This study should enable the generation of patient-derived lung cancer organoid lines, which can be used to further the understanding of lung cancer pathophysiology and to assess drug response in personalized medicine.Lung cancer is an extremely heterogeneous disease, and its treatment remains one of the most challenging tasks in medicine. Few existing laboratory lung cancer models can faithfully recapitulate the diversity of the disease and predict therapy response. Here, we establish 12 patient-derived organoids from the most common lung cancer subtype, lung adenocarcinoma (LADC). Extensive gene and histopathology profiling show that the tumor organoids retain the histological architectures, genomic landscapes, and gene expression profiles of their parental tumors. Patient-derived lung cancer organoids are amenable for biomarker identification and high-throughput drug screening   •Generation of a living biobank of patient-derived lung adenocarcinoma organoids•Organoid biobank encompasses most of known subtypes of adenocarcinoma•Organoids maintain the histological and mutational spectrum of original tumors•Tumor organoids provide a tool for biomarker identification and drug testing Tissue Engineering; Biotechnology; Cancer Lung cancer is the leading cause of cancer-related mortality globally. Every year, an estimated 1.82 million people are diagnosed with lung cancer and 1.56 million die from the disease . Lung cain vitro models in which the efficacy of candidate therapeutic regimens can be assessed.Despite the considerable advances in the understanding of LADC, medical management is largely empirical and based on clinical and pathological features. However, owing to tumor heterogeneity among patients, clinical outcomes are usually unsatisfactory. The overall 5-year survival rate for lung cancer is 18% for all cancer stages combined in the United States . PrecisiThe most commonly used LADC models are two-dimensional (2D) cell lines and patient-derived xenografts (PDXs). Although lung cancer cell lines, such as the LC2/ad cell line, PC-9 cell line, and VMRC-LCD cell line, have enabled advances in our understanding of lung cancer pathogenesis, they cannot fully recapitulate the three-dimensional (3D) structure of original tumors and do not retain the mutational profiles of their parental tumors . The PDXOver the past few years, three-dimensional cancer organoid culture systems have been established from various cancers, including prostate cancer, colorectal cancer, pancreatic cancer, liver cancer, breast cancer, bladder cancer, and gastric cancer . These cIn this study, we established 12 LADC organoid lines from tumor resections. In addition, we provided a thorough phenotypic and molecular characterization of LADC organoid lines and their parental tumors, including histological architecture, clinical marker expression, genomic landscape, and expression profile. Furthermore, we demonstrated the utility of LADC organoid lines as a model for drug testing to identify new therapeutic targets and advance personalized medicine. This study should be complementary to previous reports by providing a biobank of LADC organoids that covers a large spectrum of different LADC subtypes, including acinar (ACI), solid (SOL), papillary (PAP), and enteric (ENT) adenocarcinoma. We demonstrate that mechanical dissociation method is more suitable for the passage of LADC organoids. By comparing the transcriptome of LADC organoids with that of normal lung tissue-derived organoids, we identified several potential biomarkers associated with survival status when their expression levels were altered.Surgically resected lung adenocarcinoma samples were obtained from untreated patients under informed consent. Each sample was split into four parts for organoid culture, histological analysis, genomic analysis, or transcriptomic analysis A. To isoThe majority of lung adenocarcinoma are classified into acinar (ACI), solid (SOL), papillary (PAP), micropapillary (MIP), or lepidic (LEP) subtypes based on their predominant histologic pattern .To test + samples  and Napsin A , as well samples .Figure 2in vitro.In summary, these results demonstrate that the histological architecture and marker expression of the parental LADC tissues were recapitulated in the LADC organoids, even after long-term culture TP53, the most commonly mutated gene in LADC, was mutated in most LADC organoid lines and their corresponding tumors  of the organoid lines and compared the results with those of the corresponding parental tumors. We filtered for variants and excluded polymorphisms present in organoid lines and parental tumors by comparing them to the analysis of matched patients' normal blood. The 12 LADC organoid lines displayed a heterogeneous set of cancer driver genes affected by missense mutations, splice site mutations, or frameshift mutation, some of which showed a variable pattern of alteration A. Comparg tumors A. We alsl tumors . We notil tumors A. This iTo further determine the extent to which LADC organoid lines maintain the mutation spectrum of their parental tumors, we analyzed somatic substitutions in both tumor samples and organoids. The proportion of base substitutions was well retained among the LADC tissues and the corresponding cancer organoids B. AdditiCCER1 missense mutation and a CNTNAP3 missense mutation existed in the second part of sample ACI-7 but not in the other two subregions.Intra-tumoral heterogeneity is a feature of LADC and may impact patient response to therapy. To assess whether intra-tumoral heterogeneity exists in LADC samples, we collected several parts of tumor tissues from one patient and sent them for whole-exome sequencing. Each tumor tissue was divided into three parts, and two tumor samples were collected. Genomic landscapes analysis revealed that subregion-specific genomic variants existed in different areas of the same tumor . For exaTP53 mutation seen in the tumor. In addition to global gene expression, we also evaluated the gene expression overlap in LADC-specific upregulated genes. These genes were identified by comparing the transcriptomes of LADC tumor samples with normal lung tissues from TCGA . The top 50 upregulated genes were selected in this study. The results showed that LADC organoids largely maintained the expression signatures of LADC-specific genes  on LADC organoid lines and the corresponding tumor tissues. We compared the RNA-seq data of the LADC organoids with 541 LADC expression datasets from The Cancer Genome Atlas (TCGA) to determine whether our organoids were representative of the overall population of LADC. The results show that our organoids were distributed randomly throughout the dataset, suggesting representative gene expression profiles A. Gene eic genes .Figure 4In addition to LADC organoids, we also established three normal lung organoid lines  from normal wild-type lung samples. To explore the potential of LADC organoids as a model to identify tumor biomarkers, we compared the transcriptomes of all LADC organoid lines to those of all normal lung organoid lines. First, we performed principal component analysis (PCA) to determine the contribution of the cancerous state to the difference in gene expression between LADC organoids and normal lung organoid lines. The resulting data showed that normal lung organoids clustered together, whereas the LADC organoids were scattered all around, showing the tumor heterogeneity among patients C. Most LCA9, NT5E, EFCC1, and SERPINE1  analysis to search for signaling pathways altered in LADC organoids. We found that the most enriched pathways in LADC organoids were the PI3K-Akt signaling pathway, pathways related to focal adhesion, complement and coagulation cascades, and the ECM-receptor interaction-associated pathways D.50) and by the area under the dose-response curve (AUC).In order to evaluate the utility of lung adenocarcinoma organoids as a platform to predict patient-specific sensitivities to anticancer drugs, we performed high-throughput drug dose-response screens in 12 lung adenocarcinoma organoid lines. LADC organoid cultures were gently collected and plated in low-attachment 96-well plates in 2% Matrigel/growth medium. Organoids were treated with drugs 1 day after plating and incubated for 6 days before measuring the cell number using Cell Titer-Glo 3D reagent. For each lung adenocarcinoma organoid line, we tested its sensitivity to a library of 24 anti-cancer drugs using dose titration assays with technical replicates and biological triplicates . Drugs were selected based on their clinical relevance for lung cancer treatment, including standard chemotherapy drugs and targeted agents against signaling pathways or molecules of interest. Drug sensitivity is shown by the half-maximal inhibitory concentration . Compared with the conventional enzymatic digestion method, we demonstrate that the mechanical dissociation method in which the organoids are smashed into small fragments using 10-mL Stripette Serological Pipets is a better option for the passage of LADC organoids.in vitro.Comprehensive characterization of the LADC organoid lines confirm that they recapitulate the features of the corresponding parental tumors in terms of histological architecture, cancer driver gene mutations, copy number alterations (CNVs), single-nucleotide variants (SNV), and global gene expression profiles, even after long-term culture RHOF, SLC16A3, HOXB6, ANXA10, and CDHR1, which was not reported to be altered in clinical LADC samples, was associated with survival status of patients with LADC and could be used as prognostic factors. The advantage of using LADC organoids over clinical LADC samples to search for tumor biomarkers is that LADC organoids are purer, so the differences in gene expression between tumor cells and nontumor cells are not affected by the presence of stromal cells and immune cells. Furthermore, the mechanism by which these tumor biomarkers affect tumor behaviors could be studied at the organoid level when combined with molecular biology techniques.As LADC organoids preserve the genomic characteristics and global genes expression profiles of their corresponding parental tumors, we used LADC organoids as a model to identify tumor biomarkers. We found that the expression of KRAS (G12D) mutation in AT2 cells generates multifocal, clonal adenomas in transgenic mice (+ AT2 cells can give rise to LADC in response to KRAS (G12D) activation in mice  mutation, suggesting that BASCs may be the cell of origin for LADC .Correspondence and requests for materials and reagents should be directed to and will be fulfilled by the Lead Contact, Weiren Huang (All organoid lines generated in this study will be available from the Lead Contact.The WES and transcriptome data generated during this study are available at Sequence Read Archive (SRA):SRR12059123-SRR12059164, SRR12072311-SRR12072333.All methods can be found in the accompanying


Cytomegalovirus (CMV) is a threat to human health in the world, particularly for immunologically weak patients. CMV may cause opportunistic infections, congenital infections and central nervous system infections. CMV infections are difficult to treat due to their specific life cycles, mutation, and latency characteristic. Despite recent advances, current drugs used for treating active CMV infections are limited in their efficacy, and the eradication of latent infections is impossible. Current antiviral agents which target the UL54 DNA polymerase are restricted because of nephrotoxicity and viral resistance. CMV also cannot be prevented or eliminated with a vaccine. Fortunately, letermovir which targets the human CMV (HCMV) terminase complex has been recently approved to treat CMV infections in humans. The growing point is developing antiviral agents against both lytically and latently infected cells. The nucleic acid-based therapeutic approaches including the external guide sequences (EGSs)-RNase, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system and transcription activator-like effector nucleases (TALENs) are being explored to remove acute and/or latent CMV infections. HCMV vaccine is being developed for prophylaxis. Additionally, adoptive T cell therapy (ACT) has been experimentally used to combate drug-resistant and recurrent CMV in patients after cell and/or organ transplantation. Developing antiviral agents is promising in this area to obtain fruitful outcomes and to have a great impact on humans for the therapy of CMV infections.  Herpesvirus in the order Herpesvirales, in the family Herpesviridae, and in the subfamily Betaherpesvirinae. There are nine distinct human herpesvirus (HHV) species known to cause human diseases such as HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6A, HHV-6B, HHV-7, HHV-8 [Cytomegalovirus (CMV) is a genus of 7, HHV-8 .CMV structure mainly consist of DNA core, capsid, tegument and envelope from inside to outside. The genome is complexed helically to form a DNA core, which is enclosed in a capsid composed of a total of 162 capsomere protein subunits. The capsid with a diameter of 100 nm is surrounded by the tegument. The tegument is enclosed by a lipid bilayer envelope containing viral glycoproteins to give a final diameter of about 180 nm for mature infectious viral particles (virions) . The virCMV infection will start once a virion attaches a host cell with specific receptors on the cellular surface. For a lytic infection pathway, following linking of viral envelope glycoproteins to host cell membrane receptors, the virions enter the hosts by receptor-mediated endocytosis and membrane fusion. The viral capsid decomposes to release viral DNA genome to manipulates host enzyme systems to make new virions. During symptomatic infection, infected cells express lytic genes to demonstrate a lytic pathway . NeverthViral latency can be divided into two models, namely proviral latency and episomal latency. CMV is defined as the episomal latency model which is essentially quiescent in myeloid progenitor cells. It can be reactivated by differentiation, inflammation, immunosuppression or critical diseases . LatencyMost CMV infections are silent and CMV rarely causes signs or symptoms in healthy people. Though CMV infection is usually ignored in healthy people, the diseases can be life-threatening for the immunocompromised, immunosuppressed and immunonaive patients, such as newborn infants, the elderly, the sick, acquired immunodeficiency syndromes (AIDS) patients, and organ transplant recipients. A mother who acquires an acute CMV infection during pregnancy can transmit viruses to her baby, and thereby the baby might experience signs and symptoms. People at higher risk of CMV infections encompass newborns infected through their mothers before birth, babies infected through breast milk and people with weakened immune systems such as organ transplantation recipients or immunodeficient patients. The major signs, symptoms and complications which CMV influence all individuals including healthy adults, people with weakened immunity and babies are shown in CMV is transmitted by close interpersonal contact such as saliva, semen, urine, breast milk, or vertically transmission which viruses pass the placenta and directly infect the fetus ,16. CMV At present, some antiviral drugs have been approved for the treatment of CMV infections clinically. Current available drugs for antiviral therapy of CMV infections include the inhibitors of viral DNA polymerase, such as the nucleoside analog ganciclovir, the nucleotide analog cidofovir, and the pyrophosphate analogue foscarnet . All theCurrently, ganciclovir is still the first treatment of choice for CMV infections. Letermovir has been approved for the prophylaxis of CMV infections in patients. Several new drugs were developed but still failed in the phase III and more clinical trials would be needed, including maribavir and brincidofovir . ValnoctLetermovir is a novel antiviral drug which has been approved by the USA Food and Drug Administration (FDA) through a fast track procedure and granted as an orphan drug by the European Medicines Agency (EMA). The drug was tested in CMV infected patients and likely be useful for other patients who had organ transplantation or human immunodeficient virus (HIV) infections . It has Maribavir is a promising anti-HCMV compound which is administered orally; however, it is still under advanced clinical trials. The drug targets the viral kinase UL97 which is crucial for the formation of viral teguments and assembly complexes for virion releasing . HoweverMaertens et al. used maribavir (dose-blinded) to treat cytomegalovirus reactivation preemptively for recipients of hematopoietic cell or solid organ transplants (SOT) (≥18 years old) with CMV reactivation in a phase II and open-label clinical trial . The resPapanicolaou et al. used dose-blinded maribavir 400, 800, or 1200 mg twice-daily for up to 24 weeks to treat hematopoietic-cell or SOT recipients (≥12 years old) with refractory or resistant CMV infections in a phase II and double-blind clinical trial . The resThe treatment of diseases caused by CMV is quite challenging because of high mutation rates and latency. Thus, infection is still a serious threat to humans. Fortunately, external guide sequences (EGSs), transcription activator-like effectors nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPRs)/CRISPR-associated 9 (Cas9) nuclease system might provide effective therapeutic strategies to treat diseases caused by CMV through designing a specific DNA or RNA sequence that target essential genes for viral growth. However, the effective modification of the viral genome avoiding off-target effects and the option of escape variants ignoring the editing of these approaches are required for successful clinical application.Ribonuclease P (RNase P) is a unique RNases in that it is a ribozyme – an RNA that acts as a catalyst which is somewhat like a protein enzyme. Its function is to cut an extra or precursor sequence of RNA on transfer RNA (tRNA) molecules; that is, to catalyze the cleavage of precursor tRNA into active tRNA without any protein component. RNase P has the activity in cleaving the 5’ leader sequence of precursor tRNA. EGSs signify the short RNAs that induce RNase P to specifically cleave a target mRNA by forming a precursor tRNA-like complex. Therefore, EGS technology probably acts as an effective strategy for gene-targeting therapy. Deng et al. reported that engineered EGS variants induced RNase P to efficiently hydrolyze target mRNAs which code for HCMV major capsid protein . In vitrLi et al. explored the antiviral effects of an engineered EGS variant in targeting the shared mRNA sequence which codes for capsid scaffolding proteins (mCSP) and assemblins of murine CMV (MCMV) in the animals . In vitrIn CRISPR/Cas9 system, CRISPR is used to build RNA-guided genes drives to target a specific DNA sequence. By the Cas proteins and a specifically designed single-guiding RNA (sgRNA), the genome can be cut at most locations with only the limitation of a protospacer adjacent motif (PAM) sequence (NGG) existing in the target site . CRISPR/Gergen et al. designed two CRISPR/Cas9 systems which contain three sgRNAs to target the HCMV UL122/123 gene crucial for the regulation of lytic replication and reactivation from latency . Both syvan Diemen et al. observed that the clear depletion emerged in the anti-HCMV sgRNA expressing cells targeting essential genes UL57 and UL70 , compared with the sgRNAs targeting the nonessential genes US7 and US11  . The seqTranscription activator-like effectors (TALEs) are crucial virulence factors that function as transcriptional activators in the cell nucleus of plants, where they directly bind to DNA via a central domain of tandem repeats . CurrentChen et al. utilized three pairs of TALEN plasmids  to target the MCMV M80 and M80.5 overlapping (M80/80.5) sequence to test their efficacy in blocking MCMV lytic replication in NIH3T3 cells . Using lThe vaccines against HCMV are still being developed, no licensed vaccine is available so far. It is necessary to have a specific and strong antibody and cell-mediated immunity to confer protection against HCMV primary infection through the analysis of the immune response to HCMV. Many efforts have been made to produce an HCMV vaccine for years, but a successful vaccine candidate has not yet to be developed, probably due to what immune responses needed for protecting against HCMV infections are still poorly understood . To deveChoi et al. used the guinea pig which is a small-animal model to develop a CMV vaccine. A glycoprotein pentamer complex encoded by guinea pig cytomegalovirus (GPCMV) is essential for viral entry into non-fibroblast cells to enable congenital CMV . Like HCLiu et al. hypothesized that a vaccine candidate able to elicit immune responses analogous to those of HCMV-seropositive subjects may confer protection against congenital HCMV . The V16It is known that the infection-related morbidity and mortality will be increased, if the T cell-mediated immune responses are impaired in transplantation recipients. Virus-specific T cells capable of targeting a variety of pathogens in patients after hematopoietic stem cell transplantation (HSCT) have demonstrated potential efficacy for multiple viruses such as CMV, Epstein-Barr Virus (EBV) and adenovirus . AdoptivFaist et al. used the peptide specific proliferation assay (PSPA) to study CMV specific central memory T cells (TCM) repertoires and determined their functional and reproductive abilities in vitro . In the Smith et al. applied high-throughput T cell receptor Vβ sequencing and T cell functional profiling to demonstrate the influence of ACT on T cell repertoire remodeling in the pretherapy immunity and ACT products . The cliCMV is the most frequent etiological factor for congenital infections and its infections are still global health problems of humans. Though CMV infections are often opportunistic, they sometimes cause serious diseases in healthy adults with weakened immunity and babies. Currently, no drugs are available for asymptomatic infants and for infants with CMV congenital infections to reduce related morbidity during the neonatal period. Some traditional antiviral drugs  are applied for the treatment of CMV acute infections, however, their efficacy is limited by side effects, cross-resistance and others. There is no effective cure for CMV infections, especially for latent infections. Promisingly, many novel antiviral drugs/agents and preventive/therapeutic strategies have been approved for clinical application  or are being developed . We should investigate the interactions between CMV and hosts thoroughly to understand how antiviral agents or therapeutic strategies affect CMV infection outcomes. Moreover, the development and implications of novel antiviral agents and preventive/therapeutic strategies should be explored as extensively as possible. The future research tendency and application of these new insights should also be a highlight and could potentially become a promising milestone in the development of therapeutic strategies for CMV infections.


Chronic infection with HBV is a major cause of advanced liver disease and hepatocellular carcinoma. Nucleos(t)ide analogues effectively control HBV replication but viral cure is rare. Hence treatment has often to be administered for an indefinite duration, increasing the risk for selection of drug resistant virus variants. PEG-interferon-α-based therapies can sometimes cure infection but suffer from a low response rate and severe side-effects. CHB is characterized by the persistence of a nuclear covalently closed circular DNA (cccDNA), which is not targeted by approved drugs. Targeting host factors which contribute to the viral life cycle provides new opportunities for the development of innovative therapeutic strategies aiming at HBV cure. An improved understanding of the host immune system has resulted in new potentially curative candidate approaches. Here, we review the recent advances in understanding HBV–host interactions and highlight how this knowledge contributes to exploiting host-targeting strategies for a viral cure. Chronic hepatitis B (CHB), caused by hepatitis B virus (HBV), is a major cause of advanced liver disease and hepatocellular carcinoma (HCC), the second leading cause of cancer death worldwide ,2••. HBVAlthough available drugs against CHB including PEG-interferon-α-based therapies and nucleos(t)ide analogues (NUCs) can effectively control HBV replication, viral cure is extremely rare, for NUCs even upon decade-long treatment . NUCs inCHB is characterized by the persistence of the episomal covalently closed circular DNA (cccDNA) form of the HBV genome which persists as a stable minichromosome in the nuclei of infected hepatocytes . After tTargeting host factors involved in the viral life cycle is a promising and increasingly successful therapeutic approach to overcome resistance by new inhibitors that are characterized by a high genetic barrier. Several host targeting agents (HTA) have already been transferred into clinic. Notably, Maraviroc is a specific antagonist of the human immunodeficiency virus-1 (HIV-1)CCR5 chemokine receptor. It inhibits HIV-1 from entering host cells and is clinically approved for anti-HIV treatments . FurtherDeveloping new HTA to control and/or possibly cure viral infection requires a comprehensive characterization of virus-host molecular interactions, including targetable cell factors playing key roles in the viral life cycle. The recent development of high-throughput functional genomics applied to virus infection systems paved the way for the identification and characterization of many such factors. Numerous genome-scale loss-of-function screens via RNAi knockdown and CRISPR/Cas9 knock-out have been successfully applied to hepatitis viruses, HIV-1, influenza A and Zika virus , 15–17. In this review, we present the cellular functions known to be involved in key steps of the HBV life cycle, discuss the therapeutic potential of individual HBV-related host factors, and highlight the molecules currently in development for novel therapeutic strategies that may lead to HBV cure.Hepadnaviridae family, characterized by infectious virions carrying a 3.2 kb relaxed circular DNA (rcDNA) genome which is produced by reverse transcription. Viral entry initiates with the attachment to heparan sulfate proteoglycans including glypican 5 (GPC5) [′ end of the minus-strand in rcDNA is covalently linked to the viral polymerase [′ ends. While the details of rcDNA to cccDNA conversion are far from being understood it is highly plausible that HBV usurps host DNA repair factors to remove the non-DNA moieties and surplus sequences, and to fill-in and eventually ligate the gaps to generate cccDNA as a proper template for viral transcription. As each nt in the hepadnaviral genome has coding function in one or even two open reading frames all the repair reactions must proceed with single nt accuracy [HBV is an enveloped hepatotropic DNA virus belonging to the 5 (GPC5)  and is f5 (GPC5) ,20. Inte5 (GPC5)  is media5 (GPC5) . How the5 (GPC5)  to the n5 (GPC5) ,25, rece5 (GPC5) , rcDNA clymerase  and carraccuracy .in vitro rcDNA to cccDNA conversion: proliferating cell nuclear antigen, the replication factor C complex, DNA polymerase δ, flap endonuclease 1 and DNA ligase 1 [Meanwhile various DNA repair associated factors have indeed been identified as hepadnaviral host dependency factors. Using duck HBV (DHBV) in human hepatoma cells as a high-copy cccDNA model for HBV, Königer and collaborators found Tyrosyl-DNA-phosphodiesterase 2 (TDP2) as capable of releasing the viral polymerase from rcDNA . DNA polligase 1 , and theligase 1 . Likely ligase 1 ; these mIn vivo, cccDNA associates with cellular histones and nonhistone-proteins, and likely also viral proteins, into a minichromosome as the actual transcription template. Similar to host DNA its activity is regulated by epigenetic modifications . From ccin vitro  [de novo HBV infection and suppress intra-hepatic viral spreading, prompted clinical trials [As the first step in the viral life cycle entry is a conceptually attractive target for the prevention and, if chronicity involves reinfection, also treatment of virus infection. Targeting HBV entry into hepatocytes was extensively studied following the discovery of NTCP as a receptor for HBV and HDV which hijacks the HBV envelope to produce infectious particles . A largetonavir) . Cyclosptonavir) ,44. Meantonavir) . Alreadytonavir) . The deml trials . Althougl trials . The entl trials  and may Elimination of HBV cccDNA, the persistence reservoir in infected hepatocytes, is the ultimate approach towards an HBV cure. As this review focuses on HTAs we will only briefly consider direct strategies such as sequence-specific CRISPR/Cas targeting of cccDNA. Though seemingly straightforward, there a various caveat beyond classical off-target effects. Particular attention will have to be paid to chromosomally integrated HBV DNA; recent data suggest that although integration is a nonproductive pathway for the virus it occurs already early after infection . Hence CA less demanding alternative to cccDNA eradication is silencing of its transcriptional activity. As for CRISPR/ Cas we will here not discuss RNAi mediated silencing of HBV transcripts  althoughAs mentioned the transcriptionally active form of cccDNA is a minichromosome that is subject to epigenetic modifications  Figure . cccDNA On the virus side, the multifunctional HBx protein has been ascribed to modulate recruitment to cccDNA of epigenetic host factor such as p300, HDAC and SIRT1  and thusHBx counteracts this restriction by usurping, through interaction with DNA damage binding protein 1 (DDB1), a cellular E3 ubiquitin ligase which then ubiquitylates Smc5/6 and tags it for proteasomal degradation. Targeting these protein interactions also opens a wide range for drug design. Nitazoxanid reportedly inhibits the HBx-DDB1 interaction, resulting in inhibition of HBV transcription and viral protein production in human hepatocytes . Pevonedde novo synthesized HBc [HBV features an icosahedral capsid formed mostly by 120 dimers of the about 180 amino acid long core protein (HBc). Its most obvious role is to provide a stable container for the viral genome yet HBc is dynamically involved in nearly all steps of the viral life cycle . After iized HBc .Regarding interference with functional capsid formation, most advanced are small molecules which directly target the core protein and capsid assembly. The first such capsid assembly modulators (CAMs) were AT130, a phenylpropenamide , and BAYFor instance, unresolved issues are the fate and effects of the misdirected or prematurely assembled HBc multimers which could cause hepatocyte damage and/or even be beneficial for therapy, perhaps by inducing specific immune responses against the cells which harbor them.HBV produces several types of particles , includiet al., The Hsp40 chaperone DNAJB12 is involved in the morphogenesis of HBV spherical subviral particles and is selectively targeted by nucleic acid polymers. AASLD The Liver Meeting, LP42). Inhibition of HBsAg secretion by NAPs can be reproduced in a human hepatoma cell line which may help to clearly identify the host factor(s) in question [More advanced is the inhibition of HBsAg secretion. While not immediately crucial for progeny virus formation as long as enough envelope proteins remain for virion formation the enormous excess of SVPs over virions secreted from HBV infected cells certainly has a benefit for the virus, for example, via a long-assumed decoy activity against virus-neutralizing anti-envelope antibodies and/or by contributing to the inactivation of virus-specific T cell responses during CHB . SVPs arquestion . A more Perhaps the broadest class of HBV-relevant host factors are those from the innate and adaptive immune response and various approaches exploiting immune-mediated antiviral pathways are being pursued  Figure . As summin vitro and in vivo through production of antiviral cytokines such as IFNs and activation of natural killer (NK) and T cells [ClinicalTrials.gov). Another actively pursued principle is the use of immune checkpoint inhibitors, that is, anti-programmed cell death-1(PD-1), anti-PD-Ligand1 (anti-PD-L1) and anti-CTLA-4  [We here focus on only a few select aspects. Activation of TLR3, TLR7/8 and TLR9 can reduce HBV replication  T cells –108. Sev T cells . Notably T cells ,110. How T cells . If this T cells . Not the T cells ,102. Sev T cells , all aimphase 1) . These aphase 1) , and HBVphase 1)  which maThe finding that patients can spontaneously eliminate HBV infection suggests that the development of curative therapies is an achievable goal. Recent knowledge in virus–host interactions from studies in cell-based and animal model systems has uncovered novel options to exploit host-dependency factors and the innate immune system to target the numerous steps in the HBV life cycle. Many host dependency factors targeting approaches are still in the discovery or preclinical stage, but several immune targeting therapies have already reached clinical development.The increased barrier to pathogen resistance development upon targeting host factors, and the proneness to side-effects of host factor inhibition are two sides of the same medal. It is therefore difficult to predict which host factor targets are the most promising and under which circumstances. Clearly, limiting the duration and localization of host factor inhibition would help to minimize side effects but for most of the host factor targets outlined above much more data are required for strong judgements. Reassuringly, though, one of the host factors discussed above, NTCP, already provides clinical proof that the benefit of antiviral activity can outweigh minor side effects ,20. VirtHowever, in combination regimens Bulevirtide also showed some promise regarding CHB treatment (see below). Of the other host factors outlined above as potential HBV targets those involved in cccDNA biogenesis might be closest to the sterilizing cure goal. This view is supported the positive impact of entry blockade on viral titers which implies that HBV persistence relies not only on cells stably harboring, or intracellularly recycling, cccDNA but on infection of naive hepatocytes. If cccDNA did not undergo turnover and cccDNA harboring hepatocytes lived forever, blocking rcDNA to cccDNA conversion would be fruitless. If, however, the pool of cccDNA is constantly replenished by infection, then inhibiting cccDNA neogenesis can be expected to have therapeutic benefit.Most of the host factors identified as important for rcDNA to cccDNA conversion, especially the five factor core set , are essGenerally, however, while the efficacy of monotherapies may be high, as for NUCs, it is still insufficient to warrant complete viral suppression. Hence combination therapies appear to have great potential. One reason is that while it is biologically feasible to achieve 99% inhibition, reaching 100% is very difficult if not impossible. In combination therapy this is accounted for by ‘synergy’ of two or more components, that is, that the whole is greater than the sum of the individual parts . An examIndeed, several clinical studies suggest such an added benefit. Earlier studies concluded that combining NUCs, including ETV , with tyClinicalTrials.gov Identifier: NCT04820686).The overall goal will be to maximize interference with viral multiplication, prevent viral escape, and help the host immune system to regain control. As different modes of action may be most advantageous combining an effective direct antiviral agent  with a hIn the light of the increasing number of targets for each class of antiviral we are likely to see many more double and triple combination trials in the near future. While this increases the number of options to test it also boosts chances that one or more of these approaches will bring us closer to a cure of CHB, likely with a significant contribution from host factor targeting agents.Given the complexity of the HBV life cycle, it is likely that only such combination therapies, for example, the integrated use of direct-acting antiviral(s) and immune-activating approaches, will be able to achieve the eventual goal of cccDNA elimination. Hence further studies are needed to understand and assess the efficacy and safety of the therapeutic strategies that are already in clinical trials and develop advanced treatment modalities ,87,118;


Histopathologic factors predictive of nintedanib efficacy in idiopathic pulmonary fibrosis have not been studied. We aimed to describe the characteristics, focusing on histopathology, of idiopathic pulmonary fibrosis patients who did and did not respond to nintedanib.This study retrospectively examined the clinicoradiopathologic features of 40 consecutive patients with surgical lung biopsy-confirmed idiopathic pulmonary fibrosis treated with nintedanib. Additionally, we compared the histopathologic scoring of 21 microscopic features between patients with functional or radiological progression and those with non-progression during 12 months of treatment.The histopathologic evaluation showed edematous changes in the interlobular septum as the only histologic finding observed more frequently in patients with both functional and radiological progression than in those without . Regarding per-year change, patients with edematous changes in the interlobular septum showed greater progression in median changes in spared area  and reticular shadow  on computed tomography. Functional and radiological progression-free survival were shorter in patients with edematous changes in the interlobular septum than in those without .Edematous changes in the interlobular septum may indicate poor nintedanib efficacy in idiopathic pulmonary fibrosis. Further studies are needed to validate these findings and address the mechanism behind ECIS. Predictive biomarkers of nintedanib efficacy in patients with idiopathic pulmonary fibrosis (IPF) have not been addressed previously but are urgently needed for optimal clinical outcomes. As a study of serum markers is already underway , a histoAlveolar epithelium and fibroblasts are thought to be key players in the pathogenesis of IPF because recurrent epithelial injury (EI) leads to aberrant repair of the injured alveoli and deposition of interstitial fibrous tissue by myofibroblasts . The 201Another clinical problem associated with IPF is its unpredictable response to antifibrotic treatment. Although antifibrotics showed a 50% reduction of FVC decline in 1 year, compared with the placebo group in a phase 3 trial and recent network meta-analysis, the individual response to antifibrotics is unpredictable , 10. PreThus, the aim of this study was to describe the histopathologic characteristics of patients with IPF who did and did not respond to nintedanib treatment.Consecutive patients with IPF, who started nintedanib (200 or 300 mg/day) treatment by January 2017, were retrospectively identified from electronic medical records at two tertiary care hospitals. IPF was diagnosed by a multidisciplinary team using multimodal information, including chest computed tomography (CT) scans and surgical lung biopsy (SLB) findings. The exclusion criteria were: (1) loss to follow-up within 6 months from starting nintedanib treatment; (2) concomitant medication  use when starting nintedanib treatment; (3) missing data within 6 months prior to starting nintedanib treatment and at 12 months after the start of nintedanib treatment, except for cases of death or acute exacerbation; and (4) unresectable lung cancer.This was a two-centre, retrospective, cohort study of patients with IPF in Japan that assessed the characteristics of patients with IPF who did and did not demonstrate functional or radiological progression. Functional progression was defined as the presence of indicators based on the following composite factors after 12 months of nintedanib treatment: 1) all-cause death; 2) acute exacerbation, reported by pulmonary physicians; 3) relative FVC decline of ≥10% . RadioloBaseline clinical, pulmonary function test (PFT), and chest CT data were obtained from the electronic medical records of both hospitals. Clinical data included patient age, sex, smoking status, and comorbid diseases. PFT and chest CT data were obtained before the start of nintedanib treatment (within 3 months), as well as at a variable point of 6 to 12 months after starting nintedanib treatment.Chest CT scans were obtained with 1- or 2-mm collimation at 1- or 10-mm intervals and fitted under a high spatial frequency algorithm. Two blinded board-certificated chest radiologists, with 30 and 16 years of experience reviewed the chest CT data. The images were independently evaluated to score the extent of reticular opacity, HC, traction bronchial-bronchiolectasis, ground-glass opacity, airspace consolidation, cyst formation, emphysema (Em), and spared lung area in gradations of 5%, as previously described by Fujisawa et al. . ConsensWhole-section SLB specimens were stained using haematoxylin and eosin and Elastica van Gieson or Elastica–Masson staining. All evaluations were performed using digitized images by slide scanners at 20× magnification . The SLB images were independently evaluated by four board-certificated pulmonary pathologists, with 9–24 years of experience, blinded to the clinical data. The histopathological findings were rated from 0 to 3 points  and further dichotomized as negative (0–1 points) or positive (2–3 points). Consensus was established when the positive or negative judgements were discrepant among the reviewers; the inter-rater variability was assessed. The histopathological findings of interest  were: dense fibrosis (DF), cellular interstitial pneumonia, marked organizing pneumonia, fibroblastic focus (FF), destroyed architecture (DA), HC, smooth muscle hyperplasia, elastosis, lymphoid follicles, cellular/fibrinous pleuritis, plasma cell infiltration, constrictive bronchiolitis, vascular intimal thickness, loose granuloma, interstitial giant cells, peribronchiolar metaplasia, airway-centred changes, EI, Em, alveolar exudate, and edematous changes in the interlobular septum (ECIS). All pathologists had a group session to standardize the morphologic features prior to their review of the study specimens.2 test was performed to analyse categorical variables and the Mann–Whitney U test was performed to analyse continuous variables. Logistic regression analysis was performed as multivariate analysis. Statistical significance was indicated by a P-value <0.05. All analyses were performed using the R statistical software package  with the add-on EZR package  . In. In20]. CO , lower extent of spared area on chest CT , and marginally significantly greater proportion of honeycomb pattern on chest CT . Multivariate analysis including sex, age, the proportion of honeycomb pattern on chest CT, and initial %DLCO revealed that only age and sex had statistically significant correlation to patients with ECIS.The baseline characteristics of patients with and without ECIS are presented in Functional and radiological PFS were both significantly shorter in patients with ECIS than in those without . Kaplan–Meier curves and comparisons of outcomes for both groups are shown in This study described the characteristics of patients with IPF undergoing nintedanib treatment, including histopathological findings associated with poor response to therapy. We found that most patients developed functional and radiological progression during the 12-month follow-up period. In patients showing disease progression, ECIS in SLB specimens was significantly more frequently observed; very few patients without functional and radiological progression showed ECIS. In patients with ECIS, PFS was significantly shorter.To the best of our knowledge, our study is the first to report the histopathological characteristics of patients with IPF who did not respond to nintedanib treatment; we also determined ECIS as an important predictive marker of disease progression. Although the evaluation of some pathological findings is challenging and the level of diagnostic agreement of IPF in previous reports has been very low  , the κ-vAccording to previous publications, 24–30% of IPF patients with nintedanib developed progression at a rate similar to that of patients without any treatment and showed reduced FVC values during treatment . The curMUC5B or MUC2 [To ensure that ECIS was not artifactually induced, we matched tissue processing protocols at both institutions and found that the stepwise specimen processing and reagents used were the same. We also consistently observed dilated lymphatics associated with ECIS , which p or MUC2 , but by This study had some limitations that should be addressed. First, this was a two-centre study based on a relatively limited sample size due to the rare prevalence of the disease  and striConversely, the strengths of the study are obvious. It is the first of its kind to examine in detail the histopathological changes (21 parameters scored semiquantitatively) of IPF patients treated with nintedanib. We found that ECIS, a previously underappreciated histological finding, was correlated with poor prognosis in the study cohort. Hence, certain histopathologic parameters might be of prognostic significance and a careful evaluation of SLB during IPF treatment should be considered.In conclusion, the current study described characteristics of patients with SLB-confirmed IPF who demonstrated disease progression during nintedanib treatment. ECIS was the key factor predictive of poor treatment efficacy contributing to shortened PFS. Further studies involving larger series of patients are needed to validate the factors predictive of nintedanib efficacy.S1 Table(DOCX)Click here for additional data file.S1 Dataset(CSV)Click here for additional data file.


The leaf economics spectrum (LES) describes consistent correlations among a variety of leaf traits that reflect a gradient from conservative to acquisitive plant strategies. So far, whether the LES holds in wetland plants at a global scale has been unclear. Using data on 365 wetland species from 151 studies, we find that wetland plants in general show a shift within trait space along the same common slope as observed in non-wetland plants, with lower leaf mass per area, higher leaf nitrogen and phosphorus, faster photosynthetic rates, and shorter leaf life span compared to non-wetland plants. We conclude that wetland plants tend to cluster at the acquisitive end of the LES. The presented global quantifications of the LES in wetland plants enhance our understanding of wetland plant strategies in terms of resources acquisition and allocation, and provide a stepping-stone to developing trait-based approaches for wetland ecology. Leaf economics spectrum theory has greatly advanced understanding of plant functional ecology, but it is unclear whether its predictions hold in wetland communities. Here, Pan and colleagues analyse leaf economics traits in wetland plants, showing that their trait relationships deviate from fully terrestrial plants, particularly by clustering towards acquisitive plant strategies. The LES has been shown to be present across different plant life forms and varied habitat types at a global scale and to a large extent independent of climate3. Along the LES, species with higher leaf mass per area (LMA) generally have a longer leaf life span (LL), but a lower leaf nitrogen content , and lower photosynthetic rates, at least on a mass basis (Amass). This conservative strategy usually prevails in less fertile habitats. On the other hand, species with lower LMA, shorter LL, higher leaf N and photosynthetic rate have a faster return on investment of resources, commonly coinciding with nutrient-rich areas. Such trait-trait coordination in LES traits may be caused by underlying physiological and structural trade-offs4.During the past two decades, trait-based ecology has advanced considerably. The leaf economics spectrum (LES) is an important component thereof. The LES provides convincing evidence of a consistent and continuous relationship among the leaf economics traits, reflecting a gradient of slow (conservative) to fast (acquisitive) strategies in terms of investment and use of nutrients and other resources5 or on global analyses6. However, whether the general LES also exists in global wetland ecosystems still remains unclear. This gap prevails despite the fact that leaf economics traits have been widely measured in wetland plants to study local plant functioning, community structure, growth and competition7.Studies on trait-trait relationships, including those on LES, have focused mainly on non-wetland terrestrial plants from a variety of ecosystems, such as forests or grasslands8. Important ecological processes in wetlands such as methane emission and denitrification are linked to wetland plant functional traits10. LES traits in wetlands are likely to play a role in these ecosystem processes and services12. While the wide fertility gradient across different wetland types theoretically provides a natural gradient for the expression of LES from the acquisitive to conservative strategies13, additional constraints induced by adverse environmental conditions in wetlands compared to non-wetland systems mean that it cannot be taken for granted that LES traits will show similar patterns.A better understanding of trait-based relationships in wetlands is profoundly needed in light of the important ecosystem services provided by wetlands, including their role as the major carbon sink at a global scale2+) and sulphide  in the substrates, as well as a less efficient way of producing ATP in cells experiencing an O2 deficit14. In addition, reactive oxygen species (ROS), which can cause cellular macromolecule and membrane damage, accumulate in plant tissues especially upon return to aerobic conditions after flooding15. To survive in such an adverse environment, wetland plants have developed a suite of adaptive strategies15. Whether the LES also exists in wetlands depends, to a large extent, on whether the prevalent adaptive strategies of plants to environmental stressors are generally costly or cheap13. If adaptations are cheap, the LES should be unaffected and similar to non-wetland ecosystems. But if adaptive traits are costly, the LES should be shifted along the same axes (or even shifted in trait space entirely) to compensate this cost13. Moreover, leaf mass per area  seems to also be directly involved in flooding tolerance of wetland plants16, which may also lead to deviations within the LES.The varied environmental stressors unique to wetland ecosystems constrain plants that inhabit these systems. For example, intermittent/permanent flooding causes altered biogeochemical processes and the production of phytotoxic compounds such as ferrous iron  content (wt/wt). This would naturally lead to faster photosynthesis in the day and higher dark respiration rate in the night. Assuming the trade-off between LMA and leaf longevity that exists in non-wetland plants17, artificial waterbodies, bogs, estuaries, fens, forested/shrub wetlands, mangrove swamps, marsh, rivers and lakes, temporary brackish/saline non-forested wetlands and temporary non-forested wetlands; see details in Appendix S1 of Pan et al.18). These habitat types occupy different positions along the gradients of two dominant drivers: hydrological regime (flooding depth and duration) and fertility (from oligotrophic to eutrophic)19. The wetland plant species analyzed in this study represent a full spectrum of plant characteristics and belong to eight life-form categories . To take the effect of submergence on wetland plants into account, we carefully separated traits measured on plants of which only the root-zone or part of the stem was flooded of which tissues emergent above the water table were measured  vs. traits measured on plant tissues that were submerged .To test these hypotheses, we collected the LES traits measured in 365 wetland species of 184 families from 151 studies of both published and unpublished sources from a global scale. These wetland species are mainly from 10 wetland habitat types , while three out of four relationships for submerged plants had a lower R2 than those of non-wetland plants (Table R2 of four non-significant trait-trait relationships of both plants of waterlogged and of submerged conditions showed no overlap with the (significant) R2 observed for the corresponding trait-trait relationship of non-wetland plants. In combination, these results indicate weaker trait-trait relationships between wetland plant traits than corresponding relationships among non-wetland plants  and submerged wetland plants (R2 = 0.31). The SMA analysis revealed that there was no significant difference in slopes of leaf P-leaf N associations between non-wetland plants and wetland plants . However, the parallel slopes of both waterlogged and submerged wetland plants were elevated compared to non-wetland plants (both P < 0.001), which indicates that at a given leaf N, wetland plants tended to have a higher leaf P than non-wetland plants. Moreover, there was a significant shift along the common slope towards higher values in wetland plants , while submerged wetland plants had a significantly steeper slope (P < 0.001). Thus, as LMA decreases, the increase in leaf N was less pronounced in waterlogged wetland plants, while such increase of leaf N was steeper in submerged wetland plants, compared to non-wetland plants  compared with non-wetland plants. This indicates that even though leaf P and LMA maintained similar relationships in non-wetland and wetland plants, wetland plants maintained a higher value of leaf P but a lower value of LMA  and had an elevated parallel slope (P < 0.001) compared to non-wetland plants, indicating that at given leaf N, waterlogged wetland plants had a higher photosynthetic rate. This suggests that waterlogged wetland plants had a higher nitrogen use efficiency (photosynthesis per unit investment of leaf N). No significant shift along the common slope nor elevation differences among parallel slopes were detected for submerged wetland plants .The slopes of photosynthetic rate-leaf N in wetland plants were similar to those of non-wetland plants . This suggests a similar proportional change between leaf P and photosynthetic rate of both wetland plants and non-wetland plants, while wetland plants had higher values of photosynthetic rate and leaf P than non-wetland plants. No elevation differences among parallel slopes were detected , suggesting that wetland plants and non-wetland plants have a similar photosynthetic rate per unit leaf P.There were no significant differences in slopes of photosynthetic rate-leaf P between wetland plants and non-wetland plants (P < 0.001) along the common slope towards the corner of lower LMA values but higher photosynthetic rates. This suggests that waterlogged wetland plants generally had lower LMA, but a higher photosynthetic rate. For submerged wetland plants, the photosynthetic rate-LMA slope was significantly steeper than for non-wetland plants (P < 0.01). This shows that the decrease of photosynthetic rate with an increase per unit of LMA was stronger in submerged wetland plants, indicating that the effect of changed leaf structure on the photosynthesis was bigger in submerged wetland plants. In other words, the photosynthetic rate of submerged wetland plants was even more reduced by an increase of LMA  than non-wetland plants. This suggests that submerged wetland plants maintained their respiration rate to a lower level as leaf N increases than non-wetland plants , LMA (P < 0.001) or photosynthetic rate (P < 0.01)  Fig. . In combP = 0.15), nor a shift along the common slope (P = 0.06), nor elevation differences among parallel slopes (P = 0.42) between the waterlogged wetland plants and non-wetland plants  was observed, but waterlogged wetland plants showed a significant shift along the common slope towards higher dark respiration rate and leaf P values (P < 0.001)  Fig. . In boths P = 0.4 between nts Fig. . Finallynts Fig. .n = 3 for LL-leaf N and LL-leaf P, and the absence of data linking LL-LMA, LL-photosynthetic rate and LL-dark respiration rate). For waterlogged wetland plants, we found a significant lower parallel slopes between LL-leaf N than non-wetland plants  in submerged wetland plants remains uncertain, because of the limited number of data points  of trait-trait relationships) suggest that alternative strategies exist among wetland plants to deal with the complex and adverse wetland conditions with specific stressors. It may also suggest that besides nutrients and light, other limitations in wetlands also influence the LES and require alternative strategies and consequently the special leaf structure and function of wetland plants. This would cause a higher variation in LES traits. Besides habitat N and P fertility, leaf N can be driven by various factors, including potassium (K), temperature, phytotoxins, or the plants’ intrinsic maximal growth rate7. Habitat wetness may also drive leaf N through two indirect mechanisms. On the one hand, denitrification caused by prolonged soil flooding may decrease nitrate availability, thus reducing leaf N20. On the other hand, species living in wet habitats usually have a lower LMA, and thus tend to have a higher leaf N22. The more variable leaf N may further affect the expression of trait-trait associations in wetland plants, such as the leaf N-photosynthetic rate associations23 and the leaf N-dark respiration rate associations24.We compared leaf economics spectrum (LES) trait associations of wetland and non-wetland plants and found that the LES does exist in wetland plants, but with weaker and often deviating/shifting trait-trait associations relative to the non-wetland LES. The weaker trait-trait associations  submerged conditions29. Therefore, besides its leaf economics aspect, LMA should be considered also as a key wetland trait.Secondly, a major deviation in LES trait-trait relationships of wetland plants compared to non-wetland plants occurs in the leaf N-LMA relationship Fig. . The dif30. Our results suggest that such protection of leaves is not feasible in wetlands. In addition, the higher leaf N in wetland plants may also cause an increased risk of herbivory31. Together, these processes partially explain the higher herbivory rates in wetland ecosystems compared to non-wetland terrestrial ecosystems31. One way to compensate for the higher losses is to become more acquisitive. Such a strategy is supported by the shift in LES traits along the common slope, but may also relate to the elevated leaf P at a given LMA. The results on the leaf N to leaf P relationships suggest that leaf P is even more elevated in wetland plants than leaf N  expresses the combination of photosynthesis-related active nutrients and those nutrients used for storage and protection33. Low oxygen can restrict aerobic respiration, both in roots34 and in leaves35. The relatively low dark respiration rate in leaves of wetland plants may be due to a lower investment of resources in leaf construction and maintenance, and related reductions of energy requirements and respiration during the night24. The lower respiratory demand allows to more readily face hypoxia when leaves become submerged. In addition, leaves with porous tissues will enhance the oxygen status of the innermost cells. Note that, although the adaptive formation of aerenchyma will significantly decrease the cell oxygen consumption on a tissue volume basis36, the data analysed here are measurements expressed on a tissue mass basis. Hence, aerenchyma formation per se does not explain the patterns found in this study.Lastly, leaves of submerged wetland plants have a lower dark respiration rate (mass basis) than expected from a comparison with the non-wetland LES. Oxygen can decline to hypoxic levels during submergence, and especially in shallow water bodies during the nightmass-LMA relationship  in this study. This threshold was chosen to help ensure that the most ecologically relevant relationships were detected in relatively large datasets . However, for those relationships with smaller sample sizes (in particular in relation to dark respiration rate and leaf life span), this approach may have resulted in overly conservative interpretation. This indicates that deviations in the LES of wetland plants may include even more trait-trait relationships than identified here. Future research could serve to quantify whether these patterns are robust in the face of more data.All of the described significant changes in the slope of trait-trait relationship, in the position along the slope or due to shifted parallel slopes were detected based on a rather conservative 37, also exists in wetland plants. However, such a relationship is expressed differently in wetland plant species compared with non-wetland plants. The results from our analysis show that submerged wetland plants are capable of having lower dark respiration rate at a given photosynthetic rate than non-wetland plants.Altogether, our analysis suggests that the direct link between photosynthetic rate and dark respiration rate, as evidenced from non-wetland plants to complement N-rich enzymatic and other metabolic components that lead to a higher respiration cost when maintaining a high photosynthetic rate13. In addition, there are some environmental stressors that rarely happen in wetlands. For example, drought stress, which is a common problem in terrestrial ecosystems, is less constraining in most wetlands, and might move LES traits of wetland plants to the optimum end with lower LMA with higher leaf nutrient content38. The combination of the high productivity in wetlands and the retarded biochemical cycling rate in the anoxic environments of the substrates together make wetlands the largest contributor to the terrestrial biological carbon pool8.When upscaling the findings to wetland ecosystem functioning, we ascribe the generally high productivity in wetland ecosystems globally to the adaptation of wetland plants by having generally fast-return strategies and a higher payback rate. In this way, the adverse wetland conditions may have very limited impact on the wetland plant functioning in terms of resource accumulation. The assumed trade-offs between the cost of adaptation to wetlands and plant function from the leaf economics spectrum perspective are therefore not profound in general17. We summarized the 3 major groups including 42 sub-groups wetland habitat types in Ramsar Convention to be 12 categories . We collected leaf economics traits for wetland plants on a global scale including those plants exposed to intermittent/permanent wetland conditions (waterlogged or flooded) from both field and experiment measurements. The wetland plant leaf economics trait dataset was compiled based on a systematic search in Web of Science and Google Scholar (last updated on the 5th June 2018). The literature search included permutations of the following keywords: wetland plants, marsh plant, bog plant, isoetid, aquatic plants, macrophytes, submerged plants, floating-leaved plants, emergent plants, mangroves, leaf economics traits, leaf economics spectrum, leaf nitrogen, leaf phosphorus, SLA, LMA, leaf life span, photosynthetic rate, underwater photosynthetic rate, dark respiration rate. Additionally, our network of wetland experts from around the world contributed recommendations for possible literature that we had overlooked. Finally, we added unpublished data of our own and of our network. We did not include data from other trait databases that are dominated by terrestrial records, including TRY, because the few records available for wetland plants in these databases do not have a sufficiently detailed habitat description that would allow the differentiation between waterlogged and submerged required for our analysis.We defined wetland plants as plants that mainly occur in (or are exposed to) wetland habitats as described by the Ramsar Conventionhttp://www.theplantlist.org) to unify all plant synonyms names from the original references to a unique and consistent accepted name.We followed the nomination system in The Plant List . We took trait values for the same six traits for non-wetland plant traits (of 1569 species) from the GLOPNET database for comparison3. For a consistent analysis of trait-trait trade-offs, we expressed all leaf economics traits on a mass basis. Mass-based and area-based traits can be interconverted via a division by LMA. The mean value for each trait of each species was used  to strictly aquatic plants (level 12)R2) of each trait pair within the six LES traits of waterlogged and submerged wetland plants at the species level was calculated by a standardized major axis (SMA) analysis40. The slopes and R2-values were compared to those of trait-trait relationships of non-wetland plants as derived from the GLOPNET3. The evaluation was based on the comparison between waterlogged wetland plants and submerged wetland plants, with non-wetland plants, respectively.The slope and its associated coefficient of determination , a second to assess shift along the common slope (i.e. a more predominant position of wetland plants on either the conservative or acquisitive end of LES), and a third to assess whether trait associations of wetland and non-wetland plants can be characterized as having elevation differences among parallel slopes (suggesting a specific trait would be more -or less- costly in wetland conditions)Test A: sma(y~x*groups) tests for differences in slopes fitted for different groupsTest B: sma tests for a shift along the common slopeTest C: sma(y~x+groups) tests for elevation differences among parallel slopes between groups41, tests B and C were only run if there was no significantly different slope detected in Test A. If all three tests were non-significant, we conclude that wetland and non-wetland plants have similar trait-trait relationships.A significant difference in slope (Test A) implies a difference in the direction and location of the relationship in trait space. Since the location and direction of lines with different slopes are not comparableP-value is strongly depended on sample size, and it does not measure the size of an effect or the importance of a result42. In this study, we set a rather conservative P-value threshold (P < 0.01) for our tests. This was done to help reducing type I errors and to ensure that the most ecologically relevant relationships (with a reasonable effect size) were detected in these datasets43.The 44. The major axes analysis was conducted with the sma and ma function in the smatr package (version 3.4–8)40.The statistical analysis used R softwareFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Deep-seated slow slip and tremor (SST), including slow slip events, episodic tremor and slip, and low-frequency earthquakes, occur downdip of the seismogenic zone of numerous subduction megathrusts and plate boundary strike-slip faults. These events represent a fascinating and perplexing mode of fault failure that has greatly broadened our view of earthquake dynamics. In this contribution, we review constraints on SST deformation processes from both geophysical observations of active subduction zones and geological observations of exhumed field analogues. We first provide an overview of what has been learned about the environment, kinematics and dynamics of SST from geodetic and seismologic data. We then describe the materials, deformation mechanisms, and metamorphic and fluid pressure conditions that characterize exhumed rocks from SST source depths. Both the geophysical and geological records strongly suggest the importance of a fluid-rich and high fluid pressure habitat for the SST source region. Additionally, transient deformation features preserved in the rock record, involving combined frictional-viscous shear in regions of mixed lithology and near-lithostatic fluid pressures, may scale with the tremor component of SST. While several open questions remain, it is clear that improved constraints on the materials, environment, structure, and conditions of the plate interface from geophysical imaging and geologic observations will enhance model representations of the boundary conditions and geometry of the SST deformation process.This article is part of a discussion meeting issue ‘Understanding earthquakes using the geological record’. E–3. E1–3]°C, which are conditions commonly associated with hot and young subducting oceanic plates, downdip of the ‘seismogenic zone’ hosting regular earthquakes of up to M > 9 (figures The ‘SST zone’ is located within depth and temperature ranges of about 25–55 km and approximately 350–550 figures . DowndipM < 2.5) low-frequency earthquakes (LFEs) and M 3–4 very low-frequency earthquakes embedded in the tectonic tremor signal allows for more detailed investigation of underlying source properties and the spatio-temporal distribution of fault slip. Thus, accurate locations and source properties of tremors and LFEs are essential ingredients for improved understanding of the slow slip process.There is abundant evidence that indicates a close temporal and spatial association of tremor and slow slip ,15. TremSo, what are the rocks and structures preserved in the geologic record that could represent deep tremor and slow slip? An essential aspect of understanding the SST deformation process is the examination of rocks exhumed from SST source depths, which are exposed in subduction complexes in a wide range of tectonic settings around the globe . Exhumed2.Deep-seated SST represents only a small part of the wide spectrum of seismic and aseismic slip processes that we now recognize to occur across the whole width of the subduction plate interface ,19,20. I(a)P-wave and S-wave velocities (Vp/Vs), the low-velocity layer (LVL) )(ii)Given the frequency at which SST events recur , whereak record –202). ThOne of the most widely documented potential markers of transient deformation in subduction complexes are ‘melange belts’, which we loosely define here as localized high strain shear zones in which blocks of higher viscosity material are embedded in a less viscous matrix –206. TheAn additional possible form of transient frictional-viscous creep that does not necessarily require significant lithological variations are localized shear zones that appear to have deformed by some combination of pressure solution, frictional sliding on phyllosilicate laminae or cleavage planes, and dilational micro-cracking ,215. Pre(iii)a). There are also examples of mutually cross-cutting relationships between veins opened both perpendicular and parallel to the shear zone fabric, suggesting transient switches in the orientation of the maximum compressive stress, also indicative of very high pore fluid pressures (figures 10c) )(4)What distinguishes short slow slip events that are accompanied by abundant tremor and LFEs (e.g. as observed ETS in Cascadia and Nankai) from long-term, sometimes `tremorless’ SSEs ? And what distinguishes portions of ETS slip zones that apparently produce little or no tremor e.g. ,182)? Ar? Ar182])(5)Can the wide range of geophysical and geological observations inform the development of meaningful new approaches to modelling fault slip in the SST zone? Ultimately, improved constraints on what’s down there; i.e. the rocks, macro- and micro-structures, fluids and reactions, need to be distilled down into meaningful fault-zone model parameters, such that we can better represent the (time-dependent) boundary conditions and geometry of the ETS system. Detailed information about the dynamic evolution of slow slip and tremor failures and their response to changing conditions and external forcing should allow for improved characterization of the spatio-temporally variable deformation, dominant deformation processes and relevant rheological properties.The preceding overview of the geophysics and geology of the deep subduction interface illustrates the considerable progress being made toward understanding the structure, materials and environment of deep-seated episodic tremor and slow slip (cf.figures


High-grade serous carcinoma (HGSC) is the most prevalent and aggressive subtype of ovarian cancer. The large degree of clinical heterogeneity within HGSC has justified deviations from the traditional one-size-fits-all clinical management approach. However, the majority of HGSC patients still relapse with chemo-resistant cancer and eventually succumb to their disease, evidence that further work is needed to improve patient outcomes. Advancements in high-throughput technologies have enabled novel insights into biological complexity, offering a large potential for informing precision medicine efforts. Here, we review the current landscape of clinical management for HGSC and highlight applications of high-throughput biological approaches for molecular subtyping and the discovery of putative blood-based biomarkers and novel therapeutic targets. Additionally, we present recent improvements in model systems and discuss how their intersection with high-throughput platforms and technological advancements is positioned to accelerate the realization of precision medicine in HGSC. BRAF, KRAS and PTEN mutations. Type II cancers, on the other hand, comprise of HGSC, which account for the vast majority of all EOCs, carcinosarcomas and undifferentiated carcinomas. HGSCs have a high degree of genetic instability and are characterized by the presence of acquired or inherited mutations in different DNA repair pathways including TP53, BRCA1/2 and other defects in homologous recombination repair genes [The American Cancer Society estimates that in 2020, 21,750 women will be newly diagnosed with ovarian cancer in the USA and ~ 13,940 will die from the disease [ir genes .Early-stage disease is typically asymptomatic, and currently there are no proven screening strategies for HGSC that reduce mortality , 10. TheBRCA1/2 mutations is an example of the shift toward precision medicine in HGSC; however, additional work is still necessary. Biomarkers that are reflective of tumor burden and therapies which target specific tumor characteristics are needed to improve patient outcomes. Advancements in high-throughput biological techniques have provided new opportunities for the discovery of biomarkers and therapeutic targets. These platforms allow for the simultaneous profiling of thousands of molecules and the subsequent generation of a wealth of biological data. Together with large cohorts of well-annotated patient samples and improved model systems, these approaches have facilitated novel insights into biological heterogeneity at an unprecedented scale. In this review, we provide an overview of how high-throughput approaches have contributed to the molecular profiling of patient heterogeneity within HGSC and highlight the utility of these technologies in the discovery of putative blood-based biomarkers and therapeutic targets as a step toward enabling precision medicine as a reality for all HGSC patients  network, where 489 tumor samples were subjected to genomic and transcriptomic analyses . Exome sC tumors , suggest in HGSC . Serous  process . GermlinA cohort . Seven oA cohort . The TCGA cohort . Indeed,A cohort . Other fnd FOXM1 . In an aThe profiling of mRNA expression in HGSC tumors has identified four overlapping transcriptional subtypes of HGSC: C1—mesenchmyal, C2—immunoreactive, C4—differentiated and C5—proliferative , 23. IndAside from delineating molecular heterogeneity between patients, high-throughput tumor profiling can also be used to elucidate the diversity within a tumor. Deconvolution of bulk HGSC transcriptional data has revealed that individual tumors often display multiple subtype signatures , accentuA biomarker is a measurable feature that is reflective of biological processes and can provide information regarding the disease state of an individual. Cancer biomarkers are used for various purposes throughout the course of disease progression, including assessing the likelihood of developing cancer, diagnosing malignancies, determining prognosis, predicting patient responses to specific therapies and monitoring residual disease post-treatment and during remission. In contrast to directly examining tumor tissue, liquid biopsies can facilitate minimally invasive tumor assessments to guide clinical decisions. Blood is an attractive biological fluid for biomarkers in clinical practice due to the standardized collection procedures and abundant availability. In this section, we briefly review the current landscape of HGSC blood-based biomarkers and discuss the utility of high-throughput approaches in the discovery of novel biomarkers to help improve clinical management of HGSC.In the context of HGSC, and EOC in general, serum biomarkers are currently used for the differential diagnosis of a pelvic mass prior to surgery, monitoring response to treatment and detecting recurrent disease. Although definitive diagnosis of EOC currently requires histological examination, differential diagnosis of a pelvic mass determines preoperative referral . This isMUC16 and was identified as a tumor marker for EOC in 1983 [CA125 is a large membrane glycoprotein encoded by the gene  in 1983 . Signifi in 1983 . Despite in 1983 . Further in 1983 . The low in 1983 . Given t in 1983  and the  in 1983  were dev in 1983  demonstrNevertheless, CA125 offers clinical utility when evaluating treatment response and monitoring remission. A decrease in serum CA125 is indicative of treatment response, whereas a persistence of abnormally elevated CA125 or increases may suggest treatment resistance and/or residual disease . Many poWFDC2, is a secreted glycoprotein that is overexpressed in serous and endometrioid ovarian cancers [Human epididymis protein 4 (HE4), encoded by the gene  cancers . Hellstr cancers  initiall cancers –61. This cancers . Serum H cancers . Serum H cancers . In a pi cancers  found th cancers , 67. The cancers , but datIn response to the promising data regarding the complementary potential between CA125 and HE4 as diagnostic markers, the Risk of Malignancy Algorithm (ROMA) was developed and is currently approved for differential diagnosis . ROMA coBRCA1/2 are the strongest genetic risk factors for nonmucinous EOC [BRCA1 and BRCA2 carriers ranges from 40 to 59% and 16 to 18%, respectively [BRCA1/2 mutations are considered predictive biomarkers due to strong associations with favorable outcomes following both platinum-based chemotherapy [BRCA1/2-deficient women with recurrent disease [BRCA1/2 mutations is recommended for all newly diagnosed ovarian cancer patients to aid in therapy selection and determining cancer risk for family [Germline deficiencies in nous EOC . The cumectively –82. As sectively  and willectively , 85. In otherapy , 87 and otherapy , 89. PAR disease , 91. Henr family .Though blood is an attractive source of biomarkers, limitations in the throughput of highly sensitive molecular measurements have been a challenge, especially for heterogeneous diseases such as HSGC. Advancements in ‘omics’ approaches have enabled the ability to characterize and evaluate various classes of circulating molecules as potential blood-based biomarkers Fig. . A biomaTP53 mutations are ubiquitous in HGSC, detection of TP53 mutants in cfDNA has been preferentially used when investigating ctDNA as a biomarker [TP53 mutations in longitudinal plasma samples from HGSC patients undergoing treatment to evaluate the value of ctDNA in determining prognosis and response to treatment. This study revealed that the abundance of TP53 mutant ctDNA fractions prior to treatment significantly correlated with volumetric measurements of tumors from CT images, unlike CA125, and that a decrease of > 60% of TP53 mutant ctDNA fractions following treatment was a predictor of time-to-progression. Christie et al. [BRCA1/2 mutations can be detected in ctDNA, as this molecular alteration is known to correspond with acquired chemo-resistance. Reversion mutations were detected in the plasma of three out of five patients with reversion mutations observed in tumor samples, all of whom were resistant to platinum-based therapy or PARP inhibitors. The authors noted that detection of reversal mutations was associated with the fraction of ctDNA out of total cfDNA, measured by the presence of TP53 mutant alleles. Certainly, a limiting factor of utilizing ctDNA as a tumor marker is that current detection strategies may not be sensitive enough to detect rare mutants in early-stage disease where the ctDNA fraction is low [Circulating cell-free DNA (cfDNA) are short fragments of DNA released into the bloodstream from apoptotic or necrotic cells . The quaiomarker –101. Pariomarker  analyzede et al.  recentlyn is low .In addition to investigating specific genes, examination of genome-wide chromosomal aberrations in ctDNA can be promising for the discovery of novel tumor markers. Harris et al.  used whomicroRNAs (miRNAs) are a class of short (19–25 nucleotides) noncoding RNAs that are involved in gene regulation. miRNAs can function as oncogenes or tumor suppressors depending on cellular context, and expression has been shown to be deregulated in several cancers . miRNAs Proteins are the primary functional elements of most biological processes, and thus, protein expression is often deregulated in disease states. Mass spectrometry (MS) is a powerful approach for protein measurement as current MS-based proteomic experiments can detect thousands of proteins in a single sample. MS has already proven to be fruitful in EOC biomarker discovery as the four markers in OVA1 (excluding CA125) were discovered using MS-based approaches . In the N-glycosylation is a posttranslational modification that plays an important role in the stability, solubility and localization of proteins to the cell surface [N-glycosylated proteins can be enriched from biological samples using chemoproteomic- and lectin-based approaches [N-glycosylation is highly prevalent among extracellular proteins (including secreted proteins) and is not present on several high-abundance blood proteins , the N-glycoproteome represents a clinically relevant sub-proteome for liquid biomarker discovery. Sinha et al. [N-glycoproteomics-based approach for detecting biomarkers of HGSC relapse. N-glycosylated peptides were enriched from the sera and tumors from recurrent HGSC patient-derived xenograft (PDX) mice and from sera of non-engrafted mice. Species mapping was used to distinguish between peptides of human (tumor) and mouse (stroma) origin, and comparative analysis was used to select a set of candidate markers. Subsequently, PRM was used to quantify the candidates in longitudinal HGSC patient serum samples, revealing four candidates that demonstrated an earlier rise between the remission and the recurrence time points than CA125. Although large-scale clinical validation of the markers is warranted, this study is a proof-of-concept for the use of N-glycoproteomics and PDX models in serum protein biomarker discovery for HGSC.Detection of blood-based protein markers is challenging due to the large dynamic range and high sample complexity of serum/plasma. Considering that the 22 most abundant proteins in plasma account for 99% of the total mass of protein , detecti surface . N-glycoproaches , 119. Asa et al.  recentlyN-glycome profiles between serous EOC patients and healthy women. This glycomics study revealed a marker panel comprising 11 differentially abundant glycans that demonstrated an improved specificity for distinguishing patients from healthy controls compared to CA125. Moreover, as metabolic alterations have been implicated in tumorigenesis [Posttranslational modifications and metabolic processes are important determinants of cellular signaling and modulating phenotypes. As such, these molecular classes also represent promising candidates for blood-based biomarker discovery. Considering that aberrant glycosylation occurs during malignant transformation , one sucigenesis , metaboligenesis  used MS Aside from investigating freely circulating molecules in blood, molecular profiling of extracellular vesicles (EVs) and circulating tumor cells (CTCs) are alternative approaches for blood-based biomarker discovery. EVs, such as exosomes, are secreted from most cell types, play a role in intercellular communication and contain molecular content from the cell-of-origin . In a piEpCAM and ERBB2 expressions in CTCs were correlated with platinum resistance and overall survival. Emerging technologies in microfluidics-based CTC isolation and single-cell molecular analysis present new avenues for high-throughput examinations of individual CTCs. Single-cell RNA sequencing of CTCs has been shown to be promising in understanding clonal resistance and metastasis to potentially inform therapeutic decisions in other cancers [CTCs are tumor cells that are shed into vasculature and play an important role in metastasis . Althoug cancers , 136. Fu cancers , 138. AlTargeted therapies are therapeutic agents that act on specific molecular targets, pathways or aspects of the tumor microenvironment that drive the cancer phenotype, in an effort to reduce harm in normal cells. Contemporary systemic management of EOC has progressed from chemotherapy to combination treatments and frontline targeted therapy, when appropriate. In this section, we review the current application of targeted therapies in HGSC clinical practice and describe high-throughput biological workflows for therapeutic target discovery.Although there are several emerging therapies under clinical investigation for EOC  [BRCA1/2 mutation. More recently, the PRIMA trial has shown that niraparib is also effective in the overall population regardless of the HRD status, but the post hoc subanalysis has clearly shown that those patients with BRCA1/2 mutations and other HRDs benefited most from maintenance treatment with niraparib [In the SOLO-1 trial, olaparib was tested as a frontline maintenance treatment in women with newly diagnosed FIGO stage III–IV ovarian cancer with germline or somatic < 0.001) . The FDAiraparib .For patients at high risk for recurrence/progression, there is currently a lack of evidence to suggest the superiority of either anti-angiogenic agents or PARP inhibitors over the other. Interim data from the ongoing PAOLA study investigating the combination of PARP inhibitors and bevacizumab suggest a significant benefit in PFS from concurrent use of both agents . Future TP53) and extensive heterogeneity, there remains a vast potential for uncovering unanticipated vulnerabilities as novel targeted therapies. High-throughput experiments for therapeutic target identification can be classified in one of two broad themes: molecular profiling to detect aberrantly expressed molecules in tumors or phenotypic screening to determine the molecules important for cancer cell survival.High-throughput experiments can serve as useful means of discovery in targeted therapy development. These large-scale molecular examinations enable the generation of novel hypotheses regarding putative therapeutic targets to help select candidates for further validation. As HGSC is characterized by a lack of discernible drivers , may inform the understanding of disease pathology. As such, the discovery experiments are typically followed by analyses designed to reveal these differences, including differential expression analyses and ontologically informed pathway analyses. Molecular profiling experiments on patient cohorts enable matching clinical data to molecular phenotype which can be especially apt for identification of candidates for targeted therapies. In one such study, Coscia et al.  comparedMolecular expression profiling experiments offers the advantage of not requiring a priori information regarding the pathology of the disease. However, a significant hurdle which lays between molecular expression profiling experiments and novel therapeutics is the potential need to develop novel drug compounds. While there are some measures for druggability which can inform therapeutic target selection, drug development remains a costly process with uncertain success . One strSurface proteins are another especially useful class of molecules as their accessibility renders them favorable therapeutic targets—evidenced by the fact that over 58% of the known protein targets of FDA-approved drugs are cell surface proteins . HoweverPhenotypic screening approaches can be used to identify tumor-specific molecular dependencies as putative targets for therapeutic inhibition. Technological advancements have enabled large-scale molecular perturbations which allow for the functional examination of thousands of molecules in a single experiment. Functional genomic screens entail the use of RNA interference (RNAi) or more recently, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 systems coupled with NGS to characterize the genes associated with a phenotype of interest , 171. InBRCA2 deficient tumors, Mengwasser et al. [BRCA2 gene and the screen targeted 380 genes that are involved in DNA damage repair. Interestingly, the authors determined two candidates that not only demonstrated synthetic lethality with BRCA2 deficiencies but also BRCA1, as evident through subsequent investigations. The candidates identified in this study represent potential therapeutic targets for BRCA-deficient tumors that are resistant to PARP inhibitors. Likewise, Baratta et al. [MYCN overexpressing tumors.In contrast to genome-wide interrogations, functional genomics screens can also be conducted on a subset of experimentally relevant genes. To identify novel synthetic lethal targets in r et al.  conductea et al.  designedMYC; an oncogene essential when overexpressed in HGSC yet pharmacologically undruggable. This screen revealed a compound that suppressed MYC expression through simultaneous inhibition of three specific cyclin-dependent kinases, hence identifying putative targets for MYC overexpressing HGSC tumors. A caveat associated with drug screens is that target discovery is restricted to those proteins with existing small molecule inhibitors, thus ignoring the potential of other therapeutic classes such as monoclonal antibodies.Although functional genomics experiments are advantageous approaches for identifying molecular vulnerabilities for potential inhibition, exploiting these candidates as therapeutic targets can be hindered by the druggability of proteins. The use of high-throughput drug screens is an alternative phenotypic screening approach to identify actionable dependencies. In these experiments, numerous small-molecule compounds, typically with known mechanisms of action, are simultaneously tested against cells to identify novel vulnerabilities. Kenny et al.  recentlyKRAS and in parallel, conducted a targeted CRISPR knockdown screen of ~ 1600 membrane proteins to functionally characterize the oncogenic KRAS surfaceome. Integration of both datasets resulted in the discovery of CDCP1 as a therapeutic antibody target for KRAS-driven cancer cells. Considering the limited success in translating promising targets into beneficial therapies, comprehensive characterization through both expression profiling and functional analyses early in the discovery process may help ensure the selection of robust targets for therapy development.The use of the aforementioned high-throughput experiments is beneficial as initial steps in therapeutic target discovery. Both approaches enable the concurrent screening of numerous molecules to select potential candidates for further validation experiments. However, as technological advancements improve the capabilities of these platforms, these large-scale experiments can identify hundreds of hits and given the time-consuming nature of molecular biology interrogations, it is often not feasible to individually follow up on every single hit. The use of bioinformatic tools that prioritize targets  or miniIdeal experimental models should accurately reflect tumor biology to ensure maximum translational utility. Given the ease of use and accessibility, immortalized human cancer cell lines are the most widely used models for experimental interrogations of HGSC . HoweverAnother caveat of in vitro cell line models is the artificial microenvironment invoked by monolayer growth on plastic and the lack of multicellularity. Three-dimensional (3D) culture has emerged as a step toward bridging the gap between in vitro and in vivo experiments. 3D culture more closely resembles the tumor microenvironment by restoring 3D cell–cell and cell–ECM interactions , 187. MoTP53, BRCA1, RB1 and PTEN, in fallopian tube epithelial cells has resulted in a new generation of HGSC GEMMs [Genetically engineered mouse models (GEMMs) offer the potential for in vivo tumor investigation. Various molecular biology techniques can be used to introduce genetic modifications in a spatial and temporal manner for in vivo modeling of genetic defects contributing to tumorigenesis . HoweverSC GEMMs –197. ThePDXs are an alternate approach for in vivo experimental models, in which minced fragments of patient tumors are transplanted into immunodeficient mice . The priApart from potential unsuitable experimental models, there are several other factors that can impede the utility of high-throughput studies in precision medicine discovery efforts. Provided that several preanalytical variables  have been shown to influence molecular profiles , 204, a BRCA1/2 and other HRDs as an indication for the use of PARP inhibitors is an example of the adoption of precision medicine into the clinical management of HGSC, yet the dismal five-year survival rate suggests that more work is still needed. The ability to simultaneously examine thousands of molecules in a single experiment has fueled the discovery of numerous putative tumor markers for liquid biopsies in HGSC. Considering the limited utility of single markers  due to tumor heterogeneity, the use of high-throughput tools has enabled the potential for uncovering multimarker panels with improved clinical performance. Large-scale biological experiments have also been utilized for the identification of novel therapeutic targets, and integration of orthogonal approaches can be promising for the detection of actionable vulnerabilities in HGSC.Through applications in molecular subtyping, liquid biopsies, and targeted therapies, advancements in high-throughput technologies have opened new avenues for precision medicine discovery in HGSC. Large-scale tumor profiling has provided insights regarding the molecular complexity underlying tumorigenesis. Appreciation of this vast heterogeneity has warranted diverging from the one-size-fits-all approach traditionally used for the management of HGSC and EOC as a whole. The use of genetic testing for Despite the alluring potential of high-throughput approaches, failure to appreciate the intricate nature of HGSC biology in research design and experimental models can stifle the translational utility of the findings from these experiments. Considering that HGSC and the other histological subtypes of EOC are distinct diseases characterized by differences in molecular profiles, clinical progression and pathogenesis, EOC is often still examined as a single entity without subtype-specific stratification in preclinical and clinical validation studies, thus acting as a potential confounder of findings. Furthermore, despite increasing evidence indicating fallopian tissue epithelium as the primary tissue of origin for HGSC, many studies continue to use ovarian surface epithelium as ‘normal’ tissue for comparative experiments, resulting in the potential identification of biologically irrelevant biomarkers and therapeutic targets. As such, it is imperative to incorporate our evolving understanding of HGSC biology in research design to leverage the full potential of emerging high-throughput applications in precision medicine.