The gadolinium, gallium, aluminum garnet doped with cerium and co-doped with dysprosium ions were prepared using sol gel method. The SEM images show that after synthesis, the grains are below 100 nm. The powders were ultrasonically mixed with graphene nanoflakes and ceramics were prepared using the high pressure low temperature sintering technique. A series of the ceramics was prepared using different graphene content. The structure of the samples was examined using X-ray diffraction (XRD), scanning electron microscope (SEM) and Raman techniques. The spectroscopic properties were checked using conventional and persistent luminescence spectra measurements. The thermoluminescence glow curves and fading time of persistent luminescence measurements were performed to check how the graphene presence affects the electron traps number and depth. It was found that the addition of graphene improved the thermal conductivity of co-doped samples. This resulted in faster release of deeper traps and an increase in fading of persistent luminescence. The possibility of releasing energy from deep traps without additional stimulation may allow the use in different applications, the matrices and luminescent ions, which so far did not show persistent luminescence at room temperature. Persistent luminescence is a phenomenon where material emits light for a long time after ceasing the irradiation. This type of luminescence as an irradiation source can be used for visible light, gamma, X-ray or UV radiation. The long-time luminescence decay is the effect of releasing the electrons from the traps with the help of thermal energy . The eneDifferent additives can increase thermal conductivity of the composites, but the most well-known and recently used is graphene. The allotropic form of carbon, called graphene, consists of one plain layer of carbon atoms that are organised in a honeycomb lattice. In this structure, the low-lying electrons behave like massless relativistic Dirac fermion, which are the cause of many interesting properties such as conductance quantization feasibilities of inducing a band gap through the lateral quantum confinement and unusually high room temperature carrier mobility. One of the most interesting properties of graphene is its large thermal conductivity. The diamond-like carbon value of thermal conductivity is about 1.7 W/mK, and for graphene this value increases to 5300 W/mK ,5 or met3Al5O12) [3Al5O12 (LuAG) [3+/Gd3+ ions by other lanthanide ions  [3Al5O12 emits in the green/yellow range after excitation with blue radiation. The most desirable result is to obtain perfectly white light, so we need to use a phosphor, which has a wide emission band covering green and red regions after a blue light excitation. By doping the garnet lattice with some cations, we can modulate the emission colour. The smaller ions shift the emission band towards blue, and larger ones shift the emission band towards red [3Ga3Al2O12 garnet (GGAG), doped with Ce3+, focuses the attention of researchers due to the possibility of using this matrix as a good quality scintillator unique light yield where energy resolution can reach up to 50,000 photons/MeV. Because of the strong crystal field splitting, the emission of Ce3+ doped GGAG has a maximum of about 565 nm and can be used for warm white LEDs [Garnets are the most common materials used for lightening or various optical applications due to their important physical properties such as good thermal conductivity, large thermal stability and optical isotropy. In recent years, two of the most widely used garnets are yttrium aluminium garnet Y3Al5O12)  and Lu3A  and Lu32 (LuAG) ,14,15, b+, Eu3+) ,17,18. I+, Eu3+) . For exaards red . Gd3Ga3Aite LEDs ,22.This work was conducted to find out whether the addition of graphene flakes will change the thermal conductivity of the ceramics and in consequence their persistent luminescence properties. It was checked whether the modification of the thermal conductivity has an impact on the release of electrons from shallow and/or deep energy traps. Better thermal conductivity should reduce the amount of energy needed to release charges from energy traps and thus modify the properties of persistent luminescence. The ability to control persistent luminescence is of great importance in many applications. First of all, such emission is currently used in emergency marking and bioimaging. For such materials, it is important to obtain emissions in the red or infrared range. Unfortunately, some matrices and luminescent ions that could be useful here have traps too deep to observe emission at room temperature. In order to allow their emptying and to observe luminescence at the same time, various actions were taken that could release the trapped energy without additional stimulation. To verify our assumption, ceramics showing persistent luminescence and composites containing an admixture of graphene flakes were prepared and characterized. For all materials, their structure, morphology, spectroscopic properties, thermoluminescence and thermal conductivity were examined. Based on the obtained results, the mechanism of persistent luminescence was constructed, taking into account the impact of the graphene flakes addition and its relationship with the thermal conductivity of composites.2O3, powder, 99.995%, Stanford Materials Corporation, Lake Forest, IL, USA), dysprosium oxide , hydrated gallium nitrate (Ga(NO3)3∙xH2O, Onyxmet, Olsztyn, Poland), aluminium nitrate nonahydrate (Al(NO3)3∙9H2O, 98%, Alfa Aesar, Haverhill, MA, USA) and cerium nitrate hexahydrate (Ce(NO3)3∙6H2O, 99,99%, Sigma Aldrich, Saint Louise, MO, USA), citric acid  and ethylene glycol  were used to synthesize garnet powders.The gadolinium oxide (GdThe powders were prepared by the Pechini sol-gel method . The fir1 radiation (1.5406 Å) and step 0.03°. The morphology and homogeneity of the samples were checked using SEM images taken using FEI Helios G4 PFIB CXe DualBeam microscope . All measured samples were placed on aluminum holders using copper tape. The elemental composition was determined and mapped using an EDS detector: Bruker Nano GmbH XFlash Detector 630 . The excitation spectra of Gd3Ga3Al2O12 doped with cerium and co-doped with dysprosium were measured using FLS980 Fluorescence Spectrometer from Edinburgh Instruments  equipped with a holographic grating of 1800 lines/mm, flashed at 300 mm focal length monochromators in Czerny Turner configuration. To obtain the excitation and emission spectra a 450 W Xenon lamp was used and as a detector the R928P side window photomultiplier tube from Hamamatsu  was used. The persistent luminescence (PersL) spectra of the GGAG were measured at room temperature after 5 min laser diode irradiation  with a 445 nm wavelength using SilverNova CCD spectrometer  with 200 µm slit and 1 s integration time. The same system was used for conventional luminescence measurements. The thermoluminescence (TL) and PersL decay curves were measured using a lexsygresearch TL/OSL reader . The signal was detected by a R13456PMT detector  monitoring emission in whole spectral range (from 185 to 980 nm) with an integration time of 0.1 s. The TL curve was registered from room temperature to 300 °C. As an irradiation source, a blue laser diode PL 450B  was used. Before measurements, each sample was heated to 350 °C and kept for 1 min to traps cleaning. Thermal conductivity measurements were carried out using Physical Property Measurement System (PPMS®) operating in the Thermal Transport Option. The temperature range was from 2 K up to 295 K, the thermal conductivity coefficient was determined in a continuous mode of the device with an accuracy of ±5%.The XRD were collected between 10 and 60 2θ degrees at room temperature by an X’PERT PRO PANalytical  diffractometer using CuKα2.994Ce0.006Ga3Al2O12 and Gd2.964Ce0.006Dy0.03Ga3Al2O12 powders and ceramics . This peak is assigned to the graphite structure whose presence is related to the effect of high pressure on the graphene flakes [2O4) [2.994Ce0.006Ga3Al2O12@xGraphene and Gd2.964Ce0.006Dy0.03Ga3Al2O12@xGraphene ceramics.The XRD diffraction technique was used to study the structure of the Gdceramics . Resultee flakes . The abs2.994Ce0.006Ga3Al2O12 and Gd2.964Ce0.006Dy0.03Ga3Al2O12 ceramics with different graphene content are shown in The representative SEM images of the Gd2.994Ce0.006Ga3Al2O12 and Gd2.964Ce0.006Dy0.03Ga3Al2O12 ceramics were taken (see To check how the graphene (carbon—“C”) is distributed through the ceramics volume, the EDS maps of the Gdaken see . It can 2.994Ce0.006Ga3Al2O12 and Gd2.964Ce0.006Dy0.03Ga3Al2O12 nanoceramic samples with different graphene content show two broad bands at 344 and 441 nm  observed due to 4f-5d2 and -5d1 transitions of Ce3+ and sharp peaks at 275 and 310 nm  assigned to transitions from 6IJ and 6PJ excited level to the 6S7/2 ground state of Gd3+, respectively (21) and their redshift is a result of a higher disorder of the surrounding of Ce3+ ions and displacement from the cubic polyhedron to disordered square anti-prism (dodecahedron) [2 and redshift for 5d1 levels was observed (2 and 5d1 bands (Δ21) increases when the graphene concentration increases. It was also observed that full width at half maximum (FWHM) of the bands for the pure ceramic are broader in case of 5d1 and shallower in case of 5d2 level compared to the samples with graphene addition. A large value and a deviation from the FWHM change tendency observed for the sample co-doped with Ce3+/Dy3+ with the highest concentration of graphene (1.6%), is related to the fact that it was difficult to register the excitation spectrum for this sample with the same slits as for other samples and therefore in this case they were opened wider. It can also be noticed that FWHM of 5d1 decreases with an increase in graphene content and for the 5d2 level the tendency is the opposite. As in the GGAG matrix, the optically active ions may occupy several positions [The excitation spectra of Gdectively . For allahedron)  that theobserved . The dis2.994Ce0.006Ga3Al2O12 nanoceramics doped and co-doped with cerium and dysprosium ions were measured under 445 nm laser diode excitation  may lead to a reduction in residual Ce4+ to Ce3+ and, as a result, a shift of the emission wavelength toward lower wavelengths. The decrease in the emission intensity is related to the dark gray color of the ceramics after the addition of graphene and their strong light absorption.The emission spectra of the Gdcitation . An inte of Ce3+  can be o ligands ,32. Herr ligands  showed t2.994Ce0.006Ga3Al2O12 and Gd2.964Ce0.006Dy0.03Ga3Al2O12 ceramics were registered after ceasing 445 nm laser diode irradiation  of the decay was assigned to the bright emission in the initial time, and was observed due to releasing of the electrons from the shallow traps. As the carriers from deeper traps need more energy to be released, they are transferred more slowly to the excited states of Ce3+ and, therefore, in the fading exponent a long component (τ2) is also observed. It can be seen that for both types of ceramics, the decay related to the shallow traps became longer with the increase in the graphene content and at the same time the longer component dropped instantly  for 5 min  limits the thermal conductivity by three @RT. Below 140 K the difference between different graphene concentration on thermal conductivity is negligible. An entirely opposite process is observed for the Gd2.964Ce0.006Dy0.03Ga3Al2O12 sample. For the cerium/dysprosium co-doped case, adding the graphene enhances the heat flow as desired. Graphene acts here as a catalysator. The 0.1% graphene addition results in a third increase in thermal conductivity from 1.5 W/mK up to 2 W/mK at room temperature. The highest graphene addition (1.6%) lifts the thermal conductivity to 4.25 W/mK. The examined ceramics have demonstrated that improvement of heat flow cannot be straightforwardly achieved by just a graphene addition. The most important factor is the way that the graphene is incorporated into the matrix structure, and as a result, if it acts as a scatterer or carrier of heat.The impact of the graphene content in Gdmeasured . For allty value . The mec3Ga3Al2O12 matrix has relatively small activation energy [3+ through CB (4). As the energy of the traps is relatively small (below 1 eV), the captured electrons may already be released at room temperature and due to improved thermal conductivity and greater traps distribution, it is also easier to release electrons from deeper traps. When the charge carriers are released, they move back to the emission center and then to the ground state giving persistent luminescence (4). As the charge carriers are gradually released from trapping centers, the persistent luminescence may take up to tens of minutes.The mechanism of persistent luminescence is proposed by adopting the energy transfer diagram already described by us  supplemen energy  the rema2.994Ce0.006Ga3Al2O12 and Gd2.964Ce0.006Dy0.03Ga3Al2O12 ceramics was investigated. The XRD measurements show that in the ceramics containing graphene, the structure is maintained, but small changes in the crystallographic environment of luminescent ions may appear. SEM and EDS maps and their analysis show that the graphene added to the ceramics does not form a uniform network, which in some cases may hinder the interpretation of the results. In the ceramics excitation spectra, an increase in the splitting of the 5d states (Δ21) with the changing content of graphene can be observed, due to displacement from the cubic polyhedron and an increase in disorder in the environment of Ce3+ ions. The fading time of the persistent luminescence shows that after graphene addition, the shallower traps are emptied slower, while carriers from deeper traps are released faster, which shows that the presence of graphene mainly affects the traps with higher energy. These observations are confirmed by thermoluminescence measurements, where a shift of the glow curve towards higher temperatures (higher energies) is observed. The relationship between the depth of the traps, the presence of the Dy co-dopant and the changes observed in persistent luminescence are related to the increase in thermal conductivity after the addition of graphene, which was confirmed by thermal transport measurements. It can be concluded that the addition of graphene can improve the process of released electrons from deeper traps and have an influence on persistent luminescence, mainly in the materials where the charge carriers binding traps are deeper. However, for the best effect, low concentrations of graphene (about 0.1–0.2% by weight) and its homogeneous distribution between the grains should be preserved.The influence of graphene addition on the persistent luminescence of the Gd


In contrast, females show higher capacity for remodelling, with peak plasticity corresponding to highest estrogen levels during the estrous cycle. Estrogen manipulation through ovariectomies and estrogen replacement with 17β estradiol in vivo was found to significantly alter spine density and mitigate disease severity. Collectively, these findings reveal that synpatic plasticity is reduced in ALS, which can be amelioriated with estrogen, in conjuction with improved disease outcomes.Amyotrophic lateral sclerosis (ALS) attacks the corticomotor system, with motor cortex function affected early in disease. Younger females have a lower relative risk of succumbing to ALS than males and older females, implicating a role for female sex hormones in disease progression. However, the mechanisms driving this dimorphic incidence are still largely unknown. We endeavoured to determine if estrogen mitigates disease progression and pathogenesis, focussing upon the dendritic spine as a site of action. Using two-photon live imaging we identify, in the The online version contains supplementary material available at 10.1007/s12035-022-02742-5. Amyotrophic lateral sclerosis (ALS) exists on a disease spectrum with frontotemporal dementia (FTD), sharing pathological and genetic features, and clinical overlap –3. In ALALS presents with sex differences in incidence, severity, and progression. Younger females appear to be more protected than males to ALS onset ; howeverNeuroplasticity of dendritic spines is critical for processing information, with changes in synaptic structure and function being determinants of cognition, behaviour, and circuit integrity . The genprpTDP-43A315T (TDP-43) mouse  mice were all purchased from Jackson Laboratories, with all lines maintained on a C57/BL6 background. To capture high-resolution fluorescence of layer V pyramidal neurons residing in layer V and extending into layer II/III the TDP-43 model was crossed with Thy1-YFPH mice. For tissue collection, animals were injected with an intraperitoneal (i.p.) terminal anaesthesia  and transcardially perfused with 0.01 M phosphate-buffered saline (PBS). The cortex was immediately dissected and frozen in liquid nitrogen, and stored at − 80 °C prior to experimental processing. Male and female mice were included.Experimental procedures utilised male and female mice and were approved by the Animal Ethics Committee of the University of Tasmania (A0016593). Experiments were performed in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (2013) and in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) Guidelines. Mice were housed in individually ventilated cages at 20 °C with littermates unless otherwise stated, on a 12-h light–dark cycle, with access to food and water ad libitum. C57/BL6 , Thy1-YFPH  , 32, andThe mouse estrous cycle is divided into four stages  cycling every 4–5 days . For exp2, 2 mM MgSOa) applied regularly. Dexapent  was topically applied to the dura surface after removal of the bone island. A coverglass  was placed over the dura flush with the skull, and Loctite® adhesive (Henkel Adhesives) applied around window edges. The cranial window was sealed with dental cement  covering the exposed cranium, wound margins, and lip of the coverglass. At the time of the first 2PLSM imaging round, a titanium steel bar  was fastened with Loctite® adhesive to the dental acrylic over the intact hemisphere for animal stabilisation. Mice were subsequently monitored for 2 weeks for any adverse effects, as a standard for all surgical interventions.Open skull cranial windows were surgically implanted over the motor or somatosensory cortices of male and female WT and TDP-43 mice at post-natal day (P) 50, following the protocol previously described by Holtmaat et al. . Mice wez-plane coordinate was saved, and objective changed to a W Plan-Apo 40 × /1.0 water immersion objective (Nikon), after which the same z-plane coordinate was initiated to capture a high-powered region of interest to identify the same position in consecutive imaging sessions. The MaiTai laser captured blue/green emissions (Channel 1 PMT 550–565 nm) at 910 nm/1700mW of power at a maximum of 25% equivalent to 70 mW [z-stacked images (20 μm thick at 1-μm slices). To locate layer II/III for in vivo imaging, z-stacks were begun at a depth of 150 μm from fluorescence first visible from the cortical surface to a maximum depth of 300 μm. Landmark dendrites were identified during the initial session and returned to in consecutive sessions, to follow the same dendritic spines over time.In vivo two-photon microscopy was performed with a custom-built microscope (Scientifica) equipped with a Ti–sapphire laser  and a × 20 water immersion objective , controlled by ScanImage software underMATLAB® software (MathWorks) . Power dto 70 mW . The denTo capture dendritic spine turnover and survival, mice were anaesthetised with an i.p. injection of 0.12 mg/g ketamine to 0.01 mg/g xylazine  and imaged every 24 h. Approximately 10 dendrites from each dendritic compartment per animal (for density) or 4 dendrites per animal , per group were quantified and manual counts were performed under blinded conditions in Neurolucida™, with layer II/III spines annotated along the segment as mature or immature according to their morphology . BrieflyN(t)/N0, where N0 is the number of spines at 0 h and N(t) is the number of spines surviving after 24 h [Ngained + Nlost)/(N(t1) + N(t2)) where Ngained is the number of spines gained over 24 h; Nlost is the number of spines lost over 24 h; and N(t1) is the total number of spines present at the first time point and N(t2) is the total number of spines present at the second time point [Nlost) and a mature spine ‘gained’ (included in Ngained) (and vice versa). Ex vivo two-photon microscopy was used to quantify dendritic spine densities (p/μm) in layer II/III from layer V pyramidal neurons of the motor cortex within whole brains at P60. Layer II/III corresponds to the depth which in vivo 2PLSM was undertaken, with our previous studies having identified that spine pathology is present in the male TDP-43 motor cortex in this region in 20-μm fixed sections [The same dendritic segments were selected and imaged for analysis over the experimental timeline. Spines were identified on the dendritic segments and tracked over time. Survival fraction  was calculated as ter 24 h . Turnoveme point , 39. In sections .Tissue stored at − 80 °C was weighed and placed in RIPA buffer  with EDTA free protease inhibitor tablets (Roche), then homogenised using a T10 basic Ultra-Turrax homogeniser (IKA) for 3–5-s bursts. The homogenate/lysate was centrifuged at 13,000 rpm at 4 °C for 10 min. Protein concentration of whole brain/motor cortex protein fractions was determined using the DC Protein Assay (Bio-Rad). Western blots were blocked and probed for antibodies listed in Table z-spacing of 0.5 µm were collected. Standard excitation and emission filters were used.Mice were terminally anesthetised with an intraperitoneal injection overdose of sodium pentobarbitone (Jurox USA) and transcardially perfused with 4% (w/v) paraformaldehyde  in 0.01 M phosphate-buffered saline . Coronal sections containing primary motor cortex were collected from each animal and a blocking solution containing 1% normal goat serum and 0.3% triton-X (Sigma-Aldrich USA) in 0.01 M PBS was added for 1 h. Sections were incubated with anti-GFP and anti-ERß (Table −1 O2), mice were maintained on isoflurane (1.5–2%) and lay supine on a heatpad kept at 37 °C for cleaning using chlorhexidine (4%) on the dorsal portion. Using scissors, a small incision was made laterally into the skin, approximately above the site of the body cavity and ovarian fat pad. After blunt dissection of the fascia, a second small incision was made in the muscle, creating an opening into the body cavity. The fat pad of the ovaries was located using blunt forceps, and pulled out of the incision to be clamped and lain over the back of the mouse. The ovarian bursa was torn using fine-tipped forceps, releasing the fallopian tubes and the ovary. A low-heat cauteriser was then used to remove the ovary from the fat pad, without creating large bleeds or damage to the fallopian tubes. Once removed, the fat pad was released and carefully reinserted into the body cavity. These steps were then repeated for the other side of the mouse, removing both ovaries. After ovarian excision, the muscle incision was closed with a single surgeon’s knot using absorbable synthetic sutures and the skin incision closed using Michel clips. Mice were given a post-surgical s.c. injection of metacam (meloxicam 5 mg/ml dosed at 1 mg/kg) and 1 ml of saline, and were monitored for normal activity upon recovery. Mice with slower healing incisions were housed individually.Female WT and TDP-43 mice underwent ovariectomy surgery at P40, with female mice typically becoming sexually mature by P42-56 . SurgeryOsmotic mini pumps (ALZET® Model 2004) were inserted at the time of ovariectomy in female WT and TDP-43 mice at P40, containing either saline in 50% dimethyl sulfoxide (DMSO) (vehicle control) or 10 mg/ml 17β Estradiol  in 50% DMSO under blinded conditions [Disease severity was assessed using a neurological scoring system to evaluate phenotypic progression of hind limb weakness (Supplementary Table 1) –46. The t-tests, one-way, two-way, or three-way ANOVA, and Spearman’s correlation coefficient. For western blot and pathological analysis, each point on a graph represents a separate animal. For spine plasticity over time, each data point represents an individual dendrite imaged over time. P < 0.05 was considered significant and data reported as mean ± standard error of mean (SEM) unless otherwise stated. Where appropriate, neurological scoring and quantification of dendritic spine data was performed blinded.Statistical analyses were performed in GraphPad Prism (GraphPad Software) using tests of normality prior to The ability of spines to be plastic is crucial for network function, and disruption to their finely tuned structural dynamics can induce pathological conditions. The balance between dendritic spine persistence and dendritic spine turnover is influenced by many factors, and is in constant flux. The TDP-43 mouse carries a familial ALS mutation, and develops an ALS-FTD spectrum phenotype of motor and cognitive dysfunction , 44, 47,F = 12.55, p < 0.001), dendritic spine turnover was significantly decreased in TDP-43 males in comparison to WT males (p = 0.0019) and HE TDP-43 females (p < 0.001). Spine turnover in the TDP-43 BE female motor cortex was significantly decreased in comparison to WT BE female (p < 0.001). However, TDP-43 HE female spine turnover did not demonstrate the loss that TDP-43 BE, and TDP-43 males did, with no significant differences between TDP-43 HE and WT HE. There were no significant differences in spine dynamics in the somatosensory cortex  = 6.733, p = 0.0016), dendritic spine stability was significantly increased in TDP-43 male mice in comparison to WT male mice (p = 0.0021) and the TDP-43 HE female (p < 0.001). Spine stability in the TDP-43 BE female motor cortex was significantly increased in comparison to WT BE female (p = 0.0002) and TDP-43 HE female (p < 0.0001). However, TDP-43 HE female spine stability was not significantly different to WT counterparts. There were no significant differences in spine stability in the somatosensory cortex , and analysed using a two-way ANOVA with Tukey’s post hoc. In the motor cortex  = 20.07, p < 0.0001) and sex- and spine-type interaction  = 3.142, p = 0.043); yet, not a spine-type and genotype interaction  = 0.3960, p = 0.5296). Tukey’s multiple comparisons tests identified that the percent of both immature and mature dendritic spine turnover was significantly decreased in the male TDP-43 in comparison to WT (immature—p = 0.0031 and mature—p < 0.0001). Immature and mature dendritic spine turnover was also significantly decreased in the TDP-43 BE in comparison to WT BE (immature—p = 0.0003 and mature—p < 0.0001). TDP-43 HE was significantly increased in comparison to TDP-43 male (immature—p = 0.0020 and mature—p < 0.0001) and TDP-43 BE (immature—p = 0.0135 and mature—p < 0.0001). There were also significant differences in the percentage of immature to mature spine turnover between WT females (p = 0.0285) and TDP-43 HE females (p < 0.0001). This data indicates that mutant TDP-43 impacts both immature and mature spines' plasticity, and that whilst the protective effect of estrogen was not restricted to a certain morphological type, it was more pronounced in the mature spine types.To elucidate whether altered dendritic spine dynamics differentially impacts morphological subsets in the motor cortex, the turnover rates for spines identified to have a mature (mushroom or stubby) or immature (thin) morphology were quantified Fig. A. DendriTo investigate the relationship between estrogen levels and structural changes at the dendritic spine, we correlated dendritic spine survival and turnover to estrous cycle stage during imaging in the female WT and TDP-43 motor cortex. The stages of the estrous cycle were given a score of 1–4—with 1 being the lowest estrogen levels and 4 being the estrogen peak—based on previous work demonstrating the levels of estrogen present over the cycle  = 136.0, p < 0.0001) was revealed. Tukey’s multiple comparisons test found significantly increased global hTDP-43 in both BE (p = 0.0002) and HE (p = 0.0001) TDP-43 females, as compared to WT BE and HE females, with WT females found to express negligible disease protein levels  protein, taken at P60. Following a two-way ANOVA a significant main factor effect of genotype  = 7.45, p = 0.0259), Tukey’s multiple comparisons test did not reveal any significant differences in ERβ between specific groups  and beta (ERβ) are widely distributed throughout the brain and their differential activation influences neuronal physiology and neuroprotective genes –51. We uups Fig. J, K. ThuF = 18.08), Tukey’s multiple comparisons test revealed that whilst there were no significant differences in WT, WT + OVX, and TDP-43 spine density, TDP-43 + OVX females displayed a significant reduction (p < 0.0001) in dendritic spine density compared to WT, WT + OVX, and TDP-43 females n = 6 and + OVX with vehicle control pump) had a significantly increased disease severity compared to intact TDP-43 females. The vehicle control pump had no effect on disease score compared to the + OVX mice alone  and long-term depression (LTD) studies , 90, 91.In summary, disturbances in excitatory transmission have long been associated with ALS pathogenesis. The motor cortex plays an important role in this excitability dysfunction, as evidence now indicates that excitability alterations are in fact initiated in this brain region and move through the corticomotor system. Here, we demonstrate that TDP-43-altered dendritic spine plasticity specifically affects the motor cortex, and we have uncovered a mechanism for how females are conferred protection in the FTD-ALS spectrum—with a role for estrogen in structural plasticity. Future research into targeted estrogenic pathways directed at plasticity are relevant not only to the ALS-FTD, but to neurodegenerative diseases as a whole, with the potential to be harnessed as potent neuroprotectants in these devastating diseases.Supplementary file1 (DOCX 756 KB)Below is the link to the electronic supplementary material.


Objective: The aim of this study is to review current evidence regarding the correlation between GERD and IPF and to evaluate the current studies regarding treatments for GERD-IPF. Methods: A review to identify research papers documenting an association between GERD and IPF was performed. Results: We identified several studies that have confirmed the association between GERD and IPF, with an increased acid exposure, risk of gastric aspiration and bile acids levels in these patients. Few studies focused their attention on GERD treatment, showing how antiacid therapy was not able to change IPF evolution. Conclusions: This review investigating the correlation between GERD and IPF has confirmed the hypothesized association. However, further large prospective studies are needed to corroborate and elucidate these findings with a focus on preventative and treatment strategies.Background: Idiopathic pulmonary fibrosis (IPF) is a rare and severe disease with a median survival of ∼3 years. Several risk factors have been identified, such as age, genetic predisposition, tobacco exposure, and gastro-oesophageal reflux disease (GERD). Prevalence of GERD in IPF is high and may affect 87% of patients, of whom only half (47%) report symptoms.  Idiopathic pulmonary fibrosis (IPF) is the most common form of progressive and irreversible interstitial chronic fibroinflammatory pneumonia, accounting for approximately 50–60% of all idiopathic interstitial pneumonias (IIPs) ,2. The eThe pathogenesis of IPF is highly complex and still not fully clear. IPF pathogenesis is correlated by an increase in the synthesis and release of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-1, as well as some fibrous factors such as transforming growth factor (TGF)-β and platelet-derived growth factor (PDGF) ,7,8,9,10IPF usually occurs in adults over 50 years old. IPF prognosis is poor, with a median survival of usually 2 to 3 years, which is comparable with that of some aggressive cancers ,13,14,15Traditionally, IPF treatment was based on immunosuppressants, glucocorticoids, oxygen therapy, and palliative measures. However, the PANTHER-IPF study showed that treatment with azathioprine, N-acetyl cysteine, and prednisone was associated with increased hospitalizations and mortality ,7,8,9,10The purpose of this study is to review the current evidence regarding the correlation between GERD and IPF by examining a group of studies treating the topic. Recent literature was also evaluated for an update on current treatments for GERD-IPF.Currently, antifibrotic therapies  are neither able to cure nor abort or reverse the progression of the disease, although they have proven effective in slowing down the progression, independently of age, sex, and the stage of the disease ,9. BenefHowever, there is little information on the pharmacological interactions of these two agents in IPF patients who are usually polymedicated ,13,14,15Pirfenidone belongs to the group of immunosuppressive agents. Pirfenidone (5 methyl-1-phenyl-2-[1H] pyridone) is an agent that combines anti-inflammatory and antifibrotic effects, acting on the regulation of TGF-5 activity, TNF-α and -β pathways, and cellular oxidation ,7,8,9,10Pirfenidone reduces the accumulation of inflammatory cells and attenuates the proliferation of fibroblasts, the production of cytokines and proteins related to fibrosis, and the increased synthesis and accumulation of extracellular matrix ,7,8,9,10Nintedanib is an intracellular tyrosine kinase inhibitor developed for the treatment of various types of cancer , as well as an antifibrotic agent ,7,8,9,10The most common adverse events (AE) of antifibrotic therapy involve the gastrointestinal tract ,7,8,9,10Gastroesophageal reflux disease (GERD) can be described as the symptoms complex (occurring at least twice a week) or complications due to the ascent of gastric contents into the esophagus and higher parts of the digestive system ,16,17,18Helicobacter pylori infection could provoke reflux esophagitis [In the last few years, the prevalence of GERD has increased all over the world, including 27.8% of the general population of the USA and 25.8% of European citizens. The reasons for the apparent increase in the prevalence of GERD symptoms and erosive esophagitis are not clear ,20. Incrphagitis ,22. Morephagitis .There isphagitis ,22,23.Patients with IPF, systemic sclerosis (SSc), asthma, and COPD are affected by GERD with higher prevalence than in the general population ,28,29,30Common symptoms of GERD are chest or epigastric pain, nausea, regurgitation, and bloating. We also mention cough, throat pain or burning, wheezing, and sleep anomalies among extraesophageal symptoms. GERD can be completely asymptomatic and coincidentally diagnosed. Therefore, an accurate (and timely) diagnosis of GERD remains difficult. There are a range of available diagnostic tests , although they all have important limitations  ,26,27,28is GERD a consequence of IPF or is it a risk factor for it? Some studies have hypothesized that GERD in IPF is more frequent because of potential confounding factors, such as advancing age and smoking [The number of newly diagnosed GERD patients included a steady increase in those with IPF in the last few years; we can also say the same for the prevalence of hiatal hernia, associated with erosive esophagitis ,42,43,44 smoking ,46,47,48Although knowledge of the pathogenesis of IPF remains incomprehensive, various individual genetic and epigenetic factors have been correlated with the development of fibrosis and potential contributions of some environmental exposures  have been presumed. In particular, several studies have hypothesized that GERD-associated microaspiration may lead to persistent inflammation impairing lung infrastructure, thereby possibly accelerating the progression of IPF. Repeated microinjury to alveolar epithelial tissues has been revealed as the first trigger of an aberrant repair process in which several lung cells develop abnormal behaviors that promote the fibrotic process ,46,47,48Currently, antacid therapy  for IPF patients with GERD symptoms has only a conditional recommendation according to IPF guidelines ,38,49,50Laparoscopic fundoplication is another treatment option for patients with IPF and GERD because theoretically it may help to control reflux and avoid aspiration, with the same effects mentioned above on the progression of the disease. ,53,54,55Hiatus hernia (HH) is an alteration of the integrity of the LES, a frequent finding by both radiologists and gastroenterologists, which can be associated with episodes of microaspiration, LES pressure reduction, and an increased risk of esophagitis . As for The prevalence of HH increases with both age—particularly the 6th decade—and obstructive airway physiology, like asthma. The risk factors of HH include increased abdominal pressure , coughing, and tobacco smoking ,60,61.A barium esophagram, esophageal endoscopy, and high-resolution CT can help to describe the anatomy of the esophagus in relationship to the diaphragm and find the location of the LOS ,64,65,66It is fundamental to understand whether HH in IPF patients is a comorbidity, a consequence, or a driver of IPF . Probably, extensive fibrosis causes a reduction in lung compliance, shifting the diaphragmatic pillars and increasing the prevalence of HH in IPF patients . CT fibrIt is still not clear whether HH can be considered a predictor of mortality from respiratory causes in IPF patients . Indeed,The therapeutic options to address GERD in adults include: diet and lifestyle changes  ,69; mediFocusing our attention on GERD treatment in patients with IPF, in 2015 an American preclinical study analyzed the effect of PPIs in mitigating lung injury and fibrosis in IPF, suppressing acid reflux, and arresting cytokines release. This study started from the evidence that PPI use is associated with increased longevity in IPF patients and analyzed esomeprazole action in reducing lung inflammation and fibrosis in rats, concluding that probably through GERD treatment it is possible to find a therapeutic effect on pulmonary fibrosis ,103,104.Nevertheless, at the same time, a systematic review  publishe® in order to identify relevant full-text manuscripts treating the GERD-ILD relationship from different points of view. The search was performed using the following keywords or synonyms: ‘GERD ILD’, ‘GERD IPF’, and ‘GERD Idiopathic pulmonary fibrosis’, without limitation on the date of publication, up to the 12th of March 2022.We performed a review of the literature in PubMedn > 20) of patients examined. We included reviews, meta-analyses, case studies, and retrospective studies, without any limitation on the language of the original text.Records were selected only if they were strictly treating the specified topic and in particular those with a focus on pulmonary perspective and with a larger number (p < 0.01) oesophageal acid exposure and risk of gastric aspiration. In particular, IPF patients had more bile acids ) and a more severe GERD—which can be a reason for lung damage and fibrotic evolution, the same evolution described by Baqir et al. [We selected 14 papers, of which 6 were original studies. Four were case–control studies; the two others were a retrospective study and a meta-analysis. We chose these six papers for their different designs, for the large number of examined patients , and in order to show that, if the association of GERD-IPF is well known, clinical implications still need to be discussed. r et al. —with a 1(n = add), identified an association between GERD and IPF ; homogeneity < 0.0001), although the potential for confounding exists, e.g., smoking. It is also remarkable that this meta-analysis was based exclusively on case–control studies, so the confidence in the estimate of association is low, as the authors themselves clarified.The 2019 meta-analysis,  which inThe retrospective study conducted from the analysis of the IPF Australian Registries  showed tThe possible etiologic role of reflux in pulmonary fibrosis has been advanced on the basis of the results of several studies that have demonstrated the high frequency of GERD in the IPF population. A major challenge is that gastroesophageal reflux is frequently asymptomatic, but a diagnosis is typically based on the presence of common symptoms and therefore often inaccurate.A major unresolved issue is to elucidate whether GERD is a direct driver of IPF or a simple consequence or innocent bystander, potentially due to a decreased pulmonary compliance during respiratory ventilation secondary to fibrosis. Manometric study of esophageal motor function in IPF patients has not demonstrated particular patterns of body dysmotility, and with only a few studies indicating a higher frequency of ineffective peristalsis ,55,56. RFrom a therapeutic point of view, the role of proton pump inhibitor (PPI) in GERD was studied particularly in the last decade ,50,55,56r=0.50, p=0.03) and FVC. The authors concluded that low distal and proximal MNBI, as well as an abnormal PSPW, are predictors of a more severe decline in lung function over 1 year on pulmonary function tests among pre-lung transplant IPF patients [Regarding GERD diagnosis, esophageal MII-pH is the current gold standard. Two abstracts have shown the correlation between mean nocturnal baseline impedance (MNBI) and postreflux swallow-induced peristaltic wave (PSPW) values and changes in pulmonary function testing parameters over 1 year in IPF patients ,50,55,56patients ,50,55,56patients ,50,55,56Another possible application of EndoFLIP could be the identification of those patients presenting EGJ (esophagogastric junction) dysfunction that could benefit from surgery. All these new diagnostic tools, in addition to a more diffuse and frequent use of MII-pH, should improve our ability to establish a more accurate cause–effect relationship between IPF and GERD and to assess the real effectiveness of both medical and surgical antireflux therapies ,50,55,56The examined data strongly support an association between GERD and IPF. Therefore, all IPF patients should be objectively screened for GERD by pathophysiological studies, mainly using HRM (high-resolution manometry) and potentially MII-pH. Moreover, bronchoscopy with analysis of BALF by dosing pepsin and/or bile acids could represent a diagnostic option in order to increase diagnostic accuracy, but it is seldomly performed in routine clinical practice for IPF due to an increased risk of exacerbations; therefore, it cannot be considered in the diagnostic routine for GERD-IPF. Furthermore, the efficacy of PPI therapy, criticized in the latest IPF guidelines, should be evaluated in prospective and randomized clinical trials against placebo, aimed to assess lung function parameters and the clinical response of respiratory symptoms and to improve oesophageal pH and impedance values. Antireflux surgery, particularly LARS, appears to be an effective and safe option for these patients. In conclusion, our current uncertainty about the GERD-IPF relationship and, consequently, about its therapeutic management, is due to the poor knowledge of the real pathogenic mechanisms of the pulmonary damage in IPF . Several


It was a van der Waals (vdWs) stacked configuration composed of gold (Au) film, few quintuple layer (QL) Bi2Se3 and few-layered graphene. In particular, the proposed biosensor was created by Goos-Hänchen (GH) shift rather than phase, resulting in a more sensitive biosensing response. Under the excitation of 632.8 nm, significant sensitivity enhancement performance was obtained via varying the thickness of Bi2Se3-Graphene heterostructures. The best configuration was 32 nm Au film−2-QL Bi2Se3-3-layer graphene, generating the largest GH shift, as high as −1.0202 × 104 µm. Moreover, the highest detection sensitivity was determined to be 8.5017 × 106 µm/RIU, responding to a tiny refractive index (RI) change of 0.0012 RIU . More importantly, our proposed biosensor has shown a theoretical feasibility of monitoring virus samples. For example, there was an efficient linear detection range for severe acute respiratory syndrome coronavirus 2 ) and its Spike (S) glycoprotein (0~59.74 nM), respectively. It is expected that our proposed plasmonic biosensor has a potential application in performing sensitive detection of SARS-CoV−2.This study provided a theoretical insight for designing novel plasmonic biosensors using bismuth selenide (Bi Thus, Bi2Se3 film shows an enhanced sensitivity for RI variation [Surface plasmon resonance (SPR) biosensors are a class of important optoelectronic devices widely employed in the biomedicine science field. To date, significant breakthroughs in plasmonic biosensing are focused on the following two studies: (i) novel plasmonic materials and (ii) optimization of biosensing process . For plaariation . In addiariation . It is p2X3. Here, M stands for the element Bi or Sb, and X denotes the element Te or Se. Bi2Se3 is the most common 2D topological insulator material, whose crystal lattice consists of quintuple layers (QL) orderly arranged by a Se–Bi–Se–Bi–Se unit [2Se3 has shown many unique features, such as a large bandgap of ~0.3 eV [2Se3 films exhibited the maximum photon response [2Se3 films with a thickness less than 22 nm is 50~200 cm2V−1s−1 [2Se3/Graphene heterostructures was observed as high as 5000~6000 cm2V−1s−1 [2Se3/graphene heterostructures at room temperature can be optimized to be 3400 cm2V−1s−1 [It is well-known that TIs family has several prototypical members, which can be described as M–Se unit ,17. In a ~0.3 eV , well-de ~0.3 eV , high ph ~0.3 eV ,20 and e ~0.3 eV . It is presponse . At roomm2V−1s−1 . Howeverm2V−1s−1 ,25. Morem2V−1s−1 . To the 2Se3-Graphene heterostructures to linearly detect SARS-CoV−2 and its S glycoprotein. Rather than phase-modulation, our proposed SPR biosensor was designed by GH shift, showing a higher biosensing performance. Both photon absorption and energy dissipation were optimized to be a balance state by varying thickness of Bi2Se3/Graphene heterostructures. The optimal configuration: 32 nm Au film−2-QL Bi2Se3–3-layer graphene could provide the largest GH shift up to −1.0202 × 104 µm. For a tiny RI variation of 0.0012 RIU, the highest detection sensitivity of 8.5017 × 106 µm/RIU can be achieved. Moreover, the proposed biosensor could be employed to simulate the adsorption behavior of SARS-CoV−2 and its S glycoprotein. A good linear response interval for monitoring SARS-CoV−2 was from 0 to13.44 nM. For S glycoprotein, a theoretical linear detection interval was from 0 to 59.74 nM. In all, the optimal biosensor is a potential candidate in quantitatively monitoring SARS-CoV−2 and other infectious viruses in practical clinical applications.Inspired by these observations, we theoretically proposed a novel plasmonic biosensor using vdWs Bi2Se3 interlayer and graphene overlayer, as shown in 2Se3/Graphene heterostructures having high carrier mobility, it was introduced to work as an important plasmonic layer, significantly developing the energy dissipation. Then, Bi2Se3 interlayer was vdWs stacked onto the top of plasmonic gold film. After this, few layered graphene overlayer was further stacked onto Bi2Se3 interlayer to form Bi2Se3/Graphene heterostructures. The topmost layer was a sealed cell containing work solutions. It is worth noting that the work solutions consisting of 10 mM HEPES (4-(2-hydroxyethyl)−1-piperazineethanesulfonic acid) and 120 mM NaCl (sodium chloride) was utilized to contain SARS-CoV−2 and its S glycoprotein in this simulation. To really calculate the binding interaction between SARS-CoV−2 and sensing interface of Bi2Se3/Graphene heterostructures, the work buffer consisting of 10 mM HEPES and 120 mM NaCl solutions was injected from left entrance. Once the target analytes in running buffer were bound to the sensing interface, the change in local RI would be different. Generally, the tiny variation in RI could be readout by measuring the change in intensity of reflected light, SPR angle and differential phase. In this work, p-polarized incident light having a wavelength at 632.8 nm was applied to resonate with SPPs wave in Au film/Bi2Se3/Graphene heterostructures. Unlike previous SPR biosensors created by phase modulation, our proposed biosensor was designed by measuring GH shift. It is well-known that GH shift is an interesting displacement behavior of reflected light relative to geometric reflection theory, because practical incident lights generated by lasers are not ideal monochromatic electromagnetic plane waves [The theoretically proposed SPR biosensor consists of an SF11 prism, an Au thin film, few layered Bine waves . Moreovene waves , indicatne waves ,30, whicne waves ,32. Unde2Se3 nanosheets and graphene overlayer is 1.7786 [2Se3 and monolayer graphene is 0.9725 nm [C) can be determined to be 1.3345 [2Se3/Graphene configuration was assumed to be proportional to the concentration of S glycoprotein, which can be calculated by Equation (2):An is the RI due to the adsorption interaction of S glycoprotein and Cn is the RI of running buffer. Ac is the concentration of S glycoprotein solution. In addition, the parameter (dn⁄dc) denotes the increment of refractive index and the value of dn⁄dc is usually ~0.186 cm3•g−1 for studying protein-protein binding interactions [Ac) of S glycoproteins can be calculated according to the binding parameter (dn⁄dc).Both photon absorption and energy dissipation in our proposed configuration highly depend on optical dielectric constant of each layer. Prior to biosensing simulation, the dielectric constant of each layer needs to be determined. Under the excitation light of 632.8 nm, the refractive indices of SF11 prism layer, Au film layer, Bis 1.7786 . 0.1838 s 1.7786  and 3.00.9725 nm  and 0.34.9725 nm , respectractions . For a f1 = 0, and the relationship between the last boundary ZN−1 and first boundary Z1 could be given as:In addition to SPR reflectivity, change in incident angle and differential phase, change in GH shift generated by plasmonic enhancement in our proposed SPR configuration was systematically simulated using transfer matrix method (TMM) and Fresnel equations in a N-layer stacked model. Theoretically, each layer was supposed to parallel in Z-direction and each layer was considered to be isotropic and non-magnetic. It was assumed that the number of antibody sites on graphene layers was much more than that of S protein. The adsorption sites on graphene overlayer were S protein antibody, which can be employed for specifically capture SARS-CoV−2 using antibody-antigen interactions. Generally, the electromagnetic fields at the first boundary along the tangential direction was supposed to be Zk could be described as:kβ and kq can be determined using the following two Equations (6) and (7):kd stands for the thickness of the k-th layer. Additionally, kε represents the dielectric constant of the k-th layer. In Equation (7), 1θ and 1n are the incident angle and RI of the first stacking layer, respectively.Here, M is a transfer matrix, which can be obtained by the following equation for p-polarized light:kq was described by Equation (8):For s-polarized light, these relationships described above are still practicable. However, pR) in our proposed configuration can be obtained using Equation (9):Prior to calculating GH shift, the reflectivity : sφ stands for the phase of s-polarized light.Therefore, the differential phase ΔGHS) can be calculated by Equation (12):According to the stationary phase theory, the GH shift (dGHS) between p- and s-polarized light can be calculated as follows:For p-polarized light, the GH shifts produced by metal films are negative. However, the GH shift excited by s-polarized light in metal films are usually positive . MoreoveφS) and GH shift sensitivity (SGHS), were introduced. Both of them can be determined as follows: bion denotes a tiny variation of RI in sensing interface of Bi2Se3/graphene heterostructures due to strong adsorption binding interaction.To evaluate the biosensing performance of our proposed configuration, two parameters, such as phase sensitivity , as shown in 2Se3/graphene heterostructures, the plasmonic Au film (32 nm) has a reflectivity of 0.2786 under the illumination of 632.8 nm  and GH shift (ΔdGHS) response for a defined RIU variation (Δbion = 0.0012) were plotted, as shown in 2Se3/3-layer graphene. dGHS) by changing the number of Bi2Se3 and graphene layers. The configuration of 32 nm Au film-Bi2Se3 (2 QL)-graphene (3 layer) provides the highest differential GH shift of 1.0202 × 104 µm (green curve), which is almost 324 times larger than the second highest differential GH shift of 31.466 µm (blue curve) generated by the configuration of 32 nm Au film/2-QL Bi2Se3/2-layer graphene. When the number of Bi2Se3 QLs is 2, the phase detection sensitivity obtained by varying the number of graphene overlayer are almost on the same order of magnitude of 104°/RIU, as shown in To study the biosensing ability of our proposed biosensor, both differential phase (ΔSPRθ) for a defined RIU variation (Δbion = 0.0012), as shown in 4 degree/RIU, as shown in 6 µm/RIU. It is generally agreed that such a large GH shift can be easily detected by common optical devices. Compared with other reported GH shift-based plasmonic configurations  and graphene overlayers (0–5) were studied, as shown in 2Se3 layers was less than 2, there was a good linear response for change in differential GH shift by changing the RI of sensing interface. Obviously, the obtained GH shift was relative weak, indicating that there was no significant SPR enhancement effects, as shown in 2Se3 layers is 2, the addition of 3-layer graphene can precipitously produce an ultrasensitive response, as shown in 2Se3 layers was larger than 2, the addition of graphene overlayer could not provide a positive response because of excessive energy loss. It can be concluded that the configuration of 32 nm Au film coated with two-QL Bi2Se3 and three-layer graphene has the strongest GH shift response, showing great promise for performing ultrasensitive biosensing for a tiny RI variation.Next, the detailed changes in differential GH shift for the RI from 1.3345 to 1.3357 RIU by tuning the number of Bi2Se3-3-layer graphene. −6 RIU. In contrast to 32 nm Au thin film, 3-layer graphene coated on 32 nm Au film, the optimal configuration: 32 nm Au film−2-QL Bi2Se3-3-layer graphene can provide an enhancement factor (EF) of 2 × 105. It was assumed that significantly local field intensity enhancement should contribute to obtain such a high EF. It is well-known that typical nanoscale heterogeneous configurations, such as C-shaped [2Se3-3-layer graphene was further simulated via finite element analysis method, as shown in 2Se3/graphene sensing interface. Moreover, the electric field intensity decayed exponentially into a running buffer, resulting in a penetration depth of 150 nm. In addition, the charge-transfer mechanism generated by difference in work function (Fermi level) was proposed to contribute to giant electric field enhancement. It has been reported that the work function of Au film is ~5.54 eV and the work function of 2-QL Bi2Se3-3-layer graphene is ~4.75 eV [2Se3/Graphene heterostructures to Au thin film.To quantitatively monitor the concentrations of target analytes, it is necessary to determine a linear response interval for our optimal plasmonic configuration: 32 nm Au film−2-QL BiC-shaped , Φ-shapeC-shaped , bowtie-C-shaped  and ringC-shaped  nanostru~4.75 eV . The lar2Se3-3-layer graphene, the feasibility of monitoring SARS-CoV-2 and its S protein was theoretically studied. It was predicted that the molecular weight (MW) of SARS-CoV-2 is approximately 800 kDa [dGHS = 30.1047 *cSprotein. Here, the Sproteinc denotes the concentration of S protein solution. More importantly, our proposed biosensor also exhibited an excellent response interval (0–13.44 nM) for quantitatively monitoring SARS-CoV−2. The linear response can be expressed by such an equation ΔdGHS = 133.7987 *SARS-CoV−2c, where SARS-CoV−2c stands for the concentration of SARS-CoV−2 solution. Therefore, our proposed plasmonic biosensor has shown great feasibility in quantitatively monitoring SARS-CoV-2 and its S protein.Employing the linear response interval of 32 nm Au film-2-QL Bi 800 kDa , which i 800 kDa . Accordi2Se3/graphene heterostructures with conventional plasmonic Au film. The biosensing performance of our proposed configuration was designed by GH shift, superior to phase modulation. The energy loss could approach zero by introducing the Bi2Se3/graphene heterostructures. The best configuration employed to achieve the highest photon absorption is 32 nm Au film/2-QL Bi2Se3/3-layer graphene, producing an ultralow (approaching zero) reflectivity of 5.4402 × 10−9 and a highest GH shift of −1.0202 × 104 µm. Responding to a small RI variation in 0.0012 RIU, the proposed biosensor was able to provide an ultra-high sensitivity of 8.5017 × 106 µm/RIU. To extend the potential application in biosensing, our proposed biosensor was employed to theoretically predict the adsorption response of SARS-CoV−2 in a tiny RI variation as low as 0.000002 RIU. For SARS-CoV−2, a linear relationship (0~13.44 nM) was determined by an equation ΔdGHS = 133.7987 *SARS-CoV−2c. For its S protein, another linear relationship was described by an equation ΔdGHS = 30.1047 *S proteinc and there was a linear detection interval (0~59.74 nM). In view of these observations, we concluded that, our optimal plasmonic biosensor shows great promise in quantitatively monitoring SARS-CoV−2 and other infectious viruses for practical applications.In this work, we theoretically proposed a sensitivity enhanced SPR biosensor by incorporating Bi


Despite advances in neonatal nutrition, very preterm infants remain at increased risk of extrauterine growth faltering. This prospective study aimed to examine the effect of hindmilk, the milk at the end of a breast expression session, on growth and plasma fatty acids (FAs) of infants born <30 weeks’ gestation who had been on full enteral feeds for ≥2 weeks and had a weight gain of <15 g/kg/day despite optimizing energy and protein intakes. Weight and plasma FAs were assessed before and two weeks after feeding hindmilk. Growth anthropometrics were assessed weekly for four weeks. Paired t-tests and multiple linear regression were used for statistical analyses of data from 34 infants and their 29 mothers. There was a significant increase in weight gain in the two weeks after feeding hindmilk . Weight Z-scores were larger at two weeks  and onwards. Head circumference Z-scores were larger at three weeks  and onwards. Plasma linoleic acid (LA) and α-linolenic acid (ALA) increased after feeding hindmilk. In conclusion, hindmilk may improve weight and head growth and increase LA and ALA in very preterm infants with suboptimal growth. A large randomized controlled trial is required to examine and validate the potential benefits of hindmilk. Despite advances in neonatal nutrition, very preterm infants remain at increased risk of extrauterine growth faltering ,2. Poor Fat constitutes the main energy source in human milk and accounts for 40–50% of caloric intake ,7,8. HinBreastmilk is also the main source of essential fatty acids (EFAs) in the postnatal period. EFAs are essential for brain development, visual functions, and growth . FeedingThe primary aim of this study was to assess the effect of feeding hindmilk on the growth velocity of very preterm infants with suboptimal growth velocity. The secondary aim was to examine the impact of feeding hindmilk on infants’ plasma fatty acid profile. This was a prospective cohort study conducted between January 2019 and June 2022 in the 39-bed level III NICU at Foothills Medical Center in Calgary, Alberta, Canada. The study was approved by the Conjoint Research Ethics Board of the University of Calgary . Written informed consent was obtained from mothers before enrollment. Infants born <30 completed weeks gestation and weighing <1500 g to mothers with enough milk supply (≥150% of infant’s daily needs) were on full enteral feeds for more than 2 weeks and that had slow weight gain , despite optimization of energy and protein intakes, were eligible. Optimization of nutritional intakes was achieved by our two neonatal registered dietitians to meet recommended daily intakes of energy intake  and protein (4.0−4.5 g/kg/day for infants weighing <1000 g and 3.5−4.2 g/kg/day for infants weighing 1000−1500 g) [The primary outcome of the study was the rate of weight gain in the two weeks after feeding hindmilk. Weight gain (g/kg/day) was calculated using the average 2-point method [(W2 − W1)/([(W2 + W1)/2]/1000) /number of days] (W = weight expressed in grams) . The secMother’s own milk (MOM) was the exclusive source of enteral nutrition for study subjects. None of the subjects required pasteurized donor human milk in the 2 weeks prior to hindmilk fractioning or during the study. Weight-based enteral feeding tables were used as per our NICU guidelines, and daily feeding goals were posted at each infant’s bedside. Nutrient fortification commenced at 80 mL/kg/day, and parenteral nutrition was stopped once feeding volume reached 100–120 mL/kg per day and tolerance was observed. Fortification began at 1 package/50 mL human milk, and when the infant reached 100 mL/kg/day it was increased to 2 packages/50 mL human milk . Similac Human Milk Fortifier Extensively Hydrolyzed Protein Concentrated Liquid  was used for nutrient enrichment. In infants with protein intake less than recommended values, Similac Extensively Hydrolyzed Protein Concentered Liquid  was prescribed at 0.5–1.5 g/kg/day amounts after nutrient fortification had reached 0.78 kcal/mL. Enteral feeds were further concentrated if weekly weight gain was <15 g/kg/day. Powdered hydrolyzed-protein formula  was prescribed to further concentrate feeds to approximately 0.92 kcal/mL  as needed. Infants identified to have suboptimal growth velocity were followed daily by registered dietitians.Probiotics were prescribed in infants weighing ≥1000 g as per our NICU protocol. Standardized multivitamin drops were provided daily, containing 375 IU of vitamin A, 200 IU of vitamin D, and 17.5 mg of vitamin C, with an additional 400 IU of vitamin D until the infant reached 37 weeks corrected gestational age. Iron supplement was prescribed at 2–3 mg elemental iron/kg/day beginning at 14 days of life. All infants were weighed daily on electronic scales with a precision of 10 grams by nursing personnel, using the In-Bed scale . Other anthropometric measurements, length, and head circumference were taken by nursing personnel on admission and then weekly till discharge with a newborn length board  and measuring tape, respectively. Infants were classified as being small for gestational age at admission to the NICU if they were below the 10th centile using Fenton’s Growth Curves . Levels Once the mother’s consent was obtained, mothers were counseled by the study lactation support consultant on techniques to separate their milk during routine pumping sessions. All mothers used the double-pumping system for milk expression . Foremilk was defined as the milk collected for 3 min after the flow has begun . HindmilTwo fresh milk samples, 6 mL each, were collected once for milk content analysis at the start of the study. The first sample was from composite milk and the second sample was from hindmilk. Milk samples were analyzed using a near-infrared (NIR) instrument . Milk samples for fatty acid profiles and dried spots  were collected at the same time. Blood samples for plasma fatty acid profiles were collected on dried spots and on the same day as milk sampling; the second sample was collected after 2 weeks. Dried blood and milk spots were initially placed in a refrigerator for <12 h and then stored in a −80 °C freezer. The collection of blood samples was coordinated with other blood tests obtained for clinical purposes. Data on fluid volumes, feeds, macronutrient intakes, and any change in the nutrition plan were collected from electronic charts. Average weight gain was collected weekly for 2 weeks before and 2 weeks after starting hindmilk. The clinical team was encouraged to not change the nutrition plan for 2 weeks after starting hindmilk. Daily weight and weekly length and head circumference were collected from the electronic charts. Furthermore, weight, length, and head circumference were used to calculate Z scores using Fenton Z scores completed gestational week calculator . Our local data (unpublished) indicated that very preterm infants with suboptimal growth velocity had an average weight gain of 13 g/kg/day (standard deviation (SD) = 7). A previous small study of 15 preterm infants showed a 7 g (SD = 4.4) increase in weight gain when hindmilk was fed [p value < 0.05 was considered statistically significant. STATA software was used for all analyses.Descriptive statistics using mean, median, SD, and interquartile range (IQR) were used to describe the study population. A paired t test was used to compare average weight gain before and after hindmilk use. Linear regression analysis was used to assess the association between weight gain and milk content, and to adjust for potential confounding factors that included changes in measured milk protein, gestational age at birth, infant sex, exposure to low-dose postnatal dexamethasone to facilitate extubation (DART protocol) , and BPDn = 82), parental refusal to participate (n = 27), and other reasons (n = 21). Thirty-four infants completed the study . One hundred twenty-nine infants were excluded for; not meeting the inclusion criteria . The median age at starting hindmilk was 33.5  days. Total fluid intake slightly decreased in weeks 3 and 4 A. Total There was a significant increase in the rate of weight gain in the 2 weeks after starting hindmilk compared with the prior 2 weeks . Because the nutritional intakes and growth during the transitional period after reaching full enteral feeds were unstable in some infants, we compared weight gain between the latest week when the infants were on composite milk with the 2 weeks after hindmilk introduction. The detailed nutrient intakes during this period are summarized in Linear regression analysis showed a significant association between the difference in fat content (Δ fat) between composite milk and hindmilk, and weight Z-score catch-up (Δ) between the start of hindmilk and 4 weeks later , Oleic (C18:1 n9), palmitic (C16:0), and linoleic acid  were the dominant fatty acids in milk  and plasOur study indicates that feeding hindmilk to very preterm infants with growth faltering may result in larger weight gain and higher weight and head circumference Z-scores after four weeks. Concentrations of fat and energy were significantly higher in hindmilk and were associated with improved weight and head size growth. Feeding hindmilk resulted in higher plasma EFAs, LA, and ALA. There was no difference in milk fatty acids distribution between hindmilk and composite milk. Although the pathogenesis of suboptimal extrauterine growth is multifactorial, variations in energy intakes could explain approximately 45% of the growth deficits in preterm infants . MeetingFractionating breastmilk in our study resulted in approximately 1.4- and 1.16-fold increases in fat and energy of hindmilk content, respectively, compared with composite milk. This is in line with previous studies on the hindmilk of mothers of preterm infants ,8. PreviOur finding of increased weight gain is consistent with that in a previous study that used similar pre- and post-study design . ValentiIt is worth mentioning that the effect of hindmilk in our study was temporary. The discharge weight z-score returned to values that were presented at the time of hindmilk introduction. The transitional period between 33–34 weeks corrected gestational age, which matches the endpoints in our study, and hospital discharge is complex in extremely preterm infants on respiratory support. Healthcare providers typically try weaning infants off nasogastric tubes and non-invasive respiratory support during this period. In addition, the type of fortification and other nutritional support are usually modified to facilitate hospital discharge. Some infants in our cohort required PN after the study endpoints due to morbidities including late-onset sepsis, NEC, and the need for surgical ligation to close the patent ductus arteriosus. Furthermore, most of the preterm infants in our cohort were transferred to level II NICUs before discharge. Unfortunately, we were not able to track nutritional intakes after the transfer. Using a mathematical approach, a 1.3 g fat/dL difference between analyzed composite milk and hindmilk results in an actual nutritional difference of 1.07 g of fat (83% of milk volume in 100 mL of fortified HM) and 9.6 Kcal/dL, after considering the fortification with liquid HMF and assuming relatively equal fluid intakes between pre- and post-hindmilk. Because the fat absorption in preterm infants is significantly lower than the fat absorption rate used to define metabolizable energy of the Atwater factor (70–80% in preterm infants versus 96% in adults) , the metHindmilk is an attractive source of non-protein energy for improving growth in preterm infants because of its high energy density, lower osmolality compared with other fortification strategies, and its role in providing higher concentrations of soluble vitamins . HoweverStudies of optimal nutrition in preterm infants have primarily focused on the need for adequate protein intake to improve linear growth ,30. Use There was a significant improvement in head circumference Z-score three and four weeks after feeding hindmilk. Previous studies on hindmilk did not reveal a significant change in head growth after hindmilk; however, these studies monitored head circumference for shorter durations ,25,26. In-6 and n-3 long-chain polyunsaturated fatty acids (LCPUFAs). Dietary intakes of LA and ALA, and the balance between them, have the potential to affect the LCPUFA status and impact neural functions and tissue adiposity in preterm infants [n-6: n-3 ratio of 4.5:1.0. The use of human milk fortifier in both phases of our study may partly explain the lack of difference in plasma DHA and ARA levels with hindmilk.Plasma LA and ALA in our study increased by 1.1 and 1.3-fold, respectively, with no change in plasma ARA and DHA. LA and ALA are precursors for  infants ,36. Inte infants . This is infants . It is i infants . The con infants . The humOur study included more infants than previous studies and followed growth velocity for a longer duration. The study is the first to report plasma fatty acid distribution after feeding hindmilk. Our study has several limitations to acknowledge. First, the study did not have a control group to compare growth anthropometrics and fatty acids, so it is not known how much of the infants’ growth improvements were due to the hindmilk intervention and how much might have occurred without the intervention. Randomization or having a control group for the infants with suboptimal growth velocity was not logistically feasible or ethical in our NICU settings, because hindmilk was part of standardized practice guidelines that aim to optimize postnatal growth. It is worth noting that the only change for the infants in our cohort was the hindmilk, and there were no other changes to their diet. Second, due to logistical challenges, we analyzed the samples of composite milk and hindmilk at the beginning of the study only and before fortification. A more frequent and comprehensive analysis is required in future studies. Third, we did not perform body composition or magnetic resonance imaging to identify if hindmilk alters the body’s fat mass, fat-free mass, or brain volume and connectivity. Fourth, we did not follow up regarding the long-term growth or neurodevelopmental outcomes. Long-term follow-up studies are needed to further understand the impact of feeding fortified hindmilk.In conclusion, hindmilk produced by mothers of very preterm infants born <30 weeks’ gestation has higher concentrations of fat and energy compared with composite milk. Feeding very preterm infants with suboptimal growth velocity their mothers’ fortified hindmilk may improve weight and head growth trajectories and plasma concentrations of LA and ALA. Whether the infants’ accelerated weight and head growth and increasing plasma LA and ALA improve long-term neurodevelopment and brain growth parameters remains to be explored. Larger studies are required to examine the benefits of hindmilk and validate our results.


This activity, known as retrotransposition, may induce genomic alterations, such as insertions and deletions. Interestingly, L1s can retrotranspose and generate more de novo L1 copies in brains than in other somatic tissues. Here, we describe for the first time interchromosomal translocation triggered by ectopic L1 retrotransposition in neural progenitor cells. Such an observation adds to the studies in neurological and psychiatric diseases that exhibited variation in L1 activity between diseased brains compared with controls, suggesting that L1 activity could be detrimental when de-regulated. LINE-1 (L1) elements are a class of transposons, comprising approximately 19% and 21% of the mouse and human genomes, respectively. L1 retrotransposons can reverse transcribe their own RNA sequence into a  About 20% of the human, rat and mouse genomes are composed of LINE-1 (L1) retrotransposons . Althougin vitro and in vivo, resulting in a L1 genetic mosaic nervous system . HCN cells represent a heterogeneous population composed of neural stem cells and more differentiated NPCs. An episomal retrotransposition-competent human active L1 element (LRE3) controlled by its endogenous promoter and carried an EGFP reporter construct was used to obtain the Clone C6+ . BrieflyPreviously characterized L1 clones in NPCs showed a neuronal differentiation tendency using a mixed differentiation protocol . Then, win situ hybridization) reaction revealed the presence of two signals in two independent chromosomes in HCN cells, suggesting the presence of two X chromosomes, as expected for a female-derived cell line. However, clone C6+ cells displayed an extra signal in a third chromosome (Inverse PCR was used to characterize the post-integration site from clone C6+ . The insromosome . Finallyromosome . Interesde novo L1 insertion using a similar system described here was previously described to map a putative interchromosomal translocation in HeLa cells (Chromosomal translocations caused by L1 retrotransposons were proposed as a mechanism to explain the evolution of recent rodent L1 subfamilies and the occurrence of promoters swapping for non-LTR retroelements in certain avian species . A de noLa cells . The facThe L1 target in the clone C6+ was inside the Loc317538 in chromosome Xq22, which is a region like the dystrophin major muscle isoform. Alterations in gene expression in this region do not seem to cause the strong neurogenic effect observed in this clone. However, the SKY experiment also revealed other, less frequent chromosomal translocations that may contribute to the final phenotype. While our data was observed using an ectopic L1 reporter construct, it is currently unknown if these other interchromosomal translocations were caused by endogenous L1 retrotransposition.A chromosomal inversion involving parental L1 retrotransposon polymorphism has been correlated to autism in siblings . At a so


Letermovir (LTV) for CMV prophylaxis in allogeneic hematopoietic cell transplant (HCT) recipients has decreased utilization of broad-spectrum antivirals for CMV, which may result in more frequent reactivation of herpesviruses not targeted by LTV. We hypothesized that the cumulative incidence of HHV-6 and EBV reactivation, and associated diseases, within 100 days after HCT would increase following the introduction of LTV.We conducted a retrospective study of CMV-seropositive adults who received an allogeneic HCT at our center prior to (5/2015 – 9/2018) and after (10/2018 – 12/2021) routine use of LTV. We reviewed medical records for antiviral use, viral testing, and virus-associated end-organ disease within days 0-100 after HCT. Testing was performed at the discretion of healthcare providers or according to treatment protocols. We computed cumulative incidence estimates treating death and subsequent HCT as competing risks.Figure 1). Two patients in each cohort (0.5%) developed proven or probable HHV-6 encephalitis. Median peak plasma HHV-6 viral load was nearly 1 log10 higher in the LTV cohort . The incidence of tests for and detection of EBV was higher in the LTV cohort, but the proportion of patients with ≥ 1 positive test among tested patients was similar (14% pre-LTV vs 20% post-LTV); of note, EBV testing in both cohorts was mainly protocol driven . There were no cases of PTLD by day 100 in either cohort.We identified 403 patients prior to and 378 patients after routine use of LTV. Characteristics were similar between cohorts except for more frequent use of post-transplant cyclophosphamide for GVHD prophylaxis in the LTV cohort. Broad-spectrum antiviral use, as estimated by available data, was 68% pre-LTV versus 23% post-LTV. The incidence of tests for and detection of HHV-6 were similar in both cohorts (Despite a 66% decrease in broad-spectrum antiviral use after the introduction of LTV prophylaxis in CMV-seropositive allogeneic HCT recipients, there was no evidence for increased HHV-6 or EBV reactivation and associated end-organ diseases. Whether the higher peak HHV-6 viral load in the LTV cohort has clinical implications requires further study.Danielle M. Zerr, MD MPH, AlloVir: Advisor/Consultant Michael J. Boeckh, MD PhD, Allovir: Advisor/Consultant|Amazon: Grant/Research Support|Ansun Biopharma: Grant/Research Support|EvrysBio: Advisor/Consultant|Gates Ventures: Grant/Research Support|Gilead Sciences: Advisor/Consultant|Gilead Sciences: Grant/Research Support|GlaxoSmithKline: Advisor/Consultant|GlaxoSmithKline: Grant/Research Support|Helocyte: Advisor/Consultant|Janssen: Advisor/Consultant|Janssen: Grant/Research Support|Kyorin Pharmaceuticals: Advisor/Consultant|Merck: Advisor/Consultant|Merck: Grant/Research Support|Moderna: Advisor/Consultant|Moderna: Grant/Research Support|Regeneron: Grant/Research Support|ReViral: Advisor/Consultant|Symbio: Advisor/Consultant|Takeda: Grant/Research Support|Vir Biotechnology: Advisor/Consultant|Vir Biotechnology: Grant/Research Support Joshua A. Hill, MD, Allovir: Advisor/Consultant|Allovir: Grant/Research Support|Covance/CSL: Advisor/Consultant|CRISPR: Advisor/Consultant|Deverra: Grant/Research Support|Gilead: Grant/Research Support|Karius: Advisor/Consultant|Karius: Grant/Research Support|Merck: Grant/Research Support|Octapharma: Advisor/Consultant|OptumHealth: Advisor/Consultant|Oxford Immunotec: Grant/Research Support|Pfizer: Advisor/Consultant|Symbio: Advisor/Consultant|Takeda: Advisor/Consultant.


It is widely accepted that some types of learning involve structural and functional changes of hippocampal synapses. Cell adhesion molecules neural cell adhesion molecule (NCAM), its polysialylated form polysialic acid to NCAM (PSA-NCAM), and L1 are prominent modulators of those changes. On the other hand, trace eyeblink conditioning, an associative motor learning task, requires the active participation of hippocampal circuits. However, the involvement of NCAM, PSA-NCAM, and L1 in this type of learning is not fully known. Here, we aimed to investigate the possible time sequence modifications of such neural cell adhesion molecules in the hippocampus during the acquisition of a trace eyeblink conditioning. To do so, the hippocampal expression of NCAM, PSA-NCAM, and L1 was assessed at three different time points during conditioning: after one , three , and six (complete acquisition) sessions of the conditioning paradigm. The conditioned stimulus (CS) was a weak electrical pulse separated by a 250-ms time interval from the unconditioned stimuli . An acquisition-dependent regulation of these adhesion molecules was found in the hippocampus. During the initial acquisition of the conditioning eyeblink paradigm (12 h after 1 and 3 days of training), synaptic expression of L1 and PSA-NCAM was transiently increased in the contralateral hippocampus to the paired CS-US presentations, whereas, when the associative learning was completed, such increase disappeared, but a marked and bilateral upregulation of NCAM was found. In conclusion, our findings show a specific temporal pattern of hippocampal CAMs expression during the acquisition process, highlighting the relevance of NCAM, PSA-NCAM, and L1 as learning-modulated molecules critically involved in remodeling processes underlying associative motor-memories formation. Acquired motor abilities are believed to be stored in the form of structural and/or functional changes in synaptic efficiency . The neuThe role of NCAM, its polysialylated form, and L1 in synaptic plasticity and cognitive processes has been based on several lines of evidence. First, interference with the expression and/or function of these molecules in knock-out mice models or pharmacologically by the administration of antibodies, peptides, enzymes, or antisense oligonucleotides results in impaired long term potentiation (LTP)  and learClassical conditioning of eyelid responses is a well-known and widely used associative motor learning task. In trace eyeblink conditioning paradigms, the conditioned stimulus (CS) is separated from the unconditioned stimulus (US) presentation by a stimulus-free interval (trace), and the hippocampus is thought to play a major role in the formation of this memory trace , 2015. IGiven the critical role of the hippocampus in associative learning , and thead libitum. All experimental procedures were reviewed and approved by the Ethical Committee for Use of Laboratory Animals of the National Distance Education University (UNED) and conducted according to the European Union guidelines (2010/63/EU) and the Spanish regulations for the use of laboratory animals in chronic experiments . All efforts were made to minimize animal suffering.Fifty-seven adult male Wistar rats , group housed three per cage, were used for all experiments. Animals were kept in their home cages at 22 ± 2°C and with a 12 h light-dark cycle (lights on at 7:00 a.m.). Food and water were provided All surgical procedures were carried out under sodium pentobarbital anesthesia (40 mg/kg i.p.) and using aseptic surgical techniques. Four teflon-coated stainless-steel wires  were implanted in the subcutaneous tissue of the right upper eyelid to be used as bipolar stimulating electrodes of the right supraorbital branch of the trigeminal nerve and as bipolar electromyographic (EMG) recording electrodes in the ipsilateral orbicularis oculi (OO) muscle . Electroswivel, Plastics One, US) which allowed the rat to move freely during training.Classical conditioning training was developed using an experimental design that allowed the animals to freely move during acquisition sessions . For this purpose, electrical stimulation of the supraorbital sensory nerve was used as conditioned (CS) and unconditioned (US) stimuli, respectively . ElectriClassical conditioning was achieved using a trace paradigm . BrieflyThe EMG activity of the OO muscle was recorded using a GRASS P511 differential amplifier with a bandwidth of 1 Hz to 10 kHz . Data was stored directly on a computer with an analog/digital converter , at a sampling frequency of 11–22 kHz and an amplitude resolution of 12 bits. Data was analyzed off-line for quantification CRs. The presence of EMG activity during the CS-US period which lasted >10 ms and was initiated >50 ms after CS onset was considered a CR; thus, we avoided including putative alpha responses in the quantification of true CRs. Those recordings presenting EMG activity in the 250 ms preceding CS presentation (change in baseline), or with an evident startle response (response beginning 4 ms after CS and expanded until 100 ms) or artifactual recordings were excluded from the quantitative analysis. The percentage of trials containing a CR in a given session was calculated based on the number of valid trials.g for 5 min. The supernatant was then centrifuged at 15,000 g for 15 min, and the pellet was resuspended in Krebs buffer, containing protease inhibitors, for further use. Protein concentration for each sample was measured by the Bradford method  12 h after the end of the 1st, 3rd, or 6th training session and decapitated. A total of 4–9 animals per group were used. Immediately after decapitation, brains were kept on ice and both right and left hippocampi  were dissected. Tissue samples were coded and stored at −80°C until use. Crude synaptosomal pellets were obtained according to a modified protocol from d method .2CO3 0.1 M/NaHCO3, 0.1 M) for 2 h at room temperature (RT). The solution was removed and 50 μl of pellet samples were added at a concentration of 10 μg/ml to each well of polystyrene flat-bottom ELISA plates. Plates were incubated overnight at 4°C and then washed three times with 1 M phosphate-buffered saline (PBS) containing 0.05% Tween 20, pH 7.4. Additional binding sites were blocked with BSA (3%) for 2 h at RT. Wells were rinsed three times as described above, and incubated with 50 μl aliquots of the corresponding first antibody for 20–24 h at 4°C. Then, wells were washed and 50 μl aliquots of peroxidase-conjugated secondary antibody were added for a 2 h incubation period. Afterward, 50 μl of citrate buffer  containing 1 mg/ml o-phenylene diamine and 0.06% H2O2, added just before use, were placed in each well, and the peroxidase allowed to react for 10 min at RT. The reaction was terminated by the addition of 50 μl of 10 M H2SO4 to each well. Optical density was determined by measuring absorbance at 492 nm with a Microplate Reader .Total NCAM , PSA-NCAM, and L1 were quantified by enzyme-linked immunosorbent assays (ELISAs) according to a previously described protocol . In brieThe following primary antibodies were used: a polyclonal rabbit anti-rat NCAM IgG ; a monoclonal mouse IgM antibody that recognizes specifically a-2–8-linked PSA with chain length superior to 12 residues, and binds with high specificity to PSA on NCAM ; and a monoclonal rat anti-rat L1 IgG . As secondary antibodies, an anti-rabbit IgG peroxidase conjugate , an IgM anti-mouse peroxidase conjugate , and an anti-rat Ig-POD Fab fragments (from sheep immunoglobulin) to IgG-rat peroxidase conjugated  were used, respectively.post-hoc analysis. Biochemical data were analyzed with three-way ANOVAs , followed by Tukey’s post-hoc analysis. Statistical significance was set at p < 0.05. If the Levene’s test for normal distribution was significant then data were normalized by square root transformation (percentage of conditioned responses). All analyses were performed using the SPSS software v.28  and final figures were prepared using CorelDraw X8 Software .Data was expressed as mean ± SEM. Statistical differences between percentages of CRs were determined by repeated measures two-way ANOVA, followed by Tukey’s n = 26), pseudo-conditioning , or context exposure without stimulation . Animals were sacrificed after the 1st , 3rd , or 6th  training sessions.Animals were randomly assigned to one of the following three experimental paradigms: classical eyeblink conditioning  of the supraorbital nerve evoked an early (6–7 ms) activation of the OO muscle, followed by a second, more variable (15–25 ms) EMG activation (not illustrated). These successive muscle activations corresponded to the R1 and R2 components previously described in rats , as wellF = 318.802; p < 0.001; post-hoc analysis revealed that the different was present in all training sessions except the first one.Then the learning capabilities of conditioned and pseudo-conditioned rats were compared using a trace conditioning paradigm. As illustrated in In order to evaluate the effect of classical eyeblink conditioning on the expression of neural cell adhesion molecules in the hippocampus, expression levels of NCAM, PSA-NCAM, and L1 in hippocampal crude synaptosomes were determined 12 h after the 1st , 3rd , and 6th conditioning sessions (complete acquisition) .F = 1.967; p = 0.168], PSA-NCAM , or L1  expression levels at any of the training session studied (The two control groups submitted to either pseudo-conditioning (unpaired CS/US training) or context exposure (non CS or US stimulation) did not significantly differ in neither their synaptic ipsi- nor the contralateral hippocampal NCAM  and post-hoc analysis indicated that it was specifically an upregulation of L1 expression in the contralateral hippocampus of conditioned rats after sessions one and three. Moreover, there was also an hippocampal side effect , since that increase in the contralateral hippocampus was not present in the ipsilateral area, and a session effect  as the difference between groups was lost in the last session. Thus, L1 levels seems to be regulated by conditioning learning in an acquisition-dependent way in the contralateral hippocampus, being overexpressed specifically in this side during initial and partial learning acquisition but not when the conditioning was completely acquired.Regarding L1 levels , a threeF = 4.113; p = 0.0452] in the contralateral hippocampus after one and three training sessions. Once again, there was a significant side effect , specifically in the contralateral hippocampus. After one and three conditioning session PSA-NCAM values were enhanced, but they did not differ to control ones after six sessions . As it happened with L1, PSA-NCAM was overexpressed in the contralateral hippocampus during the initial and partial acquisition sessions but decreased to normal values after complete conditioning acquisition.A similar pattern of learning-induced modulation was found for synaptic PSA-NCAM levels in the paired  group . ConditiF = 47.53; p < 0.0001]. The regulation was not only session-dependent, but also training-dependent. Hence, synaptic NCAM levels from conditioned rats were higher than those of both pseudo-conditioned and context-exposed control groups after the sixth conditioning session, when asymptotic learning levels had been reached . As shown in F = 3.195; p = 0.0772]. Therefore, NCAM seems to be bilaterally upregulated by conditioning learning only after complete acquisition of the association.Finally, total NCAM expression in hippocampal crude synaptosomes, as assessed by ELISA using an antibody that recognizes all NCAM isoforms, was bilaterally increased 12 h after submitting rats to six conditioning sessions [The hippocampus has been one of the brain areas tightly related to the acquisition of new motor abilities. Many studies have proven that this area facilitates, reinforces, and organizes the proper achievement of CRs. According to available information, the full integrity of the hippocampus is needed in order to acquire the conditioning .In the present work, classical eyeblink conditioning in rats was achieved using a trace paradigm in which both the CS and the US were electric pulses. Although rats in both paired (conditioned) and unpaired (pseudo-conditioned) groups were presented with CS and US electrical stimuli, only rats assigned to the paired group did establish an association between both stimuli and therefore learned the task , achieviThen, to evaluate the effect of classical eyeblink conditioning on the expression of cell adhesion molecules in the hippocampus, levels of L1, PSA-NCAM, and NCAM in hippocampal crude synaptosomes were determined. A few rodent studies have evaluated the possible regulation on NCAM, its polysialylation and/or L1 by hippocampal-dependent training experiences  but noneAdditionally, to determine the evolution of the expression of these molecules throughout the learning curve, the measurement was carried out after 1st , 3rd , and 6th (complete acquisition) conditioning sessions.We report here that L1 and PSA-NCAM upregulation is transient in the hippocampus. Thus, both CAMs were overexpressed after first and third sessions in the contralateral hippocampus of the conditioned animals compared to both its ipsilateral hippocampus and the hippocampi of the control group. However, after the 6th conditioning session both L1 and PSA-NCAM returned to basal levels. This finding is in accordance with other studies relating learning to changes in cell adhesion molecules ,b or theOn the other hand, our data showed a completely different pattern for NCAM expression. During the initial and partial conditioning, there were no alterations of its levels, while after conditioning completion (six sessions), a bilateral enhancement of NCAM was observed in the paired group compared to control animals. Previous studies have related NCAM with the progression from a labile memory to a stable, consolidated memory . Our resMoreover, NCAM is composed of three molecular components with relative molecular masses of 120, 140, and 180 kD. NCAM-120 does not appear to be expressed in synapses, whereas NCAM-140 is located on both pre- and post-synaptic membranes and NCAM-180 is restricted mostly to post-synaptic sites . In the In conclusion, the temporal evolution of the three studied adhesion molecules in the hippocampus along the conditioning eyeblink paradigm  could account for initial facilitation of structural changes during learning acquisition (by PSA-NCAM and L1) and late stabilization of previously restructured synapses to progress from a labile to a stable motor memory (by NCAM). These results also suggest that CAMs are learning-modulated molecules whose expression needs to be orchestrated in a coordinated manner in the hippocampus for the proper acquisition of hippocampal-dependent associative motor learning.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.This animal study was reviewed and approved by Ethical Committee for Use of Laboratory Animals of the National Distance Education University (UNED).LJ-D, JD-G, AG, and CS were responsible for the initial conceptualization. LJ-D, AH, KT, KC, and CV performed the experiments. LJ-D, JN-L, and AC analyzed the data, did the writing—review and editing, and were responsible for writing the original draft. CS contributed to the materials. LJ-D, JN-L, JD-G, and AG were responsible for funding acquisition. All authors contributed to the article and approved the submitted version.