Albendazole (ABZ) is a weakly basic drug that undergoes extensive presystemic metabolism after oral administration and converts to its active form albendazole sulfoxide (ABZ_SO). The absorption of albendazole is limited by poor aqueous solubility, and dissolution is the rate-limiting step in the overall exposure of ABZ_SO. In this study, PBPK modeling was used to identify formulation-specific parameters that impact the oral bioavailability of ABZ_SO. In vitro experiments were carried out to determine pH solubility, precipitation kinetics, particle size distribution, and biorelevant solubility. A transfer experiment was conducted to determine the precipitation kinetics. A PBPK model for ABZ and ABZ_SO was developed using the Simcyp™ Simulator based on parameter estimates from in vitro experiments. Sensitivity analyses were performed to assess the impact of physiological parameters and formulation-related parameters on the systemic exposure of ABZ_SO. Model simulations predicted that increased gastric pH significantly reduced ABZ absorption and, subsequently, ABZ_SO systemic exposure. Reducing the particle size below 50 µm did not improve the bioavailability of ABZ. Modeling results illustrated that systemic exposure of ABZ_SO was enhanced by increasing solubility or supersaturation and decreasing the drug precipitation of ABZ at the intestinal pH level. These results were used to identify potential formulation strategies to enhance the oral bioavailability of ABZ_SO. Physiologically-based pharmacokinetic (PBPK) modeling has been widely adopted as an experimental approach for predicting the pharmacokinetic behavior of a drug in humans by using preclinical data. Applications of PBPK modeling include in vitro–in vivo correlation (IVIVC) ,2,3,4, dBioavailability enhancement of low soluble drugs requires the development of supersaturated formulation strategies, such as nanoparticle amorphous solid dispersions (ASDs), lipid formulations, and cyclodextrin complexes ,15,16,17Characterization of ASDs, with respect to supersaturation and precipitation kinetics, and incorporating these parameters into PBPK absorption models allow for more realistic and reliable predictions of the in vivo behaviors of these formulations. Precipitation kinetics is a critical determinant for absorption of a weak base low soluble drug, and researchers have relied on in vitro transfer model experiments to mimic the in vivo transit of drugs from the stomach into the intestine, where precipitation is inevitable, thereby, limiting the amount of the drug that is available for systemic absorption. This in vitro experimental approach has proved to be an important screening tool for oral formulations of weak base drugs and it has also been used to inform PBPK model development ,18,19,20In addition to its demonstrated utility in predicting the in vivo performance for oral formulations of low soluble compounds, several researchers have used PBPK modeling to guide formulation development and optimization. For example, Kuentz demonstrated the utilization of drug absorption modeling as a rationale for the formulation selection of several investigational compounds by performing parameter sensitivity analysis to evaluate the impact of dose and particle size on pharmacokinetic parameters . LikewisAlbendazole (ABZ), a small molecule benzimidazole derivative, was first marketed in 1982 as an anthelmintic drug for clinical use. It is on the World Health Organization’s list of essential medicines . After oThe current research explored the capability of PBPK absorption modeling to guide the formulation development of albendazole. To the best of our knowledge, there is no research available where the capabilities of PBPK absorption modeling have been explored with albendazole. Based on its weak basic nature, in vitro transfer model experiments were utilized to determine the precipitation kinetic parameters upon the transfer of the drug from the stomach to the small intestine. These parameters, along with data generated from solubility studies, were used to inform PBPK modeling. This study aimed to investigate and understand the important physiologic and formulation-related parameters affecting ABZ oral absorption and the systemic exposure of its active metabolite, ABZ_SO, using the Simcyp™ Simulator. The results enabled the identification of specific potential formulation techniques, which can enhance ABZ_SO bioavailability and maximize efficacy by using an approach that can reduce the resources and time spent in various phases of drug development.2HPO4, Lot No. BJ2212), and sodium dihydrogen phosphate  were purchased from Spectrum Chemical . A marketed immediate-release tablet formulation of albendazole  was purchased from Mankind Pharmaceuticals . Biorelevant media powder (Fasted State Simulated Gastric Fluid (FaSSGF)/Fasted State Simulated Intestinal Fluid (FaSSIF)/Fed State Simulated Intestinal Fluid (FeSSIF), Lot. No. FFF-0519-B) was purchased from Biorelevant Company . Methanol and concentrated hydrochloric acid (36.5–38.0% w/w) were purchased from Sigma-Aldrich . All chemicals and reagents used in this research were of analytical grade.Albendazole (ABZ) USP (Lot No. ASA000124A), sodium hydrogen phosphate (NaThe equilibrium solubility of ABZ was measured by the shake flask method at 37 ± 2 °C in different pH solution systems: 0.1 N HCl (pH 1.20 ± 0.01 and pH 2.0 ± 0.01), 0.02 M acetate buffer (pH 4.5 ± 0.02), and 0.05 M phosphate buffer (pH 6.8 ± 0.01). Solubility was also determined in FaSSGF (pH 1.6 ± 0.01), FaSSIF (pH 6.5 ± 0.01), and FeSSIF (pH 5.0 ± 0.01) containing bile concentrations of 0.08 mM, 3 mM, and 15 mM, respectively . For eac10, d50, and d90, representing the volume percentage or portion of particles below the 10th, 50th, and 90th percentiles, respectively. PSD was characterized as a Weibull distribution function, and the data were analyzed to estimate the Weibull parameter values and mean radiuses, which served as the input parameters in the model development [Particle size distribution (PSD) data for the marketed formulation are not publicly available. Hence, as a reference, pure ABZ was used for these experiments. The particle size distribution of ABZ was measured by laser diffraction using a Mastersizer 3000 equipped with Aero S (dry dispersion feeder unit). The instrument software was used to calculate delopment .These experiments were carried out in order to characterize the precipitation kinetics of ABZ in a system designed to mimic the physiologic conditions in the GI tract as the drug moves from the stomach to the small intestine, where precipitation is inevitable and limits the availability of the soluble drug for absorption. To simulate the impact on solubility when the drug transits from the stomach into the intestine, the experimental system  consisteThe gastric compartment was sampled at 5, 15, and 30 min, at which time the transfer to the intestinal compartment was initiated. Then, samples were collected from the intestinal compartment for an additional 90 min . Samples were filtered through 0.45 µm PTFE filters and immediately diluted with the mobile phase before quantitative analysis by HPLC. All dissolution experiments were performed in triplicate.® analytical software on an Agilent 1100 system , equipped with a Zorbax SB-CN  column . The temperature of the column was set at 25 °C and the samples were analyzed at 308 nm. The mobile phase consisted of acidified methanol: phosphate buffer (60:40% v/v). Acidified methanol comprised methanol and HCl (99:1), while the phosphate buffer consisted of 1.375% w/v monobasic sodium phosphate dissolved in deionized water. The injection volume was 20 µL, and the mobile phase flow rate was 1.5 mL/min. The detection wavelength was 308 nm. The method was validated according to USP guidelines. The assay was linear over a concentration range from 1 to 200 μg/mL The limits of detection and quantification were 0.24 μg/mL and 0.81 μg/mL.Quantitative analysis of ABZ was performed by reverse-phase gradient high-performance liquid chromatography, in accordance with the method described in the United States Pharmacopoeia (USP) monograph for ABZ, with slight modification . AnalysiPBPK modeling and simulation were performed using the Simcyp™ Simulator . The models for ABZ and its active metabolite ABZ_SO were built using the “middle-out” approach . A summa2 cell permeability was used to predict the in vivo absorption parameters fraction absorbed (fa) and the first-order absorption rate constant (ka) [TM was used and the parameter estimates for Vmax and Km CYP3A4- and flavin-containing monooxygenase 3 (FMO3)-mediated enzymatic pathways were obtained from the literature [TM Simulator captures the intestinal metabolism based on the default abundance of both intestinal enzymes.ABZ absorption was described using the Advanced Dissolution, Absorption, and Metabolism (ADAM) model. A literature-derived value for CaCOant (ka) . The meaterature . The FMOterature . There iFor the active metabolite, ABZ_SO, a minimal PBPK model was utilized. ABZ_SO absorption was modeled using ADAM. Permeability of the converted ABZ_SO was predicted using the mechanistic passive regional permeability predictor in Simcyp™, which calculates the drug permeability based on the log P of the drug. The distribution of ABZ_SO was modeled in Simcyp™ using the minimal PBPK model. Volume at steady state (Vss) and oral clearance were optimized using the Simcyp™ Parameter Estimation tool and data from a published clinical study.Clinically observed plasma profiles of ABZ and ABZ_SO were digitized from a published study using WebPlotDigitizer , and the systemic exposure parameters  were used to verify the model ,42. PBPKThe sensitivity analysis tool in Simcyp™ was used to assess the impact of physiologic (gastric pH and gastric MRT) and drug-specific  model parameters on ABZ systemic exposure. PBPK model sensitivity for each parameter was carried out over a wide range of values (10 steps within each range) and assessed the impact on Cmax and AUC of ABZ_SO. The results were plotted as response surface 3D graphs using Originpro . Among the parameters tested were gastric pH (pH range: 1.2 to 4.5), mean gastric residence time , particle size (range: 0.2 µm to 200 µm), and intrinsic solubility (0.012 mg/mL and 0.12 mg/mL). For the extreme solubility values  the effect of varying the supersaturation and precipitation rates on ABZ-SO systemic exposure was also studied. This sensitivity analysis included a critical supersaturation ratio  and a precipitation rate constant . Sensitivity analysis of particle size was performed by fixing the initial value to the mean of the Weibull distribution (7.52 µm) and upper and lower bound values of distribution were assigned as the range for sensitivity analysis.2 = 0.9771). The intrinsic solubility was estimated to be 0.012 mg/mL, and this parameter value was used in the PBPK model (2 = 0.7448). Based on this biorelevant solubility data, SIVA estimated values for log K m:w  and log K m:w (ion) as 2.92 and 5.42, respectively. These bile-micelle partition coefficients for neutral and ionized species are required for the estimation of ABZ total solubility [PK model . For biolubility The measured d10, d50, and d90 for ABZ were 0.95 ± 0.10 µm, 4.1 ± 0.64 µm, and 22 ± 1.3 µm, respectively. These values were utilized to define the alpha and beta parameters of the Weibull distribution function, which were, then, used as an input for the ADAM model in Simcyp™.The parameter for the critical supersaturation ratio (CSR), an indicator of the degree of supersaturation, is calculated as follows:The concentration of ABZ never exceeded the equilibrium solubility upon transfer to the intestinal compartment (FaSSIF), indicating immediate precipitation. Therefore, PBPK model parameter values for CSR and precipitation rate constant (PRC) were set to 1 and 1000 (1/h), respectively.The predictive performance of the PBPK model was demonstrated by comparing the simulated systemic exposure of ABZ and ABZ_SO to published clinical data . As demoA series of sensitivity analyses were carried out to evaluate the impact of various model parameters on the systemic exposure metrics  of the active metabolite, ABZ_SO. This approach can potentially help guide the development of an improved oral formulation for the compound by identifying the critical parameters impacting in vivo performance. The results of these analyses are described below.The sensitivity analysis showed that increasing gastric MRT was associated with higher systemic exposure, as demonstrated in −1  were associated with increases in both AUC (80%) and Cmax (40%)—based on the experimentally determined solubility profile. When the analysis was repeated with the ‘10X’ solubility profile, Cmax and AUC increased by 400% and 200%, respectively.Additionally, the impact of varying degrees of supersaturation over a range of precipitation rate constants on ABZ_SO Cmax and AUC is presented as response surface 3D graphs . This seF) on these processes can be described by the following equation:af represents the fraction of the administered dose absorbed into the intestinal cell, gf is the fraction of the dose reaching the intestinal cell (af) that escapes gut metabolism, and hf is the fraction of the dose absorbed into the bloodstream (af × gf) that escapes first-pass hepatic metabolism.The general goal of oral drug administration is to deliver a sufficient amount of drug to the systemic circulation, where the drug can then distribute to its site of action. For a drug to reach systemic circulation, it must first absorb across the intestinal lumen, and this process depends on numerous factors, including aqueous solubility and membrane permeability. Once in the intestinal cell, the drug is susceptible to intestinal metabolism mediated by CYP3A4 and other enzymes before entering the bloodstream. Finally, the fraction of the dose absorbed into the bloodstream must first pass through the liver and escape hepatic metabolism (termed the “first-pass effect”) before becoming systemically available. The dependence of bioavailability (af). Since ABZ is highly permeable, aqueous solubility is the critical determinant of ABZ absorption. Although increasing ABZ absorption would be expected to have a limited impact on ABZ bioavailability (due to extensive presystemic metabolism), it would be expected to increase the exposure to the active metabolite, ABZ_SO.ABZ is an anthelmintic drug used to treat systemic infections. The low absolute oral bioavailability (<5% in humans) of albendazole is due in part to extensive presystemic metabolism by the liver and small intestine , where Awww.pearrl.eu), and OrBiTo  are examples of the efforts undertaken by academia and the pharmaceutical industry to develop new biopharmaceutics tools, which better predict in vivo performance, and thereby, facilitate drug development [Solubility and dissolution testing using biorelevant media has become a critical tool for predicting the in vivo performance of oral dosage forms. This approach more reliably mimics gastric and intestinal fluid composition  . PEARL  after reaching the critical supersaturation concentration (CSC). Physiological factors , physiochemical properties , and formulation excipients can all influence the precipitation kinetics of a drug ,28,31,48Several studies reported decreased and varying absorption of weakly basic drugs under exposure to high gastric pH conditions leading to subtherapeutic exposure. This phenomenon has been attributed to the slow and incomplete dissolution of these drugs when stomach pH is high ,50,51. ESensitivity analysis of mean gastric residence time showed that ABZ_SO exposure (Cmax and AUC) increased with increasing MRT. This can be attributed to the increased dissolution of ABZ in the acidic environment resulting from the prolonged residence of the drug in the stomach. At an MRT of 2.0 h, the model predicted a slight decrease in Cmax (with no change in AUC) reflecting the impact of delayed absorption (increased Tmax).A gastro-retentive drug delivery system (gastric mucoadhesive tablet or multiunit particulate system (MUPS)) strategy is a potential option for weak basic compounds by allowing for extended residence on the stomach to promote solubilization of the drug before its transits to the intestine ,55,56,57N is the number of particles, S is the scalar factor, effD and effh are the effective diffusion constant and effective diffusion layer thickness, respectively, r is the radius of the particle, and (surface)S and b(bulk)C are the solubilities at the particle surface and in the bulk, respectively. Dissolution from the drug particles depends on the surface area of the particle, and the diffusion layer thickness, which is modeled by the Hintz–Johnson relationship [The particle size (PS) of the API is a critical quality attribute, and changes in this parameter may affect drug performance in vivo . This istionship . Absorpttionship ,64. DecrSensitivity analysis confirmed that increasing ABZ solubility would improve ABZ_SO systemic exposure. PBPK modeling further predicted that exposure metrics for ABZ_SO would be significantly improved with increased supersaturation and decreased precipitation of ABZ in the intestine, the primary site of drug absorption. Hence, increasing the solubility of ABZ in the intestinal fluids accompanied by increased supersaturation and reduced precipitation creates a larger concentration gradient driving absorption. Kourentas et al. observed significant supersaturation of ABZ in the duodenum using an HPMC-based ABZ suspension formulation . This suAlternatively, model simulations demonstrated that ABZ_SO systemic exposure could be improved by increasing the equilibrium solubility of ABZ. Accordingly, formulation strategies such as amorphous solid dispersions (ASDs) or lipid formulations may be appropriate to achieve this goal. ASDs incorporate surfactants/solubilizing agents and polymers that increase the drug solubility and prolong the supersaturation state ,66. ASDsFor a weak base such as ABZ, conventional solubility, and dissolution experiments will only provide a comparative understanding among prototype formulations. However, PBPK modeling simulations for ABZ demonstrate that in order to anticipate the in vivo performance of a formulation, it is extremely important to study the degree of supersaturation and precipitation rate because these factors determine both the amount of soluble drug and the time period that is it available for absorption in the intestine. Some of the major advantages of PBPK absorption modeling are that it captures the dynamic pH changes that occur throughout the GI tract and also considers the precipitation kinetics of the drug, which are critical in understanding the impact of formulation on the bioavailability of weak basic compounds. The approach used in this study builds upon previous investigations that combined in silico modeling with in vitro transfer experiments ,28,30.Overall, these analyses based on PBPK absorption modeling provided critical insight into which parameters to target when developing oral formulations for ABZ. Model predictions provide a foundation for future studies that can further validate the model by preparing prototype formulations of ABZ using various techniques  and characterizing their performance in terms of solubility and precipitation kinetics.Understanding the critical quality attributes affecting the in vivo performance of a potential drug candidate prior to clinical testing would be beneficial in designing an oral formulation that can maximize efficacy by improving the rate and extent of absorption. In this research, absorption modeling combined with in vitro experiments provided an increased understanding of the complex in vivo performance of a low soluble prodrug, ABZ. PBPK absorption modeling with the Simcyp™ Simulator identified two critical parameters that drive the systemic exposure of its active metabolite, ABZ_SO: (1) gastric pH and (2) drug precipitation in the basic environment of the upper small intestine. Model sensitivity analysis inferred that reducing particle sizes would not improve ABZ_SO bioavailability and this strategy should be eliminated from the formulation decision tree. Alternatively, formulations containing excipients that either maintain the microenvironmental pH around ABZ to mitigate the impact of gastric pH changes on dissolution or can delay ABZ precipitation upon its transit into the intestinal environment should be considered.


Studies that examined sex differences in first-episode patients consistently show that males compared to females have poor premorbid adjustment, earlier age of onset, worse clinical characteristics, and poorer outcomes. However, little is known about potential mediators that could explain these sex differences.n = 105; 77%) with a mean age of 22.1(s.d. = 4.1) years and mean education of 12.5(s.d. = 1.7) years. At entry, patients were within 2 years of their first psychotic episode onset. Baseline assessments were conducted for premorbid adjustment, symptoms, cognitive functioning, insight, and at 6-months for role and social functioning.Our sample consisted of 137 individuals with first episode schizophrenia , an earlier age of onset , more negative symptoms (p = 0.044), poorer insight (p = 0.031), and poorer baseline and 6-month role (p = 0.002) and social functioning (p = 0.034). Several of these variables in which males showed impairment were significant predictors of 6-month role and social functioning. Premorbid adjustment and insight mediated the relationship between sex and role and social functioning at 6-months, but not negative symptoms.Males as compared to females had poorer premorbid adjustment across several key developmental periods (Males compared to females were at lower levels across several key premorbid and clinical domains which are strongly associated with functional outcome supporting the hypothesis that males might have a more disabling form of schizophrenia. The relationship between sex with role and social functioning was mediated through premorbid adjustment and insight suggesting pathways for understanding why females might have a less disabling form of schizophrenia. The general conclusion from studies of multi-episode schizophrenia is that males compared to females appear to be at greater risk of developing schizophrenia and have a more severe form of the illness with regard to onset, symptoms, and psychosocial functional outcome  years, the mean education was 12.5 (1.7) years, and most patients were single . All participants received outpatient psychiatric treatment at the UCLA Aftercare Research Program and were participants in the fourth phase of the Developmental Processes in Schizophrenic Disorders Project  and 32 females (23%). The mean age at study entry was 22.0 (Entry criteria were: (1) an onset of psychotic illness within 2 years of study entry; (2) a DSM-IV diagnosis of schizophrenia, schizoaffective disorder-depressed type, or schizophreniform disorder; (3) 18 to 45 years of age; (4) no evidence of a known neurological disorder; (5) no evidence of significant and habitual drug abuse or alcoholism in the 6 months prior to study entry and that the psychosis was substance-induced; (6) premorbid IQ not less than 70; (7) sufficient fluency in English to avoid invalidating research measures; and (8) treatment with risperidone was not contraindicated.Normal comparison subjects were recruited through the use of flyers and advertisements which were placed in community sites and local newspapers. Normal comparison participants were chosen on the basis of age, sex, race/ethnicity, handedness, parental education, and community of residence that were comparable to those of the patient sample. Potential normal comparison subjects were assessed using the SCID-IV, sections of SCID-II for Personality Disorders, and the Brief Psychiatric Rating Scale (BPRS). The eligibility criteria included: no major psychiatric disorder except for a single episode of major depression, no personality disorder, no family history of a major psychotic disorder, and no current of substance use or history substance dependence.Study Entry was defined as the time point in which the patient enrolled in the study. Baseline occurred about 2–3 months after study entry and the 6-month point occurred subsequent to the baseline assessment.UCLA Psychiatric and Social History Schedule (P&SH). Demographic and premorbid history data were collected at study entry using a comprehensive form developed by the study team. P&SH data that were collected included age, sex, marital status, race, age of onset, patient and parental years of education, first appearance of prodromal and psychotic symptoms, and treatment history as well as additional psychiatric history variables.Cannon-Spoor Premorbid Adjustment Scale (PAS)  , Negative Symptoms , and Depression-Anxiety . The BPRS ratings were administered at Study Entry.Scale for the Assessment of Negative Symptoms (SANS)  and Global Functioning Scale: Social   was administered at Baseline to provide a standardized assessment of cognitive functioning  Part III uses video clips depicted individuals either lying or expressing sarcasm  is a 75-item paper-and-pencil assessment of an individual's perception of various aspects of relationships. The RAD has good internal consistency in schizophrenia patients and normal controls  is a self-report measure consisting of 141 items and 8 subscales that evaluate four components (branches) of emotion processing   were used to evaluate whether the relationship between sex and role functioning (school and work) was mediated by these predictor variables . We alsoF or t test values, and p values.Demographic information and a statistical analysis of sample characteristics can be found in p = 0.015), Early Adolescence (p = 0.019), Late Adolescence (p = 0.010), and Adulthood (p = 0.004), and had an earlier age of onset, M = 21.7 (s.d. = 3.2) v. 23.7 (s.d. = 4.8), p = 0.004 (p = 0.002) and Social Functioning (p = 0.034) and at 6 months (p = 0.044), but that there were no differences between the two groups in positive symptoms (p = 0.684) or mood symptoms (p = 0.372). In addition, we found that at baseline females had better insight than males  × Sex  ANOVAs  ANOVAs . We founGiven the significant sex differences in premorbid adjustment, and baseline negative symptoms and insight we conducted correlational analyses which indicated that better total premorbid adjustment was predictive of better role and social functioning at 6 months . Lower nWe conducted analyses to determine if the relationship between sex and role functioning (work or school) at 6 months was mediated by any of three potential mediators: premorbid adjustment, negative symptoms, and insight , while negative symptoms were not. Specifically, the mediation analyses indicate that pathways from the patient's sex to role and social functioning (school and work) go through premorbid adjustment and insight, suggesting that higher levels of functioning observed in females can partially be explained by the influence of sex on premorbid adjustment and insight. Although both males and females with schizophrenia performed more poorly than did healthy controls on measures of neurocognition and social cognition, no statistically significant diagnosis by sex interactions were found in cognitive performance.The important role of premorbid functioning is consistent with prior studies which show an association with a better prognosis in first episode patients (Albert et al., Males typically have an earlier age of onset (Immonen, Jaaskelainen, Korpela, & Miettunen, The observation of a relationship in our sample between greater baseline insight and higher role and social functioning at 6-months suggests that insight is an important predictor of the early course of schizophrenia. The finding that insight is a mediator of the relationship between premorbid adjustment and role functioning is important because there are sex differences in insight early in the course of schizophrenia and better insight is linked to lower levels of symptoms and better functioning (Cobo et al., Broader issues in sex-related cultural stereotypes in expectations of males compared to females might interact with the clinical mediators we identified to influence the course of the illness. Congruent with gender stereotype theory, adolescent men may feel greater pressure to leave home and begin a career at an earlier age than do females (Hentschel, Heilman, & Peus, Biological factors may also act as a mediator which could be an additional influence on the more favorable course observed in females compared with males with schizophrenia. In fact, the level of estrogen has been suggested as a protective factor (da Silva & Ravindran, We did not find any Diagnosis by Sex interactions in neurocognitive or social cognitive performance. Some prior studies that found sex differences in cognition among schizophrenia patients did not include a healthy control group and did not use cognitive scores that were corrected for sex differences in the general population. Thus, many of the prior reports of sex differences in cognitive performance among schizophrenia patients might be due to general population differences rather than to the impact of schizophrenia.v. females to produce the sex differences we found is not obvious.The study had some limitations. Some patients may not accurately recall how long ago their psychotic symptoms began. We believe that our use of parental and professional reports as well as medical records to minimize this issue. As is the case with many studies of individuals with schizophrenia, our sample size for females is smaller than for males. However, we were able to detect significant sex differences with this sample size of females. Because we received most of our referrals from hospitals at which patients were acutely ill, sample selection may favor those patients who required a hospitalization. Men or women who never needed hospitalization may therefore be underrepresented. Our sample consists of individuals who are willing to participate in clinical research. However, how these factors would differentially impact participation of males To summarize, in examining sex differences between males and female individuals with schizophrenia, we found many elements of consistency with the prior literature which indicates that males have poorer premorbid adjustment, an earlier age of onset, higher negative symptoms, and lower levels of role and social functioning. We found that predictors of functioning, such as premorbid adjustment and insight partially mediated the relationship between sex and role functioning and fully mediated the relationship between sex and social functioning. These findings underscore that males are more impaired than females and have prognostic implications for the impact of sex on the early course of schizophrenia.


Organelles play important roles in human health and disease, such as maintaining homeostasis, regulating growth and aging, and generating energy. Organelle diversity in cells not only exists between cell types but also between individual cells. Therefore, studying the distribution of organelles at the single-cell level is important to understand cellular function. Mesenchymal stem cells are multipotent cells that have been explored as a therapeutic method for treating a variety of diseases. Studying how organelles are structured in these cells can answer questions about their characteristics and potential. Herein, rapid multiplexed immunofluorescence (RapMIF) was performed to understand the spatial organization of 10 organelle proteins and the interactions between them in the bone marrow (BM) and umbilical cord (UC) mesenchymal stem cells (MSCs). Spatial correlations, colocalization, clustering, statistical tests, texture, and morphological analyses were conducted at the single cell level, shedding light onto the interrelations between the organelles and comparisons of the two MSC subtypes. Such analytics toolsets indicated that UC MSCs exhibited higher organelle expression and spatially spread distribution of mitochondria accompanied by several other organelles compared to BM MSCs. This data-driven single-cell approach provided by rapid subcellular proteomic imaging enables personalized stem cell therapeutics. Organelles cooperate to form a network of interactions that enable different cellular activities2. Therefore, studying organelle interactions is key to understanding how cells function more comprehensively. Cell-to-cell variability is observed not only between cell types but also between cells of the same type, resulting in molecularly and functionally distinct cells3. Such differences may contribute to the health and function of the entire organism. Multiple factors, such as microenvironment variability, differences in the cellular stages, genetics or epigenetics, or fluctuations in gene expression levels, can cause this heterogeneity. Single-cell analysis approaches are thus useful in investigating aspects of cellular mechanisms that are not revealed in bulk-level studies5.Cells perform different functions like providing structure and support, facilitating growth, producing energy, etc. to support and sustain life. These activities are handled by various subcellular structures termed organelles such as the nucleus, mitochondria, endoplasmic reticulum, and Golgi apparatus11. Therefore, analyzing the spatial organelle networks within MSCs can lead to a better understanding of cell functions to design appropriate treatment methods16. Although there have been recent studies focusing on spatial organelle analysis, these studies have used spatial data of each organelle from different cells, which reduces the accuracy of the resulting spatial information17. As such, a highly multiplexed protein imaging and analysis method is needed to obtain spatial information on organelles within the same cell, which can then be used to compare and understand differences in spatial organization between different cell types. In addition to intra-population variability, the most commonly used18, readily available stem cells sourced from bone marrow (BM) and postnatal umbilical cord (UC) introduce additional variables for the study. These cells display different molecular profiles, differentiation potential, and therapeutic efficacy19, and there is little consensus on how much, if any, the effect that MSC source has on outcome20.The use of mesenchymal stem cells (MSCs) has become a promising therapeutic method for treating a variety of diseases, as they can repair damaged cells by differentiating into replacement cells and modulating immune responses21 and multiplexed ion beam imaging (MIBI)22 can image up to 36 proteins using isotope-labeled antibody libraries and specialized equipment. However, these measurements are limited by their low resolution of about 0.5 to 1-μm. High-dimensional fluorescence imaging methods can map up to 50 proteins and include DNA-barcoded co-detection by indexing (CODEX) imaging23, multiplexed immunofluorescence microscopy (MxIF)24, and cyclic and sequential IF techniques28. While conventional immunofluorescence (IF) is time-consuming, rapid multiplexed immunofluorescence (RapMIF) provides multiplicity through quick multiple rounds of immunostaining and fluorophore inactivation. RapMIF enables high-throughput in situ proteomic analysis using conventional microscopes.Highly multiplexed imaging technologies enable the detection of multiple proteins in a single cell. Imaging mass cytometry (IMC)Herein, we established a rapid protein analysis pipeline for deciphering spatial organelle networks within a single cell. To achieve this, proteins in key organelles in mesenchymal stem cells (MSCs) such as the nucleus, mitochondria, Golgi, and endoplasmic reticulum (ER) have been targeted using antibodies. Multiplexed protein imaging was performed to examine the spatial organization of organelles and the interactions between organelles in MSCs. The analysis of organelle interactions on a single-cell level can eventually aid the stem-cell field in better understanding cell functions and exploring treatment methods using MSCs. Differences in organelle localization, interactions, and associated energy highlight the MSC heterogeneity from donor sources that will better inform therapeutic cell designs.To measure single-cell organelle distributions, we profiled subcellular localization of organelle proteins in the bone marrow and umbilical cord MSCs using RapMIF Fig. a. MultipFrom the 10-plex data, up to 25 cells (BM and UC MSCs combined) were selected for each marker. The scatter plots of intensity were calculated by random sampling of 50,000 pixel intensity values from all the cells for each marker pair targeting the same organelle. The scatter plots showed similar distribution for BM and UC MSCs for mitochondria targeting markers, implying similar interactions between the mitochondria markers in the two cell types   Fig. , and 4.  Fig. , a05) Fig. . While t29. While TOM20 and HSP60 both colocalize in mitochondria, HSP60 can also be found in extramitochondrial regions such as the cytosol, vesicles, and the cell membrane32. Similarly, Sortilin can be found in regions other than the Golgi, such as endosomes and lysosomes, whereas GOLPH4 is predominantly localized in the Golgi34.Because BM and UC MSCs expressed different spatial organelle patterns, we reasoned these differences could be recapitulated in their single-cell distributions. Single-cell analysis and comparisons were performed by selecting 4 organelle protein markers  for 7 BM and UC MSCs. DAPI was used as an additional marker to locate the nucleus. These markers were selected based on their specificity to the target organelle  hypothesis test to highlight significant differences between the spatial distribution of organelle marker pairs, including GOLPH4/Sortilin and TOM20/HSP60 Fig. . Concern26. The goal of performing pixel-level clustering is to identify a subset of pixels that share a unique multiplexed intensity profile. These clusters could indicate an organelle pattern or regions of similar functionality, and their hierarchical relationship reveals communication among them. The K-Means clustering algorithm was used to group the pixels into 10 clusters. The two cell types were clustered independently, and the resultant clusters were colored back on the images of the single cells. The spatial mapping of a single cell’s clustered regions was compared with the organelle colocalization calculated as the product of markers targeting that organelle , and the different texture features were calculated for each marker  were visualized together to observe regions of colocalization ion Fig. . Histogrion Fig. b to examion Fig. b. On the40. For example, the ER-mitochondria linkage has been implicated in lipid and energy metabolism as well as apoptosis signaling41. Since organelle phenotypes indicate changes in cell energy that affect cell function, it is important to study these phenotypes to improve future therapeutics.Image analysis technology for investigating cell features in biological research has advanced significantly over the past several decades. Finding protein associations within cells through spatial image analysis has the potential to deepen our understanding of their microenvironment and how their structural organization changes as cells transition from normal to abnormal cell behaviors. This work focused on observing spatial organelle distribution in two types of MSCs–BM and UC—and studying their differences. RapMIF was performed using antibodies that target the organelles to understand colocalization across the proteins. Multiplex analysis of organelle markers indicated differences in the spatial organization of different organelle proteins in BM and UC MSCs. Interactions between the proteins were compared using Pearson’s correlation coefficients and colocalization, which revealed differences between BM and UC cells. Pixel-level clustering, morphology, and texture analysis methods also showed a considerable organelle networking difference between the two cell types. Organelle phenotypes and their energy changes have been linked to stem cell fate, signaling, and therefore functionThis study demonstrated a RapMIF protocol to perform spatial profiling of organelles in single cells using 10 protein markers. The targets of these proteins were limited to key organelles, namely mitochondria, the Golgi, ER, the nucleus, and the nucleolus. To obtain a comprehensive understanding of cell behavior, proteins targeting other organelles, such as the peroxisomes, need to be included in the study. An important point that needs to be considered while selecting these proteins is the target organelle. While some proteins colocalize in a single organelle, some are chaperones and therefore move between different cellular components. These proteins can be useful to understand communication between organelles, providing insights into the functionality of the cells. However, it is important to consider multiple roles in the spatial and temporal coordination of such proteins to answer questions about the structural organization of the organelles.A limitation of this study is the number of cells considered for analysis. Imaging of the sample performed manually at high magnification (60×) is time-consuming, and therefore the number of regions captured was limited. Discarding cells that had image quality issues resulted in a small dataset of 14 cells (7 BM and 7 UC MSCs). Automated imaging is a potential way to overcome this limitation, as it can assist with capturing more regions, and therefore more cells. This study considered two-dimensional (2D) data, but using three-dimensional (3D) high-resolution data could improve organelle network mapping and provide more insights into interactions and colocalization. Additionally, organelle contacts and interdependency between the locations of different organelles could be better understood by using 3D data.45. Our study examines the organelle-specific activity differences among single cells from each tissue source. The detected activity differences can be attributed to energetic demand differences from their in situ origin. For example, since UC MSCs are typically derived from infants and BM MSCs originate from older, more mature adults, the difference in each population’s energy needs can be observed in the MSC population. UC MSCs were observed to exhibit higher organelle colocalization patterns  and protein markers indicating proliferation or apoptosis . Also, to relate gene expression with the change at the proteome level, RNA targets could be included in the staining panel. With the multiplexed experiments, the proteomic and transcriptomic phenotyping of cell lines and primary cells could provide a complete relationship between phenotypic differences and cellular function.To evaluate the mechanisms of how different protein/organelle phenotypes contribute to cell-specific functions, the transcriptome and proteome levels of primary cells and cell lines could be compared. Cell line experiments are a well-established approach because they are readily available and cost-effective. However, culturing cells in the media makes them lack tissue architecture and heterogeneity. Cells in culture can have different molecular phenotypes from cells in vivo54. Subcellular organelle analysis using proteomics is a useful method for obtaining spatial cellular maps that can reveal details about the structure and functionality of organelles and the cell. The complex spatial organelle interaction is yet to be fully understood in the context of organelle-related diseases56 such as cancer57, aging, and regenerative medicine58 to assess their potential in precision medicine and therapeutics.An accurate, reproducible, multiplexed protein imaging and analysis method could aid in better visualizing and understanding various disease states and microenvironments in single cells, which could then be used to find or design drugs and therapy methods that work best depending on patient needsBone marrow‐derived MSCs (BM MSCs) and umbilical cord-derived MSCs (UC MSCs) were obtained from RoosterBio, Inc. The culture media was prepared using 89% ɑ-MEM media (Cat # 12561-049) with L-glutamine, 10% heat-inactivated fetal bovine serum (HI-FBS), and 1% penicillin–streptomycin (Cat # P4333). The culture media was mixed and filtered before use. BM and UC MSCs were cultured in T-75 flasks with 10 mL of culture media. Cell passages were performed when cells reached 75% confluency using Trypsin LE cell detachment media (Cat # 12605-010) at 37 °C. The cells were resuspended in respective culture media after centrifugation at 280 g for 6 min and then seeded on collagen-coated glass coverslips. The cells were then cultured on coverslips for 24 h before fixation. Cells were then fixed in 1.6% paraformaldehyde in PBS for 10 min at room temperature, followed by another PBS washing and multiplexed staining of organelle protein markers. Duplicate experiments were used for each measurement. Cells were thawed at passage 2 and cultured until passage 20. BM and UC cells were from similar passage numbers.The primary antibodies considered for this study were ATF6 , β-tubulin , GOLPH4/GPP130 , HSP60 , Nucleolin , Sortilin , Tom20 , Phalloidin , Wheat Germ Agglutinin , and Concanavalin A . They were used for cell segmentation and additional organelle markers . For each 10µL of primary antibody, 1µL of modifier reagent was added and mixed gently. The lyophilized powder was dissolved in 10µL PBS , and 1µL of this solution was added to each antibody and mixed gently. The mixture was incubated at room temperature for 15 min in the dark. After incubation, 1µL of quencher reagent was added for each 10µL of antibody used and was then gently mixed. In this experiment, the antibodies were conjugated to Alexa Fluor 647, which is a bright dye with less background fluorescence2O2 and 20 mM NaOH in PBS for 1 h at RT with white light. The sample was imaged to ensure that the fluorescence signal had fallen to background levels , 555 nm (TRITC), and 647 nm (Cy5), respectively. The exposure time varied between the markers and the cells. The sample was imaged with a 60X oil lens, resulting in a high-resolution acquisition at 0.1083 µm/pixel. Each imaging region was imaged across 16 z-stacks with 0.5 µm/stack. The best focus z slices of each image were used for analysis. The images were stored as 16-bit multichannel images in ND2 format. They were converted to TIFF format using ImageJ and saved as single or multi-channel grayscale images. Each marker was thresholded with the Otsu method for all analyses60. DAPI channel images of each cycle were aligned using the DAPI channel image of the first cycle as a reference to obtain the registration transforms, which were then applied to the remaining channels of each cycle. After registration, the best cells were manually selected through visual inspection based on image quality. For example, unreliable regions with intensity values too close to the local background (insufficient contrast) or saturated intensity values (making it hard to distinguish features due to high intensity) were filtered out. Cells that had overlapping cytosol and cells which were touching the image borders were also discarded. This quality control step resulted in 7 BM MSCs and 7 UC MSCs for analysis. Background subtraction was performed using a rolling ball algorithm in ImageJ to remove the background signal from the images. Finally, the pixel intensities were thresholded by calculating the lower 20 percentile (background pixels) and the upper 99.9 percentile (saturated pixels), rescaling the intensities using the two percentiles between 0 and 1 and clipping the values that were lower than 0 and greater than 1. The cells were segmented in ImageJ using cell masks obtained from the Phalloidin marker outline  hypothesis test was conducted between organelle marker pairs targeting the Golgi (GOLPH4 and Sortilin) and mitochondria (TOM20 and HSP60). For each cell, the center of mass was computed from the segmented mask. Then, each marker’s pixel distance to the center of mass was calculated to convert 2D spatial image coordinates to 1D spatial distributions  and K-Means clustering into superpixels37. Texture features were calculated on the superpixels: pixel intensity, energy Laplacian, modified Laplacian, diagonal Laplacian, variance Laplacian, and gray level variance. Pixel intensity computes the mean of the intensities within each superpixel. Energy Laplacian computes the square of the Laplacian of the image from skimage61. Modified Laplacian also uses a Laplace function but with two kernels in which one is the transpose of the other; then, the resulting images are summed. Diagonal Laplacian uses convolution with 4 kernels to produce the resulting images that are then summed. Variance Laplacian consists of the square of the difference between the image Laplace and the mean of the image Laplace. Gray level variance involves the square of the difference between the image and the mean of the image. The Python code that contains all the texture functions is labeled “texture_analysis_functions.py” and is available on the Github. The superpixels and texture features were plotted as images and heatmaps were obtained for each marker to compare BM and UC MSCs.The images were segmented into superpixels using scikit-image38. These files were opened in ConfocalVR software to visualize them in an immersive 3D format62. The pixel data was also visualized in another VR software, Genuage, and histograms of the pixel counts were obtained using histogram bins drawn using controllers63.The images of organelle markers in BM and UC cells stored as TIF files were converted into Neuroimaging Informatics Technology Initiative file format (NIfTI or .nii) using ImageJ64, and were then plotted as heatmaps with hierarchical clustering. To first identify and segment the nuclei of cells, “IdentifyPrimaryObject” was used on the DAPI marker with the global two-class Otsu thresholding method. Using the segmented nuclei, the “IdentifySecondaryObject” module was then used to segment the general shapes of the organelles marked by each marker using the same thresholding method. After images of every marker were segmented, all the size/shape features were measured using the “MeasureObjectSizeShape'' module for each of the markers and exported into an excel file. The data stored in an excel spreadsheet was manually sorted to extract and analyze only the features of interest, which were the area, major axis, and minor axis of each marker. The measurements of interest were converted into CSV files for visualization. Heatmaps were generated using Python with dendrograms to compare the biophysical measurements and to determine any close relationships or associations between markers and between the two cell types (UC and BM). The z-score was calculated to normalize the output data.Morphological features, such as area, major axis, and minor axis, were obtained for each cell using a CellProfiler pipelineSupplementary Information.


This study introduces and explores the use of supersaturated solutions of calcium and phosphate ions to generate well-defined hydroxyapatite coatings for orthopaedic implants. The deposition of hydroxyapatite is conducted via several solutions of metastable precursors that precipitate insoluble hydroxyapatite minerals at a substrate–solution interface. Solutions of this nature are intrinsically unstable, but this paper outlines process windows in terms of time, temperature, concentration and pH in which coating deposition is controlled via the stop/go reaction. To understand the kinetics of the deposition process, comparisons based on ionic strength, particle size, electron imaging, elemental analyses and mass of the formed coating for various deposition solutions are carried out. This comprehensive dataset enables the measurement of deposition kinetics and identification of an optimum solution and its reaction mechanism. This study has established stable and reproducible process windows, which are precisely controlled, leading to the successful formation of desired hydroxyapatite films. The data demonstrate that this process is a promising and highly repeatable method for forming hydroxyapatites with desirable thickness, morphology and chemical composition at low temperatures and low capital cost compared to the existing techniques. Orthopaedic implant fixation is hindered by fibrosis encapsulation, which is the body’s natural response to a foreign object ,2,3,4. CBeing the main ceramic component of bones and teeth in the human body, with properties of rigidity and compressive strength and a brittle nature, hydroxyapatite (HA) coatings are used to improve the osteointegration of orthopaedic implants. The use of synthetic HA coating aids orthopaedic recovery since it mimics natural bone, and when deposited in a porous structure, it encourages new bone-ingrowth directly onto an implant . HA coat10(PO4)6(OH)2 and hexagonal crystal structure of space group P63/m. Biological HA is a product of the nucleation and crystallisation of calcium and phosphate ions in vivo, but these ions in solution can also synthetically initiate HA formation. Synthetic HA can enhance bone growth in vivo, and its deposition can be achieved on a wide range of materials [HA is a crystalline phase of calcium, phosphate and hydroxyl groups of chemical formula Caaterials ,12,13. Caterials ,15. 2+ and PO43− ions out of a solution onto a surface to form a HA layer has been achieved with relevant precursors under specific conditions of pH and temperature on collagen or bone interfaces [However, synthetic methods of coating metallic parts with HA are far from facile. Solution-based HA processes highlight a requirement to regulate the ionic strength of solutions ,17,18,19terfaces ,27. In gterfaces ,29.9(PO4)6, which are formed from the nucleation of phosphate-based fluid in contact with calcium semi-solid interfaces [HA crystallisation in vivo is thought to start with 10–25 nm sized amorphous apatite particles . Studiesterfaces ,32,33. Tterfaces ,35,36. Fterfaces . HoweverThis method of depositing hydroxyapatite (HA) on orthopaedic implant-type substrates mimics biological-like self-assembly growth processes. One novelty of this process lies in the use of chemical precipitation directly onto a substate to be coated without intermediate steps. Using these saturated solutions of calcium and phosphorous gives rise to coatings with adequate coverage, thickness and morphology control that are relevant to manufacturing requirements. This method also allows for a non-line of sight HA deposition, which is also another of its advantages over the existing techniques ,39,40. IOur results detail a proposed nucleation and growth pathway for HA within this deposition system. With the aim of identifying the ideal process solutions, analysis is carried out on pH, ionic strength, and dynamic light scattering (DLS) properties. Subsequently, HA films are formed with different solutions and are analysed. Weight analysis, scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) of the deposited films reveals the solutions which generated HA films with coverage, morphology and chemical structure best aligned to osetoinduction HA coatings. 2PO4) United States Pharmacopeia (USP) reference standard, Honeywell Fluka hydrochloric (HCl) acid solution 6 M, tris(hydroxymethyl)-aminomethane (TRIS) ACS reagent, 99.8% sodium chloride (NaCl) BioXtra, and 99.5% calcium nitrate tetrahydrate (Ca(NO3)2·4H2O) ACS reagent were all purchased from Sigma Aldrich . A calibrated benchtop pH meter with a temperature-enabled probe  was used for pH measurements with an accuracy of 0.001 pH. Titanium coupons of Ti-6Al-4V alloy were used as substrates. All substrates were submerged in hot basic solutions to increase roughness and yield a negatively charged surface for calcium ion attachment [All materials and reagents were used as received. Monobasic potassium phosphate  to yield a supersaturated phosphate concentrate. See 3)2·4H2O was mixed with DIW to yield a supersaturated calcium concentrate. For deposition process runs, the supersaturated concentrates were combined and diluted to various concentrations.KHv/v, which is the percentage of concentrate volume to process solution volume. Solutions were warmed to 40–50 °C for the deposition of hydroxyapatite. Solutions of varying dilution are identified with %Ionic strength is a well-established modifier in hydroxyapatite formation since it increases or decreases the ions present in a solution ,49. Ioni−1 and Z = electric charge.Equation (1): Ionic Strength (I) formula: Despite the ratio of components remaining fixed, ionic strength of the supersaturated solutions is increased via sodium chloride addition (increasing the ratio of concentrate in solution); see A custom-designed experimental apparatus was used for deposition. A sample holder, a thermometer, and a pH probe for in situ temperature and acidity measurements, and an overhead stirrer to agitate the solution were inserted into the vessel. High agitation rates of 1000 RPM prevented gross precipitation of mineral from the solution. Substrates were placed in the reaction vessel for deposition, and then removed and rinsed with DIW; this process was repeated several times with fresh solutions to grow a coherent layer of HA at the solution–substrate interface of desired thickness. Between deposition steps, substrates were removed from solution and allowed to dry for a minimum of 15 min in ambient conditions (to ensure that active sites are available for subsequent process runs). More data behind the drying rational are provided in Dynamic Light Scattering (DLS) was performed using Malvern Zetasizer Nano ZS , with 633 nm HeNe laser. Samples were added to glass cuvette which had a 10 mm pathlength and allowed to equilibrate for 120 s at 47 °C prior to sampling. The machine was operated in backscatter mode at an angle of 173°. Scanning Electron Microscopy (SEM) images were recorded on a Zeiss Ultra Plus system  with accelerating voltages in the range of 5–10 kV, at a working distance between 3 and 10 mm and with an in-lens detector. Energy-dispersive X-ray spectroscopy (EDX) spectra were acquired at 15 kV on an Oxford Inca EDX detector . The solution reactions are complex but may be thought of using the following reaction schemes which detail the formation of hydroxyapatite (HA). At equilibrium, calcium and phosphate ions in solution form a tricalcium phosphate (TCP) mineral product as shown in Equation (2). Equation (2): Equilibrium chemical reaction for the formation of tricalcium phosphate (TCP) from calcium and phosphate ions.2+ or Ca(OH)2 are required, Equation (3). At stoichiometric proportions and in the presence of hydroxyl ions, the formation of TCP is thermodynamically favoured. To form pure hydroxyapatite (HA) under solution, excess calcium ions as CaEquation (3): Example of one reaction where additional calcium ions with tricalcium phosphate and hydroxyl ions can form hydroxyapatite (HA).In this work, as the concentration of solutions are equally supersaturated with respect to calcium and phosphate ions, they drive the precipitation of thermodynamically unstable phases of octacalcium phosphate (OCP), as shown in Equations (4) and (5) ,51,52.Equation (4): Formation of calcium-deficient (CDHA) precursor to octacalcium phosphate (OCP) particles in aqueous solution.Equation (5): Nucleation of octacalcium phosphate (OCP) particle in aqueous solution.Whilst the experimental solutions at room temperature contain metastable intermediaries , their nucleation into stable precipitates only occurred above a critical temperature, and deposition only occurred between 44 and 50 °C. There is an upper temperature limit because of redissolution. − concentration. Within this study, pH dropped by 0.12 ± 0.02 from 0 to 30 min with almost no change after that; therefore, process runs were ceased at 30 min for all solutions, Precipitation of Ca-P phases from solution should be associated with a pH drop as a result of decreased calcium ion concentration . As EquaWhile all constituent parts were accounted for in the ionic strength calculations, 95% of the ionic strength value for each solution was due to the NaCl component, see v/v has particle size with a mean of 5.2 µm, whereas concentrations between 6 and 9%v/v have a particle size of around 100 nm, and the concentration of 3%v/v have 35% of particles that are <8 nm. DLS data from various process solutions shows that the particle size in solution varies significantly with dilution becoming smaller with increased dilution, When a substrate is added to the solutions at the correct temperature, heterogeneous nucleation occurs at the substrate, thereby forming a deposit. A side-by-side photograph of samples post 7 deposition runs reveals a colour gradient of the HA film across solutions; see The porosity of an HA film is fundamental to the success of the coating in vivo, and it should be on the few hundred nm to micron scale ,55. SEM Solution 4 showed the beginning of a porous network after 2 process runs, with mineral clusters forming a porous layer amidst needle-like interconnectivity; see EDX analysis enhanced SEM observations. Lower titanium signals indicated higher HA coverage, and it can be seen in It was possible to quantify the amount of film deposited from the weight of the samples after each process runs; see 2+ from the solution. For solutions 1 and 2, there are not enough available ions to form TCP or OCP mineral as per Equations (1) and (2). After 2 process runs, both solutions 3 and 4 show a higher Ca/P, aligning with Equation (2), since a TCP content would increase the Ca/P. However, as deposition progresses , the OCP phase dominates; see The calcium to phosphate at% ratio (Ca/P) and the oxygen at% are widely used to identify hydroxyapatite phases; see Together, these data sets reveal that solutions 1 and 2 had minimal apatite film attachment across all process runs, implying that their concentration and ionic strength are too low. Solutions 3 and 4 showed slower apatite film growth kinetics, but they showed the most adhered layer with a suitable mg of material added per run; also, they had the best morphological development after 7 process runs, mirrored through EDX data of the relevant elements at 2, 4 and 7 process runs. Compared to other studies, this morphology and phase composition are advantageous since the presence of TCP can accelerate coating degradation, whereas the mix of ACP and OCP allows for ease of integration and the structure encourages bone growth ,61. SoluThis study has shown that HA film can be deposited and controlled using a colloidal solution process. The repeatability of the process at 47 °C is confirmed through consistent dropping of pH in 30 min runs. Bonding of calcium ions to the substrate leads to a drop in pH, as explained in the proposed chemical pathway. DLS data show various particle sizes in solutions, with ~100 nm particles being preferred for porous and coherent films (because of the denser packing). SEM topography showed that various solutions used gave rise to a morphologically porous and chemically consistent HA coating. The concentration levels of solutions influence film thickness, coverage, and coating rate; NaCl was shown to have no role in HA bonding. Lower concentration solutions formed very thin films, with calcium and phosphate contents being <5 at% after 7 deposition runs. While some films contained carbonates at higher concentrations, all films had similar oxygen content. Formation of HA films at >1 mg per run is a strong indication that the phase of HA formation is consistent in all solutions. Ca/P ratio data strongly indicated an OCP phase; additionally, the oxygen atomic percentage indicated the presence of water and ACP. This HA phase aligns with the proposed nucleation and growth pathway from Equations (2)–(5).Based on our results, it can be inferred that the optimal process solution is between solutions 3 and 4, as they clearly followed the proposed chemical nucleation and growth process. The advantage of this slower crystal growth is that it allows for excellent porosity and morphology, which can be seen via SEM, and the adhesion of the layer can be also be conveniently ratified with the photographs.Overall, our study has shown that colloidal solutions can be used as a novel HA coating process, wherein crystallisation occurs in a timely manner at low temperatures.


We conducted a retrospective statistical analysis of clinical and physical dosimetric factors of lung cancer patients who had previously undergone lung and/or mediastinal radiotherapy and died of or survived severe acute radiation pneumonitis (SARP). Our study was the first to reveal the heterogeneity in clinical factors, physical dosimetric factors, and SARP onset time that determined the clinical outcomes of lung cancer patients who developed SARP.U test was used for data with skewed distribution. Qualitative data were represented using frequencies (%), and Fisher’s exact test or χ2 test was used for intergroup comparison of nonparametric data. Binary logistic analysis was used for univariate and multivariate analyses. Differences with a P < 0.05 were considered statistically significant.The clinical characteristics, physical dosimetry factors, and SARP onset time of deceased and surviving patients were retrospectively analyzed. SPSS 20.0 was used for data analysis. Student’s t-test was used for intergroup comparison, and a Mann–Whitney Univariate analysis revealed that the potential predictors of SARP death were as follows: ipsilateral lung V5 and V30, contralateral lung V5, V10, and V30, total lung V5, V10, and V30, mean lung dose, mean heart dose, and maximum spinal cord dose. Multivariate analysis showed that ipsilateral lung V5 and total lung V5 were predictors that determined the final outcome of SARP patients. In addition, we analyzed the time from the completion of radiotherapy to SARP onset, and found significant difference between the two groups.There was no decisive correlation between clinical characteristics and SARP outcome  in lung radiotherapy patients. Ipsilateral lung V5 and total lung V5 were independent predictors of death in SARP patients. Lung cancer is still one of the leading malignancies that threaten human health and life globally. The incidence and mortality rates of lung cancer are ranked first among all malignancies in males and second in females –3. RadioThe Radiation Therapy Oncology Group (RTOG) classified RP into grades 0 to 5 based on the clinical symptoms, lung radiologic presentation, and corticosteroid treatment results. After discontinuing radiotherapy, some patients develop severe acute RP (SARP), i.e., grade 3 or above . Most prClinical and dosimetric factors that determine the clinical outcomes of lung cancer patients with SARP are still unknown. There have been no related studies that compared the radiation dosimetric parameters between patients with different outcomes and evaluated their role in treatment-related toxicity. Therefore, we focused on the final outcome of SARP patients and attempted to determine the heterogeneity in clinical factors and radiotherapy dosimetric parameters in lung cancer patients with grade 3 and above RP who had different outcomes. This approach could provide a theoretical research foundation for further optimization of lung radiotherapy plan and decreasing the incidence of fatal RP in lung cancer patients in clinical practice.Data were collected from 66 lung cancer patients who had undergone lung and/or mediastinal radiotherapy and developed SARP from 2013 to 2019 in our center. There were 31 deaths and 35 survivors. The inclusion criteria were as follows: (1) clinicopathologically confirmed lung cancer; (2) lung and/or mediastinum received 40–65 Gy radiotherapy; (3) complete and available clinicopathological and physical dosimetric parameters; (4) satisfactory general condition of the patient during radiotherapy. The exclusion criteria were as follows: (1) severe cardiac, hepatic, or renal impairment; (2) discontinuation of treatment due to severe complications during and after radiotherapy ; (3) previous history of chest radiotherapy; (4) lack of complete and periodic radiologic diagnosis when RP occurred; (5) acute infection or autoimmune disease. This study was approved by the Ethics Committee of the hospital. Every subject was given an informed consent form for participating in this study, and approval was granted for data collection.Clinical information, including the general condition, personal history, tumor stage, pathological and radiologic information, and treatment status of the patient, was obtained from the doctor’s workstation (Haitai) and the imaging system (PECS) of this center. Clinical data included patient’s age, gender, ECOG (Eastern Cooperative Oncology Group) score, history of smoking, history of COPD, history of diabetes mellitus, pathological diagnosis, Ki67, tumor site, TMN stage, whether surgery was performed, whether chemotherapy was performed, and progression. Radiologic data included enhanced lung computed tomography (CT) within 6 months after the completion of RT and evaluation of radiologic characteristics of SARP within half a year after radiotherapy.All lung cancer patients received three-dimensional conformal RT (3D-CRT) or intensity-modulated radiation therapy (IMRT). All radiotherapy plans were generated by a therapist on Monaco  or Eclipse  treatment planning systems. The X-ray beam energy was 6 MV. The requirements were as follows: the prescribed dose is achieved in at least 95% of the PTV; ≥110% dose hot spots are absent outside of the PTV. The dose constraints were defined for organs at risk as follows: Mean lung dose(MLD) < 20 Gy; total lungs: V5 < 60%, V20 < 30%, V30 < 20%; Heart: V30 < 40%, V40 < 30%; Maximum point dose of spinal cord < 45 Gy. The delineation of target volumes and organs at risk was in accordance with the Radiation Therapy and Oncology Group guidelines . PhysicaAll lung cancer patients received personalized treatment strategy of synchronous or sequential chemotherapy based on the pathological diagnosis. The chemotherapy regimens included cisplatin combined with etoposide, pemetrexed combined with carboplatin, or cisplatin combined with paclitaxel. These regimens are widely used in clinical practice , 16. DosWe retrospectively analyzed the clinical data and radiologic examinations of all lung cancer patients before and after radiotherapy. Most patients underwent one chest CT follow-up every 3 months. The diagnosis of RP was mainly confirmed through clinical symptoms, physical examination, and chest radiologic examinations. Pneumonitis was defined as follows: (1) localized pneumonitis in which the timing and symptoms were consistent with radiotherapy and required the use or extension of steroid treatment; (2) radiologic presentation of pneumonitis consistent with the Irradiation volume. RP was graded in accordance with RTOG and/or Common Terminology Criteria for Adverse Events Version 5.0 (CTCAE v5.0) . SARP waU test was used for data with skewed distribution. Qualitative data were presented as frequencies (%), and Fisher’s exact test or χ2 test was used for intergroup comparison of nonparametric data. A Mantel–Haenszel χ2 and Mann–Whitney U test were used for intergroup comparison of ordinal data. A binary logistic regression model was used for univariate and multivariate analysis to evaluate the relationship between SARP patient outcomes  and clinical factors and physical dosimetric characteristics. The hazard ratio, 95% confidence interval, and P value were reported. All data were computed using the Statistical Package for Social Science program  and R software (version 3.5.3). P < 0.05 was considered statistically significant.Student’s t-test was used for intergroup comparisons, while a Mann–Whitney In this study, the clinical characteristics of lung cancer radiotherapy patients who developed SARP were retrospectively analyzed. Table SARP had developed in 31 patients who died and in 35 surviving patients. To further analyze the differences between deceased patients and surviving patients with SARP, we conducted a statistical analysis of physical dosimetric parameters. Table The clinical factors and physical dosimetric parameters of deceased and surviving SARP patients were considered for univariate and multivariate analyses. Table To further analyze whether there are differences in the time of SARP onset between deceased and surviving patients, we compared the time from RT completion to SARP onset between the two groups. We found that the P value was 0.033, which was statistically significant Fig. . The medRadiation pneumonitis(RP) is a common and potentially fatal dose-limiting toxicity that occurs in lung cancer patients undergoing lung and/or mediastinal radiotherapy or synchronous radiochemotherapy, and its incidence is 14.6–37.2%. Most cases occur around 6 months after radiotherapy , 19. RTOIn this study, the data of 31 patients who had undergone lung and/or mediastinal radiotherapy and died of SARP from January 2013 to December 2019 were collected. In addition, data were collected from 35 patients who developed SARP and survived during the same period. It is worth noting that we tried as much as possible to collect patients who had undergone chest and/or mediastinal radiotherapy and died of SARP. However, we did not include all patients who developed SARP and survived because equal sampling was employed in this study and the surviving patients that were included to the analysis could represent the overall characteristics of all patients who developed SARP and survived. Therefore, the number of patients in this analysis does not represent the mortality rate of SARP in our center. In the existing studies on lung cancer patients who develop RP after radiotherapy, some clinical characteristics have been considered important risk factors for RP progression. Previous studies have shown that performance status, history of smoking, nutritional status, past lung disease, tumor stage, pathology and site, concurrent chemotherapy, pre-radiotherapy lung function, and surgery are associated with the occurrence and severity of RP , 21–27. It has been widely observed and reported that DVH dose parameters are associated with RP. Multiple doses, including mean lung dose and the percentage volume that received a fixed dose, are associated with an increased risk of SARP. Previous studies have found that MLD shows a significant positive correlation with RP . HernandThe lungs are “parallel organs” as they consist of multiple parallel functional units. Other functional units are not affected when one functional unit is damaged . TherefoMany studies have shown that the mean V20 and V30 of patients with RP were 42.0% and 38.0%, respectively, which were higher compared with patients who did not develop RP . CurrentWith advances and developments in the IMRT era, researchers have paid more attention to the effect of low dose regions in the lungs on the development of radiation pneumonia, particularly V5 . Many reOur study showed that the median time to SARP onset in the survivor group and the death group was 64 days and 98 days, respectively. The difference in the time of SARP onset between the two groups was statistically significant, and the median time of SARP onset in the survivor cohort was significantly earlier than that in the death cohort. This may be due to the pathophysiological developmental trends of RP, and patients with earlier SARP onset have significant treatment advantages as they receive treatment earlier. Therefore, these patients have better outcomes. We also found that the median time from SARP onset to death was 159.5 days in the death group, and the median time from SARP onset to outcome was 93.5 days in the survivor group. Although this study was the first to reveal the heterogeneity in clinical factors, physical dosimetric factors, and time of SARP onset in SARP patients, there are several limitations that need to be urgently solved. First, the sample size of this study was small, and the sample size should be expanded in future studies to further validate our prediction results. Furthermore, this was a retrospective study, and selection bias may be present. Lastly, we only analyzed basic clinical factors and physical parameters. In subsequent studies, bioinformatics, genome information, and radiomics information will be added to improve the prognosis determination criteria for SARP patients.In this study, there was no decisive correlation between clinical characteristics and SARP outcome  in lung cancer patients who had undergone radiotherapy. Physical dosimetric factors, such as ipsilateral lung V5 and V30, contralateral lung V5, V10, and V30, total lung V5, V10, and V30, MLD, mean heart dose, and maximum spinal cord dose may be potential predictors of death in SARP. Multivariate analysis showed that ipsilateral lung V5 and total lung V5 were independent predictors of death in SARP patients. These ultimately determine the outcome of patients after SARP occurrence, i.e., death or survival.the median time of SARP onset in the survivor cohort was earlier than that in the death cohort. In summary, this study is the first to provide a theoretical research foundation for further optimization of chest radiotherapy plan and decreasing the incidence of fatal RP in lung cancer patients.


Malignant hyperthermia (MH) is an inherited skeletal muscle disorder caused primarily by a genetic mutation, usually in the calcium channel gene of the muscle. This mutation can lead to muscle hypersensitivity to volatile anesthetics (such as sevoflurane) and the depolarizing muscle relaxant succinylcholine, resulting in hyperthermia, muscle stiffness, metabolic disturbances, and other severe physiological reactions. This condition may prove fatal unless it is recognized in its early stages and treatment is administered promptly and aggressively. We report a 13-year-old adolescent who underwent laparoscopic appendectomy and developed MH after the use of inhalational anesthetics, manifested by unremitting hyperthermia with a maximum temperature of 44.2°C, muscle rigidity, tachycardia, hypercapnia; and malignant arrhythmias, cardiogenic shock, hyperkalemia, metabolic, and respiratory acidosis. After early and timely recognition, multidisciplinary management and administration of dantrolene, the case was successfully treated. Exome sequencing revealed a point mutation (amino acid change) on the RYR1 gene: c.12700G>C. Due to the lack of ready-made dantrolene in our hospital, the patient in this case received dantrolene treatment only 6 h after the first observation of high body temperature. We review the development of the disease and summarize the success of treatment and what can be done to improve the chances of saving the patient’s life if dantrolene is not available in time. In most patients, MH is triggered by conventional inhalation anesthetics or depolarizing muscle relaxants (succinylcholine), typically resulting in systemic muscle rigidity, an abnormally rapid increase in body temperature, and hypermetabolic state. Although the incidence of MH is low, the disease progresses rapidly and has a high mortality rate if not diagnosed and treated promptly . The exaIn May 2022, we successfully treated a patient with fulminant MH, when dantrolene was not available in time, and herein summarize the case.2A healthy 13-year-old Chinese (Han ethnicity) boy presented for an emergency laparoscopic appendicectomy weighing 46 kg and 160 cm tall with an American Society of Anesthesiologists class I. He had a history of attention-deficit/hyperactivity disorder and premature birth (1 month premature). Prior to surgery he was clinically stable with a febrile temperature of 38.1°C and heart rate (HR) was 117 beats/min.2) and nasopharyngeal temperature (NT) were continuously measured. The airway pressure was 14 mmHg, PetCO2 was 35 mmHg, and temperature was 37.3°C. Anesthesia was maintained with sevoflurane inhalation, propofol infusion, and remifentanil infusion.The patient was admitted to the operating room with the following preoperative vital signs: noninvasive blood pressure of 89/48 mmHg, HR of 85 beats/min, and pulse oxygen saturation of 99%. General anesthesia was induced with midazolam (2 mg), propofol (100 mg), sufentanil (15 µg), and cisatracurium (10 mg) followed by endotracheal intubation. Mechanical ventilation was set up and the end-tidal carbon dioxide  continued to rise from 48 to 81 mmHg. At the same time, his temperature rose to 38.5°C, his HR increased to 120 beats/min, and he developed mixed metabolic and respiratory acidosis on arterial blood gas (ABG) analysis , his temperature began to drop and recovered to 38.1°C. PetCO2 levels also decreased to 81 mmHg and acidosis improved  cooling therapy ; (2) correction of acidosis (hyperventilation and intravenous administration of sodium bicarbonate); (3) correction of electrolyte disturbances, mainly hyperkalemia  and monitoring of blood glucose; (4) appropriate use of vasoactive drugs (norepinephrine and epinephrine) to maintain hemodynamic stability; and (5) administration of high-dose methylprednisolone shock therapy (500 mg) to alleviate systemic inflammation. Intravenous administration of heparin (2 mg) was also given to prevent disseminated intravascular coagulation (DIC). During this period, the body temperature continued to rise, reaching a maximum of 44.2°C and PetCOimproved . After sAfter obtaining dantrolene, the initial dose of 2 mg/kg  was immediately administered, followed by intermittent bolus of 1 mg/kg every 4–6 h. Intravenous dantrolene was continued for another 2 days, during which time he did not have any signs of recrudescence, renal impairment, or other overt complications of MH. During his ICU stay, he initially developed rhabdomyolysis, resulting in elevated creatine kinase (CK) and myoglobinuria. His CK and myoglobinuria levels both decreased after peaking on postoperative day (POD) 5 . Liver fWith the consent of the patient’s family, the patient underwent whole-exome sequencing. The sequencing revealed a point mutation (amino acid change) on the RYR1 gene: c.12700G>C . HoweverConsent for publication: The authors have obtained the patient’s father written informed consent for print and electronic publication of this case report.32 of >55 mmHg with sufficient minute ventilation, 15 points; abnormally rapid increase in perioperative body temperature, 15 points; abnormal tachycardia, 3 points; the base excess more negative than −8 on ABG analysis, 10 points; pH of <7.25 on ABG analysis, 10 points).MH is an abnormal metabolic hyperactivation of skeletal muscle spasms that is induced by exposure to inhaled anesthetics and depolarizing muscle relaxants. As medical treatment has improved, the number of operations performed under general anesthesia has increased annually; additionally, with the progress of anesthesia monitoring methods and the improvement of doctors’ knowledge of MH, the number of reported cases of MH has increased in recent years . The curRYR1) abnormalities are the molecular basis for MH in most patients [RYR1 gene results in abnormalities in the sarcoplasmic reticulum of skeletal muscle, leading to a high calcium concentration when the patients touched trigger factor such as inhalation anesthetics, followed by continuous tonic contraction of skeletal muscle [The patient underwent whole-exon sequencing to identify the mutations in the RYR1 gene. This variation locus has been classified in the ClinVar database as suspected pathogenic, and it has been detected in multiple patients with MH episodes or individuals with a family history. But this is the first time that the point mutation has been reported in the general East Asian population, and the prediction result of both REVEL software and ClinPred software was “harmful” . Studies have confirmed that ryanodine receptor type 1 gene  Improved monitoring is very important for the early detection and diagnosis of MH, and early diagnosis and early treatment are the basis of successful rescue of MH. (2) It is critically important to quickly convene the whole hospital for multidisciplinary joint diagnosis and treatment. (3) It is important to use a variety of cooling techniques. In this case, a variety of cooling methods are used to bring the body temperature down below the danger range as quickly as possible. Extracorporeal circulation can be used for cooling if necessary. (4) CRRT is beneficial to maintain internal environmental homeostasis, correct electrolyte disturbance and acidosis, maintain hemodynamic stability, and more effectively remove myoglobin and part of inflammatory mediators by means of convection and adsorption. It can lower the body temperature of patients with hyperthermia by lowering the temperature of replacement fluid, and provide nutritional support under high metabolic state. Prevent and treat renal failure. (5) Early application of heparin therapy reduces the consumption of coagulation factors and platelets by DIC. Ensure hemodynamic stability and give adrenocorticosteroids and diuretics if necessary. (6) Extracorporeal membrane oxygenation (ECMO) may be used as a last resort in patients with sustained cardiac arrest. (7) MH is rare, and many hospitals have insufficient experience in rescue. Timely and effective contact with anesthesia experts in other hospitals to guide the treatment is also important for successful rescue.4The characteristics of MH are rapid onset, high mortality rate, and numerous complications. Dantrolene can greatly improve the prognosis of patients. However, if dantrolene is not routinely available in the hospital, it is necessary to take correct symptomatic treatment measures, including CRRT, lowering body temperature, brain protection, maintaining stable vital signs, ECMO, etc., to save valuable time for obtaining dantrolene. Furthermore, dantrolene should be routinely equipped in every hospital or region whenever possible.ABGarterial blood gasCKcreatine kinaseCRRTcontinuous renal replacement therapyDICdisseminated intravascular coagulationHRheart rateICUintensive care unitMHmalignant hyperthermia2PCOpartial pressure of carbon dioxide2PetCOthe end-tidal carbon dioxideRYR1ryanodine receptor type 1 gene


There is controversy about the indication for nonalcoholic fatty liver disease (NAFLD) screening in patients with type 2 diabetes mellitus (T2D). The present study aims to contribute to NAFLD surveillance in patients with T2D, assessing the association of clinical and biological variables with hepatic stiffness and steatosis.A cross-sectional design was used, with data collection from electronic medical records, including adults with T2D who underwent transient elastography (TE) between June 2018 and December 2019. Liver stiffness and steatosis were evaluated using TE and controlled attenuation parameter (CAP), respectively, with cutoff points > 8 kpa for increased stiffness and > 275 dBm for steatosis. The relationship between clinical variables and elastography results were evaluated by bivariate correlation and multivariate analysis, using SPSS 27. Seventy-nine patients (n = 79) met the inclusion and exclusion criteria.Advanced fibrosis and hepatic steatosis were detected in 17,7% and in 21,5% of the patients, respectively. There was a direct and significant correlation between CAP and BMI, waist circumference, HbA1c, triglycerides levels, and insulin doses and an inverse correlation with HDL. The waist circumference, low levels of HDL cholesterol and the insulin dose maintained a significant association with CAP values in multivariate analysis. Elastography values showed an inverse correlation with HDL and a direct correlation with BMI and insulin dose. The association was only maintained for the insulin dose in multivariate analysis.Our results suggest that clinical factors such as insulin dose, waist circumference, and HDL cholesterol levels could identify T2D patients more likely to present NAFLD. Nonalcoholic fatty liver disease (NAFLD) is defined by the fat accumulation in hepatocytes without a secondary cause, such as alcohol consumption, use of steatogenic drugs or genetic syndromes . It is aThere is a bidirectional relationship between diabetes and NAFLD. People with T2D and NAFLD have a higher risk of progressing to steatohepatitis, cirrhosis and death. On the other hand, NAFLD seems to be associated with a higher risk of progressing to diabetes and its macro and microvascular complications . The preThe scientific society's guidelines are controversial about NAFLD screening in patients with T2D ,13 due tThere are different methods used to assess NAFLD. Liver biopsy is the “gold standard” for the assessment of steatosis and fibrosis and the only method capable of identifying the presence of steatohepatitis . HoweverThe present study aims to contribute to NAFLD surveillance in patients with T2D, assessing the association of clinical and biological variables with hepatic stiffness and steatosis.This is a cross-sectional study with electronic medical data of the Diabetes Unit outpatients from Policlínica Piquet Carneiro, Rio de Janeiro State University (UERJ). The study included patients with T2D who underwent TE between June 2018 and December 2019. The exclusion criteria included previous history of liver disease, history of alcohol abuse or use of steatosis-inducing drugs described in the medical record . The stu® . The median value of 10 successful acquisitions, with a success rate of at least 60% and an interquartile range of less than 30% expressed in kilopascals (kpa), was used to represent hepatic stiffness. Cutoff points for the definition of steatosis and advanced fibrosis were CAP > 275 dB/m and hepatic stiffness ≥ 8 kpa, respectively  and its classification as normal (18.5 to <25), overweight (25 to <30) or obesity (≥30) , degree of cardiovascular risk (CVR) , waist cty (≥30) . Accordity (≥30) .®, TRC NW 8 ImageNet Lite system, with a single ophthalmologist reading the image. DR was defined as present when at least one eye was affected by any degree of injury. Diabetes neuropathy and protective sensitivity of the feet were evaluated by the distal polyneuropathy assessment scale, as validated by Moreira and cols. (Glomerular filtration rate (GFR) was calculated by CKD-EPI formula . The urind cols. , and witnd cols. , respectStatistical analysis was performed using SPSS version 27 . Distribution of variables was tested for normality using the Kolmogorov-Smirnov test. An exploratory analysis was performed and continuous variables with normal distribution were expressed as mean and standard deviation, but those with asymmetric distribution were expressed as median (interquartile range). Categorical variables were expressed as frequencies. Student's t-test or Mann-Whitney test was used to compare means or medians between groups, when indicated. When comparing three groups, one-way analysis of variance (F) or Kruskal-Wallis (Z) was used according to the normality of the analyzed variable. In the same way, a bivariate Pearson or Spearman correlation was used to assess the correlations between the studied variables and the CAP and elastography values. Variables with p value < 0.1 were included in the linear regression multivariate analysis, in which variables without normal distribution were converted to logarithmic scale to fit the model. The multicollinearity test between variables included in the multivariate analysis was performed. A two-sided p value < 0.05 was considered statistically significant.Eight two patients underwent liver elastography between June 2018 and December 2019. Seventy-nine met the inclusion criteria. vs. 209.6 ± 50.0, p = 0.04, respectively) and in those with MS compared to those without . When comparing patients with normal BMI, overweight and obesity, significant differences were observed in CAP means between groups . Patients with GFR < 60 had lower CAP means compared to patients with GFR ≥60 . Individuals of normal weight or overweight had lower values of elastography than those with obesity [5 (vs. 5(vs. 6 (p = 0.012)]. No significant difference was observed when the elastography values were compared between the groups described above.There were no significant differences in CAP values according to sex; diabetes duration (< 10 or ≥10 years); HbA1c values (<7% or ≥7%); hypertension; neuropathy or altered protective sensitivity of the feet; and subgroups with ACR <30 or ≥30 mg/g or ACR <300 or ≥300 mg/g. Higher mean CAPs were observed in patients using insulin compared to those who did not  was higher in patients with advanced fibrosis (31 and Lombard and cols. found a higher risk of CVD and microvascular complications in patients with hepatic fibrosis (NAFLD has been associated with an increased risk of microvascular complications and CVD in T2D patients, but published data are heterogeneous. Ciardullo and cols.  found thfibrosis . In our The present study has several limitations. The associations obtained are at most moderate, the sample size is small and the exclusion of other causes of liver disease was based only on patient's medical records. The patients of the present study were from a specialized outpatient clinic at a tertiary care center, with prolonged disease duration, high prevalence of microvascular complications and high cardiovascular risk. Therefore our findings may not apply to populations with different characteristics. No causal relationship can be ascribed to the associations found, which requires prospective studies with larger samples. On the other hand, the associations found between clinical characteristics easily identifiable in medical practice and the presence of steatosis and/or fibrosis can help to determine which patients with T2D benefit the most from NAFLD diagnostic tests.Our study contributes to the study of NAFLD in people with T2D with the use of CAP and elastography, considering that those methods have been increasingly used in literature. As controversies still exist about the screening of NAFLD in T2D patients, our results suggest that clinical factors such as insulin dose, waist circumference, and HDL cholesterol levels could identify T2D patients more likely to present NAFLD. However, studies with a larger number of patients are needed to confirm these results and possibly identify the cutoff points for each of the variables that would indicate screening for NAFLD.