In recent years, it has been demonstrated that extracellular vesicles (EVs) can be released by almost all cell types, and detected in most body fluids. In the tumour microenvironment (TME), EVs serve as a transport medium for lipids, proteins, and nucleic acids. EVs participate in various steps involved in the development and progression of malignant tumours by initiating or suppressing various signalling pathways in recipient cells. Although tumour-derived EVs (T-EVs) are known for orchestrating tumour progression via systemic pathways, EVs from non-malignant cells (nmEVs) also contribute substantially to malignant tumour development. Tumour cells and non-malignant cells typically communicate with each other, both determining the progress of the disease. In this review, we summarise the features of both T-EVs and nmEVs, tumour progression, metastasis, and EV-mediated chemoresistance in the TME. The physiological and pathological effects involved include but are not limited to angiogenesis, epithelial–mesenchymal transition (EMT), extracellular matrix (ECM) remodelling, and immune escape. We discuss potential future directions of the clinical application of EVs, including diagnosis (as non-invasive biomarkers via liquid biopsy) and therapeutic treatment. This may include disrupting EV biogenesis and function, thus utilising the features of EVs to repurpose them as a therapeutic tool in immunotherapy and drug delivery systems. We also discuss the overall findings of current studies, identify some outstanding issues requiring resolution, and propose some potential directions for future research.Video abstractSupplementary information accompanies this paper at 10.1186/s12964-020-00643-5. Extracellular vesicles (EVs) are small cell-derived membranous structures, serving as conduits for exchange of significant information between cells , 2. The EVs have been demonstrated to take part in managing tumour spread and medication resistance , 5. TumoAside from the relatively well-researched proteins, lipids, mRNAs, and miRNAs, emerging evidence demonstrates that LncRNAs and circRNAs participate in managing the microenvironment and tumour progression –12. CircIn this literature review, we provide a brief introduction to EVs and the tumour microenvironment (TME), present findings on the influences of T-EVs on neighbouring cells and the TME, and describe EVs from non-malignant cells (nmEVs) and their influences on the TME. We explore the functions of EVs which are potentially important for the next generation of diagnosis and therapy in the field of malignant tumours, discuss the breakthroughs and shortcomings of current research, and suggest possible future directions of research in this field.The malignant properties of tumours and their advancement are not solely regulated by the tumour cells , but alsDuring the onset of tumourigenesis, the microenvironment presents anti-tumour immunity and moderates tumour growth , but as The exchange of information in the TME may influence tumour incidence and advancement, in addition to intrusion, metastasis, and various other malignant biological actions . To explIt has been determined that EVs are intercellular messengers , 26, 27.Theoretically speaking, EVs can be classified  as eitheHowever, this classification system is known to be confusing. The definitions of the terms Exos or MVs in the classification criteria are based on EV biogenesis, but in many studies, researchers use these terms based on particle size distributions rather than their true biogenesis. Top-level researchers in the field of EV research, including Clotilde Thery, indicated that despite the fact that Exos and MVs have disparate biogenic mechanisms, the current technology for EV isolation is not able to precisely subdivide these EV sub-populations . SmallerExos biogenesis consists of a sequence of cellular activities Fig. a. The doMV biogenesis differs from that of MVB-derived Exos , 35, 36.EVs manage many different cellular procedures, such as cell proliferation, survival, and transformation via autocrine and paracrine intercommunication , 37. It There are several procedures through which EVs and their consignments could be transmitted to recipient cells. EVs can dock at the plasma membrane of a target cell , 39, andTypical functions of EVs include promoting development and growth , 41 and The fast growth of the field of EV study has illuminated unfamiliar mechanisms involving the innate intercellular correspondence systems occurring during malignant tumour commencement and progression , 49. NevT-EVs are able to transmit oncogenic molecules between tumour cells Fig. . Glioma In hepatocellular carcinoma (HCC) invasive cell lines, in vitro and in vivo resistance to Sorafenib is caused by the distribution of hepatocyte growth factor (HGF), with the assistance of T-EVs and the subsequent activation of the HGF/c-MET/PI3K/AKT signalling pathway . AdditioMalignant tumour cells are able to transfer resistance via horizontal transmission of T-EVs containing “drug outflow pumps” . AmongstThe existence of selective P-gp/MDR-1 mRNA in T-EVs discharged from doxorubicin-resistant osteosarcoma cells suggests that resistant tumour cells employ methods for spreading drug resistance to sensitive cells. This may occur via delivery of MDR proteins directly to sensitive cells or by delivering the mRNA which encodes them .LncRNA also plays an important role in this process. Lnc-ARSR is strongly expressed in sunitinib-resistant renal cell cancer (RCC) cells compared to sunitinib-sensitive RCC cells. EV-carrying Lnc-ARSR competitively binds miR-34 and miR-449, triggering the improved expression of AXL/c-MET and re-activation of STAT3, AKT, and ERK signalling. Triggered AKT causes the transcriptional de-repression of Lnc-ARSR via destruction of FOXO1/FOXO3a, developing a positive feedback loop . T-EVs cCirc-PDE8A was found to be a highly-expressed circRNA in pancreatic ductal adenocarcinoma (PDAC) –70. CircAngiogenesis is the formation of new blood vessels from pre-existing vessels under particular physiological circumstances, including development or in response to tissue damage . In healThe upregulation of heparanase in myeloma and breast cancer cells is connected with enhanced release of syndecan-1, vascular endothelial growth factor (VEGF), and HGF in T-EVs. This results in increased endothelial infiltration via the extracellular matrix (ECM), and hence enhanced angiogenic activity . T-EVs gT-EVs regulate angiogenesis in tumours through the release of non-coding RNAs. For instance, T-EVs transporting miR-9 stimulate EC migration and tumour angiogenesis via the reduction of suppressor of cytokine signalling 5 (SOCS5) levels and activation of the JAK/STAT pathway . FurtherVascular leakiness is a characteristic of PMN formation , 85. T-EMore research is required to accurately identify the mechanism by which T-EVs influence the stability of the endothelial barrier, as well as the specificity of this particular targeting within the vasculature of various body organs.Tumour-associated FBs ) comprise a large part of the responsive tumour stroma, and carry out essential functions in tumour development. These cells are reprogrammed stromal cells that play a role in malignant tumour initiation, ECM remodelling and advancement, PMN development, and metastasis , 93. In T-EVs deliver EMMPRIN to FBs, causing the production of MMPs and allowing tumour invasion and metastasis . T-EVs cT-EVs, but not those released by non-malignant cells , contain crucial enzymes associated with miRNA biogenesis. These enable cell-independent miRNA maturation inside EVs . InhibitCompared to normal FBs, ovarian tumour-adjacent tFBs constantly downregulate miR-31 and miR-214 while upregulating miR-155. Transfecting miRNA mimics (miR-31 and miR-214 mimics) and miRNA inhibitors (miR-155 inhibitors) induces a functional shift of normal FBs into tFBs, while the reverse transfection causes the opposite result, the reversion of tFBs into normal FBs . SuccessT-EVs are able to stimulate MSCs to differentiate into tumour-supportive cells Fig.  by delivIn numerous cell types, epithelial–mesenchymal transformation (EMT) pertains to tumour intrusion and metastasis , 107. EpT-EVs also take part in EMT Fig. . EVs isoTwo miRNAs (miR-191 and let-7a) have been shown to contribute to melanoma cell-derived T-EV-mediated EMT . A colleSNAI1, TWIST1, ZEB1, ZO1, MMP1, LAMB3, and LAMC2. Utilising shHOTAIR in a pair of human bladder cancer cell lines showed that expression of the master regulator of EMT (SNAI1) was dramatically decreased [Primary urothelial bladder cancer (UBC) cells were determined to affect the expression of EMT genes by means of EV-carrying LncRNA-HOTAIR. These include ecreased . LncRNA-ecreased .A recent study determined that circRNA-circPRMT5 was upregulated in serum and urine EVs from UBC patients. Further investigation determined that circPRMT5 supports the UBC cell EMT by serving as a miR-30c “sponge”. As a result, the expression of its own target gene SNAIL1 and E-cadherin is enriched, allowing the cells to become more invasive .It is commonly acknowledged that metastatic growth is associated with the risk of thrombotic issues, which is a major cause of death in malignant tumour patients . CoagulaEVs associated with coagulation can originate from platelets, inflammatory cells, and malignant tumour cells . Raised Fascinatingly, mutated KRAS and TP53 are associated with raised levels of TFs in T-EVs secreted by human colorectal tumour cells . PancreaThe TME is penetrated by a range of immune cells, including lymphocytes  cells, and T regulatory (Treg) cells), dendritic cells (DCs), monocytes, Mφs, myeloid-derived suppressor cells (MDSCs), and granulocytes . The major function of these cells is to ensure immune supervision. Nevertheless, tumour cells are efficient in regulating signalling pathways within these immune cells, turning them into an immunosuppressive entity and resulting in improved malignant tumour cell survival and proliferation . There iOne study determined that T-EVs induce immunosuppression by promoting apoptosis of hematopoietic stem cells (HSCs), DCs, and peripheral blood lymphocytes (PBLCs) . Many T-T-EVs carry TGF-β externally and transport it to T cells, suppressing their proliferation in response to IL-2 and altering their phenotype to Treg cells , 135. FuThese T-EVs have been revealed to trigger DCs and cause IL-6 secretion, which enhances tumour invasion by increasing MMP-9 metalloproteinase expression . T-EVs aA previous study confirmed that PD-L1 exists in EVs derived from the urine or blood of patients with early IgA nephropathy . ResearcTumour-released miRNAs have similarly been associated with immunosuppression. For example, miR-214 carried by T-EVs was effectively transported into recipient T cells. An in vivo study has shown that miR-214 mediates Treg cell expansion, causing increased immunosuppression and tumour growth in mice .Mφs are multifunctional antigen-presenting cells characteristically classified into a pair of polarised phenotypes: pro-inflammatory (M1) and anti-inflammatory (M2) . Tumour-Throughout the process of malignant tumour progression, the molecular and cellular environments of stromal cells and their extracellular proteins and enzymes are dynamically changing as a result of the influence of T-EVs Fig. . ECM remResearch has revealed that EVs participate in invasion and metastasis by means of invadopodium formation , 158. InEVs could be used as carriers by malignant tumour cells to promote drug resistance via drug sequestration and banishment Fig. . SheddenMalignant tumour cells can also make use of EVs as “fake targets”, thus weakening targeted treatments Fig. . T-EVs tThis is exemplified by B-cell lymphoma, when the existence of CD20 on the surface of EVs protects targeted lymphoma cells from rituximab (an anti-CD20 mAB) . Both inImmune checkpoint blockade therapies feature anti-CTLA-4 monoclonal antibody (mAb), anti-PD-1 mAb, and anti-PD-L1 mAb . It is lMetastasis is a multi-step procedure resulting in the spread of primary tumour cells to distant body organs. T-EVs have been associated with all steps of tumour invasion and metastasis , 170–172T-EVs possess their own protein “postal code” of specific integrin profiles Fig. b. This dCircRNAs in blood EVs, called ciRS-133, are closely associated with the light browning of white adipose tissue (WAT) and malignant tumour-associated cachexia. After being provided to pre-adipocytes, ciRS-133 reduce miR-133 expression, activate PRDM16, and promote the differentiation of preadipocytes into brown-like cells. It has been demonstrated that ciRS-133 KD may prevent tumour-implanted mice from struggling with malignant tumour-related cachexia, demonstrating the contribution of EV-circRNAs in tumour pathogenesis .Tumour expansion and drug resistance are not only decided by malignant tumour cells but are also sustained by non-tumour cells inside the TME. Hence, it is quite reasonable to think that nmEVs also play an important role in affecting the TME Fig. . Thus tFMSC-derived EVs (MSC-EVs) may generate drug resistance in gastric cancer cells by activating the CaM-Ks/Raf/MEK/ERK signalling pathway . EVs carHRAS overexpression in EpiCs boosts the packing of mesenchymal markers (including vimentin and MMPs) in EVs, possibly causing EMT in recipient cells .Transfer of miR-365 in Mφ-derived EVs (Mφ-EVs) causes pancreatic adenocarcinoma cells to become resistant to gemcitabine in vitro and in vivo . M2-Mφs The transfer of miRNAs specifically targeting PTEN expression from astrocyte-derived EVs to invading tumour cells in the brain microenvironment supports brain metastasis, despite the fact that other autocrine and paracrine signalling may also be coordinated throughout tumour development .CircRNAs are commonly expressed in individual cells such as blood cells , 186. ReGiven that they are incredibly stable, abundant, and tumour-specific, T-EVs have numerous unique advantages as biomarkers . One intEV integrins (as a counterpart to tumour-expressed integrins) can act as biomarkers to predict the probability of malignant tumours, in addition to determining metastatic tendencies in specific organ sites . SpecifiCirculating T-EVs from patients with stage IV melanoma carry a protein signature composed of the melanoma-specific protein tyrosinase-related protein-2 (TYRP2), very late antigen 4 (VLA-4), HSP70, and MET oncoprotein . T-EVs fResearch has illustrated that the detection of PD-L1(+) EVs in serum is correlated with poor prognosis in individuals with pancreatic ductal adenocarcinoma . New finAdditionally, EVs transport single-stranded DNA (ssDNA), which summarises genomic eccentricities such as oncogene amplifications (such as MYC) in the primary tumour . In the Tumour-specific mRNA isolated from T-EVs from the serum and tissue of glioblastoma patients reflects the mutational condition of EGFRvIII , 201. MiLncRNAs associated with T-EVs are also appealing as prospective biomarkers. Nevertheless, LncRNAs in EVs may work as biomarkers in various other malignant tumours, such as LncRNA-p21 in prostate cancer and LncRNA-HOTAIR in bladder cancer . In coloNonetheless, the features of circRNAs mark these molecules as a better option for detecting disorders due to their closed conformation and resistance to RNase. Compared to the 48-h half-life of the majority of circRNAs, the ordinary half-life of miRNAs is normally less than 10 h . ScientiWith recent technological improvements, microfluidic technology has been introduced into the field of EV study. Compared with traditional methods, microfluidic technology can carry out EV-based diagnosis more easily, efficiently, and economically .EV biogenesis is a major target for EV-targeting therapy in malignant tumour treatment –219. A n. In a mouse xenograft model of melanoma, they demonstrated that therapy with a proton pump inhibitor reduces the release of EVs and enhances tumour cell sensitivity to cisplatin [Federici et al. carried out therapy with a proton pump inhibitor to observe the effects of both cisplatin uptake and EV release in vitro and in vivoisplatin . Numerouisplatin , preventisplatin . Inhibitisplatin .While many of the agents specifically blocking T-EV release from malignant tumours lack specificity, some inhibitors target tumour-specific enzyme isoforms. This is the case for peptidylarginine deiminase (PAD)2 and PAD4 inhibitors, which are overexpressed in prostate and ovarian malignant tumour cells. Their inhibition by chloramidine minimises T-EV production, thus increasing the sensitivity of malignant tumour cells to chemotherapy drugs . In a moAlthough these treatments have had success in vitro and sometimes in vivo, their lack of selectivity for malignant tumour cells restricts their therapeutic usage. This is not the case for the specific elimination of circulating T-EVs from plasma.In a technique quite similar to haemodialysis, extracorporeal hemofiltration with cartridges composed of hollow fibres (with a size cut-off of 200 nm) combined with an affinity matrix allows specific elimination of ultra-filtered EVs. This procedure is known as Adaptive Dialysis-like Affinity Platform Technology (ADAPT™), and was first developed by Aethlon Medical Inc. for eliminating Hepatitis C virus (HCV) particles from the bloodstream of contaminated patients . The expActivation of anti-tumour T cell reactions by DC-derived EVs (DC-EVs) has been determined to be critical in reducing the expansion of well-established tumours . LoadingEVs may be therapeutically targeted to supply anti-tumour cargos to malignant cells . Based oCapitalising on EVs, researchers have the ability to target medications to tumour cells. EVs may raise the therapeutic index of doxorubicin (DOX). EVs carrying doxorubicin (EV-DOX) avoid cardiac toxicity by partly restricting the crossing of DOX via myocardial ECs . AnotherAn in vivo study revealed that neuron-targeted EVs packed with Bace1 siRNAs specifically and significantly decreased Bace1 mRNA (60%) and protein (62%) in nerve cells . SimilarThese results suggest that EVs may indeed be beneficial as drug delivery tools. Although several anti-tumour therapies have been investigated/tested in preclinical models and phase I clinical trials, these studies have reinvigorated the desire for novel anti-cancer therapies.In the previous sections, we introduced the role of EVs in the TME, including the role of T-EVs and EVs from non-malignant cells. Some representative contents of EVs, their functions and mechanism are shown in Table The physiological and pathological study of tumours can be mainly categorised into two levels: (1) single molecule studies, which study a specific molecule in EVs and its role; and (2) functional observational studies, which determine the changes and possible roles of EVs and their cargo in the TME. There is a very large gap between the functional observational studies and the single molecule studies, as well as between in vivo and in vitro studies.EVs through their cargo play a variety of different roles, but it is important to determine which of these play primary or secondary roles, to what percentage this function is carried out, and whether there is synergy or antagonism between these molecules or EVs. Current research is not sufficient to provide satisfactory answers. There is also a gap between molecules or single source EVs and the TME. This is because the TME is complex, containing many cells and molecules carrying out different functions. Rather than a single molecule or single source of EVs contributing to their functions, there is an interplay between various molecules and all sources of EVs.Moreover, cellular communication has not been well studied. The interaction between cells is bidirectional, and tumour cells and non-malignant cells in the body continuously interact with each other in dynamic equilibrium to form the TME. Many in vitro experiments are carried out using EVs from one type of cell to stimulate another type of cell. The effect of EVs on cells in the TME is not a simple and direct effect, but rather an effect similar to the “iterative effect”, such that the influence of T-EVs on non-malignant cells can also affect the content of nm-EVs; and the influence of affected nm-EVs on tumours and T-EVs is changed. When this process is continuously repeated, the features of EVs will completely deviate from the simple in vitro model. Although it is not clear what kind of new model will be most appropriate, organ-on-a-chip may be a more appropriate option for future research .Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018) endorses EVs as the standard terminology for vesicles which are released naturally by the cell and enclosed within a lipid bilayer without replication capacity  . Over thFinding discriminating markers for T-EVs versus normal stromal EVs has been an important research direction in this field for a long time. Although a number of markers on EVs have been identified for a particular type of tumour , so far, no universal markers have been found. If one or more universal/general markers, which can cover various kinds of tumours not just one or a few, can be found on T-EVs, then the use of T-EVs for tumour diagnosis will become even more meaningful. If no universal markers can be found, then compromise tactics will need to be used. EVs from various tumour sources can be analysed, and the data can be uploaded to a database. Through bioinformatics technology, a minimal set of genes could be found, which can cover as many tumours as possible, and the database should be updated as often as possible.New evidence shows that EV-circRNAs could possess important biological functions in different pathological and physiological procedures. EV-circRNAs are confirmed to be extremely stable . FurtherLiquid biopsy is based on the detection and analysis of biomarkers , cell-free nucleic acids (cfNAs), and EVs) in readily-available body fluids such as peripheral blood –246. TheAnalysis of EVs is a promising potential new star in the field of liquid biopsy, and an approach that has attracted more and more attention in recent years is single-EV analysis, which is of crucial significance for the precise analysis and diagnosis of diseases . A multiEVs are involved in various pathophysiological conditions such as development and progression of disease , 249. SeAlthough many of the EV-based therapeutic approaches performed well in pre-clinical models and phase I clinical trials, they exhibited many notable issues in subsequent phase II clinical trials. In patients with advanced non-small cell lung carcinomas, interferon-γ (IFN-γ)-DC-EV treatment ceased to be effective, revealed by long-term clinical observation . TherefoOn account of their good stability, long circulating half-life and relatively good bio-safety, EVs are considered to be potential drug delivery systems with high delivery efficiency and low toxicity . MoreoveAptamers are RNAs or single-stranded DNAs (ssDNAs) folded into particular 3D structures with high specificity and affinity through a similar mechanism to antigen–antibody binding . AptamerCRISPR/Cas9 treatment through EVs is also a very promising research prospect. At present, this technology is mainly used as a research tool. The most typical application of this treatment is to confirm the spread of EVs as reporters. It would be interesting to compare EV-based CRISPR/Cas9 with known methods using CRISPR/Cas9 for gene therapy, in addition to determining the differences in the scope of application, treatment effects, and the advantages and disadvantages of this approach.EVs function as a transport medium for various molecules in the TME, and therefore have a variety of potential uses in the diagnosis and treatment of cancer. EVs also participate in the progression of various processes involved in malignant tumour development. Tumour cells and non-malignant cells typically communicate with each other, together determining the progress of the disease. Although T-EVs are known for orchestrating tumour advancement via systemic pathways, nmEVs also contribute substantially to malignant tumour development.In this review, we have summarised the features of both T-EVs and nmEVs, and their roles in tumour progression, metastasis, and EV-mediated chemoresistance in the TME. This review discusses recent and current research regarding the clinical applications of EVs, the findings of these studies, and how this information can be used to repurpose EVs as a therapeutic tool. This sound and current overview of the present research, questions to be addressed, and potential directions for future research in the field makes a significant contribution to the literature.


Currently, approved first-line treatment options of metastatic hormone-sensitive prostate cancer (mHSPC) include (1) androgen deprivation therapy (ADT) alone, ADT plus one of the following: (2) docetaxel, (3) abiraterone, (4) enzalutamide, and (5) apalutamide. The high cost of novel androgen receptor pathway inhibitors warrants an understanding of the combinations’ value by considering both efficacy and cost.This study aimed to compare the cost-effectiveness of these five treatment options in mHSPC from the US payer perspective to guide treatment sequence.A Markov model was developed to compare the lifetime cost and effectiveness of these five first-line treatment options for mHSPC using outcomes data from published literature. Health outcomes were measured in life-years and quality-adjusted life-years (QALYs). Drug costs were obtained from the Veterans Affairs Pharmaceutical Catalog. We extrapolated survival beyond closure of the trials.Life-years, QALYs, lifetime costs, and incremental cost-effectiveness ratios (ICERs) were estimated. Univariable, 2-way, and probabilistic sensitivity analyses were performed to evaluate parameter uncertainty. A willingness-to-pay (WTP) threshold of US$100,000 per QALY was used.Compared to ADT alone, docetaxel plus ADT provided a 0.28 QALY gain at an ICER of US$12,870 per QALY. Abiraterone plus ADT provided an additional 1.70 QALYs against docetaxel plus ADT, with an ICER of US$38,897 per QALY. Compared to abiraterone plus ADT, enzalutamide plus ADT provided an additional 0.87 QALYs at an ICER of US$509,813 per QALY. Apalutamide plus ADT was strongly dominated by enzalutamide plus ADT. Given the WTP threshold of US$100,000 per QALY, abiraterone plus ADT represented high-value health care.Abiraterone plus ADT is the preferred treatment option for men with mHSPC at a WTP threshold of US$100,000 per QALY. In the US, prostate cancer accounts for one in five new cancers, making it the most diagnosed cancer in men; it is also the second most common cancer-related death in men . DespiteIn 2015, STAMPEDE  and CHAAThere is no head-to-head comparison of different agents and a randomized controlled trial comparing all agents is unlikely feasible. The emergence of these treatment options in the castration-naïve setting has led to new challenges in finding the best treatment sequence for mHSPC. Network meta-analyses reported so far offered no clear answer. Sathianathen et al. suggested that different combination strategies were statistically comparable with none being clearly superior .Our group previously reported that abiraterone appears to be more effective than other treatment options in reducing risk of death and preventing disease progression .With an aging population and improved survival outcomes, mHSPC represents an increasing economic burden to healthcare systems in developed countries. Previous cost-effectiveness analysis comparing ADT alone, ADT in combination with docetaxel or abiraterone from a US private payer perspective demonstrated that docetaxel plus ADT was cost-effective with an incremental cost-effectiveness ratio (ICER) of US$34,723, compared to abiraterone plus ADT with an ICER of US$295,212 . With thA Markov state-transition model was developed to evaluate the costs and health outcomes of treating mHSPC with one of the following: (1) ADT alone; (2) docetaxel plus ADT; (3) abiraterone plus ADT; (4) enzalutamide plus ADT; or (5) apalutamide plus ADT. ADT alone was the referent strategy, as it was the control arm across mHSPC trials. The model considered a hypothetical cohort of 70 kg 60-year-old men with newly-diagnosed mHSPC transitioning through three discrete health states: progression free, progression, and death , and quality-adjusted life years (QALYs), which were used to calculate the ICERs. Costs were calculated from the US health payer perspective. Costs, LYs and QALYs were accumulated over a lifetime horizon at one-month cycles and discounted at 3% per year. Willingness-to-pay (WTP) threshold of US$100,000 was used . Model cThe transition probability of progression-free to CRPC for each treatment strategy was estimated based on the progression-free survival (PFS) curves in respective clinical trials  Pharmaceutical Catalog . The cosQALYs are estimated by adjusting survival time with health-related quality of life. Health utilities for patients on ADT, abiraterone and docetaxel are derived from published literature , 18–20. We performed sensitivity analysis for all key variables to explore how results vary across plausible ranges. In univariable sensitivity analysis, all key variables were varied based on reported confidence intervals or with a variance of 20% from base-case values. We examined the uncertainty of the fitted PFS curves by expressing into median PFS. We performed 1,000 Monte Carlo simulations to conduct probabilistic sensitivity analysis, with the variables simultaneously varied with specified distributions. In the base-case, we assumed the proportion of patients receiving subsequent line therapy to be approximately 65% to 80%. Based on clinical observation and literature, more patients who had first-line docetaxel plus ADT or ADT alone would receive subsequent line therapy, since they would more likely be candidates for second-line targeted therapy, as compared to patients who had first-line abiraterone, enzalutamide, or apalutamide, whose second-line treatment would preferably be docetaxel. As an exploratory scenario analysis, we evaluated the impact of having a lower proportion of patients receiving subsequent line therapy after first-line docetaxel , similar to that for abiraterone, enzalutamide, and apalutamide plus ADT.Simulated results for the base-case are outlined in Results of univariable sensitivity analysis showed that the variables with greatest influence on the ICER included drug costs of abiraterone and enzalutamide, median PFS of all strategies, and utilities of receiving abiraterone and enzalutamide Figure 1The results of the two-way sensitivity analysis demonstrated that abiraterone plus ADT remained the preferred option over docetaxel plus ADT (To our knowledge, this study is the first cost-effectiveness analysis of all approved drug options for mHSPC. Our analysis was based on US data, but we believe that our results can be applied to healthcare systems in other developed countries. Based on our results, abiraterone plus ADT is the preferred treatment at a WTP threshold of US$100,000 per QALY gained. The results from our study differs from a previously reported analysis , which sFirst, with longer follow-up data of abiraterone from CHAARTED trial  and publThere are several limitations to our analysis. First, outcomes data were derived from randomized controlled trials and a network meta-analysis. The patient selection criteria under these trial settings may limit the generalizability of our results in real-world clinical practice. Secondly, drug costs vary across health care systems and are expected to change over time. For instance, the introduction of generic abiraterone has evidently led to a lower incremental cost per QALY gained, making it a cost-effective treatment option. Moreover, drug costs in the model were derived from the VA, which are generally lower and better reflect the actual economic value of the drug, while prices paid by private payers and Medicare tend to be inflated. We performed sensitivity analyses to account for the wide variability in cost values. Thirdly, we did not include the use of radiotherapy in our comparison despite its proven role in low-volume disease. Fourthly, the follow-up duration of ENZAMET , ARCHES From the US payer perspective, abiraterone plus ADT costs US$38,897 per QALY gained and is the preferred treatment option at a WTP threshold of US$100,000 per QALY gained in the first-line treatment of mHSPC.The original contributions presented in the study are included in the article/WS, TS, and HC conceived and designed the experiments. WS, HC, PL, and TS performed the experiments. WS, HC, PL, and TS analyzed the data. WS, TS, and HC wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bartonella bacilliformis, a Gram-negative bacteria found in the Andean valleys. The spread of resistant strains underlines the need for novel antimicrobials against B. bacilliformis and related bacterial pathogens. Carrion’s disease (CD) is a neglected biphasic illness caused by B. bacilliformis. The main aim of this study was to integrate genomic-scale data to shortlist a set of proteins that could serve as attractive targets for new antimicrobial discovery to combat  We performed a multidimensional genomic scale analysis of potential and relevant targets which includes structural druggability, metabolic analysis and essentiality criteria to select proteins with attractive features for drug discovery.fabI, folA, aroA, trmFO, uppP and murE genes, meet an important number of desirable features that make them attractive targets for new drug development. This data compendium is freely available as a web server (http://target.sbg.qb.fcen.uba.ar/). We shortlisted seventeen relevant proteins to develop new drugs against the causative agent of Carrion’s disease. Particularly, the protein products of B. bacilliformis drug discovery. This work represents an effort to reduce the costs in the first phases of  Bartonella bacilliformis (Bb) is a Gram-negative pathogenic bacterium responsible for Carrion’s disease (CD) which causes haemolytic anaemia and skin lesions.in vitro, such as beta-lactams , aminoglycosides and quinolones,Salmonella). Alternative drugs include beta-lactams , tetracyclines (doxycycline), macrolides , trimethoprim-sulfamethoxazole, and fluoroquinolones .While research has found that Bb, and another genus’ species, are highly susceptible to a wide-set of drugs in-silico efforts to select drug targets in clinically relevant pathogens such as Streptococcus pneumoniae,Mycobacterium tuberculosis,Pseudomonas aeruginosa,Klebsiella pneumoniae,Salmonella enterica subsp,P. aeruginosaS. epidermidisShigella flexneri.The World Health Organization (WHO) has been warning that simple infections treatment is facing an alarming crisis in the health system due to the diverse resistant mechanisms emerging and spreading globally in the last decades. Despite this critical situation, we are facing a low output antibiotic development pipeline that, coupled with regulatory challenges and unattractive costs, led to many pharmaceutical companies exiting the field. The main limitation of traditional high-throughput screening (HTS) is that only a limited set of chemicals are available in any given HTS library. As this chemical space limitation would hardly be overcome, new approaches are urgently needed to deal with the increasing problem of bacterial resistance. Nowadays, omic approaches have created new opportunities for antibiotic discovery, aiding the evaluation of protein suitability to be used as a drug target, which allows to significantly reduce the costs of the initial phases of drug discovery. Thus, the initial detection of new targets can be done entirely computationally by integrating data and applying filters to select those with better features for the later steps. An example of a filter that can be used to select potential targets would be to consider only proteins that have an inhibition site with adequate structural characteristics to bind a drug-like compound. We and others have previously used To our knowledge, this is the first report that uses an exhaustive application of a multidimensional data integration strategy to prioritise drug targets in Bb. Combining several layers of genomic-scale information which included genomic, metabolic, and protein structural data sources, we were able to describe a set of proteins with attractive features for target selection.Bacterial strain - Bb USM-LMMB 07 was first isolated in 2011 by our research group in Laboratorio de Microbiología Molecular y Biotecnología - Facultad de Ciencias Biológicas - Universidad Nacional Mayor de San Marcos (UNMSM) - Lima, Perú, during an outbreak in Carmen de la Frontera district, Huancabamba province, Piura. Its genome was previously sequenced and annotated by our group.Obtaining B. bacilliformis protein sequences and preliminary analysis - All open reading frames (ORFs) were derived from the proteome downloaded from the Uniprot database . All ORFs were analysed with the HMMER software (http://hmmer.org/), which assigned them to families or domains, grouped according to PFAM (https://pfam.xfam.org/) classification.Generation of models based on structural homology - Since no crystallised Bb protein structures were found deposited in the Protein Data Bank (PDB) (https://www.rcsb.org/) homology-based models were built for all proteome sequences using MODELLER  when the adequate template was available as described before.,,-05) against the UniRef50 . Once a position-specific scoring matrix (PSSM) was obtained, it was used to search against the PDB95 (non-redundant PDB proteins with 95% sequence identity threshold) using PSI-BLAST with E-value ≤ 10-5 (-e 1e-05) as a cutoff threshold. Up to five recovered PDB structures were used as templates for homology-based modeling using MODELLER. MODELLER was used without modifying the parameters implemented as default by the software. Five models per template for each Bb protein were built. Models were classified by the GA341 score. Only those models with GA341 above 0.7 and over 60% coverage were retained. At last, one representative model which maximises QMEAN Z-score was chosen for further analyses. To ensure the good quality of the models only those models with QMEAN-Z between - two and two were kept. With this strategy, we were able to build a total of 882 structural models from a total of 1143 ORF present in the Bb genome.Druggability assessment - Druggability is a concept used to describe proteins’ ability to bind drug-like compounds, leading to protein modulation in the desired way.Off-target criteria - All Bb USM-LMMB07 proteins were submitted to BLASTp analysis against the human proteome (NCBI assembly access GCF_000001405.36) to identify non-human-homologous targets. Hits with an E-value lower than 10−5 and identity of 40% or above were discarded, as they can share a high degree of structural preservation that could produce adverse effects if the bacterial protein is used as a target.−5, identity ≥ 40%). This is aimed at identifying candidate proteins for which the developed drugs show an improved selectivity towards the bacterial pathogen, thus minimising the impact on the commensal gut microbiota.Similarly, protein inhibition of the normal flora is likely to result in adverse effects. To mitigate this possibility, Bb USM-LMMB07 proteins were compared to the proteins of the 226 representative microorganisms of human intestinal flora sequenced by the Human Microbiome Project.Construction of the metabolic network of Bb USM-LMMB07 - The PathoLogic module from Pathway Tools v. 20.0B. bacilliformis, NCBI taxonomy ID = 774). This metabolic reconstruction is mainly based on the EC number corresponding to each enzyme. EC number annotation has been previously performed manually during the genomic annotation of the Bb USM-LMMB07 strain (https://www.ncbi.nlm.nih.gov/assembly/GCF_001624625.1).After manual curation of the metabolic network reconstruction was completed, it was exported in SBML format for further analysis. In particular, those reactions called “small molecules”,  were exported to reduce the complexity of the network when evaluating topological measures and choke-point reactions.The resulting metabolic network was represented as a reaction graph using Cytoscape_v3.7.0,Data integration and target prioritisation - All previously calculated data were integrated into our own developed platform Target-Pathogen (TP),Bartonella.Characterisation of druggable proteins for Bb USM- LMMB07 - After target prioritisation, the selected proteins were better characterised by making further analysis. PSORTbv3.0 (https://www.psort.org/), running in Bacterial- Gram-negative mode, was used to predict subcellular localisation. Gene Ontology terms were obtained from Unpriot KB. Compute pI/Mw-5 as the cutoff threshold. DEG proteins are classified as essential proteins based on experimental trials. If an essential homologous protein is found for a given protein, it is also likely to be essential since functions encoded by essential genes are widely conserved in microorganisms.Bb protein structures USM-LMMB07 are enriched in druggable pockets - Our analysis began by classifying all obtained domain structures of Bb USM-LMMB07, according to their structural druggability. For this purpose, structural domains were first grouped into two categories considering only the structural models obtained from our work scheme, since the Protein Data Bank (PDB) does not feature any information on experimentally crystallised Bb USM-LMMB07 proteins.The first category includes structures modeled from a template that was co-crystallised in the presence of an inhibitor or a drug-like compound (MD+), thus the proteins of this group are likely to be druggable. The second category groups all modeled proteins whose structure was resolved from proteins deposited in the PDB with no drugs or associated inhibitors present (MD-).,Supplementary data(Table I)].For all structures, all the possible pockets and their corresponding drug-related score (DS) were calculated using the fpocket software. Using the DS, we classified all structures in each category into four drug groups, according to the criteria set out above . The resReconstruction of the metabolic network of Bb USM- LMMB07 - In the next step of our prioritisation procedure, we used Pathway tools and manual revision to build a comprehensive metabolic network of Bb USM- LMMB07 from its annotated genome. A total of 898 reactions  composed the MN. These enzymatic reactions are distributed as part of 148 metabolic pathways, which groups 1143 proteins.K. pneumoniae metabolic networkFor comparison, our previously constructed After the construction of the metabolic network, it was exported in systems biology markup language (SBML) format for downstream analyses. Reactions involving macromolecules  were filtered out, and only reactions involved in small-molecules metabolism were considered. The rationale for this strategy was that since most of the current antibiotics target macromolecules , the focus on enzymatic activities that are not related to these types of molecules would comprehend an unexplored universe suitable for drug discovery.After exporting the Bb-MN, we calculated the frequency with which all compounds were involved in the predicted reactions. The most frequent compounds were considered a potential ubiquitous compound . After manual inspection, a total of 33 compounds were filtered out to avoid artificial links creation on the reaction graph. Cytoscape v. 3.7.0 was then used for graphic visualisation and topological metrics calculation, particularly the betweenness centrality (BC) . The preIntegration of metabolic data, druggability information, and off-target criteria make it possible to identify and prioritise potential molecular targets in drug development against B. bacilliformis - All previously calculated data was integrated into Target-Pathogen.Supplementary data(Table II)]. Afterward, we further filter these proteins by taking into account its importance from the metabolic point of view. In this sense, we kept only those proteins involved in high centrality and choke-point reactions. The resulting 17 proteins  and a structural criteria (DS > 0.7) match. Moreover, in six  of a total of seven proteins that fulfill these criteria, the predicted druggable pocket overlaps with the site where the drug binds the protein in the co-crystallised structure . Table IBartonella bacilliformis targets characterisation - Various layers of information were combined, including whole-structural, metabolic, and genomic data that allowed a shortlisting of target proteins with characteristics from a druggability standpoint. The six highlighted proteins are druggable, don’t have close homologs in the human genome, and are relevant from the metabolic point of view.To further characterise these proteins, we analysed their essentiality, ontology , subcellular localisation, isoelectric point, and molecular weight .fabI is an enoyl-[acyl-carrier-protein] reductase and was found to be located in the cytoplasmic membrane. This protein develops fatty acid biosynthesis processes in the bacterium. The homology analysis through BLASTp against the DEG database shows a high degree of similarity to Sphingomonas wittichii RW1 and Caulobacter crescentus FabI . This protein was also reported to be essential in many other bacteria such as E. coli and M. tuberculosis.fabI is also essential since functions encoded by essential genes are broadly conserved in microorganisms. Dihydrofolate reductase (DHFR) FolA is a protein with binding NADP activity that participates in the metabolism of drugs, in the biosynthetic process of amide, and the biosynthetic process tetrahydrofolate. PSORTb analysis reveals that FolA is found in the cytoplasm. Protein search using DEG reveals that there are essential genes, homologous to Bb folA , present in more than ten different organisms, such as Caulobacter crescentus and P. aeruginosa.The protein product of B. bacilliformis. This protein intervenes in aromatic amino acids and chorismate biosynthesis as revealed by ontology analysis. High identity was found against Rhodopseudomonas palustris CGA009 and C. crescentus AroA (53.3% and 50.9% respectively). As this protein is essential in both bacteria, it may also be essential for Bartonella. Protein FADH(2)-oxidising methylenetetrahydrofolate--tRNA-(uracil(54)-C(5))-methyltransferase (TrmFO) is involved in tRNA processing, performs methyltransferase activity and participates in the binding of flavin adenine dinucleotide. This protein was found to be located in the cytoplasm. Staphylococcus aureus N315 trmFO (gid) essential gene was found to be homologous to this Bb protein . Undecaprenyl-diphosphatase UppP is an integral protein of B. bacilliformis cytoplasmic membrane. This protein has been reported as involved in the synthesis of the carrier lipid undecaprenyl phosphate that is essential for the biosynthesis of peptidoglycan in other bacterial species. The enzyme catalyses the dephosphorylation of undecaprenyl diphosphate (UPP). The reaction product is a lipid molecule, that transports the peptidoglycan precursors from the cytoplasm to the periplasm through the cytoplasmic membrane. However, we did not identify homologous genes in DEG. The last prioritised protein is the cytoplasmic UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-2, 6-diaminopimelate ligase (MurE). This protein is involved in the cell cycle, the biosynthetic process of peptidoglycan, cell division, and cell wall organisation. After performing the homology analysis against the DEG database, we noted that it presents an identity greater than 50% with the essential proteins of Agrobacterium fabrum str. C58 and R. palustris CGA009 . It is thus inferred that it may be essential for Bartonella’s survival.3-phosphoshikimate 1-carboxyvinyltransferase AroA; it is also located in the cytoplasm of B. bacilliformis strain USM- LMMB07, collected in 2011 in northern Perú  and identified as the causal agent of Oroya fever. Different layers of omic information were combined, including genomic, structural, and metabolic data which allowed a shortlisting of targets with desirable characteristics from a druggability point of view. Out of 1143 predicted proteins that form the proteome of Bb USM- LMMB07, we obtained 882 structural models, and we could predict high druggable pockets for 235 of them. Metabolic network reconstruction of this strain allowed metabolic complement and enzymatic activities identification performed by Bb, as well as important topological metrics in the metabolic network. This information allowed us to contextualise functional aspects of the proteins identified as promising targets. All this information, along with other genomic features calculated for each protein, were loaded into an openly available web-server (Target Pathogen),In this work, we make an integrative analysis framework for protein target prioritisation using as model organism K. pneumoniae.6 of the pteridine ring. Tetrahydrofolate is an important intermediate in the biosynthesis of proteins and nucleic acids, it is biosynthesised de novo by bacteria and is involved in important biosynthesis pathways such as methionine, purines, thymidylate, and pantothenate. Humans are entirely dependent on nutritional sources of folate, transforming it into an essential vitamin. Because dihydrofolate reductase is essential for cell division and growth, it is an attractive target for drug development. Antimicrobial agents against FolA, such as 5-substituted-2, 4-diaminopyrimidine (TMP), are widely used to treat Streptococcus pyogenes, S. pneumoniae, Escherichia coli, and K. pneumoniae infections.By applying a set of filters, we were able to delineate a unique set of six druggable proteins with no close homologs in humans (and its microbiome) and that are of interest for drug discovery. One of them, FabI participates in fatty acid synthesis (FAS) processes. The proteins of this pathway, such as FabA, FabB, FabD, FabI, and FabH, have an essential role during the biosynthesis of phospholipid membranes, lipoproteins, and LPS and represent attractive targets due to the structural differences between the human and bacterial proteins.aroA, 3-Phosphoshikimate-1-carboxyvinyltransferase (EPSP synthase), is another target identified through our prioritisation pipeline. This protein participates in the biosynthesis of aromatic amino acids, siderophores, and metabolites such as folate, ubiquinone and vitamin K. EPSP synthase is involved in the 6th step of the chorismate pathway, catalysing the transfer of the enolpyruvoyl moiety from phosphoenolpyruvate to the hydroxyl group of carbon 5 of shikimate 3-phosphate with the phosphate elimination to produce 5-enolpyruvoyl shikimate 3-phosphate (EPSP).e.g. Salmonella typhimurium and E. coli), are inhibited at low concentrations of glyphosate.aroA gene product make it attractive as an antimicrobial target, including essentiality, druggability, and the lack of a human homolog.The protein product of gidA. Furthermore, this gene deletion has produced defective cell growth, decreasing the growth by approximately 10-30% in several bacteria like S. enterica, P. aeruginosa, and E. coli.,,gidA could be considered a novel and potential target in B. bacilliformis. Another attractive target found by our analysis is Undecaprenyl-diphosphatase UppP. This protein has been reported as essential in other bacteria, however, in vitro analysis is required to determine its essentiality in B. bacilliformis. Overexpression of undecaprenyl-diphosphatase generates resistance to bacitracin.in-silico as a druggable molecular target in Streptococcus suisClostridium botulinum.B. bacilliformis; regarding it as a potential target of new targeted drugs to destabilise the cell wall to disrupt bacteria growth.Our analysis also revealed methylenetetrahydrofolate-tRNA-(uracil-5-)-methyltransferase (TrmFO) as a promising target to combat Bb. TrmFO is a ﬂavin dinucleotide (FAD) binding protein, which has been identiﬁed as a tRNA modiﬁcation enzyme, responsible for the folate-dependent m5U-54 biosynthesis. In bacteria, RNA modification enzymes have been reported to exhibit a moonlighting function associated with many cell processes like virulence, stress response, morphology, growth, antibiotic susceptibility, and others.murE, the UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-L-lysine/2,6-diaminopimelate ligase, is a bacterial enzyme belonging to the ATP-dependent Mur ligases, which are essential for peptidoglycan (PG) synthesis. PG is a key component of the cell wall of almost all eubacteria, it is responsible for rigidity and shape of bacterial cells, serves as a platform for anchoring other cell envelope components and is essential for growth and survival of bacteria. The enzymes that catalyse PG are considered one of the best sources of antibacterial targets. Similar to the other Mur class enzymes, MurE has been targeted in efforts to produce novel antimicrobials. Different chemical compounds are being studied taking into account their SAR  studies and inhibitory potential. Such is the case of hyperenone A which is obtained from hexane and chloroform extracts of the different aerial parts of plant Hypericum acmosepalum and quinolone derivatives as the novel N-methyl-2-alkenyl-4-quinolone are capable to inhibit MurE ligase of M. tuberculosis and Methicillin-resistant S. aureus (MRSA).The protein product of With regards to the cellular compartment where these candidates are found, most of the top-ranked proteins are located either in the cytoplasm or in the cell membrane and are a priori unavailable for external binding. This apparent inappropriateness of cytoplasmic proteins to serve as a target should not hamper their further exploration, as it is well-known that many antibiotics are capable of efficiently crossing bacterial membranes (either by diffusion or through porin channels),Further studies are warranted to follow-up experimentally on our elicited targets, and we invite the scientific community dedicated to this subject to help pursue these goals, thus strengthening the ongoing fight against pathogenic bacteria.


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide causing a global health emergency. Pa-COVID-19 aims to provide comprehensive data on clinical course, pathophysiology, immunology and outcome of COVID-19, to identify prognostic biomarkers, clinical scores, and therapeutic targets for improved clinical management and preventive interventions.Pa-COVID-19 is a prospective observational cohort study of patients with confirmed SARS-CoV-2 infection treated at Charité - Universitätsmedizin Berlin. We collect data on epidemiology, demography, medical history, symptoms, clinical course, and pathogen testing and treatment. Systematic, serial blood sampling will allow deep molecular and immunological phenotyping, transcriptomic profiling, and comprehensive biobanking. Longitudinal data and sample collection during hospitalization will be supplemented by long-term follow-up.Outcome measures include the WHO clinical ordinal scale on day 15 and clinical, functional, and health-related quality-of-life assessments at discharge and during follow-up. We developed a scalable dataset to (i) suit national standards of care, (ii) facilitate comprehensive data collection in medical care facilities with varying resources, and (iii) allow for rapid implementation of interventional trials based on the standardized study design and data collection. We propose this scalable protocol as blueprint for harmonized data collection and deep phenotyping in COVID-19 in Germany.We established a basic platform for harmonized, scalable data collection, pathophysiological analysis, and deep phenotyping of COVID-19, which enables rapid generation of evidence for improved medical care and identification of candidate therapeutic and preventive strategies. The electronic database accredited for interventional trials allows fast trial implementation for candidate therapeutic agents.Registered at the German registry for clinical studies (DRKS00021688)The online version of this article (10.1007/s15010-020-01464-x) contains supplementary material, which is available to authorized users. Lower respiratory tract infections cause several millions of deaths every year and remain the most deadly communicable disease as reported by WHO  data of the WHO, 2018). Comprehensive phenotyping of diseases caused by new infectious agents is required to enhance the understanding of their pathophysiology, to improve clinical management, and to develop therapeutic agents. In the past 20 years alone, zoonotic viruses have caused multiple outbreaks, which all involved respiratory infections. In November 2003, Severe Acute Respiratory Syndrome (SARS) virus caused a global disease outbreak with > 8000 infections and 774 deaths . The locRapid generation of meaningful evidence for improved clinical management of COVID-19 requires harmonized phenotyping and comparable data collection in a well-defined patient population. Besides decade-long experience in multicentric phenotyping and clinical studies of community acquired pneumonia  , Berlin This unique local expertise together with the global burden of COVID-19 implies a special responsibility in assessing all available information drawn from medical care and human biomaterial within Charité as well as gathering patient data nationwide to enable fighting the outbreak of SARS-CoV-2 infection in a systematic and effective way.The aim of Pa-COVID-19: harmonized deep clinical, molecular, and immunological phenotyping in patients with COVID-19 to (i) elucidate the pathophysiology of the disease, (ii) identify diagnostic and prognostic scores and biomarkers for improved clinical management, (iii) identify putative therapeutic targets, (iv) produce evidence regarding short- and long-term clinical outcomes, and (v) identify correlates of protective immunity. Longitudinal data- and bio-sampling as well as post-mortem analysis will enable for characterization of clinical features of distinct disease courses and identify determinants of severity and transmission.Pa-COVID-19 is a prospective observational cohort study. All patients diagnosed with COVID-19 at Charite - Universitätsmedizin Berlin are eligible for inclusion. The protocol was developed in accordance with and extends upon the standardized protocol for the rapid, coordinated clinical investigation of severe acute infections by pathogens of public health interest published by the International Severe Acute Respiratory and Emerging Infection Consortium (ISARIC) .We aimed to establish a study protocol that can serve as blueprint and central platform for harmonized deep clinical, molecular, and immunological phenotyping studies in COVID-19 patients in Germany. The protocol supports data sharing efforts through adherence to international data harmonization standards. Data are collected longitudinally from patients with confirmed COVID-19 three times per week during their hospitalization and at follow-up visits. Data include epidemiological and demographic parameters, medical history and potential risk factors, documentation of standard of care procedures, and clinical course, including different patterns of organ involvement, quality of care, morbidity, and quality of life. Moreover, extensive serial high-quality bio-sampling consisting of various sample types with deep molecular, immunological, and virological phenotyping through single-cell- and bulk- multi-omics analysis is performed.Here, we provide a comprehensive protocol for the interdisciplinary characterization of the syndrome caused by infection with SARS-CoV-2, a newly-emerged zoonotic virus and poorly characterized human pathogen. Therefore, the sample size is not prospectively determined. Recruitment of participants will depend on the emergence and spread of the disease in Berlin, Germany, and on the number of patients presenting at Charite - Universitätsmedizin Berlin. With the evolving outbreak, recruitment in other study centers will be considered to facilitate the establishment of a comprehensive clinical and molecular database. The study has no set end date.Proven infection with SARS-CoV-2 (positive pathogen testing). Willingness to participate in the study.Refusal to participate by patient, parent, or appropriate legal representative.Any conditions that prohibit supplemental blood sampling.https://studycenter.charite.de/covid_19/.This protocol outlines the methodology of the Berlin prospective cohort for capturing data of COVID-19. The parameters were selected and adapted to local standards through a multidisciplinary expert review board consisting of clinical researchers at Charité, Department of Infectious Diseases and Respiratory Medicine, Institute of Virology, Departments of Anaesthesiology and Operative Intensive Care Medicine, Department of Psychosomatic Medicine, Department of Pediatrics, Departments of Cardiology, Department of Neurology and Center for Stroke Research, Department of Nephrology and Medical Intensive Care, Department of Endocrinology and Metabolism, Department of Medicine, Division of Gastroenterology, Infectious Diseases and Rheumatology, Department of Hepatology and Gastroenterology, Institute of Tropical Medicine and International Health, Central Biobank Charité (ZeBanC), and the Clinical Study Centre at the Berlin Institute of Health, based on available preliminary data and in accordance with the WHO supported case report form (CRF) proposed by the International Severe Acute Respiratory and Emerging Infection Consortium (ISARIC). Items of the ISARIC–CRF were translated to German using the standardized Functional Assessment of Chronic Illness Therapy (FACIT) translation methodology . Data el® (PROMIS), targeting for precise assessments of dyspnoea and physical function [®-29 Profile at discharge, 6 weeks, 3 months, 6 months, and 12 months after inclusion. In addition, lung function, blood gases, cardiac function, renal function, occurrence of Post-Intensive Care Syndrome, neuropsychiatric function, exercise capacity, and cognitive function will be assessed in selected patient groups at follow-up visits [In addition to medical history and history of present illness, we aim for detailed longitudinal documentation of (i) clinical symptoms, (ii) vital signs and blood gases, (iii) laboratory parameters , (iv) imaging data , (v) microbiological and virological tests, (vi) supplementary diagnostic data , (vi) concomitant medication, and (vii) details of clinical interventions  Table . Patientfunction . Overallfunction  at studyp visits .Table 1SWe will also recruit a representative cohort of outpatients with mild symptoms of COVID-19, in whom study visits and biosamplings are only performed at enrolment (V1) and at day 15 (V7) plus follow-up, provided that no hospitalization becomes necessary during the course of disease. Telephone interviews will be performed with outpatients to assess patient-reported health status between V1 and V7.The molecular basis of the widely variable clinical course of COVID-19 is unknown. Many risk factors for severe disease, prognostic biomarkers, or correlates of protective immunity remain unclear. Several reports have indicated a central role for improper immune responses in COVID-19-associated lung damage and severe disease . To elucAs scientific data on clinical, cellular, and molecular aspects of COVID-19 are rapidly evolving, we will preserve high-quality biosamples, including cryopreserved immune cells, through a large-scale biobanking effort. An overview of the sample flow is shown in Fig. To ensure maximal safety for medical staff involved in this clinical study, SOPs for clinical sample handling are defined. Personal safety equipment for laboratory staff includes tightly closed protective gown, face shield or protective glasses, surgical face mask, and protective gloves. All blood samples are handled under a bio-safety level (BSL) 2 hoods and primary tubes are discharged. Analyses of infectious specimen will be handled according to the current regulation under BSL2 safety conditions.Post-mortem analysis under strict safety considerations will be considered for patients succumbing to COVID-19 in this study. A detailed SOP has been devised at our institution. Autopsy will provide in-depth insights into viral tropism and organ pathologies of severe disease. Additional analysis of infected tissue using spectral confocal microscopy will provide insights into the pathology of fatal COVID-19. Samples will be stored for future analysis.®. All data will be pseudonymized using a six-digit alphanumerical patient code and the online records kept will not include any information that allows patient identification. An enrolment log list will be used to match patient codes in the database to individual patients to record clinical outcomes and supply any missing data points. The enrolment log and study data will be kept separately. Access to the database is protected by username and password. The electronic database set up in this study is suitable for the use in interventional trials and can, therefore, rapidly be modified for future clinical testing of therapeutic agents.Clinical and laboratory data will be collected from patient charts, through patient interviews or from examinations and submitted online to a purpose-built eCRF using the electronic data capture system SecuTrialA detailed data dictionary will define data to be collected on eCRF. Quality checks will be built into the data management system, including the possibility to generate queries, to ensure standardization and validity of the data collected. Any information that is not available to the investigators will be considered as missing. No assumptions will be made for missing data. Data monitoring of a randomly selected subset of collected data will be performed in the course of the study. Discussion of data collection techniques throughout the course of the study with investigators will ensure data quality and allow for adjustments.Access to samples for additional analyses, open access, and data sharing via the Global Research Collaboration for Infectious Disease Preparedness (GloPID-R) will be governed by a research committee comprising the clinical lead investigators and scientific investigators for this study. It is a fundamental principle of this study that all contributors and researchers who have access to samples commit to unrestricted data sharing to provide timely insights into this new disease. In addition, all clinical investigators contributing to this research efforts will be given full recognition for their efforts and the opportunity to access data and samples. Data management adheres to the FAIR data principles, stating that all data collected shall be findable, accessible, interoperable, and re-usable . SamplesThis study is conducted in compliance with the principles laid down in the 1964 Declaration of Helsinki and its later amendments. Where applicable, the principles of Good Clinical Practice  and other applicable regulations and guidelines will be used to guide procedures and considerations. The study was reviewed and approved by the Charité Ethics Committee (EA2/066/20). All patients enrolled will give written informed consent in person, or by a legal guardian. Pa-COVID-19 is registered at the German Clinical Trials Register and WHO International Clinical Trials Registry Platform (DRKS00021688).Medical management of participants in this study must never be compromised by study procedures. At all times, priority will be given to samples required for medical management and day-to-day care of patients. Study blood samples will be taken together with routine blood sampling for clinical monitoring to minimize the individual risk connected with phlebotomy or manipulation of intravascular catheters. There is no direct benefit for patients participating in the study, but results from the study might improve clinical care in future patients and benefit public health.Specific ethical consideration includes the recruitment of critically ill patients who might not be able to consent, the strong moral obligation to participate in a public health emergency, keeping the balance between public health and research, and the potential risk for research staff involved in this study.The enrolment of patients, who are unable to consent, is a common challenge in critical care, acute research, and minors. The process follows a clear legal framework with consent being obtained from legal guardians as soon as possible. In addition, all efforts will be made to obtain informed consent from patients retrospectively after recovery. For patients refusing retrospective informed consent, all data and biosamples will be deleted. For patients succumbing to COVID-19 before informed consent can be obtained, all data will be anonymized.The patient information clearly outlines potential benefits and limitations. The autonomy and well-being of the individual patient and the medically indicated procedures and treatments are always paramount. All staff is trained in infection control measures and has ready access to appropriate personal protective equipment. Dedicated research staff will be available to support the study activities.This study protocol of the observational prospective cohort study Pa-COVID-19 that has recently started recruitment at Charite - Universitätsmedizin Berlin may serve as a blueprint for data and biosample collection in patients with COVID-19 in Germany. The protocol combines a comprehensive clinical and translational approach with tools for implementation in different clinical settings. It is scalable to local resources, and by design, this platform allows for rapid initiation of interventional trials, which are urgently needed to investigate effective treatments for COVID-19. Supplemental material: Data collection elements for scalable data collection. Data elements are categorized to allow scalability to local research resources. Categories are: core data elements (1), recommended data elements (2) and supplemental data elements (3). (XLSX 393 kb)Below is the link to the electronic supplementary material.


CPAchecker.In theory, software model checkers are well-suited for automated test-case generation. The idea is to perform (non-)reachability queries for the test goals and extract test cases from resulting counterexamples. However, in case of realistic programs, even simple coverage criteria  force model checkers to deal with several hundreds or even thousands of test goals. Processing each of these test goals in isolation with model checking techniques does not scale. Therefore, our tool HybridTiger builds on recent ideas on multi-property verification. However, since every additional property  reduces the model checker’s abstraction possibilities, we split the set of all test goals into different partitions. In Test-Comp 2019, we applied a random partitioning strategy and used predicate analysis as model checking technique. In Test-Comp 2020, we improved our technique in two ways. First, we exploit domination information among control-flow locations in our partitioning strategy to group test goals being located on (preferably) similar paths. Second, we account to inherent weaknesses of the predicate analysis by applying a hybrid software model-checking approach that switches between explicit model checking and predicate-based model checking on-the-fly. Our tool HybridTiger is integrated into the software analysis framework


Pseudomonas aeruginosa. The protocol enables bimodal image fusion in the in-house software CycloBranch, as demonstrated by SEM and MALDI-MSI.A procedure for processing frozen rat lung tissue sections for scanning electron microscopy (SEM) from deeply frozen samples initially collected and stored for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was developed. The procedure employed slow thawing of the frozen sections while floating on the surface and melting in a fixative solution. After the float-washing step, the sections were dehydrated in a graded ethanol series and dried in a critical point dryer. The SEM generated images with well-preserved structures, allowing for monitoring of bacterial cells and fungal hyphae in the infected tissue. Importantly, the consecutive nonfixed frozen sections were fully compatible with MALDI-MSI, providing molecular biomarker maps of  Imaging of human and animal tissues, individual cells, and subcellular structures plays a key role in biomedicine and cell biology research. A combination of data recorded by various imaging microscopy platforms has been developed into correlative approaches, such as correlative light electron microscopy , or intoMerging visual morphology information with molecular or elemental mass spectrometry data is another challenge. Mutually different scales in lateral resolution and sampling with compatible sample preparation represent the main hurdles for scanning electron microscopy (SEM)/mass spectrometry imaging (MSI) data fusion. From a mass spectrometry point of view, the most common MALDI-MSI method allows for label-free mapping of molecules, including proteins, peptides, lipids, metabolites, and small molecules, directly in tissue. The physicochemical information in a single spot may involve the molecular formula, relative ion abundance, and organic structure-specific product ion mass spectrum. To reveal structure- and function-specific information, it is possible to combine MALDI-MSI molecular maps of tissue sections with classical histology ,4 and flAnalogous to MSI, wide-field optical microscopy also does not deliver images with high lateral resolution. On the other hand, SEM is capable of visualizing large samples with unprecedented resolution in a complex environment and a considerable depth of field  to show Pseudomonas aeruginosa and the fungus Aspergillus fumigatus.There is no information in the literature on SEM of the same or consecutive tissue sections combined with MALDI-MSI. SEM analysis has been used to monitor the quality of the matrix deposition procedure ,21 and iAll animal experiments were conducted following the regulations and guidelines of the Czech Animal Protection Act No. 246/1992) and with the approval of the Czech Ministry of Education, Youth, and Sports of the Czech Republic (MSMT-21275/2016-2 and MSMT-9487/2019-5) and the institutional Animal Welfare Committees . Animal studies were performed using female 2–3-month-old Lewis rats . During infection models, research staff followed the general guidelines for the protection  to minimize animal suffering according to the protocol [Fungal infection was studied in a rat model, as described previously . Brieflyprotocol . The aniAspergillus hyphae in sections were stained by Grocott’s methenamine silver (GMS) [P. aeruginosa detection. All reagent solutions were prepared from analytical grade chemicals  and double-distilled water. Safranin  counterstained dehydrated sections  were cleared in xylene and mounted in DPX medium. Permanent histological mounts of the whole-lung lobe slices were scanned in Minolta DiMAGE Scan Multi PRO  at 4800 dpi.Dried, thaw-mounted, 15 er (GMS)  using aner (GMS)  Gram staGMS-stained sections were examined under a DN45 light microscope  equipped with a Canon EOS 700D Digital SLR camera . The camera’s built-in white balance correction was used for all recorded images. Spatial calibration of the recorded images was performed with the Fiji software suite  using a A six-well plate  was filled with 2 All tissue samples were sputter-coated with 3 nal lens ,32,33 waProprietary software delivered with microscopes was used unless otherwise stated. In some cases, Fiji software suite, a clone of ImageJ, which is an open source image processing program ,36, was P. aeruginosa was cryosectioned, thaw-mounted onto indium-tin-oxide (ITO) glass slides, and vacuum-dried in a desiccator (https://ms.biomed.cas.cz/cyclobranch/ (accessed on 8 May 2012)) [Liquid nitrogen snap-frozen rat lung tissue infected with y 2012)) .A. fumigatus infectious life cycle, e.g., dissemination, uncontrolled hyphal growth through the pulmonary parenchyma, and severe tissue damage [Aspergillus in the infected tissues [By using GMS staining, the eccentric growth of fungal hyphae in the alveolus is shown in the dark in e damage , were al tissues , were viP. aeruginosa cells in the Gram-stained lung sections, their morphology could barely be judged chemical point of view ,18, whicSurface tension forces allowing the frozen section to float on the fixation and washing solutions were the critical factors of our procedure. In this new procedure, glutaraldehyde fixed the sections during thawing on the surface of the melting frozen fixative solution at 4 Aspergillus hyphae ranged from 1.8 μm to 3.8 μm with an average value of Aspergillus hyphae in such a large tissue section without it.All infected tissues were processed by the float-fixation/washing method. ure data . GMS staP. aeruginosa in the alveolar space. The arrows point to a cluster of bacterial cells, apparently formed by active bacterial growth in the tissue  with a umigatus . Howeverumigatus . One musumigatus ,44. HoweAspergillus in the infected tissues [Optical microscopy has not been able to provide sufficiently detailed structural features but served as a quick tool for examination of the extent of fungal or bacterial infection in rat lung tissue. Nevertheless, eccentric growth of fungus in alveolus A and oth tissues ,40,45 ar tissues . ImportaAt a higher magnification than that shown in A. fumigatus or P. aeruginosa was sufficient for detailed SEM analysis of alveolar space colonized with both microorganisms. The uncomplicated flow-fixation/washing method is not limited to lung tissue and can be used to process frozen sections from other soft biological tissues. It does not require any specialized tools or sophisticated equipment and is feasible for any standard laboratory with occasional access to an electron microscopy facility. Of final note, the float-fixation procedure worked even for relatively large sections with an area exceeding 50 mm2. In other words, sections through the whole rat lung lobe can be processed.To conclude, preservation of the lung tissue structure in the samples infected with


Furthermore, an independent validation cohort (n = 40), including healthy controls, SSc and SSc-PAH patients, was used for further validation by quantitative real-time polymerase chain reaction. The results showed that four key genes, including IFIT2, IFIT3, RSAD2, and PARP14, may serve as potential biomarkers in SSc-PAH. Also, they could be independent risk factors for SSc-PAH. In conclusion, the four key genes can be expected to become the potential therapeutic targets and early biomarkers for accurate therapy and diagnosis of SSc-PAH in the future, which also provides promising insights into the pathogenesis of SSc-PAH at the molecular level.Systemic sclerosis-associated with pulmonary arterial hypertension (SSc-PAH) is still a major cause of SSc related deaths. Early diagnosis and prompt treatment are crucial to reduce the mortality of patients with SSc-PAH. To screen the candidate biomarkers and potential therapeutic targets for SSc-PAH, we analyzed the data set (GSE33463 and GSE19617) for confirming key genes in peripheral blood mononuclear cells from SSc-PAH patients. A total of 105 SSc patients from gene expression omnibus (GEO) were included as discovery cohort ( Systemic sclerosis (SSc) is an intractable autoimmune disease characterized by immune dysfunction, vascular disease, cellular inflammation, and dermal fibrosis and involvement of multiple organs including heart, kidney, and lung with high clinical heterogeneity and mortality than other autoimmune diseases . AlthougEpidemiological investigations have indicated that age (≥47) at the diagnostic time of SSc , diffusiThe occurrence of SSc-PAH is characterized by progressive remodeling of the small- to medium-sized pulmonary vasculature . SeveralRSAD2), interferon-induced protein with tetratricopeptide repeats 2 (IFIT2), interferon-induced protein with tetratricopeptide repeats 3 (IFIT3), and poly (ADP-ribose) polymerase family member 14 (PARP14) in an independent validation cohort by quantitative real-time PCR (qRT-PCR) and receiver operating characteristic curve (ROC). Our findings may point to the potential candidate genes for accurate therapy of SSc-PAH and provide powerful evidence for a better understanding of the pathogenesis of SSc-PAH.In this study, weighted gene co-expression network analysis (WGCNA) was utilized to discover correlation patterns among genes and identify the relationship between co-expressed modules and SSc-PAH. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and Gene Ontology (GO) analysis were also applied to explore potential mechanisms. Specifically, we validated the expression levels and diagnostic accuracy of the four genes including radical S-adenosyl methionine domain containing 2   . Patientn = 10) . Twenty –ΔΔCT [ΔΔCT = (CT of gene) − (CT of β-actin) − (CT of HCs)], in which CT represents the threshold cycle for each transcript.A 2–3 ml sample of blood was collected and PBMCs were isolated by human PBMCs separation medium . TRIzol Reagent  was utilized to extract the total RNA from PBMCs samples. The total RNA was reversely transcribed into cDNA by PrimeScript RT Master Mix  according to the manufacturer’s instructions. One microliter of cDNA was used for qRT-PCR with TB Green Premix Ex Taq  and specific primer pairs . The exp1  were acquired from Gene Expression Omnibus (GEO)1 . GSE33461 .First, ranked by diminishing standard deviation (SD), the top 25% of genes were selected for further analysis. Second, the pickSoftThreshold function was utilized to reckon the soft-thresholding power β for constructing modules. The function provided an appropriate β value ranging from 1 to 30 and the value closest to 0.8 was selected by the result of the scale-free topology fit index and the mean connectivity. Once a suitable β value was determined, co-expressed modules were built by the WGCNA algorithms in R software. Next, the adjacency was converted into a topological overlap matrix for calculating the network connectivity of each gene. Then, genes with similarly expressed patterns were classified as diverse modules with the smallest gene size of 50 based on the topological overlap matrix similarity . FinallyP-value of <0.05 were considered statistically significant.To further understand potential mechanisms and functions, we performed the GO and KEGG enrichment analysis of the genes in the key modules by using clusterProfiler of R software . The ter2 and visualized the top 30 hub-genes in each module by Cytoscape  network, we imported the genes in the key modules into the STRING databaseytoscape .2FC > |1|, P-value < 0.05. Among them, we selected 386 genes with the most obvious up-regulation and down-regulation (P < 0.05), which can better show the difference between sample groups. Next, to overlap DEGs and the genes in key modules, we constructed the Venn diagram by using jvenn3  of each hub gene to evaluate the abilities for the diagnosis of SSc-PAH by SPSS 22.0. The student’s To construct the weighted co-expression networks, the top 12200 genes ranked diminishing SD were chosen for WGCNA. Then, cluster analysis was performed . One obvP value  and purple module  was significantly correlated with gene significance for SSc-PAH.To explore the similarity between co-expressed modules, we calculated the association between modules and clinical traits associated with pulmonary phenotypes (PAH and ILD), and clustered eigengenes based on their correlation. As a result, 18 modules were primarily classified into two clusters. PAH and ILD clustered together, indicating similarity between PAH and ILD. The heatmap plotted according to adjacencies showed similar results . MoreoveP value . We founP value . The resGene Ontology enrichment analysis revealed that genes in saddlebrown module were mainly distributed in GO:0051607 (defense response to virus), GO:0009615 (response to virus) and GO:0060337 , and which were positively correlated with PAH. Genes in the purple module were mainly enriched in GO:0005622 (intracellular), GO:0043170 (macromolecule metabolic process) and GO:0005488 (binding), which played an important role in metabolic functions . MoreoveP < 0.05 to obtain DEGs. We overlapped the DEGs and genes in the saddlebrown module by Venn diagram. As a result, four overlapped hub genes  were shown in DEGs and saddlebrown modules, indicating that the four genes we are looking for are highly related to the SSc-PAH  and clinical risk factors % predicted, DLCO% predicted) in patients with SSc-PAH, we applied the ULR analysis in duplication cohort. We found that four genes, age, and DLco (% predicted) could become independent risk factors for SSc-PAH  for the combination of these four genes , which raiseed the possibility of overfitting their classifier. However, our study identified the key genes in SSc-PAH in three independent cohorts, and determined the key genes as potential biomarkers by different methods.Compared with the previous studies, our study provides new insights into the pathogenesis of SSc-PAH. oreover,  used GSEA limitation of this study is that despite the value of validation cohort for reliability, our limited samples from largely middle-aged female patients of Chinese ancestry cannot represent the general population. Therefore, our findings may not generalize total SSc or SSc-PAH patients. Further studies with large sample size are needed for the validation, justification, and generalization of our results. Moreover, lack of the phenotyping data of SSc-PAH is a major limitation in this study. Due to the different phenotypes of SSc-PAH, pathological the mechanisms of SSc-PAH are highly heterogeneous. However, datasets included SSc-PAH patients’ samples are very few in the public databases, not to mention detailed phenotyping data of SSc-PAH patients. Therefore, it can be challenging to determine the dominant mechanism in a particular patient due to the limitation of phenotyping data. Despite these limitations, these findings highlight candidate genes and underlying mechanisms for further research.IFIT2, IFIT3, RSAD2, and PARP14) associated with SSc-PAH. The upregulation of these genes may be the independent risk factors for SSc-PAH. Our current study provides reliable evidence that these genes are associated with the occurrence of SSc-PAH. Bioinformatics analysis revealed these key genes were involved in response to virus, cellular response to type I interferon and type I interferon signaling pathway. In the future, there remains a strong need for well-designed clinical studies with more exhaustive strategies, more homogenized populations, and larger samples size to study the underlying function of these genes in the development of SSc-PAH.In conclusion, we identified the key genes (http://www.ncbi.nlm.nih.gov/geo/).Publicly available datasets were analyzed in this study. Discovery cohort and duplication cohort (GSE33463 and GSE19617) were downloaded from GEO  for the publication of any potentially identifiable images or data included in this article.J-NZ, YL, and QW designed the study. W-QS collected the blood samples. J-NZ, YL, and T-TZ did the statistical analyses. Y-MY and HS prepared the figures. J-NZ, YL, Y-MY, HS, W-QS, and QW reviewed the results and wrote the manuscript. All authors have made an intellectual contribution to the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hand gesture recognition (HGR) takes a central role in human–computer interaction, covering a wide range of applications in the automotive sector, consumer electronics, home automation, and others. In recent years, accurate and efficient deep learning models have been proposed for real-time applications. However, the most accurate approaches tend to employ multiple modalities derived from RGB input frames, such as optical flow. This practice limits real-time performance due to intense extra computational cost. In this paper, we avoid the optical flow computation by proposing a real-time hand gesture recognition method based on RGB frames combined with hand segmentation masks. We employ a light-weight semantic segmentation method (FASSD-Net) to boost the accuracy of two efficient HGR methods: Temporal Segment Networks (TSN) and Temporal Shift Modules (TSM). We demonstrate the efficiency of the proposal on our IPN Hand dataset, which includes thirteen different gestures focused on interaction with touchless screens. The experimental results show that our approach significantly overcomes the accuracy of the original TSN and TSM algorithms by keeping real-time performance. Hand gesture recognition (HGR) plays a central role in Human–Computer Interaction (HCI). Recently, HGR systems using vision-based interaction and control have become more common ,2,3, andIn recent years, accurate and efficient deep learning models have been proposed for HGR ,11,12,13In this paper, we propose to use hand segmentation masks as an alternative to depth and optical flow modalities. Specifically, we extend our FASSD-Net  algorith(SpyNet) .To validate our proposal, we composed the IPN Hand dataset  for handWe propose an alternative to the expensive dense optical flow estimation, and the extra sensor requirement of depth images, by using semantic segmentation images of the hand for real-time hand gesture recognition.By using a simple input modality combination, we demonstrate that our RGB-S proposal outperforms state-of-the-art methods of TSN and TSM. We also present a statistical analysis of both approaches’ results, which shows the type of gestures that gets more benefit from different input modalities.github.com/GibranBenitez/IPN-hand.We extend our new IPN Hand dataset , includiIn summary, the main contributions of this paper are:HGR methods can be divided into two groups based on the way for obtaining the gesture features, i.e., e hand-crafted and deep-network based approaches ,8,21. HaOn the other hand, previous works focused on learned feature extraction methods tend to use deep convolutional neural networks (CNNs) ,12,26 anThe multi-stream technique has been prevalent in recent years as shown in the ChaLearn Look At People (LAP) gesture recognition challenge , where aIn this section, we introduce the FASSD-Net  algorithFASSD-Net  is basedThe pioneering work of Wang et al.  proposesV, we divide it into K segments . Following the original open-source implementation of FASSD-Net [We train the FASSD-Net  model wiASSD-Net , we use K). Specifically, for We used the same ResNet-50  backbonen of TSM , the traV with duration N is based on the average prediction scores of 10 clips of K frames, sparsed by a stride of size S, where All the dataset videos are segmented into isolated gesture samples based on the beginning and ending frames manually annotated. So that, the classification problem resided on predicting the class label for each gesture sample. In this paper, the percent of correctly labeled samples (Accuracy), and the confusion matrix of all isolated gesture predictions are used as evaluation metrics. Nonetheless, the number of segments and the number of frames of each gesture sample restricts the one-to-one evaluation. Therefore, a common practice is to sample 10 random multiple clips per video ,43. HoweK) with RGB and RGB-S modalities. From this table, we can see that the proposed RGB-S outperforms RGB’s accuracy for all the choosing segments. The improvement is more significant when choosing 32 segments for the process. In this way, RGB-S with 32 frames achieves the best accuracy when using TSM.To clearly show the improvement achieved of RGB-S, In this sub-section, we compare the results of TSM and TSN when using different input modalities with 32 segments. Interestingly, the accuracy of TSN is significantly better than that of TSM. To clearly analyze each method’s performance, we present its confusion matrices with the different modalities. Comparing In this experiment, we train the models analyzed in the previous sub-section with only the 11 action classes of the dataset. In this way, we can evaluate the performance of both HGR methods, as well as the improvement gained by different multimodality. In the previous section, we have shown that our proposed RGB-S modality helps to improve the accuracy performance of both TSM and TSN methods. However, we obtain interesting results when comparing both methods using the same modality. TSM  was propFirst of all, as mentioned in On the other hand, the problem of repetitive gestures mentioned in In this paper, we proposed an alternative to the expensive dense optical flow estimation used in the multimodal approaches for hand gesture recognition (HGR). We based our proposal on a combination of RGB frames with hand segmentation masks. To this end, we employed a light-weight semantic segmentation method (FASSD-Net) to boost the accuracy of two real-time HGR methods: Temporal Segment Networks (TSN) and Temporal Shift Modules (TSM). We demonstrated the efficiency of RGB-S modality over RGB-OF and RGB with extensive experimental tests. Furthermore, we analyze both HGR methods’ results, showing that TSM is better at recognizing gestures that rely on temporal information, while TSN stands out on static gestures. As future work, we plan to combine TSM and TSN in a single architecture to recognize both types of hand gestures in real-time.