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Human immunodeficiency virus (HIV) co-infection is a major challenge for visceral leishmaniasis (VL) control, particularly in Ethiopia where the incidence of both pathogens is high. VL-HIV often leads to high rates of antileishmanial treatment failure and recurrent VL disease relapses. Considering the high prevalence of HIV and Leishmania in the Ethiopian population, preventing the progression of asymptomatic Leishmania infection to disease would be a valuable asset to VL disease control and to the clinical management of people living with HIV (PLWH). However, such a strategy requires good understanding of risk factors for VL development. In immunocompetent individuals living in Brazil, India, or Iran, the Human Leukocyte Antigen (HLA) gene region has been associated with VL development. We used NanoTYPE, an Oxford Nanopore Technologies sequencing-based HLA genotyping method, to detect associations between HLA genotype and VL development by comparing 78 PLWH with VL history and 46 PLWH that controlled a Leishmania infection, all living in a VL endemic region of North-West Ethiopia. We identified an association between HLA-A*03:01 and increased risk of VL development (OR = 3.89). These data provide candidate HLA alleles that can be further explored for inclusion in a potential Leishmania screen-and-treat strategy in VL endemic regions., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: W.A. received a travel grant by Omixon to participate in the European Federation for Immunogenetics conference to present the preliminary results of this work. Omixon had no role in study design, data collection and analysis, preparation of the manuscript, nor the decision to publish., (Copyright: © 2024 de Vrij 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.)

A large proportion of HIV-coinfected visceral leishmaniasis (VL-HIV) patients exhibit chronic disease with frequent VL recurrence. However, knowledge on immunological determinants underlying the disease course is scarce. We longitudinally profiled the circulatory cellular immunity of an Ethiopian HIV cohort that included VL developers. We show that chronic VL-HIV patients exhibit high and persistent levels of TIGIT and PD-1 on CD8 + /CD8 - T cells, in addition to a lower frequency of IFN-γ +  TIGIT - CD8 + /CD8 - T cells, suggestive of impaired T cell functionality. At single T cell transcriptome and clonal resolution, the patients show CD4 +  T cell anergy, characterised by a lack of T cell activation and lymphoproliferative response. These findings suggest that PD-1 and TIGIT play a pivotal role in VL-HIV chronicity, and may be further explored for patient risk stratification. Our findings provide a strong rationale for adjunctive immunotherapy for the treatment of chronic VL-HIV patients to break the recurrent disease cycle., (© 2024. The Author(s).)

Global soil acidification is increasing, enlarging aluminum (Al) availability in soils, leading to reductions in plant growth. This study investigates the effect of Al stress on the leaf growth zones of Rye (Secale cereale, cv Beira). Kinematic analysis showed that the effect of Al on leaf growth rates was mainly due to a reduced cell production rate in the meristem. Transcriptomic analysis identified 2272 significantly (log2fold > |0.5| FDR < 0.05) differentially expressed genes (DEGs) for Al stress. There was a downregulation in several DEGs associated with photosynthetic processes and an upregulation in genes for heat/light response, and H 2 O 2 production in all leaf zones. DEGs associated with heavy metals and malate transport were increased, particularly, in the meristem. To determine the putative function of these processes in Al tolerance, we performed biochemical analyses comparing the tolerant Beira with an Al sensitive variant RioDeva. Beira showed improved sugar metabolism and redox homeostasis, specifically in the meristem compared to RioDeva. Similarly, a significant increase in malate and citrate production, which are known to aid in Al detoxification in plants, was found in Beira. This suggests that Al tolerance in Rye is linked to its ability for Al exclusion from the leaf meristem., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Importance: Malaria is caused by parasites of the genus Plasmodium , and reached a global disease burden of 247 million cases in 2021. To study drug resistance mutations and parasite population dynamics, whole-genome sequencing of patient blood samples is commonly performed. However, the predominance of human DNA in these samples imposes the need for time-consuming laboratory procedures to enrich Plasmodium DNA. We used the Oxford Nanopore Technologies' adaptive sampling feature to circumvent this problem and enrich Plasmodium reads directly during the sequencing run. We demonstrate that adaptive nanopore sequencing efficiently enriches Plasmodium reads, which simplifies and shortens the timeline from blood collection to parasite sequencing. In addition, we show that the obtained data can be used for monitoring genetic markers, or to generate nearly complete genomes. Finally, owing to its inherent mobility, this technology can be easily applied on-site in endemic areas where patients would benefit the most from genomic surveillance., Competing Interests: The authors declare no conflict of interest.

Background: Plasmodium vivax is the second most important cause of human malaria worldwide, and accounts for the majority of malaria cases in South America. A high-quality reference genome exists for Papua Indonesia (PvP01) and Thailand (PvW1), but is lacking for South America. A reference genome specifically for South America would be beneficial though, as P. vivax is a genetically diverse parasite with geographical clustering., Results: This study presents a new high-quality assembly of a South American P. vivax isolate, referred to as PvPAM (P. vivax Peruvian AMazon). The genome was obtained from a low input patient sample from the Peruvian Amazon and sequenced using PacBio technology, resulting in a highly complete assembly with 6497 functional genes. Telomeric ends were present in 17 out of 28 chromosomal ends, and additional (sub)telomeric regions are present in 12 unassigned contigs. A comparison of multigene families between PvPAM and the PvP01 genome revealed remarkable variation in vir genes, and the presence of merozoite surface proteins (MSP) 3.6 and 3.7. Three dhfr and dhps drug resistance associated mutations are present in PvPAM, similar to those found in other Peruvian isolates. Mapping of publicly available South American whole genome sequencing (WGS) data to PvPAM resulted in significantly fewer variants and truncated reads compared to the use of PvP01 or PvW1 as reference genomes. To minimize the number of core genome variants in non-South American samples, PvW1 is most suited for Southeast Asian isolates, both PvPAM and PvW1 are suited for South Asian isolates, and PvPAM is recommended for African isolates. Interestingly, non-South American samples still contained the least subtelomeric variants when mapped to PvPAM, indicating high quality of the PvPAM subtelomeric regions., Conclusions: Our findings show that the PvPAM reference genome more accurately represents South American P. vivax isolates in comparison to PvP01 and PvW1. In addition, PvPAM has a high level of completeness, and contains a similar number of annotated genes as PvP01 or PvW1. The PvPAM genome therefore will be a valuable resource to improve future genomic analyses on P. vivax isolates from the South American continent., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Overexpression of the transmembrane mucin MUC13, as seen in inflammatory bowel diseases (IBD), could potentially impact barrier function. This study aimed to explore how inflammation-induced MUC13 disrupts epithelial barrier integrity by affecting junctional protein expression in IBD, thereby also considering the involvement of MUC1. RNA sequencing and permeability assays were performed using LS513 cells transfected with MUC1 and MUC13 siRNA and subsequently stimulated with IL-22. In vivo intestinal permeability and MUC13-related signaling pathways affecting barrier function were investigated in acute and chronic DSS-induced colitis wildtype and Muc13 -/- mice. Finally, the expression of MUC13 , its regulators and other barrier mediators were studied in IBD and control patients. Mucin knockdown in intestinal epithelial cells affected gene expression of several barrier mediators in the presence/absence of inflammation. IL-22-induced MUC13 expression impacted barrier function by modulating the JAK1/STAT3, SNAI1/ZEB1 and ROCK2/MAPK signaling pathways, with a cooperating role for MUC1. In response to DSS, MUC13 was protective during the acute phase whereas it caused more harm upon chronic colitis. The pathways accounting for the MUC13-mediated barrier dysfunction were also altered upon inflammation in IBD patients. These novel findings indicate an active role for aberrant MUC13 signaling inducing intestinal barrier dysfunction upon inflammation with MUC1 as collaborating partner.

Aims: To gain insights into the underlying mechanisms of NTP therapy sensitivity and resistance, using the first-ever NTP-resistant cell line derived from sensitive melanoma cells (A375)., Methods: Melanoma cells were exposed to NTP and re-cultured for 12 consecutive weeks before evaluation against the parental control cells. Whole transcriptome sequencing analysis was performed to identify differentially expressed genes and enriched molecular pathways. Glucose uptake, extracellular lactate, media acidification, and mitochondrial respiration was analyzed to determine metabolic changes. Cell death inhibitors were used to assess the NTP-induced cell death mechanisms, and apoptosis and ferroptosis was further validated via Annexin V, Caspase 3/7, and lipid peroxidation analysis., Results: Cells continuously exposed to NTP became 10 times more resistant to NTP compared to the parental cell line of the same passage, based on their half-maximal inhibitory concentration (IC50). Sequencing and metabolic analysis indicated that NTP-resistant cells had a preference towards aerobic glycolysis, while cell death analysis revealed that NTP-resistant cells exhibited less apoptosis but were more vulnerable to lipid peroxidation and ferroptosis., Conclusions: A preference towards aerobic glycolysis and ferroptotic cell death are key physiological changes in NTP-resistance cells, which opens new avenues for further, in-depth research into other cancer types., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Introduction: Digital health interventions may support physical activity among solid organ transplant recipients. These interventions should be designed with users in mind, including healthcare professionals who counsel transplant recipients on physical activity to ensure acceptance and to promote an optimal user experience. The purpose of this study was to explore the perspectives of health care providers on the features of digital health interventions that would be useful in the promotion, implementation, and maintenance of physical activity among solid organ transplant recipients. Methods: This qualitative, cross-sectional study used semistructured interviews that were conducted remotely, via videoconferencing software, with providers who worked with transplant recipients. Interviews were transcribed, and an iterative-inductive, thematic analysis was used to identify common themes. Data were coded using NVivo software. Findings: Thirteen providers participated in this study. Four main themes were identified: (a) physical activity and exercise features (eg, physical activity guidelines, and exercise instructions); (b) credibility; (c) self-management; and (d) user engagement. Potential barriers to using digital health interventions included staffing requirements, professional regulatory issues, cost, perceived low patient motivation to use, and lack of technological literacy or access. Discussion: Digital health interventions were perceived to be a potential adjunct to current physical activity counseling practices, and part of an innovative strategy to address identified barriers to physical activity participation in solid organ transplant recipients.

Reverse vaccinology (RV) marked an outstanding improvement in vaccinology employing bioinformatics tools to extract effective features from protein sequences to drive the selection of potential vaccine candidates (Rappuoli, Curr Opin Microbiol 3(5):445-450, 2000). Pioneered by Rino Rappuoli and first used against serogroup B meningococcus, since then, it has been used on several other bacterial vaccines, varying during time the adopted bioinformatics tools. Based on our experience in the field of RV and following an extensive literature review, we consolidate a lean RV pipeline of publicly available bioinformatic tools whose usage is described in this contribution. The protein features, whose extraction is reported in this contribution, can be also the input in a matrix format for machine learning-based approaches., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Radiotherapy is the standard of care for breast cancer. However, surviving radioresistant cells can repopulate following treatment and provoke relapse. Better understanding of the molecular mechanisms of radiation resistance may help to improve treatment of radioresistant tumours. To emulate radiation therapy at the cellular level, we exposed MCF7 breast cancer cells to daily radiation doses of 2 Gy up to an accumulated dose of 20 Gy. Fractionally irradiated cells (FIR20) displayed increased clonogenic survival and population doubling time as compared with age-matched sham-irradiated cells and untreated parental MCF7 cells. RNA-sequencing revealed a core signature of 229 mRNAs and 7 circular RNAs of which the expression was significantly altered in FIR20 cells. Dysregulation of several top genes was mirrored at the protein level. The FIR20 cell transcriptome overlapped significantly with canonical radiation response signatures and demonstrated a remarkable commonality with radiation and endocrine therapy resistance expression profiles, suggesting crosstalk between both acquired resistance pathways, as indicated by reduced sensitivity to tamoxifen cytotoxicity of FIR20 cells. Using predictive analyses and functional enrichment, we identified a gene-regulatory network that promotes stemness and inflammatory signalling in FIR20 cells. We propose that these phenotypic traits render breast cancer cells more radioresistant but may at the same time serve as potential targets for combination therapies., (© 2022 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The Plasmodium vivax reticulocyte invasion process is still poorly understood, with only a few receptor-ligand interactions identified to date. Individuals with the Southeast Asian ovalocytosis (SAO) phenotype have a deletion in the band 3 protein on the surface of erythrocytes, and are reported to have a lower incidence of clinical P. vivax malaria. Based on this observation, band 3 has been put forward as a receptor for P. vivax invasion, although direct proof is still lacking. In this study, we combined functional ex vivo invasion assays and transcriptome sequencing to uncover a band 3-mediated invasion pathway in P. vivax and potential band 3 ligands. Invasion by P. vivax field isolates was 67%-71% lower in SAO reticulocytes compared with non-SAO reticulocytes. Reticulocyte invasion was decreased by 40% and 27%-31% when blocking with an anti-band 3 polyclonal antibody and a PvTRAg38 peptide, respectively. To identify new band 3 receptor candidates, we mRNA-sequenced schizont-stage isolates used in the invasion assays, and observed high transcriptional variability in multigene and invasion-related families. Transcriptomes of isolates with low or high dependency on band 3 for invasion were compared by differential expression analysis, which produced a list of band 3 ligand candidates with high representation of PvTRAg genes. Our ex vivo invasion assays have demonstrated that band 3 is a P. vivax invasion receptor and confirm previous in vitro studies showing binding between PvTRAg38 and band 3, although the lower and variable inhibition levels observed suggest the involvement of other ligands. By coupling transcriptomes and invasion phenotypes from the same isolates, we identified a list of band 3 ligand candidates, of which the overrepresented PvTRAg genes are the most promising for future research., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 De Meulenaere, Prajapati, Villasis, Cuypers, Kattenberg, Kasian, Laman, Robinson, Gamboa, Laukens and Rosanas-Urgell.)

Aneuploidy causes system-wide disruptions in the stochiometric balances of transcripts, proteins, and metabolites, often resulting in detrimental effects for the organism. The protozoan parasite Leishmania has an unusually high tolerance for aneuploidy, but the molecular and functional consequences for the pathogen remain poorly understood. Here, we addressed this question in vitro and present the first integrated analysis of the genome, transcriptome, proteome, and metabolome of highly aneuploid Leishmania donovani strains. Our analyses unambiguously establish that aneuploidy in Leishmania proportionally impacts the average transcript- and protein abundance levels of affected chromosomes, ultimately correlating with the degree of metabolic differences between closely related aneuploid strains. This proportionality was present in both proliferative and non-proliferative in vitro promastigotes. However, as in other Eukaryotes, we observed attenuation of dosage effects for protein complex subunits and in addition, non-cytoplasmic proteins. Differentially expressed transcripts and proteins between aneuploid Leishmania strains also originated from non-aneuploid chromosomes. At protein level, these were enriched for proteins involved in protein metabolism, such as chaperones and chaperonins, peptidases, and heat-shock proteins. In conclusion, our results further support the view that aneuploidy in Leishmania can be adaptive. Additionally, we believe that the high karyotype diversity in vitro and absence of classical transcriptional regulation make Leishmania an attractive model to study processes of protein homeostasis in the context of aneuploidy and beyond., Competing Interests: The authors have declared that no competing interests exist.

Studies have shown that variants in bedaquiline-resistance genes can occur in isolates from bedaquiline-naive patients. We assessed the prevalence of variants in all bedaquiline-candidate-resistance genes in bedaquiline-naive patients, investigated the association between these variants and lineage, and the effect on phenotype. We used whole-genome sequencing to identify variants in bedaquiline-resistance genes in isolates from 509 bedaquiline treatment naive South African tuberculosis patients. A phylogenetic tree was constructed to investigate the association with the isolate lineage background. Bedaquiline MIC was determined using the UKMYC6 microtiter assay. Variants were identified in 502 of 509 isolates (98.6%), with the highest (85%) prevalence of variants in the Rv0676c ( mmpL5 ) gene. We identified 36 unique variants, including 19 variants not reported previously. Only four isolates had a bedaquiline MIC equal to or above the epidemiological cut-off value of 0.25 μg/mL. Phylogenetic analysis showed that 14 of the 15 variants observed more than once occurred monophyletically in one Mycobacterium tuberculosis (sub)lineage. The bedaquiline MIC differed between isolates belonging to lineage 2 and 4 (Fisher's exact test, P  = 0.0004). The prevalence of variants in bedaquiline-resistance genes in isolates from bedaquiline-naive patients is high, but very few (<2%) isolates were phenotypically resistant. We found an association between variants in bedaquiline resistance genes and Mycobacterium tuberculosis (sub)lineage, resulting in a lineage-dependent difference in bedaquiline phenotype. Future studies should investigate the impact of the presence of variants on bedaquiline-resistance acquisition and treatment outcome.

Melanoma remains a deadly cancer despite significant advances in immune checkpoint blockade and targeted therapies. The incidence of melanoma is also growing worldwide, which highlights the need for novel treatment options and strategic combination of therapies. Here, we investigate non-thermal plasma (NTP), an ionized gas, as a promising, therapeutic option. In a melanoma mouse model, direct treatment of tumors with NTP results in reduced tumor burden and prolonged survival. Physical characterization of NTP treatment in situ reveals the deposited NTP energy and temperature associated with therapy response, and whole transcriptome analysis of the tumor identified several modulated pathways. NTP treatment also enhances the cancer-immunity cycle, as immune cells in both the tumor and tumor-draining lymph nodes appear more stimulated to perform their anti-cancer functions. Thus, our data suggest that local NTP therapy stimulates systemic, anti-cancer immunity. We discuss, in detail, how these fundamental insights will help direct the translation of NTP technology into the clinic and inform rational combination strategies to address the challenges in melanoma therapy., (© 2022 The Authors. Bioengineering & Translational Medicine published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.)

The implementation of prospective drug resistance (DR) studies in the research-and-development (R&D) pipeline is a common practice for many infectious diseases but not for neglected tropical diseases (NTDs). Here, we explored and demonstrated the importance of this approach using as paradigms Leishmania donovani, the etiological agent of visceral leishmaniasis (VL), and TCMDC-143345, a promising compound of the GlaxoSmithKline (GSK) "Leishbox" to treat VL. We experimentally selected resistance to TCMDC-143345 in vitro and characterized resistant parasites at the genomic and phenotypic levels. We found that it took more time to develop resistance to TCMDC-143345 than to other drugs in clinical use and that there was no cross-resistance to these drugs, suggesting a new and unique mechanism. By whole-genome sequencing, we found two mutations in the gene encoding the L. donovani dynamin-1-like protein (LdoDLP1) that were fixed at the highest drug pressure. Through phylogenetic analysis, we identified LdoDLP1 as a family member of the dynamin-related proteins, a group of proteins that impacts the shapes of biological membranes by mediating fusion and fission events, with a putative role in mitochondrial fission. We found that L. donovani lines genetically engineered to harbor the two identified LdoDLP1 mutations were resistant to TCMDC-143345 and displayed altered mitochondrial properties. By homology modeling, we showed how the two LdoDLP1 mutations may influence protein structure and function. Taken together, our data reveal a clear involvement of LdoDLP1 in the adaptation/reduced susceptibility of L. donovani to TCMDC-143345. IMPORTANCE Humans and their pathogens are continuously locked in a molecular arms race during which the eventual emergence of pathogen drug resistance (DR) seems inevitable. For neglected tropical diseases (NTDs), DR is generally studied retrospectively once it has already been established in clinical settings. We previously recommended to keep one step ahead in the host-pathogen arms race and implement prospective DR studies in the R&D pipeline, a common practice for many infectious diseases but not for NTDs. Here, using Leishmania donovani, the etiological agent of visceral leishmaniasis (VL), and TCMDC-143345, a promising compound of the GSK Leishbox to treat VL, as paradigms, we experimentally selected resistance to the compound and proceeded to genomic and phenotypic characterization of DR parasites. The results gathered in the present study suggest a new DR mechanism involving the L. donovani dynamin-1-like protein (LdoDLP1) and demonstrate the practical relevance of prospective DR studies.

The next-generation, short-read sequencing technologies that generate comprehensive, whole-genome data with single nucleotide resolution have already advanced tuberculosis diagnosis, treatment, surveillance, and source investigation. Their high costs, tedious and lengthy processes, and large equipment remain major hurdles for research use in high tuberculosis burden countries and implementation into routine care. The portable next-generation sequencing devices developed by Oxford Nanopore Technologies (ONT) are attractive alternatives due to their long-read sequence capability, compact low-cost hardware, and continued improvements in accuracy and throughput. A systematic review of the published literature demonstrated limited uptake of ONT sequencing in tuberculosis research and clinical care. Of the 12 eligible articles presenting ONT sequencing data on at least one Mycobacterium tuberculosis sample, four addressed software development for long-read ONT sequencing data with potential applications for M. tuberculosis. Only eight studies presented results of ONT sequencing of M. tuberculosis, of which five performed whole-genome and three did targeted sequencing. Based on these findings, we summarize the standard processes, reflect on the current limitations of ONT sequencing technology, and the research needed to overcome the main hurdles. The low capital cost, portable nature and continued improvement in the performance of ONT sequencing make it an attractive option for sequencing for research and clinical care, but limited data are available on its application in the tuberculosis field. Important research investment is needed to unleash the full potential of ONT sequencing for tuberculosis research and care.

Disease relapse and therapy resistance remain key challenges in treating multiple myeloma. Underlying (epi-)mutational events can promote myelomagenesis and contribute to multi-drug and apoptosis resistance. Therefore, compounds inducing ferroptosis, a form of iron and lipid peroxidation-regulated cell death, are appealing alternative treatment strategies for multiple myeloma and other malignancies. Both ferroptosis and the epigenetic machinery are heavily influenced by oxidative stress and iron metabolism changes. Yet, only a limited number of epigenetic enzymes and modifications have been identified as ferroptosis regulators. In this study, we found that MM1 multiple myeloma cells are sensitive to ferroptosis induction and epigenetic reprogramming by RSL3, irrespective of their glucocorticoid-sensitivity status. LC-MS/MS analysis revealed the formation of non-heme iron-histone complexes and altered expression of histone modifications associated with DNA repair and cellular senescence. In line with this observation, EPIC BeadChip measurements of significant DNA methylation changes in ferroptotic myeloma cells demonstrated an enrichment of CpG probes located in genes associated with cell cycle progression and senescence, such as Nuclear Receptor Subfamily 4 Group A member 2 (NR4A2). Overall, our data show that ferroptotic cell death is associated with an epigenomic stress response that might advance the therapeutic applicability of ferroptotic compounds.

Background: The multifactorial nature of non-alcoholic fatty liver disease cannot be explained solely by genetic factors. Recent evidence revealed that DNA methylation changes take place at proximal promoters within susceptibility genes. This emphasizes the need for integrating multiple data types to provide a better understanding of the disease's pathogenesis. One such candidate gene is paraoxonase-1 (PON1). Substantial interindividual differences in PON1 are apparent and could influence disease risk later in life. The aim of this study was therefore to determine the different regulatory aspects of PON1 variability and to examine them in relation to the predisposition to obesity-associated fatty liver disease., Results: A targeted multi-omics approach was applied to investigate the interplay between PON1 genetic variants, promoter methylation, expression profile and enzymatic activity in an adult patient cohort with extensive metabolic and hepatic characterisation including liver biopsy. Alterations in PON1 status were shown to correlate with waist-to-hip ratio and relevant features of liver pathology. Particularly, the regulatory polymorphism rs705379:C > T was strongly associated with more severe liver disease. Multivariable data analysis furthermore indicated a significant association of combined genetic and epigenetic PON1 regulation. This identified relationship postulates a role for DNA methylation as a mediator between PON1 genetics and expression, which is believed to further influence liver disease progression via modifications in PON1 catalytic efficiency., Conclusions: Our findings demonstrate that vertical data-integration of genetic and epigenetic regulatory mechanisms generated a more in-depth understanding of the molecular basis underlying the development of obesity-associated fatty liver disease. We gained novel insights into how NAFLD classification and outcome are orchestrated, which could not have been obtained by exclusively considering genetic variation., (© 2021. The Author(s).)

In this meeting overview, we summarise the scientific program and organisation of the 16th International Society for Computational Biology Student Council Symposium in 2020 (ISCB SCS2020). This symposium was the first virtual edition in an uninterrupted series of symposia that has been going on for 15 years, aiming to unite computational biology students and early career researchers across the globe., Competing Interests: No competing interests were disclosed., (Copyright: © 2021 Cuypers WL et al.)

Auranofin (AF) is an FDA-approved antirheumatic drug with anticancer properties that acts as a thioredoxin reductase 1 (TrxR) inhibitor. The exact mechanisms through which AF targets cancer cells remain elusive. To shed light on the mode of action, this study provides an in-depth analysis on the molecular mechanisms and immunogenicity of AF-mediated cytotoxicity in the non-small cell lung cancer (NSCLC) cell line NCI-H1299 (p53 Null) and its two isogenic derivates with mutant p53 R175H or R273H accumulation. TrxR is highly expressed in a panel of 72 NSCLC patients, making it a valid druggable target in NSCLC for AF. The presence of mutant p53 overexpression was identified as an important sensitizer for AF in (isogenic) NSCLC cells as it was correlated with reduced thioredoxin (Trx) levels in vitro. Transcriptome analysis revealed dysregulation of genes involved in oxidative stress response, DNA damage, granzyme A (GZMA) signaling and ferroptosis. Although functionally AF appeared a potent inhibitor of GPX4 in all NCI-H1299 cell lines, the induction of lipid peroxidation and consequently ferroptosis was limited to the p53 R273H expressing cells. In the p53 R175H cells, AF mainly induced large-scale DNA damage and replication stress, leading to the induction of apoptotic cell death rather than ferroptosis. Importantly, all cell death types were immunogenic since the release of danger signals (ecto-calreticulin, ATP and HMGB1) and dendritic cell maturation occurred irrespective of (mutant) p53 expression. Finally, we show that AF sensitized cancer cells to caspase-independent natural killer cell-mediated killing by downregulation of several key targets of GZMA. Our data provides novel insights on AF as a potent, clinically available, off-patent cancer drug by targeting mutant p53 cancer cells through distinct cell death mechanisms (apoptosis and ferroptosis). In addition, AF improves the innate immune response at both cytostatic (natural killer cell-mediated killing) and cytotoxic concentrations (dendritic cell maturation)., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

In high-throughput omics disciplines like transcriptomics, researchers face a need to assess the quality of an experiment prior to an in-depth statistical analysis. To efficiently analyze such voluminous collections of data, researchers need triage methods that are both quick and easy to use. Such a normalization method for relative quantitation, CONSTANd, was recently introduced for isobarically-labeled mass spectra in proteomics. It transforms the data matrix of abundances through an iterative, convergent process enforcing three constraints: (I) identical column sums; (II) each row sum is fixed (across matrices) and (III) identical to all other row sums. In this study, we investigate whether CONSTANd is suitable for count data from massively parallel sequencing, by qualitatively comparing its results to those of DESeq2. Further, we propose an adjustment of the method so that it may be applied to identically balanced but differently sized experiments for joint analysis. We find that CONSTANd can process large data sets at well over 1 million count records per second whilst mitigating unwanted systematic bias and thus quickly uncovering the underlying biological structure when combined with a PCA plot or hierarchical clustering. Moreover, it allows joint analysis of data sets obtained from different batches, with different protocols and from different labs but without exploiting information from the experimental setup other than the delineation of samples into identically processed sets (IPSs). CONSTANd's simplicity and applicability to proteomics as well as transcriptomics data make it an interesting candidate for integration in multi-omics workflows., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The natural course of chronic hepatitis B virus (HBV) infections follows distinct clinical disease phases, characterized by fluctuating levels of serum HBV DNA and ALT. The immune cells and their features that govern these clinical disease transitions remain unknown. In the current study, we performed RNA sequencing on purified B cells from blood (n = 42) and liver (n = 10) of healthy controls and chronic HBV patients. We found distinct gene expression profiles between healthy controls and chronic HBV patients, as evidenced by 190 differentially expressed genes (DEG), but also between the clinical phenotypes of a chronic HBV infection (17-110 DEG between each phase). Numerous immune pathways, including the B cell receptor pathway were upregulated in liver B cells when compared to peripheral B cells. Further investigation of the detected DEG suggested an activation of B cells during HBeAg seroconversion and an active regulation of B cell signalling in the liver., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Multiple myeloma (MM) is a hematological malignancy characterized by plasma cells' uncontrolled growth. The major barrier in treating MM is the occurrence of primary and acquired therapy resistance to anticancer drugs . Often, this therapy resistance is associated with constitutive hyperactivation of tyrosine kinase signaling. Novel covalent kinase inhibitors, such as the clinically approved BTK inhibitor ibrutinib (IBR) and the preclinical phytochemical withaferin A (WA), have, therefore, gained pharmaceutical interest. Remarkably, WA is more effective than IBR in killing BTK-overexpressing glucocorticoid (GC)-resistant MM1R cells. To further characterize the kinase inhibitor profiles of WA and IBR in GC-resistant MM cells, we applied phosphopeptidome- and transcriptome-specific tyrosine kinome profiling. In contrast to IBR, WA was found to reverse BTK overexpression in GC-resistant MM1R cells. Furthermore, WA-induced cell death involves covalent cysteine targeting of Hinge-6 domain type tyrosine kinases of the kinase cysteinome classification, including inhibition of the hyperactivated BTK. Covalent interaction between WA and BTK could further be confirmed by biotin-based affinity purification and confocal microscopy. Similarly, molecular modeling suggests WA preferably targets conserved cysteines in the Hinge-6 region of the kinase cysteinome classification, favoring inhibition of multiple B-cell receptors (BCR) family kinases. Altogether, we show that WA's promiscuous inhibition of multiple BTK family tyrosine kinases represents a highly effective strategy to overcome GC-therapy resistance in MM.

Aim : The purpose of this manuscript is to study the potential characteristics of acquired nutlin-3 resistant non-small cell lung cancer cells (NSCLC). Nutlin-3 is an inhibitor of the murine-double minute 2 protein, the main negative regulator of wild type p53, of which several derivatives are currently in clinical development. Methods : A549 NSCLC cells were exposed to increasing concentrations of nutlin-3 for a period of 18 weeks. Monoclonal derivates were cultured, and the most resistance subclone was selected for whole transcriptome analysis. Gene set enrichment analysis was performed on differentially expressed genes between A549 nutlin-3 resistant cancer cells and the parental A549 p53 wild type cancer cells. Relevant findings were validated at the gene, protein and/or functional level. Results : All nutlin-3 resistant subclones acquired mutations in the TP53 gene, resulting in overexpression of the mutant p53 protein. The most resistant subclone was enriched for genes related to epithelial to mesenchymal transition (EMT), resulting in increased migratory and invasive potential. Furthermore, these cells were enriched in genes related to inflammation, tissue remodelling, and angiogenesis. Importantly, expression of several immune checkpoints, including PD-L1 and PD-L2, was significantly upregulated, and cisplatin-induced cell death was reduced. Conclusion : Transcriptome analysis of a highly nutlin-3 resistant A549 subclone shows the relevance of studying (1) resistance to standard of care chemotherapy; (2) secretion of immunomodulating chemo- and cytokines; (3) immune checkpoint expression; and (4) EMT and invasion in nutlin-3 resistant cancer cells in addition to acquired mutations in the TP53 gene., Competing Interests: All authors declared that there are no conflicts of interest., (© The Author(s) 2021.)

Susceptibility for leishmaniasis is largely dependent on host genetic and immune factors. Despite the previously described association of human leukocyte antigen (HLA) gene cluster variants as genetic susceptibility factors for leishmaniasis, little is known regarding the mechanisms that underpin these associations. To better understand this underlying functionality, we first collected all known leishmaniasis-associated HLA variants in a thorough literature review. Next, we aligned and compared the protection- and risk-associated HLA-DRB1 allele sequences. This identified several amino acid polymorphisms that distinguish protection- from risk-associated HLA-DRB1 alleles. Subsequently, T cell epitope binding predictions were carried out across these alleles to map the impact of these polymorphisms on the epitope binding repertoires. For these predictions, we used epitopes derived from entire proteomes of multiple Leishmania species. Epitopes binding to protection-associated HLA-DRB1 alleles shared common binding core motifs, mapping to the identified HLA-DRB1 amino acid polymorphisms. These results strongly suggest that HLA polymorphism, resulting in differential antigen presentation, affects the association between HLA and leishmaniasis disease development. Finally, we established a valuable open-access resource of putative epitopes. A set of 14 HLA-unrestricted strong-binding epitopes, conserved across species, was prioritized for further epitope discovery in the search for novel subunit-based vaccines.

Cytosine C5 methylation is an important epigenetic control mechanism in a wide array of eukaryotic organisms and generally carried out by proteins of the C-5 DNA methyltransferase family (DNMTs). In several protozoans, the status of this mechanism remains elusive, such as in Leishmania , the causative agent of the disease leishmaniasis in humans and a wide array of vertebrate animals. In this work, we showed that the Leishmania donovani genome contains a C-5 DNA methyltransferase ( DNMT ) from the DNMT6 subfamily, whose function is still unclear, and verified its expression at the RNA level. We created viable overexpressor and knock-out lines of this enzyme and characterized their genome-wide methylation patterns using whole-genome bisulfite sequencing, together with promastigote and amastigote control lines. Interestingly, despite the DNMT6 presence, we found that methylation levels were equal to or lower than 0.0003% at CpG sites, 0.0005% at CHG sites, and 0.0126% at CHH sites at the genomic scale. As none of the methylated sites were retained after manual verification, we conclude that there is no evidence for DNA methylation in this species. We demonstrated that this difference in DNA methylation between the parasite (no detectable DNA methylation) and the vertebrate host (DNA methylation) allowed enrichment of parasite vs. host DNA using methyl-CpG-binding domain columns, readily available in commercial kits. As such, we depleted methylated DNA from mixes of Leishmania promastigote and amastigote DNA with human DNA, resulting in average Leishmania: human enrichments from 62× up to 263×. These results open a promising avenue for unmethylated DNA enrichment as a pre-enrichment step before sequencing Leishmania clinical samples.

Only recently it was discovered that haemoglobin (Hb) belongs to the standard gene repertoire of insects, although their tracheal system is used for respiration. A classical oxygen-carrying function of Hb is only obvious for hexapods living in hypoxic environments. In other insect species, including the common fruit fly Drosophila melanogaster, the physiological role of Hb is yet unclear. Here, we study recombinant haemoglobin from the European honeybee Apis mellifera (Ame) and the malaria mosquito Anopheles gambiae (Aga). Spectroscopic evidence shows that both proteins can be classified as hexacoordinate Hbs with a strong affinity for the distal histidine. AgaHb1 is proposed to play a role in oxygen transport or sensing based on its multimeric state, slow autoxidation, and small but significant amount of five-coordinated haem in the deoxy ferrous form. AmeHb appears to behave more like vertebrate neuroglobin with a complex function given its diversified distribution in the genome., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Visceral leishmaniasis (VL) treatment in HIV patients very often fails and is followed by high relapse and case-fatality rates. Hence, treatment efficacy assessment is imperative but based on invasive organ aspiration for parasite detection. In the search of a less-invasive alternative and because the host immune response is pivotal for treatment outcome in immunocompromised VL patients, we studied changes in the whole blood transcriptional profile of VL-HIV patients during treatment., Methods: Embedded in a clinical trial in Northwest Ethiopia, RNA-Seq was performed on whole blood samples of 28 VL-HIV patients before and after completion of a 29-day treatment regimen of AmBisome or AmBisome/miltefosine. Pathway analyses were combined with a machine learning approach to establish a clinically-useful 4-gene set., Findings: Distinct signatures of differentially expressed genes between D0 and D29 were identified for patients who failed treatment and were successfully treated. Pathway analyses in the latter highlighted a downregulation of genes associated with host cellular activity and immunity, and upregulation of antimicrobial peptide activity in phagolysosomes. No signs of disease remission nor pathway enrichment were observed in treatment failure patients. Next, we identified a 4-gene pre-post signature (PRSS33, IL10, SLFN14, HRH4) that could accurately discriminate treatment outcome at end of treatment (D29), displaying an average area-under-the-ROC-curve of 0.95 (CI: 0.75-1.00)., Interpretation: A simple blood-based signature thus holds significant promise to facilitate treatment efficacy monitoring and provide an alternative test-of-cure to guide patient management in VL-HIV patients., Funding: Project funding was provided by the AfricoLeish project, supported by the European Union Seventh Framework Programme (EU FP7)., Competing Interests: Declaration of Competing Interest The authors declare that there are no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

The C-terminal variants G1 and G2 of apolipoprotein L1 (APOL1) confer human resistance to the sleeping sickness parasite Trypanosoma rhodesiense, but they also increase the risk of kidney disease. APOL1 and APOL3 are death-promoting proteins that are partially associated with the endoplasmic reticulum and Golgi membranes. We report that in podocytes, either APOL1 C-terminal helix truncation (APOL1Δ) or APOL3 deletion (APOL3KO) induces similar actomyosin reorganization linked to the inhibition of phosphatidylinositol-4-phosphate [PI(4)P] synthesis by the Golgi PI(4)-kinase IIIB (PI4KB). Both APOL1 and APOL3 can form K + channels, but only APOL3 exhibits Ca 2+ -dependent binding of high affinity to neuronal calcium sensor-1 (NCS-1), promoting NCS-1-PI4KB interaction and stimulating PI4KB activity. Alteration of the APOL1 C-terminal helix triggers APOL1 unfolding and increased binding to APOL3, affecting APOL3-NCS-1 interaction. Since the podocytes of G1 and G2 patients exhibit an APOL1Δ or APOL3KO-like phenotype, APOL1 C-terminal variants may induce kidney disease by preventing APOL3 from activating PI4KB, with consecutive actomyosin reorganization of podocytes., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Aims: Structural and functional characterization of the globin-coupled sensors (GCSs) from Azotobacter vinelandii ( Av GReg) and Bordetella pertussis ( Bpe GReg). Results: Ultraviolet/visible and resonance Raman spectroscopies confirm the presence in Av GReg and Bpe GReg of a globin domain capable of reversible gaseous ligand binding. In Av GReg, an influence of the transmitter domain on the heme proximal region of the globin domain can be seen, and k ' CO is higher than for other GCSs. The O 2 binding kinetics suggests the presence of an open and a closed conformation. As for Bpe GReg, the fully oxygenated Av GReg show a very high diguanylate cyclase activity. The carbon monoxide rebinding to Bpe GReg indicates that intra- and intermolecular interactions influence the ligand binding. The globin domains of both proteins ( Av GReg globin domain and Bpe GRegGb with cysteines (Cys16, 45, 114, 154) mutated to serines [ Bpe GReg-Gb*]) share the same GCS fold, a similar proximal but a different distal side structure. They homodimerize through a G-H helical bundle as in other GCSs. However, Bpe GReg-Gb* shows also a second dimerization mode. Innovation: This article extends our knowledge on the GCS proteins and contributes to a better understanding of the GCSs role in the formation of bacterial biofilms. Conclusions: Av GReg and Bpe GReg conform to the GCS family, share a similar overall structure, but they have different properties in terms of the ligand binding. In particular, Av GReg shows an open and a closed conformation that in the latter form will very tightly bind oxygen. Bpe GReg has only one closed conformation. In both proteins, it is the fully oxygenated GCS form that catalyzes the production of the second messenger.

This article describes the motivation, origin and evolution of the student symposia series organised by the ISCB Student Council. The meeting series started thirteen years ago in Madrid and has spread to four continents. The article concludes with the highlights of the most recent edition of annual Student Council Symposium held in conjunction with the 25th Conference on Intelligent Systems for Molecular Biology and the 16th European Conference on Computational Biology, in Prague, in July 2017.

Leishmania donovani is the responsible agent for visceral leishmaniasis (VL) in the Indian subcontinent (ISC). The disease is lethal without treatment and causes 0.2 to 0.4 million cases each year. Recently, reports of VL in Nepalese hilly districts have increased as well as VL cases caused by L. donovani from the ISC1 genetic group, a new and emerging genotype. In this study, we perform for the first time an integrated, untargeted genomics and metabolomics approach to characterize ISC1, in comparison with the Core Group (CG), main population that drove the most recent outbreak of VL in the ISC. We show that the ISC1 population is very different from the CG, both at genome and metabolome levels. The genomic differences include SNPs, CNV and small indels in genes coding for known virulence factors, immunogens and surface proteins. Both genomic and metabolic approaches highlighted dissimilarities related to membrane lipids, the nucleotide salvage pathway and the urea cycle in ISC1 versus CG. Many of these pathways and molecules are important for the interaction with the host/extracellular environment. Altogether, our data predict major functional differences in ISC1 versus CG parasites, including virulence. Therefore, particular attention is required to monitor the fate of this emerging ISC1 population in the ISC, especially in a post-VL elimination context., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

The flame retardant, 9,10-dihydro-9-oxa-10-phosphaphenanthrene 10-oxide (DOPO), has been receiving great interest given its superior fire protection properties, and its predicted low level of persistence, bioaccumulation, and toxicity. However, empirical toxicological data that are essential for a complete hazard assessment are severely lacking. In this study, we attempted to identify the potential toxicological modes of action by transcriptome (RNA-seq) profiling of the human liver hepatocellular carcinoma cell line, HepG2. Such insight may help in identifying compounds of concern and potential toxicological phenotypes. DOPO was found to have little cytotoxic potential, with lower effective concentrations compared to other flame retardants studied in the same cell line. Differentially expressed genes revealed a wide range of molecular effects including changes in protein, energy, DNA, and lipid metabolism, along with changes in cellular stress response pathways. In response to 250 μM DOPO, the most perturbed biological processes were fatty acid metabolism, androgen metabolism, glucose transport, and renal function and development, which is in agreement with other studies that observed similar effects of other flame retardants in other species. However, treatment with 2.5 μM DOPO resulted in very few differentially expressed genes and failed to indicate any potential effects on biology, despite such concentrations likely being orders of magnitude greater than would be encountered in the environment. This, together with the low levels of cytotoxicity, supports the potential replacement of the current flame retardants by DOPO, although further studies are needed to establish the nephrotoxicity and endocrine disruption of DOPO.