CDK4/6 inhibitor-mediated cell overgrowth triggers osmotic and replication stress to promote senescence.

Abnormal increases in cell size are associated with senescence and cell cycle exit. The mechanisms by which overgrowth primes cells to withdraw from the cell cycle remain unknown. We address this question using CDK4/6 inhibitors, which arrest cells in G0/G1 and are licensed to treat advanced HR+/HER2- breast cancer. We demonstrate that CDK4/6-inhibited cells overgrow during G0/G1, causing p38/p53/p21-dependent cell cycle withdrawal. Cell cycle withdrawal is triggered by biphasic p21 induction. The first p21 wave is caused by osmotic stress, leading to p38- and size-dependent accumulation of p21. CDK4/6 inhibitor washout results in some cells entering S-phase. Overgrown cells experience replication stress, resulting in a second p21 wave that promotes cell cycle withdrawal from G2 or the subsequent G1. We propose that the levels of p21 integrate signals from overgrowth-triggered stresses to determine cell fate. This model explains how hypertrophy can drive senescence and why CDK4/6 inhibitors have long-lasting effects in patients.

A conserved metabolic signature associated with response to fast-acting anti-malarial agents.

IMPORTANCE: In malaria drug discovery, understanding the mode of action of lead compounds is important as it helps in predicting the potential emergence of drug resistance in the field when these drugs are eventually deployed. In this study, we have employed metabolomics technologies to characterize the potential targets of anti-malarial drug candidates in the developmental pipeline at NITD. We show that NITD fast-acting leads belonging to spiroindolone and imidazothiadiazole class induce a common biochemical theme in drug-exposed malaria parasites which is similar to another fast-acting, clinically available drug, DHA. These biochemical features which are absent in a slower acting NITD lead (GNF17) point to hemoglobin digestion and inhibition of the pyrimidine pathway as potential action points for these drugs. These biochemical themes can be used to identify and inform on the mode of action of fast drug candidates of similar profiles in future drug discovery programs.

Alignment of multiple metabolomics LC-MS datasets from disparate diseases to reveal fever-associated metabolites.

Acute febrile illnesses are still a major cause of mortality and morbidity globally, particularly in low to middle income countries. The aim of this study was to determine any possible metabolic commonalities of patients infected with disparate pathogens that cause fever. Three liquid chromatography-mass spectrometry (LC-MS) datasets investigating the metabolic effects of malaria, leishmaniasis and Zika virus infection were used. The retention time (RT) drift between the datasets was determined using landmarks obtained from the internal standards generally used in the quality control of the LC-MS experiments. Fitted Gaussian Process models (GPs) were used to perform a high level correction of the RT drift between the experiments, which was followed by standard peakset alignment between the samples with corrected RTs of the three LC-MS datasets. Statistical analysis, annotation and pathway analysis of the integrated peaksets were subsequently performed. Metabolic dysregulation patterns common across the datasets were identified, with kynurenine pathway being the most affected pathway between all three fever-associated datasets.

Using Organic Contaminants to Constrain the Terrestrial Journey of the Martian Meteorite Lafayette.

A key part of the search for extraterrestrial life is the detection of organic molecules since these molecules form the basis of all living things on Earth. Instrument suites such as SHERLOC (Scanning Habitable Environments with Raman and Luminescence for Organics and Chemicals) onboard the NASA Perseverance rover and the Mars Organic Molecule Analyzer onboard the future ExoMars Rosalind Franklin rover are designed to detect organic molecules at the martian surface. However, size, mass, and power limitations mean that these instrument suites cannot yet match the instrumental capabilities available in Earth-based laboratories. Until Mars Sample Return, the only martian samples available for study on Earth are martian meteorites. This is a collection of largely basaltic igneous rocks that have been exposed to varying degrees of terrestrial contamination. The low organic molecule abundance within igneous rocks and the expectation of terrestrial contamination make the identification of martian organics within these meteorites highly challenging. The Lafayette martian meteorite exhibits little evidence of terrestrial weathering, potentially making it a good candidate for the detection of martian organics despite uncertainties surrounding its fall history. In this study, we used ultrapure solvents to extract organic matter from triplicate samples of Lafayette and analyzed these extracts via hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS). Two hundred twenty-four metabolites (organic molecules) were detected in Lafayette at concentrations more than twice those present in the procedural blanks. In addition, a large number of plant-derived metabolites were putatively identified, the presence of which supports the unconfirmed report that Lafayette fell in a semirural location in Indiana. Remarkably, the putative identification of the mycotoxin deoxynivalenol (or vomitoxin), alongside the report that the collector was possibly a student at Purdue University, can be used to identify the most likely fall year as 1919.

Microalgae show a range of responses to exometabolites of foreign species.

Studies on microalgae interspecific interactions have so far focused either on nutrient competition or allelopathic effects due to excreted substances from Harmful Algal Bloom (HAB) species. Evidence from plants, bacteria and specific microalgae groups, point to a range of responses mediated by sensing or direct chemical impact of exometabolites from foreign species. Such processes remain under-investigated, especially in non-HAB microalgae, despite the importance of such knowledge in ecology and industrial applications. Here, we study the directional effect of exometabolites of 4 "foreign" species Heterosigma akashiwo, Phaeocystis sp., Tetraselmis sp. and Thalassiosira sp. to each of three "target" species across a total of 12 treatments. We disentangle these effects from nutrient competition by adding cell free medium of each "foreign" species into our treatment cultures. We measured the biomass response, to the foreign exometabolites, as cell number and photosynthetic biomass (Chla), whereas nutrient use was measured as residual phosphorus (PO4) and intracellular phosphorus (P). Exometabolites from filtrate of foreign species were putatively annotated by untargeted metabolomics analysis and were discussed in association to observed responses of target species. Among others, these metabolites included L-histidinal, Tiliacorine and dimethylsulfoniopropionate (DMSP). Our findings show that species show a range of responses with the most common being biomass suppression, and less frequent biomass enhancement and intracellular P storage. Filtrate from the green microalgae Tetraselmis caused the most pronounced negative effects suggesting that non-HAB species can also cause negative chemical interference. A candidate metabolite inducing this response is L-histidinal which was measured in high abundance uniquely in Tetraselmis and its L-histidine form derived from bacteria was previously confirmed as a microalgal algicidal. H. akashiwo also induced biomass suppression on other microalgae and a candidate metabolite for this response is Tiliacorine, a plant-derived alkaloid with confirmed cytotoxic activity.

Multi Platforms Strategies and Metabolomics Approaches for the Investigation of Comprehensive Metabolite Profile in Dogs with Babesia canis Infection.

Canine babesiosis is an important tick-borne disease worldwide, caused by parasites of the Babesia genus. Although the disease process primarily affects erythrocytes, it may also have multisystemic consequences. The goal of this study was to explore and characterize the serum metabolome, by identifying potential metabolites and metabolic pathways in dogs naturally infected with Babesia canis using liquid and gas chromatography coupled to mass spectrometry. The study included 12 dogs naturally infected with B. canis and 12 healthy dogs. By combining three different analytical platforms using untargeted and targeted approaches, 295 metabolites were detected. The untargeted ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) metabolomics approach identified 64 metabolites, the targeted UHPLC-MS/MS metabolomics approach identified 205 metabolites, and the GC-MS metabolomics approach identified 26 metabolites. Biological functions of differentially abundant metabolites indicate the involvement of various pathways in canine babesiosis including the following: glutathione metabolism; alanine, aspartate, and glutamate metabolism; glyoxylate and dicarboxylate metabolism; cysteine and methionine metabolism; and phenylalanine, tyrosine, and tryptophan biosynthesis. This study confirmed that host-pathogen interactions could be studied by metabolomics to assess chemical changes in the host, such that the differences in serum metabolome between dogs with B. canis infection and healthy dogs can be detected with liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) methods. Our study provides novel insight into pathophysiological mechanisms of B. canis infection.

Transcriptional Shift and Metabolic Adaptations during Leishmania Quiescence Using Stationary Phase and Drug Pressure as Models.

Microorganisms can adopt a quiescent physiological condition which acts as a survival strategy under unfavorable conditions. Quiescent cells are characterized by slow or non-proliferation and a deep downregulation of processes related to biosynthesis. Although quiescence has been described mostly in bacteria, this survival skill is widespread, including in eukaryotic microorganisms. In Leishmania, a digenetic parasitic protozoan that causes a major infectious disease, quiescence has been demonstrated, but the molecular and metabolic features enabling its maintenance are unknown. Here, we quantified the transcriptome and metabolome of Leishmania promastigotes and amastigotes where quiescence was induced in vitro either, through drug pressure or by stationary phase. Quiescent cells have a global and coordinated reduction in overall transcription, with levels dropping to as low as 0.4% of those in proliferating cells. However, a subset of transcripts did not follow this trend and were relatively upregulated in quiescent populations, including those encoding membrane components, such as amastins and GP63, or processes like autophagy. The metabolome followed a similar trend of overall downregulation albeit to a lesser magnitude than the transcriptome. It is noteworthy that among the commonly upregulated metabolites were those involved in carbon sources as an alternative to glucose. This first integrated two omics layers afford novel insight into cell regulation and show commonly modulated features across stimuli and stages.

Mapping immune variation and var gene switching in naive hosts infected with Plasmodium falciparum.

Falciparum malaria is clinically heterogeneous and the relative contribution of parasite and host in shaping disease severity remains unclear. We explored the interaction between inflammation and parasite variant surface antigen (VSA) expression, asking whether this relationship underpins the variation observed in controlled human malaria infection (CHMI). We uncovered marked heterogeneity in the host response to blood challenge; some volunteers remained quiescent, others triggered interferon-stimulated inflammation and some showed transcriptional evidence of myeloid cell suppression. Significantly, only inflammatory volunteers experienced hallmark symptoms of malaria. When we tracked temporal changes in parasite VSA expression to ask whether variants associated with severe disease rapidly expand in naive hosts, we found no transcriptional evidence to support this hypothesis. These data indicate that parasite variants that dominate severe malaria do not have an intrinsic growth or survival advantage; instead, they presumably rely upon infection-induced changes in their within-host environment for selection.

Veterinary trypanocidal benzoxaboroles are peptidase-activated prodrugs.

Livestock diseases caused by Trypanosoma congolense, T. vivax and T. brucei, collectively known as nagana, are responsible for billions of dollars in lost food production annually. There is an urgent need for novel therapeutics. Encouragingly, promising antitrypanosomal benzoxaboroles are under veterinary development. Here, we show that the most efficacious subclass of these compounds are prodrugs activated by trypanosome serine carboxypeptidases (CBPs). Drug-resistance to a development candidate, AN11736, emerged readily in T. brucei, due to partial deletion within the locus containing three tandem copies of the CBP genes. T. congolense parasites, which possess a larger array of related CBPs, also developed resistance to AN11736 through deletion within the locus. A genome-scale screen in T. brucei confirmed CBP loss-of-function as the primary mechanism of resistance and CRISPR-Cas9 editing proved that partial deletion within the locus was sufficient to confer resistance. CBP re-expression in either T. brucei or T. congolense AN11736-resistant lines restored drug-susceptibility. CBPs act by cleaving the benzoxaborole AN11736 to a carboxylic acid derivative, revealing a prodrug activation mechanism. Loss of CBP activity results in massive reduction in net uptake of AN11736, indicating that entry is facilitated by the concentration gradient created by prodrug metabolism.

Cell-based and multi-omics profiling reveals dynamic metabolic repurposing of mitochondria to drive developmental progression of Trypanosoma brucei.

Mitochondrial metabolic remodeling is a hallmark of the Trypanosoma brucei digenetic life cycle because the insect stage utilizes a cost-effective oxidative phosphorylation (OxPhos) to generate ATP, while bloodstream cells switch to aerobic glycolysis. Due to difficulties in acquiring enough parasites from the tsetse fly vector, the dynamics of the parasite's metabolic rewiring in the vector have remained obscure. Here, we took advantage of in vitro-induced differentiation to follow changes at the RNA, protein, and metabolite levels. This multi-omics and cell-based profiling showed an immediate redirection of electron flow from the cytochrome-mediated pathway to an alternative oxidase (AOX), an increase in proline consumption, elevated activity of complex II, and certain tricarboxylic acid (TCA) cycle enzymes, which led to mitochondrial membrane hyperpolarization and increased reactive oxygen species (ROS) levels. Interestingly, these ROS molecules appear to act as signaling molecules driving developmental progression because ectopic expression of catalase, a ROS scavenger, halted the in vitro-induced differentiation. Our results provide insights into the mechanisms of the parasite's mitochondrial rewiring and reinforce the emerging concept that mitochondria act as signaling organelles through release of ROS to drive cellular differentiation.

Genomic instability at the locus of sterol C24-methyltransferase promotes amphotericin B resistance in Leishmania parasites.

Amphotericin B is an increasingly important tool in efforts to reduce the global disease burden posed by Leishmania parasites. With few other chemotherapeutic options available for the treatment of leishmaniasis, the potential for emergent resistance to this drug is a considerable threat. Here we characterised four novel amphotericin B-resistant Leishmania mexicana lines. All lines exhibited altered sterol biosynthesis, and hypersensitivity to pentamidine. Whole genome sequencing demonstrated resistance-associated mutation of the sterol biosynthesis gene sterol C5-desaturase in one line. However, in three out of four lines, RNA-seq revealed loss of expression of sterol C24-methyltransferase (SMT) responsible for drug resistance and altered sterol biosynthesis. Additional loss of the miltefosine transporter was associated with one of those lines. SMT is encoded by two tandem gene copies, which we found to have very different expression levels. In all cases, reduced overall expression was associated with loss of the 3' untranslated region of the dominant gene copy, resulting from structural variations at this locus. Local regions of sequence homology, between the gene copies themselves, and also due to the presence of SIDER1 retrotransposon elements that promote multi-gene amplification, correlate to these structural variations. Moreover, in at least one case loss of SMT expression was not associated with loss of virulence in primary macrophages or in vivo. Whilst such repeat sequence-mediated instability is known in Leishmania genomes, its presence associated with resistance to a major antileishmanial drug, with no evidence of associated fitness costs, is a significant concern.

Microfluidic Devices for Drug Assays.

In this review, we give an overview of the current state of microfluidic-based high-throughput drug assays. In this highly interdisciplinary research field, various approaches have been applied to high-throughput drug screening, including microtiter plate, droplets microfluidics as well as continuous flow, diffusion and concentration gradients-based microfluidic drug assays. Therefore, we reviewed over 100 recent publications in the field and sorted them according to their microfluidic approach. As a result, we are showcasing, comparing and discussing broadly applied approaches as well as singular promising ones that might contribute to shaping the future of this field.

Metabolomic profiling of macrophages determines the discrete metabolomic signature and metabolomic interactome triggered by polarising immune stimuli.

Priming and activating immune stimuli have profound effects on macrophages, however, studies generally evaluate stimuli in isolation rather than in combination. In this study we have investigated the effects of pro-inflammatory and anti-inflammatory stimuli either alone or in combination on macrophage metabolism. These stimuli include host factors such as IFNγ and ovalbumin-immunoglobulin immune complexes, or pathogen factors such as LPS. Untargeted LC-MS based metabolomics provided an in-depth profile of the macrophage metabolome, and revealed specific changes in metabolite abundance upon either individual stimuli or combined stimuli. Here, by factoring in an interaction term in the linear model, we define the metabolome interactome. This approach allowed us to determine whether stimuli interact in a synergistic or antagonistic manner. In conclusion this study demonstrates a robust approach to interrogate immune-metabolism, especially systems that model host-pathogen interactions.

Non-invasive visualisation and identification of fluorescent Leishmania tarentolae in infected sand flies.

Background: The leishmaniases are neglected diseases that affect some of the most vulnerable populations in the tropical and sub-tropical world. The parasites are transmitted by sand flies and novel strategies to control this neglected vector-borne disease are needed. Blocking transmission by targeting the parasite inside the phlebotomine vector offers potential in this regard. Some experimental approaches can be best performed by longitudinal study of parasites within flies, for which non-destructive methods to identify infected flies and to follow parasite population changes are required. Methods: Lutzomyia longipalpis were reared under standard insectary conditions at the Wellcome Centre for Molecular Parasitology. Flies were artificially infected with L. tarentolae expressing green fluorescent protein (GFP. Parasite counts were carried out 5 days post-infection and the percentage of infected flies and survival of infected females was established up to days 5 post-infection. Whole living females were visualised using an epifluorescence inverted microscope to detect the presence parasites inferred by a localised green fluorescent region in the upper thorax. Confirmation of infection was performed by localised-fluorescence of dissected flies and estimates of the parasite population. Results : Leishmania tarentolae was successfully transfected and expressed GFP in vitro. L. tarentolae-GFP Infected flies showed similar parasite populations when compared to non-transfected parasites ( L. tarentolae-WT). Survival of non-infected females was higher than L. tarentolae-infected groups, (Log-rank (Mantel-Cox) test, p<0.05). L. tarentolae-GFP infected females displayed an intense localised fluorescence in the thorax while other specimens from the same infected group did not. Localised fluorescent flies were dissected and showed higher parasite populations compared to those that did not demonstrate high concentrations in this region (t-test, p<0.005). Conclusion : These results demonstrate the feasibility of establishing a safe non-human infectious fluorescent Leishmania-sand fly infection model by allowing non-destructive imaging to signal the establishment of Leishmania infections in living sand flies.

Microfluidics-Based Approaches to the Isolation of African Trypanosomes.

African trypanosomes are responsible for significant levels of disease in both humans and animals. The protozoan parasites are free-living flagellates, usually transmitted by arthropod vectors, including the tsetse fly. In the mammalian host they live in the bloodstream and, in the case of human-infectious species, later invade the central nervous system. Diagnosis of the disease requires the positive identification of parasites in the bloodstream. This can be particularly challenging where parasite numbers are low, as is often the case in peripheral blood. Enriching parasites from body fluids is an important part of the diagnostic pathway. As more is learned about the physicochemical properties of trypanosomes, this information can be exploited through use of different microfluidic-based approaches to isolate the parasites from blood or other fluids. Here, we discuss recent advances in the use of microfluidics to separate trypanosomes from blood and to isolate single trypanosomes for analyses including drug screening.

CRISPR/Cas9 Genome Editing Reveals That the Intron Is Not Essential for var2csa Gene Activation or Silencing in Plasmodium falciparum.

Plasmodium falciparum relies on monoallelic expression of 1 of 60 var virulence genes for antigenic variation and host immune evasion. Each var gene contains a conserved intron which has been implicated in previous studies in both activation and repression of transcription via several epigenetic mechanisms, including interaction with the var promoter, production of long noncoding RNAs (lncRNAs), and localization to repressive perinuclear sites. However, functional studies have relied primarily on artificial expression constructs. Using the recently developed P. falciparum clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, we directly deleted the var2csa P. falciparum 3D7_1200600 (Pf3D7_1200600) endogenous intron, resulting in an intronless var gene in a natural, marker-free chromosomal context. Deletion of the var2csa intron resulted in an upregulation of transcription of the var2csa gene in ring-stage parasites and subsequent expression of the PfEMP1 protein in late-stage parasites. Intron deletion did not affect the normal temporal regulation and subsequent transcriptional silencing of the var gene in trophozoites but did result in increased rates of var gene switching in some mutant clones. Transcriptional repression of the intronless var2csa gene could be achieved via long-term culture or panning with the CD36 receptor, after which reactivation was possible with chondroitin sulfate A (CSA) panning. These data suggest that the var2csa intron is not required for silencing or activation in ring-stage parasites but point to a subtle role in regulation of switching within the var gene family.IMPORTANCEPlasmodium falciparum is the most virulent species of malaria parasite, causing high rates of morbidity and mortality in those infected. Chronic infection depends on an immune evasion mechanism termed antigenic variation, which in turn relies on monoallelic expression of 1 of ~60 var genes. Understanding antigenic variation and the transcriptional regulation of monoallelic expression is important for developing drugs and/or vaccines. The var gene family encodes the antigenic surface proteins that decorate infected erythrocytes. Until recently, studying the underlying genetic elements that regulate monoallelic expression in P. falciparum was difficult, and most studies relied on artificial systems such as episomal reporter genes. Our study was the first to use CRISPR/Cas9 genome editing for the functional study of an important, conserved genetic element of var genes-the intron-in an endogenous, episome-free manner. Our findings shed light on the role of the var gene intron in transcriptional regulation of monoallelic expression.