A rapid and accurate diagnosis is a crucial strategy for containing the coronavirus disease (COVID-19) pandemic. Considering the obstacles to upscaling the use of RT–qPCR, rapid tests based on antigen detection (Ag-RDT) have become an alternative to enhance mass testing, reducing the time for a prompt diagnosis and virus spreading. However, the performances of several commercially available Ag-RDTs have not yet been evaluated in several countries. Here, we evaluate the performance of eight Ag-RDTs available in Brazil to diagnose COVID-19. Patients admitted to tertiary hospitals with moderate or mild COVID-19 symptoms and presenting risk factors for severe disease were included. The tests were performed using a masked protocol, strictly following the manufacturer’s recommendations and were compared with RT–qPCR. The overall sensitivity of the tests ranged from 9.8 to 81.1%, and specificity greater than 83% was observed for all the evaluated tests. Overall, slight or fair agreement was observed between Ag-RDTs and RT–PCR, except for the Ag-RDT COVID-19 (Acro Biotech), in which moderate agreement was observed. Lower sensitivity of Ag-RDTs was observed for patients with cycle threshold > 25, indicating that the sensitivity was directly affected by viral load, whereas the effect of the disease duration was unclear. Despite the lower sensitivity of Ag-RDTs compared with RT–qPCR, its easy fulfillment and promptness still justify its use, even at hospital admission. However, the main advantage of Ag-RDTs seems to be the possibility of increasing access to the diagnosis of COVID-19 in patients with a high viral load, allowing immediate clinical management and reduction of infectivity and community transmission. Initially described in China in December 2019, coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), rapidly spread worldwide, becoming a major global health concern, and causing massive socioeconomic disruption [Real-time reverse transcription polymerase chain reaction (RT–qPCR) using respiratory specimens has been recommended as a reference diagnostic test for acute SARS-CoV-2 infection and has been widely implemented . ConsideAntigen-based tests (Ag-RDTs) quickly emerged as a viable alternative to large-scale testing for SARS-CoV-2. Although they are inferior to RT–qPCR in terms of sensitivity and specificity , 7, theiThis study was performed according to the Standards for Reporting of Diagnostic Accuracy (STARD) statement .This prospective study was conducted in a tertiary hospital setting in the municipality of Belo Horizonte, state of Minas Gerais, Brazil. Patients presenting a clinical suspicion of COVID-19 were consecutively enrolled and tested on hospital admission by RT–qPCR for SARS-CoV-2. Only patients with a mild clinical condition who were eligible for noninvasive treatment were included in this study. For each recruited participant, two samples were collected through consecutive paired nasopharyngeal swabs, one from each nostril. Depending on the manufacturers’ requirements, the rapid test was performed at the bedside immediately after collection (maximum of 30 minutes) or, if explicitly authorized, could be performed using the Universal Transport Medium  in the reference laboratory at a maximum of 12 hours after sampling. Thus, using the UTM, from the clinical specimen obtained from a single participant it was possible to perform several tests, limited by the total volume of the transport solution available. The UTM volume was always limited to a maximum of 2 mL to minimize sample dilution. Therefore, whenever authorized by the manufacturer, the UTM was the preferred source of performing the test. Thus, considering these two tests execution possibilities, we developed a study schedule based on two phases. In the first phase, the swab used in the first nostril was kept in transport medium and, from this solution the RT–qPCR and four Ag-RDTs were performed (randomly chosen among the six Ag-RDTs UTM-enabled). The swab used in the second nostril was directly used in the execution of one Ag-RDT . As the same way, in the second phase, the swab used in the first nostril was immersed in UTM, which was used in the execution of the RT–qPCR and the other two Ag-RDTs UTM-enabled. The swab used in the second nostril was directly used in the execution of one Ag-RDT, and the patients were submitted to a new collection, this time covering only the nasal mucosa for execution of a commercial test based on exclusive nasal swab collection . The number of participants varied depending on the total patients recruited at each study phase, which ended when the minimum number of samples required  was reached at the end of the day. At the end of each study phase, depending on the availability of Ag-RDTs, two additional days of recruitment and testing strategy were maintained in order to gather some patients to be tested simultaneously using dry swab and UTM sources, a secondary analysis. During all the study, RT–qPCR results were masked to all researchers involved in the rapid test reading.Ethical approval was obtained from Instituto René Rachou, Fundação Oswaldo Cruz, CAAE: 30960120.0.0000.5091 (Number 4.001.133) and Eduardo de Menezes Hospital, Fundação Hospitalar do Estado de Minas Gerais, CAAE 42314921.0.3001.5124 (Number 4.595.768). The samples were used only after written formal acceptance of the participants over than 21 years old.For a comparison based on agreement with the reference test (RT–qPCR), the minimum population required for one test validation was estimated as 52 patients, according to Arifin et al. (2021) . The preAll manufacturers of the 58 Ag-RDTs for COVID-19 registered at the Brazilian National Health Surveillance Agency (ANVISA) until March 2021 were contacted and invited to participate in this validation study. Of the 16 tests whose manufacturers agreed to participate in this validation, eight were selected based on the availability of supplying the kits within 40 days and possibility of execution from transport medium using a minimum required volume. All the tests were performed strictly according to the manufacturer’s instructions using the buffer provided in each kit or UTM. The main characteristics of the selected Ag-RDTs are shown in For tests performed immediately after sampling, the nasopharyngeal swab was immersed in the buffer solution and then dripped onto the test plate. For tests performed using samples stored in UTM, this medium was diluted in the buffer, and the final solution was dripped in the appropriate place on the reagent strip. In both cases, after the recommended waiting time, the control and test bands were observed in the test membrane. The test was considered positive if the control band was reactive and any intensity band was observed at the test band.RNA extraction was performed using a MagMax Viral/Pathogen Nucleic Acid Isolation Kit  or a Chemagic Viral DNA/RNA H96 kit , and amplification was performed using a commercial rRT-qPCR kit  containing ORF1ab, Nucleocapsid (N) and Spike (S) as target sequences for SARS-CoV-2. All RT-qPCRs were performed using QuantStudio 5 . A cycle threshold (CT) value of 37 was designated the cutoff value for positive results. Amplifications in at least two target regions of SARS-CoV-2 were considered positive, and the absence of an amplification signal was considered negative. Any other RT–qPCR results were considered inconclusive.Descriptive statistics were used to present the main characteristics of the population. We used the Shapiro–Wilk normality test to evaluate whether the data were normally distributed. Continuous variables were presented as medians and interquartile range (IQR 25–75%), and the Mann–Whitney U test was used to compare medians. Categorical variables, expressed as numbers (percentages), were compared by chi-squared test or Fischer’s exact test as appropriate. The accuracy analyses of Ag-RDT tests were determined according to RT-qPCR result using MedCalc Software (Version 20.015). Based on a two-by-two contingency table, sensitivity was considered as the number of true positive patients on the Ag-RDTs divided by the total of positive patients on the RT-qPCR, and specificity was considered the number of true negative patients on the Ag-RDTs divided by the total of negative patients on the RT-qPCR. Finally, accuracy was determined by the number of RT–qPCR and Ag-RDT concordant results divided by the total number of tested patients. RT–qPCR was defined as the reference standard, and the agreement was calculated using the Kappa index and interpreted following the criteria of Landis and Koch (1977) [A total of 162 nonvaccinated participants were included from March 22 to April 21, 2021. The flow diagram showing the patients included in the study and its primary outcome according to the test is presented in the ® Ag-RDT (Nanosens) that presented slight agreement (K = 0.04).Overall, the sensitivity of the Ag-RDTs ranged from 9.8 to 81.1%, and the specificity was higher than 83.3% for all evaluated tests . The agrThe agreement between the results of three commercial tests (COVID-19 Ag ECO teste (Eco Diagnostica), Coris Bioconcept Ag-RDT (Nanosens) and SARS-CoV-2 Ag-RDT (SD BIOSENSOR)) performed on the same patients using directly obtained respiratory secretions and MTU source is shown in  and was The indirect method of quantifying viral load expressed by the Ct value, obtained by RT–qPCR, was correlated with Ag-RDT positivity. False negative Ag-RDT results were mostly observed in patients with high Ct values for all Ag-RDTs evaluated  published by WHO, Ag-RDTs should achieve ≥ 80% sensitivity and ≥ 97% specificity compared with a nucleic acid amplification test . NotablyThe performance of Ag-RDTs may be related to the intrinsic characteristics of the patients, such as the viral load, disease severity and length of symptoms as well as the characteristics of the tests, quality of the specimen and proper handling , 22. OveRegarding sample processing, several Ag-RDT manufacturers allow the use of UTM for the temporary storage of nasopharyngeal samples. Besides the additional time gained between the sample collection and testing, the transport medium uses the same sample for the execution of different tests, which could be a strategy to allow the execution of RT–qPCR on the same specimen already collected in the case of a negative Ag-RDT test.Even performed in accordance with the manufacturer’s instructions, one possible limitation of this strategy would be the potential of the transport medium to influence the performance of the test by promoting dilution of the clinical specimen. Here, the comparison between results obtained with the same test but performed using dry swab or UTM source revealed a worryingly weak agreement. Similar results were described by Cubas-Atienzar et al. (2021), who observed a lower sensitivity analytical limit of detection for Ag-RDT using UTM than dry swabs .™ Ag-RDT Device Nasal (Abbott Rapid) clearly stated that the virus nucleocapsid (N) protein was used as the specific antigenic target. This structural protein is often used because of its relative abundance and because it presents the least amount of variation in the gene sequence, indicating that it is a stable protein [All evaluated Ag-RDTs are based on a sandwich immunodetection methodology with intrinsic characteristics that may affect their performance. Only two of the test manufacturers (CORIS Bioconcept Ag-RDT (Nanosens) and Panbio protein , 24. How protein , 26.Several limitations may affect the test’s accuracy. This study was conducted under the same controlled laboratory conditions (temperature and lighting). Samples were collected, tests were performed by trained staff, and a unique Ag-RDT batch was used throughout the study. The total sample of patients to be included was supported by sample calculation; however, subgroup analysis should be interpreted carefully considering the heterogeneity of the population.Although Ag-RDTs presented lower sensitivity than RT–qPCR, those tests may be a useful diagnostic tool for COVID-19, rapidly detecting patients with high viral loads. These results confirm that the performance of rapid tests based on the antigen search for SARS-COV-2 in the routine of laboratories or health services may be inferior to that described by the manufacturers and that marked differences exist between commercial brands. Viral load seems to be the main determinant of test positivity, explaining the influence of symptom duration on observed performance. Even so, a positive Ag-RDT remains useful to diagnose symptomatic cases at hospital admission, particularly in terms of the speed of results, considering that a negative result does not rule out SARS-CoV-2 infection. The main benefit of Ag-RDTs would be to confirm the COVID-19 diagnosis in patients with higher viral shedding and possibly greater infectivity, reducing the number of cases for RT–qPCR. Thus, diagnostic algorithms combining tests with different methodologies must be evaluated in cost-effectiveness studies to confirm the best strategy for using rapid tests. Therefore, the various tests must be performed in different populations, justifying further studies in real-life scenarios, such as this one.S1 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Protozoan pathogens lead complex life cycles, from one host to another, surviving in a variety of morphotypic forms through this complex continuum. The developmental stages can be free-living, extracellular parasitic or intracellular parasitic. The key trigger identified for such transitions has often been stresses such as the alteration of temperature or pH, hypoxia, oxidative burst, or nutrient depletion (2+) which modulate chemotaxis, antioxidant defence of the parasite  and reactive nitrogen intermediates (RNI). Santos Moura et al. describe a novel aspect of posttranscriptional regulation of mitochondrial superoxide dismutase (SODA), a major antioxidant enzyme of the parasite. With an aim to explore the biological significance of their earlier profiling of lysine-acetylated proteins in procyclic and bloodstream forms of T. cruzi epimastigotes, where a number of anti-oxidant proteins were identified (•–) resulting from K97 acetylation. In this context, a mitochondrial lysine deacetylase- TcSir2rp3 was explored for possible involvement in the process. TcSir2rp3 interacts with TcSODA and overexpression of TcSir2rp3 resulted in elevated SOD activity with reduced ROS levels both in mitochondria and cytosol. Using mutated versions of TcSODA and TcSir2rp3 the possible cross talk in the context of K97 acetylation was confirmed, which also affects responsiveness to benznidazole and nifurtimox. The study for the first-time evidenced significance of acetylation in modulating antioxidant enzyme action in Trypanosomatids and reinforced the importance of PTM in modulating stress response.Chagas disease, endemic in 21 countries across Latin America, registers significantly higher morbimortality compared to other parasitic diseases . The cauentified , the groDas et al. here describe the development of a novel nano-formulation using a liposomal curcuminoid HO-3867 which poses cytotoxicity on the extracellular promastigotes and intracellular amastigotes of L. donovani. The work is of particular interest in formulating a safe, specific, and effective therapeutic treatment, still an unaccomplished mission against visceral leishmaniasis. HO-3867 was encapsulated with phosphatidylcholine (PC) with stearylamine (SA) by thin film rehydration to prepare PC-SA/HO-3867 liposomes. The formulation induced four-fold upsurge in abrupt ROS production in the parasite coupled to altered lipid metabolism in the promastigotes as reflected by enhanced accumulation of lipid bodies. When tested on L. donovani promastigotes, the liposomal preparation induced substantial alteration of morphology and prompted stress induced apoptosis like event typically marked by cell cycle arrest with an increase of sub-Go population, mitochondrial depolarization, and DNA fragmentation. Stress induced activation of metacaspase and poly [ADP]-ribose polymerase-1 (PARP-1) have recently been linked to impaired sirtuin (SIR2) function with depletion of NADH  for cross-resistance to paromomycin (PMM), AmB or MIL identified six isolates to be cross-resistant to MIL. The association of intracellular thiol content and antimony resistance, as projected earlier , grant numbers 37324 and 38858. Work in the ABi lab was supported by DST-INSPIRE Funding (IFA-12 LSBM-22).This study was partially funded by the SERB, Govt. of India (SRG/2020/000702) granted to AB-lab. Work in the CFP-Lab was supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant RGPIN-2017-04480 and by the Canada Foundation for Innovation (We thank the authors of this special issue for their valuable contributions.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be considered as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A safe and effective vaccine is urgently needed to bring the current SARS-CoV-2 pandemic under control. The spike protein (SP) of SARS-CoV-2 represents the principal target for most vaccines currently under development. Despite the presence of a CoV proof-reading function in viral replication, SP protein from SARS-CoV still extensively mutates, which might have an impact on current and future vaccine development. Here, we present analysis of more than 1600 SP unique variants suggesting that vaccine candidates based on the Wuhan-Hu-1 reference strain would be effective against most of currently circulated SARS-CoV-2 viruses, but that further monitoring of the evolution of this virus is important for identification of other mutations, which could affect the effectiveness of vaccines. There is a consensus that development of an effective and safe vaccine represents the best strategy to control the COVID-19 pandemic, and consequently, this endeavor remains a top public health priority. Researchers and drug companies around the world are working hard to develop a COVID-19 vaccine, and currently 282 vaccine candidates are in clinical trials or will enter clinical investigation soon . The spike glycoprotein (SP) of SARS-CoV-2 is the principal target for most these vaccine candidates. Despite the presence of a CoV proof-reading function in viral replication, SARS-CoV still extensively mutates, which might have an impact on current and future vaccine development. In the GISAID database more than 1,600 SARS-CoV-2 strains with unique combinations of mutations in SP are deposited. In order to fully understand the impact of the mutations on the SP properties , advanced laboratory studies are required. These investigations take time and are done in close collaboration between different research groups, institutions and academia. However, some important information about the biological effects of mutations could and should also be obtained by thein silico analysis of SP, which can be performed quickly and with a minimal experimental foreknowledge at the time of analysis.The current Coronavirus Disease 2019 (COVID-19) pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), represents an unprecedented health threat resulting in over 1.1 million deaths globally  of mutations in SP from SARS-CoV-2 virus on the effectiveness of the COVID-19 vaccines and/or vaccine candidates. The presented results showed that VE of vaccine candidates that are based on SP from the reference SARS-CoV-2 strain YP_009724390 remains between 76% and 94%, despite an extensive mutation rate in this important and likely protective antigen. Our analysis also suggests that further close monitoring of SP mutations might be essential in order to be able to respond to changes that may influence VE in the future.Previously, a novel bioinformatics approach, which is based on electronic biology, was used in the assessment of the effect of mutations on the vaccine effectiveness (VE) for seasonal flu vaccines. Most recently, this approach allowed successful prediction of VE for two successive flu seasonshttps://www.epicov.org/epi3/cfrontend#18c7c7) from January to October, 2020, and SP1 from viruses isolated from mink that are presented in the same database. Prototype virus Wuhan-HU-1 (YP_009724390) is used in the analysis as the wild type (WT) and as the vaccine virus.We analyzed the non-redundant subunit 1 of S proteins (SP1) from human SARS-CoV-2 viruses deposited in the GISAID (. This bioinformatics approach encompasses three basic steps: (i) the representation of the primary structure of the protein as the numerical sequence by the assignment to each amino acid of the corresponding value of the electron-ion interaction potential (EIIP), (ii) the transformation of the obtained numerical sequence into the informational spectrum (IS), and (iii) the calculation of the cross-spectrum (CS) between interacting proteins.The ISM is the virtual spectroscopy method for the analysis of the protein biological properties. This molecular descriptor is defined by the following equation:The EIIP is the physical parameter that determines the long-range interactions of biological molecules (interactions at distances >5Å)where Z* is the average quasivalence number (AQVN):N is the total number of atoms,in is the number of atoms of the i-th component,iZ is the valence number of the i-th atomic component in the molecule and m is the number of components. The EIIP values calculated according to thewhereThe numerical sequence, representing the primary structure of protein, is transformed into the IS by the discrete Fourier transformationx(m) is them-th member of a given numerical series,N is the total number of points in this series, andX(n) are discrete Fourier transformation coefficients. In this way, the information defined by the sequence of amino acids is represented as the series of frequencies and their amplitudes. The frequencies in IS correspond to the distribution of the structural motifs with defined physicochemical properties determining a biological characteristics of a protein. When comparing proteins, which share the same biological or biochemical function, the ISM technique allows detection of code/frequency pairs that are specific for their common biological properties, or which correlate with their specific interactions.where.d defined on the ratio between specific frequencies F(0.257) and F(0.479) in IS of SP1 protein, which characterize its interaction with host factors.The algorithm of the ISM-based phylogenetic analysis, which was used for the assessment of the biological effect of mutations in SP1 proteins from SARS-CoV-2, was previously described in detailExtended data: Figure 1S) calculated for 1643 nonredundant SP1 proteins from SARS-CoV-2 viruses collected from January to October, 2020. Analyzed SP1s in this tree are grouped into four separated clusters. The largest cluster A, encompassing 78% of analyzed proteins, contains two sub-clusters, which includes the Wuhan-Hu-1 reference sequence for the Spike protein that is also the basis for different vaccine candidates (sub-cluster A2) and viruses with SP1 with the most abundant mutation D614G (sub-cluster A1). According to the IS concept, proteins that are grouped in the same cluster in the ISM-base phylogenetic tree have similar interacting and immunological profiles. This indicates that a vaccine based on SP from the Wuhan-Hu-1 reference strain will be effective against viruses that are grouped together with this virus in the sub-cluster A2. It was suggested that a single vaccine candidate based on the reference SARS-CoV-2 strain would likely match most of currently circulating variants, including the mutant D614G. Recently, it also showed that sera from D614-infected hamsters exhibit modestly higher neutralization titers against G614 virus than against D614 virus, indicating that this mutation may not reduce the ability of vaccines in clinical trials to protect against COVID-19. This suggests that viruses which are grouped together with the mutant D614G in the sub-cluster A1 would also be responsive to the vaccine candidates that are based on the Wuhan-Hu-1 reference strain. In contrast to the cluster A, viruses that are grouped into other three small clusters B, C and D do not perfectly match the vaccine virus and might be resistant to the vaccine.Phase-3 clinical trial of the candidate COVID-19 vaccine. This study, which enrolled 43,538 subjects, showed that the tested vaccine candidate to be more than 90% effective in preventing COVID-19 in participants.InAlthough potentially vaccine non-responsive viruses in the clusters B, C and D represent only 22% of all 1,643 viruses with non-redundant SP1, these viruses could negatively affect VE. Mass vaccination, which is expected in the near future, creates an immune pressure in the population that would lead to increase in mutations in SP1 as well as in natural selection and preservation of escape mutants. Moreover, this fraction of mutant viruses that can escape vaccine protection and be efficiently transmitted could significantly increase among circulating SARS-CoV-2 once vaccination starts. For this reason, real-time monitoring of virus evolution with different methods, including the ISM-based phylogenetic tool for detection of “functional” changes in the SP1 of SARS-CoV-2 is needed.12 human cases of COVID-19 have been identified with SARS-CoV-2 unique variants associated with farmed minks. Viruses isolated from these patients had a combination of mutations, or changes that have not been previously observed and the implications of the identified changes in this variant are not yet well understood. Preliminary findings indicate that this particular mink-associated variant identified in both minks and the 12 human cases could negatively affect VE because mutations in these viruses moderately decreased sensitivity to neutralizing antibodies. Danish authorities have undertaken different actions to limit the further spread of this variant of the virus among mink and human populations, including culling of all farmed mink in Denmark (more than 17 million).In September 2020 in Denmark,In order to assess the effect of these mutations on VE we calculated the ISM-phylogenetic tree for 5 non-redundant SP1 sequences from SARS-CoV-2 viruses from mink deposited in GISAID . As presThe presented analysis of SP1 proteins, performed by the electronic biology tool, suggest (i) that vaccine candidates based on the Wuhan-Hu-1 reference strain would be effective against most of currently circulated SARS-CoV-2 variants, and ii) further ISM-based monitoring of the evolution of SARS-CoV-2 is important for identification of other mutations, which could affect the effectiveness of vaccines against this virus. Therefore, the scientific community needs to be proactive in submitting genetic sequences into databases such as GSAID. For example, we have States such as Wisconsin, South and North Dakota with low mortality rate 0.8, 1.2, 1.2, respectively , but no ability to check genetic sequences of viruses from those States as none were deposited. It is very important to support genetic data collection and to share that data in real time in order to organize successful monitoring using different approaches including the one published in this paper.GISAID Database. To access the database each individual user should complete the “Registration Form For Individual Users”. This form, together with detailed instructions, are available on the website. After submission of the Registration form, the user will receive a password. There are no other restrictions for access to GISAID. Conditions of access to, and use of, the GISAID Database and Data are defined byTerms of Use.Sequence data of the viruses were obtained from thehttps://doi.org/10.7910/DVN/JLXWWP.Harvard Dataverse: The ISM-based phylogenetic tree of the non-redundant SP1 proteins from SARS-CoV-2 viruses deposited in GISAID from January to October 2020,This project contains the following extended data:- Figure 1SCreative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).Data are available under the terms of the Dear Editor, I want to inform you that this article is an exciting piece of work. The study presented the analysis of SP unique variants using the electronic biology tool whereas SP is an important target for developing most of the SARS-CoV-2 vaccines. The authors found that the vaccine candidates based on the Wuhan-Hu-1 reference strain would be effective against most of the currently circulated SARS-CoV-2 variants and that ISM-based monitoring of the evolution of SARS-CoV-2 is important for the identification of other mutations, which could affect the effectiveness of vaccines against this virus.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Clinical Pharmacy, Respiratory system diseases and COVID-19I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. et al. describes the application of an in silico method previously developed by the group to monitor the "functional" evolution of viral sequences based on the informational spectrum method (ISM). This method is based on the primary amino acid sequences of proteins, from which informational spectra are being derived that ultimately inform about protein-protein interactions. This method allows a rapid assessment of functional alterations in evolving proteins like changes in antigenicity or receptor usage and has been validated in previous studies of the Veljkovic group. A key study was the validation of mutations in the hemagglutinin sequences of H5N1 Influenza viruses predicted by ISM to alter receptor specificity in functional in vitro studies by Schmieret al. (2015), which should be mentioned in the introduction.The article by Veljkovic Here this method is applied to analyze more than 1600 non-redundant sequences of the spike glycoprotein of the SARS-CoV-2 actually circulating and deposited in the GISAID database over time to derive their immunological profiles and to detect potential escape from the actual vaccine strains. The derived phylogenetic tree uncovers 2 major clusters corresponding to the Wuhan-Hu1 reference group and a derived D614G mutant covered by the actual vaccines and 3 minor clusters representing 22% of sequences, of which vaccine coverage is not known. These results nicely show how potential candidates for vaccine escape mutants can rapidly be identified by the ISM method, which can then be proven experimentally. The authors also identified by ISM a subgroup of mink SARS-CoV-2 sequences transmitted to humans in Denmark, which represent outliers compared to the actual vaccine strains and should thus be watched carefully in the future. Overall, this paper nicely underlines the value of the ISM method, which can rapidly be performed on a huge set of related sequences to identify potentially "dangerous" outliers, which may be functionally altered in terms of virulence or antigenic escape. This sets the ground to focus experimentally on these outlier strains for functional implications of the predicted mutations, thus limiting long-lasting experimental work to essential strains. As such, the manuscript potentially contributes essentially to the control of the most acute viral health problem we are actually facing.  The paper is generally well written, minor linguistic issues should be corrected in the manuscript.Minor issues: Abstract: "....most vaccines currently in clinical applications or under development"; "Here we present computational analysis of more than...."; "effective against most of currently circulating SARS-CoV-2 viruses. However, further monitoring....".Introduction:  "...in silico analysis of SP, which can be performed much more rapidly and with minimal experimental knowledge at the time of analysis. Furthermore, in a proof of principle study, it was shown that HA mutations predicted in silico to alter avian receptor specificity of H5N1 Influenza A viruses indeed result in enhanced specificity for human receptors .Results: In Fig. 2 it should be indicated, what percentage of sequences has been assigned to the different groups . Also in Table 1, it should be indicated in an additional column, what percentage of sequences belongs to the minor groups  over the months to detect a potential increase over time.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?Not applicableAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Virology, HIV-1, Influenza Viruses, vaccine development, virus entry, antigenicityI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Taxonomy illustrates that natural creatures can be classified with a hierarchy. The connections between species are explicit and objective and can be organized into a knowledge graph (KG). It is a challenging task to mine features of known categories from KG and to reason on unknown categories. Graph Convolutional Network (GCN) has recently been viewed as a potential approach to zero-shot learning. GCN enables knowledge transfer by sharing the statistical strength of nodes in the graph. More layers of graph convolution are stacked in order to aggregate the hierarchical information in the KG. However, the Laplacian over-smoothing problem will be severe as the number of GCN layers deepens, which leads the features between nodes toward a tendency to be similar and degrade the performance of zero-shot image classification tasks. We consider two parts to mitigate the Laplacian over-smoothing problem, namely reducing the invalid node aggregation and improving the discriminability among nodes in the deep graph network. We propose a top-k graph pooling method based on the self-attention mechanism to control specific node aggregation, and we introduce a dual structural symmetric knowledge graph additionally to enhance the representation of nodes in the latent space. Finally, we apply these new concepts to the recently widely used contrastive learning framework and propose a novel Contrastive Graph U-Net with two Attention-based graph pooling (Att-gPool) layers, CGUN-2A, which explicitly alleviates the Laplacian over-smoothing problem. To evaluate the performance of the method on complex real-world scenes, we test it on the large-scale zero-shot image classification dataset. Extensive experiments show the positive effect of allowing nodes to perform specific aggregation, as well as homogeneous graph comparison, in our deep graph network. We show how it significantly boosts zero-shot image classification performance. The Hit@1 accuracy is 17.5% relatively higher than the baseline model on the ImageNet21K dataset. Automatic species classification is a problem of great concern in computer vision and pattern recognition. Visual phenomena in practical scenarios generally follow a long-tail distribution ,2, whichIn recent years, GCN-based zero-shot learning (ZSL) has shown great potential. Similar to the operation of traditional convolution on Euclidean space, graph convolution enables convolutional operations on the data of non-Euclidean structure by introducing Laplacian matrices as convolutional kernels for weight sharing. This approach allows nodes in the graph to share statistical strength, providing ways for knowledge transfer from the known categories to the unknown in ZSL. Specifically, the GCN-based method is accomplished with both the implicit node feature and explicit hierarchical information. Graph convolution transfers the semantic and structural priors of unlabeled data from source domains, and trains a classifier for classifying unlabeled data in the target domain.Existing works have pointed out the limitations of graph convolutional neural networks ,9,10,11.We propose a novel graph encoder, GUN-2A, for performing zero-shot image classification individually or applied as an effective graph encoder in the graph contrastive learning module.We introduce a Structural Symmetric Knowledge Graph for zero-shot image classification. The additional knowledge graph enhances the representative power of the nodes in the embedding space.We propose a graph contrastive learning framework, CGUN-2A. We test it on the most challenging zero-shot image classification dataset, ImageNet-21K, and the result shows that our method significantly outperforms the baseline methods.To balance the insufficient message passing of shallow graph networks and the severe over-smoothing of deep graph networks, we propose GUN-2A and CGUN-2A. We first propose GUN-2A, a graph convolutional network with five graph convolutional layers. Our intuition is that some nodes after shallow GCN aggregation are analogous to their neighbors and should be discarded before deeper aggregation. We design a novel U-Net-like graph convolutional framework adapted to zero-shot learning. In this method, a new top-k graph pooling scheme based on the self-attention mechanism, Att-gPool, is proposed to highlight under-aggregated nodes. The proposed framework introduces additional implicit knowledge to enhance the representation power of the model without destroying the original topological structure of the knowledge graph. Second, we consider that contrastive learning effectively encourages the intra-class compactness for positive sample pairs and inter-class separability for negative sample pairs. This paradigm seems naturally suitable for node-wise representation in a hybrid knowledge graph and, to some extent, resists the over-smoothing problem. In this case, we propose CGUN-2A. We seamlessly integrate the mentioned graph encoder GUN-2A into a novel graph contrastive learning framework, which utilizes a dual knowledge graph—Hierarchy Knowledge Graph (HKG) and Structural Symmetric Knowledge Graph (SSKG)—to generate visual classifiers for unknown categories via a contrastive loss. The proposed method incorporates various priors for global structural information and fine-grained semantic representation. Experimental results show that our proposed method consistently outperforms existing multi-layer GCN models, which validates the effectiveness of the proposed GUN-2A and contrastive learning modules. Our contributions are as follows:With the availability of image sensors, ecologists can easily get access to a large number of ecological pictures or videos based on monitors. However, processing data from surveys remains a major bottleneck in ecology. Deep learning (DL) algorithms have been increasingly used in ecological monitoring. The success of traditional supervised learning methods often depends on a training dataset with a large number of examples. Reference  discusseKnowledge transfer is the key to ZSL, which is used to establish relationships between seen and unseen classes. The relationship can be established through implicit semantic information or explicit hierarchical structure. Early works used semantic information, such as manually defined attributes ,18, wordThe over-smoothing problem has been extensively discussed in previous works, mainly concentrating on node classification tasks. DropEdge  randomlyRecently, contrastive learning has renewed a surge of interest ,37,38 inIn zero-shot learning settings, assume that set In this section, we summarize some preliminary works, mainly including three parts: Graph Convolutional Learning, Graph U-Nets and Graph Contrastive Learning.Graph convolutional layers are the basis for extensive aggregation in graph networks. We employ the random walk regularization Laplacian matrix  as a graWe observed the application of the Graph U-Nets (GUN) proposed in , which aMost of the previous GCN methods accomplish the aggregation of node features with a single knowledge graph, usually a Hierarchy Knowledge Graph (HKG). HKG is constructed by concepts and hierarchical relationships in WordNet, where concepts are transformed into semantic embeddings by a GloVe text model train on the Wikipedia dataset. Recent developments in contrastive learning have shown the great potential of this approach to regularize consistent representations of different inputs. In particular, this method usually works in a self-supervised manner. The work of  introducIn this section, we will start by describing the framework of GUN-2A, as the improved GUN model fitting for ZSL tasks and the fundamental graph encoder utilized throughout the CGUN-2A in our experiments. Then, we explain the attention-based graph pooling (Att-gPool) layer in detail. Last, the improvement of GUN-2A on GUN will be discussed.The complete architecture of the proposed encoder, GUN-2A, is shown in In this section, we explain our attention-based graph pooling (Att-gPool) layer on graph data. The layer closely follows the work of , but theSuppose a graph We take the graph data as input. Given nodes tting of .By calculating the attention coefficients of all nodes, we build the attention coefficient matrix Differences between GUN-2A and GUN: Compared with the semi-supervised node classification task, the zero-shot learning classification task is more like a regression that needs to output real-valued weights for each node in a fine-grained manner. In this case, the previous GUN may not have enough potential to classify every node in the knowledge graph. Our practice in In this section, to begin with, we present the overall framework of CGUN-2A. Next in importance, the Structural Symmetric Knowledge Graph is described for maintaining the dual semantic knowledge for contrastive learning. Once again, we explain how the CGUN-2A is used for ZSL. In the end, differences between DKG  and CGUNBesides the widely used GolVe-encoded knowledge graph nsformer  with thensformer  to buildIn addition to the widely spread hierarchy knowledge graph, we further explore the potential of CLIP  used in In the graph contrastive learning module, the inputs of our module are two topological correlation knowledge graphs,ation of , we intrlearning  is formuarameter  that conOur model builds upon the Contrastive Graph U-Net. Inspired by ,29, the During the inference phase, we use the obtained classifier to replace the last layer of the pretrained CNN model for prediction.Differences between CGUN-2A and DKG: The main distinctions are summarized in three aspects: knowledge graphs as input, graph encoder and loss function. First, we apparently use different dual knowledge graphs. DKG seems to use an HKG and a skip-connected HKG, which can be thought of as a contrast between SGCN and DGP. In our dual knowledge graph, we preserve the original structure of the graph while introducing additional semantic information in a loose coupling manner. Second, based on the proposed graph aggregator GUN-2A, we construct a graph encoder with higher efficiency, which makes full use of the hierarchical information in the knowledge graph. Last, in our task, we want to generate stronger intra-class compactness and inter-class separability for the node representations of the two outputs, q and k. We take the InfoNCE loss, which is designed to have a low value when the query is similar to its positive key and dissimilar to all other keys. We draw a simple comparison of the two modules we mentioned in We performed a comparative evaluation of our experiments on ImageNet , currentConsistent with previous works ,28,29, wThe results of our comparative evaluation experiments on three datasets are summarized in In the test involving seen classes, namely generalized zero-shot learning (GZSL), we add the weights of both seen and unseen classes to the final predictive classifier, and the results are shown in We quantify our two proposed frameworks based on the mean average distance (MAD)  metric. We conduct ablation studies to verify the effectiveness of each component in our proposed method. As is shown in We perform an empirical evaluation to verify that our intuition is correct and that additional hidden layers in GUN-2A indeed aggregate more information from the global structure in the knowledge graph. In this paper, we propose a new zero-shot image classification method in ecological monitoring. The proposed method explicitly exploits the hierarchy among natural creatures and explores multiple relations between different classes for learning the weight of a visual classifier. The proposed encoder GUN-2A achieves broad cross-node knowledge propagation. The introduced structural, symmetric knowledge graph and graph contrastive learning module substantially alleviate the Laplacian over-smoothing problem in a single knowledge graph and enhances the distinguishability of the predicted classifier. To validate the potential of our method in real-world environments, we tested it on several large-scale benchmark datasets. Our experimental results demonstrate the effectiveness of the proposed zero-shot learning framework.


Ultrathin carbon nanomembranes (CNMs) are two-dimensional materials (2DM) of a few nm thickness with sub-nm intrinsic pores that mimic the biofiltration membranes found in nature. They enable highly selective, permeable, and energy-efficient water separation and can be produced at large scales on porous substrates with tuned properties. The present work reports the mechanical performance of such CNMs produced by p-nitrobiphenyl phosphonic acid (NBPS) or polyvinylbiphenyl (PVBP) and their composite membranes of microporous supporting substrates, which constitute indispensable information for ensuring their mechanical stability during operation. Measuring the nanomechanical properties of the ultrathin material was achieved by atomic force microscopy (AFM) on membranes both supported on flat substrates and suspended on patterned substrates (“composite membrane”). The AFM analysis showed that the CNMs presented Young’s modulus in the range of 2.5–8 GPa. The composite membranes’ responses were investigated by tensile testing in a micro-tensile stage as a function of substrate thickness and substrate pore density and diameter, which were found to affect the mechanical properties. Thermogravimetric analysis was used to investigate the thermal stability of composite membranes at high temperatures. The results revealed the structural integrity of CNMs, while critical parameters governing their mechanical response were identified and discussed. Among the different developed technologies ,2, waterinterest ,9,10.latent pores”, at the locations where polymeric bonds in the PET are broken. Since the surface remains continuous, CNMs can still be created on these ion-beamed PET films. The tracks can be subsequently etched into open pores while the CNM, which is chemically inert, is not attacked. In fact, the membrane acts similar to an etch-stop and remains free-standing over the track-etched micron-sized pores in the PET support, while the production of such composite membranes is highly scalable [CNMs are two-dimensional materials (2DMs) created by the cross-linking of self-assembled monolayers (SAMs) of aromatic precursor molecules using low-energy electron irradiation . The thiscalable ,18.Due to the unique characteristics of their anisotropic atomic structure and bonding, 2D materials, such as CNMs, display a wide range of unusual characteristics, while their exploitation relies greatly on the establishment of their mechanical attributes ,21. In cIn the present work, CNMs were suspended over patterned substrates, and their mechanical properties were evaluated quantitatively and directly at the nanoscale by AFM. Additionally, the mechanical properties of CNM-composite membranes supported on track-etched PET substrates were studied by tensile experiments using a micro-test tensile stage as a function of substrate thickness and pore density. The thermal stability of the composite membranes was studied at high temperatures by thermogravimetry. The current study gives insights into the acquired mechanical integrity of nanomembranes before and after their suspension over patterned substrates, thus highlighting the importance of a transfer-free procedure for the creation of nanocomposites for pressure-driven applications, as well as their thermal and mechanical stability as polymeric 2D active layers. The importance of the current work relies upon the use of AFM to collect quantitative and qualitative data on the mechanical properties of these nanomembranes, to offer a wide picture of their mechanical behavior more easily and with high accuracy. It could be shown that CNM-composite membranes have mechanical properties sufficient for pressure-driven water separation processes.−5 mbar) with low-energy electrons of 50–500 eV at a dose of 50–100 mC/m2, which yields a highly cross-linked CNM that is mechanically, thermally, and chemically stable, and can be transferred from the initial substrate to virtually any other substrate, including porous ones, without collapsing. In this study, two types of membranes were prepared; namely, NBPS-CNMs were created by cross-linking self-assembled monolayers of p-nitrobiphenyl phosphonic acid (NBPS)   precursod (NBPS)  on SiO2 CNM-composite membranes were prepared directly on top of PET substrates according to the following transfer-free recipe ,25. PET 2/Si substrates as well as Si3N4 substrates perforated with 0.8 μm holes for testing the membranes in a suspended state. The transfer process was assisted by a sacrificial PMMA layer which stabilized the ultrathin CNM [2/Si growth were performed by etching the SiO2/Si growth substrate, which was achieved in hydrofluoric acid.As reliable nanomechanical measurements on nanometer-thick membranes in the AFM require flat-ideally, atomically flat-supporting surfaces , CNMs wethin CNM ,27; majothin CNM ). Transf® instrument residing in a noise-isolation chamber. Peak force-quantitative nano-mechanical (PF-QnM) mapping was used for the investigation of nanomechanical properties. The applied force was carefully selected so that the deformations occurring during the scan were purely elastic. PF-QnM offers the ability to acquire and analyze individual force curves from each tap on the material under investigation during the imaging process and extract the local mechanical properties [R = 8 nm, the spring constant of k = 37 N/m, and resonance frequency of f = 320 kHz were used for topographic imaging. To obtain the optimal visualization of even the finer sample features, the forces exerted were kept to the lowest level possible. Nanomechanical data were also acquired and correlated to topography. To allow the acquisition of quantitative nanomechanical properties, the tip had to be calibrated for deflection sensitivity on a sapphire standard, while the Sader method was used for the force constant [All AFM characterizations were performed under ambient conditions in a Bruker Dimension Iconoperties . To sepaconstant . The samTM micro-tensile stage, which is especially designed for measuring the mechanical properties of thin films [The mechanical response of neat PET substrates and composite membranes in tension was recorded by corresponding testing on a Debenin films . The sta® instrument . Small pieces of the composite membranes were heated at 550 °C in a nitrogen atmosphere at a rate of 10 °C/min.The performance of polymeric materials, such as the substrate of the composite membranes, which constitutes the largest portion of the materials’ volume, was strongly influenced by the operational temperature and thermal transitions in the material. To investigate the thermal stability of the produced composite membranes at high temperatures, thermogravimetric analysis (TGA) experiments were performed in a TA Q502/Si and patterned Si3N4 substrates for topographic assessment and the measurement of mechanical properties at the nanoscale by AFM. Topography was obtained with both the contact and tapping mode AFM measurements, and the results showed good repeatability. 2/Si substrate were evident in all acquired channels and could be easily distinguished from the CNM due to their difference in nano-mechanical properties  and the outside of the hole exhibit similar values. Young’ s modulus of the active CNM layer of the composite membrane ranged within 2.5–3 GPa. Similar behaviors were recorded for suspended PVBP CNMs, while those membranes of Young’s modulus were found to vary around a mean of 6 GPa. As evident from the mappings, the outer ring of the suspended material displays a different mechanical performance than the suspended region. This effect can be rationalized by the unequal tension applied by the rim to the suspended membrane, which differentiates it mechanically from the inside of the “drum”. Tip-sample adhesion appearing lower at the top-right regions inside the “drum” in The very high modulus of elasticity that was found for NBPS (8 GPa) and PVBP (6 GPa) compared with common polymers can be attributed to their molecular structure. NBPS  and PVBPAs entailed from the AFM results of transferred CNMs, the transferring process on top of already-patterned substrates induce points of failure in the material system. In fact, this was the primary reason behind the need for the development of a transfer-free manufacturing process of CNM-composite membranes. Typical topographies of the front and back sides of the composites are shown in By observing 6 pores/cm2 to 1.5 × 106 pores/cm2, did not appear to affect the mechanical performance of the material albeit a slight effect only on the plastic region of the curves (The effect of the sample thickness on mechanical performance is presented in e curves b. The typical TGA response of composite membranes and the PET reference sample is presented in Two types of CNMs as model systems for pressure driven membrane water separation applications, each originating from different precursor molecules, NBPS and PVBP, were synthesized and tested in terms of their mechanical stability. NBPS-CNMs resemble, regarding their water separation performance, TPT-CNMs, which have been shown to be highly permeable for water while exhibiting an extraordinarily high rejection of organic molecules, anions, and cations; PVPB-CNMs are structurally similar to the active CNM-layer of our CNM-composite membranes ,15. AFM


Transplantation is an effective approach for treating end-stage organ failure. There has been a long-standing interest in xenotransplantation as a means of increasing the number of available organs. In the past decade, there has been tremendous progress in xenotransplantation accelerated by the development of rapid gene-editing tools and immunosuppressive therapy. Recently, the heart and kidney from pigs were transplanted into the recipients, which suggests that xenotransplantation has entered a new era. The genetic discrepancy and molecular incompatibility between pigs and primates results in barriers to xenotransplantation. An increasing body of evidence suggests that innate immune responses play an important role in all aspects of the xenogeneic rejection. Simultaneously, the role of important cellular components like macrophages, natural killer (NK) cells, and neutrophils, suggests that the innate immune response in the xenogeneic rejection should not be underestimated. Here, we summarize the current knowledge about the innate immune system in xenotransplantation and highlight the key issues for future investigations. A better understanding of the innate immune responses in xenotransplantation may help to control the xenograft rejection and design optimal combination therapies. The inadequate supply of human organs for allografting has prompted interest in xenografts. Xenotransplantation is one of the most attractive strategies for alleviating the critical worldwide shortage of donor organs for clinical transplantation. Therefore, pig-to-human xenotransplantation has become a promising field of research to overcome this problem. There are three main types of immune rejection after xenotransplantation. Hyperacute rejection (HAR) occurs within minutes or hours. An acute humoral xenograft rejection (AHXR) evolves within days or weeks, causing organ xenograft dysfunction. Additionally, an acute cellular rejection is another common type of allogeneic rejection . In addiThe first major obstacle to xenotransplantation is the hyperacute rejection of the transplanted organ due to natural antibodies. After that, cellular immunity, including T and B lymphocytes, natural killer (NK) cells, and macrophages may become involved as well. Currently, because of the strong immune response to xenografts, success will probably depend upon the new strategies for immunosuppression.Histopathological studies have demonstrated that the mechanisms involved in xenogeneic graft rejection are significantly different from those associated with allogeneic graft rejection. Cytotoxic T lymphocytes may play a major role in allograft rejection, whereas xenografts mainly induce the infiltration of NK cells and macrophages ,3. ConseInnate immunity is an important system in animals and humans that is precisely regulated and used to maintain homeostasis and prevent a microbe invasion. Innate immunity is coordinated by immune cells, natural antibodies, and complements, along with cytokines and chemokines secreted by innate immune cells under physiological and pathological conditions ,5. The aThe reviews published in the past few years mainly discuss the functions of the adaptive immune system and the initiation of the allo-immune response in general. The current armamentarium of immunosuppressive drugs is primarily designed to limit the adaptive immune response. The role of innate immunity as a significant driver of the xeno-immune response is increasingly recognized. However, only a few foci were placed on the immune effects and immune treatment strategies of the innate immune system after xenotransplantation. In this review, we summarize recent advances in the role of the innate immune system in xenotransplantation, as well as the innate immune system-based immunotherapies strategies to overcome xenogeneic rejection.As we know, the complement system still plays a vital role in xenogeneic rejection . ComplemThe activation of a complement is controlled in part by complement regulatory proteins (CRPs) that are the integral components of cell membranes. EC activation in the xenograft setting is a key step in the pathophysiology of AVR, and the data suggest that the classic complement pathway plays a major role in the human serum-induced activation of porcine endothelial cells (ECs) . The firThe inhibition activation of the complement system can prevent the occurrence of HAR in pig to non-human primate (NHP) xenotransplantation. The complement regulatory proteins play a role in a species-specific manner to discriminate between the self and non-self to prevent the over-activation of the complement system and complement-mediated damage. These proteins include the decay-accelerating activator , the membrane inhibitor of reactive lysis (CD59), and the membrane cofactor protein . CD46 is a cofactor for the factor I-mediated inactivation of C3b and C4b . Human cChronic rejection is also a potential cause of clinical allograft failure. Indeed, xenograft vasculopathy has been reported in the hearts transplanted from hCRP transgenic pigs to primates, which was considered to be a characteristic feature of chronic rejection ,24. The Studies have shown that dextran sulfate (DXS), which inhibits the classical and alternative complement pathway, can delay hyperacute rejection in xenotransplantation models ,30. SimiNatural antibodies (Nab) are produced early in life and constitute life-long high titers of circulating antibodies. Most complement-fixing xenoreactive Nab in humans is directed against αGal, a saccharide expressed in the cells of lower mammals . The αGaIn pig-to-primate organ transplantation, HAR can also be prevented by depleting the recipient of the anti-αGal or anti-non-Gal antibody . SeveralThe addition of multiple hCRPs and HT to GTKO further reduced the incidence of initial graft dysfunction by constraining the amplification of the antibody-mediated classical complement cascade, inhibiting the formation of the MAC, and better preventing an endothelial injury relative to either hCPRP or GTKO alone ,40. In aAt the same time, the study has shown that by supplementing the primate blood coagulation components, massive hemorrhage in the setting of a liver xenotransplantation can be avoided . In 2017The primary interaction of macrophages with human cells appears to be mediated by lectins expressed by the human macrophages, which are stimulated by endothelial cell saccharides . Once stThe first indications of the involvement of macrophages in xenorejection came from studies demonstrating that the cellular infiltrates in rejected xenogeneic heart grafts in hamster- or guinea-pig-into-rat or pig-into-baboon models contained a prominent macrophage population ,56,57. AThe macrophages activated by T cells were attracted from the peripheral circulation and were capable of the specific targeting and destruction of porcine islet grafts . O’ConneThe pathways by which macrophages recognize and destroy xenogeneic tissues are gradually being discovered. Macrophages can be activated through Fc receptors (FcRs) and complement receptors, and hence participate in a complement activation and the xenoreactive natural antibody-dependent mechanisms of xenograft rejection . Signal-In addition, studies suggested that the decreased clearance of porcine cells in primates may be achievable by the transgenic expression of primate CD47. For example, the transgenic expression of human CD47 (hCD47) on porcine cells substantially increased the duration of the transient xenogeneic chimerism following the transplantation of hematopoietic cells (HCs) to baboons . SimilarBy contrast, thrombospondin 1 (TSP-1) is an adhesive glycoprotein that mediates cell behavior by engaging with molecules in the extracellular matrix and with receptors on the cell surface and contributes to platelet aggregation. CD47 is a high-affinity TSP1 receptor. TSP1-CD47 signaling has been indicated to accelerate the rejection of vascularized allografts . TSP-1 aCD200 is another inhibitory molecule involved in the regulation of macrophage proinflammatory immune responses. CD200 induces an immunosuppressive function through the binding to CD200R, which contains an inhibitory intracellular Asn-Pro-X-Tyr (NPXY) signaling motif. Human CD200 on porcine cells was found to suppress macrophage-mediated phagocytosis and cytotoxicity. Yan et al. demonstrated that the overexpression of CD200 in pECs suppressed the xenogeneic immune response of human macrophages and improved the survival of pEC xenografts in humanized mice . ExpressThe surfactant protein (SP)-D molecules recognize PAMPs on microorganisms to induce innate immunity and contribute to the pathogen removal and pro-inflammatory responses to infection. SP-D can also bind SIRPα, decreasing the cytokine production by monocytes/macrophages. Jiaravuthisan et al. prepared cDNA for the membrane-type protein, collectin placenta 1 (CL-P1) with the C-terminal heads of SPD, and transfected it into EC, then found that this molecule was able to suppress macrophage-mediated xenobiotic cytotoxicity . In addiCD33-related sialic acid-binding immunoglobulin-like lectins (Siglecs) belongs to the immunoglobulin superfamily that typically exerts their functions by binding to sialylated ligands ,77. HumaEvidence indicates that the mechanism of the cytotoxicity of NK cells to xenogeneic cells has at least three separate pathways . First, NK cells can destroy xenogeneic tissues through direct lysis and ADCC. AHXR is characterized by multiple microvascular thrombosis and massive NK-cell and macrophage infiltration. Additionally, the results of the study of Chen et al. indicated that in the absence of intravascular thrombosis, xenograft rejection in the mouse-to-rat heart transplantation model is mediated by NK cells . In addiNK cells are tightly regulated through the signals mediated by inhibiting and activating receptors expressed on their cell surface. Many inhibitory NK receptors recognize MHC class I molecules, and in the absence of sufficient inhibitory signals, the binding of activating receptors induces NK cytotoxicity  Figure . The in In addition to a direct NK cell interaction with the vascular endothelium leading to an endothelial injury, NK cells can migrate to the graft, providing cross-species interactions between NK cells, and pEC-related adhesion molecules can occur. Here, it is worth noting that the lack of Gal on the ECs on GalT-KO pigs resulted in a significant reduction in complement-mediated lysis and ADCC, but did not eliminate NK cells adhesion and direct anti-porcine NK cytotoxicity, indicating that αGal is not a major target for direct human NK cytotoxicity against porcine cells . There iAnalyzing adhesive interactions between purified hNK cells and pECs under static conditions by the blocking of common adhesion receptors demonstrated the involvement of CD2, CD11a (LFA-1 integrin-αL chain), CD11b (Mac-1 integrin-αM), CD18 (integrin-β2), and CD49d (VLA-4 integrin-α4) on hNK cells and CD106 on pEC . After bThe activating receptor NKG2D has been found to be capable of mediating human anti-pig NK cytotoxicity. ULBP1 is the primary, functional porcine ligand for human NKG2D, which plays a critical role in the cytotoxic lysis of pECs by hNK cells through the recognition of ligands ULBP1. Lilienfeld et al. indicated that using anti-ULBP1 polyclonal completely blocked hNK cytotoxic lysis against pEC .Further, an investigation tested a range of NK receptors, including NKp46, 2B4, CD49d, CD48, CD2, and NKG2D, and found that only CD2 and NKG2D were involved in the cytotoxicity and cytokine (IFN-γ and TNF-α) release against porcine cells . It furtHLA-E and HLA-G are immunoregulatory molecules that belong to the HLA-Ib family. The main function of HLA-Class Ib molecules is to regulate the immune response by interacting with the specific inhibitory receptors expressed on different immune effector cells . PreviouCD154, an inflammatory mediator also known as CD40 ligand, has been identified as a novel binding partner for some members of the integrin family. Interleukin (IL)-2-activated human NK cells can express CD154 (CD40 ligand), which is an inflammatory mediator of the TNF family. NK cells also interact with marginal zone B cells in the spleen via the CD40-CD154 pathway and the production of T cell-mediated αGal-independent antibodies, thereby facilitating antibody-mediated rejection . TherefoIt has been shown that prenylated quinolinecarboxylic acid-18 (PQA-18), a novel immunosuppressant, could suppress the production of IL-2, IL-4, IL-6, and TNF-α production in human peripheral lymphocytes . In addiTaken together, the implementation of strategies to inhibit NK cytotoxicity and other yet to be defined NK cell responses, such as the cytokine release and enhancement of adaptive immune responses, may promote both a successful clinical xenotransplantation. Paradoxically, despite the above studies elucidating a role for NK cells in rejection, in some animal models, they are also involved in immune regulation and are necessary for allograft tolerance . SpecifiNeutrophils are generally viewed as nonspecialized effector cells that are recruited into tissues as a response to inflammatory mediators and through the formation of C3bi on foreign cell surfaces. Indeed, there may also exist primary interactions between neutrophils and xenogeneic cells that might precede inflammation. For example, human neutrophils attach to pECs and activate these cells independently of xenoreactive antibodies, complement, or other plasma mediators . NeutropActivated neutrophils can induce xenograft damage primarily by the production of reactive oxygen species (ROS), the release of tissue digestive enzymes, or neutrophil extracellular traps .The complement is thought to increase the adhesion of neutrophils . The incThe activation of human neutrophils by pECs is independent of αGal structures . Human nThe cluster of differentiation 31 (CD31), also known as the platelet EC adhesion molecule 1, is a member of the Ig-ITIM family and is expressed on hemopoietic and ECs where it functions as a homophilic adhesion and signaling receptor . Recent TLRs are key PRRs of the immune system required to initiate an effective innate immune response at an early stage of infection, recognizing PAMPs or danger-associated molecular patterns (DAMPs) ,110. TLRA serious ischemia/reperfusion (I/R) injury is the main relevant factor in solid organ transplant failure. TLRs are activated by endogenous ligands from damaged/stressed cells in an I/R injury. The activation of TLRs-carrying cells triggers the release of inflammatory cytokines and chemokines as well as the recruitment of macrophages, neutrophils, and T cells, causing a full-blown I/R injury. Additionally, neutrophils are the major leukocytes found in I/R injury and promote the progression of I/R tissue damage.A TLR4 activation can signal through a MyD88-independent pathway . In the MyD88-independent pathway, TLR4 may recruit the TRIF-related adaptor molecule (TRAM) and TRIF which activates TBK1, thus allowing the activation of interferon-regulatory factor-3 (IRF3). This pathway may also signal through the signaling of NF-κB through the recruitment of TRAF6 recruitment. The activation of the TRIF-dependent pathway induces IFN-β and increases inflammatory cytokine gene expression, causing damage to xenografts .TLR2 and TLR4 trigger the same pathway, the MyD88 pathway, resulting in the release of NF-κB from IκB, which subsequently stimulates the expression of pro-inflammatory cytokines and pro-apoptotic molecules within the recruited immune cells ,114 Fig. FurtherThe activation of TLR in porcine islets induced both a pro-inflammatory and procoagulant response and thereby contributed to a xenograft rejection . In addiThe interaction of transmembrane receptor TLR with a high mobility group box 1 (HMGB1) has been the focus of research into the pathogenesis of inflammatory diseases and cell damage ,122. HMGIn the last months, different teams have performed clinical transplantations of porcine organs to humans. It was a major milestone in organ transplantation. At the end of 2021, the first kidney xenotransplantation of brain-dead patients was performed by the Robert Montgomery team from New York University (NYU) . The kidFurther work was done on the first heart xenotransplantation. The University of Maryland Medical Center (UMMC) has received the food and drug administration (FDA) approval to perform xenotransplantation in a patient with heart failure without liver or kidney failure. The group conducted a transplant of a porcine heart into a 57-year-old patient, whose immunosuppression was based on a CD40 blockade . The heaXenotransplantation has not yet been fully developed clinically, mainly because xenoreactive antibodies remain the cause of graft failure . SeveralIn addition, anti-CD154 mAb is one of the main therapies used in cardiac or renal pig to NHP transplantation and helps to significantly prolong solid-organ survival ,138,139.Furthermore, a tolerance induction by mechanisms such as mixed chimerism and thymus transplantation has been explored as a way to overcome the xenogeneic immune barrier.A tolerance induction with combined renal and hematopoietic cell transplantation has been used in the clinical field. Researchers from Stanford University injected donor hematopoietic stem cells with a fixed number of donor T cells after the kidney transplantation . This haThe use of a xenoantigen reduction method without the use of transgenes that interfere with the ability of the recipient’s immune system to interact with the xenograft preserves protective immunity within the xenograft intact, allowing the recipient’s immune system to be protected from harmful antibodies in the event of an infection. Taken together, since xenoreactive antibodies are still the cause of xenograft failure, only recipients with negative crossmatches are considered for an initial transplantation in xenografts, which is essential to achieve an extended xenograft survival in human patients.Knowledge of the role of innate immunity in xenograft rejection is rapidly evolving. In most organs, an I/R injury is initiated by the activation of natural antibodies and complements. Moreover, a cellular xenogeneic rejection has also become an important immune barrier to overcome in the clinical use of xenogeneic organs. It is emphasized that innate immunity by macrophages, NK cells, and neutrophils causes severe rejections during xenotransplantation.Activated innate immune cells destroy allografts not only through direct cytotoxicity but also by promoting the initiation of T cell responses against the graft. The rejection of allografts by the innate immune system involves multiple effector mechanisms that cannot be prevented by a single approach. Our understanding of the mechanism of action of innate immunity to xenoantigen helps to identify the molecules used for the genetic manipulation in donor pigs to suppress the innate alloimmune responses. For example, adding complement regulatory proteins, along with the regulation of coagulation factors, appears to be the basis for a successful xenotransplantation. Furthermore, the transgenic expression of HLA molecules and hCD47 was used to suppress a xenograft rejection by NK cells and macrophages, respectively, and prolong the survival time of the xenograft organs. However, simply the inhibition of antibody-and receptor-mediated xenograft immune responses in animals may not be sufficient without considering the other factors.In addition, innate immune cells are required for an acute and chronic rejection in certain animal transplant models and by extension perhaps in clinical transplantation. Therefore, the development of strategies for suppressing innate immune cells has considerable potential for the practical applications of xenotransplantation. Activated innate immune cells destroy grafts not only through a direct cytotoxicity but also by promoting the initiation of T cell responses against the graft. Consequently, the tight control of innate immunity such as macrophages, to control the movement of lymphocytes, will be important as the next step in the era of xenotransplantation is realized.HMGB1 has since been revaluated as a classic DAMP protein that activates various graft cells, leading to dysregulated inflammatory responses and macrophage depletion ,164. RecSo far, this genetic engineering selectively addresses only major interspecific molecular differences, such as eliminating highly immunogenic epitopes, modulating the complement expression, or blocking major immune cell activation pathways. It is worth noting that an interspecific blastocyst complementation and the in vivo generation of organs derived from xenogeneic donor pluripotent stem cells have potential . The Nak


Identification of tumor cell surface targets is vital for chimeric antigen receptor-T cell (CAR-T) therapies and antibody drug conjugates (ADCs). This study utilized the Cancer Genome Atlas (TCGA) database to perform a series of conditional screenings of tumor-specific surface proteins. Accordingly, we found a tumor tissue-specific gene set associated with the survival of cancer patients. Furthermore, these tumor-specific surface proteins can function to render the ability of tumor cells to metastasize. Correlation analysis revealed that these overexpressed membrane proteins were positively correlated, which suggests they maybe potential dual-drug targets. Our findings reveal the significance of tumor cell surface targets in CAR-T- and ADC-related drug development.Tumor cells can be recognized through tumor surface antigens by immune cells and antibodies, which therefore can be used as drug targets for chimeric antigen receptor-T (CAR-T) therapies and antibody drug conjugates (ADCs). In this study, we aimed to identify novel tumor-specific antigens as targets for more effective and safer CAR-T cell therapies and ADCs. Here, we performed differential expression analysis of pan-cancer data obtained from the Cancer Genome Atlas (TCGA), and then performed a series of conditional screenings including Cox regression analysis, Pearson correlation analysis, and risk-score calculation to find tumor-specific cell membrane genes. A tumor tissue-specific and highly expressed gene set containing 3919 genes from 17 cancer types was obtained. Moreover, the prognostic roles of these genes and the functions of these highly expressed membrane proteins were assessed. Notably, 427, 584, 431 and 578 genes were identified as risk factors for LIHC, KIRC, UCEC, and KIRP, respectively. Functional enrichment analysis indicated that these tumor-specific surface proteins might confer tumor cells the ability to invade and metastasize. Furthermore, correlation analysis displayed that most overexpressed membrane proteins were positively correlated to each other. In addition, 371 target membrane protein-coding genes were sifted out by excluding proteins expressed in normal tissues. Apart from the identification of well-validated genes such as GPC3, MSLN and EGFR in the literature, we further confirmed the differential protein expression of 23 proteins: ADD2, DEF6, DOK3, ENO2, FMNL1, MICALL2, PARVG, PSTPIP1, FERMT1, PLEK2, CD109, GNG4, MAPT, OSBPL3, PLXNA1, ROBO1, SLC16A3, SLC26A6, SRGAP2, and TMEM65 in four types of tumors. In summary, our findings reveal novel tumor-specific antigens, which could be potentially used for next-generation CAR-T cell therapies and ADC discovery. Cancer is the second leading cause of death all over the world with 9.96 million deaths every year . AlthougNowadays, cancer immunotherapies. such as immune checkpoint blocking therapy (ICB) have been introduced into multiple lines of cancer treatment with great success . RecentlHere, we exploited the publicly available Cancer Genome Atlas (TCGA) databases to identify specific TSAs in various cancer types. Firstly, we identified highly expressed genes present on the tumor cell surface. Secondly, we performed a survival analysis to select genes that were significantly associated with survival outcomes in cancer patients. In addition, we analyzed the function and correlation of these genes and the prognostic value of the correlated genes with survival rates of cancer patients. To sum up, our work identified specific TSAs that might serve as useful targets for CAR-T cell therapies, ADCs, or co-targeting strategies for the treatment of solid tumors.https://xenabrowser.net/ (accessed on 1 September 2020)) in fragments per kilobase million (FPKM) (See https://membranome.org/ (accessed on 1 September 2020)) [https://www.uniprot.org/ (accessed on 1 September 2020)) [Gene expression of 17 types of tumor samples were collected from TCGA on the University of California Santa Cruz (UCSC) Xena website (r 2020)) ,28 and Ur 2020)) . Human ir 2020)) . The RNAr 2020)) .The protein expression abundance data of a total of 7 types of cancer tissues were obtained from the CPTAC database. The detailed cancer types and sample numbers are shown in p value was less than 0.01.A rank-sum test was used to analyze the differential expression of membrane protein-coding genes between tumor and normal samples. A threshold of Log2FoldChange (Log2FC) was greater than 1.00 and the adjusted Taking into account the missing values of protein abundance in the CPTAC data, we first deleted genes whose expression was missing in more than 10% of samples of the tumor , and then filtered the missing values through the K-nearest neighbor method (k = 10). Finally, the relative normalized protein expression abundance profiles of 7 cancers were obtained. Since the protein abundance levels were normalized and log transformed, the difference in the expression abundance was calculated as the abundance in the tumor tissue minus the abundance in the normal tissue via the rank-sum test.A hypergeometric test was used to analyze the enrichment relationship between high-expression and high-risk membrane protein-coding genes in the tumor tissues according to the ten cancer hallmarks .The tumor patients were divided into a high-expression group and a low-expression group by the mean value of membrane protein expression. A log-rank test was used to compare the survival rates of these two groups of patients using the R package “Survfit” .Multivariate Cox regression was used to analyze the impact of membrane protein pairwise combinations on the survival rates of the cancer patients. Hazard ratios (HR) of each membrane protein based on the expression levels of the protein in the sample and the prognostic information of the patient were analyzed through univariate cox regression. Among them, the genes with HR > 1.00 were considered to be poor prognostic factors for the cancer patients.exp) multiplied by its regression coefficient (β) obtained from the univariate Cox regression model. Each patient’s prognostic risk-score was calculated as the sum of two gene scores; the formula is as follows [Each gene score was constructed as the selected gene expression level RiBased on this formula, the risk-score of each sample was calculated . AccordiWe analyzed the correlation between every two membrane proteins by Pearson correlation. The visualization process was depicted using the R package “corrplot”.To analyze the TSAs in tumor tissues, we first identified the membrane protein-coding genes with up-regulated expression levels through a series of pan-cancer screenings. Firstly, the expression profiles of mRNA were obtained from the TCGA database by excluding the data from the cancer types with less than three normal controls A. SecondIn the first step of the analysis, we found that 3919 membrane proteins were differentially expressed in most tumor samples, compared with their corresponding adjacent normal tissues . SpecifiTo further explore the impact of these highly expressed membrane proteins of the tumor tissues on the prognosis of the tumor patients, Cox risk regression analysis of these genes was applied to the patients from TCGA . AdditioTo gain deeper insight into the potential functions of these selected membrane proteins, we comprehensively analyzed their functions according to the ten cancer hallmarks, such as invasion and metastasis, etc. . MembranSEZ6 and ULBP1, ULBP2 and MAFA, and PCDHD1 and MAFA, compared with the results when the genes were individually analyzed.To further investigate the combinatorial effects of these genes on the survival of the cancer patients, we established a risk scoring system using a formula containing the gene expression levels and the regression coefficients from the univariate Cox regression model . We founTo investigate whether these membrane proteins were correlated, we calculated the association among the membrane proteins in the 17 tumor types through a Pearson’s correlation test. As shown in To further investigate the potential “on-target, off-tumor toxicity” effect of these proteins, we obtained the expression levels of the 371 target membrane protein-coding genes See . We defiTo further validate our above findings, we checked the protein expression levels of the obtained genes from the CPTAC tumor protein database . We identified the expression levels of 23 proteins in four types of tumors  . It was The identification of antigens specifically expressed on the surface of tumor cells is vital for the design of adoptive T cell therapy and ADCs. Our study found that there were various highly expressed membrane proteins in a variety of tumor types (17 cancer types), which were also significantly associated with the survival rate of patients. These findings expand the recent document that identified 200 genes as breast cancer subtype-specific targets by differential expression analysis of RNA-seq data from TCGA .In our discovery, 184 genes were lowly expressed in normal tissues, which further supports the advantage of our strategy in identifying potential targets. Some of them were proved to be successful targets with significant curative effects on some malignant hematological tumors, such as anti-CD19 CAR-T therapy for the treatment of chronic lymphocytic leukemia , and antOur enrichment analysis identified the relationship of these highly expressed surface proteins and the human immune status, which has a great advantage over the findings from Schreiner et al. Although they could predict the surface antigens of several hematological tumors which may be more applicable across cancer types, they did not exclude the potential adverse effects on immune cells, which may dampen the efficacy of the targets discovery . For exaCorrelation analysis further demonstrated that the surface proteins with poor prognosis were significantly correlated with each other, which suggests that the effects of the surface proteins on tumor progression may not work independently but coordinate with each other. In general, the paired target membrane proteins could be good potential candidates for dual-target CAR-T therapy and ADCs with fewer side effects .Since the selected surface protein-coding genes were closely associated with each other, we speculated that the selected surface proteins could share a similar transcription pattern. When choosing specific types of surface proteins as drug targets, the off-targets side effects may be prevented by controlling the activation of engineered T cells using integrated multi-input signals ,73. BispIn spite of the limited data in the protein database, we still validated the protein expression levels of some of the selected genes. These results make it more convincing that the identified tumor surface protein genes may be potential targets for CAR-T cell therapies and ADCs. Further integration of proteomic information may boost the discovery of TSAs because the post-modification of proteins such as glycosylation, and lipidation, have also been identified as a source of tumor surface antigens by liquid chromatography–mass spectrometry ,77.In sum, our study revealed some potential tumor-specific surface proteins for the rational design of TSA-based immunotherapies. These findings might pave the way for a comprehensive and efficient approach to construct novel CAR-T cells and ADCs in pre-clinical animal studies and clinical practice by utilizing tumor-specific surface proteins as multi-target binding sites.


Glaucoma is a group of eye diseases afflicting more than 70 million people worldwide. It is characterized by damage to retinal ganglion cells (RGCs) that ultimately leads to the death of the cells and vision loss. The diversity of RGC types has been appreciated for decades, and studies, including ours, have shown that RGCs degenerate and die in a type-specific manner in rodent models of glaucoma. The type-specific loss of RGCs results in differential damage to visual and non-visual functions. One type of RGC, the intrinsically photosensitive retinal ganglion cell (ipRGC), expressing the photopigment melanopsin, serves a broad array of non-visual responses to light. Since its discovery, six subtypes of ipRGC have been described, each contributing to various image-forming and non-image-forming functions such as circadian photoentrainment, the pupillary light reflex, the photic control of mood and sleep, and visual contrast sensitivity. We recently demonstrated a link between type-specific ipRGC survival and behavioral deficits in a mouse model of chronic ocular hypertension. This review focuses on the type-specific ipRGC degeneration and associated behavioral changes in animal models and glaucoma patients. A better understanding of how glaucomatous insult impacts the ipRGC-based circuits will have broad impacts on improving the treatment of glaucoma-associated non-visual disorders. Glaucoma affects more than 70 million people worldwide and causes 10% of those afflicted to be bilaterally blind, making it one of the main causes of blindness . GlaucomSince their discovery, a plethora of studies have been published on ipRGCs, and we now know that this group of cells is heterogeneous  open reading frame  of the IPL. Their somata are located in the GCL and significantly larger than the M1 somata . They arCentral targets of ipRGCs have been described in macaque . Using aCre±;EYFP+/+Opn4 mouse line exhibited a uniform density, but M1 and M2 ipRGC types labeled by melanopsin antisera showed a superior-inferior gradient with almost one-fold difference. The M4 and M5 cells collectively exhibited the reverse gradient, where significantly higher density was found in the inferior retina , where the axons bundles exit the eye . This stThe optic nerve crush (ONC) and optic nerve transection (ONT) models have been used to study rapid RGC degeneration. ipRGCs have been found to survive better than general RGCs in ONC and ONT models . In miceThough an elevated IOP is associated with a majority of glaucoma cases, some patients do not exhibit elevated IOP , suggestIndeed, many factors may contribute to ipRGC survival in glaucoma. In various axonopathies where axons degenerate in a manner whereby they die back, longer axons tend to suffer more damage or die more quickly than shorter ones, possibly due to higher metabolic demands . While tIntrinsically photosensitive retinal ganglion cells may also receive more trophic support than other RGC types in a diseased condition. Neurotrophic factors (NTFs) are diffusible molecules that bind tropomyosin related kinase (Trk) receptors and p75 receptors (p75NTR) [reviewed by Studies have begun to characterize the downstream signaling pathways of ipRGC survival in glaucoma. For example, ipRGCs maintain a relatively high level of mTOR activity, which is associated with axon regeneration . This hiIt is known that the majority of retinal afferents to the SCN, arises from M1 ipRGCs , suggestAs degeneration of ipRGCs including M1s are also observed in human glaucoma patients  and in oIn normal tension glaucoma (NTG) patients, sleep quality as measured by the Pittsburgh Sleep Quality Index did not differ significantly from controls . This phvia the geniculohypothalamic tract  . TherefoIntrinsically photosensitive retinal ganglion cells are also involved in other non-image-forming visual functions that are affected in glaucoma. Brn3b-positive M1 ipRGCs project to OPN , a regioThough a great number of ipRGC studies focus on the non-image-forming aspects of vision, increasing attention is being directed to the contributions of ipRGCs to image-forming vision. M2, M4, M5, and M6 cells and a small percentage of M1 cells send their axons to the dLGN, the principal relay for visual information to the visual cortex . Such ciIn conclusion, ipRGC-mediated image-forming and non-image-forming visual circuits and behaviors are affected in glaucoma. A better understanding of ipRGC type-dependent degeneration and the corresponding ipRGC circuits that account for the behavioral changes is needed. The results will help to improving patient care and treatment with glaucoma development and progression in the future.All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.