. The tIn this paper we start by briefly outlining what is known about the sites based on limited previous research and more recent excavation and research conducted by the authors. Many questions remain however, regarding the megaliths of Laos such as, the date when the megaliths were placed in their current location  and the likely provenance of the stone used to create the jars at Site 1.To establish a chronological framework for the sites and mortuary activity we present radiocarbon dates from Site 2 (none were viable for Site 52), and compare these with those previously obtained from Site 1 . In an etriandra) . TheSite 21 (N 19°28’50” E 103°05’15”), c. 8 km from Site 1 has been identified as a quarry site and may have been the source of stone for the jars at Site 1 . The quaThrough the combination of radiocarbon dating of charcoal and bone, OSL dating of sediments and U-Pb dating of zircons within the stone from which the jars are made, we have sought to expand our knowledge regarding the provenance of the stone from which the megaliths are fashioned, the timing of their placement and the relationship between the megaliths and the surrounding mortuary activity. Permits to work at the megalithic sites were obtained from the Lao Department of Heritage, Ministry of Information Culture and Tourism, Lao PDR.At Site 1, excavations in 2016 were conducted over three units ,6. In 2013C measured by AMS [Charcoal samples from Sites 1 and 2 were submitted to the Australian National University (ANU), Canberra. After physically cleaning individual fragments with a scalpel, charcoal was pretreated using an acid-base-acid (ABA) protocol. For bone samples, collagen was extracted and purified according to an ultrafiltration protocol. An HCl solution was used to remove the bone mineral and exogenous carbonates, and alkali was used to remove humic substances. The sample was then gelatinized by heating in a weak HCl solution. The resulting soluble gelatin allows for larger insoluble particles to be removed with a pre-cleaned filter, followed by a second filtering with a pre-cleaned >30K Dalton molecular weight cutoff size ultrafilter to remove smaller-sized exogenous material . Both bod by AMS . Calibrad by AMS ,19,20.terminus post quem for jar emplacement, OSL samples were taken from sediments underneath two jars at Site 52 in 2017 (052030064 located at N19.49553698 E103.4319365 and 052020052 located at N19.49593697 E103.4328666) and two jars at Site 2 in 2019 (W0013 located at N19.31881937 E103.15284144 and W0021 located at N19.31880992 E103.15278109). Such dating provides an estimate of when sediment was last exposed to light, which, in near surface sediments, will reflect the age of primary sediment deposition or cessation of reworking [In an effort to establish a eworking  or a ble2O2), followed by sedimentation in acetone, a further acid digestion  evaluation using the Single-Aliquot, Regenerative-Dose (SAR) protocol [e values. Repeat regenerative-doses [e values for each sample were calculated using the Central Age Model [r) values [r modulation forced by grain size [Owing to the clayey-silt texture of the deposits, quartz in the fine silt (5–15 μm) fraction was isolated; this involved acid and alkaline digestion  values , accountain size , presentr values were calculated on the basis of sample depth, geomagnetic latitude and matrix density [e and Dr and expressed relative to the year of sampling. Since the number of grains in the fine silt aliquots is many magnitudes greater than a fine sand aliquot, there is almost no overdispersion/difference in age between aliquots, and overdispersion in the data is not a problem.To assess the influence of jar material on the gamma dose rate within 15cm of the sediment-artefact interface, a basal fragment of jar 052020052 at Site 52 (above sediment sample GL17084) was collected. The geometry of the jar’s base  suggests c. 50% of the gamma radiation field interacting with the OSL sample immediately beneath would be located within the jar. The fractional gamma dose rate function in the R Luminescence package ,37 was u density . Age est238U to 206Pb, half-life 4460 Ma; 235U to 207Pb, half-life 700 Ma) provides two independent chronometers that help to assess the integrity of the parent-daughter systems and ages. Importantly, U-Pb dating of zircons can be applied to rocks from the Earth’s earliest history to rocks only a few hundreds of thousands of years old.U-Pb dating of zircon has long been a preferred and reliable method for establishing igneous and metamorphic ages of crustal (felsic) and—in the rare instances where zircons exist–in mantle (mafic) rocks. Zircon is the ideal mineral for U-Pb dating as it includes significant amounts of uranium but, crucially, excludes initial Pb in the original structure. The decay of the U to radiogenic Pb through two separate decay schemes  [e.g. [The development of  SHRIMP)  and subs SHRIMP)  instrumeP) [e.g. ), includet al. [Although geologists have used detrital zircon U-Pb dating for several decades, this approach has only recently been used to establish provenance of ceramic and stone sources in the archaeological environment. In 2010, author R. Armstrong and M. Leclerc (unpublished) unequivocally established a local source for ceramic sherds in Vanuatu using zircons extracted from volcanic tuff horizons and small sherds. Temper sands were sourced in the Solomon Islands, southwest Pacific  and moreet al.  used U-POne of the unsolved issues in studies of the Plain of Jars is the source of the carved rock jars and how these were transported to their present sites. As the vast majority of the jars are carved from sandstones or other sediments of similar mineralogical composition, it was clear that use of the U-Pb zircon ages in the manner described above, could help to match jars to their original sources or outcrops.in situ, incomplete jar (sample 12) measuring 9 x 8 x 6 cm and a further piece of natural sandstone (sample 13), measuring 18 x 12 x 8 cm, from the same locale.The quarry for Site 1 jars has been suggested to be Site 21 (Poukeng). In order to test this supposition we collected samples from both sites. With permission from the Lao Department of Heritage, a geological hammer was used to remove a small, 5 x 11 x 4 cm sample from a damaged jar, 1020102, at Site 1 (sample 15). Two samples were taken at Site 21; one from a cracked, but 204Pb and the relevant common Pb compositions from the Stacey and Kramers model [204Pb corrected 206Pb/238U system because of the errors that are associated with low yields of 204Pb, 207Pb and 208Pb from relatively young zircons. All ages older than 800 Ma are reported using 204Pb corrected 207Pb/206Pb ratios. The 207Pb/206Pb ages that are >15% discordant are not plotted. The ages are plotted on cumulative probability plots  excavated from beneath one of the jars at Site 1, Group 2 (Jar 01020061 located at N19.431023 E103.152399) returned a date of 1168–1264 calAD providing a possible Charcoal obtained from Site 2 rendered dates ranging from 7583–7483 calBC (ANU62920) to recent . A charcr function in the R Luminescence package [r at the jar-sediment interface is 2.2% lower than that at 0.3 m depth beneath the jar, well within the bounds of uncertainty for gamma Dr associated with GL17084. Therefore, the radiochemistry, breadth and thickness of the jar’s base yields a relatively homogenous gamma field enveloping the OSL sample located directly beneath the jar.OSL age estimates and associated data are summarised in Tables  package ,37, the r alone. Assuming the radiochemistry of the sandstone comprising the jars is similar between Sites 2 and 52, the assessment of total Dr should be considered a minimum value and the corresponding OSL age a maximum value.The jar bases could not be sampled at Site 2, so the OSL age estimates of these samples are based on lithogenic and cosmogenic DThe source of the fine quartz silt beneath the jars at Sites 2 and 52 was probably wind and rain-borne dust. This is consistent with the slow accumulation rate at Site 2 (<0.05 mm/yr) demonstrated by the difference in age between the samples from 20 cm and 30 cm depth , even though the reliability of the age estimates is compromised by poor dose recovery ratios (significantly less than unity) and, for sample GL17084, significant U disequilibrium. These ages are far in excess of those previously reported for human activity associated with jar sites; the jars were likely worked with metal tools that were not in use in mainland Southeast Asia until the second millennium BC ,51. The At Site 2, the consistent age of the sediment at 20 cm depth beneath the two jars 6.3 m apart  from Site 21, which is c. 8 km to the northwest of Site 1 and is the closest quarry to the site –6. Zirco Isoplot  it is poThe zircon age distributions in all three samples show that they have a very similar provenance. These data also show that the jar 01020102 from Site 1 matches that from the sandstones and unfinished/abandoned jars from Site 21, suggesting this outcrop is the likely source of the material . Minor din situ preserved inside a jar.Archaeological research including excavation and survey conducted since 2016 has increased our understanding of the enigmatic megalithic culture of northern Laos. Through comparative typological analysis of the megaliths, similarities in morphology and stylistic treatment such as mouth shape, rim form  and geneWith excavation we have established that the sites are associated with mortuary ritual, and have documented divergent mortuary practices at Site 1 comprising primary, secondary and ceramic burial jar interments ,6. DatinThe dating of the placement of the stone jars has, until recently, proven difficult not least because of the lack of associated organic material. A radiocarbon date obtained from charcoal from beneath one of the megalithic jars (W0018) at Site 2 and the OSL dates obtained from beneath two other jars (W0013 & W0021) at the same site, indicate that the jars were likely placed in their current location potentially as early as the late second millennium BC. A radiocarbon date obtained from charcoal from beneath one of the jars at Site 1 suggests a later event, in the 12th or 13th century AD . This laThe data presented here strongly suggests that the placement of the megaliths preceded the mortuary activity around the jars, indicating re-use of the sites and enduring ritual significance. While a human cranial sample obtained by Sayavongkhamdy  returnedThe U-Pb dating undertaken on the zircons from a jar from Site 1 matches the dates obtained from rock and an unfinished jar at Site 21 (Phoukeng quarry), the presumed quarry. While this does not preclude that sandstone of similar age exists in other areas accessible to the site, it is the only known quarry site in close proximity to have been identified to date. Extensive geological mapping of the region is hindered by the lack of high-resolution maps and foot survey is not possible in many areas around the site due to UXO. How the jars, some estimated to weigh more than 30 tonnes, were transported from the quarry to their final position is unknown, though is likely to have required a substantial workforce. Phoukeng quarry is separated by 8 km of undulating terrain from Site 1. Whether the completed jars were dragged on some form of wooden rollers or sledge remains speculative.The megalithic sites of Laos have seen sporadic efforts in archaeological research since the 1930s, with more recent excavations at three of the main sites, namely Sites 1, 2 and 52. This research has created a deeper understanding of the prehistoric and historic cultures of Xieng Khouang Province. Dating for the placement of the jars and that of skeletal material found buried around the megaliths indicates multiple use of the sites and enduring ritual significance. While Colani  posits aAlthough the original purpose of the megalithic jars remains to be determined, the present research indicates a long history of activity at the sites. The evidence provided by OSL dating has provided the first ever dates for the original placement of the jars at Site 2–1240 BC to 660 BC. While the broad similarity in megalith morphology across Laos might suggest contemporaneity and the expression of a unique, yet to be identified, cultural group, more research needs to be conducted. Future studies might usefully be directed at obtaining further samples from under, and at the sediment-artefact interface with the megalithic jars, at other sites and from across the geographic extent of the culture, using OSL to refine the earliest jar emplacement date. Efforts to ascertain the original purpose of the megaliths themselves should be directed at undisturbed sites or newly recorded sites and those with concealed contents or buried megalithic jars, instances of which are known.S1 FigThe locations of the SHRIMP spots are shown by the yellow ellipses and the labels refer to the data in the relevant tables.(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table*Relative misfit.(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Atmospheric immersion freezing (IF), a heterogeneous ice nucleation process where an ice nucleating particle (INP) is immersed in supercooled water, is a dominant ice formation pathway impacting the hydrological cycle and climate. Implementation of IF derived from field and laboratory data in cloud and climate models is difficult due to the high variability in spatio-temporal scales, INP composition, and morphological complexity. We demonstrate that IF can be consistently described by a stochastic nucleation process accounting for uncertainties in the INP surface area. This approach accounts for time-dependent freezing, a wide range of surface areas and challenges phenomenological descriptions typically used to interpret IF. The results have an immediate impact on the current description, interpretation, and experiments of IF and its implementation in models. The findings are in accord with nucleation theory, and thus should hold for any supercooled liquid material that nucleates in contact with a substrate. As such, the available liquid volume or ice nucleating surface of an ice nucleating particle (INP)3 that initiates ice nucleation is miniscule. Predicting ice crystal formation on INPs remains a great challenge in climate models due to large uncertainties,5 despite the importance of ice formation in atmospheric processes and climate.The formation of ice crystals in the atmosphere has important consequences for the radiative and precipitation properties of clouds, thus for the climate system and the hydrological cycle.3 The dominant ice formation pathway in mixed-phase clouds, where ice crystals and supercooled droplets coexist, is immersion freezing (IF) where an insoluble INP is immersed in a supercooled water droplet.7 IF is also significant for cirrus cloud conditions.8 A conundrum in cloud microphysics studies is that the observed number of INPs does not match the observed number of ice crystals in clouds6 as is the case, e.g., for long-lived Arctic mixed-phase boundary layer clouds.9 Secondary ice formation processes may account for enhanced ice crystal numbers in some instances.10 However, laboratory and field observations are limited, and these secondary processes may only occur at specific temperature and droplet size ranges.16 Detailed cloud modeling studies indicate that ice activation should persist over long time to maintain the observed cloud structure and the amount of glaciation.19 In other words, continual ice nucleation may explain the observed discrepancy between INP number concentration and the actual ice crystal number concentrations with potential consequences for explaining observed cloud structure and precipitation amount. Online measurements of INP concentrations in a volume of air are commonly conducted by instantly exposing aerosol particles to supersaturated conditions with respect to water or ice at some temperature, T, for a short time (~10 s).21 This leads to cooling or humidification rates that are significantly faster than those experienced in the atmosphere, and lead to nucleation time scales much smaller than typical cloud formation time scales. Therefore, neglecting the time dependence of ice nucleation is not possible.Different pathways exist for heterogeneous ice nucleation in the atmosphere.ns, in units of cm−2. This mathematical normalization describes the number of observed nucleation events per unit INP surface area (ISA) at a given temperature, ns(T), and assumes that each nucleation event equates to one ice-active site.7 However, this framework neglects that nucleation is fundamentally a stochastic process based on fluxes of molecules to and from a critical cluster, and thus is inherently time dependent24 (see Laboratory ice nucleation data are often interpreted using a time-independent concept of ice nucleation active site (INAS) densities, nt24 see .ns-based description employs an ad hoc parameter that can be used to compare studies, but in fact is not a physically-rooted parameter.25 As such, ns is not a theoretically testable dependent variable. After decades of advancements in ice nucleation experiments and molecular dynamics simulations, a scientifically accepted framework that represents IF by INAS or describes which microscopic physicochemical parameters define an ice-active site has not yet emerged.8 Nanoscale characterization of particles is certainly difficult and has been the subject of entire studies.27 Despite great advances in spectro-microscopic analyses,30 observation of ice nucleation in situ on the nano-level  to unambiguously characterize ice-active sites has not been accomplished.31 Recent X-ray and electron spectro-microscopic observations of ice crystal formation on surfaces provide varying evidence. Ice crystals formed on preferable crystal faces of laboratory standard mineral particles28 and in surface defects on cleaved mineral sheets31 support the active site description. In the atmosphere, INPs and ice crystal residues are more physicochemically complex, and cover a range of particle types present in significant numbers in the ambient particle population.34 This supports the notion that the ice formation is also related to particle abundance or the total surface area. This exemplifies the challenge in linking surface characteristics of INPs to underlying ice nucleation physics. Clearly, different surfaces can interact with the surrounding water molecules differently.36 However, application of an arbitrary variation of these properties in the form of different ns values to describe the temperature or time dependence of IF of same INP type is not theoretically established, thus eliminating to have a predictive framework for atmospheric ice nucleation.The Jhom in units of cm−3s−1, and Jhet in units of cm−2s−1, respectively, to account for the dependence of nucleation probability on liquid volume or ISA and time.23 As a direct result one obtains when increasing ISA, the probability to nucleate ice increases exponentially.23 Classical nucleation theory (CNT) extends nucleation theory by assuming a macroscopic density and surface tension of water molecular clusters with their energy following a Boltzmann distribution.23 In the case of pure water droplets, Jhom and Jhet depend on T, while in aqueous solutions, they additionally depend on solute concentration or water activity.39 In other words, for a given temperature, water activity, and INP type, one Jhet value exists.Nucleation theory invokes a homogeneous and heterogeneous ice nucleation rate coefficient, 39 shown conceptually in Jhet. It is, hence, suggested that application of Jhet that captures the time dependence of nucleation would be a superior approach to describe ISO experiments. From observations of nucleation, one derives25Nufz is the number of unfrozen droplets, Ntot is the total droplet number examined, A is the ISA in a droplet, and t is the time the droplets stay liquid. For the same ISA in each droplet and a fixed Jhet, Nufz/Ntot) versus time should follow a straight line for ISO experiments as illustrated in 25 have been observed to be curved as indicated by the green and red curves in 8 However, UnF results deviating from 25Jhet, and ns.There is a clear experimental evidence for a time-dependent behavior of IF,39 when ISA increases, the probability to nucleate ice also increases. Contrary to assumptions of previous studies, we put forth that any experiment examining freezing droplets with immersed INPs cannot prepare them with identical ISA. In other words, the distribution of ISA per droplet will have an uncertainty or variability and therefore, the ice nucleation probability will also vary between the droplets. We only assume that the material- specific surface dominating features that make one material a better or worse INP compared to others, increase by the same amount as ISA increases and nucleation occurs randomly. We do not consider varying ice-active sites on the ISA per droplet. This alternative approach to interpret data from ISO and CCR experiments avoids empirical distributions of INAS . In our model, we executed 105 simulations of each ISO and CCR experiment. In each simulation, we sampled the ISA per individual droplet and the number of droplets frozen at each time or temperature interval. UnF and FF were calculated for each single simulation, which is analogous to a single experiment with the same Ntot. Due to random sampling of ISA, UnF, and FF for one simulation was never the same as another and, therefore, the 5th and 95th percentiles of the simulations were used for comparison with our data. The model uses the measured Brunauer–Emmett–Teller (BET) ISA  and  ISA see  as the c ISA see . We alsoJhet values.Jhet following the water activity-based immersion freezing model (ABIFM)39 with fitted constants given in Jhet in the model. However, any other parameterization of Jhet would also be applicable  in accordance with nucleation theory. ISO experiments using relatively lower ISA result in freezing at lower temperature, and, expectedly, our model concurrently yields higher Jhet values. The fiducial limits shown in Jhet values but more importantly, they represent the maximum allowable range of freezing events in a given time and temperature interval, ultimately defined by the number of droplets used in the experiment , smaller relative uncertainties in ISA result from larger droplets. This implies that experiments using micrometer-sized droplets or sub-micrometer aerosol particles would be subject to greater ISA variability. However, typical experimental procedures result in other sources of ISA variability for stock solutions, large volume aliquots, and small droplet volumes, which include particle settling and aggregation, and non-uniform or standardized sampling. The ISA variability may exacerbate when working with ambient samples, either when washed off a substrate or after droplet formation on a substrate. Particle sizes collected on filters can span orders of magnitude. Impaction also leads to a significant size variability depending on cut-point, knowledge of particle aerodynamic diameter, and particle bounce.43 Fundamental to this approach is that particles are given a bipolar charge distribution, and assuming identical ISA will influence data interpretation potentially leading to incorrect conclusions44 due to aerosol multiple charging. 45 when selecting particles with an electrical mobility diameter of 250 nm at 5 × 104 cm−3. Due to multiple charging, particles larger than 1000 nm are also present and the total ISA is twice that of ISA calculated when assuming a monodisperse PSD at 250 nm. This represents an idealized case of perfectly spherical particles with the same charging efficiencies and thus represent the minimal PSD width.46 Of course, solid and crystalline particles are likely non-spherical, thus, enhancing PSD width. One study attempted to remove multiply charged particles with a cascade impactor before attempting to establish a bipolar equilibrium charge distribution with a charge neutralizer.44 The authors also measured the size distribution of particles by a different method based on the supersaturation threshold to act as a cloud condensation nuclei. When comparing the measured PSD with the expected bipolar charge distribution, the ISA distribution width was much larger than expected despite employing an impactor.25T = 258.15 K). Using Jhet = 107 cm−2 s−1 and an activation time of 10 s, which is typical of continuous flow diffusion chambers,47 we obtain FF = 0.3. We plot the contribution of each multiple charged particle size to the FF in Jhet or ns, inaccurate assumptions about ISA lead to substantial miscalculation and misinterpretation of nucleation kinetics. We conclude that identical particle size should not be assumed when using mobility-selected particles in a continuous flow for derivation of ice nucleation kinetics. This demonstrates that it is important to measure the actual PSD or at a minimum, to use the bi-polar charge distribution to derive the ISA per particle.Selecting a monodisperse aerosol using an electrostatic particle classifier is a typical approach in ice nucleation studies.Jhet, ns, and similar types of parameterizations that depend on ISA. Furthermore, application of FF data to infer nucleation kinetics is prone to uncertainties. FF and UnF data represent a cumulative dataset, by definition. As such, uncertainties in single data points result in large uncertainties due to error propagation . If CNT describes IF correctly, UnF from ISO experiments for a perfect INP would be log-linear and FF from CCR experiments would have a typical sigmoidal curve (“S” shape) as a function of temperature. However, as shown in To improve our understanding of the underlying processes of ice nucleation, a reference INP with defined ISA has to be developed.ns approach, a perfect ice-active particle would have sites that all activate at an identical characteristic temperature, Tc. Freezing for T > Tc would be impossible as shown in the CCR simulation in Tc = 261.5 K. In Tc = 261.5 K are very different from our current experimental findings.Following the singular 31 used highspeed optical microscopy to identify ice nucleation on solid substrates in contact with millimeter-sized droplets and observed that crystallization occurred mostly in the same spot for sequential freezing and melting cycles. Using scanning electron microscopy and atomic force microscopy, they found that micrometer-sized morphological defects were present where ice nucleation occurred.31 We follow the authors and interpret this single defect as hosting a single ice-active site. The measured FF of a single site31 is plotted in Tc but deviates around 2 K. The authors suggest that there is some freezing variability around Tc, however, this is only a phenomenological description without yet a physical basis.52 Therefore, the FF of this single site is more in line with a time-dependent and stochastic freezing process.The question remains, do well-defined, favorable ice-active sites exist on INPs and if found, would one of them always nucleate ice at the same temperature? Recently, Holden et al.het parameter as a function only of temperature. This finding challenges the role of active sites and the application of a distribution of active sites with varying ice formation potential to interpret the freezing trends in heterogeneous ice nucleation experiments. We also challenge future heterogeneous ice nuclea- tion studies to strictly characterize their data variance and encourage its quantitative explanation. The major advantage of this approach is that it depends on a rigorous uncertainty analysis of the ice nucleating surface area, ice nucleation statistics and quantifying the nanoscale-characterization of INP surfaces, all of which can be (in principle) directly measured. Doing so will greatly enhance the understanding of ice nucleation and move into new and practical directions to improve prediction of ice nucleation using a fundamental theory, developing new theoretical extensions based on physical parameters as done in previous literature,57 and foster new technological innovation in surface science and engineering. We expect that our results also hold when analyzing nucleation from any supercooled liquid when in contact with a nucleating substrate, thereby greatly extending the applicability and falsifiability of our proposed approach.We presented an IF interpretation that unambiguously demonstrates that the nucleation kinetics analysis has to consider the stochastic nature of freezing and varying ISA among droplets. The insight gained from our analysis challenges several aspects commonly employed in IF studies, including its mathematical description and the lack of a time-dependent freezing process. All of our data scatter is explained by stochastic freezing and a J58 The WISDOM setup and validation are described in Reicher et al.5959 and Zipori et al.16The WeIzmann supercooled droplets observation on a microarray (WISDOM) is a microfluidic device based on the design of Schmitz et al.−1 to ~8 K higher than the expected start of the droplets’ freezing. Subsequently, a slower cooling rate of 1 K min−1 was applied to monitor the individual droplet freezing events. After freezing, the droplets were heated and the ice melting point was measured at a heating rate of 1 K min−1. In ISO freezing experiments, the droplets were cooled from 273.15 at 5 K min−1 to the temperature at which ~10% of the drops froze. From this point on, the temperature was held constant for 2 h. For direct comparison of nucleation data, we conducted both CCR and ISO freezing experiments with the same droplet samples.We performed two types of experiments: CCR and ISO freezing experiments. In CCR experiments, the droplets were cooled at 20 K min25 is a statistical Monte Carlo model made up of numerous simulations. Nucleation is represented by binomial statistics following Koop et al.22 Model input includes the number of droplets, ISA, and Jhet as a function of temperature parameterized following ABIFM.39 Modeled UnF or FF were fitted to all datasets globally with five optimizable parameters, three for ISA distribution width and two for the ABIFM39 shown in Jhet, the droplet ISA, and the simulation time, which is equal to the observed nucleation time. At any given time step, the freezing probability is used in a binomial random sample. If freezing is registered, the frozen droplet is not considered in subsequent time steps. A Monte Carlo method of 105 simulations is used for a complete model run, each initialized with a random sample from ISA distributions, which are either lognormal, uniform, or a Dirac. The latter is when identical ISA per droplet was used. Mean values of UnF and FF with 5th and 95th percentiles are derived. A record of ISA in every droplet and the time and temperature that droplet froze is kept, thereby allowing the mean Jhet and upper and lower fiducial limits following Poisson statistics to be derived.The model used here, based on Alpert and Knopf,61 in a 5 wt% aqueous stock suspension. The stock solution was diluted to yield examined suspension concentrations of 1 wt%, 0.1 wt%, and 0.005 wt%. After preparation of the illite/water stock solution, it was stirred for 24 h. We performed all freezing experiments within 8 days from the time of the fresh stock solution preparation.In this study, we use illite-NXSupplement


Resting-state fMRI has the potential to help doctors detect abnormal behavior in brain activity and to diagnose patients with depression. However, resting-state fMRI has a bias depending on the scanner site, which makes it difficult to diagnose depression at a new site. In this paper, we propose methods to improve the performance of the diagnosis of major depressive disorder (MDD) at an independent site by reducing the site bias effects using regression. For this, we used a subgroup of healthy subjects of the independent site to regress out site bias. We further improved the classification performance of patients with depression by focusing on melancholic depressive disorder. Our proposed methods would be useful to apply depression classifiers to subjects at completely new sites. Depressive disorder is a mental disorder characterized by long-lasting low mood. The diagnosis of depressive disorder has traditionally been made through the interaction between patients and doctors. It is important to develop more objective ways to diagnose depressive disorder in order to increase the reliability and accuracy of the diagnosis.The combination of machine learning and functional magnetic resonance imaging (fMRI) has been used to diagnose or to find the physiological characteristics of psychiatric disorders –11. ReceHowever, functional connectivity is affected by site bias , 12. To In addition to site bias, the heterogeneity of major depressive disorder would be a problem when the classifier is applied to new data. The abnormality of functional connectivity of depression might be different depending on the subtype of the depression. It would be possible to improve classification performance by focusing on a typical severe depression, called melancholic depressive disorder. In this paper, we propose the methods to improve the performance of diagnosis of major depressive disorder (MDD) at an independent site by reducing the site bias effects. In addition, we investigated the performance depending on the classification algorithms and the heterogeneity of the major depressive disorder.One hundred sixty-three patients with MDD  were recruited by five sites  with no history of mental or neurological disease were recruited from the local community. All control subjects underwent the same self-assessment and examination administered to the MDD group. Thereafter, for the melancholic MDD classifier, the dataset was limited to have the subtype of melancholia (based on M.I.N.I.). The number of patients and healthy controls were set to be equal for each site, except for the subjects from the National Institutes for Quantum and Radiological Science and Technology, in order to develop a classifier unbiased toward either group  sequences. High-resolution T1-weighted magnetization-prepared rapid gradient echo images were also acquired before scanning the functional data. In the scan room with dimmed lights, participants were instructed not to think of anything in particular, not to sleep, and to keep looking at a cross mark at the center of the monitor screen. The first four to seven images were discarded to allow magnetization to reach equilibrium. All participants underwent an approximately 5 to 10 min resting-state scan. The scanners and imaging parameters are different depending on the site  were linearly regressed out from the fMRI time series , 23. Herhttp://brainvisa. Info) , anatomically defined in the Brainvisa Sulci Atlas  , 24 coveFor functional connectivity data, we applied the Fisher z-transformation to each correlation coefficient. We then linearly regressed them on training subjects' ages, sex, and dummy variables for each site. The resulting residuals of the regression are used for the subsequent procedures as the features controlling for age, sex, and site effects. We also used the regression coefficients to remove nuisance variables such as age, sex, and site from test subjects. Features like functional connectivities were high-dimensional. We then used a rank-sum test for dimensional reduction; we used the threshold of 0.05 unless noted. The procedures are summarized in We then applied machine learning classification using the above features. We used ensemble learning (Random Forest and AdaBoost), support vector machine (SVM), or sparse logistic regression (SLR)  to classWe implemented both ensemble learning methods using MATLAB fitcensemble function and each decision tree for the weak classifier was constructed using the standard CART algorithm .The hyperparameters adjusting tree depth (minimum leaf size and the maximum number of splits), and the other hyperparameters such as the number of ensemble learning cycles and learning rates were optimized using Bayesian optimization described in the following section. Ensemble learning has advantages in dealing with small sample size, and high-dimensionality . SVM is The performance of the classification was evaluated using Leave-One-Subject-Out cross-validation, Leave-One-Site-Out cross-validation within the discovery cohort or an independent dataset from a replication cohort.For Leave-One-Subject-Out cross-validation, one subject was used for the test and the rest of the subjects were used for the training and this process was repeated until all subjects were used as test data. The hyperparameters of the AdaBoost , Random Forest , and SVM (box constraint (a regularization factor (often denoted as parameter C), controlling maximum penalty imposed on margin-violating observations), kernel scale , and kernel functions  were optimized with Bayesian optimization using the Gaussian process with Expected Improvement acquisition function . Random For the optimization, the whole data was divided into 9-folds, keeping an equal amount of diagnosis, gender, and site combinations per fold. The optimization was performed using the 8-folds with 8-folds cross-validation . The test data was selected in the outer 1-fold. The rest from outer 1-fold and the outer 8-folds were used for the training of the classifier . While the GSR can minimize global signal drifts, it may cause an additional bias by moving FC distribution to a negative direction –39. To iWe used Brainvisa Sulci Atlas to calculate the functional connectivity according to . DiffereFinally, we should bear in mind that the condition of medication may affect our classification performance. To minimize the medication effect, we designed this study so that all patients underwent the fMRI scan before or immediately (less than two weeks) after starting medication of selective serotonin reuptake inhibitors (SSRIs). However, acute neural effect of SSRIs was possible as reported in , 41, andIn this study, we did not consider the interaction between site and other attributes such as age and sex. There is a possibility that the removal of the confounding bias could be more efficient if we consider the interaction. Actually, when we performed ANOVA for the interaction, the distribution of p-values of all FCs significantly differed from uniform distribution . We expected that SLR could be an alternative approach to coping with both generalization and feature selection simultaneously. However, the result was negative. Accordingly, the reasonable strategy to discover necessary and sufficient FCs for depression diagnosis is still an open question.When we consider an application for clinical diagnosis, site bias is a large problem. If traveling subjects, who visit multiple sites to undergo fMRI with the same protocol, are available, we would be able to perform more effective calibration and thereby achieve better performance for the diagnosis of patients with MDD . Even ifyamawaki@hiroshima-u.ac.jp). IRB imposing these restrictions on our data is Ethical Committee for Epidemiology of Hiroshima University (contact: Shoji Karatsu kasumi-kenkyu@office.hiroshima-u.ac.jp). Additional contact information is available from: https://www.hiroshima-u.ac.jp/en/pharm/contact.The original data for this study will not be made publicly available due to the involvement of patient data. It can be obtained upon request to the Department of Psychiatry and Neurosciences, Hiroshima University, Japan , Health, Labour and Welfare Sciences Research Grants, Ministry of Health, Labour and Welfare Japan.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ancylonema nordenskioeldii, a species that has never previously been quantitatively documented in the Alps and that dominates algal blooms on the Greenland Ice Sheet. Our results show that, at the end of the ablation season, the concentration of Ancylonema nordenskioeldii on the glacier surface is higher than that of other algal species (i.e. Mesotaenium berggrenii). Using field spectroscopy data, we identified a significant correlation between a reflectance ratio (750 nm/650 nm) and the algae concentration. This reflectance ratio could be useful for future mapping of glacier algae from remote sensing data exploiting band 6 (740 nm) and band 4 (665 nm) of the MultiSpectral Instrument (MSI) on board Sentinel-2 satellite. Here we show that the biological darkening of glaciers  is also occurring in the European Alps, and thus it is a global process that must be taken into account when considering the positive feedback mechanisms related to glacier melting.The melting of glaciers and ice sheets is nowadays considered a symbol of climate change. Many complex mechanisms are involved in the melting of ice, and, among these processes, surface darkening due to organic material on bare ice has recently received attention from the scientific community. The presence of microbes on glaciers has been shown to decrease the albedo of ice and promote melting. Despite several studies from the Himalaya, Greenland, Andes, and Alaska, no quantitative studies have yet been conducted in the European Alps. In this paper, we made use of DNA sequencing, microscopy and field spectroscopy to describe the nature of glacier algae found at a glacier (Vadret da Morteratsch) of the European Alps and to evaluate their effect on the ice-albedo feedback. Among different algal species identified in the samples, we found a remarkable abundance of  It has been demonstrated that several species of microorganisms, algae and small arthropods find their optimal environment on melting ice and snow. These organisms are also able to shape their environment, through a feedback cycle that involves the albedo. In fact, since these organisms are darker than snow and ice, their presence increases the light absorption and promotes the melting of underlying snow or ice4. On the surface of glaciers, such extremophiles often aggregate with inorganic material  to form cryoconite6. Cryoconite is a dark sediment7 that increases the absorption of visible radiation8, and promotes the formation of characteristic cryoconite holes9 which are globally recognized as an hot spot of biodiversity on ice10. Besides living organisms, cryoconite is known to concentrate pollutants such as contaminants and radionuclides12. This may represent a problem in the future, with a possible secondary release of these substances to the environment14.Glaciers and ice sheets are not lifeless17. This feedback is associated with the spatial distribution of organic material that increases the absorption of light, promotes the phase transition of ice, produces a film of meltwater on snow- and ice-fields, and allows the growth of the algal population19. The decrease of ice albedo has important consequences on glacier mass balance21 and represents an active field of research23. Recent literature focused on the impact of algae on snow and ice in the Greenland Ice Sheet26, Iceland27, Norway28, Himalaya17, Alaska30, Sierra Nevada31, Andes33. These studies highlighted the presence of different species of algae on snow and ice, and their role in the albedo decrease. While recent literature focusing on the southwest margin of the Greenland Ice Sheet identified a strong impact of glacier algae on the optical properties of ice35, this phenomenon remains largely unexplored in the European Alps.Cryoconite accumulated in cryoconite holes has a limited impact on glacier and ice sheet surface mass balance. Instead, the presence of distributed organic material on the margin of ice sheets recently motivated the formalization of the concept of “bioalbedo feedback”36. In contrast, studies of glacier surfaces (i.e. bare ice after snow melting) are sparse. Recently, the glacier alga Mesotaenium berggrenii was described in Austrian glaciers38. Kol39 first reports about the filamentous alga Ancylonema nordenskioeldii in a glacier close to the Mont Blanc. The latter species is well known from polar icefields40, but its presence in Europe was questionable41.In the Alps, most of the studies deal with the characterization of communities living in melting snow24 and from aerial26 and satellite sensors25. While field spectroscopy data are fundamental for assessing the local impact of glacier algae on the optical properties of ice24, remotely sensed data can provide a synoptic view of the phenomenon. In particular, the launch of new satellite missions such as Sentinel-2 and Sentinel-3, from the European Space Agency (ESA) Copernicus program, has created new opportunities for the study of the cryosphere from space43. The spatial, spectral and temporal resolution of these missions allows the monitoring of changes in both alpine and polar glaciers. Sentinel-2 is particularly suited for mapping spatial and temporal variability of the cryosphere at fine scale44, while Sentinel-3 allows a broader perspective43. Some studies have already exploited these data for mapping algae distribution in Maritime Antarctica33 and Southwest Greenland35.The impact of glacier algae on ice albedo can be studied through spectroscopy data collected both in the field46. In particular, Oerlemans et al.21 identified a decreasing trend in the albedo of bare ice and attributed it to the accumulation of dust from lateral moraines. More recently, Di Mauro et al.4 demonstrated that the presence of high concentrations of elemental and organic carbon may have contributed to the albedo decrease on this glacier. Here, we report the identification of glacier algae through DNA sequencing of samples collected on the Morteratsch Glacier at the end of the ablation season on September 2016. Furthermore, we measured the impact of glacier algae on the optical properties of ice using near surface reflectance measurements collected with a field spectrometer, and we identified a reflectance ratio that was correlated with algae density. The spectral measurements were then resampled at the same spectral resolution as the ESA MultiSpectral Instrument (MSI) onboard Sentinel-2 to evaluate the potential of global satellite mission observations for mapping the spatial and temporal distribution of algae in the alpine environment.The objectives of this paper are to identify the algae living on the surface of an Alpine glacier, and to determine their impact on the optical properties of ice. We addressed these objectives with data collected during a survey at the Vadret da Morteratsch Glacier in the Swiss Alps. This glacier has been the focus of numerous studies in recent decadesAncylonema nordenskioeldii, Mesotaenium berggrenii and Sanguina nivaloides were the most representative. We point out the rediscovery of A. nordenskioeldii  from those of the polar regions, whose cells have smaller lengths47, and from the Chilean ones, which feature larger lengths32. M. berggrenii was present with two distinct non-filamentous varieties  is comparable with those found in South-West Greenland50. Thus, we might expect that the algae density observed on Vadret da Morteratsch Glacier will have an effect on optical properties similar to that already observed on polar glaciers35.The average algal cell density found in this work  are reported in Table axa Fig. . Redundaaxa Fig. .Figure 251. Given the complexity of the supraglacial environment, even the relatively low explained variance (i.e. 36.8%) can be considered satisfactory52. We remark that RDA analysis was conducted using only the variable “supraglacial habitats” (i.e. ice surface or cryoconite hole).Indeed, the RDA plot Fig.  shows thAncylonema nordenskioeldii, Mesotaenium berggrenii var. alaskana and the Unclassified Trebouxiophyceae changed according to the type of sample. In particular, Ancylonema nordenskioeldii , the opposite occurred for the algae belonging to the Trebouxiophyceae . The taxonomic affiliation of the most abundant OTUs is reported in Table Generalized linear models (GLMs) performed on the three most abundant algae showed that the abundance of dii Fig.  and Mesoana Fig.  were moreae Fig.   to 2.9 × 105 cell/mL (sample ID: SP20). Photographs of the area measured with the field spectrometer and then sampled for the analysis are showed in Fig. The presence of algae on bare ice causes a decrease of the reflectance at wavelengths shorter than 750 nm because of the absorptions by several intracellular pigments 55 Fig. . As prev2~0.6). We compared our correlation hot spot with other indices proposed by Takeuchi et al.30  was the one corresponding to Sentinel-2 band 6 (centered at 740 nm) and band 4 (centered at 665 nm) . This result fosters the use of this reflectance ratio for mapping glacier algae. Reflectance ratios of wavelengths across the red edge position are established methods for mapping autotrophic life from satellite data57. We here propose the use of the spectral ratio between the reflectance at 740 nm and 665 nm as a useful tool for mapping the presence of algae on ice using remote sensing data collected by different platforms . The relatively weak correlation found between the reflectance ratio and the algae concentration  using the bandwidth of the MSI on board the Sentinel-2 platform. The reflectance ratio that best correlated with the algal density (Rnm) Fig. . No signion Fig.  may be e2 = 0.52, p-value = 0.001) between the continuum removal and the concentration of algae in surface ice samples. The absorption feature of Chlorophyll-a due to algae is very narrow  of the European Alps. In our samples, we found an average algal concentration of 14.2 × 10The presence of algae on glacier ice increases the absorption of solar radiation, fostering the ice-albedo feedback during the melting season. Our dataset represents the first direct evidence of the impact of glacier algae on the optical properties of ice in the European Alps, and it is intended to pave the way for future studies on the bioalbedo feedback in the Alps. The identification of all the players involved in ice darkening is a fundamental task for understanding surface glacier melt, and for predicting the response of Alpine glaciers to future climate change.th 2016, a campaign was conducted on the ablation zone of the Vadret da Morteratsch Glacier , an alpine valley glacier located in the Bernina Massif . Morteratsch is a large glacier (area ~7.5 km2) in the Bernina range, with an altitudinal range of ~2000 m. The glacier snout is located at 2100 m a.s.l. The glacier is characterized by a continental climate, with ablation seasons that can last up to three months during warm summers. This glacier has been extensively studied in recent years46. Morteratsch Glacier has been rapidly retreating21, and it represents the perfect test bed for studying the impact of glacier algae in the European Alps.On September 1360. Spectral reflectance was acquired at 18 sampling points distributed on bare ice in the ablation zone of the glacier. For each sampling point, we collected surface material in correspondence of the spectrometer field of view. Since a strong variability of ice properties can be found on the ablation area of Alpine glaciers, the sampling methodology represents a complexity in spectroscopy of ice. For this reason, we paid much attention in sampling ice exactly from the area measured by the ASD field spectrometer.During the campaign, field spectroscopy data were measured with a HandHeld Analytical Spectral Devices (ASD) Field Spec . The hemispherical conical reflectance factor (HCRF) was calculated by normalizing the reflected radiance with the incident radiance measured from a calibrated Spectralon© panel. Each acquisition was the average value of 15 spectra. A levelled bare optical fiber (field of view = 25°) was used to collect data at 80 cm from the ice surface (footprint diameter = 35 cm). All measurements were collected around midday for minimizing the effect of the changing solar illumination. Further details on the methodology can be found in previous papers60, and it is useful for identifying hot spot of correlation in spectral data. We resampled (i.e. averaged) the reflectance measured with the ASD spectrometer to the spectral resolution of Sentinel-2. This sensor features a high spatial resolution (up to 10 m in the visible spectrum)61 and it is promising for mapping glacier algae distribution from space33.From reflectance data, we calculated all possible spectral ratios and we created a series (n = 423801) of linear regression models between different spectral indexes and the algal density. This variable selection analysis has been already used for other type of impurities31. The continuum removal quantifies the absorption features at specific wavelengths, normalizing the reflectance spectra to a common baseline62. This is achieved by approximating the continuum between local spectral maxima through straight-line segments: a value of 1 is assigned to the local maxima, and a value between 0 and 1 is obtained in correspondence of the absorption features. The continuum removal calculated at 680 nm was then directly compared with algal concentration through linear regression analysis.Furthermore, we calculated the continuum removal (between 655 and 700 nm) at the Chlorophyll-a absorption feature located at 680 nm63 and literature65 were used to identify the algae. The organisms were counted by inverted microscopy (400× magnification) and a camera, pictures were analyzed using AxioVision software, density and biovolume were estimated as reported in literature66. In order to characterize the size of the identified organisms, we measured the length and width of at least 50 cells for each species67. Using a Whatman GF/C (1.2 µm) glass fiber filter we also quantified the total inorganic sediments contained in samples collected on the Morteratsch Glacier.Eighteen samples of ice were placed inside 50 ml conical bottomed phials. These were kept in freezing condition and transported to Milano-Bicocca University, where they were preserved at −30 °C. For the laboratory analysis, the samples were completely thawed at room temperature. The algal classification by light microscopy and count were performed on fresh material. Identification keysIce was melted and centrifugated at 12000 × g for 2 minutes and the supernatant was discharged. The pellet was resuspended in 978 µL of Sodium Phosphate Buffer and 122 µL of MT Buffer of the FastDNA Spin for Soil kit  and the DNA was then extracted according to the manufacturer’s instructions. Samples volume varied from 9 to 24 mL of melted ice. Further samples were collected from the bottom of nine cryoconite holes found on the Morteratsch Glacier. The DNA composition of these samples was analyzed and compared with samples from surface ice DNA extraction from the cryoconite holes samples was performed with the same kit, as for surface ice samples, from 0.7 g of sample, according to the manufacturer’s protocol.68. A first DNA amplification was performed for each sample to evaluate its quality on the original ad on the 1:10 dilution to identify inhibition or insufficient sample. The regions were sequenced with MiSeq Illumina  with a 2 × 250 bp paired-end protocol and Operational Taxonomic Units (OTUs) were defined with an aggregative clustering of sequences with 99% of sequence identity for the 18S rRNA gene fragment. To prepare the libraries for sequencing, a PCR was performed on the samples with GoTaq® Green Master Mix  and 1 μM of each primer, for a final volume of 50 × 2 µL each. Illumina adapters (6 bp) were added at 5′ end.The V4-V5 hypervariable region of the 18S rRNA gene was amplified using the eukaryotic primers 528F and 706 RThe cycling conditions for the 18S rRNA fragment were: initial denaturation at 95 °C for 4 min; 30 cycles at 94 °C for 30 s, 57 °C for 30 s, and 72 °C for 30 s and a final extension at 72 °C for 5 min. Then amplicons were purified with  and quantified with Qubit® . Library preparation with the addition of standard Nextera indexes  and sequencing were carried out at Parco Tecnologico Padano .69 assigning the sequences to the “best hit”. The ten most abundant OTUs were assigned by BLAST70. A. nordenskioeldii was the most predominant alga in all the samples, but conventional clustering methods sometimes were not able to distinguish between A. nordenskioeldii and Mesotaenium species54. Thus, the oligotyping pipeline71 was used on the A. nordenskioeldii sequences. Singletons (OTUs present once in one sample only) were removed from the analyses because their inclusion could inflate variance explained by multivariate analysis72. Analyses were performed with R 3.4.2 , with the VEGAN, BIODIVERISTYR, MULTTEST, MULTCOMP packages.The obtained reads were demultiplexed according to the indexes. Forward and reverse reads were merged only if without mismatches. Taxonomic assignation of the 18S rRNA sequences was performed by BLAST  comparison against the SILVA 132 SSURef Nr 99 database73.We normalized algal OTUs abundance to 100 to compare sequences of different samples and Hellinger-transformed them. Redundancy Analyses (RDA) were performed to see which variables significantly explained the communities variation, correcting P-values for multiple testing according to the false discovery rate (FDR) procedureSupplementary Information.Supplementary Information2.Supplementary Information3.