Gadolinium aluminate was synthesized by the sol-gel self-combustion method. This method allowed us to obtain a highly porous structure in which open pores prevail, a structure favorable to humidity sensors. Most of the materials studied as capacitive/resistive humidity sensors have significant sensitivities only with respect to one of these types of sensors. In the case of the studied gadolinium aluminate with p-type electric conductivity, the relative humidity of the air has a significant influence on both capacitive and resistive types of electric humidity sensors. The capacity increases about 10,000 times, and the resistance decreases about 8000 times as the relative humidity increases from 0 to 98%. The investigated gadolinium aluminate can be used successfully to obtain high-sensitivity capacitive and/or resistive humidity sensors.This paper presents the synthesis of gadolinium aluminate (GdAlO Humidity is a physical parameter that describes the amount of water vapor found in the atmosphere. It is an extremely important factor in many industrial sectors ,2. Thus,resistive electrical method, based on the variation in the effective electrical conductivity of a material in the presence of water vapor, and the capacitive electrical method, based on the variation in the effective dielectric constant of a porous dielectric material in the presence of water vapor.Due to low costs and good thermal and chemical stability, ceramic sensors are in the spotlight of current research . The opeAmong the oxide compounds, perovskites  occupy a very important place in terms of applications concerning metal–air batteries , superca3, NdFeO3, LaFeO3—in the form of nanofibers synthesized by the electrospinning method as resistive humidity sensors within the range of a relative humidity (RH) of 11–95%. All the studied humidity sensors exhibit optimal working frequency of 100 Hz at which the sensors display good linearity and high response. Compared with other sensors, the SmFeO3 nanofiber humidity sensor shows the best sensitivity, and the impedance variation is more than five orders of magnitude, with the relative humidity changing from 11% to 95%.Zou et al.  studied xCrx1−Oδ3− where x = 0.0, 0.2, 0.4, 0.6, 0.8 and 1), obtained by the sol-gel method, for resistive humidity sensor applications. Measurements of resistance versus relative humidity within the range of 5–98% were performed, and the humidity-sensing factors (Sf = R5%/R98%) were calculated. Chromite doped with magnesium ions (x = 0.4) had the highest humidity-sensing factor of 21,407, while chromite without magnesium (x = 0) had the lowest sensitivity factor, namely 27.27. The response and recovery characteristics for magnesium-doped (x = 0.4) chromite exhibited a good linearity and a very narrow hysteresis loop.Josephine et al.  studied x1−LaxSnO3, where x = 0.0–0.1), synthesized by the classical method (ceramic), for both capacitive and resistive humidity sensor applications. This study shows that the working frequency has very little influence on the resistivity of the sensor, while the capacity is very much influenced. As the value of the relative humidity of the environment increases from 10 to 95%, the resistivity of barium stannate doped with La ions changes considerably compared to its capacity.Upadhyay and Kavitha  have perA very important class of perovskites from an applicative point of view is that of aluminates doped with lanthanide elements ,17,18,193) perovskite, as well as the investigation of this compound for capacitive and/or resistive humidity-sensing device applications. Gadolinium aluminate was synthesized via the sol-gel self-combustion method with polyvinyl alcohol (PVA) as a colloidal medium [This work presents the synthesis of gadolinium aluminate . The surface morphology of the heat-treated powders and the elemental surface composition were examined by scanning electron microscopy (SEM-EDX), using a JEOL-200CX microscope equipped with an EDX module. The values regarding the pore volume, the specific surface area and the average particle size were determined by the Brunauer, Emmet and Teller (BET) method, with a Nova-2200 instrument .To obtain information on the values of the transition temperatures during the formation of perovskite-type phases, the calcined powder obtained through self-combustion has been characterized appealing to thermogravimetric and differential thermal analysis (TG-DTA) techniques, using Netzsch STA 449 F1 Jupiter apparatus . Information on the structural properties of the heat-treated powders was obtained by X-ray diffraction (XRD) measurements using a PANALYTICAL X’ PERT PRO MPD diffractometer  with CuKThe sensing element (both capacitive and resistive) was made of as-synthesized pressed powders in the form of discs with a diameter of 17 mm and a thickness of 2 mm, to which porous silver metal electrodes were deposited on both surfaces using the “screen printing” method.The values of the capacity and electrical resistance of the sensor element were determined using an impedance analyzer  that operates in the frequency range of 40 Hz–110 MHz.2), while the values of RH between 11% and 98% were obtained by using saturated solutions of some salts [For the measurement of humidity, the sensing element was inserted into a thermostat chamber maintained at 25 °C under different humidity conditions. The value of 0% RH was obtained by using dry calcium chloride  than required using a classical method ,24,25. C3 and of some secondary phases . In addition, the X-ray diffractogram for the powder heat treated at 1000 °C for 7 h .a = 5.250 Å; b = 5.302 Å; c = 7.447 Å) of the powder heat treated at 1000 °C/7 h are in accordance with the values found by other authors [a = 5.285 Å; b = 5.371 Å and c = 7.511 Å, which also represented the spatial structure of the unit cell, where Gd3+ ion is bonded in an eight-coordinate geometry to eight O2− ions, and Al3+ ion is bonded to six O2− ions to form a corner-sharing AlO6 octahedra , the average particle size (DBET) and the total pore volume (VBET). 2 adsorption/desorption isotherms at 77 K. According to the IUPAC classification [3-type hysteresis loop. The inflexion point of isotherms reflects the stage of complete monolayer coverage, when multilayer adsorption begins to take place. From BET analysis, the specific surface area, SBET = 10.0 m2/g, the average particle size, DBET = 81.0 nm, and the total pore volume, VBET = 0.0018 cm3/g, were obtained. The value of the average particle size obtained from the BET analysis falls within the value ranges obtained from the SEM analysis.From the BET analysis for gadolinium aluminate with a crystal structure without secondary phases (heat-treated powder at 1000 °C/7 h) valuable information was obtained concerning the specific surface area  or B/(A + B) ratio has a value of about 0.5, proving a homogeneous distribution of the elements in the sample (A is Gd at. % and B is Al at. %).Energy-dispersive X-ray spectroscopy (EDX) provides an accurate determination of the atomic concentrations of different elements present in the surface region of the samples. For the sensor element obtained from the heat-treated powder at 1000 °C for 7 h, the capacitance and resistance were measured depending on the relative humidity of the environment as well as working frequency.In S) can be calculated using the equation [maxM and minM representing the capacitances (C) or resistances (R) measured for the maximum and minimum values of the relative humidity interval, respectively.Humidity sensitivity (equation :(1)S=MmaIn C) of the material can be expressed as follows [ε*, C0 and rε represent the complex dielectric constant, capacitance and relative dielectric constant of an ideal capacitor, respectively; ω represents the angular frequency, γ represents the conductance and ε0 represents the permittivity of free space. This equation shows that the value of the capacity decreases with the increase in the working frequency as well as with the increase in the humidity value. Moreover, conductance increases with the increase in humidity, which implies the increase in the capacitance with the increase in humidity as a function of the angular frequency [Regarding the capacitive humidity sensor, when the sensor material is exposed to humidity, the water molecules are absorbed and the material presents a leak conduction (γ) . The ele follows ,31:(2)C=requency ,31.In the case of low working frequencies, the capacity variation with humidity can be explained by the phenomena of adsorption and absorption of water molecules, and their effect on the variation in the system capacity formed by porous material and water ,33.When several layers of water molecules are adsorbed on the material surface, they will be linked together by a single hydrogen bond, leading to increased mobility of the molecules and, therefore, to an increase in the complex dielectric constant. If the absorption of liquid water also takes place, the water molecules are extremely mobile and the relative dielectric constant becomes the maximum, corresponding to the water .C vs. RH curve.At high working frequencies, energy losses do occur due to the ionic conductivity that is achieved by hopping transfer of protons between adjacent hydroxyl groups . The enen or p).Regarding the resistive humidity sensor, when the sensor is exposed to humidity, the electrical conduction nature can be of two types: ionic, in which the sensor resistivity decreases with the increasing RH due to absorption and water vapor condensation in capillary pores; and electronic, in which the sensor resistivity depends on the semiconductor type . The hysteresis value is very low (0.3–3%) for both types of sensors on the entire RH range. Displaying a small hysteresis, when the relative humidity of the environment will decrease, desorption of water molecules will be achieved with lower additional energy.The gadolinium aluminate studied for humidity capacitive/resistive sensor application exhibits good reversibility  in the low-frequency range, at increasing and decreasing RHs, within the investigated humidity range. In Another important factor for a humidity sensor consists of the response time. When the sensor at a certain humidity value is exposed to a higher humidity value, we can define a response time to absorption. When returning to the initial value, we can define a response time to desorption. The time required for the response value to reach 90% of the variation value for absorption and desorption, respectively, was considered the response time.The gadolinium aluminate was studied as the humidity capacitive/resistive sensor was investigated regarding the absorption and desorption response time, for the RH range of 0–98% and the frequency range of 40 Hz–10 MHz. The sensor element in both regimes, both as a capacitive sensor and as a resistive sensor, had a response time of around 45 s for absorption and around 60 s for desorption at a humidity variation of about 11%. It was found that the response time is not considerably influenced by the working frequency for both regimes of the sensor element (capacitive/resistive).The response time characteristics at room temperature (25 °C) for the sensor element (capacitive/resistive) within an RH range between 43% and 85%  at frequencies of 40 Hz, 100 Hz and 1 kHz are shown in Gadolinium aluminate with a perovskite structure, synthesized by the sol-gel self-combustion method, has been investigated for applications as a capacitive and/or resistive humidity sensor.The sensor’s electric capacity increases about 10,000 times within a 0–98% interval of relative humidity, and the electric resistance decreases about 8000 times within the same RH interval at the optimum working frequency of 40 Hz. Used as a capacitive sensor, its sensitivity diminishes quickly with the working frequency up to 1 kHz. Used as a resistive sensor, its sensitivity exponentially decreases up to the working frequency of 10 MHz.C and Log R vs. RH characteristics of the sensor show good linearity at all working frequencies. The sensor shows a small hysteresis (<3%) and a response time of around 180 s.The Log The investigated gadolinium aluminate can be used successfully to obtain high sensitivity capacitive or resistive humidity sensors.


Thompson sampling). The interplay between optional stopping and prior mismatch is examined. We propose a novel partitioning of regret into peri/post testing. We further show a strong dependence of the parameters of interest on the assumed prior probability density.The connection between optimal stopping times of American Options and multi-armed bandits is the subject of active research. This article investigates the effects of optional stopping in a particular class of multi-armed bandit experiments, which randomly allocates observations to arms proportional to the Bayesian posterior probability that each arm is optimal ( Sequential testing procedures allow an experiment to stop early, once the available/streaming data collected is sufficient to make a conclusion. The benefits of reducing the duration of e.g. Web based A/B tests, clinical trials or the monitoring of adverse events are obviously very attractive in terms of saving costs and lives. Wald  proposedOne consequence of unplanned optional stopping rules for Null Hypothesis significance testing (NHST) are severely inflated type-I error rates. Bayesian hypothesis testing (BHT) is frequently hailed as a win-win alternative to traditional A/B testing. While there is a growing literature in favor of Bayesian stopping criteria based on the sequential collection of data , other aOur work touches upon previous research on American Options  that demAs compactly described in , the namterature  but moreMultiple algorithms have been proposed to optimize bandit problems: Upper Confidence Bound (UCB) methods  for whica up to time t  a uniform  as the percent difference between the optimal arm and the one chosen by the bandit through a stopping rule: TR fined in . The totThe numbers confirm the pattern gleaned from While MABs make no guarantees on type-I/II errors, stopping rules I and II suggest precise probabilistic statements which a user might want to rely upon. We now show that the combination of optional stopping and mismatched priors leads to poor calibration of inferential expectations.k (fixed at All simulations up to this point have been “fully Bayesian” in the sense that the true The left panel of We treat the special case of Our contribution is to demonstrate a heightened sensitivity to prior assumptions in MABs when data-dependent stopping rules are used; a problem which has been known to be true in simple Bayesian inference for a few decades by now . We haveWe further worry about the inability to easily set priors for the differences in Bernoulli probabilities rather than just the parameters themselves. The additional flexibility would allow to optimize for much more realistic experimental situations.Of course, it is true that the cost of an experiment can be substantially reduced by deploying RPM based bandits instead of pre-committing to a fixed sample size. Allowing to rapidly detect large differences and not waste resources sticking to an unnecessarily rigid protocol is obviously a benefit of most sequential testing algorithms. One difference being that optimization attempted by Bayesian bandits is not concerned with a null hypothesis but with minimizing the posterior expected loss. The implicit assumption that Type-I errors incur no additional cost is likely to be wrong in realistic applications: it seems more reasonable that switching campaigns for no good reason should be avoided. These insights complement recent literature on frequentist-oriented bandit algorithms implementing optional stopping .


Circulating soluble programmed death-1 ligand (sPD-L1) is measurable in the serum of cancer patients. This study aimed to investigate the significance of sPD-L1 in cancer patients receiving immune checkpoint inhibitor therapy. Blood samples were obtained before and after immune checkpoint inhibitor therapy (January 2015 to January 2019). The study cohort consisted of 128 patients who were diagnosed with non-small cell lung cancer (n = 50), melanoma (n = 31), small cell lung cancer (n = 14), urothelial carcinoma (n = 13), and other cancers (n = 20). Patients with a high level (> 11.0 pg/μL) of sPD-L1 were more likely to exhibit progressive disease compared with those with a low level . High sPD-L1 was also associated with worse prognosis; the median PFS was 2.9  months versus 6.3 (95% CI 3.0–9.6) months (p = 0.023), and the median OS was 7.4 (95% CI 6.3–8.5) months versus 13.3 (95% CI 9.2–17.4) months (p = 0.005). In the multivariate analyses, high sPD-L1 was an independent prognostic factor for both decreased PFS  and OS . sPD-L1 levels did not correlate with tissue PD-L1 expression. However, sPD-L1 levels were positively correlated with neutrophil to lymphocyte ratios and negatively correlated with both the proportion and the total number of lymphocytes. We found that high pretreatment sPD-L1 levels were associated with progressive disease and were an independent prognostic factor predicting lower PFS and OS in these patients. Therefore, it is important to identify predictors of this negative response. Some biomarkers that predict treatment response have already been identified. For example, programmed death ligand 1 (PD-L1)2 expression in tumor tissue is predictive of a higher response rate to programmed death 1 (PD-1) or PD-L1 inhibitor therapy in patients with non-small cell lung cancer (NSCLC)5. It is also a marker of poor prognosis in some solid tumors8. However, inter-assay discordance and tumor heterogeneity hinder the standardization of PD-L1 testing and interpretation10. Researchers have tried to standardize the methods used to measure PD-L1 expression, but no clinically validated assays are currently available11. Microsatellite instability or deficient mismatch repair (dMMR) is one of the biomarkers studied as a predictor of ICI response2. dMMR is associated with favorable clinical outcomes in patients with colorectal cancer13. PD-1 blockade results in a robust response in some subjects with dMMR-positive solid tumors14. Tumor mutational burden, tumor infiltrating lymphocytes, and genetic signatures are also predictive factors. However, these markers lack standardization and exhibit high variability across tumor types and study settings2.Since the first immune checkpoint inhibitor (ICI), ipilimumab, was approved in 2011, the treatment paradigm for solid tumors has changed greatly. ICIs play important roles in the treatment of various types of solid tumors. However, in clinical practice, 16–55% of patients receiving ICI treatment suffer from severe toxicities16. The use of small biopsies can also result in misclassification of up to 35% of PD-L1 assessments in patients with advanced NSCLCs17. Therefore, circulating blood biomarkers are being investigated to predict the response to PD-1/PD-L1 blockade. These potential markers include, among others, circulating immune cells and circulating PD-L123. In previous studies, CD8+ T cells showed a proliferative burst or functional reinvigoration after PD-1 blockade25. Another study showed that functionally active CD8+ T cells or NK cells are associated with good prognosis after PD-1 blockade26.Lack of sufficient tissue for examination is another common and important factor limiting biomarker utility. A shortage of tumor tissue is especially problematic in patients with NSCLC because diagnostic biopsies often yield only tiny pieces of tissue. However, approximately five molecular genetic tests are required to select the appropriate therapeutic agents for an individual NSCLC patient29. Compared with healthy subjects, circulating sPD-L1 concentrations are elevated in the plasma of patients with cancer. In patients with lymphoma, these concentrations return to normal levels after a complete response30. High concentrations of sPD-L1 are associated with poor prognosis in patients with hepatocellular carcinoma, gastric cancer, and NSCLC34. Here, we examined circulating sPD-L1 and its role as a prognostic and predictive marker in patients with cancer who received ICI treatment. We analyzed pretreatment and posttreatment levels of circulating sPD-L1 and investigated the relationship between these levels and clinical outcomes in patients with advanced solid tumors.PD-L1 is present in a membrane-bound form in tumor cells and immune cells. However, it may also be secreted as a truncated form called soluble PD-L1 (sPD-L1) which may mediate immunosuppression or resistance to PD-L1 blockade therapyA total of 128 patients with stage IV solid tumors were included in this study. Samples were obtained between January 2015 and January 2019. The characteristics of the study population are presented in Table t test). However, the mean sPD-L1 concentration was numerically higher in cancer patients and we have to consider the possibility that there was no statistical significance because of the small number of healthy volunteers (n = 20). We used a receiver operating characteristic (ROC) curve to determine the optimal sPD-L1 cutoff level for the prediction of progressive disease after ICI treatment . The ORRs were not significantly different . However, the disease control rates were 79% versus 58% in patients with low and high sPD-L1 levels, respectively (p = 0.013) . The mean sPD-L1 value in the patients with cancer was not significantly different from the mean level in healthy volunteers  versus 2.9 months (95% CI 2.1–3.7 months); this difference was statistically significant  varied Fig. e. A sharThe analyses to examine relationships between ΔsPD-L1 and treatment response did not reveal any apparent patterns . We also examined the correlation between sPD-L1 levels and NLR or circulating immune cell numbers  suggested that these two factors were not correlated. The mean sPD-L1 levels in the negative/low PD-L1 expression group were also not significantly different from those in the moderate/high expression group 45. In contrast, preliminary results indicate that in patients with biliary tract cancer, increased sPD-L1 levels after chemotherapy are associated with longer PFS46. In patients with advanced pancreatic cancer receiving cytotoxic chemotherapy, sPD-L1 dynamics correlate with the disease course39. Several studies have been performed to investigate the dynamics of sPD-L1 changes over time in patients receiving ICI treatment. One study of patients receiving ipilimumab-based treatment for advanced melanoma found that many who had a ≥ 1.5-fold increase in sPD-L1 within 4.5 months after treatment experienced progressive disease44. A study analyzed PD-L1 mRNA expression in plasma-derived exosomes in melanoma and NSCLC patients at baseline and 2 months after PD-1 inhibitor treatment. They showed that exosomal mRNA copies of PD-L1 were correlated with tumor response in melanoma (n = 18) and NSCLC (n = 8) patients47. Another study of 21 patients with lung, gastric, or bladder cancer who underwent anti-PD-1 therapy found that a reduction in plasma sPD-L1 levels is significantly correlated with tumor size reduction48. The significance of sPD-L1 in our NSCLC population was inconsistent with other studies. It seems too early to draw conclusions, as the sample size of all previously published studies was very small.We also investigated whether changes in sPD-L1 levels after ICI treatment are associated with response or prognosis. We were not able to acquire the sample sizes required to identify correlations between the extent of sPD-L1 changes and tumor response or PFS. Because each tumor type had very different patterns of change in sPD-L1 levels, the effects of changes in sPD-L1 on PFS were likely diluted. This possibility was supported by our observation of the opposite pattern when each cancer type (melanoma and NSCLC) was analyzed separately. Few studies have examined the relationships between sPD-L1 dynamics and prognosis in patients receiving radiation or chemoradiation, and the results of those limited studies have been inconsistent. Increased sPD-L1 levels after chemoradiation are associated with a poor prognosis in patients with rectal cancer49. The results of our study suggest that sPD-L1 is derived from cells identified as neutrophils in routine complete blood count tests. Additionally, in a previous study that analyzed peripheral immune cells from 28 cancer patients, 5 to 35% of peripheral blood myeloid-derived suppressor cells (MDSCs) expressed PD-L1. The expression of PD-L1 is highest in granulocytic MDSCs (35.8%) and lower in T cells (< 1%), most NK cells (< 1%), and B cells (11%)50. Therefore, we can carefully assume that sPD-L1 originates from granulocytic MDSCs. Considering the limited information currently available, however, the main source of sPD-L1 should be explained in future studies.We also examined the association between PD-L1 expression levels in tumor tissue and circulating sPD-L1 levels. PD-L1 expression in tumor tissue did not correlate with sPD-L1 levels. This was an unexpected finding, as we expected the levels of sPD-L1 to reflect the expression of tissue PD-L1. Therefore, we explored circulating immune cells as a possible source of sPD-L1. sPD-L1 levels were positively correlated with NLR and negatively correlated with total lymphocyte numbers. The results of previous studies suggest that sPD-L1 originates mainly from a membrane-bound form of PD-L1 present in cancer cells or immune cells49.This study had some limitations. The study population was heterogeneous with respect to cancer type, blood sampling times, ICI administration timing, and post-ICI treatment regimens. This heterogeneity might have reduced the power to detect the effects of various characteristics for individual types of cancer. There were also no reference levels for sPD-L1, and the results between assay kits seemed to be quite different. There are also no pre-established cutoff levels that predict response or prognosis. To overcome this problem, some researchers are investigating reproducible, standardizable methods that can be used instead of ELISAIn summary, high pretreatment sPD-L1 levels were associated with low disease control rates. sPD-L1 levels were an independent predictor of PFS and OS in patients receiving ICI treatment for advanced cancer. sPD-L1 was likely derived from peripheral blood neutrophils, and levels generally increased following ICI administration. The amplitude of sPD-L1 change after ICI treatment was associated with PFS in patients with NSCLC and melanoma but in the opposite direction for each cancer type. A future, larger study should be undertaken to reveal the significance of changes in sPD-L1 levels for each carcinoma type.53. Posttreatment samples were obtained from 67 patients at either the next visit or a later visit. Patients were eligible for the study if they (1) were 18 years of age or older, (2) had a histologically confirmed malignancy, (3) received ICI treatment at Seoul National University Hospital, (4) had study samples taken before and/or after ICI treatment, and (5) completed a written consent form for research using human derivatives, which allowed for secondary utilization of samples (IRB No. 1104-086-359). A patient was excluded from the study if they had a diagnosis of two or more types of malignancy within the previous 5 years, withdrew consent before or during the study, or not enough samples were stored for analysis. Retrospective clinical and follow-up information was obtained from the medical records. Pre- and posttreatment samples from patients who received molecularly targeted agents were also analyzed. Blood sampling and analyses were performed after the study protocol was approved by the Institutional Review Board. All patients provided written informed consent to participate in this study. The study protocol was approved by the Institutional Review Board of Seoul National University Hospital (IRB No. 2002-070-110). All study procedures were performed in accordance with the Helsinki Declaration and its later amendments or comparable ethical standards.Blood samples were taken from each cancer patient before they received ICI treatment. Some patients participated in other, previously published studiesg for 10 min) and then aliquoted and stored at − 80 °C until study analysis. sPD-L1 was assayed using a commercially available ELISA Kit  following the manufacturer’s instructions. Samples were analyzed in duplicate for each marker.Serum was obtained by centrifugation  and Excel 2019  software. A p value < 0.05 was considered statistically significant.Demographic and clinical parameters were analyzed using descriptive statistics. Differences in sPD-L1 distribution and median values in healthy donors and cancer patients were compared using nonparametric Mann–Whitney Supplementary Information.


SPP1hi and FABP4hi macrophages, cytotoxic T cells, and natural kill cells of IPF, while type I interferon signaling and production was upregulated in the corresponding SSc-ILD populations. Plasmacytoid dendritic cells were found in diseased lungs only, and exhibited upregulated cellular stress pathways in SSc-ILD compared to IPF. Alveolar type I cells were dramatically decreased in both IPF and SSc-ILD, with a distinct transcriptome signature separating these cells by disease. -/KRT17+KRT5 aberrant basaloid cells exhibiting markers of cellular senescence and epithelial-mesenchymal transition were identified in SSc-ILD for the first time. In summary, our study utilizes the enriched capabilities of scRNA-seq to identify key divergent cell types and pathways between IPF and SSc-ILD, providing new insights into the shared and distinct mechanisms between idiopathic and autoimmune interstitial lung diseases.Idiopathic pulmonary fibrosis (IPF) and systemic sclerosis-associated interstitial lung disease (SSc-ILD) differ in the predominant demographics and identified genetic risk alleles of effected patients, however both diseases frequently progress to respiratory failure and death. Contrasting advanced SSc-ILD to IPF provides insight to the role dysregulated immunity may play in pulmonary fibrosis. To analyze cell-type specific transcriptome commonalities and differences between IPF and SSc-ILD, we compared single-cell RNA-sequencing (scRNA-seq) of 21 explanted lung tissue specimens from patients with advanced IPF, SSc-ILD, and organ donor controls. Comparison of IPF and SSc-ILD tissue identified divergent patterns of interferon signaling, with interferon-gamma signaling upregulated in the  Pulmonary fibrosis can occur as a consequence of autoimmunity, environmental exposures, or genetic mutations, as well as idiopathic disease. Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease (ILD) of unknown etiology primarily occurring in the elderly, invariably progressing to respiratory failure, resulting in lung transplant or death. While some overlap exists between the clinical features, pathogenesis, and more recently the approved anti-fibrotic therapies for IPF and the connective tissue disease-associated ILDs, significant heterogeneity exists amongst the incidence and morbidity and mortality of the various autoimmune ILDs –3. In syvia the expansion of aberrant myofibroblasts, overproducing extracellular matrix  and surfactant-encoding genes , along with single nucleotide polymorphisms (SNPs) identified by genome-wide association studies (GWAS) in 20 independent loci currently account for 25-30% of the sporadic IPF disease risk  pattern, but not SSc-ILD  of peripheral parenchymal lung tissue obtained at the time of lung transplant from patients with IPF and SSc-ILD, and control lung tissue from organ donors without pre-existing lung disease. Comparison of IPF and SSc-ILD tissue identified divergent patterns of interferon signaling in IPF and SSc-ILD, increased cellular stress pathways in the plasmacytoid dendritic cells of SSc-ILD, a profound loss of alveolar type 1 (AT1) cells in both IPF and SSc-ILD with a distinct transcriptome signature defining each AT1 subset by disease, as well as identified aberrant basaloid cells in SSc-ILD for the first time.The University of Pittsburgh Institutional Review Board approved procedures involving human samples. Explanted subpleural peripheral lung tissue was digested and scRNA-sequencing performed as previously described , 32. AllIn order to determine commonalities and differences between IPF and SSc-ILD, we performed single-cell RNA-sequencing (scRNA-seq) on 21 peripheral lung tissue specimens obtained at the time of lung transplant from patients with IPF (n=8 samples) and SSc-ILD (n=8) and control lung tissue from organ donors without pre-existing lung disease whose lungs were declined for transplant (n=5). Separate upper and lower lobe samples were included for each IPF and SSc-ILD patient and from one control patient, with only lower lobe samples available from the other 3 controls. For these analyses we added 2 not previously analyzed IPF samples with previously described samples , 32. TheIn total, we analyzed 85,756 cells, with individual cell types identified by multiple distinct markers 3 and saSPP1hi macrophages, FABP4hi macrophages, and a population of monocyte-derived macrophages (FCN1hi)  fluid of the healthy human lung, FABP4hi macrophages comprised the majority of BAL macrophages, and likely most closely approximate the traditionally designated alveolar macrophages  , 32. A srophages . ProlifeSPP1hi and FABP4hi macrophages in IPF, compared to SSc-ILD  was primarily expressed by natural killer cells and to a lesser extent by T lymphocytes, with macrophages its primary target via their expression of the heterodimeric receptor comprised of IFNGR1 and IFNGR2  mediated signaling was distinctly upregulated amongst ine lung .SPP1hi and FABP4hi macrophages in SSc-ILD , CLEC4C, CLIC3, and NRP1  was also increased in SSc-ILD pDCs compared to IPF  expressing MUC5B, goblet cell marker SCGB1A1 and ciliated markers FOXJ1 and RSPH1  cells and increase in cells originating from the bronchial epithelium was consistent in both diseases 5B. SampRNF168, TRIM37, RNF40, and HUWE1, as well as the deubiquitinase BAP1 were upregulated in IPF AT1 cells, implicating the histone ubiquitination pathway which has been linked to double-stranded DNA break repair as well as transcription repression and activation  mediated unfolded protein response was upregulated in IPF. IRE1 is an endoplasmic reticulum (ER) stress sensor previously identified to signal through the transcription factor XBP1, as well as the JNK and NF-κB pathways . Murine nfection , 48.-/KRT17KRT5+ cells), with no cells sharing this distinct transcriptome amongst the control samples , and markers of cellular senescence (including CDKN2A(p16), CDKN1A(p21), and GDF15)  as well as the presence of a small control only WIF1hi population clustering within the myofibroblasts, mirrored those defined in our previous analysis of fibroblasts in SSc-ILD and control lungs  of our SSc-ILD samples showed UIP on histopathology, we identified significant systematic differences compared to IPF, supporting the notion that many distinct disease mechanisms remain despite the shared histopathologic pattern. Thus, all UIP is not the same at the molecular level, and there is potential loss of scientific knowledge and treatment opportunities in the inclination to think of it as such. The similarities and differences identified between diseases could lead to additional hypotheses regarding treatments that will have a benefit in both diseases versus only IPF or SSc-ILD. We believe that even greater differences could be present in a comparison of early stage disease tissue, however due to the associated risks, patients with SSc-ILD rarely undergo surgical lung biopsy, resulting in no early disease tissue available for research studies.hiSPP1 and hiFABP4 macrophages and cytotoxic T and natural killer cells, while Type 1 IFN signaling was amplified in the analogous SSc-ILD populations. In up to 50% of patients with SSc, even early in disease, a type I IFN signature is identifiable in the peripheral blood and correlates with other mediators of fibrosis and inflammation   lungs , as wellr group) . The abepression , 50, nowpression . The relAkin to what other scRNA-seq analyses have observed in IPF , 65, ourOur study was limited by its small sample size and exclusive use of advanced disease tissue. As patients with IPF and SSc-ILD now rarely undergo surgical lung biopsy, there was no early-stage disease tissue available for analysis. Pulmonary fibrosis, in particular IPF, often develops along an apicobasal gradient, thus we instead included samples from both the upper and lower lobes to capture a greater spectrum of disease. The graded shifts in cell populations most pronounced in the lower lobes, such as the loss of alveolar type 1 and natural killer cells, suggest that this method may enable the identification of progressive changes in the disease course for at least some samples. All IPF and SSc-ILD samples are from patients with end-stage disease receiving care at a tertiary medical center and may not reflect the comprehensive IPF and SSc-ILD population. Due to the limited number of samples, we were unable to perform matching for age, sex, or the degree of pulmonary hypertension. Specifically, the sizeable difference in mean pulmonary artery pressure between the IPF and SSc-ILD patients (20.25 vs 39.75 mmHg) precluded us from determining if differences identified in the endothelial cells, smooth muscle cells, and pericytes are truly disease-specific versus secondary effects of increased pulmonary vascular pressures. To limit confounding from differences in experimental technique, we have chosen to limit our analysis to samples processes with the same digestion protocol and reagent chemistry. Due to the limited number of alveolar type 1 and plasmacytoid dendritic cells recovered from IPF and SSc-ILD samples, analysis of differentially expressed genes may have a higher false positive rate in these populations.In summary, our analysis utilizes the enhanced capabilities of scRNA-seq to discriminate disease-specific alterations in explanted SSc-ILD and IPF lungs. Macrophages exhibit distinct interferon signatures in IPF versus SSc-ILD, with cytotoxic T cell and natural killer cells expressing similar patterns. The striking loss of alveolar type 1 cells in both diseases likely results from distinct mechanisms, with the presence of aberrant basaloid cells transcriptionally similar to those in IPF now confirmed in SSc-ILD as well. Focusing on shared and unique mechanisms of pathology in IPF and SSc-ILD provides improved insight to identifying treatments that may benefit both diseases. Improved phenotyping of patients with IPF and SSc-ILD to indicate those at highest risk for progressive disease, as well as those with a predominance of specific pathologic mechanisms  is needed to better guide treatment choices throughout the disease course. Expanding existing lung scRNA-seq datasets and linking these to peripheral blood biomarkers may be a unique opportunity to link pathologic alterations in the tissue to clinically accessible biomarkers, allowing such improved phenotyping.https://www.ncbi.nlm.nih.gov/geo, GSE 128169, GSE 128033, and GSE 156310.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: EV: design of study, data generation, data analysis, writing of manuscript. TT, AP, JS, HB, MR: data generation and analysis. RL: design of study, interpretation of data and editing manuscript. All authors contributed to the article and approved the submitted version.Research reported in this publication was supported by the National Institutes of Health NIAMS 2P50AR060780 (RL) and NHLBI 2T32HL007563-31 (EV). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.RL reports grants from Bristol Myers Squibb, Corbus, Formation, Elpidera, Regeneron, Pfizer, and Kiniksa outside the submitted work; personal fees from Bristol Myers Squibb, Formation, Sanofi, Biocon, Boehringer Mannheim, Merck, and Genentech/Roche outside the submitted work.The remaining 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.


To solve the inspection problems in cotton storage, as well as the need for environmental monitoring in the process of modern cotton bale storage, an intelligent inspection and temperature and humidity intelligent monitoring system based on RFID cotton bale was developed by adopting RFID (Radio Frequency Identification) technology, wireless temperature and humidity real-time monitoring technology and handheld terminal intelligent inspection technology. The system was composed of RFID positioning inspection module and temperature and humidity real-time monitoring and transmission module. The artificial neural network (ANN) based on the particle swarm optimization (PSO) algorithm was used to process the monitoring data of the system by Gaussian filtering, and an accurate classification model of RSSI and label position was established. The test results showed that: Through the comparative analysis of the RFID indoor positioning algorithm, the positioning error of the PSO-ANN algorithm was small. In the actual cotton bale warehouse test, the relative error of positioning and monitoring for RFID cotton bale intelligent inspection and monitoring system was less than 6.7%, which effectively improved the working efficiency of inspection personnel and the security of cotton bale storage. The relative error of temperature and humidity was less than 8% and less than 7%, which could display the temperature and humidity information in real time and meet the real-time demand. This study improved the management personnel's effective positioning and inspection of the cotton bale, prevented the loss of cotton bale, reduced the deterioration probability of cotton bale, and effectively improved the storage management level of the cotton bale. It was of great practical significance to realize the networking, automation, and intelligence of cotton bale storage management. For a long time, for the safety of cotton in the warehouse, bale warehouse still adopts the methods of manual periodic inspection, which requires a large number of inspectors to put into work. Due to the variability of manual inspection4, it is difficult to ensure the quality of decision management, especially the means and methods of large and medium-sized intelligent cotton storage urgently need to be improved. It is very important to carry out real-time inspection and temperature and humidity monitoring of cotton stacks in the process of cotton storage.The number and scale of warehousing enterprises in China are showing a trend of rapid growth. At the same time, due to national conditions and development reasons, as well as the physical characteristics and special storage environment of cotton, there are problems such as mildew, fire, theft, etc.6, domestic and foreign experts and scholars have carried out research on multi-sensor fusion technology8, wireless communication technology13, and optimization algorithm technology16, which have effectively improved the intelligence level of critical operation monitoring17. Since RFID19 has the advantages of non-contact reading of information, large data storage capacity, and small volume22, it could be better applied in monitoring and management of cotton stack storage23, and the optimization processing algorithm was adopted to improve positioning25 and environmental temperature and humidity monitoring accuracy26. For example, Ullah et al.27 developed a mathematical model, compared with the cost of the card reader, the proposed RFID system has more significant sensing power Omer et al.28 developed a new procedure to estimate the indoor distance of passive UHF RFID tags, which effectively improved positioning monitoring accuracy. Srbinovska et al.29 designed a practical low-cost greenhouse monitoring system based on a wireless sensor network technology to monitor key environmental parameters such as temperature, humidity, lighting. Wu et al.30 proposed a new differential received signal strength (RSS) positioning algorithm, which can better achieve device tracking accuracy compared with the existing RFID positioning method. Badia-Melis et al.31 adopted WSN and RFID technology to realize temperature and humidity detection and positioning of the cold chain, which can monitor temperature and position changes at a high rate. According to the physical and chemical characteristics of cotton, JU32 proposed a set of fire prevention measures for cotton logistics warehouses, and obtained the optimal temperature and humidity threshold in the cotton storage environment. To sum up, the application of sensors has been effectively studied in various fields34, but the development and application of the existing storage monitoring system36 can only realize intelligent collection and management in the process of logistics and warehousing37 and the corresponding technologies39 have not been applied in the collection of cotton stack storage, it is imperative to develop a terminal for intelligent monitoring inspection of cotton bale.For the monitoring and processing of positioning informationTo improve the management of cotton warehouse, this research broke through key technologies of real-time monitoring of cotton bag storage environment and artificial inspection positioning. Aiming at Cotton stack positioning and storage environment temperature and humidity monitoring, an RFID intelligent inspection system for cotton bag storage was developed, which realized the informationization and intelligence of cotton bale inventory management, and integration of host computer service platform and handheld terminal information. The system ran stably and reliably. The research results provided reference for related cotton bale storage.As shown in Fig. As shown in Fig. As shown in Fig. As shown in Fig. As shown in Fig. The location server is shown in Fig. As shown in Fig. As shown in Fig. The distance between the electronic tag and the antenna is calculated by measuring the field strength of the tag wireless signal. The relationship between the distance and the wireless signal strength is parabolic. As shown in Fig. In the formula, D is the distance converted according to the signal strength, in cm; RSSI is the signal strength, and the absolute value is in dBm; P is the parameter, which is related to the antenna; L is the parameter, which is related to the antenna.40 optimized the structure of ANN, determined the best vector network, improved the stability of the ANN algorithm, reduced the positioning error, but did not filter out the noise data in the experiment. Therefore, to improve the positioning accuracy, the data was processed by Gaussian filtering in this paper, and a positioning algorithm based on PSO optimization ANN(PSO-ANN) was proposed, and environmental noise was preprocessed by the Gaussian filtering to compensate for the positioning defects of ANN, thus improving the prediction effect of ANN and the learning speed of ANN.ANN is a highly complex algorithm inspired by biology, which can solve complex nonlinear problems through modeling. ReferenceIn this study, PSO was used to optimize the initial weight and threshold parameters of ANN, and Gaussian filtering was used to preprocess the noise barriers in the storage environment, which reduced the label positioning error and made up for the ANN positioning defect. In the real-time positioning of cotton bales, the signal strength will reflect, diffract and scatter in the storage environment and the common model of signal path loss in the storage environment isPSO-ANN has outstanding performance in terms of efficiency and positioning accuracy. The PSO particle continuously updates its speed and position. The update formula isFor cotton bale storage location, it is necessary to construct the mapping relationship between To reduce the noise error, the collected data were processed by Gaussian filtering with the formula as follows:2 is the variance, A is the expected value, andIn the formula, σAccording to the 2The signal sent by the RFID reader to the tag decreases with the increase of the propagation distance. Since the RSSI value measured at a certain point during positioning will fluctuate constantly, and the fluctuation of the RSSI value generally follows the Gaussian distribution, the positioning result is related to the Euclidean distance of the signal space between the positioning tags. The layout of the card reader during storage can be equivalent to plane optimization, and the position of the reader on the plane distribution can be optimally solved by PSO algorithm. The solution process is shown in Fig. Set the relevant parameters according to the actual input samples of the ANN: the number of nodes in the input layer is set to n, the number of nodes in the hidden layer is set to s, and the number of nodes in the output layer is set to m.Set the PSO-related parameters: the initial position of the particles is Calculate the fitness value of each solution according to formula , and obtOutput the optimal global solution of the PSO algorithm, and calculate the current fitness value fi of all particles. If The optimal solution, where According to Eqs.  and 4),,4), updaDetermine whether to terminate: If the number of iterations reaches the expected specified value, terminate the process and output the optimal solution , otherwise, return to step (3).The specific steps of PSO-ANN are as follows:As shown in Fig. oT is the temperature data read from SHT20 by the main control chip; d1 and d2 are temperature conversion coefficients. Relative humidity conversion formula:linear is the relative humidity; The humidity data read by SoRH from 1-SHT20 by the main control chip; c1, c2 and c3 are humidity conversion coefficients. Because the measurement of humidity is affected by temperature, the temperature compensation of the sensor should also be considered when measuring humidity. The compensation formula is:true is absolute humidity; t1, t2 are temperature compensation coefficients. Combining the design of the temperature and humidity module, the temperature accuracy SoT is 14 bits, and the humidity accuracy SoRH is 12 bits. Substituting the coefficient results into Eqs. ,  are the coordinates of two points A and B, respectively, where point A is the position of the RFID positioning tag, and B is the origin of the coordinates, The test record data is shown in Table To further verify the practical application effect of RFID positioning inspection system in cotton bag warehouse, a performance test was carried out in December 2020 in Baoding in YIN XIANG cotton industry co., LTD., Hebei province. Figure The test showed that the RFID cotton bale positioning monitoring system, based on the test data of the RFID tag positioning information, showed that the relative error of the system monitoring was less than 6.7%. The reason for the error was that the positioning distance was based on the positioning of the tag by the base station. The accurate distance was calculated by the signal field strength. In the case of a long cotton pile, a slight error would occur. After debugging for many times, the error can be effectively improved by arranging multiple base stations, and the monitoring requirements can be met based on performance and technical indicators required by the system.As shown in Fig. Through the temperature and humidity monitoring effect test of the temperature and humidity monitoring sensor, the test results showed that the temperature monitoring error of the system was less than 8%, which met the monitoring requirements. The error was caused by the uneven temperature value in the temperature and humidity constant temperature and humidity box. By adjusting the time, the error can be effectively improved, effective monitoring can be realized within the expected index range, and temperature monitoring met the monitoring performance index requirements.By adjusting the humidity value of the constant temperature and humidity box, the humidity of the temperature and humidity sensor of the system was monitored. The handheld terminal for inspection was wirelessly connected with the sensor through Bluetooth, and the data was transmitted to the bale inspection system in real-time, and the humidity of the temperature and humidity sensor was tested. When using a constant temperature and humidity box and the temperature and humidity sensor humidity monitoring comparison, testing in different environments. By adjusting the actual humidity value of the constant temperature and humidity box and the measured value monitored by the system temperature and humidity sensor, it can be concluded whether the humidity measurement of the system is normal and accurate. The test results are shown in Table Through the temperature and humidity monitoring effect test of temperature and humidity monitoring sensor, the test results showed that: temperature measurement error was less than 8%, and relative humidity error of the system was less than 6%. The temperature and humidity sensor monitoring device can monitor real-time temperature and humidity monitoring values, and the system ran normally and met the design requirements. The error is caused by uneven temperature and humidity in the temperature and humidity constant temperature and humidity box. By adjusting the time, effective monitoring within the range of expected indicators could be achieved and the requirements of monitoring performance indicators could be met.A scheme based on RFID warehousing intelligent inspection system was proposed in this paper, and the overall scheme design of the system was carried out, mainly including hardware design and software design. The real-time sensor technology, wireless transmission technology, and intelligent monitoring technology was applied to Cotton stack warehousing in the system, which achieved the purpose of monitoring the temperature and humidity of circulation in the cotton stack storage warehouse. Many cotton stack warehouse can be inspected in real time through hand-held terminals, and cotton package information can be uploaded to the platform in real time, which reduced the wiring cost and maintenance difficulty, also liberates the labor, and has a broad application prospect in the cotton industry.An RFID intelligent inspection terminal was developed in the system, which integrated RFID positioning technology and wireless temperature and humidity monitoring technology in the system platform, and adopted the method of particle swarm optimization (PSO) algorithm to optimize artificial neural network (ANN) to process the system monitoring data by Gaussian filtering, and established an accurate classification model of RSSI and tag position. Through the comparison and analysis of the algorithms, the positioning error of the PSO-ANN algorithm was generally smaller than that of the ANN algorithm, that was, PSO-ANN had better positioning accuracy of cotton stacks.Through the demonstration application and experiment of the National Quality Testing Center, the RFID cotton bale positioning inspection and temperature and humidity monitoring system were tested respectively. The test results showed that the positioning accuracy error was less than 6.7% and the temperature and humidity monitoring accuracy error was less than 8% and 7%. The system can monitor the temperature and humidity of the warehouse and the positioning information of the cotton stack, with high reliability and low cost, and effectively realize the automation, information, and intelligence of the storage management of cotton bale.


Integrative Fine-Mapping, to pinpoint causal SNPs for diversity outbred (DO) mice eQTL by integrating founder mice multi-omics data including ATAC-seq, RNA-seq, footprinting, and in silico mutation analysis. We demonstrate INFIMA’s superior performance compared to alternatives with human and mouse chromatin conformation capture datasets. We apply INFIMA to identify novel effector genes for GWAS variants associated with diabetes. The results of the application are available at http://www.statlab.wisc.edu/shiny/INFIMA/.Genome-wide association studies reveal many non-coding variants associated with complex traits. However, model organism studies largely remain as an untapped resource for unveiling the effector genes of non-coding variants. We develop INFIMA, The online version contains supplementary material available at (10.1186/s13059-021-02450-8). Vast majority of disease and complex human trait-associated single nucleotide polymorphisms (SNPs) identified through genome-wide association studies (GWAS) are non-coding . This crThe recent transcriptome-wide association studies (TWAS) that leverage reference expression panels led to notable progress in identifying candidate disease-associated genes , 9. HoweAbcc8  that leverages multiple multi-omics data modalities to elucidate causal variants underpinning the DO-islet eQTLs. INFIMA exploits differences of the candidate genetic variants in terms of their multi-omics data support such as the chromatin accessibility of the variant locations, correlations of chromatin accessibility, and transcriptome with variant genotypes and DO mice allelic expression patterns. As a result, it maps genetic variants within the DO founder strains to eQTL genes by quantifying how robustly the multi-omics data explains the allelic patterns observed in the eQTL analysis. Application of INFIMA to islet eQTLs identified in DO mice  measures the correlation between ATAC-seq signal of the peak harboring SNP k and the genotype effect of SNP k on founder expression; |cor|∈ similarly quantifies the correlation between ATAC-seq signal and gene expression in the founder strains.Here, each component of Yg is then given by The combined generative model for DO-eQTL effect size ax and by. In summary, INFIMA model takes as input DO-eQTL results, summarized functional data from RNA-seq and ATAC-seq analysis of founder strains, as well as ATAC-seq-based comparative footprint and in silico mutation analysis of SNPs and outputs SNP-level quantifications , varying levels of informativeness in the multi-omics data yield improved area under receiving operating characteristics and precision recall curves, with an average of 0.61 ± 0.079% improvement in power from moderately to highly informative setting . The NI, MI, and HI settings yielded 33.90%, 22.22%, and 14.04% credible proportions, respectively  around DO-eQTL markers across all the G=10,936 gene-marker associations . This resulted in a right-skewed distribution for the number of candidate local-ATAC-MVs within a window , INFIMA reduces the size of the set of candidate local-ATAC-MVs by 75.0% |, contributes the most at 33.0% . Next, we compared INFIMA effector gene predictions for these human GWAS SNPs with two baseline strategies: (1) linking mouse ATAC-seq peaks syntenic to GWAS variants to their nearest genes instead of INFIMA predictions and (2) linking human GWAS SNPs to their nearest genes without going through model organism data and INFIMA predictions. We observed that INFIMA predictions were markedly better supported by the pcHi-C data  and (2), respectively; “Next, we used INFIMA to predict effector genes of diabetes-associated GWAS SNPs. Among the 1532 mouse ATAC-seq peaks syntenic to GWAS variants, 737 contained local-ATAC-MVs. Of these, 548 were causally linked to at least one DO-eQTL gene, with 18.1% linked to a single gene  as well as three KCNJ11 intronic GWAS SNPs rs5215, rs5219, and rs757110 (type 2 diabetes) by pcHi-C data. These three human SNPs are syntenic to rs25937937, rs230081777, and rs227822836 in mouse and are identified by INFIMA as causal for a Abcc8 DO-eQTL, the homolog to human ABCC8  and, furthermore, rs5215 also results in a better sequence motif for YY1  as well as novel candidates . While the ability to infer susceptibility genes for only 3.5% of the GWAS SNPs might appear low, this is due to several potential limiting factors. First, by utilizing multi-omics data from islets, we are aiming to identify effector genes of diabetes-associated GWAS variants in islets. This will inherently exclude SNPs that might be exerting their effects in other tissues. Second, the set of candidate regulatory regions  that we have defined in founder strain islets excludes other known potential regulatory mechanisms  tharactions , and, furactions .https://github.com/keleslab/INFIMA. The web application for INFIMA results are available at http://www.statlab.wisc.edu/shiny/INFIMA/.Model organism studies provide extensive resources for human GWAS; however, effective model organism data integration methods as well as reliable cross organism transfer learning frameworks are lagging behind. INFIMA provides a general framework for fine-mapping model organism molecular quantitative loci by integrating multiple functional data modalities. The availability of such fine-mapping results enables their transfer to the human genome to identify putative effector genes of GWAS variants. The current implementation of INFIMA excludes trans-eQTLs. As the ability to measure inter-chromosomal interactions matures, incorporating trans-eQTLs into INFIMA framework would be a natural extension. The INFIMA software is released at GitHub under the MIT license , https:/g for 1 min. Three hundred microliters of ATAC Lysis buffer  was used to resuspend the islets. The islets were incubated for 20 minutes on ice. After incubating, the islets were lysed by trituration with a 25-gauge needle until intact islets were no longer visible, usually 6 triturations. The lysate was centrifuged at 500 ×g for 10 min at 4C. This generated a crude nuclei pellet and a supernatant. The supernatant was discarded and the nuclei pellet was washed with 100 µL of ATAC Lysis buffer in order to reduce cytoplasmic and mitochondrial contamination. This mixture was centrifuged at 500 ×g for 10 min at 4C and the supernatant was removed. Per ATAC-seq sample, a mixture of 25 µL 2x TDE buffer, 22.5 µL nuclease-free water, and 2.5 µL TDE1 transposase enzyme  was applied and incubated for 30 min in a 37 ∘C water bath. The samples were then purified using a MinElute Reaction cleanup kit (Qiagen) and eluted using two sequential aliquots of 10 µL EB buffer. After purification all ATAC samples were kept at −80 ∘C. All ATAC-seq samples were transposed and frozen prior to preparing all libraries. Libraries were amplified using 20 µL of ATAC sample, 2.5 µL Primer-1 , 2.5 µL Primer-2 , and 25 µL of NEBNext High Fidelity 2x PCR Master Mix. Each ATAC sample was amplified by 12 cycles which was determined by qPCR to be saturating for the libraries. The PCR thermocycler was set to 72 ∘C for 5 min, 98 ∘C for 30 s, and then 12 total cycles of 98 ∘C for 10 s, 63 ∘C for 30 s, 72 ∘C for 1 min. After amplification the libraries were purified using MinElute PCR purification cleanup kit (Qiagen). The libraries were sequenced to a depth of 134.8 ± 8.2 million reads using paired-end 125 bp reads on a HiSeq2500 (Illumina) at the University of Wisconsin Biotechnology Center DNA Sequencing Facility.The ATAC-seq samples were prepared using a selection of 50 average sized mouse islets. The islets were washed with 500 µL of PBS at 4C and pelleted by centrifugation at 100 ×Illumina Nextera adapters were trimmed with cutadapt (version 2.0)  using thp-value” outputs from IDR analysis as measures of peak-level signal to noise. The “SignalValue” output was normalized across strains by multiplying 108/(# of reads) to adjust for differences in the sample sequencing depths. IDR identified peaks from the pooled peak sets were trimmed to exclude peaks with the lowest 10% “SignalValue” for each strain and then merged to form the master peak list across all strains. “SignalValue” and “ − log10” columns were aggregated as “MeanSignal” and “MeanP” in the master peak list.Peaks from individual and pooled samples across sexes of each strain were identified using MOSAiCS , 28 at FStrain-specific ATAC-seq peaks tended to have lower ATAC-seq signals compared to peaks present in multiple strains  the binding site, (2) 25 bp flanking regions of the binding site, and (3) 26–50 bp flanking regions of the binding site. We adapted the footprint depth (FPD) metric  and subtracted the allelic expression effect of the reference strain B6. We then trinarized the entries with values > 0.2, <− 0.2 to 1, − 1 respectively, and set other entries to 0 to obtain Eg, if the effect size from the marginal regression of gene expression on the genotype was significant at level 0.05, we replaced the effect size with 1 or - 1 depending on the sign of the effect size; otherwise, the effect size was replaced by 0. Therefore, we obtained For the DO-eQTL data W equal to 1 Mb and defined the distance score function as D(x)=0.5 if x≤0.25 Mb; x>0.25 Mb, where x is the distance between local-ATAC-MV and DO gene promoter. As a component of the prior Πg, the maximum value of distance score Dg is 0.5, which serves as a “tie-breaker” rather than overwhelming the other three components |+|cor|+1 according to the following three settings: NI:Πg,k is set to be 1 for all candidate SNPs, corresponding to an uninformative prior.The prior parameter MI:Πg,k set to its observed value in the actual data and accommodates multiple SNPs with Fg,k= 1. Multiple SNPs are affecting footprints under this setting. Causal SNPs are distinguished by other components of the prior parameter.The prior parameter HI:Fg,k is set to 10 for a randomly selected SNP k and 0 for other SNPs. Under this setting, the SNPs that affect footprints are more likely to be chosen as causal due to the dominant contribution of the footprint component.In order to simulate realistic data for our evaluations, we leveraged the parameters estimated by the INFIMA on the DO-eQTL data fit with all the summarized data from ATAC-seq, local-ATAC-MVs, and RNA-seq data. We used these parameter values as well as the actual summarized ATAC-seq, local-ATAC-MVs, and RNA-seq to simulate Vg was calculated at FDR of 0.05 by using a direct posterior probability approach [Statistical power for approach .The peak-based lift-over consisted of two steps: 1) direct and (2) indirect  by considering the local-ATAC-MVs with aggregated posterior probability of being causal larger than 0.80 and with a credible set less than 50% of the all the candidate SNPs. We leveraged 175,784 significant promoter capture Hi-C contacts from  for valiiPA v3.3 ) of the Additional file 1 Supplementary Information. Additional file 1 contains Supplementary Notes, Supplementary Figs.S1-S50 and Supplementary Tables S1-S6.Additional file 2 Review history


Aggregates of TAR DNA-binding protein (TDP-43) are a hallmark of several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Although TDP-43 aggregates are an undisputed pathological species at the end stage of these diseases, the molecular changes underlying the initiation of aggregation are not fully understood. The aim of this study was to investigate how phase separation affects self-aggregation and aggregation seeded by pre-formed aggregates of either the low-complexity domain (LCD) or its short aggregation-promoting regions (APRs). By systematically varying the physicochemical conditions, we observed that liquid–liquid phase separation (LLPS) promotes spontaneous aggregation. However, we noticed less efficient seeded aggregation in phase separating conditions. By analyzing a broad range of conditions using the Hofmeister series of buffers, we confirmed that stabilizing hydrophobic interactions prevail over destabilizing electrostatic forces. RNA affected the cooperativity between LLPS and aggregation in a “reentrant” fashion, having the strongest positive effect at intermediate concentrations. Altogether, we conclude that conditions which favor LLPS enhance the subsequent aggregation of the TDP-43 LCD with complex dependence, but also negatively affect seeding kinetics. Transactivation response element (TAR) DNA-binding protein 43 (TDP-43) is a DNA/RNA-binding protein implicated in transcriptional and post-transcriptional regulation of gene expression. Under normal conditions, the protein is primarily located in the nucleus, while it also shuttles between the nucleus and the cytoplasm. TDP-43 regulates RNA metabolism, RNA transport and RNA splicing ,2. The lRecently, many ALS-related, aggregation-prone proteins have been shown to undergo liquid–liquid phase separation (LLPS), a process where biomolecules de-mix from a homogeneous solution and form liquid-like, high-density droplets within the low-density protein solution, potentially leading to the formation of aggregates as summarized in the following reviews ,12. LLPSTDP-43 contains two tandem RNA-binding domains (RRMs) and a low-complexity domain (LCD) at the C-terminal end (TDP-43 LCD). LCDs are characterized by a low diversity of amino acids biased towards polar and charged ones, although the LCD of TDP-43 is not rich in charged residues . The TDPRecent results showed that the formation of liquid droplets by LLPS is often followed by their “maturation”, when they undergo a gradual transition to a less dynamic, gel-like state followed by solidification, such as aggregation ,28,29. FWhereas the connection between LLPS and aggregation, and the effect of RNA thereof, have been assessed under some conditions , and the two processes are apparently mechanistically connected, their exact mechanistic link is difficult to dissect, because they rely on different elementary steps and ionic strength, for example, may affect them differently. As the interplay between LLPS and seeding aggregation of TDP-43 LCD had not been studied in detail, our aim was the detailed characterization of this relationship via a broad buffer screen to find a range of distinct conditions and thus uncouple LLPS from aggregation. To investigate seeding, we chose peptides corresponding to aggregation-prone regions (APRs) of LCD and applied them under LLPS promoting (LLPS+) and inhibiting (LLPS−) conditions. We observed a complex effect of phase separation on both the maturation of these dynamic condensates and their seeded “prion-like” aggregation. These results highlight a previously unrecognized role of LLPS in aggregation by affecting its aggregation kinetics. http://n2t.net/addgene:98669; RRID:Addgene_98669) [Escherichia coli BL21 STAR were transformed with the TDP-43 containing plasmid, and grown overnight. Cells were lysed by sonication on ice in 500 mM NaCl, 2 mM Tris HCl, 1 mM 1,4-dithiothreitol (DTT), protease inhibitors  , 0.5 mM benzamidine hydrochloride  and 0.1 mM of phenylmethanesulfonyl fluoride (PMSF) solution (Sigma-Aldrich). The pellet was resolubilized in a denaturing buffer: 20 mM Tris HCl, 8 M urea, 500 mM NaCl, 10 mM imidazole, 1 mM DTT, pH 8.0 and sonicated in ice for 30 min. The solution was centrifuged at 20,000× g for 1 h at 4 °C. The supernatant was filtered through a filter of 0.45 μm and loaded onto a nickel-charged immobilized metal chelate affinity chromatography (IMAC) column HistrapTM HP , Buckinghamshire, UK), to which the 6x HIS tag of TDP-43 LCD binds. Bacterial debris was washed away with a denaturing buffer , after which the bound TDP-43 LCD was eluted with a linear 0 mM to 500 mM imidazole  gradient. The human TDP-43 DNA sequence (UniProtKB-Q13148 (TADBP_HUMAN)) was codon-optimized for bacterial expression, preceded by a 6X histidine tag, and located in a prokaryotic expression vector plasmid (CTD TDP-43) with a kanamycin resistance gene as a selection marker, preceded by lac operon, and was a kind gift from Prof. N. Fawzi   . Escheri® 20, 3000 MWCO PES filter, Sartorius, Göttingen, Germany) and buffer-exchanged into MES buffer 20 mM, pH 5.5 to keep the protein soluble, without undergoing LLPS. Proteins were filtered through a 0.22 μm filter. Protein concentration was determined by Qubit  and absorption at 280 nm. The effectiveness of each purification step was determined by gel electrophoresis with Page Blue staining  to visualize the protein band. Pure protein samples were flash frozen and kept at −80 °C at a concentration of 0.7 mg/mL.The protein fractions were combined, and the protein was buffer-exchanged into 2-(N-morpholino) ethanesulfonic acid (MES) buffer 20 mM, 3 M urea, pH 7.0. The 6xHis-tag domain was cleaved by incubating the samples with the tobacco etch virus protease  (1:10 protein/TEV molar ratio). All samples were incubated overnight at 34 °C without shaking. The TDP-43 LCD was separated from TEV using a nickel-charged IMAC column  with 20 mM tris HCl, 8 M urea, 500 mM NaCl, 10 mM imidazole, 1 mM DTT, pH 8.0. TEV was eluted with a linear 0 mM to 500 mM imidazole gradient. With gel filtration using 8 M urea, 20 mM MES, pH 7.0 buffer, salts and other impurities were removed through the resin beads. After gel filtration, the protein was concentrated (VivaspinPeptides were generated by Genescript (The Netherlands) to total concentrations of 2–3 mg/mL per peptide. Hexafluoro isopropanol (HFIB) (Sigma-Aldrich) was added to lyophilized peptide so that samples could be maintained in coated film only to be redissolved when needed. Where specified, peptides were sonicated  with 1-min cycles (15 pulses at 10% power with 30 s pauses), until completing a 1 min sonication time.To test aggregation of TDP-43 LCD, we used the amyloid-detection dye Thioflavin T (ThT)  in a Corning Black 96-well plate . ThT is a fluorescent probe which is widely used to detect amyloid fibril formation. Upon binding to amyloid fibrils, a fluorescence signal is observed, which is indicative of cross-β-sheet formation which is due to the loss of internal aromatic ring rotation upon binding. However, ThT binding is not restricted to fibrillar cross-β-sheets, it also binds to some extent to oligomers and hydrophobic pockets via π–π interactions of aromatic residues, with low intensity . ThT howTurbidity was measured using Polar Star Optima by BMG laboratories  at an absorbance of 600 nm. The assay was conducted at 37 degrees Celsius.Total yeast RNA  was prepared in 2-(N-morpholino)ethanesulfonic acid (MES) buffer  or phosphate buffer . For fluorescence microscopy, we spiked the phase-separating mixture with TDP-43 labeled with Alexa Fluor™ 488 at 1:100 ratio. We incubated the samples for 5 min (unless otherwise stated) in incubation chambers  sealed with 1.5# coverslips coated with PEG silane. Samples were visualized by fluorescence microscopy  using a 488 laser and Apo 60X objective. Images were taken in approximately the middle of the incubation chamber to avoid wetting effects on the coverslips.For samples analyzed in air, aliquots of TDP-43 LCD time points (100 µL) were placed on a clean, freshly cleaved grade V-1 mica . After 10 min of adsorption, the solvent was removed by filter paper successively in order to remove salts. Samples were dried overnight. AFM images were acquired in PeakForce QNM mode on MultiMode 8  with ScanAsyst-air tips. AFM images were analyzed using the NanoScope Analysis 1.9. For samples analyzed in liquid, 100 µL of TDP-43 LCD in phosphate buffer and/or MES buffer were placed on clean, freshly cleaved mica sheets . After different indicated time points of adsorption, AFM images were acquired in PFQNM mode on multimode VIII (Bruker-Santa Barbara) with ScanAsyst-air tips. AFM images were analyzed using the NanoScope Analysis 1.9.Fluorescence recovery after photobleaching (FRAP) assays were performed in a black, low binding 96-well plate with clear bottom using the Leica dfc7000 microscope with Leica WF FRAP system. Protein was labeled with DyLight 488 ester . LLPS was induced, and the sample was incubated for 30 min. Stationary droplets were bleached , and observed every second. To study single bleached droplets, the fluorescence of a single droplet was quantified with ImageJ. To study droplet recovery, data were normalized using following formula:Recovery half-time values and plateau values were obtained by fitting the data with Prism one-phase association.n = 3). Data were acquired, and Wyatt Dynamics software 7.8.1.3 was used to calculate the average radius. Dynamic light scattering (DLS), also known as photon correlation spectroscopy or quasi-elastic light scattering, measures the fluctuation of intensity of scattered light with time. Using a DynaPro DLS plate reader III instrument  equipped with an 830 nm laser source, we determined the hydrodynamic radius of the particles. One hundred microliters of each sample (containing 20 µM of TDP-43 LCD) was placed into a flat-bottom 96-well microclear plate . Ten image acquisitions of particles were taken at approximately 150x of data per replicate  and incubated for 5 min. Excess protein was removed using filter paper from the edge of the grid. The grids were washed 3 times with filtered distilled water, and blotted dry on filter paper. Next, negative staining was conducted with 2% filtered uranyl acetate for 2 min. Excess dye was blotted on filter paper, washing was conducted again 3 times, and samples were left to air dry for 10 min. Micrographs were obtained using a 120 kV JEM-1400 transmission electron microscope  at accelerating voltage 80 keV.® R-250  shaker at 4 °C overnight. Next, the gel was washed with deionized water before being de-stained with de-staining solution (containing 10% ethanol and 7.5% acetic acid).For Western blotting, samples were boiled at 95 °C for 10 min. Each sample was loaded onto 10-slot 4–20% precast polyacrylamide gels . The gels were run at 100 V for approximately 1 h. Next, the gels were stained with CoomassieWestern Blots were imaged with the ImageQuant LAS 4000 Biomolecular Imager . Protein bands were quantified with ImageQuant™ TL version 7.0 software .g, and smaller species such as oligomers or shorter fibril fragments sediment more slowly under high-speed centrifugation 20,000× g. Samples at end-point (15 h) were placed in a new Eppendorf and spun at 20,000× g for 1 h at 4 °C to ensure that both fibrils and oligomers sedimented. The average soluble concentration of 3 readings was then read on Thermo Scientific Nanodrop2000 using the molar extinction of TDP-43 LCD.While complex mixtures of aggregated protein can be imaged by a variety of imaging and plate reader techniques, the separation of insoluble species can be challenging. To gain insight into how much seeded assembly contributes in comparison to spontaneous assembly, we used sedimentation techniques. Insoluble fibrils and larger aggregates pellet quickly around 14,000–16,000× LCDs of multiple proteins have been shown to undergo spontaneous LLPS in the test tube, and this process is affected by conditions such as pH, salt concentration, molecular crowding and temperature ,36. We cOur first results suggest that aggregation of TDP-43 LCD is promoted by LLPS, as it appears more prevalent in LLPS+ buffer (phosphate buffer) in line with what was shown before ,30. NextTo this end, we assessed the relationship between phase separation and aggregation in 18 different buffers of different pHs, anionic components and ionic strengths , by recoFor the underlying atomic interactions, phase separation of TDP-43 LCD has been suggested to have both hydrophobic  and electrostatic  interactions . The smaAs previously described, aggregated TDP-43 can seed its own aggregation . We alsog, and smaller species such as oligomers or shorter fibril fragments sediment more slowly even under high-speed centrifugation. The samples containing protein and seed at end-point (15 h) were centrifuged and the soluble fractions compared. The soluble fraction of the LLPS- condition is 13% less soluble than the LLPS+ buffer condition confirming that indeed there is more aggregated TDP-43 LCD in the non-LLPS condition, which corresponds to the higher ThT signal  conditions, i.e., while it aggregated faster when TDP-43 LCD is in the form of droplets, it aggregates to a higher extent without droplets being present. These results show that specific nucleating region(s) exist in TDP-43 LCD, which are within peptide 6 that roughly covers peptides 1 and 3. When comparing peptides, it should be noted that peptide 6 is of the longest length. We can compare this to Recent work implies an intricate interplay between RNA and proteins in the LLPS of RNA-binding proteins ,46. As aAs we have observed that RNA has a strong effect on the LLPS of TDP-43 LCD, and LLPS provides a mechanism of aggregation, we tested whether RNA exerts an effect on the aggregation of TDP-43 LCD induced through LLPS. First, we observed that the addition of total yeast RNA to TDP-43 LCD results in higher turbidity (OD600), compatible with the formation of more  droplets already observed before A. In agrIn this study, we observed that the formation of TDP-43 LCD droplets by LLPS enhances the formation of aggregates. This was demonstrated using three different approaches: by altering the buffer conditions, by adding seeds and by combining the protein with RNA.Placed in favorable conditions, droplets formed by LLPS of a broad range of proteins are initially highly dynamic, liquid-like , but almOne of the most intriguing proteins to study for the link between LLPS, aggregation and disease is TDP-43. This protein is found in aggregates in ALS and FTLD and readily undergoes LLPS, which very often appears to be linked to aggregation . InteresWe used a buffer screen to test the propensity of TDP-43 LCD in multiple conditions to dissect the relationship between LLPS and aggregation. We observed that every condition favoring LLPS also significantly promoted aggregation. In general, this can be interpreted in terms of two types of mechanism of aggregate formation, either by “homogeneous” (within droplets) or “heterogeneous” (on the surface of droplets) nucleation of droplets.328AALQSS333, 321AMMAAA326 and 333SWGMMGMLASQ343 (the latter (peptide 4) with the addition of one glutamine, is almost identical). 321AMMAAA326 and 333SWGMMGMLASQ343 belong to the pathogenic/mutational aggregation core. Potential mutation sites as shown in 312NFGAFSI317. While the Eisenberg group showed using EM that LARKS can form microcrystals, we observed fibers using EM. Mutations in this region are said to disrupt phase separation but not aggregation [By conducting seeding experiments using synthetic peptides and the LCD of TDP-43 itself, we obtained mechanistic details on the role of TDP-43 LCD seeding in the context of LLPS, which, to our knowledge, has not been previously addressed. While all the peptides seeded aggregation in an LLPS setting, peptide seeding  was more dramatic in non-LLPS conditions. This implies that not only do these regions probably function as APRs and promote the amyloidogenic behavior of this protein, but they depend on the protein buffer situation. TDP-43 seeding was investigated previously both in cells and in vitro, as demonstrated by the plethora of evidence comprising: human to animal propagation, human to cell propagation, cell to cell propagation, cell seeding with recombinant protein, in vitro seeding with recombinant protein, fibril formation ,51,52,53regation . We obseWe interpret these results in the context of particular structural features of the peptides assessed by the Zipper DB algorithm ,56. UsinIn a disease-related context, it is not yet clear whether RNA assemblies, such as SGs, besides their regular function, are indeed harmful intermediates of aggregates. This conjecture was favored because of the link of neuronal vulnerability to stress, which is implicated in aging. However, stress granules are not ALS-specific, and the absence of many SG proteins in inclusions has questioned this hypothesis. Many studies have been conducted to establish the number and composition of proteins in SGs, although their results heavily depended on the methods used to promote and isolate SGs ,59,60,61To further address this issue, we studied the effects of RNA on the TDP-43 LCD phase separation and aggregation ,63. RNA Furthermore, it should also be noted that TDP-43 LCD contains an arginine- glycine, -glycine (RGG) motif previously found to interact with RNA , and usiThe regions which seed aggregation used in this study could be used to generate aggregation-based models in early embryonic cells, such as induced pluripotent stem cell (iPSC)-derived motor neurons, to generate physiologically relevant models of pathology, as these models lack end-stage phenotypes attributed to their premature nature. Furthermore, peptide-specific aggregation blockers could be potentially generated as methods of therapy not just to influence aggregation but LLPS as well. However, as demonstrated in our paper, the method with which seeding for an LLPS prone protein is studied is critical. The results obtained in this study contribute to our understanding of how LLPS can accelerate aggregation and why the LCD of TDP-43 is so sensitive to environmental changes. Based on our findings, we conclude that droplets formed by LLPS behave as aggregators for TDP-43, and RNA can further enhance this effect. We conclude that the level of aggregate seeding is weakened when droplets are present, despite droplets allowing seeding to occur faster.


Streptococcus sanguinis, Actinomyces naeslundii, Porphyromonas gingivalis, and Fusobacterium nucleatum was grown for 14 days on various titanium discs: machined, sandblasted, sandblasted and acid-etched (SLA), ZnCuO-coated, and hydroxyapatite discs. Bacterial species were quantified with qPCR, and their viability was examined via confocal microscopy. Osteoblast-like and macrophage-like cells grown on the various discs for 48 h were examined for proliferation using an XTT assay, and for activity using ALP and TNF-α assays. The CSLM revealed more dead bacteria in biofilms grown on titanium than on hydroxyapatite, and less on sandblasted than on machined and ZnCuO-coated surfaces, with the latter showing a significant decrease in all four biofilm species. The osteoblast-like cells showed increased proliferation on all of the titanium surfaces, with higher activity on the ZnCuO-coated and sandblasted discs. The macrophage-like cells showed higher proliferation on the hydroxyapatite and sandblasted discs, and lower activity on the SLA and ZnCuO-coated discs. The ZnCuO-coated titanium has anti-biofilm characteristics with desired effects on host cells, thus representing a promising candidate in the complex battle against peri-implantitis.This study aims to investigate the effects of a novel ZnCuO nanoparticle coating for dental implants—versus those of conventional titanium surfaces—on bacteria and host cells. A multispecies biofilm composed of  The success of dental implants depends on the integration of the implant with the bone and mucosal connective tissue, as well as the absence of inflammation and infection in the surrounding tissues . AlthougStreptococcus spp. and Actinomyces spp. via interaction of the bacterial wall with the salivary film formed on the substrate ..28]. Bio2 and 37.5 °C). After 48 h, the cells were harvested for XTT analysis and quantified with a cell proliferation colorimetric assay . The ALP activity of the osteoblast-like cells was determined with a commercial kit , and the activity of macrophage-like cells was assessed by the level of TNF-α cytokine in an enzyme-linked immunosorbent assay (ELISA) kit , according to the manufacturer’s instructions.The osteoblast-like cell line Saos-2  and the macrophage-like RAW 264.7—a murine leukemic monocyte cell line—were grown in Dulbecco’s modified Eagle’s medium  supplemented with 15% fetal calf serum, 1% glutamine (2 mmol/L), and 1% penicillin/streptomycin (25 mg/mL), in 96-well culture plates. The osteoblast-like cells were harvested with trypsin–ethylenediaminetetraacetic acid (EDTA), while the macrophage-like cells were harvested by scraping, then seeded in the culture wells containing the discs at a density of 75,000 cells/200 mL/well, and cultured  . The morThe crystalline nature of the formed particles was studied by XRD . The anaP. gingivalis and F. nucleatum on all of the disc surfaces  on hydroxyapatite, sandblasted, SLA, and ZnCuO-coated surfaces was 1.1-, 1.23-, 1.31-, 1.4-fold higher than that on the machined control surface, respectively a. ALP acMacrophage-like cell proliferation (XTT assay) was significantly higher on the sandblasted and hydroxyapatite surfaces than on the machined surface  a. TNF-α The findings of the present study show that ZnCuO-coated titanium surface discs may reduce biofilm accumulation, enhance osteoblast activity, and modulate the immune reaction. Our aim was to evaluate the potential of a novel ZnCuO nanoparticle coating of a titanium surface suggested for dental implants. Therefore, we designed a relatively simple and, thus, repeatable and controlled model composed of an in vitro four-species biofilm, early and later colonizers, and two representative host cells to closely simulate the multiple peri-implant conditions in the oral cavity.Dental implant osseointegration is a complex phenomenon dependent on several factors and characterized by a long sequence of events. Although it is well known that micro-rough or nano-rough implant surfaces are beneficial for accelerated osseointegration, their long-term success is debatable, as there is a higher risk of peri-implantitis due to a higher propensity for plaque accumulation . Our finF. nucleatum and P. gingivalis, the dominant bacteria inhabiting the biofilm being S. sanguis and A. naeslundii. Although the biofilm grown on the ZnCuO-coated surface showed a decrease in all bacterial species, the sandblasted surface displayed an increased number of P. gingivalis and S. sanguis and a decrease in F. nucleatum in the biofilm compared with that of the machined surface.The bacterial composition of our biofilm model grown for 14 days on different surfaces showed relatively low amounts of the periopathogens The ZnCuO-coated surface exhibited a non-specific anti-biofilm effect, which was similar to the results obtained by other studies that used different coating strategies ,19. The Our findings showed that the osteoblast-like cells cultured on the ZnCuO-coated surfaces as well as on the other modified titanium surfaces tended to exhibit greater proliferation and higher ALP activity when on ZnCuO-coated and sandblasted surfaces, compared with the machined surface. Indeed, previous studies reported that the ALP activity of osteoblast-like cells was higher on rough surfaces ,35. AlthA similar proliferative effect of the sandblasted rough surface was observed on the macrophage-like cells, consistent with previous studies showing that these cells preferentially grow well on rough surfaces ,41. MacrIn conclusion, it appears that the new ZnCuO nanoparticle coating of the titanium surface suggested for dental implants has anti-biofilm characteristics, with the desired effects on host cell activity and proliferation. The ZnCuO nanoparticle coating of titanium appears to be a promising surface modification for dental implant applications in the complex battle against peri-implantitis. Nevertheless, the release of metal ions—especially Cu ions—might cause long-term cytotoxicity or side effects . Thus, f


Tattooing is an increasing trend among Western countries, with about 18% of the population undergoing the procedure once in their lifetime. The process looks simple; introduce exogenous pigment into the dermis layer of the skin, altering the skin color permanently. However, this simple procedure leads to several health issues and medical complications, both acute and chronic, and some are difficult to cure. Sarcoidosis is high on the list of severity involving almost all body organs. Multiple organ involvement makes this condition more difficult to treat. Lungs and lymphatics are the leading sites of involvement, followed by an inflammatory disease of the eye called uveitis. An additional problem is the limited confirmatory diagnostic tests and treatment options for sarcoidosis. Each patient must be considered unique based on their age, clinical presentation, and severity of involvement. Proper treatment must be tailored for better outcomes with minimum side effects and rapid cure. Here we describe two case reports of tattoo-associated sarcoidosis with severe uveitis successfully treated with mycophenolate mofetil. Sarcoidosis is a multisystem autoimmune granulomatous disease that presents with systemic noncaseating granulomas made up of clusters of different immune cells, including macrophages, mononuclear cells, CD4+ T cells, a few CD8+, and epithelioid cells. Sarcoidosis can affect any body organ, including the skin and eyes -4The etiology of sarcoidosis is still unknown despite extensive research and effort. Its occurrence differs in different ethnic groups and is 10 times more common in African Americans with a more severe presentation. The predominance in the female population is also slightly higher. African Americans are among the highest in the list with 17-43 annual cases per 100,000 people and a 2:1 female prevalence ratio, Northern Europeans with 11 to 24 cases, Hispanics and Asians with the lowest numbers of one to three per 100,000 people. The peak age group of sarcoidosis is usually between 20 and 39 years of age -7.Tattoo-associated sarcoidosis is rare, and the exact prevalence and incidence of tattoo-associated sarcoidosis is unknown. The term “tattoo sarcoidosis” emerged in the early 1940s, when patients with black tattoos presented with nodules on their tattoos. Further studies reported the increasing inflammatory trend on almost all body sites, including ocular sarcoidosis and uveitis, due to increasing tattoo and permanent makeup trends. Permanent makeup, in addition to tattooing, makes the situation more complex .Approximately one-fourth of patients with sarcoidosis present with ocular involvement, of which 3%-10% exhibit sarcoid/tattoo uveitis with or without systemic sarcoidosis and show bilateral hilar adenopathy or pulmonary reticular opacities .Here, we present two cases of tattoo-associated sarcoidosis with pulmonary involvement, uveitis, and cutaneous nodules. Both cases were successfully treated with prednisone and mycophenolate mofetil (MMF).Case 1A 26-year-old Guyanese male who worked at a warehouse presented with worsening left red-eye and blurry vision for two weeks. The ophthalmologist started on prednisolone eye drop, which initially controlled his eye symptoms. However, he noticed new, small skin papules on the left forearm of the tattoo that he had received five years ago. He had no significant medical history or family history of sarcoidosis. On physical examination, he was found to have a bilateral conjunctival injection, a tattoo with small bumps on the left forearm and upper arm, and no erythema or discharge was noted around the tattoo.Results of laboratory studies, including serum calcium, were normal; additionally, the erythrocyte sedimentation rate (ESR) was 6 mm/h, angiotensin-converting enzyme (ACE) level was 55 U/L (ref 14-82 U/L), and C-reactive protein (CRP) level was 2 mg/L .Chest X-ray was normal, and chest CT revealed bilateral axillary lymphadenopathy with the largest node noted in the left axillary region measuring 2.1 x 1.2 cm and a 2 mm nodule noted in the right middle lobe. Skin punch biopsy showed the tattoo having sarcoid granuloma . Chest CT showed a 2 mm pulmonary nodule in the superior segment of the left lower lobe and a 1 mm nodule in the left upper lobe, which did not show axillary or mediastinal lymphadenopathy. Skin punch biopsy demonstrated a tattoo with non-necrotizing epithelioid granulomas , and methotrexate is top of the list of clinician prescriptions due to the availability of extensive research, clinical trial data, and authentic literature availability. Other less prescribed DMARDs are azathioprine, leflunomide, mycophenolate, and chloroquine/hydroxychloroquine. Other biological agents are considered as third-line therapeutic options due to higher costs and limited FDA regulations. Anecdotal reports describe the efficacy of MMF in sarcoidosis treatment, recommending the continuous monitoring of CBC and renal function tests every three months .Local treatment of corticosteroid (prednisone) gives positive results in uveitis by suppressing inflammation, pain, and ocular scarring. The therapeutic option chosen for our cases was MMF, which is a successful cytotoxic drug in the case of uveitis. Both of our patients were successfully treated with MMF, labeled as second-line therapy for sarcoidosis. MMF was reportedly well-tolerated in the majority of sarcoidosis patients and helpful in reducing the prednisone dose. The main reason for its limited use is the lack of randomized control trials and evaluation outcomes in a large patient group ,15.Sarcoidosis is a systemic disease that can affect multiple organ systems. Cutaneous inflammation is one of the most common presentations. Providers should be aware of the skin involvement of sarcoidosis, as early recognition and intervention can decrease the risk of complications. The etiology of sarcoidosis is still unknown, and sarcoidal granulomatous reactions due to tattoo pigment have been reported. Systemic sarcoidosis associated with tattoos is rare and cutaneous lesions associated with tattooing are increasingly reported. Therefore, early recognition of skin involvement and intervention can help decrease the risk of complications. With the increased popularity and demand for tattooing, there is a significant need for research related to its complications and treatment. Mycophenolate mofetil is a safe option for treatment, although extensive research and investigation are needed.