Bioengineered in vitro models of the kidney offer unprecedented opportunities to better mimic the in vivo microenvironment. Kidney-on-a-chip technology reproduces 2D or 3D features which can replicate features of the tissue architecture, composition, and dynamic mechanical forces experienced by cells in vivo. Kidney cells are exposed to mechanical stimuli such as substrate stiffness, shear stress, compression, and stretch, which regulate multiple cellular functions. Incorporating mechanical stimuli in kidney-on-a-chip is critically important for recapitulating the physiological or pathological microenvironment. This review will explore approaches to applying mechanical stimuli to different cell types using kidney-on-a-chip models and how these systems are used to study kidney physiology, model disease, and screen for drug toxicity. We further discuss sensor integration into kidney-on-a-chip for monitoring cellular responses to mechanical or other pathological stimuli. We discuss the advantages, limitations, and challenges associated with incorporating mechanical stimuli in kidney-on-a-chip models for a variety of applications. Overall, this review aims to highlight the importance of mechanical stimuli and sensor integration in the design and implementation of kidney-on-a-chip devices. The kidney is a complex organ consisting of more than 20 cell types, including glomerular endothelial cells, podocytes, mesangial cells, and multiple segment-specific tubular epithelial cells. Collectively, these cells filter the blood, excrete toxins and metabolic waste, and reabsorb water and essential solutes from the glomerular filtrate, among other critical physiological functions ,2. The kMechanical forces can affect kidney cell behavior that ultimately impacts organ function. Cells sense and translate mechanical inputs to activate cellular signaling through mechanotransduction . DiseaseKidney-on-a-chip technologies hold great potential to facilitate the design of kidney models that incorporate physiologically important aspects of the tissue microenvironment in vitro for studying basic mechanisms of kidney function and disease. In addition to studying kidney physiology and pathophysiology, kidney-on-a-chip devices have been widely utilized as platforms for nephrotoxicity screening. Kidney-on-a-chip has the potential to serve as miniaturized, low-cost bridges between overly simplistic in vitro cell culture models and expensive and complicated animal models. Additionally, fabrication techniques are amenable to rapid prototyping and iterative device design.Devices can include single or multiple cell types inside a microchamber or microchannels with or without continuous flow and ECM to recapitulate the tissue microenvironment. Various kidney-on-chip devices have been developed to model the glomerulus ,10,11,12This review provides an overview of mechanical stimuli applied in kidney-on-a-chip devices and 3D in vitro models of the kidney. We provide an overview of passive biomechanical stimuli including incorporating synthetic, hybrid, and biological materials for modulating stiffness, surface topography, and confined geometry. We then discuss active stimuli, including fluid shear stress (FSS), compression, and cyclic stretch that are incorporated into kidney-on-a-chip models . We alsoIn vivo, kidney cells are subjected to complex and dynamic biomechanical stimuli that regulate cell behavior and function ,34. Mech2 [2 [2 in the proximal tubule [Active mechanical stimuli include FSS, tensile stretch, and compression. The frequency, magnitude, and duration of active stimuli vary considerably between different cell types within the kidney. Glomerular endothelial cells are subject to apical FSS from blood flow that can be 30–50 dyn/cm2 ,39,40, a2 [2 . Shear sl tubule . Each inl tubule ,44,45. AKidney cells are supported by cell-specific ECM networks or basement membranes that provide both structural support and signaling platforms that regulate cell function . GlomeruThe mechanical properties of the ECM regulate a host of cellular processes including proliferation, migration, survival, and differentiation ,53. MultMultiple studies have evaluated the effects of stiffness on various kidney cell types including podocytes and tubular epithelial cells. Hydrolyzed PAA scaffolds with stiffness ranging between 0.6–44 kPa have been used to study podocyte differentiation and morphology . The autCells cultured in vitro face the limitation of loss of differentiated function and reduced expression of cell-specific proteins ,72. EngiRecently, efforts have focused on incorporating the natural properties of the ECM or basement membrane composition and architecture into organ-on-a-chip and 3D culture models . We deveAdherent cells attach and spread on the ECM, and controlling cell spreading using micropatterned ECM has significant effects on cell behavior ,79. A loThe importance of active mechanical stimuli in models of kidney-on-a-chip is highlighted by numerous studies. Generally, these devices employ photolithography, soft lithography, or 3D printing to mold microchannels. Cells cultured on the surface of single or multiple microfluidic channels are perfused with a rotary or syringe pump to apply FSS to the apical cell surface. Other devices have been fabricated from hollow fibers with curved topography along with shear stress to simulate the physiological environment of the proximal tubule ,83. Thesτ = 6 μQ/bh2, where τ is FSS, μ is the medium viscosity, Q is the flow rate, b is the width of the channel, and h is the height of the channel. Evaluating fluid forces due to blood in the glomerulus requires more complex modeling. Different techniques have been used to evaluate fluid forces in the glomerular capillary including FSS on the glomerular endothelium [Blood flow in the kidney microvasculature or filtrate in the tubules creates a fluid force at the cell surface parallel to the direction of flow commonly referred to as fluid shear stress (FSS). FSS has been widely applied for physiological and pathological studies of the glomerulus, primary tubule, distal tubule, and collecting duct ,86,87,88othelium ,39,40. S2 resulted in podocyte loss, cytoskeletal reorganization, and the activation of specific tyrosine kinases [2 FSS, with or without retinoic acid, showed increased expression of podocyte-specific markers including synaptopodin, podocin, and WT-1 at the gene and/or protein level. This shows that mechanical stimulation with FSS effectively influences podocyte differentiation. As compared to static culture, podocyte-specific markers were significantly increased under FSS. Studies have further shown that shear stress regulates prostaglandin E2 and proteoglycan signaling in podocytes [The glomerulus is the filtering unit of the nephron and consists of fenestrated endothelial cells that line the capillary, the GBM, and podocytes on the filtrate side of the filtration barrier . Both gl kinases . Huang e kinases . FSS has kinases . Podocytodocytes ,93. Glomodocytes . Glomeruodocytes , as wellodocytes .Significant effort has focused on modeling the proximal tubule due to its important role in water and salt reabsorption and the susceptibility of the proximal tubule to drug and toxin-induced injury ,97. The 2 for 5 h. Their results showed reinforcement of peripheral actin bands and a tighter spatial distribution of ZO-1 and E-cadherin at cell junctions [2 FSS for 3 h. FSS increased apical localization in NHE3, upregulated Na/K-ATPase expression and translocation, and induced V-ATPase trafficking. Several other studies have also observed changes in actin cytoskeletal architecture in response to FSS [Several studies have shown that proximal tubule cells are highly responsive to FSS. Duan et al. quantitatively evaluated changes in the actin cytoskeleton and cell–cell junctions in response to shear stress. Proximal tubule cells were exposed to FSS at 1 dyn/cmunctions . The samunctions . Cells ce to FSS ,100. Seve to FSS ,102,103.e to FSS ,102.2 of shear stress for 5 h after culturing on the membrane for 3 days. The cells expressed specific markers: AQP2 localized at the apical side and the Na-K-pump localized at the basolateral membrane, which was not observed in the cells cultured on glass. In addition to 2D models, a 3D kidney cortical collecting duct model was developed by Rein et al. using a pin-pullout technique [2). The model exhibited tight barrier function after diffusing FITC-dextran for 1 h. The cells showed polarization and transmembrane receptor expression.There are relatively few distal tubule kidney-on-a-chip devices compared to proximal tubule models. Baudoin et al. developed a microfluidic distal tubule in vitro model by culturing MDCK cells in a PDMS microchip with a flow rate of 10 μL/min . At a hiechnique . The 3D 2 for 48 h, the expression of tip cell marker genes was upregulated, but stalk cell marker genes were downregulated. The method provides a solution for culturing several plates under shear stress. Orbital shear stress has also been used to apply shear forces to kidney epithelial cells [Most microfluidic devices apply FSS using an external syringe pump or peristaltic pump. Other pumpless techniques have been used to apply FSS to kidney cells. Kimura et al. demonstrated a pumpless microfluidic device for culturing ureteric bud cells from mouse embryonic kidneys . The mical cells ,106. ThiMicrofluidic devices precisely control the cell microenvironment and provide more reliable drug toxicity screening and modeling of pathological conditions such as renal fibrosis and proteinuria. One of the primary applications of kidney-on-a-chip devices has been for screening drug nephrotoxicity. Proximal tubule toxicity is a primary off-target effect of many drugs such as chemotherapeutics and antibiotics. Kidney-on-a-chip devices for drug screening and nephrotoxicity have been reviewed elsewhere ,107,108.2 were applied to the endothelial chamber. The results showed that with the FSS at 0.001 dyn/cm2, the barrier was restrictive to BSA and IgG transport, but permeability increased at high flow rates. The loss of selectivity was accompanied by a redistribution of F-actin, reduced CD-31 expression, and increased vWF expression in endothelial cells. Podocytes also showed cytoskeletal changes and reduced synaptopodin, nephrin, and podocin expression. These data suggest that high FSS and increased stretch increase glomerular permeability by damaging both endothelial cells and podocytes.High blood pressure is one of the most common causes of chronic kidney disease. Hypertension may cause glomerular dysfunction by increasing filtration pressure. Zhou et al. developed a functional microfluidic chip to mimic hypertensive nephropathy . The devFSS was applied within the microfluidic device to model fibrosis in the renal tubule . The dev2). The permselectivity of the filtration barrier was evaluated based on FITC-albumin transport across the filtration barrier. They used different combinations of cells and showed that the highest selectivity was achieved when podocytes and glomerular endothelial cells were co-cultured. They also demonstrated increased permeability when cells were treated with the puromycin aminonucleoside (PAN) or with serum from patients with membranous nephropathy. Xie et al. developed a glomerulus-mimicking knot with microscale hollow fibers with complex concave and convex topography to mimic the glomerulus in vitro [The glomerular filter provides a size, charge, and shape-selective barrier that filters the blood by allowing small molecules and water to cross the barrier while retaining large proteins, such as albumin (3.5 nm hydrodynamic radius), in the plasma . Podocytin vitro . PodocytThe pressure drop across the glomerular filter creates both compression and stretch in the capillary wall. Studies have shown that hypertension increases the pressure gradient to enhance mechanical stress on podocytes, resulting in podocyte loss . As incr2 FSS in the top and bottom chambers, respectively. A 10% cyclic strain (1 Hz) with shear stress exhibited a significant increase in podocyte-specific marker expression and increased VEGF-A secretion with flow and an additional increase when both flow and stretch were applied. This suggests that FSS and cyclic stretch have compounding beneficial effects on cell differentiation. They further showed that the permeability of the barrier increased with exposure to the cancer drug adriamycin.Most kidney-on-a-chip devices are designed as a single channel with applied shear stress that lacks the systematic analysis of cell functions. As the glomerulus is exposed to pulsatile blood flow, cyclic stress and shear stress need to be considered in recapitulating the human physiological response. Musah et al. developed a glomerulus-on-a-chip using stem-cell-derived podocytes to model the glomerular capillary wall. This system incorporated both FSS and cyclic stretch and showed synergistic effects of flow and strain on device function . A porouIntegrating sensors into organ-on-a-chip devices is advantageous for real-time monitoring of cellular activity on a chip. Studies have incorporated physical, electrical, electrochemical, and optical sensors into microfluidic devices ,32. For 2 FSS. An optical pH sensor was connected to the outflow of the device. After 5 days, high glucose media were used to create a pro-inflammatory environment. Then, metformin-containing media were circulated to mitigate the pro-inflammatory response. The device detected the TEER and pH value changes when the cells were cultured with high glucose media or metformin. Cohen et al. developed a kidney-on-a-chip by combining vascularized proximal tubule spheroid with tissue-embedded sensors for investigating drug-induced nephrotoxicity [2 FSS. The bioreactor outflow was connected to a biosensor array containing electrochemical sensors, and an on-chip potentiostat (PSTAT). The electrochemical sensor arrays detected glutamine, glutamate, glucose, and lactate. The sensor-integrated microfluidic device captured the loss of polarization leading to glucose accumulation and subsequent lipid buildup and toxicity. Such multifunctional devices have the potential to elucidate disease mechanisms that may guide therapeutic responses.Additional functionalities are beginning to be integrated into kidney-on-a-chip devices. Asif et al. designed a platform for the real-time monitoring of TEER and pH values of media in a proximal tubule microfluidic model . The tratoxicity . The devKidney-on-a-chip models have wide-ranging applications for studying fundamental renal physiology, drug screening, disease modeling, and tissue engineering. It has become increasingly clear that incorporating mechanical signals into organs-on-a-chip is important in many organs and tissues, including the kidney . These sOne of the challenges of recapitulating kidney function on a chip is the complexity of renal tissue and the large number of interconnected cell processes that regulate overall tissue function. Most kidney-on-a-chip devices focus on a single tissue compartment such as the glomerulus or the proximal tubule. However, complex interactions between tissue compartments ultimately control organ-level function. For example, interactions between the tubule and the glomerulus are important for regulating filtration rate based on tubular salt concentrations through reciprocal signaling between the glomerulus and the tubule . As anotWith regard to modeling the biophysical factors that regulate kidney function, better characterization of the in vivo microenvironment is likely to improve our ability to replicate these parameters in vitro. Kidney tubular fluid shear stresses are relatively well defined based on previous micropuncture studies of kidney flow rates and the more recent application of intravital imaging for the real-time monitoring of kidney function in live animals ,129. TheFinally, there is a paucity of active instrumentation and sensing mechanisms incorporated into kidney chips for real-time readouts of functional parameters. One exception is the integration of TEER as a surrogate for epithelial permeability. The ability to integrate sensors onto kidney chips to evaluate tubular epithelial drug toxicity in real time, monitor the loss of glomerular permselectivity through fluorescence analysis or albumin biosensing, or measure specific analytes in the filtrate such as creatinine or urea could add significant additional functionality to kidney-on-a-chip. This sensing technology could also provide a real-time measure of how mechanical factors applied within devices alter important cell behavior such as barrier function or monitoring cell viability. Other organ-on-a-chip systems have begun to incorporate such sensing mechanisms, and these approaches are likely to carry over to kidney-on-a-chip.


Metabolic disease encompasses excess adiposity, dysglycaemia, type 2 diabetes mellitus, dyslipidaemia, non-alcoholic fatty liver disease (NAFLD), and hypertension that translate to excess cardiovascular disease and increased mortality. The prevalence of metabolic disease is increasing in prevalence, driven by excessive intake of energy dense food and physical inactivity, and is a major challenge for healthcare services. Physical inactivity is a major contributor to the development of metabolic disease and regular exercise can prevent, delay, and control metabolic disease.Parimbelli et al., training based on short-duration high-intensity exercise improves overall metabolism and aerobic fitness in a case of carnitine palmitoyltransferase II deficiency. Also, Gao et al. reported in their first meta-analysis that focus on effect of long-term exercise on liver lipid metabolism in Chinese patients with. NAFLD  is a relatively new addition to the metabolic disease cluster. NAFLD is not only associated with liver manifestations such as steatohepatitis, cirrhosis, and hepatocellular carcinoma, but is also associated with excess cardiovascular mortality.Based on extensive evidence of health benefits, physical activity is widely recommended today for individuals with or at high risk of developing metabolic disease. Furthermore, exercise prescription, particularly aerobic exercise, has been used as an efficient approach to counteract many metabolic impairments including type 2 diabetes . UndertaPark et al. reported that above moderate-active physical activity (PA) levels were associated with lower body weight, body fat mass, percent body fat and higher free-fat mass and higher HDL-C than low-active PA. Also, above high-active PA levels were associated with lower cardiometabolic risk factors, waist circumference, total cholesterol, and triglycerides than low- and moderate-active PA. In terms of the risk of osteoporosis, sarcopenia, obesity, sarcopenic obesity, and cardiac metabolic risk factors according to PA levels, the prevalence of cardiometabolic disease was significantly lower in high-active PA (vs. low- and moderate-active); waist circumference, and HDL-C were significantly better in moderate- and high-active PA (vs. low-active), respectively, and triglycerides significantly better in high-active PA (vs. low- and moderate-active). Today, it still too much to do before being able to clarify all mechanisms involved in such improvement. A recent study of Luo et al. concluded that an exercise training accompanied by dietary fat regulation modulate TRIB3 and macrophage phenotype to attenuate insulin resistance. This latter, is widely viewed as a common basis of the etiology of metabolism diseases related to obesity . MacIntosh et al. provide a framework for understanding “moderate to vigorous” physical activity intensities and advanced strategies in terms of individual identification for exercise prescription. The authors recommend that expressing the exercise according to ventilatory threshold 1 and 2 (VT1 and VT2) or lactate threshold 1 and 2 (LT1 and LT2) is most useful for accurate exercise prescription.To summarize, current evidence suggest that long-term exercise and high intensity exercise can improve several key health indicators. Concurrent with the new evidence, establishing an updated “consensus of PA/exercise intervention”  become necessary. An accurate exercise prescription (moderate exercise/intensity vs. vigorous exercise/intensity) is still confusing to enable the determination of the exercise mode. In fact, the common practice of prescribing exercise at a fixed metabolic rate (# of METs) or percentage of maximal heart rate or of maximal oxygen uptake (V̇O2max) does not acknowledge the individual variability of these metabolic boundaries. As training adaptations occur, these boundaries will change in absolute and relative terms (Despite studies reporting numerous benefits of exercise for both the prevention and the treatment of metabolic disease, large variability in the inter-individual changes and improvement at post-intervention has been reported. It is important to highlight that one of the major challenges were in controlling for factors that differ between participants as well as in exercise prescription. Other issues are small sample sizes and lack of long-term follow-up to assess the impact of the exercise intervention. Prescribing exercise is promising for both prevention and reversal of metabolic disease. The challenge will be to incorporate this into daily lives to ensure that potential long-term benefits are realized.


Generalized estimating equation modeling analyzed the association between these changes and out-of-pocket medical cost. Out-of-pocket cost was significantly higher among older adults with multiple chronic conditions (p < 0.0001). Total out-of-pocket medical cost and out-of-pocket cost for outpatient care and prescription drugs were significantly higher for Bad→ Bad or Good → Bad changes. Older adults with cognitive decline had significantly higher total out-of-pocket medical cost and out-of-pocket cost for prescription drugs. This study demonstrates the need to improve the multiple chronic conditions management construction model to enhance the health of older adults in Korea and secure national health care finances long-term. It provides a foundation for related medical and medical expenses-related systems.This study aims to analyze the relationship between cognitive function and out-of-pocket cost of the state change of multiple chronic conditions in individuals aged 60 or older. Data from the 2014 to 2018 Korean Longitudinal Study of Aging were used for 2202 older adults who were cognitively “normal” at the start of the survey. Four status change groups were established (“Good  A common public health issue around the world is aging. In south-east Asia, it has been reported that the pace of acceleration of aging along with the decline in fertility rates is faster than in other countries . As the As the number of chronic diseases increases, the proportion of people with multimorbidity, in which one person has two or more chronic diseases, has also increased . Older aAlong with chronic diseases, many representative geriatric diseases involve cognitive decline, such as mild cognitive impairment or dementia. Dementia affected approximately 50 million people worldwide in 2018, which is projected to increase to approximately 150 million by 2050 . Older aThis study aimed to analyze the association of changes in MCC status with out-of-pocket medical cost using longitudinal data on Korean elderly people over four years (2014–2018) with a subgroup analysis of whether cognitive function declines. Our findings emphasize the need for effective management of MCCs to identify factors affecting older adults’ out-of-pocket expenses and reduce the burden.https://survey.keis.or.kr/eng/index.jsp (accessed on 14 March 2021).This study used raw data from a longitudinal study—the Korea Longitudinal Study of Aging (KLoSA)—in 2014, 2016, and 2018 . The KLoFirst, we excluded participants who (a) were aged <60 years, (b) had low cognitive impairment , and (c) were missing data on medical cost in 2014, 2016, and 2018. Additionally, only those who participated in the 2014, 2016, and 2018 surveys were included. For the final analysis, we had 2202 participants.Medical expenses were calculated by summing admission medical, outpatient medical, visit treatment, and regular prescription drug expenses. The medical expenses investigated by the KLoSA are out-of-pocket expenses with responses based on the respondent’s recollection.The main predictor variable was change in individual status of having MCCs between any two waves of the KLoSA. The chronic diseases assessed in the KLoSA were hypertension, diabetes, cancer, chronic obstructive pulmonary disease, liver disease, heart disease, cerebrovascular disease, psychiatric disease, and arthritis. The presence of each chronic disease was assessed by asking the participant whether they had been diagnosed by a physician or were currently under treatment  for these diseases. Participants with more and less than two chronic diseases were defined as having a “bad (more than two chronic diseases)” or “good (0–1 chronic disease)” medical condition, respectively. In this study, MCCs were defined in four categories: “Good → Good”; “Good → Bad”; “Bad → Good”; or “Bad → Bad.”2), normal (18.5–24.9 kg/m2), or obese (≥25 kg/m2). We also included individuals’ current smoking status (yes or no), alcohol consumption , and self-rated health , regular physical activity , activities of daily living (ADL)/instrumental ADL (IADL) status .The covariates assessed in this study were gender (men or women), age , marital status , education level (less than middle school/more than high school), employment status (employed or unemployed), household income (discretized based on quartiles), and region . To quantify health-related covariates, body mass index (BMI) was classified as underweight  model with odds ratios (ORs) and 95% confidence intervals (CIs) with log link, γ distribution, and robust standard errors to examine the relationship between MCCs and medical cost, adjusted for all covariate variables. A GEE model with log link was also performed for subgroup analysis by cognitive status. All analyses were performed using SAS software, version 9.4 . All p < 0.00001). There were significant differences in average cost for out-of-pocket total medical costs according to age, educational level, household income, BMI, self-rated health, current smoking, current alcohol consumption, and ADL/IADL status . However, there was no significant association between change in MCCs and out-of-pocket hospital admission medical costs. In terms of cognitive function, the out-of-pocket total and pharmacy medical cost were significantly higher for those with cognitive impairment than those with normal cognitive function . In terms of out-of-pocket total and pharmacy medical costs, women’s medical costs were statistically significantly lower than men’s , not only Korea but also BRICs countries such as Brazil, Russia, India, South Africa, and China reported that medical expenses per capita will increase . In the Changes in MCC status influenced all out-of-pocket medical costs  with the exception of inpatient care. Total out-of-pocket medical cost and out-of-pocket cost for outpatient care and prescription drugs significantly increased among those whose MCCs changed from Bad to Bad or Good to Bad compared to the reference group (Good → Good). Additionally, we adjusted for cognitive function status and observed that total out-of-pocket cost and out-of-pocket cost for prescription drugs were significantly higher among older adults with cognitive decline than those with normal cognitive function.In this study, total out-of-pocket medical cost was lower among women than men. Previous studies reported that MCCs common among men are chronic obstructive pulmonary disease, cardiovascular disease, hypertension, and heart failure while those common among women are mental disorders , arthritis, and varicose veins; in other words, the MCCs that are more prevalent among men are associated with greater medical needs and prescription cost ,18,19. OPrescription drugs used to treat cognitive decline are generally costly, and polypharmacy is often necessary for older adults because they require concurrent treatments for a number of conditions, including fall, depression, and MCCs . In addiOn the other hand, we observed no significant association between changes in MCC status and out-of-pocket cost for inpatient care. Contradicting our findings, Leibson et al. (2015) reported that the cost of inpatient care increases while the cost of outpatient care decreases among those with cognitive impairment . This diThere was no interaction term, only MCC status changes, and the medical cost for all variables increased when the status changed from Bad to Bad or from Good to Bad, suggesting that older adults and their families must pay more attention to the treatment of MCCs and that healthcare policies must be updated . As our While it has been reported that medical expenditure increases with age , our resThe reason for the inconsistency in study findings, including ours, pertains to the limitation in comparing results due to the lack of a clear definition and criteria for MCCs as well as the disparity in healthcare delivery systems and medical fee reimbursement systems across countries . Having Total out-of-pocket medical cost was also higher among unemployed and obese individuals, those with a poor self-rated health, and those with poor ADL/IADL, and lower among those with low income. Fostering a supportive environment that promotes health behaviors among older adults to lower the risk of a single chronic condition as well as MCCs is recommended ,29. LifeThis study has a few limitations. Older adults with MCCs have been reported to frequently utilize the emergency department (ED) and, thus, have high medical expenditure . The KLoEven in countries with very similar population distribution and health systems, long-term medical expenditures have various outcomes. This leads to lower credibility of comparability of cross-border health expenditure studies even within Organization for Economic Cooperation and Development (OECD) countries ,16. AmonSystems or programs that help older adults practice health behaviors are needed, and, for older elderly individuals who cannot engage in such practices independently, measures must be devised to promote their families and supporting organizations to reduce risk factors and practice health behaviors. According to previous studies, technologies and platforms that access drug use management through smartphones or tablets have recently been implemented . ThroughThis study aims to investigate the effects of changes in MCC status on out-of-pocket medical costs. There was no significant association between changes of MCC status and out-of-pocket cost for inpatient care, but the out-of-pocket cost for outpatient care and prescription drugs were significantly higher among patients whose MCC statuses were consistently bad and or whose statuses changed from good to bad.Patients with MCCs with high out-of-pocket medical cost are bound to experience a deterioration in quality of life and self-rated health , and theEmploying single-disease-targeting approaches to manage older adults with MCCs is ineffective, and a more systematic and comprehensive approach is required . In partFurthermore, based on these findings, more specific studies on productivity losses caused by MCCs should be conducted by examining not only direct medical costs but also indirect medical costs.


Blood-brain barrier penetrating peptides (BBPs), a class of peptides that can cross BBB through various mechanisms without damaging BBB, are effective drug candidates for CNS diseases. However, identification of BBPs by experimental methods is time-consuming and laborious. To discover more BBPs as drugs for CNS disease, it is urgent to develop computational methods that can quickly and accurately identify BBPs and non-BBPs. In the present study, we created a training dataset that consists of 326 BBPs derived from previous databases and published manuscripts and 326 non-BBPs collected from UniProt, to construct a BBP predictor based on sequence information. We also constructed an independent testing dataset with 99 BBPs and 99 non-BBPs. Multiple machine learning methods were compared based on the training dataset via a nested cross-validation. The final BBP predictor was constructed based on the training dataset and the results showed that random forest (RF) method outperformed other classification algorithms on the training and independent testing dataset. Compared with previous BBP prediction tools, the RF-based predictor, named BBPpredict, performs considerably better than state-of-the-art BBP predictors. BBPpredict is expected to contribute to the discovery of novel BBPs, or at least can be a useful complement to the existing methods in this area. BBPpredict is freely available at  Blood-brain barrier (BBB) highly protects the central nervous system (CNS) , preventBlood-brain barrier penetrating peptides (BBPs) can cross the BBB through various mechanisms without destroying the integrity of BBB . It has There have been two BBP databases published to date, Brainpeps  and B3Pdhttp://i.uestc.edu.cn/BBPpredict/cgi-bin/BBPpredict.pl. By comparing the results of the nested five-fold cross-validation and independent testing dataset of various machine learning predictors, the RF-based model showed the best prediction performance. Thus, BBPpredict was implemented by using RF. We expect BBPpredict will help researchers find more novel BBPs.In this work, we collected more BBPs from existing databases  and publIn this work, we selected experimentally validated BBPs as candidate positive samples that were collected from Brainpeps , B3PdbK, public http://i.uestc.edu.cn/BBPpredict/download.html.To evaluate the performance of our predictor and existing predictors (BBPpred and B3Pred), 99 BBPs that collected through published literatures and 99 non-BBPs randomly selected from candidate negative samples construct an independent testing dataset that was completely independent of the training dataset of the three predictor models  . The remk-spaced amino acid group pairs , pseudo-amino acid composition (PAAC) and grouped amino acid composition (GAAC) to extract the characteristics of peptide sequence. Here we set the length of a peptide to be N, and all feature extraction methods are based on 20 natural amino acids . Feature extraction was implemented by an in-house script.Feature extraction refers to the transformation of peptide sequences into fixed-length feature vectors, which is an indispensable step for the construction of predictors. In this study, we selected five feature encoding methods, including amino acid composition (AAC), dipeptide composition (DPC), composition of N(i) is the number of the amino acid type i.AAC calculates the frequency of each amino acid in the peptide sequence . It can Nrs is the number of the dipeptide consisting of amino acids r and s in the peptide sequence.DPC gives 400 descriptors  . It is dN(i) is the number of the amino acid type i.For the GAAC encoding, 20 natural amino acids are firstly divided into five categories according to their physicochemical properties: amino acid groups g1 (GAVLMI), g2 (FYW), g3 (KRH), g4 (DE) and g5 (STCPNQ). Group g1 belongs to the aliphatic group, g2 aromatic group, g3 positive charge group, g4 negative charged group and g5 uncharged group, respectively. GAAC represents the frequency of each amino acid group  and can k-spaced group pairs. And the detailed calculation of CKSAAGP can refer to  was utilized to accomplish linearSVM and rbfSVM , RF, k-nearest neighbors (KNN), adaptive boosting (AdaBoost), gentle adaptive boosting (GentleBoost), adaptive logistic regression (LogitBoost), linear support vector machine (linearSVM) and radial basis function (RBF) kernel SVM (rbfSVM) were used to build the predictive models based on the features selected by feature selection see in , respectd rbfSVM . DT, RF,d rbfSVM . The LSTTP describes the number of genuine BBPs which are predicted as BBPs. FN represents the number of genuine BBPs that are identified as non-BBPs. Denote TN as the number of true non-BBPs classified as non-BBPs and FP the number of true non-BBPs identified as BBPs. SN and SP primarily assess the ability of a predictive model to identify positive and negative samples respectively, while ACC and MCC investigate the comprehensive capacity of a prediction model to classify both positive and negative samples , sensitivity (SN), specificity (SP), Matthews correlation coefficient (MCC) and the area under the receiver operating characteristic (ROC) curve (AUC), were utilized to quantify the performance of each predictive model. The first four indicators are calculated as follows: samples . The AUCData analysis reproducibility plays a vital role for achieving an independent verification of the analysis results . In thisThe framework of this study is depicted in The performance of the nine predictive models in the nested five-fold cross-validation is shown in To determine the final model for constructing BBPpredict, performance evaluation on the independent testing dataset is much more convincing than five-fold cross-validation. According to the steps in the method section, nine classification models are established by using the training dataset. The independent testing dataset was then utilized to test the performance of these models. As depicted in We also used the RF algorithm with optimal features selected by Pearson and Lasso feature scoring methods to construct prediction model. As shown in There are two published predictors for identifying BBPs, B3Pred and BBPpred. These predictors and our predictor are based on peptide sequence information. The comparison of datasets of existing predictors and our proposed predictor can be seen in http://i.uestc.edu.cn/BBPpredict/cgi-bin/BBPpredict.pl, conveniently. The web service of BBPpredict was developed by using Perl and Html, Python and Matlab. Users can paste peptide sequences or upload a sequence file to predict BBPs, as illustrated in To facilitate users to identify BBPs, we established an online web service named BBPpredict that was implemented based on optimized features and the RF model. BBPpredict can be accessed at BBPpredict allows users to adjust the threshold of the probability value (tp) to distinguish between predicted positives and negatives, which can range from 0 to 1. As shown in In the past 30 years, many studies have demonstrated that BBPs are promising for the treatment of CNS diseases. BBPs can pass through the BBB and enter brain parenchyma without destroying BBB. Them can be used as transport carriers of DNA, RNA and protein as well as drug-assisted treatment and diagnosis of CNS diseases. However, the discovery of BBPs is still a thorny problem. Only a few hundreds of peptides have been experimentally confirmed as BBPs so far, since BBPs were discovered in 1996 . TherefoAt present, two BBPs predictors, BBPpred  and B3PrThe BBPpredict website allows users to set the tp value. We tested the performance of BBPpredict in the independent testing dataset and provided sensitivity and specificity values under different tp values, which can serve as reference for users and increases the confidence they can have about the positive predictions.We also reconstructed the BBPs/non-BBPs classification models with different machine learning methods using the new feature vectors that were generated from 16 feature extraction methods, including AAC, DPC, CKSAAGP, PAAC, GAAC, Grouped Di-Peptide Composition (GDPC) , DipeptiBBPs pass through BBB via six penetration mechanisms, including diffusion transport, carrier-mediated transcytosis, efflux transporter, receptor-mediated transcytosis, adsorptive-mediated transcytosis and cell-mediated transcytosis . The abiIn the present work, we utilized RF algorithm to construct BBP predictor. The RF is an ensemble algorithm which is composed of several weak classifiers (decision trees). Our constructed model contains 63 decision trees. We speculate that these different decision trees might cover different penetration mechanisms and it might be the reason why the RF algorithm is superior to other machine learning algorithms. In the future, if the number of BBPs with a certain transport mechanism increase, it is possible and preferable to construct new BBP predictors using BBPs with the same penetrating mechanism.http://i.uestc.edu.cn/BBPpredict/cgi-bin/BBPpredict.pl. To find the optimal classifier, eight traditional machine learning algorithms and one deep learning algorithm were used for developing models. The RF algorithm was selected to construct BBPpredict after comparing the results of nine classifiers in the five-fold cross-validation and independent test. The RF-based model reached an AUC of 0.9030 with an accuracy of 81.90% and an AUC of 0.8332 with an accuracy of 77.27% in the nested five-fold cross-validation and independent testing dataset, respectively. We also compared BBPpredict with two existing BBPs predictors, BBPpred and B3Pred. The results showed that BBPpredict was remarkably higher in accuracy, MCC, sensitivity and specificity than these two predictors. BBPpredict is a promising classification model, and we expect it to play a positive role in the discovery of BBPs to facilitate the development of drugs for CNS diseases.In this study, we proposed an RF-based predictor for identifying BBPs, called BBPpredict, which is available for free at


Non-invasive cardiac imaging has a growing role in diagnosis, differential diagnosis, therapy planning, and follow-up in children and adolescents with congenital and acquired cardiac diseases. This review is based on a systematic analysis of international peer-reviewed articles and additionally presents own clinical experiences. It provides an overview of technical advances, emerging clinical applications, and the aspect of artificial intelligence.The main imaging modalities are echocardiography, CT, and MRI. For echocardiography, strain imaging allows a novel non-invasive assessment of tissue integrity, 3D imaging rapid holistic overviews of anatomy. Fast cardiac CT imaging new techniques—especially for coronary assessment as the main clinical indication—have significantly improved spatial and temporal resolution in adjunct with a major reduction in ionizing dose. For cardiac MRI, assessment of tissue integrity even without contrast agent application by mapping sequences is a major technical breakthrough. Fetal cardiac MRI is an emerging technology, which allows structural and functional assessment of fetal hearts including even 4D flow analyses. Last but not least, artificial intelligence will play an important role for improvements of data acquisition and interpretation in the near future.Non-invasive cardiac imaging plays an integral part in the workup of children with heart disease. In recent years, its main application congenital heart disease has been widened for acquired cardiac diseases. Non-invasive imaging has gained a fundamental and increasing role as a routine diagnostic in pediatric and adolescent heart disease. Here, congenital heart disease (CHD) served as a kind of pacemaker for the increasingly broader role of these imaging techniques. Depending on national structural requirements, it might be centered at tertiary hospitals or might be offered in the broad. Typically, non-invasive imaging compasses echocardiographic, computed tomography, and MR imaging.Current guidelines provide a role of non-invasive cardiac imaging for diagnosis, differential diagnosis, and prognosis. Current hot topics in non-invasive cardiac imaging are improvements in spatial and especially temporal resolution, a decrease in contrast agent amount, and the integration of artificial intelligence (AI). The latter addresses data acquisition and data interpretation, as well as data fusion.The current article reviews established applications of non-invasive cardiac imaging in children and adolescents with a focus on congenital heart disease. Moreover, current technical innovations such as fetal cardiac MRI imaging and AI-based methods are discussed.Current guidelines emphasize the role of non-invasive imaging in pediatric cardiac diseases. Echocardiography is the primary imaging method with cardiac CT and MRI as cross-sectional imaging modalities for further analysis. Especially the latter show significant recent technical improvements.https://www.ncbi.nlm.nih.gov/pubmed/). The search was limited to original human studies in peer review journals with an available abstract. No publication date limits were applied. Our aim was to identify studies assessing the value of imaging methods in children and adolescents with heart disease, with also a focus on emerging imaging techniques. Inclusion criteria in detail were (1) original research, (2) sufficiently large enough patient groups, and (3) focus on a pediatric population.The systematic literature search was performed according to the PRISMA 2020 guidelines . In shorInitially, article screening was performed by two independent readers (M.S. and M.B.) considering only the title and abstract after the removal of duplicates. Both authors read all titles and abstracts independently. All articles that did not meet the inclusion criteria were excluded, and the remaining articles were chosen for reading of the full text.After independent reading of the full text, articles fulfilling the inclusion criteria (as mentioned above) were selected. Disagreements were resolved by consensus. Finally, references of included articles were hand-searched to check for further eligible studies.Transthoracic echocardiography represents the non-invasive tool most commonly used in the diagnosis and follow-up of patients with any suspected or known heart disease. It enables the assessment of the morphology and function of the heart and is widely used as a diagnostic imaging modality in pediatric cardiology.Compared with standard protocols in adult cardiology , the echstrain imaging and three-dimensional echocardiography.Recent developments in functional echo assessment include Strain imaging is commonly used for the assessment of ventricular function allowing additional measurement of regional differences in contraction (dyssynergy) or dyssynchrony. Strain imaging Fig.  measuresThree-dimensional echocardiography (3DE) enables detailed anatomical assessment of cardiac pathology, particularly valvular defects, and cardiomyopathies. The ability to slice the virtual heart in infinite planes in an anatomically appropriate manner and to reconstruct three-dimensional images of anatomic structures makes it unique for the understanding of the congenitally malformed heart .3DE has become important in the management of patients with congenital heart disease, particularly with pre-surgical planning (including 3D printing), to guide the location of bioptomes during right ventricular endomyocardial biopsies and for the guidance of catheter intervention  and functional assessment of the heart. 3DE is increasingly used in children because of good acoustic windows and the non-invasive nature of the technique. Novel measurements of 3D deformation, atrial and ventricular volumes, including atrio-ventricular coupling have led to the publication of normal values across a wide range of ages and body sizes , 7.Cardiac CT and especially MRI have gained a central role in the diagnosis and differential diagnosis of multiple cardiac diseases in children and adolescents, according to SCMR (Society for Cardiovascular Magnetic Resonance) and other guidelines . ApproprIn nuce, the major application for cardiac CT in children is the delineation of coronary anatomy. Here, the detection or exclusion of coronary anomalies  is the major task . For thaBesides the standard single-source CT scanners , dual-source high-end scanners (DSCT) are available since almost two decades . Dose reduction and high temporal resolution are advantages of DSCT scanner technology; however, high costs are a major drawback and limit widespread availability. Most recently photon-counting CT technology has been introduced, which might further reduce radiation dose and increase image quality and thus overall diagnostic accuracy .It is mandatory to use distinctive CT protocols for pediatric examinations. The newest CT scanner generations  plus denThe CT examination encompasses distinct steps, which are summarized in Figs. For the postprocessing, individually arranged image analyses are standard Fig. ; additioThe most established application field of cardiac MRI is congenital heart disease –19. BesiThe semi-automatic character of these new state-of-the-art cardiac MRI techniques  in pediatric cardiac MRI is and will be offered on a broader range beyond specialized centers. This will pave the way for on-site assessment of even complex cardiac conditions.Another rapidly growing field is the fusion of imaging modalities, e.g., echocardiography with cardiac MRI. Especially planning and execution of pediatric cardiac interventions significantly benefit from this kind of comprehensive imaging guidance , 24. ArtThe basic components of cardiac MRI protocols are cinematographic imaging (CINE), 2D flow and late gadolinium enhancement (LGE), and 2D/3D flow. Extended protocols also include 4D flow and mapping.Figure Especially for myocardial inflammation and cardio-oncology assessment of tissue integrity by T2-weighted imaging plays an important role besides LGE Fig. .Fig. 6 has already been cleared from the myocardium .Mapping techniques are increasingly replacing traditional LGE and T2-weighted imaging Fig. . Their aAdd-ons to standard MRI protocols are MR angiography (MRA) and 2- up to 4D flow analyses. This is especially important in CHD. State-of-the-art MRA techniques cover more than one cycle of contrast agent inflow (time-resolved angiography).Flow analyses are important in valve disease and in the assessment of shunts. Traditionally, 2D techniques are applied, which allow a fast and robust determination of flow hemodynamics , and when applied in orthogonal orientation. Recently introduced 4D flow techniques display a volume (with a future perspective of whole heart coverage). Yet, the latter has to be integrated into routine scanning.Congenital heart disease (CHD) is the major application of non-invasive imaging, especially for cardiac MRI. This is due to the challenging complex anatomy of CHD, which also affords the assessment of hemodynamic and structural parameters, a specific strength of cardiac MRI. Besides LGE and mapping as surrogates for fibrosis , 3D or even advanced 4D flow is an essential module.A first example for CHD assessment by cardiac MRI is a 6-year-old girl Figs. , and 10 More complex CHD is presented in the case vignette of a 14-year-old girl Figs.  and 12 wThe implantation of devices (for pacing etc.) in patients with CHD is an increasing challenge for cardiac MRI. Examinations in children with implanted devices afford close interaction between pediatric cardiology, partially adult cardiology, and pediatric radiology. Expert consensus statements concerning the practical handling are helpful .Coronaries and their assessment are still an unmet need by cardiac MRI. Here, cardiac CT is also in pediatric populations the method of choice for non-invasive assessment. A typical example is an 11-year-old girl Fig. , who wasA leading application of non-invasive imaging is the workup of inflammatory heart disease. Besides the “traditional” viral myocarditis, COVID-19-associated acute as well as chronic myocarditis has developed into a major challenge for clinical care. Imaging, i.e. cardiac MRI, is one of the pillows in clinical decision-making. The Lake Louise 2.0 criteria have made things simpler. A contrast-free CMR assessment is now possible for the diagnosis of myocarditis, integrating T1 native mapping sequences and T2-weighted imaging. Thus, COVID-19 gave some impetus for this development from contrast towards contrast-free cardiac MRI.A first case vignette Figs.  and 15 pThe value of the 2018 Lake Louise criteria for diagnosis and follow-up of adolescent patients has been shown by a study, demonstrating the enhancement of the diagnostic performance adding mapping techniques . MappingCompared to the workup of myocardial inflammation and CHD, the non-invasive assessment of pediatric cardio-oncology is a new emerging field. Toro-Salazar et al.  demonstrNon-invasive fetal cardiac assessment by MRI is an exciting field that has a new impetus by the advent of different fetal cardiac gating strategies . As ECG These gating approaches allow state-of-the-art cardiac imaging with the assessment of cardiac morphology and function, i.e., applying steady-state free precession (SSFP) cine imaging Fig.  or 2D upData acquisition is one major application for AI-based algorithms . A signiNon-invasive cardiac imaging in children and adolescents is one of the pillars of modern diagnostics and therapy planning in inflammatory and structural heart disease and also congenital heart disease. New areas of clinical application are cardio-oncology and fetal cardiac MRI. Available state-of-the-art techniques have decreased the amount of contrast agents and irradiation. Additionally, examination time and also data interpretation time have improved.Technical developments may help to extend applications of these non-invasive imaging technologies from specialized centers to the broad area. This will be necessary concerning the increasing number of adults with congenital heart disease with normal lifespans. AI-driven technologies will support herein.


Due to its flexible design, it supports analysis of label-free, data-dependent, data-independent and targeted proteomics datasets. protti can be run on the output of any search engine and software package commonly used for bottom-up proteomics experiments such as Spectronaut, Skyline, MaxQuant or Proteome Discoverer, adequately exported to table format.We present a flexible, user-friendly R package called https://CRAN.R-project.org/package=protti) and work on all major operating systems. The development version is maintained on GitHub (https://github.com/jpquast/protti). Full documentation including examples is provided in the form of vignettes on our package website (jpquast.github.io/protti/).protti is implemented as an open-source R package. Release versions are available via CRAN ( With novel and evolving proteomics methods such as limited proteolysis coupled to mass spectrometry , and the feature the measured intensity reports on  if different from protein intensity. The quality control, data analysis and data interpretation modules of protti can be used serially or independently.The modularity of protti enables its use in conjunction with user-defined workflows and functions. protti is written as simply as possible using tidyverse packages (We describe protti, a flexible, user-friendly R package for analysis of various bottom-up proteomics datasets. protti provides functions for quality control, data analysis on protein, peptide, precursor or fragment levels, as well as data interpretation within a single, easy-to-use package. We include examples of data analysis workflows as vignettes to illustrate the utility of the different functions for the analysis of protein- or peptide-centric datasets.


Do ART-conceived children have an increased risk for puberty disorders?Both ART-conceived boys and girls had a higher risk of puberty disorders; early puberty was more common among girls and late puberty among boys.Some physiological differences in growth and metabolism have been reported for ART-conceived children compared to non-ART-conceived children. Knowledge on pubertal development and disorders in ART-conceived children is limited.A register-based cohort study was carried out including data from 1985 to 2015. The Committee of Nordic Assisted Reproductive Technology and Safety (CoNARTaS) study population consists of all live and stillborn children, as well as their mothers, registered in the Medical Birth Registers during the study period in Denmark, Sweden, Finland and Norway.A total of 122 321 ART-conceived singletons and 6 576 410 non-ART singletons born in Denmark (1994–2014), Finland (1990–2014), Norway (2002–2015) and Sweden (1985–2015) were included. Puberty disorders were defined using International Classification of Diseases and Related Health Problems (ICD)-9/ICD-10 codes and classified in the following groups: late puberty (6268/E30.0), early puberty (2591 and 2958/E30.1 and E30.8) and unspecified disorders (V212 and V579/E30.9 and Z00.3 as well as Z51.80 for Finland). The results in Cox regression were adjusted for maternal age, parity, smoking, gestational diabetes, chronic hypertension, hypertensive disorders during pregnancy and country, and further for either gestational age, birthweight, small for gestational age or large for gestational age.There were 37 869 children with diagnoses related to puberty disorders, and 603 of them were born after ART. ART-conceived children had higher risks for early (adjusted hazard ratio (aHR) 1.45, 95% CI: 1.29–1.64) and late puberty . Girls had more diagnoses related to early puberty  and boys with late puberty .Using reported puberty disorders with ICD codes in health care registers might vary, which may affect the numbers of cases found in the registers. Register data may give an underestimation both among ART and non-ART-conceived children, especially among non-ART children, who may not be as carefully followed as ART-conceived children. Adjustment for causes and duration of infertility, mothers’ own puberty characteristics and BMI, as well as children’s BMI, was not possible because data were not available or data were missing for the early years. It was also not possible to compare ART to non-ART siblings or to study the pubertal disorders by cause of subfertility owing to a small number of discordant sibling pairs and a large proportion of missing data on cause of subfertility.This large, register-based study suggests that ART-conceived children have a higher risk for puberty disorders. However, the mechanisms of infertility and pubertal onset are complex, and ART is a rapidly advancing field with various treatment options. Studying the pubertal disorders of ART-conceived offspring is a continuing challenge.This work was supported by the Nordic Trial Alliance: a pilot project jointly funded by the Nordic Council of Ministers and NordForsk (71450), the Central Norway Regional Health Authorities (46045000), the Nordic Federation of Obstetrics and Gynaecology , the Interreg Öresund-Kattegat-Skagerrak European Regional Development Fund (ReproUnion project), the Research Council of Norway’s Centre of Excellence funding scheme (262700), the Swedish state under the agreement between the Swedish government and the county councils, the ALF-agreement (ALFGBG-70940) and FLUX Consortium ‘Family Formation in Flux—Causes, Consequences and Possible Futures’, funded by the Strategic Research Council, Academy of Finland (DEMOGRAPHY 345130). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. The authors have no conflicts of interest to disclose.N/A. The safety of ART and their potential effects on long-term health are areas of growing interest. It is known that ART-conceived babies have a higher risk of preterm birth, low birthweight, being small and large for gestational age , and perinatal death compared with non-ART babies  study population consists of all live and stillborn children, as well as their mothers, registered in the Medical Birth Registers during the study period in Denmark, Sweden, Finland and Norway  diagnosis is registered.Early puberty is defined as onset of the first sign of puberty and/or early appearance of secondary sexual characteristics before the age of 8 years in girls and 9 years in boys, or as menarche before the age of 10 years in girls . DelayedSince Norway started including identification numbers in their patient register in 2008, Norwegian children born in 2001 or earlier were excluded. The final study data contained information on 122 321 ART-conceived live born singletons and 6 574 731 non-ART live born singletons in Denmark (1994–2014), Finland (1990–2014), Norway (2002–2015) and Sweden (1985–2015).We used Cox regression adjusted for potential confounders to estimate hazard ratios (HRs) with 95% CI for puberty disorders. In the first model, we used country, maternal age, parity, maternal pregestational diabetes (type 1 and 2 diabetes) and maternal smoking during pregnancy as confounding factors (Model 1). Maternal smoking was considered to be a confounder through biological influence and was additionally used as a proxy for confounding from maternal socioeconomic position, since data on maternal educational level at childbirth were not available for all countries. We further added adjustment for potential mediators, such as maternal chronic hypertension and hypertensive disorders of pregnancy (Model 2), to account for the potential of compromised fetal blood flow leading to intrauterine growth restriction (IUGR) or SGA, as well as adding in the model either gestational age (Model 3), birthweight (Model 4) or SGA (<10th percentile) and LGA (>90th percentile) (Model 5). For variables other than maternal smoking, where we included a separate category of cases with no information, the number of missing cases was small and complete case analyses without imputations were made. Follow-up started at birth and ended at first diagnosis, death, emigration (not available for Finland) or the age of 20 years. Analyses for IVF versus ICSI and fresh versus frozen embryo transfer were done for Denmark, Norway and Sweden, excluding Finland, since these data were not available for Finland. All analyses were made separately for girls and boys. We performed analyses for known or unknown cause of infertility with data from Denmark, Norway and Sweden. Data were too sparse to be able to estimate the associations according to the cause of infertility by sex and type of ART. We also aimed to perform a sibling analysis, but our data were too small for analysis, as there were only 8944 sibling-boy-pairs and 7860 sibling-girl-pairs with discordant exposure to ART (data not shown).Approvals for data retrieval and linkage were obtained in each country. In Denmark and Finland, ethical approval is not required for scientific projects solely based on registry data. In Norway, ethical approval was given by the Regional Committee for Medical and Health Research Ethics . In Sweden, approval was obtained from the Ethical committee in Gothenburg, Dnro 214-12, T422-12, T516-15, T233-16, T300-17, T1144-17, T1071-18, T121-18 and T2019 02347 (2019-04-23). All register-keeping organizations gave their permission to use their data in this study.The mothers of ART-conceived children were older, more often primiparas and non-smokers than mothers of non-ART children . The meaThere were 37 869 children with diagnoses related to puberty disorders, of which 603 were ART-conceived children. The crude incidences of late puberty , and unspecified puberty disorders  per 1000 livebirths were lower among ART-conceived children, but the incidence of early puberty was higher among ART children  . Early pAfter adjusting for maternal age, parity, smoking, pregestational diabetes and country in Model 1, ART-conceived children had a higher risk for all studied puberty disorders compared to non-ART children . FurtherIn sex-specific analyses, the crude risks for early, late and unspecified puberty disorders were higher among ART-conceived girls than non-ART girls, as were the crude risks for early and late puberty among ART-conceived boys compared to non-ART boys . For boyThe incidences of puberty disorders did not significantly differ between children born after IVF and ICSI . HoweverThe incidence of puberty disorders among ART-conceived children was higher after fresh than after frozen embryo transfers . HoweverIn this large register-based study, ART-conceived singletons had a higher risk of puberty disorders compared with non-ART singletons. These risks were higher among both ART-conceived girls and boys. Maternal characteristics and perinatal outcomes partly attenuated the associations. Compared to non-ART girls and non-ART boys, ART-conceived girls had higher risk for early puberty and ART-conceived boys had a higher risk for late puberty, and risks after ICSI were higher than after IVF.The strengths of this study are that it is based on four large Nordic national datasets with high coverage and validity, and that it uses population-based registries, which reduce the risk of selection bias (It has been suggested that poorer health outcomes or higher use of health services by ART-conceived children might partly be explained by parental worries or higher socioeconomic position (While we adjusted for several known confounders, such as pregestational diabetes and chronic hypertension, we could not adjust for cause of infertility, duration of infertility, the mothers’ own puberty characteristics and BMI or children’s own BMI, because data were not available or data were missing for the early years. Other limitations of our study include missing information on diagnoses given in primary health care only and on paternal characteristics and a relatively short follow-up time, especially among ART-conceived children. The difference in follow-up time between the ART and non-ART groups has been considered in the aHR.In our study, incidences of late puberty were higher among boys and early puberty among girls, both among ART and non-ART children. The onset and progression of puberty are determined by genetic and epigenetic factors and are influenced by extrinsic factors such as environmental, chemical, nutritional and cultural influences , but resOur results show that ART-conceived girls have a higher proportion of diagnoses related to early puberty than non-ART-conceived girls. This is in contradiction to previous studies in which mean age of menarche and pubarche in girls were similar regardless of conception method . Early rCompared to non-ART children, ICSI-conceived children in our study had a higher risk for pubertal disorders than IVF-conceived children, especially in ICSI-conceived boys presenting with late puberty. Previously, no signs of delayed puberty in ICSI-conceived children compared to other children were observed . In a prThe higher risks for puberty disorders among ART-conceived children in our study were partly explained by the measured maternal risk factors, as well as poorer perinatal outcomes, such as low birthweight and preterm birth, as found in earlier studies regarding the pubertal disorders among female offspring (In conclusion, both ART-conceived girls and boys had a higher risk of pubertal disorders compared with their non-ART peers. Measured maternal characteristics, gestational age and birthweight partly attenuated the associations. However, as the mechanisms of infertility and pubertal onset are complex and ART is a rapidly advancing field with various treatment options, studying the pubertal disorders of ART-conceived offspring is a continuing challenge.


The identification of causative genetic variants for hereditary diseases has revolutionized clinical medicine and an extensive collaborative framework with international cooperation has become a global trend to understand rare disorders. The Initiative on Rare and Undiagnosed Diseases (IRUD) was established in Japan to provide accurate diagnosis, discover causes, and ultimately provide cures for rare and undiagnosed diseases. The fundamental IRUD system consists of three pillars: IRUD diagnostic coordination, analysis centers (IRUD-ACs), and a data center (IRUD-DC). IRUD diagnostic coordination consists of clinical centers (IRUD-CLs) and clinical specialty subgroups (IRUD-CSSs). In addition, the IRUD coordinating center (IRUD-CC) manages the entire IRUD system and temporarily operates the IRUD resource center (IRUD-RC). By the end of March 2021, 6301 pedigrees consisting of 18,136 individuals were registered in the IRUD. The whole-exome sequencing method was completed in 5136 pedigrees, and a final diagnosis was established in 2247 pedigrees (43.8%). The total number of aberrated genes and pathogenic variants was 657 and 1718, among which 1113 (64.8%) were novel. In addition, 39 novel disease entities or phenotypes with 41 aberrated genes were identified. The 6-year endeavor of IRUD has been an overwhelming success, establishing an all-Japan comprehensive diagnostic and research system covering all geographic areas and clinical specialties/subspecialties. IRUD has accurately diagnosed diseases, identified novel aberrated genes or disease entities, discovered many candidate genes, and enriched phenotypic and pathogenic variant databases. Further promotion of the IRUD is essential for determining causes and developing cures for rare and undiagnosed diseases. Identification of the causative genetic variants of hereditary diseases has revolutionized clinical medicine to enhance diagnostic accuracy, understand disease pathogenesis, and develop therapies. The major technical breakthrough behind the revolution was the development of whole-genome sequencing (WGS) and whole-exome sequencing (WES) methods employing next-generation sequencing (NGS), enabling identification of causative genetic variants by simultaneously capturing all candidate variants potentially causing diseases in affected individuals. Additionally, tremendous amounts of variant data have been generated using sequencers and remarkable advances have been made in bioinformatics exploiting such big data with powerful computational data analysis methods. Accumulation of variant data via registration in public databases has also accelerated the discovery of pathogenic variants by filtering known variants that are not associated with diseases.https://www.omim.org/), 9514 hereditary diseases were registered as of November 2021, of which 3288 diseases with (suspected) Mendelian basis are classified as having an unknown molecular basis. A complete understanding of the molecular basis of these diseases is one of the ultimate goals of human molecular genetics, which remains highly challenging even in the NGS era. The difficulty lies in identifying multiple pedigrees with pathogenic variants in the same genes to verify causality, particularly when researchers attempt to discover causative genetic variants for ultra-rare diseases, which are thought to comprise most unsolved diseases [However, there still remain many diseases for which causative genetic variants have not been identified. According to Online Mendelian Inheritance in Man (OMIM) (URL: https://irdirc.org/about-us/history/) established a worldwide network to connect individual projects to conduct international collaborative studies, further promoting the discovery of causative genetic variants [To solve the N-of-1 problem, the formation of extensive collaborative frameworks with international cooperation has become a global trend . The Undvariants . In 2015variants  and set variants . Furthervariants . One of variants . MME adovariants  and faciEven when causative genetic variants have been established, genetic diagnosis of rare diseases remains difficult in clinical settings. One reason for this is that a limited number of patients undergo WGS- or WES-based genetic diagnosis. Accessibility is among the key factors that can drive the utilization of this innovative technology in clinical genetics, necessitating a nationwide infrastructure to regionally equalize this testing. Additionally, determining pathogenic variants among a large number of variants yielded by WGS/WES and establishing the final diagnosis in which the pathogenic variants fully account for clinical manifestations is labor-intensive. Particularly, a substantial number of rare and undiagnosed diseases present with complex phenotypes with multiple affected organs, making the determination difficult by researchers in a single specialty. Therefore, it is recommended that diagnostic boards composed of physicians with a wide range of specialties and geneticists should discuss the final diagnosis based on the phenotypes and WGS/WES data.Enhancing the diagnostic accuracy of rare diseases has been vigorously pursued in Japan. Remarkable achievements have been made in the research and countermeasures for rare and intractable diseases, designated as “Nan-byo,” by the Ministry of Health, Labor and Welfare in Japan, which was established in 1972 after the subacute myelo-optic neuropathy endemic. In 2015, a new intractable disease law was enforced to expand “Nan-byo” from 56 to 333 diseases, further promoting the diagnosis of rare and intractable diseases. Nevertheless, two surveys conducted by AMED showed that more than 37,000 cases remained undiagnosed .To address these issues, the Initiative on Rare and Undiagnosed Diseases (IRUD) was launched in 2015 as a nationwide project in Japan supported by AMED . IRUD aiThe IRUD entry criteria are as follows .1. The patient remains undiagnosed for ≥6 months (not necessary for infants) and suffers from disabilities in daily life, AND2–1. Objective signs exist that cannot be attributed to a single organ; OR2–2. Direct or indirect evidence exists of a genetic etiology (e.g. similar symptom(s) found in the patient’s relatives) .Here, an undiagnosed disease is clearly distinguished from an undetermined disease in which a clinical diagnosis has been made but its causative genetic variants have not been confirmed. For example, if spinocerebellar degeneration is clinically diagnosed, although its causative genetic variants have not been analyzed and disease type has not been determined, it is classified as an undetermined disease and excluded from the IRUD. This clearly distinguishes the IRUD from genetic diagnostic services.https://annovar.openbioinformatics.org/) together with RefSeq (http://www.ncbi.nlm.nih.gov/RefSeq/), 1000 Genomes Project Database (http://www.1000genomes.org/), dbSNP135 (http://www.ncbi.nlm.nih.gov/projects/SNP/), gnomAD (http://www.gnomad.broadinstitute.org), and in-house databases.Blood samples were obtained from the participants fulfilling the criteria mentioned above with informed consent, DNA samples were extracted, and B-cell lymphoblast cell lines were established. Genomic DNA was subjected to enrichment of exonic sequences using Agilent SureSelect . Massively parallel sequencing (100-base pair paired-end reads) was performed using NGS . The sequences were aligned to the reference genome (GRCh37/hg19) using the Burrows-Wheeler Aligner or NovoAlign. Removal of potential polymerase chain reaction duplicates, recalibration of base quality values, local realignment, and variation calls were performed using SAMtools, Picard, and GATK with default parameter settings for alignment of raw reads and detection of single-nucleotide variants and short insertion/deletion variants. These variants were annotated using ANNOVAR  was established in IRUD-A as one of the leading model projects in AMED to facilitate the ethical review process in multi-institutional large-scale collaborative research. All but one of the individual institutional ethical committees in IRUD-A delegated the reviewing process to the CEC. The delegation process had been further promoted upon the integration of IRUD-A and IRUD-P.A unified research protocol for IRUD was reviewed and approved by the CEC. The ethics committee of individual institutions delegated the review process to the CEC, where approval of the protocol allowed each institute to initiate IRUD research based on the unified protocol. Amendment of the unified protocol, such as authentication of newly participating institutes, is accomplished in a one-step review process as an entire IRUD project. This study was approved by the CEC at Tohoku University on February 20, 2018 (CEC No. 2017-2-303).The most important achievement of IRUD is establishment of a unified all-Japan diagnostic and research system for rare and undiagnosed diseases covering entire geographic areas and clinical specialty/subspecialty fields. The IRUD system consists of three pillars: IRUD diagnostic coordination, analysis centers (IRUD-ACs), and a data center (IRUD-DC). IRUD diagnostic coordination consists of clinical centers (IRUD-CLs) and clinical specialty subgroups (IRUD-CSSs). In addition, the IRUD coordinating center (IRUD-CC) manages the entire IRUD system and temporarily operates the IRUD Resource Center (IRUD-RC) Fig. .Fig. 1InThe principal role of IRUD-CC is administration of the whole system through monthly IRUD-PB meetings as the highest decision-making organization. The constituents of IRUD-PB include representatives of the IRUD-CC , AMED (program officers) as the funding agency, IRUD-CLs, IRUD-CSSs, IRUD-ACs, and IRUD-DCs. IRUD-CC drafts a unified research protocol that is ratified by IRUD-PB and subjected to CEC, with the one-step approval process contributing to timely modification of the IRUD research. In addition, IRUD-CC monitors the progress of the entire research by conducting a regular survey. IRUD-CC also operates the sample and information logistics system described in detail in a subsequent section.IRUD-CL/SCL is the only contact site for patients with IRUD. Upon patient entry, parents are recruited so that trio analysis can be performed. IRUD-CL/SCL operates the IRUD Diagnostic Board (IRUD-DB), which manages the process from patient entry decisions to final diagnosis establishment. IRUD-SCLs are designed to fill the geographic gaps of IRUD-CLs, although no funding was provided. IRUD Cooperative Hospitals refer candidates for IRUD entry to IRUD-CL/IRUD-SCL. The IRUD-DB plays a central role in the regional diagnostic network with IRUD Cooperative Hospitals. The IRUD-DB is composed of pediatricians and physicians of various specialties/subspecialties for adults, clinical geneticists, genetic counselors, and data scientists. The IRUD protocol stipulates the participation of representative physicians from local medical associations in each IRUD-DB to promote regional cooperation.IRUD-DB holds regular meetings during which thorough pre-entry evaluation is conducted based on the clinical information described in a regular format on a ‘patient sheet’ to determine whether the candidate is suitable for IRUD entry and if sufficient investigation has already been completed, including whether clinical workups and available genetic tests such as chromosome analysis or gene-panel analysis have been performed. Similarly, post-analysis evaluation is conducted at the regular meetings to determine whether the pathogenic variant reported by IRUD-AC fully accounts for the clinical phenotypes leading to the final clinical diagnosis. In addition, the activities of the IRUD-DB include genetic counseling, further follow-up and reevaluation of the pedigree, public relations, and human resource development. Thus, IRUD-CL/SCL plays an essential role in the clinical aspects of the IRUD system.IRUD-CSSs are organized by assembling members of IRUD-DBs across entire IRUD-CLs according to their specialties/subspecialties. IRUD-CSSs support the activities of individual IRUD-DBs and provide professional advice based on their specialties/subspecialties for cases that cannot be resolved by IRUD-CLs alone.Thus, IRUD-CLs and IRUD-CSSs form the IRUD Diagnostic Coordination covering entire geographic areas and specialty/subspecialty fields in Japan.Each IRUD-CL/IRUD-SCL sends DNA samples via an outsourcing provider to a corresponding single IRUD-AC, which conducts comprehensive genomic analysis, identifies pathogenic variants, and reports to the IRUD-CL/IRUD-SCL via the IRUD-CC. When pathogenic variants are undetermined, further intensive research is conducted to identify novel aberrated genes/pathogenic variants using WGS, multi-omics analysis, and functional studies.IRUD-DC operates the IRUD Exchange, the data-sharing platform described above, promotes data sharing among IRUD researchers, and serves as a gateway to domestic and international collaboration. Phenotypic and genomic information has been accumulated to promote the establishment of new causative genetic variants and disease concepts. The IRUD Exchange is also used as a database to understand the overall epidemiological landscape of rare and undiagnosed diseases registered in the IRUD. All IRUD-CLs and IRUD-SCLs have a computer terminal for the IRUD-Exchange, and phenotypic and genotype data are transferred to IRUD-DC through a specific virtual private network to ensure security.IRUD-RC establishes a resource repository for clinical information and DNA samples/B cell lines and manages a utilization committee for examining the utilization of repositories. The IRUD-CC temporarily serves as the IRUD-RC, which is planned to be established as an independent facility assigned by AMED.IRUD-CC organizes a unified workflow to facilitate sample and information sharing among the IRUD-CLs/IRUD-SCLs, IRUD-ACs, and IRUD-CC and establishes a centralized repository in IRUD-RC Fig. .Fig. 2SaUpon entry into the IRUD, an individual identification (ID) number, composed of a three-alphabetical institutional code and five-digit numerical number, is allotted to each registrant, whose DNA/cell line samples, clinical information, and analysis results are handled with a specific ID. The workflow utilizes an outsourcing provider to extract genomic DNA samples and establish B lymphoblast cell lines. The genomic DNA samples are sent to the IRUD-ACs and IRUD-CC, and B lymphoblast cell lines are sent to the IRUD-CC. Each IRUD-CL/IRUD-SCL sends the samples to a specific IRUD-AC designated by IRUD-CC. Clinical information in the form of a ‘patient sheet’ is also sent to IRUD-ACs via an outsourcing provider. The analysis results and clinical information are sent from the IRUD-ACs to the IRUD-CC and reported to the corresponding IRUD-CLs. Clinical information is accumulated in the IRUD-DC through IRUD-Exchange from the IRUD-CLs. Text-based clinical data on the patient sheet delivered from the IRUD-CL/SCL via the IRUD-AC and analysis reports delivered from the IRUD-AC are also stored in the IRUD-CC. Genomic DNA samples and B lymphoblast cell lines are deposited in the IRUD-RC.In March 2021, the IRUD diagnostic system comprised 450 institutions consisting of 37 IRUD-CLs, 15 IRUD-SCLs, and 398 cooperative hospitals  Fig. . Among ties Fig.  . Particularly, approximately one-half of pedigrees with an established diagnosis possess unique aberrated genes. Importantly, such diseases are individually ‘ultra-rare’ but not as a whole, necessitating further vigorous endeavors to provide accurate diagnosis and develop therapeutic measures for individual ‘ultra-rare’ diseases.The IRUD has established an accurate diagnosis for a large number of patients and, in some cases, led to the use of specific therapies with positive effects . Pathogehttps://www.ncbi.nlm.nih.gov/clinvar/), LOVD (https://www.lovd.nl/), and MGeND (https://mgend.med.kyoto-u.ac.jp/) as well as disease databases such as OMIM and Orphanet (https://www.orpha.net/).The IRUD has had important impacts on basic research by identifying many novel aberrated genes, establishing novel disease entities, and detecting novel pathogenic variants in known aberrated genes. Disease-causing genes are the most reliable pathogenic molecules with greatest impact on the development, course and prognosis of the disease. Identification of these genes has led to an increased understanding of disease pathogenesis and identification of druggable seeds, promoting research of rare and undiagnosed diseases, common diseases, and human physiology. Furthermore, the IRUD has greatly contributed to genomic medicine by identifying a large number of novel pathogenic variants in known aberrated genes. Approximately two-thirds of the identified pathogenic variants were novel, supporting the indispensable role of the IRUD in genomic medicine in addition to its diagnostic services. Aggregation of phenotypic and pathogenic variant data can enrich variant databases such as ClinVar (https://www.matchmakerexchange.org/participants.html). The collaboration network provides a collective dataset spanning more than 150,000 cases from more than 11,000 contributors in 88 countries [A remarkable feature of the IRUD is the extensive data sharing through the IRUD Exchange, which has accumulated HPO-based phenotypic and genetic data to enable searches for similar cases automatically by pattern-matching algorithms and solving N-of-1 problems. The IRUD Exchange is compatible with MME and functions as a gateway to domestic and international collaborative networks. The IRUD is one of the principal eight nodes of international genomic research projects connected via the MME . Activities in the IRUD encompass entire fields of genome medicine or research, including phenotyping, genetic analysis, informatics, diagnosis, and counseling. Experiences in the IRUD would be of great help in individual career development. The IRUD also provides excellent educational opportunities for medical geneticists, genetic counselors, and data scientists. Therefore, the IRUD is not only a nationwide diagnostic and research system, but also a sustainable human resource development system in Japan.Notwithstanding the exhaustive WES, nearly 3000 pedigrees remained undiagnosed. Particularly, 313 pedigrees belonged to the N-of-1 category, for which discovery of another pedigree with the same candidate genes should be definitely necessary. Further promotion of international collaboration is the key to address the N-of-1 issue. On top of that, reasons for the undetermined causes are thought to include mosaicism, genomic alterations, gene regulation, and complex inheritance, all of which are difficult to capture by WES . To addrList of genes and number of novel and known pathogenic variantsNovel genes and phenotypes


Background: Dysregulation of the ubiquitin-proteasome system (UPS) can lead to instability in the cell cycle and may act as a crucial factor in both tumorigenesis and tumor progression. However, there is no established prognostic signature based on UPS genes (UPSGs) for lung adenocarcinoma (LUAD) despite their value in other cancers.Methods: We retrospectively evaluated a total of 703 LUAD patients through multivariate Cox and Lasso regression analyses from two datasets, the Cancer Genome Atlas (n = 477) and GSE31210 (n = 226). An independent dataset (GSE50081) containing 128 LUAD samples were used for validation.Results: An eight-UPSG signature, including ARIH2, FBXO9, KRT8, MYLIP, PSMD2, RNF180, TRIM28, and UBE2V2, was established. Kaplan-Meier survival analysis and time-receiver operating characteristic curves for the training and validation datasets revealed that this risk signature presented with good performance in predicting overall and relapsed-free survival. Based on the signature and its associated clinical features, a nomogram and corresponding web-based calculator for predicting survival were established. Calibration plot and decision curve analyses showed that this model was clinically useful for both the training and validation datasets. Finally, a web-based calculator (https://ostool.shinyapps.io/lungcancer) was built to facilitate convenient clinical application of the signature.Conclusion: An UPSG based model was developed and validated in this study, which may be useful as a novel prognostic predictor for LUAD. Non-small cell lung cancer (NSCLC) constitutes approximately 40%–50% of total lung cancers, which are the leading cause of tumor-associated mortality worldwide . NSCLC iThe ubiquitin-proteasome system (UPS) is a major intracellular protein degradation pathway in eukaryotic organisms, which controls a wide range of physiological processes and disease conditions, such as transcription, translation, DNA repair, and the cell cycle . The UPSHere, we investigated changes in UPSGs expression in the data downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases and then built a scoring model by classifying LUAD patients based on a multi-UPSGs signature, in combination with other clinicopathological factors, to improve our ability to predict the prognosis of LUAD patients, thereby helping to guide clinical treatment. This model might be meaningful for the development of comprehensive therapeutic approaches for LUAD patients.The transcriptomic (HTSeq-FPKM), demographic, and clinical information of patients with LUAD was collected from TCGA (training set) and GEO databases  for evaluations. First, TCGA-LUAD dataset was curated to remove any cases with incomplete survival data or a follow-up time of less than <30 days, leaving a total of 477 patients with LUAD who were then included in this study. GSE50081 were applied as validation datasets , where Ghttps://www.cbioportal.org/)  . A totalal.org/)   .To narrow the number of candidate UPSGs, we conducted univariate Cox regression analysis on TCGA-LUAD and GSE31210 datasets. The overlapping overall survival (OS)-related genes were selected for further study. These targets were then cross-validated using least absolute shrinkage and selection operator (LASSO) regression, which then produced a list of potential predictors with nonzero coefficients using the R packages “glmnet” and “survival.” Finally, we performed multivariate Cox regression analysis on TCGA-LUAD dataset to confirm the identity of highly correlated genes and construct the OS gene signature using the following risk score model:The patients in each dataset were classified as high- or low-risk, using the median risk score as the cutoff value, and the receiver operating characteristic (ROC) curves were created using the “survivalROC” package in R. The AUC values were calculated to assess the predictive potential of the UPSG signature.The prognostic UPSG model was then applied to the GSE50081 validation dataset. Validation dataset was split into high- and low-risk groups for evaluation using Kaplan-Meier curve analysis, time-dependent ROC analysis, and patient outcome distribution to assess the categorization of the UPSG signature.The clinical usefulness of the prognostic UPSG signature was evaluated through stratification analysis that was performed to investigate the differences in the prognosis of LUAD patients presenting with different clinical characteristics. Based on both characteristics and risk score, the LUAD cases were divided into low- and high-risk groups, and Cox regression was applied to analyze these subgroups.To determine whether and how UPSGs affected the tumor immune microenvironment, Cell type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT) was used to predict the fractions of immune cell types between the high- and low-risk cohorts. Eventually, 22 types of differentially expressed immune cells associated with specific clinicopathologic characteristics in the high- and low-risk cohorts were identified and depicted as a landscape map.The UPSG signature and clinicopathological predictors were combined to construct a nomogram for TCGA training set. A calibration curve was prepared to assess the agreement between the nomogram model and actual observation. The time-ROC curves and calibration plots were generated, and decision curve analyses (DCA) was used to assess the predictive accuracy of the signature.We first defined OS-related UPSGs through a univariate Cox regression analysis from TCGA-LUAD and GSE31210 datasets. The two datasets comprised 95 and 157 OS-related UPSGs, respectively. Then, we reduced the number of OS-related UPSGs by taking the intersection of TCGA-LUAD and GSE31210 datasets. Eventually, 33 overlapping OS-related UPSGs were identified . SubsequPSMD2, RNF180, UBE2V2, KRT8, and MYLIP were the most frequently mutated genes. Notably, amplification was the most common type of mutation, and UBE2V2, PSMD2, and FBXO9 were frequently amplified in LUAD .Using the risk score calculation formula derived from the multivariate Cox regression, the risk score for each TCGA-LUAD patient was calculated. Based on the median risk score, patients were separated into high- and low-risk cohorts. The distribution of OS status ranked by risk score is presented in via clinicopathologic feature revealed that this UPSG signature exhibited significant predictive value in most subgroups  for estimating the OS of patients with LUAD  to predict the prognosis of LUAD based on the established nomogram  has been reported to suppress tumor cell proliferation , an E3 ligase whose expression levels are regulated by PABPN1, is one of the most important factors involved in regulating biological function . A previtigation . FBXO9 (tigation , has beetigation . Howevertigation . Wang ettigation ; these rtigation , has beetigation . Ni et aferation . PSMD2 hferation . RNF180 feration . Previouferation . TRIM28 feration . Chen etferation . Hua et The UPS is also regarded as an important player in the regulation of immune cell function and immune response . Here, wGiven these promising results, we constructed a more reliable and individualized clinical prediction model, using our eight-UPSG signature and several other clinical features to establish a nomogram. The AUC values of the nomogram for TCGA-LUAD data at 1-, 3-, and 5-year OS were 0.776, 0.758, and 0.762, respectively, while those for the GSE31210 dataset were 0.851, 0.792, and 0.735, respectively . In addiin vitro and in vivo studies. Second, the inclusion of clinical indicators in the model seems to be insufficient; other factors such as, radiotherapy, surgery type, and targeted drug use might affect the prognosis of LUAD. Third, only two GEO datasets were applied for validation; however, further validation using larger datasets is required.To the best of our knowledge, this is the first study to establish a UPSG signature for predicting the survival of patients with LUAD. However, this study had some limitations. First, we did not investigate the detailed mechanisms of each of the eight UPSGs identified in this study; these underlying mechanisms warrant further investigation through both Compared to a signature developed on a complete set of genes, our UPSG signature has a major advantage. We made a comprehensive exploration of the prognostic role of UPSGs in LUAD from a bioinformatic perspective. Compared to the large and complex genome signature, our risk had a better clinical operability which was calculated by several simple UPSGs. However, our research also has a major disadvantage. As our research was based on UPSGs, other prognosis genes were ignored and not included in this study.In summary, we established a novel signature model for LUAD prognosis based on an eight-UPSG signature, which accurately predicted prognosis of LUAD patients. This signature may serve as an effective tool for designing personalized therapies and guiding medical decisions for LUAD patients in the future. Moreover, these UPSGs in the signature are promising targets for pharmacological and gene therapy in the future.