Two-sample Mendelian randomization (MR) allows the use of freely accessible summary association results from genome-wide association studies (GWAS) to estimate causal effects of modifiable exposures on outcomes. Some GWAS adjust for heritable covariables in an attempt to estimate direct effects of genetic variants on the trait of interest. One, both or neither of the exposure GWAS and outcome GWAS may have been adjusted for covariables.We performed a simulation study comprising different scenarios that could motivate covariable adjustment in a GWAS and analysed real data to assess the influence of using covariable-adjusted summary association results in two-sample MR.In the absence of residual confounding between exposure and covariable, between exposure and outcome, and between covariable and outcome, using covariable-adjusted summary associations for two-sample MR eliminated bias due to horizontal pleiotropy. However, covariable adjustment led to bias in the presence of residual confounding , even in the absence of horizontal pleiotropy . In an analysis using real data from the Genetic Investigation of ANthropometric Traits (GIANT) consortium and UK Biobank, the causal effect estimate of waist circumference on blood pressure changed direction upon adjustment of waist circumference for body mass index.Our findings indicate that using covariable-adjusted summary associations in MR should generally be avoided. When that is not possible, careful consideration of the causal relationships underlying the data  is required to direct sensitivity analyses and interpret results with appropriate caution. Summary genetic association results from large genome-wide associations studies (GWAS) have been increasingly used in two-sample Mendelian randomization (MR) analyses.Many GWAS adjust for heritable covariates in an attempt to estimate direct genetic effects on the trait of interest.In an extensive simulation study, we demonstrate that using covariable-adjusted summary associations may bias MR analyses.The bias largely depends on the underlying causal structure, specially the presence of unmeasured common causes between the covariable and the outcome.Our findings indicate that using covariable-adjusted summary associations in MR should generally be avoided.,Mendelian randomization (MR) uses genetic variants to assess the influence of modifiable exposures on health outcomes.Two-sample MR is an extension to the one-sample MR design, where estimates for the association of genetic variants with exposure and with outcome are derived from different (non-overlapping) samples from the same underlying population.,In GWAS, estimates for the association of genetic variants with the trait of interest are often conditioned on covariables. As an example, GWAS of waist-to-hip ratio have adjusted estimates for body mass index (BMI),The potential issue of adjusting for heritable covariates is illustrated in et al., Emdin et al., Borges et al., Au Yeung et al., Clarke et al.,,Several two-sample MR studies have used summary association data from GWAS that have estimated the effect of genetic variants on the trait of interest conditioned on heritable covariables  . This maWe performed simulations in two main ways:(i) All genetic variants have directionally consistent direct effects on the same traits (hereafter referred to as ‘homogeneous genetic variants’). For example, if there are 10 simulated genetic variants and one of them has positive direct effects on both (ii) Some genetic variants have direct effects only on some traits (hereafter referred to as ‘heterogeneous genetic variants’). This also varies according to the data-generating model (explained below).The distinction between situations (i) and (ii) is further clarified below, after presenting the causal structures underlying the simulations.Scenarios simulated under situation (i) are illustrated in IJE online), all direct effects are independent of one another. Therefore, the In scenarios A1-F1, there are no unmeasured confounders (other than R). To isolate the implications of unmeasured confounders when controlling for To simulate data under situation (ii), the same underlying causal structure used for situation (i) was used, with the exception that, in all simulations, there were four non-overlapping subgroups of genetic variants: some with direct effects on It is now easy to clarify the distinction between situations (i) and (ii). In situation (i), all genetic variants have direct effects on All simulations generated data for 40 independent candidate genetic instruments and 100 000 individuals, and the resulting dataset was divided into two halves at random: one was used to estimate instrument-exposure associations, and the other to estimate instrument-outcome associations, thus corresponding to the two-sample MR context. Briefly, for each measured phenotypic variable, all genetic variants with direct effects on the given phenotype were combined into an additive allele score which had an effect set to account for 10% of the variance of the given phenotype. For measured phenotypic and unmeasured variables with direct effects on the given phenotype, direct effects were set to account for 20% of the variance of the given phenotype. A detailed description on how effects were generated is provided in the For analyses using unadjusted variant-exposure summary association results , the outcome variables were systolic (SBP) and diastolic blood pressure (DBP) and the covariable was BMI. We selected genetic instruments of unadjusted WC and BMI-adjusted WC from the Genetic Investigation of ANthropometric Traits (GIANT) consortiumBMI was used as a covariable due to its strong correlation with WC, meaning that variants that affect WC might also affect BMI due to their effect on overall adiposity. Here, we assume that two distinct causal structures  are plauP-value <5 × 10–8. After quality control  are already available. Therefore, we selected two sets of WC genetic instruments: one using the unadjusted GWAS results; and another using the BMI-adjusted GWAS. In each case, independent genetic variants were selected as WC instruments if ,Causal effect estimates were obtained using multiplicative random effects inverse-variance weighting (IVW).IJE online, displays the number of select genetic instruments and mean IJE online). Although the absolute bias was generally larger when simulating homogeneous genetic variants, the results were generally in the same direction in both situations.In the absence of unmeasured confounders (Scenario 1), using adjusted instrument-In cases where In the unadjusted SNP-exposure and SNP-outcome analysis, each standard unit increase in WC was related to an increase of 0.06 mmHg in SBP  and of 0.12 mmHg in DBP . These effects changed in direction when only adjusting the SNP-outcome association for BMI . Effect estimates also changed direction, but were consistent with the null when adjusting only the SNP-exposure association  or using both adjusted SNP-exposure and SNP-outcome associations  .P <0.05), results were similar for the adjusted SNP-exposure analyses. The exclusion of BMI-related SNPs dramatically restricted the number of SNPs included in the unadjusted SNP-exposure analyses (two out of 37 SNPs) and, as a result, estimates are highly imprecise (IJE online).After excluding SNPs associated with BMI  between exposure-covariable, exposure-outcome and covariable-outcome, covariable adjustment eliminates bias due to horizontal pleiotropy mediated by such covariable (Scenarios E and F). However, absence of unobserved confounding is unrealistic in observational studies and is one of the primary motivations for performing MR. In the presence of unobserved confounding, mainly between the covariable and the outcome, covariable adjustment will likely lead to bias even in the absence of horizontal pleiotropy, due to collider bias when genetic instruments are marginally associated with the covariable. Therefore, minimizing horizontal pleiotropy is generally an invalid justification for using covariable-adjusted summary association results for two-sample MR.Bias was generally weaker when the true causal effect of the exposure on the outcome was null among several scenarios we evaluated. Indeed, even in the absence of any unmeasured confounding, covariable adjustment may lead to bias when the true causal effect is not null. As an example based on the directed acyclic graph (DAG) for Scenario A1 in Using covariable-adjusted instrument-outcome summary associations was also consistently related to larger biases compared with using covariable-adjusted instrument-exposure summary associations. The exception was scenarios where there was horizontal pleiotropy. However, since attempting to minimize horizontal pleiotropy via covariable adjustment is generally unjustified due to the likely presence of unmeasured confounders, we are primarily concerned with the scenarios where there is no horizontal pleiotropy.Results were generally consistent between simulations with homogeneous and heterogeneous instruments. The simulations with homogeneous instruments are useful to isolate the effect of covariable adjustment according to each underlying causal structure linking In an analysis using real data from GIANT consortium and UK Biobank, the estimated causal effect of waist circumference on blood pressure changed substantially upon adjustment for BMI. In this context, interpreting whether covariable adjustment could have affected the validity of results is particularly challenging, since adjustment for BMI  could remove bias due to horizontal pleiotropy and/or introduce bias due to conditioning on a collider (i.e. BMI) in the pathway between instrument-outcome and/or instrument-exposure. As illustrated in To our knowledge, this is the first study to systematically assess the impact of covariable adjustment in (two-sample) MR. Conditioning on a heritable covariable can introduce bias when the covariable is a collider in the pathway between instrument-exposure and/or instrument-outcome. Collider bias can also be introduced in MR studies in other settings, such as in disease progression studies which include a selected (i.e. case-only) group of individualsIt is important to emphasize that using covariable-adjusted GWAS summary association results in two-sample MR studies differs from applying multivariable MR (MVMR) on unadjusted GWAS summary association results to estimate the effect of two or more exposures on an outcome.One of the strengths of our study was that our simulations covered a wide range of scenarios, thus allowing a detailed evaluation of covariable-adjustment bias in a variety of situations. The simulation study was also complemented with a real data example illustrating the strong influence that covariable-adjustment may have not only on the magnitude, but also on the direction of the causal effect estimate. However, any simulation study is a simplification of a reality that is likely to be much more complex. It is impossible to simulate all possible scenarios that might be of relevance to this topic. Moreover some results, especially quantitative estimates of bias and coverage, are highly dependent on the data-generating model. Therefore, our results should be interpreted qualitatively, as general indications of some of the main aspects related to covariable-adjustment in MR. It was reassuring that some results (as described above) were consistent across scenarios, which indicates that they may apply generally  to covariable-adjusted MR.In conclusion, our findings indicate that using summary association results adjusted for heritable covariables may lead to bias in two-sample MR due to unmeasured confounding. We recommend avoiding adjustment for such covariables in the context of MR. When only covariable-adjusted data are available, it is important to carefully consider the causal structure underlying the research question to understand the potential impact on the results. In such cases, we recommend that researchers refrain from interpreting the causal estimate too literally . To account for horizontal pleiotropy due to measured covariables, MVMR should be preferred over non-genetic covariable adjustment.IJE online.K.T., G.D.S., D.A.L. and M.C.B. work in the MRC Integrative Epidemiology Unit at the University of Bristol which receives funding from the UK Medical Research Council . D.A.L.’s contribution is also supported by the European Research Council under the European Union's Seventh Framework Programme [FP/2007–2013] / ERC Grant Agreement  and from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 733206 (LifeCycle). M.C.B. is supported by a Medical Research Council (MRC) Skills Development Fellowship [MR/P014054/1]. D.A.L. is a UK National Institute of Health Research Senior Investigator [NF-SI-0611–10196].dyaa266_Supplementary_DataClick here for additional data file.


The conventional reconstruction method of off-axis digital holographic microscopy (DHM) relies on computational processing that involves spatial filtering of the sample spectrum and tilt compensation between the interfering waves to accurately reconstruct the phase of a biological sample. Additional computational procedures such as numerical focusing may be needed to reconstruct free-of-distortion quantitative phase images based on the optical configuration of the DHM system. Regardless of the implementation, any DHM computational processing leads to long processing times, hampering the use of DHM for video-rate renderings of dynamic biological processes. In this study, we report on a conditional generative adversarial network (cGAN) for robust and fast quantitative phase imaging in DHM. The reconstructed phase images provided by the GAN model present stable background levels, enhancing the visualization of the specimens for different experimental conditions in which the conventional approach often fails. The proposed learning-based method was trained and validated using human red blood cells recorded on an off-axis Mach–Zehnder DHM system. After proper training, the proposed GAN yields a computationally efficient method, reconstructing DHM images seven times faster than conventional computational approaches. Quantitative phase imaging (QPI) techniques quantify the phase measurements from micrometer-sized biological and non-biological samples. Because phase measurements encode sample’s topography and refractive index information, QPI methodologies have been widely utilized for diagnosing diseases and refining manufacturing processes ,2,3,4. AOver the last decade, DHM has become a mature technology thanks to thousands of research studies that have focused on investigating its optical design, phase reconstruction algorithms, and potential applications of this modality to life and materials sciences ,23,24,25As in many areas of science and engineering, deep learning strategies have been used to improve the potential of DHM technology. Deep learning and neural networks have enhanced the performance of traditional reconstruction algorithms. For example, deep learning has been utilized to automatically determine the in-focus reconstruction plane in DHM ,32 and oIn this study, we used a traditional Mach–Zehnder off-axis DHM setup operating in transmission mode . The colThe DHM hologram distribution, quencies . The secrrations . Trujillhttps://www.carolina.com/help/service-and-support?utm_source=homepage&utm_medium=banner&utm_campaign=whyshop_service (accessed on 20 November 2021)) was used to create our dataset to train and validate the proposed learning-based method. Although these RBCs present some absorption, they can be considered phase objects when no staining is applied. Unstained RBCs have been widely imaged in DHM, enabling the detection of malaria [A microscopic slide containing normal  human red blood cells (RBCs) from Carolina Biological Supply Company  . The strtraining . This patraining  with a sIn this cGAN architecture, the discriminator model is a deep convolutional neural network for binary classification. Each input to this model is a pair of an off-axis in-focus DHM hologram and its corresponding phase image concatenated horizontally in a 256 × 256 × 6 entries array. This patchGAN discriminator  processeFor each training step, these loss functions allow the tunning of the coefficients of the model , ise zero . Figure The second customized metric involved measurement of the standard deviation (STD) inside one background region of the phase maps . For simplicity, we selected a region where no RBCs were present in the reconstructed phase images. An example of the region chosen to calculate the STD value is shown in Once the generator was adjusted correctly within the GAN training, we used this learning-based model to reconstruct free-of-aberration phase images of static and dynamic human RBCs from experimental holograms recorded in an off-axis telecentric DHM system. To use our proposed learning-based method, a sample code to reconstruct quantitative free-of-aberration phase images from RBC holograms recorded in an off-axis telecentric-based DHM is freely available in . The modIn our first experiment, we compared the performance of our learning-based cGAN model, after proper training, to that of the traditional reconstruction method  to analyFor a better comparison of the differences between the reconstructed phase maps produced by the traditional reference method e–h and tt) via 0 = 532 nm is the illumination wavelength, To further validate our method performance for these static holograms, we have compared the highest- and the lowest-quality reconstructed phase images provided by the conventional reference method to those obtained by our cGAN model. ve index , and nm A straightforward comparison of the performance of our method against that of the U-Net model is presented in this subsection to validate its appropriateness in the task of retrieving complete phase compensated phase maps in transmission off-axis DHM. The implemented U-net model has the same parameters as the generator model in our proposal, depicted inside the blue rectangle in To validate the generalization ability of our proposed method in regard to system diversity, DHM holograms were recorded in a common-path DHM system using a Fresnel biprism. This DHM system was comprised of a 40X/0.74 Nikon MO lens, and the camera used had 5471 × 3648 square pixels of 2.4 μm side. The Fresnel-biprism-based DHM system still operated in the telecentric regime, and the camera was located at the microscope’s image plane. More details on this DHM system can be found in . Note thn value stands for the i-th frame in the time-lapse recording sequence. Whereas the RBC flow is marked by the blue arrow in In this experiment, we investigate the performance and the robustness of the cGAN model for reconstructing aberration-free phase images in time-lapse DHM imaging. For this study, we recorded a sequence of 300 holograms that tracked the flow of RBCs within blood plasma. These recorded holograms were used as input images of our trained cGAN model. In this study, we report on a conditional generative adversarial network (cGAN) to fully reconstruct quantitative phase images from human red blood cells (RBCs). To the best of our knowledge, this is the first learning-based method to reconstruct off-axis DHM holograms of biological samples with minimum phase distortions from raw holograms without the need for robust pre- or post-numerical procedures. The raw RBCs holograms were recorded using an off-axis DHM system operating at the telecentric regime. The proposed cGAN model was trained using two customized metrics specifically designed for tracking the imaging characteristics in DHM: (1) the number of phase discontinuities using a thresholding-and-summation metric ) and (2) the noise level measured in homogenous regions of the reconstructed phase maps using the standard deviation (STD). Because we used two customized metrics , the proposed cGAN model converges rapidly . This learning-based method was trained using 1512 pairs of raw holograms and their reconstructed phase images obtained by a traditional reference method . Figure


Numerical simulations of coupled hemodynamics and leukocyte transport and adhesion inside coronary arteries have been performed. Realistic artery geometries have been obtained for a set of four patients from intravascular ultrasound and angiography images. The numerical model computes unsteady three-dimensional blood hemodynamics and leukocyte concentration in the blood. Wall-shear stress dependent leukocyte adhesion is also computed through agent-based modeling rules, fully coupled to the hemodynamics and leukocyte transport. Numerical results have a good correlation with clinical data. Regions where high adhesion is predicted by the simulations coincide to a good approximation with artery segments presenting plaque increase, as documented by clinical data from baseline and six-month follow-up exam of the same artery. In addition, it is observed that the artery geometry and, in particular, the tortuosity of the centerline are a primary factor in determining the spatial distribution of wall-shear stress, and of the resulting leukocyte adhesion patterns. Although further work is required to overcome the limitations of the present model and ultimately quantify plaque growth in the simulations, these results are encouraging towards establishing a predictive methodology for atherosclerosis progress. Atherosclerosis consists in the progressive build-up of plaque inside an artery. Among other substances, plaque is made of leukocytes, which adhere and transmigrate through the endothelium from the artery lumen, further increasing the plaque burden2. Local hemodynamic conditions, and in particular the endothelial wall shear stress , it has a primary importance through biological signaling which regulates either athero-prone or athero-proctetive gene and protein expression in the endothelial cells4. In particular, the wall shear stress level influences the rate at which leukocytes adhere to the endothelium and the strength of their chemical bond  methods20. This accuracy provides the opportunity to develop a predictive tool for atherosclerosis growth by coupling blood-flow patterns obtained from CFD to the biochemical processes responsible for plaque growth. Recently, we have coupled our in-house CFD code with an agent-based model (ABM) to predict leukocyte adhesion22. In a previous study22, we investigated the effect of the instantaneous and time-averaged flow conditions throughout the cardiac cycle on leukocyte capture. The results emphasized the importance of accounting for the natural unsteadiness of blood flow: evaluating the rate of capture using only time-averaged information leads to inaccurate prediction of the magnitude and location along the artery of leukocyte adhesion. Our previous study22 was conducted on an idealized geometry, consisting of a straight pipe (artery) with a simplified hemispherical obstacle on the wall to represent the stenosis. The present work moves beyond this limitation.Spatio-temporal hemodynamics conditions, and hence wall shear stress, are significantly dependent upon the pulsatile nature of blood flow and the local geometry of the lumenSimulations are performed using realistic stenotic geometries reconstructed from virtual histology intravascular ultrasound (VH-IVUS) frames and bi-plane angiography. Clinical images are available for the baseline geometry and after a 6-months follow-up of the same coronary plaque. Simulations are performed for the baseline geometry, and numerical results are compared with follow-up data. The objective of this work is to present an initial verification of the numerical approach against clinical data.23. Three-dimensional spatio-temporal variations of the blood flow velocity p inside the artery are obtained from the numerical solver.For prediction of atherosclerosis progress we have developed an in-house bio-hemodynamic numerical model. Arterial hemodynamics is calculated using incompressible Navier–Stokes equations as the governing equations. The equations are discretised with a second-order conservative finite-difference approximationl’ of leukocyte is used to track the spatio-temporal leukocyte concentration in the blood 21. In Eqs.  is reduced. This reduction occurs because in the present simulations the supply of leukocytes to the wall at a location The visualizations in Fig. entclass1pt{minima24. The waveform is shown in Fig. D  are reported in the Supplementary material.The solid geometry is modeled using the immersed boundary method28. Patients were randomly selected out of a previously investigated larger cohort at Emory University Hospital11. Briefly, patients were enrolled between December 2007 and January 2009 who presented to the cardiac catherization laboratory with an abnormal non-invasive stress test or stable angina, and a non-obstructive lesion requiring physiological evaluation. Exclusion criteria included myocardial infarction, cardiogenic shock or hemodynamic instability, lesion requiring percutaneous or surgical revascularization, coronary artery bypass surgery, severe valvular heart disease, presence of visual coronary collaterals, inability to provide informed consent, serum creatinine > 1.5 mg/dL, liver disease, or significant hematologic disease. Patients underwent baseline and six-month follow-up biplane angiographic and IVUS imaging . Electrocardiogram-gated IVUS data were continuously acquired . For the adhesion, the mean Simulation results are quantitatively compared with clinical data by correlating the change in lumen area with the computed rate of adhesion. The correlation coefficient is defined as:Simulations were performed for a set of four patient-specific artery geometries. Leukocyte adhesion along the artery wall, fully coupled to the instantaneous three-dimensional hemodynamics, was computed with the numerical methodology described in the previous section. Figure s . The abscissa s is normalized by the mean artery diameter The top two plots (a and b) reports the lumen area retrieved from the VH-IVUS frames as a function of Simulation results in Fig. The correlation coefficient between the rate of adhesion and mentclass2pt{minimmentclass2pt{minimmentclass2pt{minimmentclass2pt{minimIn the following discussion, the results refer to the Patient 1 case Fig. , unless The numerical methodology does not compute plaque growth. However, it is reasonable to assume that artery segments presenting increased plaque burden will also experience significant leukocyte adhesion. The results suggest that, in spite of the modeling limitations, the current numerical framework provides a fairly accurate physiological description of the adhesion progress. The main factor in the present model is the endothelial wall shear stress , is needed for quantitative estimation. Future work will be devoted to improve the modeling assumptions towards the development of a computational model for prediction of atherosclerosis progress.Supplementary material 1 (pdf 198 KB)


The coronavirus disease-2019 (COVID-19) pandemic impacted healthcare services for kidney disease patients. Lockdown and social distancing were mandated worldwide, resulting in closure of medical services. The diagnosis of various kidney diseases may have been delayed during the COVID-19 pandemic because non-urgent tests and visits were postponed due to closure of medical services during the lockdown.We here report the impact of the COVID-19 pandemic on a total number of 209 native kidney diseases requiring renal biopsy for diagnosis in a retrospective observational study from a tertiary hospital in Germany.The lockdown period in March and April 2020 primarily affected patients admitted to the normal medical ward with a compensatory increased rate of renal biopsies in the postlockdown phase. In addition, there was a shift toward more patients admitted with hemoglobinuria during the COVID-19 pandemic. This phenomenon of an increased number of patients with hemoglobinuria during the COVID-19 pandemic was specifically observed in a subgroup with hypertensive nephropathy requiring renal biopsy and associated with increased proteinuria, not attributed to the COVID-19 lockdown period itself.To our knowledge, this is the first report of identifying a subpopulation susceptible to closure of medical services during the COVID-19 pandemic and diagnostic delay of specific kidney diseases. Therefore, the COVID-19 pandemic should be regarded as a risk factor especially in patients with diseases other than COVID-19 primarily admitted to the normal medical ward. The coronavirus disease-2019 (COVID-19) pandemic impacted healthcare services for kidney disease patients. Lockdown and social distancing were mandated worldwide, resulting in closure of medical services. The diagnosis of various kidney diseases may have been delayed during the COVID-19 pandemic because non-urgent tests and visits were postponed due to closure of medical services during the lockdown . Based oA total number of 209 renal biopsies performed on native kidneys of patients hospitalized at the University Medical Center Göttingen in 2019 and 2020 were included. The indication for kidney biopsy included clinical or serologic evidence of a systemic disease, acute nephritic syndrome, unexplained deterioration of kidney function, nephrotic syndrome, non-nephrotic proteinuria of more than 1000 mg per day and glomerular hematuria. In contrast, we do not routinely perform renal biopsies in critically ill patients with ischemic kidney injury. In addition, all patients admitted during the COVID-19 pandemic were tested negative for severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). While no formal approval was required for the use of routine clinical data, a favorable ethical opinion was granted by the local Ethics committee (no. 28/9/17). A detailed Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) flow chart of patient disposition is shown in U-test was used to determine differences in medians. Non-parametric between-group-comparisons were performed with Pearson’s Chi-square test. Data analyses were performed with GraphPad Prism .Variables were tested for normal distribution using Shapiro-Wilk test. Non-normally distributed continuous variables are expressed as median and interquartile range (IQR), categorical variables are presented as frequencies and percentages from the total number with parameters available. Statistical comparisons were not formally powered or prespecified. For group comparisons, the Mann-Whitney A total number of 209 renal biopsies were performed in 2019 and 2020 and included in our study . During We next assessed diagnoses of kidney diseases in renal biopsies during the COVID-19 pandemic in comparison to 2019. There was no significant change in diagnoses of kidney diseases based on renal biopsies , indicatBased on our observation that more patients were admitted with hemoglobinuria and proteinuria as compared to 2019, we next aimed to gain insights into distinct kidney diseases affected by increased hemoglobinuria during the COVID-19 pandemic. Interestingly, we identified that the increase in hemoglobinuria was specifically attributed to patients with hypertensive nephropathy during the COVID-19 pandemic because no such phenomenon was observed in other kidney diseases as compared to 2019 . This isPrompt diagnoses of kidney diseases requiring kidney biopsy and affected by the COVID-19 pandemic are crucial in the disease management. Although definite conclusion on long-term clinical outcomes cannot yet be drawn, our observations indicate that the COVID-19 pandemic resulted only in a minor reduction of renal biopsies in patients admitted to our tertiary center. While the lockdown period with closure of medical services was associated with an overall reduction of renal biopsies mainly attributed to patients admitted to the normal medical ward, there was a compensatory increased number of renal biopsies in this patient population during the postlockdown phase. In contrast, the number of renal biopsies in patients requiring IMC/ICU supportive care remained unaffected during the COVID-19 pandemic. This is in line with our previous report that during the lockdown period in March and April 2020, an incidence-shift with a COVID-19 gap of non-diagnosed AAV based on renal biopsy was followed by a postlockdown phase in subsequent months with a compensatory increased incidence rate . AccordiWhile strict preventive measures are necessary to protect public health, they may also have an important impact on dietary and lifestyle behaviors. Staying and working at home can impact diet and food choice, affecting lifestyle behavior including physical activity . Beside The main limitations of our study are the small patient number, no information about intended or postponed admissions from outside primary and secondary care providers and no data on long-term renal survival (increased incidence of ESRD or death). Nevertheless, this is the first report of identifying a subpopulation susceptible to closure of medical services during the COVID-19 pandemic and diagnostic delay of kidney diseases, including hypertensive nephropathy. Therefore, the COVID-19 pandemic should be regarded as a risk factor especially in patients with diseases other than COVID-19 and presenting with constitutional symptoms, primarily admitted to the normal medical ward.The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Ethics committee of the University Medical Center Göttingen (no. 28/9/17). The patients/participants provided their written informed consent to participate in this study.SH and BT conceived the study, collected and analyzed the data, and co-wrote the first draft. SH evaluated histopathological findings. DT collected and analyzed the data. DT and PK participated in the construction and editing of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This cohort study uses time-series models to estimate preexposure prophylaxis prescription trends during the first 6 months of the COVID-19 pandemic in New York State in 2020. Time-series models were created to estimate the association of the first 6 months of the pandemic with PrEP prescription trends.New York State was disproportionally affected in the early stages of the COVID-19 pandemic, with approximately 462 000 cases and 32 500 deaths by September 30, 2020.STROBE) reporting guideline.Deidentified PrEP prescription fill data in New York State from October 20, 2018, to September 27, 2020, were extracted from the Symphony Health IDV data platform by applying a validated PrEP algorithm  were developed for region (New York City and rest of New York State), sex (men and women), and race and ethnicity . Race and ethnicity data were important to include because of known disparities in PrEP uptake among Hispanic and non-Hispanic Black individuals. The COVID-19 association was calculated by comparing observed with projected total and new prescription fills during the 6-month early pandemic period. Data extraction and statistical analyses were conducted using SAS, version 9.4 (SAS Institute Inc).Interrupted time-series models based on the Box-Jenkins autoregressive integrated moving average (ARIMA) time-series methodThe characteristics of PrEP prescriptions from weeks beginning on October 21, 2018, to September 27, 2020, for observed and forecasted weekly totals are provided in the The 6 The results of this cohort study suggest an association between the COVID-19 pandemic and PrEP prescription trends in New York State.PrEP use in New York State has increased consistently since the US Food and Drug Administration approved it for HIV prevention in 2012. Continued incNew York State eases in PrEP uptake among men who have sex with men are necessary for New York State to end its AIDS epidemic.Assessing the potential association between sharp decreases in PrEP prescription fills with HIV incidence is complicated. This study’s limitations include a lack of data for a longer period during the COVID-19 pandemic and not knowing how HIV-specific risk behaviors changed among individuals who failed to fill or initiate PrEP prescriptions. Ongoing data review is necessary to understand whether PrEP use trends have stabilized to a “new normal” during the COVID-19 era. More research is needed to understand how longer-term decreased PrEP use will inform New York State policy efforts.


The acquired resistance mechanisms in patients with epidermal growth factor receptor (EGFR)-mutant lung cancer, particularly adenocarcinoma (ADC), following treatment with an EGFR tyrosine kinase inhibitor (TKI) have received extensive investigations. The phenotypic transformation to small cell carcinoma (SCCT) has been estimated to occur in approximately 3 to 10% of patients treated with an EGFR-TKI. The prognosis after SCCT is extremely poor.EGFR Del-19-mutant lung ADC treated with combined chemoradiotherapy before the era of insurance coverage for EGFR-TKIs in this country and subsequently gefitinib; deletion at codon 746–750 in exon 19 of the EGFR gene was ascertained in the original formalin-fixed paraffin-embedded lung biopsy tissue. Spinal cord compression at thoracic-12 level from SCCT was successfully relieved with neurosurgical treatment, chemotherapy with etoposide and cisplatin, and radiotherapy, while gefitinib treatment was maintained. Eleven months later, SCCT relapsed in the lung parenchyma, which was resected and was found to be sensitive to second-line weekly topotecan. Prophylactic cranial irradiation was subsequently administered. SCCT was confirmed by MALDI-TOF MS analysis of formalin-fixed paraffin-embedded tissues demonstrating the same exon 19 deletion. At the 12th-year follow-up, the patient remains relapse free with very good performance status. The novelty of this case is the successful interdisciplinary team effort to correct the spinal cord compression by maintaining the patient in an ambulatory state, non-stop use of gefitinib justified by the presence of activating EGFR mutation in SCCT tumor cells, and aggressive dose-intensive chemotherapy and radiotherapy for the SCCT that leads to an unprecedented prolonged remission and survival. This case also supports the observation that SCCT is chemotherapy sensitive, and thus, re-biopsy or complete tumor excision is recommended to understand the mutation profiles of the current tumor. Aggressive prudent administration of systemic chemotherapy obtaining optimal dose intensity leads to the successful management of the patient.We report about SCCT that occurred 45 months after the initial diagnosis of ADC in an East Asian never-smoker woman with advanced-stage  According to the literature, small cell carcinoma transformation (SCCT) from epidermal growth factor receptor (EGFR)-mutant adenocarcinoma (ADC) of the lung occurs at a rate of 3–10%  of bilateral femurs and tibias, which were compatible with bone metastases. Chest computed tomography (CT) revealed a huge lobulated lung mass measuring up to 6.87 cm in the right lower lobe, with a daughter nodule in the same lung lobe  for SCLC identified the same deletion in exon 19  and amplification of the MET proto-oncogene or the ERBB2 proto-oncogene /PD-ligand 1 (PD-L1) pathway with efficacy as demonstrated in two large prospective randomized controlled trials –15. It cIn this case report, MALDI-TOF MS was performed to diagnose the EGFR mutation in SCCT. The turn-around time for MALDI-TOF MS to deliver a quick diagnosis of EGFR mutation is as short as 4 days, and when compared with Sanger sequencing, both analytical sensitivity and specificity are 100% .EGFR Del-19 mutation in lung cancer is approximately 23.1% (In East Asia such as Taiwan, the prevalence of ly 23.1% . Patiently 23.1% . TherefoRB before transformation (RB1 implies that this may be a necessary event for SCCT to emerge (RB loss can be detected by liquid biopsy during patient treatment monitoring, we may be able to start appropriate treatment early after transformation.The molecular mechanisms underlying SCCT are beginning to be understood. A study by researchers of the Massachusetts General Hospital identified that every case of SCCT has loss of the tumor suppressor ormation . The nato emerge , 20. HenImplementation of precision oncology improves patients’ prospects after being diagnosed with EGFR-mutant ADC and even in the stage of the rare occurrence of histologic SCCT. Appropriate dose intensity salvage chemotherapy, prophylactic cranial irradiation, plus good dose radiotherapy should always be the number one option for fit patients. This case report lends strong support to the practice that should always consider rendering the most durable remission as the treatment goal while treating a patient with EGFR-mutant lung cancer having SCCT as the acquired resistance from EGFR-TKI therapy.‘It was disastrous to know that I got lung cancer in so late the stage. I trust my doctor and whose medical team from the very beginning, and I receive all the treatment plans they devise for me. I don’t think I can understand why I needed to receive those complex treatments, although the doctor has told me from time to time. I never thought that I could accompany and watch my grandson grow up for the past twelve years. I feel very happy to be able to take care of him personally. Now I can walk and prepare food for the family and perform other necessary activities of daily living. The remaining tingling sensation in my fingers and toes is negligible most of the time.’The patient provided written informed consent for academic publishing of her case.All the authors took part in taking care of the patient. VCK and CKL drafted the manuscript. VCK prepared The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Intravenous immunoglobulins (IVIGs) administration could represent a suitable immunomodulator with this respect. Here we focus on mechanisms of action of immunoglobulins in muscular dystrophies and inflammatory myopathies highlighting results of IVIGs from pre-clinical and case reports evidences.Muscular dystrophies and inflammatory myopathies are heterogeneous muscular disorders characterized by progressive muscle weakness and mass loss. Despite the high variability of etiology, inflammation and involvement of both innate and adaptive immune response are shared features. The best understood immune mechanisms involved in these pathologies include complement cascade activation, auto-antibodies directed against muscular proteins or  Growing evidences support the role of the immune system in different pathological conditions of the skeletal muscle. Immune cell infiltrate following muscle injury contributes to the pathology of various muscular dystrophies (MDs), whereas autoimmune responses specific for defined or yet undefined muscle antigens are suggested as the cause of some idiopathic inflammatory myopathies (IIMs). The initial immune response to muscle damage consists of innate immunity in which phagocytic, cytolytic, and secretory inflammatory cells (mainly macrophages and neutrophils) are rapidly mobilized and activated to identify, kill, and remove invading infectious organisms during infectious events or remove muscle fiber debris and promote muscle repair following disruption of muscle homeostasis. Innate immunity predates the adaptive immune system through the activation of professional antigen-presenting cells (APCs) that process and present muscle antigens to T-effector cells (mainly T-CD4+ and T-CD8+ cells) toward major histocompatibility complex (MHC) leading to intensive secretion of pro-inflammatory cytokines and muscle fiber necrosis . The MDsThe majority of IIMs are firstly treated with immunotherapy but often secondary and also tertiary-line agents  are necessary to allow an amelioration of the pathological muscle signs , 9. CortOur goal in this review is to present current knowledge on the immunomodulatory function of immunoglobulins in genetic (MDs) and immune-mediated (IIMs) muscle diseases, exploring similarities and differences of the mechanisms by which immunoglobulins can modulate innate and adaptive immunity and to highlight areas in which further research is needed. There are already a number of excellent reviews on IVIG and neuromuscular diseases, but they are mostly focused on immune mediated muscle diseases and in particular Myasthenia gravis. The primary literature for review on each subject was derived from searching the National Center for Biotechnology Information PubMed database using the key words IGIV, IVIG, intravenous immunoglobulin, intravenous immune globulin, subcutaneous immunoglobulin, and subcutaneous immune globulin, along with key words specific for MD and IIM disease-related topic.Immunoglobulins (Ig) are heterotetrameric glycoproteins formed by two identical heavy chains (HC) and two identical light chains (LC), linked together through di-sulfide bridge. Each Ig comprises a receptor-binding fragment  that determines the isotype and two antigen-binding fragments (Fabs). Fc components of antibody are responsible for mediating the innate and immune response. FcγRs include activating receptors and one inhibitory receptor that are widely expressed in innate immune cells as neutrophils, natural killers, monocytes and macrophages while the component of adaptive immune system, as DCs and B cells, only express selected isoforms. The FcγRI isoform possesses high affinity for the IgG .Five Ig isotypes exist: IgM, IgG, IgA, IgD and IgE that present different roles. The variability of antigen-binding sites is acquired thanks to genic rearrangement of HC gene segments: variable (V), diversity (D), and joining (J), while the V and J segments are recombined for LC chain. Variable domains are further variably joined thus this process produces a large antibody repertoire , 17. AftThe immunoglobulin found in highest concentration is IgG that is furtherly divided in four subclasses , according to the length and number of di-sulfide bridges  and to different amino acid in Fc region (varying the affinity for Fc receptors). A core glycan is present in Fc structure allowing its modification by specific saccharide . Interesvia IgG may have clinical implications to avoid immunogenicity and induce immune tolerance in MDs and IIMs.Fab- and Fc-dependent mechanisms are presumed to be involved in the immunomodulatory effects of IgG  . The FabThe therapeutic employ of immunoglobulin started over a century ago. Firstly, it was used to treat infections, later it became critical as a replacement therapy in immunoglobulin deficiency to provide passive immunity against pathogens . Nowadays IVIG gained critical importance as immunosuppressive strategies in autoimmune diseases, allowing administration of high doses (2 g/kg/month) in an easy way also in non-autonomous patients or in those with severe limitations in manual dexterity , 33. To The term myopathy can be applied to any muscle disease while the term dystrophy was classically applied by pathologists to the subset of genetic-dependent inherited myopathies in which muscle tissue destruction is the major feature. Despite etiological differences, genetic muscular dystrophy and immune-mediated inflammatory myopathy share many pathological features, namely, muscle degeneration, weakness, and different players in chronic inflammation as complement and other immune cells.The study of innate and adaptive immune response involvement in MDs has been attracting the interest of many researchers though the results are so far barely exhaustive and sometimes contradictory. The temporal regulation of these processes is necessary to determine the differentiation of muscle progenitors into mature muscle fibers. In case of muscle injury and inflammatory events, DAMPs are released from necrotic muscle fibers and activate macrophages and DCs from the innate immune system, that in turn, elicits pro-inflammatory cytokines secretion and oxidative stress. These phenomena render the cells as APCs so that they can activate the CD4+/CD8+ T-lymphocytes from adaptive immune system, reinforcing the inflammatory cycle. Not surprisingly, all these cells from immune systems were isolated from the muscles of MD and IIM patients  levels, proximal weakness and muscle necrosis with perivascular infiltrates in muscle biopsy could be difficult to differentiate anti-SRP or anti-HMGCR myopathies from LGMD. Thus, fundamental insights for patients are provided by their clinical presentation together with the presence/absence of auto-antibodies and precise evaluation of the characteristics of muscle biopsies. Moreover, systemic disease manifestations , temporal pattern and steroid responsiveness are the parameters that are analyzed together to determine the diagnosis of patients thought to have a muscular dystrophy without family history or genetically confirmed diagnosis.In MDs, the severity of muscle injury and inflammation dictates the impairment of muscle regeneration and successive replacement of myofibers with connective and adipose tissue  Table . Interesα: this condition allows membranes’ ruptures and, more importantly, fosters the recruitment of pro-inflammatory macrophages. The event that triggers the development of inflammatory phenotype is the invasion of M1 and M2 macrophages: the pro-inflammatory M1 secrete iNOS and cause the lysis of muscles while the pro-regenerative M2 regulate the activity of satellite cells. M1/M2 proliferation is regulated by the synergistic activity of cytokines  and interleukins . Since in mdx mice the intracellular signaling pathways are chronically activated, the amount of pro-inflammatory M1 macrophages is dramatically deregulated with recruitment of adaptive effectors CD4/CD8+ T-cells  is a well-known and timely process that starts early in the first two weeks of life, has a peak at 6–8 weeks and decreases at 12 weeks. Neutrophils are the first cells to be identified in dystrophic muscles where they regulate the production of superoxide and myeloperoxidase and secrete TNF T-cells , 44. End T-cells . In due  T-cells  and furt T-cells . In addi T-cells . These s T-cells –50 suggeSimilar study by Schmidt et al.  reportedNunes et al. also reported the effect of IVIG treatment in ameliorating the muscle performance and survival rate of severely affected double knockout mdx/utr− dystrophic mouse whose myopathy is more similar to DMD patients . InteresIn sum, animal models and mechanistic studies have shown promises in modifying the disease course of DMD: however, preclinical data are lacking in humans and informative trials with large patients’ cohorts are necessary to maximize beneficial outcomes.2+ handling proteins for excitation-contraction (EC) coupling at the transverse-tubules (T-tubules) in skeletal muscle  that are responsible for recessively inherited dysferlinopathy which is most pronounced in the pelvic and shoulder girdle muscles , or distal myopathy with onset in gastrocnemius and soleus muscles in cases of Miyoshi myopathy (MM or MMD1), or distal myopathy with onset in the tibialis anterior (DMAT)  , 56. Dysl muscle , 59. Thul muscle ,Confalonl muscle –64.A clinical trial was launched to try to inhibit accumulation of complement deposit by the administration of IVIG in LGMD2R patients; however, the initially encouraging results were not pursued . LerarioDMPK gene and type 2 (DM2) in the CNBP gene—and to phenotypic subtypes ; polymyositis (PM); immune-mediated necrotizing myopathy (IMNM); inclusion body myositis (IBM) . SeronegThe clinicopathological classification for dermatomyositis was introduced by Dalakas in 1991. The recent discovery of prominent type 1 interferon (IFN1) signature and the recognition of dermatomyositis-specific antibodies (DMSA) determined current understanding of dermatomyositis classification . The cliPolymyositis (PM) is an uncommon inflammatory muscle disease that preferentially affects women between 30 and 50 years old. The most common signs of PM are weakness of forearms, shoulders and back muscles with consequent difficulties in getting up, dry cough and dysphagia. Proximal muscles are invaded by CD28+ and CD8+-inflammatory T-cells that recognize muscular fibers expressing aberrantly the MHC-I . CD8+ T-The first classification of Inclusion body myositis (IBM) was done by Griggs et al. in 1995. The clinicopathological classification of IBM was later formulated into three categories: clinicopathologically defined IBM (CPD-IBM), clinically defined IBM, and probable IBM . The IBMIMNM is categorized into three subgroups according to positive antibodies: antisignal recognition particle (SRP) IMNM, anti3-hydroxy-3-methylgluaryl-coenzyme A reductase (HMGCR), and seronegative IMNM . IMNM caThe first line of treatment for IIMs is the immunotherapy performed with prednisone and/or intravenous injection of glucocorticoids, and it is often associated to physiotherapy to recover muscle force. The IVIG is used as second line of treatment in steroids refractory dermatomyositis , 97, polThe identification of dystrophin autoantibodies in DMD animal models , 107 andMost MDs and IIMs patients have a long disease duration with risk of relapse when corticosteroids or immunosuppressants are tapered or withdrawn. Taking into account the inhibitory and immune-modulatory features of IVIG, their involvement in mediating of different aspects of muscle pathologies gained importance in a clinical point-of-view. Immunoglobulins interact with complement cascade and ICAM-1 in muscle capillaries inhibiting the functions of macrophages and lymphocytes , 32, 79 Cost effectiveness of IVIGs in neuromuscular disease has also to be carefully evaluated. In fact, the cost of IVIGs is undeniably higher than corticosteroids and protocol for repeated assessments need to be developed to determine whether benefit is continued. Moreover, MD patients do not fit within strict homogeneous categories and individual responses can vary. In this sense, SCIG could represent an alternative administration of immunoglobulins allowing no need of costs of healthcare providers and hospitalization and improving patients’ quality of life. The future of IVIG therapy relies on better understanding of the mechanism of IVIGs treatment and on the possibility to engineer IVIGs preparation to allow targeted immunotherapeutic interventions in combination with mutation-based therapeutic efforts and to enhance their regulatory properties.AF wrote the paper, prepared the original draft of the manuscript that was revised by YT. All the authors stated were involved in the critical revision of the manuscript and approved the final version of the article, including the authorship list. The corresponding authors had final responsibility for the decision to submit for publication. All authors contributed to the article and approved the submitted version.This study was supported by the Associazione Centro Dino Ferrari, a French Telethon AFM grant (No. 21104), by the Italian Ministry of Health . This paper presents independent research funded by Ricerca Finalizzata 2016 (Linea di ricerca: “Theory-enhancing”).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Falls are the second most frequent cause of injury in the elderly. Physiological processes associated with aging affect the elderly’s ability to respond to unexpected balance perturbations, leading to increased fall risk. Every year, approximately 30% of adults, 65 years and older, experiences at least one fall. Investigating the neurophysiological mechanisms underlying the control of static and dynamic balance in the elderly is an emerging research area. The study aimed to identify cortical and muscular correlates during static and dynamic balance tests in a cohort of young and old healthy adults. We recorded cortical and muscular activity in nine elderly and eight younger healthy participants during an upright stance task in static and dynamic (core board) conditions. To simulate real-life dual-task postural control conditions, the second set of experiments incorporated an oddball visual task. We observed higher electroencephalographic (EEG) delta rhythm over the anterior cortex in the elderly and more diffused fast rhythms  in younger participants during the static balance tests. When adding a visual oddball, the elderly displayed an increase in theta activation over the sensorimotor and occipital cortices. During the dynamic balance tests, the elderly showed the recruitment of sensorimotor areas and increased muscle activity level, suggesting a preferential motor strategy for postural control. This strategy was even more prominent during the oddball task. Younger participants showed reduced cortical and muscular activity compared to the elderly, with the noteworthy difference of a preferential activation of occipital areas that increased during the oddball task. These results support the hypothesis that different strategies are used by the elderly compared to younger adults during postural tasks, particularly when postural and cognitive tasks are combined. The knowledge gained in this study could inform the development of age-specific rehabilitative and assistive interventions. The most significant number of fatal falls each year are reported in old adults2. Postural responses are likely influenced by the cerebral cortex directly via corticospinal loops and indirectly via communication with the brainstem centers harboring the interactions for postural responses, providing both speed and flexibility for a suitable reaction to a loss of balance3.In a normally functioning nervous system, maintaining an upright stance is predominantly controlled by cortical and spinal networks. Cortical control is challenged by cognitive loads in different ways, depending on the postural task, the individuals’ age, and their balance abilities4. CDP provided evidence that the elderly prioritize balance recovery when performing a cognitive task while responding to a balance perturbation. CDP was also performed in virtual-reality environments, where visual interference led to an increase in postural sway5. Other studies exploring the relationship among attention, posture, and gait in younger and older adults proved that postural control is more cognitively demanding in the elderly than in younger adults. Their balance is more severely affected while double tasking13. It is unclear, however, if these differences are due to split attention between the two tasks, reduced attention ability, or increasing demand due to impairments affecting the postural control system in the aging nervous system14.Complex balance tasks using different kinds of perturbations  have been assessed via computerized dynamic posturography (CDP), which consists of measuring and analyzing the center of pressure time series during various balancing tasks 15, often designed to simulate real-life situations of instability. In young, healthy volunteers walking while conversing or texting, electroencephalographic (EEG) data showed an activation of the posterior parietal cortex (PPC)15 which seems to integrate afferent inputs to facilitate changes in motor strategy during walking17. When younger adults walked without engaging in any additional tasks, the PPC displayed activity in the theta range (4–7 Hz). When a dual-task was added, a relationship could be identified between PPC activity in the alpha frequency band and walking pace: region- and frequency- specific brain activation patterns could predict gait stability during dual-task walking through a multiple regression model15. Vertical trunk acceleration was predicted by gait velocity and left PPC theta (4–7 Hz) frequency band power in single-task walking and by gait velocity and left PPC alpha (8–12 Hz) frequency band power in walking while conversing. Medio-lateral trunk acceleration was predicted by left PPC beta (15–25 Hz) frequency band power when walking and texting. Region- and frequency- specific brain activation patterns were also found to coordinate navigation performance19. The existence of neural markers of postural instability that may trigger balance compensatory adjustments was also supported by the observation that a burst of gamma activity preceded the initiation of compensatory backward posture adjustments20.Assessing the neurophysiological characteristics of the control of balance in increasingly challenging tasks  may help to clarify age-related differences. Our focus was on dual-task experiments6. The few studies addressing this issue22 showed that scalp EEG power distribution increases in elderly and young adults in the delta frequency band during challenging postural conditions. Theta rhythms were more responsive to cognitive tasks particularly in younger subjects. Gamma oscillations increased in the elderly primarily over the central and centro-parietal cortices during challenging postural tasks.Despite their potential relevance in the above-described context, there is a lack of studies investigating the EEG and electromyographic (EMG) data in the elderly during a balance task14. Beta cortico-muscular coherence in young adults was responsive to mechanical changes, but not to visual or surface changes, during standing balance tests23. An increase in frontal and parietal midline theta power was observed in association with demanding balance control tasks24. Changes in fronto–central and centro–parietal theta power were associated with balance performance24. Clusters of EEG sources obtained with Independent Component Analysis (ICA) in healthy young subjects walking heel-to-toe on a treadmill-mounted balance beam and walking on the treadmill belt at the same speed identified activations in or near the anterior cingulate, anterior parietal, superior dorsolateral-prefrontal, and medial sensorimotor cortex. These areas exhibited significantly larger mean spectral power in the theta band (4–7 Hz) during walking on the balance beam compared with treadmill walking. Spectrogram-based analyses demonstrated that the first electrocortical indication of impending loss of balance occurred in the left sensorimotor cortex at the transition from single support to double support before stepping off the beam17. In people who trained extensively to improve their balance control, such as athletes, the greater the alpha event-related desynchronization was, the more significant the reduction in sway area was25.Most studies analyzing cortical oscillations power spectrum density changes associated with postural control are based on younger adults6, findings based on EMG data are more consistent, though their interpretation is still controversial. EMG latency, amplitude and muscular co-contractions are usually more prominent in the elderly during walking and upright stance tasks31. Increased activation of antagonistic muscles appears to be associated with healthy aging in walking tasks32, and with balance impairments in balance recovery33.Despite large heterogeneity in experiments, protocols, and data analyses5.Our study aimed to investigate cortical and muscular activation patterns underlying age-related differences in postural control. Co-recording EEG and EMG data in the dual-task condition in healthy subjects mimics a real-life situation of distraction that might lead to balance instability in the elderly. We chose a visual task because visual and proprioceptive inputs appeared to play dominant roles in assuring postural stabilityConsidering the reported heterogeneous findings, our research questions are: (Q1) Which are the age-related differences in cortical and muscular activations during static and dynamic balance? (Q2) How do these differences change during dual postural-visual tasks?34 were excluded. The raw time in s for TMT-A (assessing cognitive processing speed) and TMT-B (assessing executive functioning) is reported in Fig.  35, we computed the cut-off for each participant considering these two covariates36  and in both tests in the elderly compared to the younger adults . TMT performance declines, on average, during healthy aging34, with performance on TMT-B declining significantly more than TMT-A in older adults37.We administered the Trail Making Test (TMT) A and B as a screening tool to detect visual-attention and movement speed impairments, common with aging. These deficits were considered as exclusion criteria , i.e., participants performing under the threshold of normality, based on normative values computed by taking into account the subject’s age and education levelP < 0.05) and the Confidence Ellipse Area (CEA) during the dynamic postural task with dual-tasking (P < 0.01).Visual inspection of the results in Fig. P < 0.05)  and + 22% , and in the SP in the 0% condition (P < 0.05). Significant differences for between-groups comparison  were only obtained in the core board condition and in dual-task  showed a significant difference for the Range of Motion in the antero-posterior and medio-lateral directions (ROM-AP and ROM-ML), for the Sway Path length (SP) and Confidence Ellipse Area (CEA) in the 0% condition, and for the SP in the + 22% condition in the elderly . The power spectral density in each region of interest followed the 1/f power law pattern. All comparisons of the power spectra in the same ROI among the estimated frequency bands resulted statistically significant. Results during single-task (Q1) and dual-task (Q2) conditions are reported in the following paragraphs.Figure P < 0.05) between the two groups were identified over frontal regions in the delta band  and over frontal and sensorimotor areas in the beta and gamma bands .During static balance , the elderly Figure a exhibitDuring dynamic balance tasks , the theta rhythm over the motor/occipital area displayed an increase in the elderly Fig. b,d Wilcf.Figure Figures Figures During static balance dual-tasking, the elderly showed Fig. a increasThe delta power over the centro-parietal areas significantly increased during the dynamic balance test (compared to the static one) in the younger adults. When dual-tasking, the elderly showed a decrease in the delta rhythm in the centro-parietal area parallel to an increase in the theta rhythm in the occipital area during dynamic balance compared to static balance.Comparing the performance Fig. a,b in duDuring dual-task static balance on the downward inclined plane (− 22%), the elderly showed higher cortical activations in the alpha and gamma bands over the occipital and frontal regions (compared to the younger adults). This pattern is similar to the one observed in the younger adults during the dynamic balance tests, suggesting a similar cognitive load in the elderly and the younger adults in these two different testing conditions. Higher cortical activation is observed in younger adults, during dual-task static balance on the inclined plane (+ 22%) over the frontal regions in the theta and low gamma bands, similar to the pattern observed during the static balance tests. During the same task, the elderly show higher activation on the frontal and central regions in the beta band.Results of all the statistical tests for the EEG power spectra are provided in In Fig. P < 0.01). The RMS of the GL was significantly higher in the elderly on the 0% plane and on the core-board (P < 0.01).RMS values for the ES significantly differ in all the postural conditions between the two groups: the elderly showed higher values and an increasing trend from 0% inclined surface to the core board  than in the younger adults, who appeared to prioritize the recruitment of the PL for fine movement control in the medial-lateral planeNo significant differences were found comparing EMG data during the postural task with and without the visual oddball. We obtained the same RMS trend reported in Fig. Our findings provide evidence of different motor control strategies to maintain static and dynamic balance in the elderly compared to younger healthy adults.39, e.g., the attention-based process of maintaining posture. During the dynamic balance test, the theta rhythms over the sensorimotor and occipital areas of the elderly increased. When adding a visual oddball task, the elderly showed a more robust activation over the sensorimotor areas during the static balance test compared to the single-task condition. Dynamic balance tests showed similar findings, but magnified. In the younger group, the static balance tests during dual-tasking were associated with occipital theta and gamma rhythm, and increased frontal alpha, which again are even more prominent during the dynamic balance tests.During the static balance test, the power in the delta band over the frontal regions was higher in the elderly. Younger participants had higher activation on frontal and occipital areas of the fast frequencies . Both groups showed mid-line fronto-central theta rhythms, a neural signature of focused attention22. The younger adults showed prevalent fast frequencies  over the frontal and occipital areas. This activation over the occipital lobes may suggest an ongoing attention-based task to maintain upright posture supported by a visual fixation strategy in this group.The different cortical activation strategies are more evident during the dual-tasks, but they already emerged during the quiet static balance tests. The elderly showed high delta activation over the frontal areas, a process hypothesized to contribute to maintaining postural control40, which is further reduced when adding a cognitive task, such as counting the odd stimuli 41. The same result was obtained for the elderly in all postural tasks, except for the core board. With the postural task becoming more demanding , the balance performance deteriorates in the elderly from the single- to the dual-task, showing similar median values for all variables with more significant variability.As represented in Fig. 23, we expected an increase of cortical recruitment to keep up performance in the elderly. The observed activation of the posterior areas in the theta band supports this hypothesis: it is coupled with a recruitment of sensorimotor cortices, confirmed by the muscle activation patterns hinting to an increased muscle activity to maintain posture.Dual-task is a well-known strategy to increase the cognitive load in experimental conditions and mimics real-life settings in which balance control is coupled with tasks such as speaking or texting. A standard paradigm used to simulate real-life is the visual oddball, during which the participant is asked to count the stimuli presented on a screen that differ from the more common one. In our experiment, the dual-task addressed the issue of the competing cognitive resources the elderly deploy during the stance test. In line with previous research45. This difference may also be reported to the greater difficulty the elderly display in adapting to rapidly changing environmental conditions38.In younger adults, we observed that during dual-task dynamic postural tests, the strong activation of the occipital and frontal areas (in beta and gamma bands) was consistent with the oddball task. A limitation of the study is the lack of a co-registered electrooculogram and eye tracking to ensure gaze fixation. While this may not have been an issue during the cognitive task, when target fixation is required to ensure correct execution, a wandering gaze may arise when idling. We obtained similar activation over the occipital lobes (in beta and gamma bands) during quiet standing in the younger adults. These findings suggest that the underlying neural circuits may be similar, but likely they are used with a different level of efficiency. The higher power in beta and gamma bands over the occipital cortex in the younger adults may point to a different processing strength between the two populations, with younger participants processing visual cues more efficiently, as already reported for orientation discrimination or contrast detection46 which described increased cortical activation during dual-tasking indicating a defective compensatory mechanism in the elderly. It is also possible that the different nature of the task – static vs. dynamic – may make these findings not directly comparable.The main differences in cortical activations between the two groups appear during dual-tasking and suggest that the elderly needed to recruit adaptive reserves. This finding contrasts with previous work47. This has been referred to as compensatory cognitive scaffolding, whose efficacy largely depends on each individual’s cognitive reserve48. This model explains older individual’s level of cognitive functioning: this relies on compensatory mechanisms by which supplementary higher level cortical networks are recruited to accomplish tasks supposedly be highly automatized, as the control of upright stance49. The increased allocation of attention resources to handle a challenging postural task, together with a cognitive oddball paradigm, led the older group to an increased recruitment of cortical areas. This is not needed in younger adults who rely on more automatized and less central processes, having more cognitive resources available to accomplish the task51.In normal aging, structural and functional physiological brain changes occur. Structural changes  occur together with functional maladaptive brain activity, namely decreased functional specificity and loss of functional lateralization. These phenomena trigger the activation of compensatory mechanisms52) was associated with increased postural sway and risk of falls in the elderly55. Joint stiffening is a maladaptive strategy to reduce postural sway, as a reduction in flexibility hampers an adaptive response to perturbation57. In both groups, PL shows a strong activation during the static balance test due to greater stabilization needs in the medio-lateral direction58 which shows larger range of motion of body sway than in the antero-posterior direction, especially in dynamic conditions. The elderly augment the PL activity concurrently activating TA and GL. Younger adults preferentially control the ankle joint relying on the PL activity for fine movement control in the medio-lateral plane38.The elderly showed higher muscular activity. The higher activation of the ankle plantarflexor and dorsiflexor muscles in the elderly suggests a different neuromuscular strategy. A possible stiffening action at the ankle joint in static balance becomes more evident during the dynamic tasks. The larger body sway observed in the elderly during the static and dynamic tests supports such hypothesis. Simultaneous contraction of both agonist and antagonistic muscles  and flexors (BF) during the dynamic balance in the elderly, most likely to add stiffness to the knee. The younger adults prefer lowering their center of mass to increase their stability. Besides not being significantly different, the Center of Mass (CoM) range of motion in the vertical direction was lower and less variable during the dynamic task in the younger adults than in the elderly. This supports the conclusion that the elderly implement a less efficient balance control strategy.RMS values for the ES significantly differ in all the postural conditions between the two groups: the elderly show higher values and an increasing trend from 0% inclined surface to the core board  EEG system. EMG data were simultaneously recorded using 8 wireless probes that we positioned on relevant muscles of the leg and the trunk. Subsequently, we computed: 1) for each region of interest in the brain cortex, the EEG source waveform in the time domain and its spectral power distribution in the canonical EEG frequency bands and 2) the root mean square values of the EMG data collected during various postural tasks  and dual postural-visual tasks  in nine healthy elderly  and eight healthy right-handed younger ([24-34] y) adults were recruited. Elderly were referred to as individuals 65+ years of age, according to the United Nations definition. The age range for selecting the younger adults was chosen by considering that strength and physical performance reach their peak between 18 and 39 years of ageExclusion Criteria were: (1) Diagnosis of neuropathy or sensation of tingling in the legs or feeling of having reduced sensitivity to feet or legs; (2) Diabetes mellitus; (3) Any rheumatological pathology; (4) Orthopedic problems ; (5) History of fractures of the lower limbs or feet; (6) History of spinal surgery; (7) History of hip and/or knee replacement surgery; (8) History of stroke even if completely recovered. Hypertension was not considered as a contraindication if under drug treatment and with good control.This study was carried out in accordance with the recommendations of the Ethics Committee of the Teaching Hospital of Padua (n.AOP2025). All subjects signed written informed consent.61. The TMT-B evaluates visual attention, movement speed, and cognitive impairments.Participants were evaluated using the Trail Making Test, i.e., a neuropsychological test in two parts, to ensure that elderly participants did not have any movement speed and visual attention deficits. In part A, i.e., TMT-A, the subject’s task is to quickly draw lines on a page connecting 25 consecutive numbers without lifting the pen from the paper. In case errors occur, participants are alerted, and correction is allowed. The performance is assessed using the time needed to complete the trial correctly. Part B  tests how fast the participant can connect numbers and letters in alternating increasing sequence . Part B is more challenging than Part A because it is a more difficult cognitive task requiring movement speed and visual search34, education was assessed as asking the number of years in school.To exclude participants performing under the threshold of normality based on normative values computed by taking into account each subject’s education level and age34.None of the 9 elderly participants in the study had deficits in movement speed or visual attention, as verified by TMT scores within a normative threshold considering age and education62 was used to test the null hypothesis that data come from a normally distributed population.Lilliefors test63 as suggested by65. Skin preparation was performed by removing dead skin cells  and moisturizing the skin if participants had a dry skin, or rubbing it with alcoholic wipes when they had an oily skin65. The electrode-skin impedance was maintained High-density EEG recordings were acquired at Padova Neuroscience Center inside a dimly lit sound-attenuated and electrically shielded room using the Geodesic Sensor Net system with 256 electrodes . Electrode-skin impedances were maintained leg Fig. a: Rectus69. The IMU consisted of a 3D accelerometer, a 3D gyroscope and a 3D magnetometer. For the purpose of this study, only the 3D accelerometer data were collected and sampled at 142.85 Hz.One IMU was secured on the fifth lumbar vertebra (L5) with a double-sided adhesive tape to measure the the pelvis’s movements and thus to estimate the kinematics of the body CoMSynchronization of EEG and EMG-IMU recordings was assured by physically connecting the TTL trigger out channel of the EMG-IMU receiver to the “synch-box” of the EEG system. A TTL is an electrical signal based on the transistor-transistor logic, which produces a two-level signal: a low voltage level (from 0 to 0.8 V) and a high voltage level . At the stop recording command on the EMG-IMU system, the receiver switches the TTL sent to the to the EEG synch-box from the low to the high level, and a time stamp was saved.Baseline. Three minutes of quiet upright standing (without shoes) were collected at the beginning and at the end of the experiment. Participants were instructed to keep their feet parallel and at shoulder width, while staring at a black screen.Static postural tests. Participants were standing upright without shoes on level ground or on an inclined surface at different angles. The feet position was kept consistent across trials. For the + 22% condition, the subject was instructed to stand on an inclined surface at + 12°. Thus, the participants had dorsiflexed ankle and toes pointing upward. For the − 22% condition, the fixed plane was inclined at − 12° and the participants had ankles in plantarflexion and the toes pointing downward. For the 0% condition, participants were standing upright on the floor. In this experimental condition, we used a “rocker” balance board with a flat and rectangular shape and a semi-cylindrical base. Due to its semi-cylindrical stationary base, it has a range of motion limited to one axis . Participants placed their feet parallel and at shoulder width and the core board could only rock in the antero-posterior direction. Specifically, participants were asked to keep balance on the 50 cm x 50 cm 1 degree-of-freedom wooden core board with a total of Both the static and dynamic postural tasks were tested with and without the cognitive perturbation, i.e., a visual oddball, as a dual task.Visual oddball. Participants were standing in front of a black screen and instructed to stare at it. The distance between the participant and the screen was kept constant across trials and participants . After 30 s, the presentation (block) of a sequence of repetitive stimuli  was infrequently interrupted by a deviant stimulus . Each stimulus lasted 500 ms. The inter-stimulus interval  was randomly set from 500 ms to 1 s.Each block contained circa 80 repetitive stimuli, and circa 20 deviant stimuli, for a total of 100 stimuli per block. The whole experiment included four blocks  performed in a pseudo-randomized order. Each block lasted about 3 minutes. Participants were asked to count the deviant stimuli, i.e., the yellow squares, randomly shown on the screen. Short breaks were taken when needed.EEG processing. EEG data were zero-phase-high-pass-filtered above 1 Hz through a 4th order Butterworth filter and then zero-phase-low-pass-filtered below 70 Hz through a 4th order Butterworth filter avoiding phase distortion. Channels in the cheeks and in the neck were discarded (204 channels left). EEG epochs were then extracted from the continuous dataset and time-locked from -500 to 500 ms relative to the onset of each image during the visual oddball. The 30-s recorded before and after the dual-task were divided into non-overlapping epochs of 1 s. Noisy channels were identified by visual inspection and interpolated using the nearest-neighbor spline method (average percentage of channels interpolated: 1.5%). Individual epochs containing non-stereotyped artifacts, peri-stimulus eye blinks and eye movements 76: (Right and Left:) Precentral gyrus; Superior frontal gyrus, dorsolateral; Superior frontal gyrus, orbital part; Inferior frontal gyrus, opercular part; Inferior frontal gyrus, triangular part; Inferior frontal gyrus, orbital part; Rolandic operculum; Superior frontal gyrus, medial; Superior frontal gyrus, medial orbital; Superior occipital gyrus; Middle occipital gyrus; Inferior occipital gyrus; Postcentral gyrus; Superior parietal gyrus; Inferior parietal, but supramarginal and angular gyri; Supramarginal gyrus; Angular gyrus; Paracentral lobule and Occipito-temporal gyrus; Supplementary motor area; Median cingulate and paracingulate gyri; Posterior cingulate gyrus; Calcarine fissure and surrounding cortex; Anterior cingulate and paracingulate gyri; Cuneus; Lingual gyrus; Precuneus.We applied the LAURA algorithm implemented in Cartool77.The dipoles in each ROI were represented with one unique time-series by applying a singular-value decompositionFor each time-series that represented the activity in each ROI, we computed the absolute and the relative power spectra in the canonical EEG frequency bands, i.e., delta [1-4] Hz, theta ]4-8] Hz, alpha ]8-12] Hz, beta [14-24] Hz, low gamma [30-50] Hz, high gamma ]50-70] Hz. Each 1-s EEG source-waveform epoch was multiplied by a window function, i.e., Hamming window. The periodograms of all modified epochs referred to the same experimental condition were then averaged to estimate the power spectral density of each condition. The area under the curve  was estimated for each EEG frequency band. The power of each EEG frequency band was divided by the power in the interval [1-70] Hz to compute the relative power.78.Lastly, we performed two-sided Wilcoxon rank sum tests on the relative power spectra values for each ROI under the same postural/dual-task condition between the two groups to test for significantly different activations between the elderly and the younger adults. We performed two-sided paired samples Wilcoxon signed-rank tests inside the same group to compare relative power spectra values for each ROI between the different postural/dual-task conditions. As input of the statistical tests, we used either the relative power spectral values during the baseline condition  or the difference between the relative power spectral value post-stimulus  and pre-stimulus  for the odd and frequent stimuli. Since the event-related phenomena represent frequency-specific changes of the EEG activity and consist of decreases or increases of power in given frequency bands; this pattern is meaningful only if compared to a baseline measured before the eventEMG processing EMG data (time-locked with the EEG recordings) were zero-phase-filtered in the interval [20-250] Hz through a 4th-order Butterworth filter79. EMG segments containing artifacts were identified using a threshold (We computed the Root Mean Square (RMS) value for each EMG signal. In order to compare the muscular activity between the two groups , the RMS of each muscle activation during each task was divided by the within-group median RMS value of the same muscle while performing a Maximum Voluntary Contraction (MVC). Due to the experimental setup constraints, MVCs were performed with the participants standing in the shielded room and equipped with the EEG cap. Not all the participants were able to perform a proper MVC and normalizing the RMS within-subjects would lead to unreliable results. However, RMS obtained from each muscle and within groups showed low variability. Hence, we assumed that median values are representative of the muscular activity of the elderly and the younger adults.We then compared the normalized RMS value of each muscle between the two groups: two-sided Wilcoxon rank sum tests were applied under the same postural condition 80. A roll-pitch correction was then estimated from the baseline condition to correct for possible misalignment of the sensor axes with the global anterior-posterior (AP) and medial-lateral (ML) directions of the body81. This correction was eventually applied to the acceleration signals gathered from postural tasks (static and dynamic). For the dynamic postural task only , we also estimated the vertical component (VT) of the acceleration.80. After each integration step, a high-pass 1st-order Butterworth filter  was used to account for possible drift effects of the integral. Lastly, the double integrated signal was low-pass filtered with a 5th-order Butterworth (cutoff 10 Hz). This method provides a reliable estimate of the Center of Mass trajectory of the human body82. Windows of 30 s of the estimated CoM trajectory were then retained for further analysis from the baseline and visual oddball conditions of each postural task. The range of motion in the AP and ML directions , the Sway Path length (SP) and the area of the ellipse containing the 95% of the CoM trajectory (CEA) were computed on the AP-ML plane83. The range of motion in VT direction (ROM-VT) was computed to estimate each participant’s body sway for the core board postural task.In order to estimate the Center of Mass (CoM) trajectory, a strap-down double integration was then applied to the corrected acceleration data85. In brief, the correlation between each estimated parameter and subjects’ anthropometry was tested. Then, parameters values were iteratively corrected via the linear model that best interpolates the data. As the last step, detrended data were corrected by the mean value of the estimated parameters. This method has the advantage to keep values with their original range and measurement unit, but it removes any dependencies from confounding variables84.The estimated parameters were then normalized for the participants’ height and body mass via a detrending normalization techniqueFor all variables, within-group differences between single-task and dual-task conditions for both the elderly and the younger adults, and the between-group differences in each postural condition were tested via paired and unpaired samples two-sided Wilcoxon tests (p = 0.05), respectively. We then compared each variable among the four postural conditions within-group for both single and dual-task via a paired samples two-sided Wilcoxon test . All experiments of this study were performed in accordance with relevant guidelines and regulations.Supplementary material 1 (xlsx 3673 KB)


Several impression techniques, especially in combination with computer-aided design and computer-aided manufacturing (CAD/CAM), are used in increasing the accuracy of dental implantology and decreasing patient discomfort. The study was designed to examine the accuracy of the digital impression (DI) of multiple implants with an intraoral scanner (IOS) and compared with that of a conventional impression (CI).Four dental implants were placed in teeth area #33, #36, #43 and 46# of the mandibular full-arch model. The implanted model was replicated by IOS and CI after fitting of scannable abutments over the implant screws. Then, a small hole was made on the scan region (as a reference point). Two types of CI techniques were used; dual-phase (DP) and monophase (MP). Stone casts were produced through a conventional close tray impression technique using die stone. The casts were scanned with a laboratory scanner. DI was attained by scanning the implanted model with the IOS. The control sample was accomplished by scanning the implanted model directly with a laboratory scanner. Dimensional accuracy was calculated by measuring the distances between the reference points of four measuring parameters as follows; A-B, B-C, C-D, and A-D using CAD software. The mean values and standard deviation between the four parameters of different impression techniques (CI and DI) and the control group showed convergent value. One-way ANOVA test showed all CI techniques, except IOS, showed a significant differences from the control group.Compared with CI, the IOS was more accurate because no differences were observed between its measurements and those of the control model. CI is simple and reduces patient discomfort when used in fabricating multiple implants and allowing communication with dental technicians. Key words:Dimension accuracy, conventional impressions, digital impressions, multiple implants. Dental implants have become a successful restorative treatment modality in modern clinical dentistry. Despite that surgical and restorative phases have complexities, technological advances in computer tomography (CT), guided surgery, and diagnostic tools have rendered the surgical aspect increasingly predicTable and less complex .One of the reasons for the unfamiliarity and lack of use of implants in dentistry is the perceived difficulty in making implant impressions. Dental impressions are a critical step in implant dentistry. The imprecise transfer of an implant location can lead to an ill-fitting prosthesis, which may ultimately result in both biological and mechanical difficulties .Currently, the advancement of computer-aided design and computer-aided manufacturing (CAD/CAM) has enabled the generation of digital impression from the patient’s mouth through digitizing a conventional impression (CI) with a laboratory scanner . CI is uIntraoral scanner (IOS) is a device used in capturing direct optical impressions in dentistry . SimilarThe accuracy of the impression is the main factor influencing the assembly’s fit, which is affected by impression material, impression technique, implant angulation, and the number of implants ,9. An imConventional impression (CI) techniques using tray and impression material cannot eliminate the error of expansion, shrinkage, and distortion of impression or gypsum material. An IOS provides a means to overcome or minimize such errors .This in-vitro study aimed to evaluate the accuracy of the full-arch edentulous mandible with four dental implants scanned by an IOS and compared it with two types of CI materials.The null hypothesis is that different implant impression techniques used in this study have no significant differences.-Preparation of implant model and surgical guide A mandibular implant practice model  was used in simulating the edentulous site of the patient mouth. Four dental implants were positioned on the teeth area of the edentulous model  with an implant guide kit . The model was scanned with a laboratory scanner of CAD/CAM machine for fabrication of a surgical guide, which was punctured according to previously determined implant sites.-Installation of dental implant Four implant sites were prepared according to the standard instructions used in dental implant procedure. A pilot drill (2.4 mm diameter) was inserted through each entrance point of the surgical guide hole with a depth of 10 mm, followed by two intermediate drills (3.0 and 3.5 mm in diameter) and a final drill (4 mm in diameter). A screw root-form endosseous Ti-6Al4V implants (FXS4010 (D), Dentium, Co., Ltd., Korea) was inserted and tightened using a manual ratchet in the prepared hole according to standard insertion torque recommended by the manufacturer’s instructions.-Location of scannable abutmentsFour scannable abutments were positioned and tightened into the implant analogs using 15 Ncm torque. The scan region of the scannable abutment was specified with a small notch (reference point) positioned at the mesial aspect of scan region of each scannable abutment using a low-speed handpiece and small fine round bur  condensation silicon  as a group DP, and monophase vinylpolysiloxane (addition silicone)  as a group (MP). A conventional close tray impression technique was used in both DP and MP impression materials. The impressions were poured with type IV dental die stone material  to produce a master stone model with a scannable abutment. The stone model was scanned with a laboratory scanner  for conversion of the stone cast to digital data.-Preparation of digital impression techniqueFor the DI technique, Medit-i500 IOS was used  and lower than 1000 images were made for the mandibular arch according to the manufacturer’s instructions. The control group was achieved by scanning the implanted model directly with a laboratory scanner .-Evaluation of impressions accuracy The data obtained using the laboratory scanner and IOS were imported into Exocad Dental DB 2.2 Valletta  software for the production of 3D models. The Measuring accuracy of CD and DI techniques were obtained by calculating the linear distance between the reference points of four scan bodies. The reference points were designated as A, B, C, and D which were considered standard parameters used for the measurements. The linear distance measurements were accomplished from A-B, from B-C, from C-D, and D-A respectively as shown in Figure -Statistical analysisP<0.05 using Statistical Package for Social Sciences .The calculated impression accuracy was analyzed with one-way analysis of variance (ANOVA) and post hoc multiple comparisons by Bonferroni test between groups at a significance level of -CI and DI techniques accuracy measurementsThe descriptive statistics were presented as mean values and standard deviation (SD) between reference points A to B, A to D, B to C, and C to D of different impression techniques (CI and DI). The values of control group were close to those obtained from the reference points of DI and CI .Multiple comparisons by one-way ANOVA test were conducted and the results showed a significant difference between tested groups, expect IOS group and the control group as illustrated in The mean and standard deviation of the overall reading was obtained for each impression technique . The higMultiple comparison Bonferroni test was performed on overall mean values measured. No significant difference was found between the control and IOS groups as shown in Figure The use of IOS has increased in the field of implant dentistry with the development of digital technologies. Studies focusing on edentulous models with multiple implants and demonstrating the accuracy of IOS technologies are few. The present study used a full-arch edentulous model due to increasing number of requests from edentulous patients who want to restore their teeth with fixed prosthesis, which is considered a highly accepTable treatment for a wide range of patients compared with removable complete denture ,14Four implants were placed in selected edentulous mandibular full-arch model sites according to previous studies ,16. The The linear distance differences between A-B, B-C, C-D, and D-A reference points were determined in all impression techniques . The nulMeanwhile, CI impressions showed significant differences with the control group possibly due to dimensional changes caused by potential laboratory errors, such as shrinkage, irregular thickness or detachment of impression material, and distortion of the impressions ,22. The In addition, the use of an IOS will reduces patient discomfort, time-efficient and simplify clinical procedures for dentist and laboratory technician ,27. Furtin vitro study limitation, the digital scanning systems are superior in accuracy to the conventional impression techniques and can be used in fabricating accurate long-span multiple implant-supported fixed restorations.Within this