Malaria drug resistance contributes to up to a million annual deaths. Judicious deployment of new antimalarials and vaccines could benefit from an understanding of early molecular events that promote ...the evolution of parasites. Continuous in vitro challenge of Plasmodium falciparum parasites with a novel dihydroorotate dehydrogenase (DHODH) inhibitor reproducibly selected for resistant parasites. Genome-wide analysis of independently-derived resistant clones revealed a two-step strategy to evolutionary success. Some haploid blood-stage parasites first survive antimalarial pressure through fortuitous DNA duplications that always included the DHODH gene. Independently-selected parasites had different sized amplification units but they were always flanked by distant A/T tracks. Higher level amplification and resistance was attained using a second, more efficient and more accurate, mechanism for head-to-tail expansion of the founder unit. This second homology-based process could faithfully tune DNA copy numbers in either direction, always retaining the unique DNA amplification sequence from the original A/T-mediated duplication for that parasite line. Pseudo-polyploidy at relevant genomic loci sets the stage for gaining additional mutations at the locus of interest. Overall, we reveal a population-based genomic strategy for mutagenesis that operates in human stages of P. falciparum to efficiently yield resistance-causing genetic changes at the correct locus in a successful parasite. Importantly, these founding events arise with precision; no other new amplifications are seen in the resistant haploid blood stage parasite. This minimizes the need for meiotic genetic cleansing that can only occur in sexual stage development of the parasite in mosquitoes.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Toward a chemical vaccine for malaria Phillips, Margaret A; Goldberg, Daniel E
Science (American Association for the Advancement of Science),
12/2018, Letnik:
362, Številka:
6419
Journal Article
Recenzirano
A high-throughput screen puts us on the road to protecting populations against malaria
Despite considerable progress in combating malaria, it remains one of the world's most important infectious ...diseases, with 50% of the world population at risk of developing the disease and a mortality rate of ∼0.5 million annually (
1
). The lack of an effective vaccine and the relentless ability of the
Plasmodium
parasite responsible for malaria to develop drug resistance has contributed to the continuing disease burden (
2
–
4
). Artemisinin-combination therapies (ACTs) are the mainstay of current treatment regimens, but decreased effectiveness, particularly in Southeast Asia, threatens our ability to control this disease. A global effort to develop new drugs for the treatment and prevention of malaria is under way but not guaranteed to succeed (
3
,
5
,
6
). These efforts include a systematic attempt to target all life-cycle stages of the parasite to allow combination therapies to be developed, which are also likely to reduce the development of resistance. High-throughput screens (HTSs) designed to identify small drug-like molecules that prevent growth of blood-stage parasites (
7
,
8
) and target-based approaches have identified new compounds that are currently in preclinical development and/or various stages of human clinical trials for treatment of malaria (
3
). Missing from these efforts has been a high-throughput technology to find liver stage–specific chemotypes. On page 1129 of this issue, Antonova-Koch
et al.
(
9
) report an HTS effort that has filled this gap. They identify a substantial number of new chemical starting points with potent liver-stage antimalarial activity, promising a new capacity to feed compounds through the drug development pipeline for chemoprotection.
Bladder cancer is the 10th most common cancer worldwide. For muscle‐invasive bladder cancer (MIBC), treatment includes radical cystectomy, radiotherapy, and chemotherapy; however, the outcome is ...generally poor. For non–muscle‐invasive bladder cancer (NMIBC), tumor recurrence is common. There is an urgent need for more effective and less harmful therapeutic approaches. Here, bladder cancer cell metabolic reprogramming to rely on aerobic glycolysis (the Warburg effect) and expression of associated molecular therapeutic targets by bladder cancer cells of different stages and grades, and in freshly resected clinical tissue, is investigated. Importantly, analyses indicate that the Warburg effect is a feature of both NMIBCs and MIBCs. In two in vitro inducible epithelial‐mesenchymal transition (EMT) bladder cancer models, EMT stimulation correlated with increased lactate production, the end product of aerobic glycolysis. Protein levels of lactate dehydrogenase A (LDH‐A), which promotes pyruvate enzymatic reduction to lactate, were higher in most bladder cancer cell lines (compared with LDH‐B, which catalyzes the reverse reaction), but the levels did not closely correlate with aerobic glycolysis rates. Although LDH‐A is expressed in normal urothelial cells, LDH‐A knockdown by RNAi selectively induced urothelial cancer cell apoptotic death, whereas normal cells were unaffected—identifying LDH‐A as a cancer‐selective therapeutic target for bladder cancers. LDH‐A and other potential therapeutic targets (MCT4 and GLUT1) were expressed in patient clinical specimens; however, positive staining varied in different areas of sections and with distance from a blood vessel. This intratumoral heterogeneity has important therapeutic implications and indicates the possibility of tumor cell metabolic coupling.
This work identifies aerobic glycolysis (the Warburg effect) as a general feature of both non–muscle‐invasive and muscle‐invasive bladder cancers. We further show that the glycolytic enzyme lactate dehydrogenase A is a cancer‐selective therapeutic target for bladder cancers, but that there is intratumoral heterogeneity in expression of metabolic targets. This raises the possibility of intratumoral metabolic coupling that could be therapeutically targeted.
Summary
Trypanosoma brucei is the causative agent of African sleeping sickness, putting at risk up to 50 million people in sub‐Saharan Africa. Current drug therapies are limited by toxicity and ...difficult treatment regimes and as the development of vaccines remains unlikely, the identification of better drugs to control this deadly disease is needed. Strategies for the identification of new lead compounds include phenotypic screening or target‐based approaches. Implementation of the latter has been hampered by the lack of defined targets that are both essential and druggable. In this issue of Molecular Microbiology, Jones et al. () report on the characterization of T. brucei pyridoxal kinase (PdxK), an enzyme required for the salvage of vitamin B6, an essential enzymatic cofactor. Genetic knock‐down and small molecule inhibitor studies were used to demonstrate that PdxK is essential for parasite growth both in vitro and in a mouse model, providing both genetic and chemical validation of the target. An enzyme assay compatible with high‐throughput screening (HTS) was developed and the X‐ray crystal structure solved, showing the potential for species selective inhibition. These studies add a greatly needed additional target into the drug discovery pipeline for this deadly parasitic infection.
Abstract This narrative review gathers together a range of international experts to critically appraise the existing trial-based evidence relating to the efficacy and tolerability of pharmacotherapy ...for obsessive compulsive disorder in adults. We discuss the diagnostic evaluation and clinical characteristics followed by treatment options suitable for the clinician working from primary through to specialist psychiatric care. Robust data supports the effectiveness of treatment with selective serotonin reuptake inhibitors (SSRIs) and clomipramine in the short-term and the longer-term treatment and for relapse prevention. Owing to better tolerability, SSRIs are acknowledged as the first-line pharmacological treatment of choice. For those patients for whom first line treatments have been ineffective, evidence supports the use of adjunctive antipsychotic medication, and some evidence supports the use of high-dose SSRIs. Novel compounds are also the subject of active investigation. Neurosurgical treatments, including ablative lesion neurosurgery and deep brain stimulation, are reserved for severely symptomatic individuals who have not experienced sustained response to both pharmacological and cognitive behavior therapies.
Scaling up shorter regimens for tuberculosis (TB) prevention such as once weekly isoniazid-rifapentine (3HP) taken for 3 months is a key priority for achieving targets set forth in the World Health ...Organization's (WHO) END TB Strategy. However, there are few data on 3HP patient acceptance and completion in the context of routine HIV care in sub-Saharan Africa.
The 3HP Options Trial is a pragmatic, parallel type 3 effectiveness-implementation randomized trial comparing 3 optimized strategies for delivering 3HP-facilitated directly observed therapy (DOT), facilitated self-administered therapy (SAT), or informed choice between DOT and SAT using a shared decision-making aid-to people receiving care at a large urban HIV clinic in Kampala, Uganda. Participants and healthcare providers were not blinded to arm assignment due to the nature of the 3HP delivery strategies. We conducted an interim analysis of participants who were enrolled and exited the 3HP treatment period between July 13, 2020 and April 30, 2021. The primary outcome, which was aggregated across trial arms for this interim analysis, was the proportion who accepted and completed 3HP (≥11 of 12 doses within 16 weeks of randomization). We used Bayesian inference analysis to estimate the posterior probability that this proportion would exceed 80% under at least 1 of the 3HP delivery strategies, a coprimary hypothesis of the trial. Through April 2021, 684 participants have been enrolled, and 479 (70%) have exited the treatment period. Of these 479 participants, 309 (65%) were women, mean age was 41.9 years (standard deviation (SD): 9.2), and mean time on antiretroviral therapy (ART) was 7.8 years (SD: 4.3). In total, 445 of them (92.9%, 95% confidence interval (CI): 90.2 to 94.9) accepted and completed 3HP treatment. There were no differences in treatment acceptance and completion by sex, age, or time on ART. Treatment was discontinued due to a documented adverse event (AE) in 8 (1.7%) patients. The probability that treatment acceptance and completion exceeds 80% under at least 1 of the three 3HP delivery strategies was greater than 99%. The main limitations are that the trial was conducted at a single site, and the interim analysis focused on aggregate outcome data to maintain blinding of investigators to arm-specific outcomes.
3HP was widely accepted by people living with HIV (PLHIV) in Uganda, and very high levels of treatment completion were achieved in a programmatic setting. These findings show that 3HP can enable effective scale-up of tuberculosis preventive therapy (TPT) in high-burden countries, particularly when delivery strategies are tailored to target known barriers to treatment completion.
ClinicalTrials.gov NCT03934931.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Spermine synthase is an aminopropyltransferase that adds an aminopropyl group to the essential polyamine spermidine to form tetraamine spermine, needed for normal human neural development, plant salt ...and drought resistance, and yeast CoA biosynthesis. We functionally identify for the first time bacterial spermine synthases, derived from phyla Bacillota, Rhodothermota, Thermodesulfobacteriota, Nitrospirota, Deinococcota, and Pseudomonadota. We also identify bacterial aminopropyltransferases that synthesize the spermine same mass isomer thermospermine, from phyla Cyanobacteriota, Thermodesulfobacteriota, Nitrospirota, Dictyoglomota, Armatimonadota, and Pseudomonadota, including the human opportunistic pathogen Pseudomonas aeruginosa. Most of these bacterial synthases were capable of synthesizing spermine or thermospermine from the diamine putrescine and so possess also spermidine synthase activity. We found that most thermospermine synthases could synthesize tetraamine norspermine from triamine norspermidine, that is, they are potential norspermine synthases. This finding could explain the enigmatic source of norspermine in bacteria. Some of the thermospermine synthases could synthesize norspermidine from diamine 1,3-diaminopropane, demonstrating that they are potential norspermidine synthases. Of 18 bacterial spermidine synthases identified, 17 were able to aminopropylate agmatine to form N1-aminopropylagmatine, including the spermidine synthase of Bacillus subtilis, a species known to be devoid of putrescine. This suggests that the N1-aminopropylagmatine pathway for spermidine biosynthesis, which bypasses putrescine, may be far more widespread than realized and may be the default pathway for spermidine biosynthesis in species encoding L-arginine decarboxylase for agmatine production. Some thermospermine synthases were able to aminopropylate N1-aminopropylagmatine to form N12-guanidinothermospermine. Our study reveals an unsuspected diversification of bacterial polyamine biosynthesis and suggests a more prominent role for agmatine.
During human brain development, multiple signaling pathways generate diverse cell types with varied regional identities. Here, we integrate single-cell RNA sequencing and clonal analyses to reveal ...lineage trees and molecular signals underlying early forebrain and mid/hindbrain cell differentiation from human embryonic stem cells (hESCs). Clustering single-cell transcriptomic data identified 41 distinct populations of progenitor, neuronal, and non-neural cells across our differentiation time course. Comparisons with primary mouse and human gene expression data demonstrated rostral and caudal progenitor and neuronal identities from early brain development. Bayesian analyses inferred a unified cell-type lineage tree that bifurcates between cortical and mid/hindbrain cell types. Two methods of clonal analyses confirmed these findings and further revealed the importance of Wnt/β-catenin signaling in controlling this lineage decision. Together, these findings provide a rich transcriptome-based lineage map for studying human brain development and modeling developmental disorders.
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•Single-cell RNA-seq of differentiating hESCs reveals a multitude of neural cell types•hESC-derived cells exhibit a range of early brain regional identities•Bayesian analyses reconstruct, and clonal analyses confirm, lineage relationships•Wnt/β-catenin signaling controls bifurcations between forebrain and mid/hindbrain lineages
Yao et al. perform single-cell RNA-seq during neural differentiation of hESCs. They identify many classes of neural progenitors and neurons that map to early human brain cells, computationally infer and experimentally confirm lineage relationships between them, and show that Wnt signaling influences the bifurcation between forebrain and mid/hindbrain lineages in vitro.
We have tested five distinct classes of established and experimental antimalarial drugs for their anticancer potential, using a panel of 91 human cancer lines. Three classes of drugs: artemisinins, ...synthetic peroxides and DHFR (dihydrofolate reductase) inhibitors effected potent inhibition of proliferation with IC50s in the nM- low µM range, whereas a DHODH (dihydroorotate dehydrogenase) and a putative kinase inhibitor displayed no activity. Furthermore, significant synergies were identified with erlotinib, imatinib, cisplatin, dasatinib and vincristine. Cluster analysis of the antimalarials based on their differential inhibition of the various cancer lines clearly segregated the synthetic peroxides OZ277 and OZ439 from the artemisinin cluster that included artesunate, dihydroartemisinin and artemisone, and from the DHFR inhibitors pyrimethamine and P218 (a parasite DHFR inhibitor), emphasizing their shared mode of action. In order to further understand the basis of the selectivity of these compounds against different cancers, microarray-based gene expression data for 85 of the used cell lines were generated. For each compound, distinct sets of genes were identified whose expression significantly correlated with compound sensitivity. Several of the antimalarials tested in this study have well-established and excellent safety profiles with a plasma exposure, when conservatively used in malaria, that is well above the IC50s that we identified in this study. Given their unique mode of action and potential for unique synergies with established anticancer drugs, our results provide a strong basis to further explore the potential application of these compounds in cancer in pre-clinical or and clinical settings.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Polyamines are essential for cell growth of eukaryotes including the etiologic agent of human African trypanosomiasis (HAT), Trypanosoma brucei. In trypanosomatids, a key enzyme in the polyamine ...biosynthetic pathway, S-adenosylmethionine decarboxylase (TbAdoMetDC) heterodimerizes with a unique catalytically-dead paralog called prozyme to form the active enzyme complex. In higher eukaryotes, polyamine metabolism is subject to tight feedback regulation by spermidine-dependent mechanisms that are absent in trypanosomatids. Instead, in T. brucei an alternative regulatory strategy based on TbAdoMetDC prozyme has evolved. We previously demonstrated that prozyme protein levels increase in response to loss of TbAdoMetDC activity. Herein, we show that prozyme levels are under translational control by monitoring incorporation of deuterated leucine into nascent prozyme protein. We furthermore identify pathway factors that regulate prozyme mRNA translation. We find evidence for a regulatory feedback mechanism in which TbAdoMetDC protein and decarboxylated AdoMet (dcAdoMet) act as suppressors of prozyme translation. In TbAdoMetDC null cells expressing the human AdoMetDC enzyme, prozyme levels are constitutively upregulated. Wild-type prozyme levels are restored by complementation with either TbAdoMetDC or an active site mutant, suggesting that TbAdoMetDC possesses an enzyme activity-independent function that inhibits prozyme translation. Depletion of dcAdoMet pools by three independent strategies: inhibition/knockdown of TbAdoMetDC, knockdown of AdoMet synthase, or methionine starvation, each cause prozyme upregulation, providing independent evidence that dcAdoMet functions as a metabolic signal for regulation of the polyamine pathway in T. brucei. These findings highlight a potential regulatory paradigm employing enzymes and pseudoenzymes that may have broad implications in biology.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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