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. 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. 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.
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.
Malaria is caused by parasites of the genus
, 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
DNA. We used the Oxford Nanopore Technologies' adaptive sampling feature to circumvent this problem and enrich
reads directly during the sequencing run. We demonstrate that adaptive nanopore sequencing efficiently enriches
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.
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).
Display omitted
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.
During the last decade miltefosine (MIL) has been used as first-line treatment for visceral leishmaniasis in endemic areas with antimonial resistance, but a decline in clinical effectiveness is now ...being reported. While only two MIL-resistant Leishmania infantum strains from HIV co-infected patients have been documented, phenotypic MIL-resistance for L. donovani has not yet been identified in the laboratory. Hence, a better understanding of the factors contributing to increased MIL-treatment failure is necessary. Given the paucity of defined MIL-resistant L. donovani clinical isolates, this study used an experimental amastigote-selected MIL-resistant L. infantum isolate (LEM3323). In-depth exploration of the MIL-resistant phenotype was performed by coupling genomic with phenotypic data to gain insight into gene function and the mutant phenotype. A naturally MIL-resistant L. infantum clinical isolate (LEM5159) was included to compare both datasets. Phenotypically, resistance was evaluated by determining intracellular amastigote susceptibility in vitro and actual MIL-uptake. Genomic analysis provided supportive evidence that the resistance selection model on intracellular amastigotes can be a good proxy for the in vivo field situation since both resistant strains showed mutations in the same inward transporter system responsible for the acquired MIL-resistant phenotype. In line with previous literature findings in promastigotes, our data confirm a defective import machinery through inactivation of the LiMT/LiRos3 protein complex as the main mechanism for MIL-resistance also in intracellular amastigotes. Whole genome sequencing analysis of LEM3323 revealed a 2 base pair deletion in the LiMT gene that led to the formation an early stop codon and a truncation of the LiMT protein. Interestingly, LEM5159 revealed mutations in both the LiMT and LiRos3 genes, resulting in an aberrant expression of the LiMT protein. To verify that these mutations were indeed accountable for the acquired resistance, transfection experiments were performed to re-establish MIL-susceptibility. In LEM3323, susceptibility was restored upon expression of a LiMT wild-type gene, whereas the MIL-susceptibility of LEM5159 could be reversed after expression of the LiRos3 wild-type gene. The aberrant expression profile of the LiMT protein could be restored upon rescue of the LiRos3 gene both in the LEM5159 clinical isolate and a ΔLiRos3 strain, showing that expression of LdMT is dependent on LdRos3 expression. The present findings clearly corroborate the pivotal role of the LiMT/LiRos3 complex in resistance towards MIL.
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 Ca2+-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.
Display omitted
•C-terminal helix alteration unfolds APOL1, increasing APOL1 interaction with APOL3•APOL3 binds to NCS-1, promoting NCS-1-PI4KB interaction and PI4KB activation•C-terminal APOL1 variants interfere with APOL3-NCS-1 interaction, inactivating PI4KB•PI4KB inactivation occurs in podocytes from kidney disease patients with APOL1 variants
Uzureau et al. propose a molecular explanation for the linkage between resistance to sleeping sickness and high risk of kidney disease in African individuals expressing APOL1 variants. These variants resist neutralization by T. rhodesiense SRA and kill the parasite, but they also indirectly trigger podocyte actomyosin reorganization through interaction with APOL3, inactivating the NCS-1-PI4KB complex.
It was recently hypothesized that Leishmania amastigotes could constitute a semi-quiescent stage characterized by low replication and reduced metabolic activity. This concept developed with ...Leishmania (Leishmania) mexicana and Leishmania (Leishmania) major models might explain numerous clinical and sub-clinical features of Leishmania (Viannia) braziliensis infections, like reactivation of the disease, non-response to chemotherapy or asymptomatic infections. We compared here in vitro the proliferative capability of L. (V.) braziliensis amastigotes and promastigotes, assessed the expression of key molecular parameters and performed metabolomic analysis. We found that contrary to the highly proliferative promastigotes, amastigotes (axenic and intracellular) do not show evidence of extensive proliferation. In parallel, amastigotes showed a significant decrease of (i) the kDNA mini-circle abundance, (ii) the intracellular ATP level, (iii) the ribosomal components: rRNA subunits 18S and 28S α and ribosomal proteins RPS15 and RPL19, (iv) total RNA and protein levels. An untargeted metabolomic study identified clear differences between the different life stages: in comparison to logarithmic promastigotes, axenic amastigotes showed (a) a strong decrease of 14 essential and non-essential amino acids and eight metabolites involved in polyamine synthesis, (b) extensive changes in the phospholipids composition and (c) increased levels of several endogenous and exogenous sterols. Altogether, our results show that L. (V.) braziliensis amastigotes can show a phenotype with negligible rate of proliferation, a lower capacity of biosynthesis, a reduced bio-energetic level and a strongly altered metabolism. Our results pave the way for further exploration of quiescence among amastigotes of this species.
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.
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.
Fractionated irradiation induces a treatment‐resistant stemness phenotype underpinned by interaction between competing endogenous RNAs with both tumor‐promoting and tumor‐suppressing function. Results support cross‐resistance of radioresistant MCF7 breast cancer cells to tamoxifen and presents a gene regulatory circuit associated with treatment resistance and survival in breast cancer patients, that could be evaluated for the discovery of novel targeted therapeutic strategies.
PDF
