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Background Testicular cancer (TC) is the most frequent type of cancer among young men aged between 15 and 34 years. TC is treated using cisplatin, but 3%–5% of TC patients fail to respond to cisplatin, with a very bad to fatal prognosis. Accordingly, it is most important to quickly and readily identify those TC patients who are resistant to cisplatin treatment. Methods This study seeks to investigate changes in the glycosylation associated with cisplatin resistance to TC cell lines. Results A specific glycoprofiling of human chorionic gonadotropin (hCG) was analysed in three TC cell lines and one cell line of female origin. A typical calibration curve for hCG glycoprofiling showed a dynamic range up to 50 ng/ml, with a limit of detection of 0.3 ng/ml and assay reproducibility represented by relative standard deviation of 3.0%. Changes in the glycan signatures on hCG were analysed in cisplatin‐sensitive cell lines and in their cisplatin‐resistant sub‐lines using an enzyme‐linked lectin assay (ELLA) protocol. An immobilised antibody was applied to a selective capture of hCG from a cytoplasmic fraction of cell lysates with final incubation using a lectin from a panel of 17 lectins. Conclusion The results suggest that one particular lectin Dolichos biflorus agglutinin (DBA) can selectively discriminate sensitive TC cell lines from resistant TC cell lines. Moreover, there are additional lectins which can provide useful information about the strength of cisplatin resistance.

Apoptosis mediated by Bax or Bak is usually thought to be triggered by BH3-only members of the Bcl-2 protein family. BH3-only proteins can directly bind to and activate Bax or Bak, or indirectly activate them by binding to anti-apoptotic Bcl-2 family members, thereby relieving their inhibition of Bax and Bak. Here we describe a third way of activation of Bax/Bak dependent apoptosis that does not require triggering by multiple BH3-only proteins. In factor dependent myeloid (FDM) cell lines, cycloheximide induced apoptosis by a Bax/Bak dependent mechanism, because Bax-/- Bak-/- lines were profoundly resistant, whereas FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Addition of cycloheximide led to the rapid loss of Mcl-1 but did not affect the expression of other Bcl-2 family proteins. In support of these findings, similar results were observed by treating FDM cells with the CDK inhibitor, roscovitine. Roscovitine reduced Mcl-1 abundance and caused Bax/Bak dependent cell death, yet FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Therefore Bax/Bak dependent apoptosis can be regulated by the abundance of anti-apoptotic Bcl-2 family members such as Mcl-1, independently of several known BH3-only proteins.

In African countries, cancer not only is a growing problem, but also a challenge because available funding and resources are limited. Therefore, African medicinal plants play a significant role in folk medicine and some of them are traditionally used for the treatment of cancer. The high mortality rate and adverse effects associated with cancer treatments have encouraged the search for novel plant-based drugs, thus, some African plants have been studied in recent years as a source of molecules with proven cytotoxicity. This review aims to discuss the cytotoxic activity, in vitro, of African plant crude extracts against cancer cell lines. For the period covered by this review (2017–2021) twenty-three articles were found and analyzed, which included a total of 105 plants, where the main cell lines used were those of breast cancer (MCF-7 and MDA-MBA-231) and colorectal cancer (HCT-116 and Caco-2), which are among the most prevalent cancers in Africa. In these studies, the plant crude extracts were obtained using different solvents, such as ethanol, methanol, or water, with variable results and IC50 values ranging from 200 µg/mL. Water is the preferred solvent for most healers in African countries, however, in some studies, the aqueous extracts were the least potent. Apoptosis and the induction of cell cycle arrest may explain the cytotoxic activity seen in many of the plant extracts studied. Considering that the criteria of cytotoxicity activity for the crude extracts, as established by the American National Cancer Institute (NCI), is an IC50 < 30 μg/mL, we conclude that many extracts from the African flora could be a promising source of cytotoxic agents.

Introduction: Despite promising results following targeted treatment with human epidermal growth factor receptor 2 (HER2)-inhibitors in HER2-positive gastric and esophageal adenocarcinoma (GEA), prognosis remains dismal. Many patients ultimately demonstrate progression following treatment due to resistance to HER2-targeted therapy. Here, we describe the potential primary and secondary resistance mechanisms to HER2-targeted therapy in GEA. Methods: We systematically searched PubMed/MEDLINE, EMBASE, and CENTRAL for eligible studies describing changes that were associated with drug resistance. Study quality was assessed using an adjusted version of the OHAT risk of bias tool. Quality of proposed resistance mechanisms was assessed using predefined criteria. Results: In total, 913 records were screened, of which 73 were included that investigated mechanisms of resistance against anti-HER2 treatment in cell lines, xenograft models, patient tissue samples, and publicly available datasets. HER2-targeted therapy resistance was found to be caused by HER2 receptor changes, upregulation of compensatory receptors, (re)activation of downstream signaling pathways like PI3K/AKT and MAPK, epithelial-to-mesenchymal transition, acquirement of stem cell-like properties, alterations in cell cycle related genes, cellular metabolism, and drug pharmacokinetics. Discussion: Several different mechanisms can contribute to drug resistance to anti-HER2 treatment in GEA, mainly through loss of or mutations in the HER2 receptor and upregulation of alternative receptors such as MET, HER3, and FGFRs. Despite these preclinical results, methods to overcome the proposed resistance mechanisms in the clinical setting are lacking. Therefore, further investigation of therapy resistance in GEA patients treated with HER2 targeted therapy is essential to overcome resistance and improve treatment outcome of these patients.

open access journal The development of reliable predictive models for individual cancer cell lines to identify an optimal cancer drug is a crucial step to accelerate personalized medicine, but vast differences in cancer cell lines and drug characteristics make it quite challenging to develop predictive models that result in high predictive power and explain the similarity of cell lines or drugs. Our study proposes a novel network-based methodology that breaks the problem into smaller, more interpretable problems to improve the predictive power of anti-cancer drug responses in cell lines. For the drug-sensitivity study, we used the GDSC database for 915 cell lines and 200 drugs. The theory of optimal mass transport was first used to separately cluster cell lines and drugs, using gene-expression profiles and extensive cheminformatic drug features, represented in a form of data networks. To predict cell-line specific drug responses, random forest regression modeling was separately performed for each cell-line drug cluster pair. Post-modeling biological analysis was further performed to identify potential biological correlates associated with drug responses. The network-based clustering method resulted in 30 distinct cell-line drug cluster pairs. Predictive modeling on each cell-line-drug cluster outperformed alternative computational methods in predicting drug responses. We found that among the four drugs top-ranked with respect to prediction performance, three targeted the PI3K/mTOR signaling pathway. Predictive modeling on clustered subsets of cell lines and drugs improved the prediction accuracy of cell-line specific drug responses. Post-modeling analysis identified plausible biological processes associated with drug responses.

Cache Valley virus (CVV) is a mosquito-borne virus in the genus Orthobunyavirus, family Peribunyaviridae. It was first isolated from a Culiseta inorata mosquito in Cache Valley, Utah in 1956 and is known to circulate widely in the Americas. While only a handful of human cases have been reported since its discovery, it is the causative agent of fetal death and severe malformations in livestock. CVV has recently emerged as a potential viral pathogen causing severe disease in humans. Currently, the only serological assay available for diagnostic testing is plaque reduction neutralization test which takes several days to perform and requires biocontainment. To expand diagnostic capacity to detect CVV infections by immunoassays, 12 hybridoma clones secreting anti-CVV murine monoclonal antibodies (MAbs) were developed. All MAbs developed were found to be non-neutralizing and specific to the nucleoprotein of CVV. Cross-reactivity experiments with related orthobunyaviruses revealed several of the MAbs reacted with Tensaw, Fort Sherman, Tlacotalpan, Maguari, Playas, and Potosi viruses. Our data shows that MAbs CVV14, CVV15, CVV17, and CVV18 have high specific reactivity as a detector in an IgM antibody capture test with human sera., Author summary Cache Valley virus is a mosquito-borne virus found throughout the Americas. It causes fetal death and severe malformations in livestock, and only a few cases of human viral disease have been identified. Currently, we do not fully understand the spectrum of disease in humans including its potential to cause fetal malformations. The only serological diagnostic assay available to detect recent viral infection is plaque reduction neutralization test which requires the use of live virus in biocontainment. In order to develop faster and safer serodiagnostics we generated 12 monoclonal antibodies for incorporation into new assays. These antibodies are specific to the nucleoprotein of the virus and cross-react with other closely related mosquito-borne viruses. Four of these antibodies were incorporated into an immunoassay for the detection of IgM from human sera demonstrating their utility in serodiagnosis. Rapid and higher throughput assays utilizing these antibodies will expand diagnostic capacity and facilitate research to increase our understanding of Cache Valley disease prevalence and the virus’s impact on at-risk populations.

28 p.-7 fig.-- Departamento de Biotecnología., African swine fever virus (ASFV) infectious cycle starts with the viral adsorption and entry into the host cell. Then, the virus is internalized via clathrin/dynamin mediated endocytosis and macropinocytosis. Similar to other viruses, ASF virion is then internalized and incorporated into the endocytic pathway. While the endosomal maturation entails luminal acidification, the decrease in pH acts on the multilayer structure of the virion dissolving the outer capsid. Upon decapsidation, the inner viral membrane is exposed to interact with the limiting membrane of the late endosome for fusion. Viral fusion is then necessary for the egress of incoming virions from endosomes into the cytoplasm, however this remains an intriguing and yet essential process for infection, specifically for the egress of viral nucleic acid into the cytoplasm for replication. ASFV proteins E248R and E199L, located at the exposed inner viral membrane, might be implicated in the fusion step. An interaction between these viral proteins and cellular endosomal proteins such as the Niemann-Pick C type 1 (NPC1) and lysosomal membrane proteins (Lamp-1 and -2) was shown. Furthermore, the silencing of these proteins impaired ASFV infection. It was also observed that NPC1 knock-out cells using CRISPR jeopardized ASFV infection and that the progression and endosomal exit of viral cores was arrested within endosomes at viral entry. These results suggest that the interactions of ASFV proteins with some endosomal proteins might be important for the membrane fusion step. In addition to this, reductions on ASFV infectivity and replication in NPC1 KO cells were accompanied by fewer and smaller viral factories. Our findings pave the way to understanding the role of proteins of the endosomal membrane in ASFV infection., This research was partially supported through the following awards: “La Caixa” Banking Foundation award number LCF/PR/HR19/52160012, Spain, https://fundacionlacaixa.org/en/ to CA and RD, Spanish Ministry of Science and Innovation, Spain RTI2018-097305-R- I00, https://www.ciencia.gob.es/ to CA, Instituto de Investigación Carlos III, ISCIII, Spain FIS PI 1801007, https://www.isciii.es/ to RD, European Commission, Horizon 2020 Framework Programme European Union ASFVInt ERANET-2021-862605 to CA, co-funded by Spanish Ministry of Science and Innovation, Spain PCI2021-121939 https://www.ciencia.gob.es/ to CA, VIRUSCAN FETPROACT-2016- 731868 and EPIC-CROWN-2-2021-101046084 https://ec.europa.eu/ to RD.

The global health emergency posed by the outbreak of Zika virus (ZIKV), an arthropod-borne flavivirus causing severe neonatal neurological conditions, has subsided, but there continues to be transmission of ZIKV in endemic regions. As such, there is still a medical need for discovering and developing therapeutical interventions against ZIKV. To identify small-molecule compounds that inhibit ZIKV disease and transmission, we screened multiple small-molecule collections, mostly derived from natural products, for their ability to inhibit wild-type ZIKV. As a primary high-throughput screen, we used a viral cytopathic effect (CPE) inhibition assay conducted in Vero cells that was optimized and miniaturized to a 1536-well format. Suitably active compounds identified from the primary screen were tested in a panel of orthogonal assays using recombinant Zika viruses, including a ZIKV Renilla luciferase reporter assay and a ZIKV mCherry reporter system. Compounds that were active in the wild-type ZIKV inhibition and ZIKV reporter assays were further evaluated for their inhibitory effects against other flaviviruses. Lastly, we demonstrated that wild-type ZIKV is able to infect a 3D-bioprinted outer-blood-retina barrier tissue model and disrupt its barrier function, as measured by electrical resistance. One of the identified compounds (3-Acetyl-13-deoxyphomenone, NCGC00380955) was able to prevent the pathological effects of the viral infection on this clinically relevant ZIKV infection model.

Background Cytotoxic effects of radiation play an important role in the treatment of head and neck cancer. However, irradiation is known to lead to the migration of various cancer cells, including those of head and neck cancer. Recently, fibroblasts in the cancer microenvironment have been reported to be involved in this mechanism. Nevertheless, the mechanism underlying migration of head and neck cancer cells remains unclear. Herein, we aimed to elucidate this migration mechanism induced by irradiation in terms of the interaction of head and neck cancer cells with fibroblasts. Methods We used the head and neck squamous cell carcinoma (HNSCC) cell lines SAS and FaDu as well as fibroblast cell lines. These cells were irradiated and their viability was compared. In fibroblasts, changes in interleukin-6 (IL-6) secretion caused by irradiation were measured by enzyme-linked immunosorbent assay (ELISA). The cell migration ability of cancer cells was evaluated via a migration assay using a semipermeable membrane. HNSCC cells were cocultured with irradiated and nonirradiated fibroblasts, and their migration ability under each condition was compared. We also examined the effect of IL-6 on the migration of HNSCC cells. Furthermore, to investigate the effect of fibroblast-derived IL-6 on the migration ability of HNSCC cells, we conducted a coculture study using IL-6 neutralizing antibody. Results Irradiation reduced the survival of HNSCC cells, whereas fibroblasts were resistant to irradiation. Irradiation also increased IL-6 secretion by fibroblasts. Migration of HNSCC cells was enhanced by coculture with fibroblasts and further enhanced by coculture with irradiated fibroblasts. We also confirmed that the migration of HNSCC cells was induced by IL-6. The enhanced migration of cancer cells caused by coculturing with fibroblasts was canceled by the IL-6 neutralizing antibody. Conclusion These results show that fibroblasts survive irradiation and induce the migration ability of HNSCC cells through increased secretion of IL-6.

Motivation A major challenge in cancer genomics is to distinguish the driver mutations that are causally linked to cancer from passenger mutations that do not contribute to cancer development. The majority of existing methods provide a single driver gene list for the entire cohort of patients. However, since mutation profiles of patients from the same cancer type show a high degree of heterogeneity, a more ideal approach is to identify patient-specific drivers. Results We propose a novel method that integrates genomic data, biological pathways and protein connectivity information for personalized identification of driver genes. The method is formulated on a personalized bipartite graph for each patient. Our approach provides a personalized ranking of the mutated genes of a patient based on the sum of weighted ‘pairwise pathway coverage’ scores across all the samples, where appropriate pairwise patient similarity scores are used as weights to normalize these coverage scores. We compare our method against five state-of-the-art patient-specific cancer gene prioritization methods. The comparisons are with respect to a novel evaluation method that takes into account the personalized nature of the problem. We show that our approach outperforms the existing alternatives for both the TCGA and the cell line data. In addition, we show that the KEGG/Reactome pathways enriched in our ranked genes and those that are enriched in cell lines’ reference sets overlap significantly when compared to the overlaps achieved by the rankings of the alternative methods. Our findings can provide valuable information toward the development of personalized treatments and therapies. Availability and implementation All the codes and data are available at https://github.com/abu-compbio/PersonaDrive, and the data underlying this article are available in Zenodo, at https://doi.org/10.5281/zenodo.6520187. Supplementary information Supplementary data are available at Bioinformatics online.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Currently, as dangerous mutations emerge, there is an increased demand for specific treatments for SARS-CoV-2 infected patients. The spike glycoprotein on the virus envelope binds to the angiotensin converting enzyme 2 (ACE2) on host cells through its receptor binding domain (RBD) to mediate virus entry. Thus, blocking this interaction may inhibit viral entry and consequently stop infection. Here, we generated fusion proteins composed of the extracellular portions of ACE2 and RBD fused to the Fc portion of human IgG1 (ACE2-Ig and RBD-Ig, respectively). We demonstrate that ACE2-Ig is enzymatically active and that it can be recognized by the SARS-CoV-2 RBD, independently of its enzymatic activity. We further show that RBD-Ig efficiently inhibits in-vivo SARS-CoV-2 infection better than ACE2-Ig. Mechanistically, we show that anti-spike antibody generation, ACE2 enzymatic activity, and ACE2 surface expression were not affected by RBD-Ig. Finally, we show that RBD-Ig is more efficient than ACE2-Ig at neutralizing high virus titers. We thus propose that RBD-Ig physically blocks virus infection by binding to ACE2 and that RBD-Ig should be used for the treatment of SARS-CoV-2-infected patients., Author summary SARS-CoV-2 has caused serious socio-economic and health problems around the globe. As dangerous mutations emerge there is an increased demand for specific treatments for SARS-CoV-2 infected patients. SARS-CoV-2 infection starts via binding of SARS-CoV-2 spike protein’s receptor binding domain (RBD) to its receptor, ACE2, on host cells. To intercept this binding, we generated Ig-fusion proteins; ACE2-Ig was generated to block the RBD and RBD-Ig generated to block ACE2. We showed that the fusion proteins bind to their respective target and demonstrated both in-vitro and in-vivo that it is more efficient to inhibit SARS-CoV-2 infection by blocking ACE2 receptor with RBD-Ig. We further showed that RBD-Ig does not interfere with ACE2 activity or with its surface expression. We propose that RBD-Ig physically blocks virus infection by binding to ACE2 and thus it may be used for the treatment of SARS-CoV-2-infected patients.

MicroRNAs (miRNA) are short non-coding RNAs widely implicated in gene regulation. Most metazoan miRNAs utilize the RNase III enzymes Drosha and Dicer for biogenesis. One notable exception is the RNA polymerase II transcription start sites (TSS) miRNAs whose biogenesis does not require Drosha. The functional importance of the TSS-miRNA biogenesis is uncertain. To better understand the function of TSS-miRNAs, we applied a modified Crosslinking, Ligation, and Sequencing of Hybrids on Argonaute (AGO-qCLASH) to identify the targets for TSS-miRNAs in HCT116 colorectal cancer cells with or without DROSHA knockout. We observed that miR-320a hybrids dominate in TSS-miRNA hybrids identified by AGO-qCLASH. Targets for miR-320a are enriched for the eIF2 signaling pathway, a downstream component of the unfolded protein response. Consistently, in miR-320a mimic- and antagomir- transfected cells, differentially expressed gene products are associated with eIF2 signaling. Within the AGO-qCLASH data, we identified the endoplasmic reticulum (ER) chaperone calnexin as a direct miR-320a down-regulated target, thus connecting miR-320a to the unfolded protein response. During ER stress, but not amino acid deprivation, miR-320a up-regulates ATF4, a critical transcription factor for resolving ER stress. In summary, our study investigates the targetome of the TSS-miRNAs in colorectal cancer cells and establishes miR-320a as a regulator of unfolded protein response., Author summary MicroRNA biogenesis factors, such as Drosha and Dicer, are frequently dysregulated in cancer, resulting in the disruption of miRNA-mediated gene regulation. Independent of Drosha-processing, transcription start site microRNA levels are elevated compared to most microRNAs in cancer cells with low DROSHA expression. By sequencing Argonaute-bound miRNA/mRNA pairs in colorectal cancer cells in the absence of Drosha, we identify the targetome for transcription start site microRNAs. We discovered a transcription start site miRNA, miR-320a, that down-regulates expression of the endoplasmic reticulum chaperone calnexin, activates expression of ATF4, and thus activates the unfolded protein response.

The prevalence of breast cancer in young women (YWBC) has increased alarmingly. Significant efforts are being made to elucidate the biological mechanisms concerning the development, prognosis, and pathological response in early-onset breast cancer (BC) patients. Dysfunctional DNA repair proteins are implied in BC predisposition, progression, and therapy response, underscoring the need for further analyses on DNA repair genes. Public databases of large patient datasets such as METABRIC, TCGA, COSMIC, and cancer cell lines allow the identification of variants in DNA repair genes and possible precision drug candidates. This study aimed at identifying variants and drug candidates that may benefit Latin American (LA) YWBC. We analyzed pathogenic variants in 90 genes involved in DNA repair in public BC datasets from METABRIC, TCGA, COSMIC, CCLE, and COSMIC Cell Lines Project. Results showed that reported DNA repair germline variants in the LA dataset are underrepresented in large databases, in contrast to other populations. Additionally, only six gene repair variants in women under 50 years old from the study population were reported in BC cell lines. Therefore, there is a need for new approaches to study DNA repair variants reported in young women from LA.

Mammalian adenoviruses (AdVs) comprise more than ~350 types including over 100 human (HAdVs) and just three mouse AdVs (MAdVs). While most HAdVs initiate infection by high affinity/avidity binding of their fiber knob (FK) protein to either coxsackievirus AdV receptor (CAR), CD46 or desmoglein (DSG)-2, MAdV-1 (M1) infection requires arginine-glycine-aspartate (RGD) binding integrins. To identify the receptors mediating MAdV infection we generated five novel reporter viruses for MAdV-1/-2/-3 (M1, M2, M3) transducing permissive murine (m) CMT-93 cells, but not B16 mouse melanoma cells expressing mCAR, human (h) CD46 or hDSG-2. Recombinant M1 or M3 FKs cross-blocked M1 and M3 but not M2 infections. Profiling of murine and human cells expressing RGD-binding integrins suggested that αvβ6 and αvβ8 heterodimers are associated with M1 and M3 infections. Ectopic expression of mβ6 in B16 cells strongly enhanced M1 and M3 binding, infection, and progeny production comparable with mαvβ6-positive CMT-93 cells, whereas mβ8 expressing cells were more permissive to M1 than M3. Anti-integrin antibodies potently blocked M1 and M3 binding and infection of CMT-93 cells and hαvβ8-positive M000216 cells. Soluble integrin αvβ6, and synthetic peptides containing the RGDLXXL sequence derived from FK-M1, FK-M3 and foot and mouth disease virus coat protein strongly interfered with M1/M3 infections, in agreement with high affinity interactions of FK-M1/FK-M3 with αvβ6/αvβ8, determined by surface plasmon resonance measurements. Molecular docking simulations of ternary complexes revealed a bent conformation of RGDLXXL-containing FK-M3 peptides on the subunit interface of αvβ6/β8, where the distal leucine residue dips into a hydrophobic pocket of β6/8, the arginine residue ionically engages αv aspartate215, and the aspartate residue coordinates a divalent cation in αvβ6/β8. Together, the RGDLXXL-bearing FKs are part of an essential mechanism for M1/M3 infection engaging murine and human αvβ6/8 integrins. These integrins are highly conserved in other mammals, and may favour cross-species virus transmission., PLoS Pathogens, 17 (12), ISSN:1553-7374, ISSN:1553-7366

Toxoplasma gondii is a master manipulator capable of effectively siphoning the resources from the host cell for its intracellular subsistence. However, the molecular underpinnings of how the parasite gains resources from its host remain largely unknown. Residing within a non-fusogenic parasitophorous vacuole (PV), the parasite must acquire resources across the limiting membrane of its replicative niche, which is decorated with parasite proteins including those secreted from dense granules. We discovered a role for the host Endosomal Sorting Complex Required for Transport (ESCRT) machinery in host cytosolic protein uptake by T. gondii by disrupting host ESCRT function. We identified the transmembrane dense granule protein TgGRA14, which contains motifs homologous to the late domain motifs of HIV-1 Gag, as a candidate for the recruitment of the host ESCRT machinery to the PV membrane. Using an HIV-1 virus-like particle (VLP) release assay, we found that the motif-containing portion of TgGRA14 is sufficient to substitute for HIV-1 Gag late domain to mediate ESCRT-dependent VLP budding. We also show that TgGRA14 is proximal to and interacts with host ESCRT components and other dense granule proteins during infection. Furthermore, analysis of TgGRA14-deficient parasites revealed a marked reduction in ingestion of a host cytosolic protein compared to WT parasites. Thus, we propose a model in which T. gondii recruits the host ESCRT machinery to the PV where it can interact with TgGRA14 for the internalization of host cytosolic proteins across the PV membrane (PVM). These findings provide new insight into how T. gondii accesses contents of the host cytosol by exploiting a key pathway for vesicular budding and membrane scission., Author summary Intracellular pathogens exploit their host to gain the resources necessary to sustain infection; however, precisely how the intracellular parasite Toxoplasma gondii acquires essential nutrients from its host remains poorly understood. Previous work showed that T. gondii is capable of internalizing host derived cytosolic proteins and delivering them to its lysosome-like compartment for degradation. However, the mechanism by which the material is trafficked across the membrane delimiting the replicative vacuole in which the parasite resides remained unclear. Here, we report a role for the parasite effector protein TgGRA14 in the recruitment of the host ESCRT machinery and in the uptake of host cytosolic proteins. Important human pathogens have developed strategies for exploiting the host ESCRT machinery for intracellular subsistence. Our study sheds lights on the strategy used by a eukaryotic pathogen in to exploit the host ESCRT machinery for the internalization of resources from its host cell.

Given the worldwide burden of neglected tropical diseases, there is ongoing need to develop novel anthelmintic agents to strengthen the pipeline of drugs to combat these burdensome infections. Many diseases caused by parasitic flatworms are treated using the anthelmintic drug praziquantel (PZQ), employed for decades as the key clinical agent to treat schistosomiasis. PZQ activates a flatworm transient receptor potential (TRP) channel within the melastatin family (TRPMPZQ) to mediate sustained Ca2+ influx and worm paralysis. As a druggable target present in many parasitic flatworms, TRPMPZQ is a promising target for a target-based screening campaign with the goal of discovering novel regulators of this channel complex. Here, we have optimized methods to miniaturize a Ca2+-based reporter assay for Schistosoma mansoni TRPMPZQ (Sm.TRPMPZQ) activity enabling a high throughput screening (HTS) approach. This methodology will enable further HTS efforts against Sm.TRPMPZQ as well as other flatworm ion channels. A pilot screen of ~16,000 compounds yielded a novel activator of Sm.TRPMPZQ, and numerous potential blockers. The new activator of Sm.TRPMPZQ represented a distinct chemotype to PZQ, but is a known chemical entity previously identified by phenotypic screening. The fact that a compound prioritized from a phenotypic screening campaign is revealed to act, like PZQ, as an Sm.TRPMPZQ agonist underscores the validity of TRPMPZQ as a druggable target for antischistosomal ligands., Author summary The drug praziquantel is used to treat diseases caused by parasitic flatworms. Praziquantel is an old drug, and there is a need to identify novel treatments that retain desirable features and improve weaknesses in the mode of PZQ action. One way to do this is to identify new drugs that exploit vulnerabilities in the same drug target but work in slightly differently ways. Here, we have optimized high throughput screening methods to pharmacologically profile a parasitic flatworm ion channel targeted by PZQ. We have identified several new chemical structures that interact with this channel complex. These ligands provide new opportunity for developing tools to manipulate flatworm biology and potentially new trajectories for anthelmintic drug development.

The immunological safety of drugs, nanomaterials and contaminants is a central point in the regulatory evaluation and safety monitoring of working and public places and of the environment. In fact, anomalies in immune responses may cause diseases and hamper the physical and functional integrity of living organisms, from plants to human beings. In the case of nanomaterials, many experimental models are used for assessing their immunosafety, some of which have been adopted by regulatory bodies. All of them, however, suffer from shortcomings and approximations, and may be inaccurate in representing real-life responses, thereby leading to incomplete, incorrect or even misleading predictions. Here, we review the advantages and disadvantages of current nanoimmunosafety models, comparing in vivo vs. in vitro models and examining the use of animal vs. human cells, primary vs. transformed cells, complex multicellular and 3D models, organoids and organs-on-chip, in view of implementing a reliable and personalized nanoimmunosafety testing. The general conclusion is that the choice of testing models is key for obtaining reliable predictive information, and therefore special attention should be devoted to selecting the most relevant and realistic suite of models in order to generate relevant information that can allow for safer-by-design nanotechnological developments.

Euphorbia cuneata Vahl. (Euphorbiaceae) is a plant used in folk medicine for the treatment of pain and inflammation, although the biological basis for these effects has not been thoroughly investigated. The goal of this study was to investigate the pharmacological properties and characterization of phenolic and flavonoid compounds present in the aerial parts of E. cuneata. E. cuneata aerial parts were tested for antioxidant activity (DPPH), antibacterial activity, cell viability and cytotoxic effects, and anti-inflammatory activity. Phenolic and flavonoid contents (HPLC), and volatile constituents (GC-MS) were also characterized. The methanol extract had the highest antioxidant activity, while the ether extract had the lowest. The antioxidant activity of E. cuneata extract increased from (21.11%) at a concentration of 10 µg/mL to (95.53%) at a concentration of 1280 µg/mL. S. aureus was the most sensitive organism with the highest zone of inhibition and lowest MIC, with acetone extract; whereas C. tropicalis was the most resistant, with the lowest inhibition zone. MTT assay revealed that the methanol extract of E. cuneata had significant cytotoxic effects on the A549, Caco-2, and MDA-MB-231 cell lines, respectively. Lower concentrations of methanolic extract gave anti-inflammatory activity, and those effects were compared with indomethacin as a positive control. Pyrogallol was the most abundant phenolic acid, followed by caffeic, p-coumaric, ferulic, syringic, and gallic acids, respectively. The 7-hydroxyflavone and rutin flavonoids were also found in the extract. GC-mass analysis showed that aerial parts of E. cuneata were rich in methyl 12-hydroxy-9-octadecenoate. The volatile components were also composed of considerable amounts of hexadecanoic acid, methyl ester, (9E,12E)-octadeca-9,12-dienoyl chloride, and methyl octadeca-9,12-dienoate as well as a little amount of hexanal dimethyl acetal. It can be concluded that methanolic extract of E. cuneata could be used as an available source of natural bioactive constituents with consequent health benefits.

The need to produce immortal, food-relevant cell lines is one of the most pressing challenges of cellular agriculture, the field which seeks to produce meat and other animal products via tissue engineering and synthetic biology. Immortal cell lines have a long and complicated story, from the first recognized immortal human cell lines taken from Henrietta Lacks, to today, where they are used to assay toxicity and produce therapeutics, to the future, where they could be used to create meat without harming an animal. Although work in immortal cell lines began more than 50 years ago, there are few existing cell lines made of species and cell types appropriate for cultured meat. Cells in cultured meat will be eaten by consumers; therefore, cultured meat cell lines will also require unique attributes not selected for in other cell line applications. Specifically, cultured meat cell lines will need to be approved as safe for consumption as food, proliferate and differentiate efficiently at industrial scales, and have desirable taste, texture, and nutrition characteristics for consumers. This paper defines what cell lines are needed, the existing methods to produce new cell lines and their limitations, and the unique considerations of cell lines used in cultured meat.

Gene selection in unannotated large single cell RNA sequencing (scRNA-seq) data is important and crucial step in the preliminary step of downstream analysis. The existing approaches are primarily based on high variation (highly variable genes) or significant high expression (highly expressed genes) failed to provide stable and predictive feature set due to technical noise present in the data. Here, we propose RgCop, a novel regularized copula based method for gene selection from large single cell RNA-seq data. RgCop utilizes copula correlation (Ccor), a robust equitable dependence measure that captures multivariate dependency among a set of genes in single cell expression data. We formulate an objective function by adding l1 regularization term with Ccor to penalizes the redundant co-efficient of features/genes, resulting non-redundant effective features/genes set. Results show a significant improvement in the clustering/classification performance of real life scRNA-seq data over the other state-of-the-art. RgCop performs extremely well in capturing dependence among the features of noisy data due to the scale invariant property of copula, thereby improving the stability of the method. Moreover, the differentially expressed (DE) genes identified from the clusters of scRNA-seq data are found to provide an accurate annotation of cells. Finally, the features/genes obtained from RgCop is able to annotate the unknown cells with high accuracy., Author Summary The existing approaches for gene selection which are based on high variation (highly variable genes) or significant high expression (highly expressed genes), failed to provide a stable and predictive feature/gene set. Since single cell data is susceptible to technical noise, the quality of genes selected prior to clustering is of crucial importance in the preliminary steps of downstream analysis. Here, we propose a novel regularized copula based method for gene selection that leverage copula correlation (Ccor) measure for capturing cell-to-cell variability within the data. The proposed objective function uses an l1 regularization term to penalizes the redundant co-efficient of features/genes. We got significant improvement in the clustering/classification performance of cells over the other state-of-the-art. Due to the scale-invariant property of copula RgCop is impervious to technical noise, an acute issue associated with scRNA-seq data analysis. Moreover, the selected features/genes can be able to determine the unknown cells with high accuracy. Finally, RgCop can be applicable for identifying rare cell clusters or minor subpopulations within the single cell data.

BTK is a dominant bioactive kinase expressed within both cancer and immune cells of GBM tissue. Complex cell co-cultures might better model the impact of kinase inhibitors as therapeutics in GBM., Better understanding of GBM signalling networks in-vivo would help develop more physiologically relevant ex vivo models to support therapeutic discovery. A “functional proteomics” screen was undertaken to measure the specific activity of a set of protein kinases in a two-step cell-free biochemical assay to define dominant kinase activities to identify potentially novel drug targets that may have been overlooked in studies interrogating GBM-derived cell lines. A dominant kinase activity derived from the tumour tissue, but not patient-derived GBM stem-like cell lines, was Bruton tyrosine kinase (BTK). We demonstrate that BTK is expressed in more than one cell type within GBM tissue; SOX2-positive cells, CD163-positive cells, CD68-positive cells, and an unidentified cell population which is SOX2-negative CD163-negative and/or CD68-negative. The data provide a strategy to better mimic GBM tissue ex vivo by reconstituting more physiologically heterogeneous cell co-culture models including BTK-positive/negative cancer and immune cells. These data also have implications for the design and/or interpretation of emerging clinical trials using BTK inhibitors because BTK expression within GBM tissue was linked to longer patient survival.

The rapid evolution of antibiotic resistance in Clostridioides difficile and the consequent effects on prevention and treatment of C. difficile infections (CDIs) are matters of concern for public health. Thioridazine, a compound belonging to the phenothiazine group, has previous shown antimicrobial activity against C. difficile. The purpose of this present study was to investigate the potential of a novel phenothiazine derivative, JBC 1847, as an oral antimicrobial for treatment of intestinal pathogens and CDIs. The minimal inhibition concentration and the minimum bactericidal concentration of JBC 1847 against C. difficile ATCC 43255 were determined 4 μg/mL and high tolerance after oral administration in mice was observed (up to 100 mg/kg bodyweight). Pharmacokinetic modeling was conducted in silico using GastroPlusTM, predicting low (< 10%) systemic uptake after oral exposure and corresponding low Cmax in plasma. Impact on the intestinal bacterial composition after four days of treatment was determined by 16s rRNA MiSeq sequencing and revealed only minor impact on the microbiota in non-clinically affected mice, and there was no difference between colony-forming unit (CFU)/gram fecal material between JBC 1847 and placebo treated mice. The cytotoxicity of the compound was assessed in Caco-2 cell-line assays, in which indication of toxicity was not observed in concentrations up to seven times the minimal bactericidal concentration. In conclusion, the novel phenothiazine derivative demonstrated high antimicrobial activity against C. difficile, had low predicted gastrointestinal absorption, low intestinal (in vitro) cytotoxicity, and only induced minor changes of the healthy microbiota, altogether supporting that JBC 1847 could represent a novel antimicrobial candidate. The clinical importance hereof calls for future experimental studies in CDI models.

Newly designed series of indole-containing pyrazole analogs, pyrazolinylindoles, were synthesized, and their structures were confirmed based on the spectral data of the 1H NMR, 13C NMR, and HR-MS analyses. Preliminary anti-cancer activity testings were carried out by the National Cancer Institute, United States of America (NCI, USA). Compounds HD02, HD05, and HD12 demonstrated remarkable cytotoxic activities against nine categories of cancer types based cell line panels which included leukemia, colon, breast, melanoma, lungs, renal, prostate, CNS, and ovarian cancer cell lines. The highest cytotoxic effects were exhibited by the compounds HD02 [1-(5-(1-H-indol-3-yl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-phenylethanone], HD05 [1-(3-(4-chlorophenyl)-5-(1H-indol-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)-2-phenoxyethanone], and HD12 [(3-(4-chlorophenyl)-5-(1H-indol-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone] against some of the 56 types of NCI-based cell lines in different panels. Compound HD05 showed the maximum range of cancer cell growth inhibitions against all categories of the cell lines in all nine panels. On average, in comparison to the referral standard, imatinib, at a dose level of 10 µM, the HD05 showed significant activity against leukemia in the range of 78.76%, as compared to the imatinib at 9% of cancer cells’ growth inhibitions. Molecular docking simulation studies were performed in silico on the epidermal growth factor receptor (EGFR) tyrosine kinase, in order to validate the activity.

Cellular susceptibility to SARS-CoV-2 infection in the respiratory tract has been associated with the ability of the virus to interact with potential receptors on the host membrane. We have modeled viral dynamics by simulating various cellular systems and artificial conditions, including macromolecular crowding, based on experimental and transcriptomic data to infer parameters associated with viral growth and predict cell susceptibility. We have accomplished this based on the type, number and level of expression of the angiotensin-converting enzyme 2 (ACE2), transmembrane serine 2 (TMPRSS2), basigin2 (CD147), FURIN protease, neuropilin 1 (NRP1) or other less studied candidate receptors such as heat shock protein A5 (HSPA5) and angiotensin II receptor type 2 (AGTR2). In parallel, we studied the effect of simulated artificial environments on the accessibility to said proposed receptors. In addition, viral kinetic behavior dependent on the degree of cellular susceptibility was predicted. The latter was observed to be more influenced by the type of proteins and expression level, than by the number of potential proteins associated with the SARS CoV-2 infection. We predict a greater theoretical propensity to susceptibility in cell lines such as NTERA-2, SCLC-21H, HepG2 and Vero6, and a lower theoretical propensity in lines such as CaLu3, RT4, HEK293, A549 and U-251MG. An important relationship was observed between expression levels, protein diffusivity, and thermodynamically favorable interactions between host proteins and the viral spike, suggesting potential sites of early infection other than the lungs. This research is expected to stimulate future quantitative experiments and promote systematic investigation of the effect of crowding presented here., Graphical abstract Image 1

Plant-derived natural products are significant resources for drug discovery and development including appreciable potentials in preventing and managing oxidative stress, making them promising candidates in cancer and other disease therapeutics. Their effects have been linked to phytochemicals such as phenolic compounds and their antioxidant activities. The abundance and complexity of these bio-constituents highlight the need for well-defined in vitro characterization and quantification of the plant extracts/preparations that can translate to in vivo effects and hopefully to clinical use. This review article seeks to provide relevant information about the applicability of cell-based assays in assessing anti-cytotoxicity of phytochemicals considering several traditional and current methods.

B-cell receptor (BCR) signalling is critical for the survival of B-cell lymphomas and is a therapeutic target of drugs such as Ibrutinib. However, the role of T-cell receptor (TCR) signalling in the survival of T/Natural Killer (NK) lymphomas is not clear. ZAP-70 (zeta associated protein-70) is a cytoplasmic tyrosine kinase with a critical role in T-cell receptor (TCR) signalling. It has also been shown to play a role in normal NK cell signalling and activation. High ZAP-70 expression has been detected by immunohistochemistry in peripheral T cell lymphoma (PTCL) and NK cell lymphomas (NKTCL). We therefore, studied the role of TCR pathways in mediating the proliferation and survival of these malignancies through ZAP-70 signalling. ZAP-70 protein was highly expressed in T cell lymphoma cell lines (JURKAT and KARPAS-299) and NKTCL cell lines (KHYG-1, HANK-1, NK-YS, SNK-1 and SNK-6), but not in multiple B-cell lymphoma cell lines. siRNA depletion of ZAP-70 suppressed the phosphorylation of ZAP-70 substrates, SLP76, LAT and p38MAPK, but did not affect cell viability or induce apoptosis in these cell lines. Similarly, while stable overexpression of ZAP-70 mediates increased phosphorylation of target substrates in the TCR pathway, it does not promote increased survival or growth of NKTCL cell lines. The epidermal growth factor receptor (EGFR) inhibitor Gefitinib, which has off-target activity against ZAP-70, also did not show any differential cell kill between ZAP-70 overexpressing (OE) or knockdown (KD) cell lines. Whole transcriptome RNA sequencing highlighted that there was very minimal differential gene expression in three different T/NK cell lines induced by ZAP-70 KD. Importantly, ZAP-70 KD did not significantly enrich for any downstream TCR related genes and pathways. Altogether, this suggests that high expression and constitutive signalling of ZAP-70 in T/NK lymphoma is not critical for cell survival or downstream TCR-mediated signalling and gene expression. ZAP-70 therefore may not be a suitable therapeutic target in T/NK cell malignancies.

The increasing incidence of prostate cancer (PCa) indicates an urgent need for the development of new effective drug therapy. There are limited options to treat the PCa, this study tried to determine a new therapy option for this acute cancer. Androgen-independent PCa cell lines PC3 and DU145 were treated with different melatonin concentrations (0.1~3.5 mM) for 1~3 days and assessed cell migration, cell invasion, cycle arrest in G0/G1 phase as well as apoptosis. We utilized RNA-seq technology to analyze the transcriptional misregulation pathways in DU145 prostate cancer cell line with melatonin (0.5 mM) treatment. Data revealed 20031 genes were up and down-regulated, there were 271 genes that differentially expressed: 97 up-regulated (P

Promoter DNA methylation ofMutL homolog 1 (MLH1)is considered to play a causative role in microsatellite instability (MSI) carcinogenesis in primary gastric cancer, and a high MSI status is associated with treatment sensitivity to human cancers. Nevertheless, clinicopathological analysis is defective forMLH1methylation status in a quantitative manner. We newly developed quantitative methylation specific PCR using a TaqMan probe and applied it to 138 patients with primary gastric cancer who underwent gastrectomy in addition to basic molecular features such as MSI, Epstein Barr virus, and other DNA methylation status. (1) In primary gastric cancer, median methylation value was 0.055, ranging from 0 to 124.3. First,MLH1hypermethylation was strongly correlated with MSI-High/MSI-Low status and suppressed immunostaining (P< 0.0001). (2) TheMLH1hypermethylation was associated with advanced age (P= 0.0048), antral location (P= 0.0486), synchronous multiple gastric cancer (P= 0.0001), and differentiated histology (P= 0.028). (3) Log-rank plot analysis identified the most relevant cut-off value (0.23) to reflect gentle phenotypes inMLH1hypermethylation cases (P= 0.0019), especially in advanced gastric cancer (P= 0.0132), which are designated as haploinsufficiency of MSI (MSI-haplo) phenotype in this study. (4) In synchronous multiple gastric cancer,MLH1hypermethylation was not necessarily confirmed as field cancerization. (5) MSI-haplo defined byMLH1methylation status represented distinct prognostic phenotype even after molecular classifications.MLH1hypermethylation designated as MSI-haplo may represent unique prognostic phenotype during gastric carcinogenesis.

Copyright: © 2021 Abidi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited., Background: Individuals recovering from COVID-19 are known to have antibodies against the Spike and other structural proteins. Antibodies against Spike have been shown to display viral neutralization. However, not all antibodies against Spike have neutralizing ability although they may be cross-reactive. There is a need for easy-to-use SARS-CoV-2 neutralizing assays for the determination of virus-neutralizing activity in sera of individuals. Here we describe a PCR-based micro-neutralization assay that can be used to evaluate the viral neutralization titers of serum from SARS-CoV-2 infected individuals. Methods: The SARS-CoV-2 strain used was isolated from a nasopharyngeal specimen of a COVID-19 case. The limiting dilution method was used to obtain a 50% tissue culture infective dose (TCID50) of Vero cells. For the micro-neutralization assay, 19 serum samples, with positive IgG titers against Spike Receptor-Binding Domain (RBD) were tested. After 24 hours, infected cells were inspected for the presence of a cytopathic effect, lysed and RNA RT-PCR conducted for SARS-CoV-2. PCR target Ct values were used to calculate percent neutralization/inhibition of SARS-CoV-2. Results: Out of 19 samples, 13 samples gave 100% neutralization at all dilutions, 1 sample showed neutralization at the first dilution, 4 samples showed neutralization at lower dilutions, while one sample did not demonstrate any neutralization. The RBD ODs and neutralization potential percentages were found to be positively correlated. Conclusion: We describe a rapid RT-PCR-based SARS-CoV-2 microneutralization assay for the detection of neutralizing antibodies. This can effectively be used to test the antiviral activity of serum antibodies for the investigation of both disease-driven and vaccine-induced responses., This study received funding support through a grant from the Provosts Academic Priorities Fund, The Aga Khan University. MV was supported by the European Union H2020 ERA project (No 667824 – EXCELLtoINNOV).

Endoplasmic reticulum–associated degradation (ERAD) is a protein quality control pathway of fundamental importance to cellular homeostasis. Although multiple ERAD pathways exist for targeting topologically distinct substrates, all pathways require substrate ubiquitination. Here, we characterize a key role for the UBE2G2 Binding Region (G2BR) of the ERAD accessory protein ancient ubiquitous protein 1 (AUP1) in ERAD pathways. This 27-amino acid (aa) region of AUP1 binds with high specificity and low nanomolar affinity to the backside of the ERAD ubiquitin-conjugating enzyme (E2) UBE2G2. The structure of the AUP1 G2BR (G2BRAUP1) in complex with UBE2G2 reveals an interface that includes a network of salt bridges, hydrogen bonds, and hydrophobic interactions essential for AUP1 function in cells. The G2BRAUP1 shares significant structural conservation with the G2BR found in the E3 ubiquitin ligase gp78 and in vitro can similarly allosterically activate ubiquitination in conjunction with ERAD E3s. In cells, AUP1 is uniquely required to maintain normal levels of UBE2G2; this is due to G2BRAUP1 binding to the E2 and preventing its rapid degradation. In addition, the G2BRAUP1 is required for both ER membrane recruitment of UBE2G2 and for its activation at the ER membrane. Thus, by binding to the backside of a critical ERAD E2, G2BRAUP1 plays multiple critical roles in ERAD., This study shows that a 27-amino acid domain that binds the ubiquitin conjugating enzyme UBE2G2, and which is found in two different proteins (AUP1 and gp78), is critical for endoplasmic reticulum-associated degradation (ERAD).

ERBB family members and their ligands play an essential role in embryonic heart development and adult heart physiology. Among them, ERBB3 is a binding partner of ERBB2; the ERBB2/3 complex mediates downstream signaling for cell proliferation. ERBB3 has seven consensus binding sites to the p85 regulatory subunit of PI3K, which activates the downstream AKT pathway, leading to the proliferation of various cells. This study generated a human ERBB3 knock-in mouse expressing a mutant ERBB3 whose seven YXXM p85 binding sites were replaced with YXXA. Erbb3 knock-in embryos exhibited lethality between E12.5 to E13.5, and showed a decrease in mesenchymal cell numbers and density in AV cushions. We determined that the proliferation of mesenchymal cells in the atrioventricular (AV) cushion in Erbb3 knock-in mutant embryos was temporarily reduced due to the decrease of AKT and ERK1/2 phosphorylation. Overall, our results suggest that AKT/ERK activation by the ERBB3-dependent PI3K signaling is crucial for AV cushion morphogenesis during embryonic heart development.

Members of the Old World Arenaviruses primarily utilize α-dystroglycan (α-DAG1) as a cellular receptor for infection. Mutations within the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) reduce or abrogate the binding affinity to α-DAG1 and thus influence viral persistence, kinetics, and cell tropism. The observation that α-DAG1 deficient cells are still highly susceptible to low affinity variants, suggests the use of an alternative receptor(s). In this study, we used a genome-wide CRISPR Cas9 knockout screen in DAG1 deficient 293T cells to identify host factors involved in α-DAG1-independent LCMV infection. By challenging cells with vesicular stomatitis virus (VSV), pseudotyped with the GP of LCMV WE HPI (VSV-GP), we identified the heparan sulfate (HS) biosynthesis pathway as an important host factor for low affinity LCMV infection. These results were confirmed by a genetic approach targeting EXTL3, a key factor in the HS biosynthesis pathway, as well as by enzymatic and chemical methods. Interestingly, a single point mutation within GP1 (S153F or Y155H) of WE HPI is sufficient for the switch from DAG1 to HS binding. Furthermore, we established a simple and reliable virus-binding assay, using directly labelled VSV-GP by intramolecular fusion of VSV-P and mWasabi, demonstrating the importance of HS for virus attachment but not entry in Burkitt lymphoma cells after reconstitution of HS expression. Collectively, our study highlights the essential role of HS for low affinity LCMV infection in contrast to their high affinity counterparts. Residual LCMV infection in double knockouts indicate the use of (a) still unknown entry receptor(s)., Author summary Lymphocytic choriomeningitis virus (LCMV) contributed to the fundamental understanding of immunological processes due to characteristics such as persistent and immunosuppressive infection. However, not all strains of LCMV share the same traits. Differences in tissue tropism and the course of disease led to intensive characterization of distinct LCMV variants. Point mutations within the glycoprotein of LCMV were identified that reduce or abrogate the binding affinity to its host receptor α-Dystroglycan (α-DAG1), leading to the classification of low and high affinity LCMV variants. α-DAG1-independent infection and altered tissue tropism suggested the use of an alternative, unknown receptor by low affinity LCMV variants. Applying a genome-wide knockout screen and comparing different LCMV strains by genetic, enzymatic, and chemical approaches, we identified heparan sulfate proteoglycans (HSPG) as alternative receptors favoured by low affinity LCMV variants. These findings improve the understanding of receptor usage by different LCMV variants and explain their distinct characteristics. Furthermore, residual LCMV infection of double knockout cells indicate a major role of α-DAG1 and HSPG as attachment factors for high and low affinity LCMV variants, respectively, as well as the use of (a) still unknown entry receptor(s).

Abnormal calcium absorption and iron overload from iron hyperabsorption can contribute to osteoporosis as found in several diseases, including hemochromatosis and thalassemia. Previous studies in thalassemic mice showed the positive effects of the iron uptake suppressor, hepcidin, on calcium transport. However, whether this effect could be replicated in other conditions is not known. Therefore, this study aimed to investigate the effects of hepcidin on iron and calcium uptake ability under physiological, iron uptake stimulation and calcium uptake suppression. To investigate the potential mechanism, effects of hepcidin on the expression of iron and calcium transporter and transport-associated protein in Caco-2 cells were also determined. Our results showed that intestinal cell iron uptake was significantly increased by ascorbic acid together with ferric ammonium citrate (FAC), but this phenomenon was suppressed by hepcidin. Interestingly, hepcidin significantly increased calcium uptake under physiological condition but not under iron uptake stimulation. While hepcidin significantly suppressed the expression of iron transporter, it had no effect on calcium transporter expression. This indicated that hepcidin-induced intestinal cell calcium uptake did not occur through the stimulation of calcium transporter expression. On the other hand, 1,25(OH)2D3 effectively induced intestinal cell calcium uptake, but it did not affect intestinal cell iron uptake or iron transporter expression. The 1,25(OH)2D3-induced intestinal cell calcium uptake was abolished by 12 mM CaCl2; however, hepcidin could not rescue intestinal cell calcium uptake suppression by CaCl2. Taken together, our results showed that hepcidin could effectively and concurrently induce intestinal cell calcium uptake while reducing intestinal cell iron uptake under physiological and iron uptake stimulation conditions, suggesting its therapeutic potential for inactive calcium absorption, particularly in thalassemic patients or patients who did not adequately respond to 1,25(OH)2D3.