Virus-like particles (VLPs) are increasingly used for vaccine development and drug delivery. Assembly of VLPs from purified monomers in a chemically defined reaction is advantageous compared to in vivo assembly, because it avoids encapsidation of host-derived components and enables loading with added cargoes. This review provides an overview of ex cella VLP production methods focusing on capsid protein production, factors that impact the in vitro assembly, and approaches to characterize in vitro VLPs. The uses of in vitro produced VLPs as vaccines and for therapeutic delivery are also reported. Virus-like particles (VLPs) are highly structured protein complexes that resemble a native virus capsid. VLPs typically represent gene-less virus shells but in a wider definition encompass any type of capsid-derived nanoparticle. VLPs assemble from single or multiple structural capsid proteins inside appropriate production hosts, which is often termed in vivo assembly, as the host is seen as a single-cell organism, or in defined, cell-free conditions, also termed in vitro assembly. VLPs in general can be either enveloped or non-enveloped , wherebyIn vivo-produced VLPs have been exploited for different biomedical applications, such as vaccines, drug delivery and nanomaterials ,4,5. HowAlong with progress in understanding virus assembly requirements, in vitro VLP production based on various viruses had been established. For the in vitro assembly, the capsid proteins are expressed and purified from expression hosts or cell-free protein synthesis systems (CFPS), or the proteins are obtained from in vivo VLPs via a disassembly procedure. This avoids trapping host-derived impurities, a potential source for unwanted immune response. Next, the capsid proteins are incubated under defined chemical conditions which promote VLP assembly. Compared to the in vivo VLPs, the in vitro technology offers the possibility to mix different capsid proteins or epitopes within a particle, for example resulting in a vaccine candidate for different viral genotypes ,9. For tThere are a few literature reviews on VLPs that focus on production  and variE. coli is a preferred host for high capsid-protein expression [E. coli. To increase the soluble protein yield during expression, different factors such as the choice of the E. coli strain, expression vector, codon usage, expression temperature, induction condition and medium can be optimized [E. coli owing to incorrect folding, this expression strategy may be exploited with a strong promoter to produce high yields per dry mass and ease initial purification. The inclusion bodies can be easily separated from the cell debris and solubilized using strong denaturants like 6 M guanidinium HCl or 6–8 M urea [To prepare protein monomers for a cell-free capsid assembly under defined and controllable conditions, viral capsid protein can be obtained either by expression and purification of non-assembled proteins or via a disassembly procedure from in vivo generated and purified VLPs. The choice of the hosts for capsid protein expression significantly affects all further steps. pression . Dependiptimized ,13. Furtptimized ,15,16. I8 M urea ,19,20,21Other favorable hosts for capsid protein production are Sf9 insect cells in combination with baculovirus vectors and yeast. Both organisms are amenable to scale up and offer the advantage of eukaryotic post-translational protein modification ,23,24. LE. coli were disassembled at low pH, while human papillomavirus (HPV) VLPs obtained from insect cells were dissociated with carbonate buffer at pH 9.6 [If VLPs assemble spontaneously inside the expression hosts, they may contain undesired compounds. To obtain protein subunits from VLPs formed inside the host cell, different disassembly methods exist. Bacteriophage MS2 VLPs produced in t pH 9.6 ,30. Cowpt pH 9.6 ,32,33. Ut pH 9.6 , whereast pH 9.6 . Reducint pH 9.6 ,39,40,41The capsid self-assembly is driven by Brownian motion and interactions between subunits as well as subunits and other viral or non-viral components to minimize free energy in higher structures. The capsid assembly is proposed to occur in three steps. First, a capsid oligomer nucleus is formed from capsid proteins in the so-called nucleation phase. Afterwards, building blocks  are added to the nucleus during the growth phase. Finally, the last building block is inserted to complete the capsid ,43,44. TpH plays an important role for in vitro assembly since it affects the capsid-protein charge. Most in-vitro assembly reactions were optimized at a physiological pH ,18,46. IIonic strength is another main factor for capsid assembly in vitro. Salts interact with charges on the protein surface, influence the water shell, disfavor hydrophobic exposure and ultimately affect protein stability . To optiThe effect of temperature on the capsid assembly also deserves evaluation. Low temperatures are normally favorable as they reduce protein aggregation and chemical degradation. For example, the in vitro assembly of primate erythroparvovirus 1 (B19) at 4 °C showed a better yield at 4 °C than at 37 °C . ContrarThe in vitro assembly of some capsids was highly influenced by the presence of nucleic acids. CCMV, a single-stranded RNA virus, is a well-studied model. Garmann et al. demonstrated that the assembly of CCMV depends on balanced capsid protein (CP)–CP interactions relative to CP–RNA interactions . FurtherScaffolding proteins are not a component of a mature capsid. These proteins assist during capsid formation. Providing a scaffolding protein to an in vitro capsid assembly in many cases increases the yield of fully assembled capsids. Our data on in vitro assembly of AAV2 showed that the addition of assembly-activating protein  helped to improve AAV2 capsid formation . The assSmall molecule additives can be used to aid capsid assembly. L-arginine improved the solubility of assembled VLPs by preventing aggregation during protein refolding ,65. In aTo analyze the formed in vitro VLPs, different biochemical and biophysical methods can be applied. Standard methods are sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to determine size and purity, Western blot to confirm the identity, and ultraviolet (UV) spectroscopy with light scattering compensation, which monitors amino acids  and nucleotides with an absorbance spectrum of about 240–300 nm, to determine concentrations ,72,73. OUnderstanding capsid self-assembly pathways will help to tailor the mechanisms of virus infection and replication for therapeutic applications. Different methods have been used to characterize intermediate assemblies and the assembly pathways of virus capsids, such as electron microscopy ,84,85,86VLPs are composed of protein monomer units in a repetitively ordered structure with a size range of 20–200 nm in diameter that is appropriate for vaccination . VLPs arA few VLP-based vaccines have a long and very successful tradition in the clinic and recently several new candidates progressed to the clinic. These vaccine candidates have been produced in vivo or by an in vitro system . CompareHuman papillomavirus (HPV) is a major cause of cervical cancer and associates with many human diseases . HPV capTrichoplusia ni (Hi-5) insect cell line with recombinant baculoviruses encoding the L1 proteins. The proteins are released by osmotic shock and upon a multi-step purification by filtration and chromatography the proteins assemble into spherical particles, which are mixed and formulated with adjuvant. The final product dose contains 20 µg HPV 16 L1 protein, 20 µg HPV 18 L1 protein, 500 µg aluminium hydroxide, 50 µg 3-O-desacyl-4′ -monophosphoryl lipid A (MPL) and 0.624 mg NaH2PO4·2H2O [The HPV vaccine Cervarix, which is manufactured by GlaxoSmithKline and partially based on technology from MedImmune/AstraZeneca, was approved in 2007 in the European Union (EU) and other countries and 2009 in the USA. It contains two monovalent antigen bulks of C-terminally truncated versions of the major capsid proteins L1 of either serotype 16 or 18. The proteins are produced in cells derived from the PO4·2H2O .Saccharomyces cerevisiae transformed with the pGAL110 expression vector coding for the respective proteins. Cells are harvested by filtration, frozen and then VLPs are released by homogenization. For some L1 types , a protease inhibitor is added. Purification comprises cross-flow membrane filtration for debris removal, cation exchange chromatography for host cell protein removal, and hydoxyapatite chromatography for polishing and enrichment of monodisperse VLPs. For all but type L1 serotype 18, the VLPs are disassembled using dithiothreitol (DTT) and reassembled by removing DTT, which improves VLP structure and stability. A final buffer exchange yields the final aqueous products which are then adsorbed onto the adjuvant amorphous aluminium hydroxyphosphate sulfate by in-line mixing. The individual products are then mixed by sequentially adding them to a tank with buffer and alum adjuvant, settling and decanting. The final 0.5 mL aqueous dose Gardasil 9 contains 30 µg L1 6, 40 µg L1 11, 60 µg L1 16, 40 µg L1 18, and 20 µg of L1 31, 33, 45, 52, 58 as well as 500 µg aluminium , 9.56 mg NaCl (for stability), 0.78 mg L-histidine (for buffering), 50 µg polysorbate 80 (for VLP stability and prevention of aggregation or surface adsorption), 35 µg Na-borate (for buffering) [The HPV vaccine Gardasil, which is produced by Merck Sharp and Dohme and was approved in the USA in 2010, contains the HPV L1 protein from serotypes 6, 11, 16, and 18. The vaccine Gardasil 9 , which was approved in the USA in 2014, additionally contains the HPV L1 proteins of serotypes of 31, 33, 45, 52, and 58. The proteins are individually produced in the yeast from ffering) ,110.E. coli and maintaining them in soluble pentamer form under a reducing condition (20 mM DTT). The HPV VLPs were formed by removal of the reductant during protein purification [E. coli offers a low manufacturing cost and easy scale-up compared to other licensed products [E. coli, lipopolysaccharide (LPS) might contaminate into VLPs that can enhance immune responses and cause pyrogenic and shock reactions in mammals. To reduce LPS contamination, different methods, such as size exclusion chromatography, affinity chromatography, binding to polymyxin and treatment with Triton X-114 have been used [As a biosimilar, the company Innovax produced Cecolin, a bivalent HPV16 and HPV18 vaccine, by expressing the respective L1 proteins in products . Similarproducts . As a reeen used .E. coli or insect cells as recombinant HEV vaccine candidates [E. coli and assembled into VLPs of 20–30 nm diameter via multiple purification steps [E. coli to form in vitro HEV VLPs [Hepatitis E virus (HEV), which can be quasi-enveloped, is the most acute hepatitis cause in both developing and developed countries ,132. A 7ndidates . Hecolinndidates . p239 tron steps ,112. AnoHEV VLPs .Saccharomyces cerevisiae strain DC5. Purification steps comprised cell disruption, diafiltration, size exclusion chromatography, ion-exchange chromatography, a CsCl ultracentrifugation followed by a final size exclusion chromatography. In the absence of chemical treatment, HBsAg formed spheres of about 20–22 nm containing non-glycosylated HBsAg and a lipid matrix consisting mainly of phospholipids [Plasmodium falciparum malaria parasite. In this case, the mixed particles formed already during expression [Also surface antigens from enveloped viruses have been used in mostly defined assembly reactions. The surface antigen of the hepatitis B virus (HBV) which causes chronic infection of about 3.5% world population  named HBholipids ,117. Thiholipids ,119. Thepression . Hence, Recently, the company Novavax developed the NVX-CoV2373 vaccine candidate  for preventing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the virus which spread from China around the globe in 2020 causing the coronavirus disease 2019 (COVID-19) pandemic with millions of infections and deaths ,138. TheE. coli-based cell-free protein synthesis reaction and assembled into HBV VLPs [E. coli, and the VLPs were formed by dialyzing of the proteins from a denaturing buffer (5 M GuHCl) to PBS buffer [E. coli in soluble form. The CPV VLPs were developed during SUMO tag removal and dialysis of the capsid proteins into a physiological buffer [Other vaccine candidates with different in-vitro assembly conditions are under preclinical evaluation. The C-terminally truncated hepatitis B core antigen (HBcAg) was produced using HBV VLPs . The priS buffer . A vaccil buffer .VLPs are attractive candidates to deliver drugs, small molecules or nucleic acids due to their biocompatibility, biodegradability and targeted delivery ,140. DifE. coli. The VLPs were subsequently purified and disassembled in glacial acetic acid. Since MS2 VLPs are able to reassemble and encapsulate negatively charged cargoes in vitro at neutral pH, drugs, proteins or siRNA were loaded into the capsids for therapeutic delivery [E. coli, were denatured with urea, and reassembly and drug encapsulation was achieved by dialysis of the denatured proteins with a neutral buffer. The RGD peptide was genetically incorporated to HBc VLPs to target tumors [During protein expression, some capsid proteins tend to assemble VLPs inside the hosts. To remove potential host-related impurities and encapsulate cargoes, the disassembly and reassembly of VLPs are needed. The use of bacteriophage MS2 VLPs in cargo delivery was reported by different groups. Generally, MS2 capsid proteins were produced and formed 27.5 nm MS2 VLPs in delivery ,76,143. delivery ,143. Witdelivery ,145. Anodelivery . Hepatitt tumors . In vitrt tumors ,146,147.t tumors .E. coli by expressing soluble capsid proteins, followed by purification and assembly in the presence of cargoes. To target subcutaneous cancers, folic acid (FA) was conjugated to CCMV capsid proteins [E. coli and the purified capsomeres were dialyzed resulting in the formation of VLPs containing the guest protein for delivery [E. coli, and the protein was then purified under denaturing condition (8 M urea). DOX was conjugated to denatured VP proteins and the assembly of VLPs occurred during protein dialysis into a low pH buffer [CCMV VLPs are widely tested for therapeutic delivery due to their pH-dependent capsid assembly . CCMV VLproteins ,150. Othdelivery . Zhao etH buffer .ΔC589, VP1wt or VP1N138C of SV40 were produced in Sf9 cells and pentamers were purified and assembled around magnetic nano-particles in MOPS (3-Morpholinopropane-1-sulfonic acid) buffer. The latter two particles were crosslinked with epidermal growth factor (EGF) for the targeting of EGFR expressing cells using heterobifunctional crosslinkers with N-hydoxysuccinimide and maleimide groups [E. coli have been used to encapsulate quantum dots for imaging [For diagnostic purposes such as magnetic resonance imaging (MRI), magnetic nanoparticles can also be coated with capsid proteins and additionally endowed with a targeting function. VP1e groups . Similar imaging . Brome m imaging ,154.VLPs have been widely exploited for vaccine development and therapeutic delivery with success in the clinic and promising preclinical evaluations. In vitro VLP production technology has emerged as a versatile technology besides the in vivo method. The formation of VLPs under controllable and defined conditions enables the technology to create multi-vaccine candidates by combining different antigens within a particle while avoiding the unpredictable immune response related to possible host-related contaminations. In vitro assembly of VLPs also offers a feasible tool to control the packaging amount and cargo components for therapeutic delivery.In vitro VLP assembly is a complex process and differs among viral capsids. Even though many in-vitro VLPs have been produced, it is still unclear whether all virus capsids are amenable to ex cella production. Many factors need to be optimized for each candidate to assemble VLPs in vitro, improve assembly yields, and enable large-scale production. Cell-free protein synthesis in combination with in vitro VLP assembly bears potential for the generation of various synthetic biology products at a smaller scale. In vitro VLPs have been widely used as VLP-based protein vaccines. When in vitro nucleic acid encapsidation becomes more accessible, in vitro VLPs might also become an efficient and safe technology for vector VLP vaccines, which deliver an antigen-coding sequence, or for gene therapy vectors for treating diseases. Also for other therapeutic delivery strategies, recent advances provided mostly a proof of concept. Further work is needed to fully tap the potential of in vitro VLPs, but the future looks bright.


Infliximab (IFX) and adalimumab (ADA) refer to the classic drugs to treat moderate-severe inflammatory bowel disease (IBD), which have been proven to be effective to control IBD. However, the side effects exerted by IFX and ADA should be monitored in therapies, especially the paradoxical reaction of the skin system . Psoriasis is recognized as the most common skin lesion, capable of significantly affecting the quality of patients’ life.This study searched literatures published in English language with the qualifications on PubMed, Embase, Web of Science, Google, and Geenmedical databases. Over 2 co-authors assessed the quality of the articles and extracted the data independently. The data acquired were statistically analyzed with the statistical software of Revman and Stata.P < .05); Smoking up-regulated the incidence of psoriasis ; The interval of medication was over 1 year, and the interval of medication applying IFX was longer than that of the ADA Group; most cases could be relieved by using local hormone, phototherapy, or systemic hormone therapy under the strategy of biological agents.The ADA Group achieved a higher incidence of psoriasis ; Females achieved a higher incidence of psoriasis than males (OR = 1.941, 95%CI [1.326–2.843], The frequency of reported in IBD exceeds those of other autoimmune diseases, and the ADA treatment for IBD is safer than IFX. Psoriasis is more common in females than in males. Smoking refers to one of risk factors of psoriasis. On the whole, IBD comprises ulcerative colitis (UC), Crohn disease (CD), and undifferentiated types, severely affecting the quality of life of patients and generally requiring ongoing combination therapy. Tumor necrosis factor-C (TNF-α) antagonists are critical to treating a wide range of autoimmune inflammatory diseases . As suggested from the 2018 Inflammatory Bowel Disease Consensus, biological agents should be considered to treat moderate to severe UC. Given the American Gastroenterological Association clinical guidelines, for patients with moderate to severe diseases and not responding to mesalazine, hormonal, or immunosuppressive agents, the use of biological agents should be considered. TNF-α is expressed in considerable intestinal mucosal cells in IBD patients, directly involved in the disease occurrence and progression. TNF-α-antagonists have been proven to be effective for refractory UC and CD patients with fistula and sinus formation. The efficacy of TNF-α-antagonists in respect of IBD treatment has been confirmed. As TNF-α-antagonists have been increasingly employed, the occurrence of drug-induced side effects cannot be ignored. Psoriasis refers to an autoimmune disease seriously affecting the quality of people's daily life, generally occurring after treatments with TNF-α-antagonists.,9 Psoriasis is attributed to a complex mechanism between the immune system, psoriasis autoantigens, inflammatory cytokines, as well as multiple environmental factors. Besides, there have been cases of pathogenic infection, vasculitis, drug induced lupus, eczema, erythema multiform, and a wide range of skin malignancies. TNF-α is considered the vital factor in the inflammatory reaction by regulating the inflammatory signal transduction pathway . TNF-α-antagonists have been extensively employed for treating psoriasis. However, with the increase in contradictory reactions, the safety of TNF-α-antagonists should be monitored. In addition, psoriasis is an autoimmune skin disease with abnormal T cell-mediated keratinocytes overly proliferated and abnormally differentiated.–14 According to existing studies, TNF-α-antagonist-induced psoriasis and primary psoriasis are not identical in histopathology and immunohistology. A proportion of meta-analysis was employed to study the correlation between psoriasis and IBD, as an attempt to prove the significant bidirectional correlation between them.Inflammatory bowel disease (IBD) refers to a chronic, non-specific inflammatory disease attributed to autoimmune disturbance of the intestinal mucosa, which can cause recurrent inflammatory lesions. The prevalence of TNF-α antagonists induced psoriasis between different biological agents and different genders and smokers remains controversial. In accordance with the published literatures, this study found that the smokers and ex-smokers are more prone to psoriasis and IBD. This study aimed to describe the prevalence and correlation between psoriasis and usage of biological agents and relevant risk factors in IBD patients by conducting a systematic review and meta-analysis.At present, the most extensively employed TNF-antagonists include infliximab (IFX) and adalimumab (ADA). IFX and ADA are TNF-α antagonists capable of inhibiting TNF-α production and exertion. IFX and ADA are broadly applied in autoimmune diseases . IFX is initially used for treating adult and juvenile IBD, and ADA has been primarily utilized in adult IBD.22.1This study was conducted by complying with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). By collecting 2000-to-date literatures in PubMed, Embase, Web of Science, and other databases, the key words for searching were set, that is, Inflammatory Bowel disease, IBD, Chron's disease, CD, Ulcerative colitis, UC, psoriatic, PSO, paradoxical reaction, adverse reaction, IFX, ADA, as well as biological agents. For the search in title/abstract, by searching keywords  and by using Medical Subject Headings keywords and advanced search, some references with high quality were also checked, and the literatures meeting the standard were screened out. After the preliminary screening, over 500 literatures were initially taken. The literatures were further screened based on the inclusion and exclusion criteria.2.2Literatures satisfying the inclusion criteria below were included in the meta-analysis: It was observational; the data were complete, and the incidence of psoriasis was clear; for a case-control study, there was complete information from the experimental and controls.Exclusion criteria of the literatures: subjects with other autoimmune diseases or suffering from more severe systemic diseases; the experiment poorly designed or lacking a control; psoriasis was not pathologically confirmed; the score of the article was lower than 6 after the assessment of the quality of Newcastle-Ottawa Scale (NOS); the identical article published repeatedly.By summarizing the conclusions of the respective study, an analysis was conducted on the incidence, the distribution of psoriasis, the time interval between onset and medication, as well as the prognosis of psoriasis. The required data were extracted: general data, that is, first author's name, year of publication, population nationality, inclusion sample size, gender distribution, distribution of IFX and ADA use, as well as smoking history; outcome data, that is, sample size, adjuvant therapy, lesion coverage, and management of psoriasis in the psoriasis group and the control Table . The NOS2.32 static, in which I2 denotes the total variation across studies due to heterogeneity rather than chance. The equation of I2 is expressed as:The odds ratio (OR) value was determined with the random effect model method, and the incidence ratio between the experimental group and the control was estimated. In addition, the heterogeneity between the studies was obtained by performing Cochrane Q-test and IP < .05 was considered with statistical significance.Q denotes the Cochran heterogeneity data; df represents the degree of freedom. The funnel plot was employed to analyze the publication bias. However, as impacted by the small number of articles and strict quality assessment, combined with the funnel plots, it could be considered that no publication bias was identified. Revman and Stata, version 12.0, were applied for all data analysis. 33.1Specific description of screening steps Fig. : over 503.23.2.1P < .05. The incidence rate of the IFX group was 0.658 times than the ADA group, heterogeneity: Q = 11.55, P = .172, I2 = 30.7% , 3.2.2P < .01. The heterogeneity results: Q = 19.22, P = .014, I2 = 58.4% . The subjects in this study were primarily patients with CD, and the observation groups were patients with other skin diseases besides psoriasis. Some patients might have multiple types of skin lesions simultaneously, so there may be crossed interaction between various skin lesions.The incidence of psoriasis in different gender groups of IBD including 9 studies, respectively. The incidence of different gender groups after the treatment with TNF-antagonist, and the results were statistically significant: OR = 1.941, 95%CI 1.326–2.843), 26–2.843,Adult group = 1.911, 95%CI (1.085–3.366), P < .05, heterogeneity: I2 = 72.5%, ORTeenager group = 2.087, 95%CI (1.329–3.277), P < .05, heterogeneity: I2 = 7.0% , I2 = 30.8%) -γ is maintained in the body, contributing to the regulation of the body's inflammatory response. When TNF-α is significantly inhibited, the balance between TNF-α and IFN-γ is destroyed, thereby causing an increase in the production of IFN-γ by dermal plasmacytoma dendritic cell and excessively facilitating the migration of cluster of differentiation 8 + t cells to the epidermis; as a result, dendritic cell cells are activated and maturated, thereby inducing the autoimmune response.,36 The interleukin (IL)-23/T-helper (Th)-17 axis is also vital to the pathogenesis of TNF-α-antagonist-induced psoriasis. IFN induced Th17 cell proliferation and IL-23 induced the release of large amounts of IL-17 from helper Th17 and Th1 cells, thereby disrupting cytokine balance in vita and then causing psoriasis,,29,37 which may be correlated with IFN induced gene overexpression. Besides, there have been a small number of people considering that IBD patients with cytokine receptor mutation, thereby leading to psoriasis. The treatment with TNF-α-antagonists rises the risk of infection, and exposure to infected organisms elevates the risk of psoriasis, especially pustular psoriasis. Numerous autoimmune diseases originate at the gene level, TNF-α-antagonist-induced psoriasis may be correlated with the pathogenesis of psoriasis, and patients using TNF-α-antagonists are more genetic predisposition. Furthermore, it may be correlated with the genetic variation of IFN, and the role of the X chromosome in autoimmune diseases has been evidenced.The occurrence of psoriasis is closely linked to autoimmune factors and may be correlated with genetic factors. All studies excluded subjects with personal and family history to ensure the reliability of the results. As suggested from some studies, psoriasis may be a prevalent side effect of TNF-α-antagonists, that is, class effects.3.3P = .271, I2 = 20.1% , P < .05, heterogeneity: I2 = 7.0%  administrated with IFX. Besides, in most cohort studies on pediatric patients, TNF-d antagonist induced psoriasis was more predisposed to the female population.,42 Third, IBD patients treated with TNF-α-antagonist had a higher incidence of psoriasis in smokers than that of non-smokers.,14 Smoking has been identified as a major risk factor for TNF-antagonist-induced psoriasis among IBD patients, and smokers/ex-smokers are at high risk for drug-induced psoriasis. Lauren et al, discovered obesity as another risk factor for psoriasis besides smoking through the designed case-control study. The data results of the mentioned 3 parts were pooled into Table  The change of therapy regimen is vital to prognosis in the presence of the TNF-α antagonists induced psoriasis. Ko et al concluded that the optimal choice for TNF-α antagonists induced psoriasis is discontinuation of biologic agents. Most IBD patients suffered from relapse or aggravation after the withdraw or switch of biological agents. However, some scholars considered that the continuation of biological agents would not affect the recovery of drug-induced psoriasis, and the lesions could recover under the treatment of the local hormone or topical dermatologic medicine. No adequate evidence has been found to prove that discontinuation of TNF antagonists for non-infectious skin lesions can alleviate condition. Pugliese et al initially reported that the combination of biological agents with immunosuppressors could down-regulate the risk of psoriasis.Second, the analysis concluded that females were predicted to be more prone to psoriasis than men in IBD (OR = 1.975) and had a 1.975 times higher incidence of psoriasis than males. Females are more predicted to be prone to autoimmune diseases . Moreover, women with IBD are more likely to develop psoriasis induced by TNF antagonist in considerable studies. concluded that about 50% of patients could control their conditions without stopping their medication through topical medicine, and approximately 20% should stop their medication. According to the statistics, nearly 40% of patients could relieve skin lesions through local hormones, while 20% of patients could relieve the condition through ultraviolet light therapy. Wolf et al investigated the clinical outcomes of patients in group 377 (754 cases) withdrawing or switching of TNF-α-antagonist and those continuing TNF-α-antagonist. The clinical recurrence rate was higher in 377 groups (754 cases) stopping or converting TNF-antagonist for non-medical reasons, and the frequency of in-hospital treatment, emergency treatment, and out-patient treatment was higher than that of the continued use of TNF-antagonist.Of the 12 studies, a total of 692 patients suffered psoriasis induced by TNF-antagonists, and 137 patients stopped or switched to biological agents, and over 50% of them had good prognosis. Based on a 14-year single center large sample study, Freling et al The pathogenesis has similarity between psoriasis and IBD. Drug-induced complications are reported to be more common than extraintestinal complications. In clinical practice, health education for patients with relapsed condition should be strengthened, and patients’ compliance should be improved. The long-term follow-up is required for IBD patients to ask for the effects and adverse events to make an accurate judgment and guide the subsequent regimens. Long-term management is advised to minimize the potential adverse effects of TNF antagonist therapy. The data sources all originated from the electronic medical records of different hospitals or databases which could be incomplete.It is noteworthy that psoriasis can also occur in IBD extraintestinal complications, which should be differentiated with TNF-α antagonists induced psoriasis.4.1The incidence of psoriasis induced by TNF-α antagonists is higher than other autoimmune diseases, and IFX treatment for IBD is safer than ADA. The incidence of psoriasis is higher in females than in males, and the incidence of psoriasis is significantly higher in smokers/ex-smokers than that in non-smokers. Differences exist in various TNF-α antagonists, and the therapeutic strategy of TNF antagonists is not required to be changed in the event of psoriasis.4.2All the literatures included in this meta-analysis were retrospective literatures. Although the 3 co-authors assessed the quality of the article separately through the NOS assessment list, the subjectivity of authors affected the quality assessment results. This study attempted to select the articles with complete data and screen out the low-quality literatures. Furthermore, all the studies included originate from European and American nations, and basically the studies of Asian and African nations were not contained.All authors have contributed to and approve the final version of the article. YMQ and LWX participated in the study design and wrote the main manuscript text, and DQP, LZ, and WQ participated in the data analysis. All authors reviewed the manuscript.Conceptualization: Meiqi Yang, Weixin Liu.Data curation: Meiqi Yang, Weixin Liu, Qin Wang.Formal analysis: Meiqi Yang, Weixin Liu, Qin Wang.Investigation: Meiqi Yang, Weixin Liu.Methodology: Meiqi Yang, Weixin Liu.Writing – original draft: Meiqi Yang, Qiuping Deng, Zeng Liang.Writing – review & editing: Meiqi Yang.


Heart failure with preserved ejection fraction (HFpEF) is a condition with increasing incidence, leading to a health care problem of epidemic proportions for which no curative treatments exist. Consequently, an urge exists to better understand the pathophysiology of HFpEF. Accumulating evidence suggests a key pathophysiological role for coronary microvascular dysfunction (MVD), with an underlying mechanism of low-grade pro-inflammatory state caused by systemic comorbidities. The systemic entity of comorbidities and inflammation in HFpEF imply that patients develop HFpEF due to systemic mechanisms causing coronary MVD, or systemic MVD. The absence or presence of peripheral MVD in HFpEF would reflect HFpEF being predominantly a cardiac or a systemic disease. Here, we will review the current state of the art of cardiac and systemic microvascular dysfunction in HFpEF , resulting in future perspectives on new diagnostic modalities and therapeutic strategies. Heart failure with preserved ejection fraction (HFpEF) is a health care problem of epidemic proportions, currently accounting for roughly 3 million patients in the United States alone ,2. Over The paucity of effective treatments for HFpEF creates an urge to better understand the pathophysiology of this condition. Important comorbidities associated with HFpEF such as obesity, diabetes mellitus, renal dysfunction and hypertension have been linked with a systemic-low grade pro-inflammatory state . This prBoth coronary MVD and peripheral MVD, in this review defined as MVD in vascular beds other than the heart, have previously been reported in HFpEF ,14,15,16In this narrative review, we will focus on current evidence and future perspectives of microvascular studies in HFpEF patients and review evidence for the involvement of systemic MVD in its pathogenesis. Improved understanding of the (patho)physiological relevance of peripheral MVD for HFpEF, paralleled by improved understanding of the links between systemic and coronary MVD, will aid the search for new diagnostic modalities as well as novel therapeutic targets .Adequate interpretation of HFpEF studies that focus on MVD requires knowledge of the microcirculation and its functions from the vessel network and vessel type to a molecular level. Interpretation is complicated by the absence of a gold standard to define and diagnose microvascular dysfunction. Rather, different techniques evaluate different functional and structural aspects of the microcirculation and in different tissues, and a variety of these have been used in clinical research in HFpEF. The techniques have been reviewed elsewhere , and forMicrocirculatory beds are highly dynamic networks that constantly adapt to a variety of systemic and local signals from surrounding tissue and the vascular lumen, acting both acutely and chronically . These sMVD is often used as a broadly defined term encompassing all aspects of abnormal functioning of the microcirculation. MVD is not a synonym for endothelial dysfunction since it is not limited to functional or structural alterations of ECs but can include any (cellular) component of the microcirculation such as smooth muscle cells (SMCs), matrix, or pericytes. Here, we define microvascular function as a continuum between normal function and dysfunction, instead of a binary phenomenon, and microvascular dysfunction as a state where the primary microvascular functions are suboptimal and affect the surrounding tissue.Since Paulus and Tschöpe postulated a central role for MVD in the aetiology of HFpEF, a substantial number of studies have investigated MVD in HFpEF . At firsIn addition to the various different techniques and stimuli used to assess MVD in HFpEF, an important caveat concerning the evidence relating MVD to HFpEF  is the uTo study MVD in HFpEF, most publications have focused on using various external stimuli to influence vascular tone (vasodilation or vasoconstriction) of microvascular beds, known as vasoreactivity . VasoreaImpaired endothelium-dependent vasodilation and hyperaemia of peripheral microvascular beds have been studied scarcely in HFpEF. In a subgroup of coronary artery disease patients with HFpEF, HF with reduced ejection fraction (HFrEF), and no HF, one small study featured laser Doppler imaging of the forearm skin blood flow coupled with transcutaneous iontophoresis  using acetylcholine (endothelium-dependent) and sodium nitroprusside (endothelium-independent) . ImpaireIn the heart, impaired coronary endothelium-dependent vasoreactivity has been demonstrated in HFpEF ,32,33. YA promising vascular bed to provide knowledge on endothelium-dependent and independent MVD in HFpEF is the retina, mainly because the retina allows the evaluation of both structural and functional microvascular features by direct imaging of microvessels ,52. StudSeveral other techniques have been used more frequently to assess endothelium-dependent and endothelium-independent flow responses in HFpEF, including flow-mediated dilation (FMD) of larger arteries . As thosFurthermore, reactive hyperaemia by peripheral arterial tonometry of the finger is often referred to as microvascular endothelial dysfunction in studies but is mostly investigated by vasoactive stimuli that are not endothelium specific. Data from this technique are presented in the next section, while we provide more context on the technique here. This assessed reactive hyperaemia resembles post-ischemic hyperaemia after 5 min arterial occlusion involving many microvascular metabolites, including adenosine and NO, rather than only endothelium-dependent hyperaemia, which would occur after 1–3 min occlusion . This rePeripheral endothelium-independent vasodilation has been studied in HFpEF predominantly in the digital microcirculation. Following the reported association of post-occlusive reactive hyperaemia in the finger using peripheral arterial tonometry with multiple cardiovascular risk factors in the Framingham Heart study , most HFClinical studies focusing on endothelium-independent vasodilation of the coronary microcirculation have assessed hyperaemia using different stimuli. Invasive studies have revealed impaired maximal hyperaemia in HFpEF patients compared to controls without HF, both free of epicardial stenosis. In particular, these studies reported impaired coronary flow reserve (CFR) and an increased index of microcirculatory resistance (IMR) after intracoronary adenosine infusion ,14,32,34To date, only one study assessed both coronary and peripheral (micro)vascular function in HFpEF. In their assessment, Shah et al. used novel echocardiographic tools (Doppler imaging to assess CFR of the left anterior descending artery after adenosine infusion) and observed a high prevalence of impaired maximal coronary hyperaemia in HFpEF . This was significantly but weakly correlated with impaired peripheral vasoreactivity as measured by endoPAT . This diTaking all published results regarding vasoreactivity of different (micro)vascular beds together, HFpEF patients consistently show impaired (micro)vascular vasoreactivity throughout the body, suggestive of systemic MVD. However, not all patients show identical MVD phenotypes. Nonetheless, a causative conclusion on the gradual development of MVD and HFpEF requires more robust evidence.Capillary rarefaction in HFpEF has been shown in vascular beds of the upper legs and the heart with tissue biopsies ,16. By pCoronary capillary rarefaction in HFpEF has been studied scarcely. Mohammed et al. assessed coronary capillary density by histology in autopsy samples and found that coronary rarefaction was more prevalent in HFpEF patients compared to age-matched controls. In addition, it was correlated with increased myocardial fibrosis . This siSerum endothelial biomarkers predominantly represent the microcirculation, considering that 98% of all ECs are part of the microcirculation . ResearcEndothelial leukocyte adhesion molecules seem to be the most promising markers of general microvascular function and could be used to assess the sequelae of systemic MVD longitudinally. Intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin are responsible for leukocyte adhesion to ECs . Their eElevated plasma levels of von Willebrand Factor (vWF) were shown to be of incremental prognostic value on top of routine clinical characteristics and biomarkers in HFpEF , which wIn general, there is much interest in microRNAs as biomarkers of microvascular or endothelial dysfunction, whether in blood or urine, encapsulated in exosomes or other types of vehicles ,114. MicAlterations in both cardiac and peripheral adipose tissue are present in HFpEF ,124,125,Excess epicardial adipose tissue (EAT) was found in HFpEF patients compared to HFrEF and healthy controls ,136, whiMVD has been proposed to play a role in cardiac fibrosis and cardiomyocyte stiffness in HFpEF . A numbeThe above-described microvascular alterations associated with HFpEF have been instrumental in suggesting therapeutic targets based on underlying mechanisms. Several clinical trials have been performed targeting a variety of endothelial and cardiomyocyte-based proteins that are directly or indirectly aimed at tackling (the consequences of) MVD  153,154,156,157.A large number of recent studies and ongoing trials have focused on restoring parts of the NO-sGC-cGMP-PKG pathway , particuInterventions in HFpEF targeting MVD directly or indirectly probably provide most benefits when MVD is present, but likely also when interventions have a positive effect on several aspects of the multimorbid cardiovascular system of such patients. Considering the high prevalence of glucose intolerance and other metabolic comorbidities in HFpEF and their detrimental effect on microvascular function ,159,160,Direct stimulators of sGC, such as vericiguat or praliciguat, have the capacity to activate the cGMP pathway independently from NO. A previous trial with vericiguat in HFpEF did lead to a decrease in self-reported physical limitations but not to changes in NT-proBNP or left atrial size . HoweverSacubitril-valsartan, a combination of a neprilysin-inhibitor and an angiotensin-receptor blocker, enhances natriuretic peptide bio-availability, which influences microvascular function independent of NO, but failed to show a positive effect on HF hospitalization rates or death from cardiovascular causes in a general HFpEF population . NeverthPhosphodiesterase (PDE)-5 inhibition, an established strategy to increase PKG activity, has shown conflicting results in HFpEF. A preclinical study showed protective effects of sildenafil on diastolic function by alleviating nitrosative stress in diabetic rats , but cliPhysical activity can induce skeletal muscle cells to become hypoxic, particularly when oxidative stress is present, limiting the muscle cells function . This coIn general, most trials did not include invasive cardiac measurements or peak exercise testing to evaluate the effects of the intervention. On the one hand, this could limit the ability of a trial to show an effect of the drug, especially in patients in earlier stages of HFpEF who present with fewer impairments regardless of intervention. On the other hand, studies using peak exercise or invasive methods may possibly have a selection bias by excluding frailer patients who are physically not able to participate. Moreover, aside from the nonspecific selection of HFpEF populations, most targeted trials were not powered to evaluate the effects of the intervention based on important MVD drivers.Finally, regardless of MVD or HFpEF phenotype, physical activity in HFpEF can improve exercise capacity by improving factors such as chronotropic incompetence (insufficient heart rate increase despite higher cardiac output demands) and increasing oxygen use by active muscles, which improves the symptom burden . FactorsCurrently, both MVD and HFpEF are still developing in terms of definitions and diagnosis. The number of studies has been increasing substantially, but studies to date have often provided incomplete knowledge and were difficult to compare with other studies, limiting firm conclusions. In addition, several important challenges regarding MVD and HFpEF include implications of different underlying mechanisms, the association between coronary and peripheral MVD, and confounding factors. The major limitations and knowledge gaps are summarized in MVD can be caused by different underlying mechanisms, including inflammation, oxidative stress, increased sympathetic activity, impaired glucose metabolism, and cellular and metabolic changes ,82,181. Moreover, the association between coronary and peripheral MVD in HFpEF patients and its implications remains to be further investigated. Only one study assessed this association and used different stimuli for vasoreactivity in the coronary and finger vasculature .In addition, it should be acknowledged that MVD is not specific to HFpEF, but it is associated with multiple diseases and cardiovascular risk factors. Data to date lack clear causative factors (determinants) for HFpEF or MVD in HFpEF, but associative factors (correlates) for MVD in HFpEF have been reported. Meanwhile, determinants of MVD outside the scope of heart failure have been discovered. These clinical factors are summarized in Many different microvascular abnormalities have been found in selected HFpEF populations and were associated with worse outcome ,24,25. BMoreover, assessing MVD can also aid in identifying patients at higher risk for developing HFpEF, as current data suggest structural and functional signs of MVD occur already at a presymptomatic phase of HFpEF in different microvascular beds ,120,189.In order to answer some knowledge gaps, we have started to prospectively investigate microvascular changes of multiple vascular beds in HFpEF patients and controls .Systemic MVD is present in HFpEF, based on interpretation of abundant data from many correlational studies that show impairments in microvascular function, both endothelium-dependent and endothelium-independent, in different vascular beds. MVD should be seen as a continuum between function and dysfunction, which can influence HFpEF and comorbidity progression, and vice versa. Hitherto, due to a lack of clear causative evidence, it remains unknown how systemic MVD could drive HFpEF. Furthermore, HFpEF patients unequally show different elements of MVD, which might reflect different underlying mechanisms and therapeutic targets. Future research on MVD and HFpEF is, therefore, needed to uncover the true diagnostic and therapeutic value of microvascular assessments. This will require more uniformity and confounder considerations in study design, analyses, and reporting. However, the incorporation of peripheral microvascular assessments is feasible and should be considered in clinical HFpEF trials.


Machine learning models predicting the bioactivity of chemical compounds belong nowadays to the standard tools of cheminformaticians and computational medicinal chemists. Multi-task and federated learning are promising machine learning approaches that allow privacy-preserving usage of large amounts of data from diverse sources, which is crucial for achieving good generalization and high-performance results. Using large, real world data sets from six pharmaceutical companies, here we investigate different strategies for averaging weighted task loss functions to train multi-task bioactivity classification models. The weighting strategies shall be suitable for federated learning and ensure that learning efforts are well distributed even if data are diverse. Comparing several approaches using weights that depend on the number of sub-tasks per assay, task size, and class balance, respectively, we find that a simple sub-task weighting approach leads to robust model performance for all investigated data sets and is especially suited for federated learning. Drug discovery is a costly and risky (due to high failure rates) endeavor. Time and money spent until market access are continuously increasing for various reasons, including the early-stage effort needed to discover and optimize suitable drug candidates. To overcome this, new avenues are pursued. One particularly promising approach is collaborative efforts between otherwise competing companies, e.g., Martin and Zhu , leveragA standard approach in multitask learning is to weight all tasks with a constant and equal weight, i.e., no application of a dedicated weighting scheme. However, an equal weight does not necessarily reflect the underlying circumstances, e.g., difficulty of training, relevance of the task, or scale of the task’s loss . FurtherPhase I: exploration results from the fixed scheme are discussed.Phase II: results from the continuous scheme and of further fixed scheme experiments are discussed.Phase III: results from weighing experiments based on fraction active or label balance are discussed.This work is structured in three Results and Discussion sections, which we call phases:In three phases, several weighting schemes were analyzed see . In ordeIn phase I, four different weights  for a fixed weighting scheme where all tasks with more than 1000 datapoints are down-weighted were evaluated based on a subset of all tasks. Overall, the delta AUPR values are small, in the region of −0.02 and 0.01 see a. A sligIn phase II the following approaches were investigated: (a) two continuous schemes with steps 500 and 1000 both with a weight of 0.02, fixed weighting schemes with weights 0.6 and 0.9 and (b) cutoff 1000 and (c) cutoff at the 95% quantile of the task size  as well as (d) weighting based on the number of tasks and based on the task size. These cutoffs, steps and weights were selected because they performed best in a pretest executed by one partner  are depicted in All but one (task size) weighting scheme improved the AUROC performance of the quartile task to a similar extent see . In contAn explanation for the small delta AUPR values could be that the AUPR metric is dependent on the fraction actives, which does not change through the different weighting schemes and thus masks the impact of the different weighting schemes. An analysis of the correlation of AUPR and AUROC values to several factors, e.g., fraction actives, can be found in the Subsequent to this analysis, we were curious whether other task characteristics besides task size, used to compute task weights, further improve predictive performance. Hence, in phase III other task characteristics like label balance and fraction actives were used to compute the task weights.Due to the small performance deltas in phases I–II, a significance test was used in phase III see . FurtherNo task that performs significantly better than the baseline could be identified for any of the phase III weighting schemes see . On the However, phase III also identified weighting schemes that result in only a very small percentage of significantly worse performing tasks . However, the practical value of both tasks per assay (median and lower quartile) is not equal, and a deterioration of the median-based task performance may be acceptable if the lower quartile task performance improves. Therefore, the pure quartile-based task performance was analyzed for phase III weighting schemes.The performances of weighting schemes of phase III were analyzed in more detail, as for phase 2. The delta performances for the lower quartile tasks are depicted in Interestingly, the delta AUROC performance evaluation  reveals Nevertheless, the AUPR performance evaluation , confirmThe performance evaluation based on the lower quartile tasks demonstrated phase III weighting schemes with an improved AUROC performance compared to the baseline. Interestingly, most of these beneficial weighting schemes are based on the fraction of actives. Remarkably, both directions, i.e., down-weighting an excess of actives or inactives, lead to superior performance. On the other hand, an opposite trend was observed for weighting based on balance. Here, down-weighting balanced task is clearly superior to down-weighting imbalanced tasks. Together with the results achieved for down-weighting according to the fraction of actives, one can conclude that the positive performance delta rather originates from up-weighting tasks with either high or low fraction of actives than from down-weighting the opposite, because down-weighting both  leads to a much worse performance than up-weighting imbalanced tasks. Noteworthily, the observations made by the intra-assay weighting schemes can be transferred to the global weighting schemes. In contrast to the AUROC-based performance analysis, no weighting scheme of phase III demonstrated a strong positive delta performance compared to the baseline (1) wrt. AUPR. However, preserving performance while reducing learning time would also be favorable. Thus, the impact of different weighting schemes on the speed of convergence was assessed.The speed of convergence was assessed for weighting schemes of phase II and phase III see .None of the weighting schemes with reasonable performance exhibit accelerated learning.https://github.com/melloddy/MELLODDY-TUNER, accessed on 10 November 2021]. In total six pharma companies  performed the experiments of this study. Each of the six data sets contained 100,000–2,000,000 unique compounds and 3000–26,000 tasks. The chemical space was analyzed for one private data set and shows that the majority of compounds are unsurprisingly in a drug-like chemical space with a median clogP of ~2.9 and a median molecular mass of ~390. This is in good alignment with other analyses . SparseChem is a package for machine learning models for biochemical applications capable of high-dimensional sparse input. The data were split into five folds (subsets) using locality sensitive hashing on molecular fingerprint features [The prepared data were subsequently used to train a feedforward neural network using SparseChem [The results from the best hyperparameter set were used for subsequent evaluations and analyses.The results were evaluated based on the following criteria: difference in predictive performance (between baseline performance and performance of weighting scheme under investigation) measured as area under the precision-recall or receiver operating characteristics curve , the number of statistically significant better or worse tasks based on AUROC, and the convergence speed. Convergence speed was determined as a plot of performance against epoch number. AUPR and AUROC were averaged over all tasks fulfilling the criteria, i.e., minimal number of actives and inactives and depending on the analysis, e.g., only tasks of the lower quartile threshold.p-value was calculated based on a Z-test comparing the two AUROC values. It has been shown [n1 and n0 are the number of actives and inactives, respectively, in the fold used for evaluation. Intuitively, for this binary classification setting, both the nonparametric Wilcoxon statistic and the AUROC are measures for the quality of ranking actives versus inactive samples, and depend fully on the rank between the samples.To estimate whether two tasks had statistically significant different AUROC values, a en shown  that thesd1 and sd2) have been calculated, assuming normality, a Z-test can be used to compare the difference of the mean AUROC values:Once the standard deviations of two AUROC values  exceeds the confidence level of 95%, the null hypothesis of the two AUROC values being statistically equal is rejected.Z being the z-score from which a Weighting tasks in multi-task models is a current area of considerable scientific interest. In this article, we analyzed several weighting schemes in the context of federated multi-task learning on pharmaceutical industry data in a privacy preserving setting, which was realized for the first time at this large a scale in drug discovery. In general, our models were resilient to most perturbations to the weights, indicating that the limiting factor on performance is the underlying information in the data and the model architecture. The weighting schemes that lead to a drop in performance  often would have led to extreme distributions of weights due to their reciprocal style of computation. Thus, a few very small x values would have dominated the total contribution to sums used for normalization. All analyzed weighting schemes with comparable AUPR performance also have similar speeds of convergence. Flexible and continuous weighting schemes, as well as weighting based on the fraction of actives (fractive up weight and intra assay weighting based on fraction actives), and based on the number of sub-tasks were shown to be beneficial both regarding synoptic and lower quartile task AUROC performance. Noteworthily, this result is consistent through six different pharmaceutical industry data sets. Weighting based on the number of tasks is furthermore especially suited to federated learning, because it prevents partners from artificially increasing their weight by adding more tasks.


HRPs) are a powerful design to map highly penetrant risk genes. We previously described Shared Genomic Segment (SGS) analysis, a mapping method for single large extended pedigrees that also addresses genetic heterogeneity inherent in complex diseases. SGS identifies shared segregating chromosomal regions that may inherit in only a subset of cases. However, single large pedigrees that are individually powerful (at least 15 meioses between studied cases) are scarce. Here, we expand the SGS strategy to incorporate evidence from two extended HRPs by identifying the same segregating risk locus in both pedigrees and allowing for some relaxation in the size of each HRP.High-risk pedigrees  which contains one gene, CDH20. Thirteen regions were genome-wide suggestive: 1q42.2, 2p16.1, 3p25.2, 5q21.3, 5q31.1, 6q16.1, 6q26, 7q11.23, 12q24.31, 13q13.3, 18p11.22, 18q22.3 and 19p13.12.We identified one genome-wide significant region at 18q21.33 (0.85 Mb, Our results provide novel risk loci with segregating evidence from multiple HRPs and offer compelling targets and specific segment carriers to focus a future search for functional variants involved in inherited risk formyeloma. Inherited germline susceptibility for MM is consistently supported: excess MM risk among relatives has been observed in family aggregation,4, epidemiologic case-control–9, and registry-based,11 studies. Disease rarity, short survival, clinical and locus heterogeneity challenge study ascertainment and genetic discovery. Genome-wide association studies have identified 23 loci harboring common-risk single nucleotide polymorphisms (SNPs) for MM–19. Family-based studies have identified rare germline variants in ARID1A and USP45, KDM1A, and DIS3 in exome sequencing. However, considerable missing heritability remains. Additional approaches are needed to aid the detection of the remaining risk loci and genes.Multiple myeloma (MM) is the second most common adult-onset lymphoid neoplasm and has the worst 5-year survival. Cases sharing genomic segments from a common ancestor through 15 meioses or more are unexpected at a genome-wide level, and hence a single large high-risk pedigree (HRP) can provide the power to identify novel loci with genome-wide significance. Our resource of eleven large myeloma pedigrees included several with 3–4 cases and meioses in the 8–14 range. While these remain extremely large families, they may lack sufficient power individually for genome-wide significance. Also, a multi-pedigree strategy is attractive. Evidence for the same risk locus in two extended pedigrees adds confidence to the locus and can build on the power of both. The remaining challenge for any multi-pedigree approach, however, is to adequately address heterogeneity between pedigrees.We recently described a novel strategy to map genes involved in complex disease risk using extremely large high-risk pedigrees and allowing for intra-familial heterogeneity, called Shared Genomic Segment (SGS)duo-SGS, we fix one pedigree and optimize over all pedigree pairs to balance discovery with multiple testing. Both pedigrees must have a segregating genomic segment at the same risk locus. The method is robust to allelic heterogeneity as different alleles at the same locus may be shared within each pedigree. We apply the duo-SGS approach to eleven MM HRPs to identify novel loci involved in myeloma risk.Here, we expand the SGS method based on combining evidence from pairs of HRPs, while still allowing for intra-familial heterogeneity within each pedigree. In our approach, An overview of the duo-SGS approach can be found in . Briefly, the single SGS approach identifies shared observed genomic segments by defining consecutive runs of SNPs that are identity-by-state in a group of cases  for an excessive length of sharing is established , and hence multiple testing can rapidly become an issue. Alternatively, a single analysis comprising optimization across all pedigree pairs could be considered, but this global approach may cloud individual pedigree-pair findings. To balance these two extremes, we propose a fixed-pedigree duo-SGS strategy  we model the distribution for null sharing on the distribution of the empirical P-values for each pedigree. To avoid comparing the findings to themselves or skewing to possible true-positives, the empirical-P-values are perturbed, and the distribution-fitting is performed at 1 million simulations. The latter is to avoid inappropriate distribution-fitting to extreme outliers, the few results from the alternate hypothesis if included at their final resolution. To perturb an empirical P-value we determine its Wilson score 95% confidence interval (CI) , and n is the number of simulations . The Wilson interval was selected because it always produces non-negative confidence bounds for the P-values. The genome-wide set of perturbed empirical P-values for a pedigree are considered the “null” P-values for that single pedigree. The duo-SGS procedure (described above) is performed using the single pedigree genome-wide null P-values. The result of this process is a set of optimal duo-SGS null P-values.Critical to interpreting the observed duo-SGS results are genome-wide significance duo-SGS thresholds for each pedigree , Step 6.val (CI)  and rand. Briefly, the null duo-SGS P-values are log-transformed and fitted to a gamma distribution. The shape (k) and rate  has been an NCI-supported Surveillance, Epidemiology, and End Results (SEER) Program registry since its inception in 1966. The UCR was utilized to invite all individuals with myeloma in the state to participate. Peripheral blood was collected for DNA extraction from individuals who completed informed consent.. It includes a 16-generation genealogy of approximately 5 million people with at least one event in Utah that is record-linked to the UCR and state vital records. Using the UPDB, ancestors whose descendants have an excess of disease based on internal cancer rates and years at risk can be identified and studied as HRPs. The UPDB was used to identify ancestors whose descendants showed a statistical excess of MM (P < 0.05). The expectation was based on internal disease rates based on birth cohort, sex, birthplace (in/outside Utah), and years at risk. The total number of myeloma cases in each HRP identified ranged from 4 to 37 cases. After annotating the pedigrees with those with DNA, 11 pedigrees were identified to contain 3 or 4 myeloma cases with DNA . In each pedigree, the cases were separated by 8 to 23 meioses.The Utah Population Database (UPDB) is a unique resource was used for quality control. SNPs with < 95% call rate across the 28 individuals were removed. After filtering, 678,447 SNPs remained. These SNPs were transformed to match 1000Genomes strand orientation.DNA from the 28 cases was genotyped on the Illumina Omni Express high-density SNP array at the University of Utah. Only high-quality bi-allelic SNPs and individuals with adequate call rates across the genome were included. The PLINK softwareIndividuals were removed if < 90% of the filtered SNPs are called. One myeloma case had a < 90% call rate and was eliminated from the study. We also checked for sex inconsistency based on the genotypes - all cases passed. PLINK relationship estimates were compared with the UPDB pedigree structures - no issues were found.P-values are identical, but thresholds vary by which pedigree is fixed, potentially leading to different significance levels. The most significant result was reported, and the lesser removed. If an individual resided in two pedigrees and also shared the region in both pedigrees, the region was removed. If the region spanned a centromere, it was removed. Forty-two suggestive regions were removed as duplicates, involving an overlap individual or at the centromere.The duo-SGS method was applied to the MM pedigrees to identify regions with genome-wide suggestive or significant evidence. Post-hoc, some duo-SGS regions were removed from consideration. Duplicate regions occur when the same pair of pedigrees identify the same region in both their fixed-pedigree results. In these situations, duo-SGS P-value, expected rate per genome μ(t), the two pedigrees involved, each segregating shared region in the pedigrees, and the overlapping region.Duo-SGS findings were identified for each of the eleven MM HRPs. The significance thresholds for each fixed pedigree are in P = 7.3 × 10−9, P = 2.8 × 10−5). A nested 0.8 Mb chromosomal segment  was observed in four MM cases separated by 23 meioses in pedigree UT-48833 (single pedigree P = 1.1 × 10−5). The intersecting 0.8 Mb region overlaps one gene: Cadherin 20 (CDH20). The genome-wide significant region at 18q21.33 . The 6q26 region achieves suggestive evidence in four pedigree pairs and harbors the PARK2 gene. At 5q21.3 four pedigree pairs show suggestive evidence and the locus contains the gene FBXL17. The locus at 7q11.23 is also supported by two genome-wide suggestive duo-SGS results. The remaining suggestive loci were supported by one pedigree pair: 1q42.2, 2p16.1, 3p25.2, 5q31.1, 12q24.31, 13q13.3, 18p11.22, 18q22.3 and 19p13.12. Genes in each of the duo-SGS regions are shown in Thirteen loci were found with genome-wide suggestive evidence . In four and sprinkled with sporadic cases; hence methods that require all cases to share to attain discovery power are not suitable. Here we optimize over subsets within pedigrees and consider pairing with all other pedigrees to provide the flexibility required. The method also specifically defines which pedigrees and cases share evidence at a locus, which is imperative for follow-up sequencing. Additional value may be gained by comparing demographic or clinical characteristics of the sharers in each pedigree to nuance the phenotype which may aid future gene mapping and provide insight into the nature of the mechanism of risk at a locus.We expanded the shared genomic segment method to identify segregating chromosomal segments with overlapping statistical evidence from two HRPs. The strategy allows for genetic heterogeneity within each pedigree and provides formal significance thresholds for interpretation. The approach circumvents issues of intra-familial heterogeneity that can hinder mapping in large pedigrees. For complex diseases, large HRPs are likely enriched for multiple susceptibility variantsApplication of the novel duo-SGS approach to eleven MM HRPs implicated a novel genome-wide significant region at 18q21.33 in MM risk, as well as 13 suggestive regions. Other than 6q16.1, which overlaps with our previous single pedigree SGS study, all loci identified in this study provide novel regions of interest in myeloma. None of the regions overlapped with existing genome-wide association study loci or other prior rare risk variants implicated in MM. A next step would be to investigate the loci for rare and deleterious coding variants or regulatory variants. Pedigree segregation methods can provide statistically compelling regions to concentrate efforts to identify and characterize regulatory risk variants. Also, SGS results can be used as genomic annotations of prior evidence to layer with additional omic information or provide a focused region for interrogating regulatory risk variants.CDH20, a gene that plays a role in intracellular adhesion by forming cadherin junctions. Cadherins have been suggested in solid tumor invasion, and metastasis as disruption to cell-cell junctions is a prerequisite. Solid tumors co-aggregate in MM families suggesting a shared genetic background. At 6q26, several pedigree pairs were genome-wide suggestive, and the overlapping segments fall in PARK2 which mediates proteasomal degradation. PARK2 is a tumor suppressor and the gene harbors risk variants for lung cancer.The literature supports a role of some of the genes found in our duo-SGS regions in MM. The genome-wide significant region at 18q21.33 contained .While the duo-SGS approach is useful for analyzing pedigrees smaller than those typically required for the single pedigree SGS approach, a large number of meioses are still required. The HRPs in this study are still substantially larger than those available in most family-based resources (8–23 meioses between sampled cases). Hence the method has practical limitations in other settings. Nonetheless, the interesting regions identified in large pedigrees provide evidence that can be used to narrow the search for risk variants in smaller families as well, as demonstrated in our prior studyAs in all family-based genetic studies, our results could be sensitive to inaccurate pedigree structures. However, relationship and ethnicity checks are standard protocols and mitigate the possibility of error. Another limitation to this study is the observational nature. Additional functional studies will be required to describe causation and characterize the mechanisms involved in these loci and myeloma risk.We have identified several novel loci that segregate in at least two myeloma HRPs. These loci are likely to harbor genes and rare risk variants for MM and are compelling new targets for inherited risk to MM.In conclusion, we developed a novel strategy for gene mapping in complex traits that uses multiple large high-risk pedigrees. The approach is robust to heterogeneity both within and between pedigrees and formally corrects for multiple testing to allow for statistically rigorous discovery. We applied this strategy to MM, a complex cancer of plasma cells, and identified one novel genome-wide significant locus at 18q21.33 and 13 suggestive loci. Our study offers a new technique for gene mapping and demonstrates its utility to narrow the search for risk variants in complex traits.


For the MOS outcomes, ICC2,k values ranged from 0.42 to 0.99 between two devices and 0.28 to 0.69 between two test sessions. The Kinect V2 was able to provide valid and reliable spatiotemporal gait parameters, and it could also offer accurate outcome measures for the minimum MOS. The reliability of the Kinect V2 when assessing time-specific MOS variables was limited. The Kinect V2 shows the potential to be used as a cost-effective tool for CP gait stability assessment.Children with cerebral palsy (CP) have high risks of falling. It is necessary to evaluate gait stability for children with CP. In comparison to traditional motion capture techniques, the Kinect has the potential to be utilised as a cost-effective gait stability assessment tool, ensuring frequent and uninterrupted gait monitoring. To evaluate the validity and reliability of this measurement, in this study, ten children with CP performed two testing sessions, of which gait data were recorded by a Kinect V2 sensor and a referential Motion Analysis system. The margin of stability (MOS) and gait spatiotemporal metrics were examined. For the spatiotemporal parameters, intraclass correlation coefficient (ICC Postural control is the ability to control the body’s position in space to achieve stability, requiring the centre of mass (COM) to be positioned within the boundary of the base of support (BOS) . In the About 50% of the falls happen during walking . ChildreThe margin of stability (MOS) is also one of the quantitative gait measurements that evaluate the COM’s motion relative to the BOS that determined by the foot placement . The MOSWith this advantage, MOS has already been widely applied to evaluate gait abnormality and fall risks in a wide range of clinical populations, including patients with stroke , ParkinsGenerally, parameters required for MOS computation are recorded by optoelectronic motion capture systems combined with force plates. However, these high-end infrared cameras are costly and unfriendly to nonspecialists. Its marker-based tracking strategy may cause unnatural performance for children with CP and make marker placement a challenging job. Therefore, there is a growing need to develop cost-effective and portable gait analysis systems for clinic-based gait progression monitoring and home-based gait investigation, which can empower patients to take an active role in their health management, provide convenient and accessible options for people living in remote areas , and alsThe Microsoft Kinect sensor, which was initially designed as a motion-sensing input device for interactive games, can perform real-time motion capture without any passive markers or handheld controllers. Compared to other innovative techniques, such as activity-tracking devices or wearable accelerometer-based devices, the Kinect has the advantage of providing unobtrusive investigation . PreviouTo fill the gaps mentioned above, we proposed a method to compute MOS parameters from skeleton landmarks tracked by the Kinect V2 sensor. The results were compared against data measured by a referential optoelectronic Motion Analysis system. This study aimed to assess the concurrent validity and interday reliability of the Kinect V2 when assessing MOS and spatiotemporal parameters of overground gait for children with CP. We hypothesised that the Kinect V2 could provide accurate and reliable measurements for MOS and spatiotemporal gait parameters.The rest of the paper is as follows: “Methods” section details the characteristics of ten CP participants, the definition of selected gait spatiotemporal parameters, the method of MOS calculation, as well as statistical methods implemented to determine the validity and reliability of the Kinect V2 sensor; “Results” section describes the gait spatiotemporal and MOS parameters estimated by per device on per testing day. The between-device and between-testing day comparison is given in this section. “Discussion” section discusses the findings that arose from the results, analyses possible reasons that lead to the between-device and between-testing day deviations, and discuss the Kinect’s potential implementation in gait stability management for patients with CP, followed by the conclusive remarks in the “Conclusion” section.Ten children with CP  were recruited and presented for the gait data collection. Participants were diagnosed as hemiplegia (one child), diplegia (five children), quadriplegia (two children), or dyskinesia (two children), and they were classified as Level I (three children) or II (seven children) on the Gross Motor Function Classification System (GMFCS) . ChildreAs illustrated in An eight-infrared-camera motion capture system  recorded the reflective marker trajectories at a sampling rate of 100 Hz. The length of the calibrated motion capture volume was about 4 m. The Kinect V2 sensor  was placed on a tripod with a height of 0.8 m and a distance of 5 m from the start line of the walkway. The Kinect V2 sensor was placed in front of the participant to provide a frontal view, ensuring the tracking accuracy for both kinematic and spatiotemporal parameters. It was triggered simultaneously with the Motion Analysis system, recording coordinates of twenty-five skeletal landmarks b at a flThree-dimensional gait analysis test consisted of two testing sessions. In the first testing session, participants were given sufficient time to try to walk along the walkway. After they became familiar with the testing procedures, they were asked to performed overground gait trials at a self-selected speed along the level walkway independently. Children walked from the starting line towards the Kinect V2 sensor barefoot. Each participant completed at least three successful gait trials. A successful gait trial should contain at least one complete right gait cycle with all the landmarks, and reflective markers could be seen in the motion capture interfaces of two systems. To test the interday reliability of the Kinect V2, participants were asked to attend another gait test session the following day. All the ten participants presented for the Kinect V2-based gait analysis in the second testing session. The experimental setup of the Kinect V2 for the second session was consistent with the previous one. At least three successful gait trials were obtained for each child. The criteria of a successful gait trial were consistent with what was given in the previous session.The data collected by the Kinect V2 and Motion Analysis system were filtered via a second-order Butterworth low-pass digital filter with a cut-off frequency of 6 Hz. Gait events were identified based on the distance between the sacrum and foot landmarks ,42,43,44The COM position was calculated as the average position among the sacrum, LASI, and RASI markers in the Motion Analysis ,47. The Equations (7)–(9) were used to compute the dynamic MOS based on prior studies ,23:(7)ω0ing foot . MOS at ression)  were extIn each gait trial, one right gait cycle was extracted for analysis. All the MOS and spatiotemporal parameters measured by each device on each testing day were averaged among three gait trials for each participant. The intraclass correlation coefficient (ICC) was used to assess the agreement between the Kinect V2 and Motion Analysis system and the agreement of the Kinect V2 between two testing days. ICCs were estimated, and their 95% confidential intervals were calculated based on a mean rating (k = 3), absolute-agreement, 2-way random-effects model. ICC values were interpreted as: excellent (0.75–1), modest (0.4–0.74), or poor (0–0.39) . The staom ANOVA ,51, and The relative error (in percentage) was computed to express the absolute between-device measurement difference as a percentage of the measure estimated by the referential Motion Analysis system (Equation (11)) .(11)η =Furthermore, a Bland–Altman analysis of agreement  was perf2,k with 95% confidence intervals for agreement, SEM, and relative error (in percentage) are presented in 2,k with 95% confidence intervals for agreement and SEM are presented in Mean ± 1SD values for each spatial MOS and spatiotemporal gait parameters estimated by each device, inter-device ICC2,k = 0.81) and AP  directions, and a modest agreement  was found for MOS at foot strike and midstance in both ML and AP directions between Motion Analysis and the Kinect V2. All the spatiotemporal gait parameters showed excellent agreement between the two devices . A modest agreement was found in the equivalent pendulum length between the two devices . Mean relative errors (in percentage) ranged from 1.75% to 186.4% for all the selected parameters. MOS at midstance and foot strike in the AP direction demonstrated the most deficient accuracy, with mean relative errors of 112.82% and 186.40%, respectively. All the selected spatiotemporal parameters exhibited lower relative errors (1.75–25.97%).For the MOS, an excellent agreement was observed for the minimum MOS during stance in both ML  for the Kinect V2 except for the MOS at midstance in the ML direction, which showed low reliability between two test days . All the spatiotemporal gait parameters  and equivalent pendulum length  showed excellent reliability between the two testing day.The interday reliability of most MOS variables was modest . In accordance with the present results, previous studies have reported a reasonable validity of the Kinect V2 when assessing spatiotemporal gait parameters regardless of what the walking condition (overground or treadmill-based), walk speed (at a self-comfortable or specific speed), population , and also the referential motion capture system  was [2,k = 0.78–0.88), favouring prior findings [In this study, spatiotemporal variables derived from the Kinect V2 sensor showed an excellent agreement with their referential counterparts (ICCmat) was ,44,53,54findings ,57,58,592,k = 0.81–0.99), other MOS variables did not show such a strong agreement with those obtained by the referential motion capture system . It was potentially attributed to two main reasons. Firstly, the length of the pendulum was calculated as the distance between COM and foot ground contact in a marker-based motion capture system [2,k = 0.45). Additionally, using the ankle landmark in MOS computation has the risk of underestimating BOS. A prior study reported that 66% of 492 patients with CP were affected by the intoeing abnormality, and around 25% of them had the out-toeing problem [However, except for the minimum MOS . De Jong et al. found that the test-retest ICCs of MOS are lower in healthy participants than patients with balance problems [2,k = 0.81–0.99), which are essential gait metrics to assess the risk of falls [Although the Kinect V2 could provide reliable interday spatiotemporal evaluation, it seems that the changes in clothing, accommodation to the testing procedure, and the patients’ performance over repeated gait trials may potentially result in the interday deviations in the MOS calculation  are useful features to distinguish CP gait stability from their typically developing peers . MoreoveExcept for the small sample size, some other limitations were encountered in this study. Firstly, to simplify the MOS calculation process, the “spine base” marker was used to represent the COM. However, the COM position with respect to the segmental coordinate system is generally predicted via regression equations with a full-body marker set in traditional optoelectronic motion capture systems . It was Results of this study reveal a reasonable validity and reliability level in evaluating spatiotemporal parameters and, in contrast, less strong validity and reliability when calculating MOS related variables, especially MOS at foot strike and midstance. The Kinect V2 was only firmly valid to provide minimum MOS measures, which is an essential gait metric to track the risk of falls . These r