The clinicopathological features of thrombosis with thrombocytopenia syndrome following ChAdOx1-S (AZD1222) vaccination and case outcomes in Australia: a population-based study.

Background: Thrombosis with thrombocytopenia syndrome (TTS) associated with viral vector COVID-19 vaccines, including ChAdOx1-S (AstraZeneca AZD1222) vaccine, can result in significant morbidity and mortality. We report the clinicopathological features of TTS following ChAdOx1-S vaccination and summarise the case outcomes in Australia.

Methods: In this cohort study, patients diagnosed with TTS in Australia between 23 March and 31 December 2021 were identified according to predefined criteria.

Polyamine Metabolism in Parasites: A Promising Therapeutic Target.

Parasites of the genus cause a variety of devastating and often fatal diseases in humans and domestic animals worldwide. The need for new therapeutic strategies is urgent because no vaccine is available, and treatment options are limited due to a lack of specificity and the emergence of drug resistance. Polyamines are metabolites that play a central role in rapidly proliferating cells, and recent studies have highlighted their critical nature in .

Natural Products That Target the Arginase in Parasites Hold Therapeutic Promise.

Parasites of the genus cause a variety of devastating and often fatal diseases in humans worldwide. Because a vaccine is not available and the currently small number of existing drugs are less than ideal due to lack of specificity and emerging drug resistance, the need for new therapeutic strategies is urgent. Natural products and their derivatives are being used and explored as therapeutics and interest in developing such products as antileishmanials is high.

A Comprehensive Reflective Journal-Writing Framework for Pharmacy Students to Increase Self-Awareness and Develop Actionable Goals.

To develop and evaluate the effectiveness of a structured model for reflective journal writing (RJW) and a grading rubric as part of a student portfolio designed to help Doctor of Pharmacy (PharmD) students create actionable goals. A structured, eight-domain format was developed to engage students in prioritization, identification, exploration, recollection, evaluation, and challenging/solidifying their own knowledge, while assembling an action plan for development (abbreviated using the acronym PIE-RECAP). After completing RJW using this model, students self-identified domains established by the Center for the Advancement of Pharmacy Education (CAPE) that corresponded to their entries.

A role for adenine nucleotides in the sensing mechanism to purine starvation in Leishmania donovani.

Purine salvage by Leishmania is an obligatory nutritional process that impacts both cell viability and growth. Previously, we have demonstrated that the removal of purines in culture provokes significant metabolic changes that enable Leishmania to survive prolonged periods of purine starvation. In order to understand how Leishmania sense and respond to changes in their purine environment, we have exploited several purine pathway mutants, some in which adenine and guanine nucleotide metabolism is uncoupled.

A dual luciferase system for analysis of post-transcriptional regulation of gene expression in Leishmania.

Gene expression in kinetoplastid parasites is regulated via post-transcriptional mechanisms that modulate mRNA turnover, translation rate, and/or post-translational protein stability. To facilitate the analysis of post-transcriptional regulation, a dual luciferase system was developed in which firefly and Renilla luciferase reporters genetically fused to compatible drug resistance genes are integrated in place of one allele of the gene of interest and of an internal control gene, respectively, in a manner that preserves the cognate pre-mRNA processing signals. The sensitivity and reproducibility of the assay coupled with the ability to rapidly assemble reporter integration constructs render the dual luciferase system suitable for analysis of multiple candidates derived from global expression analysis platforms.

Metabolic reprogramming during purine stress in the protozoan pathogen Leishmania donovani.

The ability of Leishmania to survive in their insect or mammalian host is dependent upon an ability to sense and adapt to changes in the microenvironment. However, little is known about the molecular mechanisms underlying the parasite response to environmental changes, such as nutrient availability. To elucidate nutrient stress response pathways in Leishmania donovani, we have used purine starvation as the paradigm.

How Trypanosoma cruzi feasts upon its mammalian host.

Trypanosoma cruzi has a complex relationship with its mammalian host in which parasite and host metabolic networks are intertwined. A genome-wide functional screen of T. cruzi infection in HeLa cells (Caradonna et al.

Purine salvage in Leishmania: complex or simple by design?

Purine nucleotides function in a variety of vital cellular and metabolic processes including energy production, cell signaling, synthesis of vitamin-derived cofactors and nucleic acids, and as determinants of cell fate. Unlike their mammalian and insect hosts, Leishmania cannot synthesize the purine ring de novo and are absolutely dependent upon them to meet their purine requirements. The obligatory nature of purine salvage in these parasites, therefore, offers an attractive paradigm for drug targeting and, consequently, the delineation of the pathway has been under scientific investigation for over 30 years.

The Leishmania donovani UMP synthase is essential for promastigote viability and has an unusual tetrameric structure that exhibits substrate-controlled oligomerization.

The final two steps of de novo uridine 5'-monophosphate (UMP) biosynthesis are catalyzed by orotate phosphoribosyltransferase (OPRT) and orotidine 5'-monophosphate decarboxylase (OMPDC). In most prokaryotes and simple eukaryotes these two enzymes are encoded by separate genes, whereas in mammals they are expressed as a bifunctional gene product called UMP synthase (UMPS), with OPRT at the N terminus and OMPDC at the C terminus. Leishmania and some closely related organisms also express a bifunctional enzyme for these two steps, but the domain order is reversed relative to mammalian UMPS.

Adaptive responses to purine starvation in Leishmania donovani.

Starvation of Leishmania donovani parasites for purines leads to a rapid amplification in purine nucleobase and nucleoside transport. Studies with nucleoside transport-deficient L. donovani indicate that this phenomenon is mediated by the nucleoside transporters LdNT1 and LdNT2, as well as by the purine nucleobase transporter LdNT3.

Characterization of amplicons that suppress the conditional lethal growth phenotype of a Leishmania donovani mutant lacking normal purine salvage mechanisms.

A conditionally lethal mutant of Leishmania donovani that lacks both hypoxanthine-guanine phosphoribosyltransferase and xanthine phosphoribosyltransferase exhibits a strikingly restricted growth phenotype, can only survive as the promastigote under pharmacological constraints, and is profoundly compromised in its ability to infect macrophages and mice. Interestingly, the conditionally lethal growth phenotype displayed by these mutant parasites can be suppressed in vitro by selection of strains that have markedly amplified the adenine phosphoribosyltransferase gene on extrachromosomal elements that are unique to these suppressor strains. Employing pulsed field gel electrophoresis, we have now determined that the amplicons in two of these suppressor lines are linear molecules by: (1) their pulse time-dependent mobility; (2) the failure of γ-irradiation to generate new discrete bands; (3) their susceptibility to λ exonuclease digestion; and (4) the presence of telomeric sequences.

Genetic evidence for the essential role of PfNT1 in the transport and utilization of xanthine, guanine, guanosine and adenine by Plasmodium falciparum.

The malaria parasite, Plasmodium falciparum, is unable to synthesize the purine ring de novo and is therefore wholly dependent upon purine salvage from the host for survival. Previous studies have indicated that a P. falciparum strain in which the purine transporter PfNT1 had been disrupted was unable to grow on physiological concentrations of adenosine, inosine and hypoxanthine.

Purine and pyrimidine metabolism in Leishmania.

Purines and pyrimidines are indispensable to all life, performing many vital functions for cells: ATP serves as the universal currency of cellular energy, cAMP and cGMP are key second messenger molecules, purine and pyrimidine nucleotides are precursors for activated forms of both carbohydrates and lipids, nucleotide derivatives of vitamins are essential cofactors in metabolic processes, and nucleoside triphosphates are the immediate precursors for DNA and RNA synthesis. Unlike their mammalian and insect hosts, Leishmania lack the metabolic machinery to make purine nucleotides de novo and must rely on their host for preformed purines. The obligatory nature of purine salvage offers, therefore, a plethora of potential targets for drug targeting, and the pathway has consequently been the focus of considerable scientific investigation.

Functional characterization of nucleoside transporter gene replacements in Leishmania donovani.

Leishmania donovani express two nucleoside transporters of non-overlapping ligand selectivity. To evaluate the physiological role of nucleoside transporters in L. donovani, homozygous null mutants of the genes encoding the LdNT1 adenosine-pyrimidine nucleoside transporter and the LdNT2 inosine-guanosine transporter were created singly and in combination by single targeted gene replacement followed by selection for loss-of-heterozygosity.

The plasma membrane permease PfNT1 is essential for purine salvage in the human malaria parasite Plasmodium falciparum.

The human malaria parasite Plasmodium falciparum relies on the acquisition of host purines for its survival within human erythrocytes. Purine salvage by the parasite requires specialized transporters at the parasite plasma membrane (PPM), but the exact mechanism of purine entry into the infected erythrocyte, and the primary purine source used by the parasite, remain unknown. Here, we report that transgenic parasites lacking the PPM transporter PfNT1 (P.

Point mutations within the LdNT2 nucleoside transporter gene from Leishmania donovani confer drug resistance and transport deficiency.

Leishmania donovani, a protozoan parasite, expresses an unusual inosine/guanosine-specific transporter, LdNT2, the gene for which was cloned by functional rescue of a drug-resistant, LdNT2-deficient (FBD5) strain. In this investigation, we have uncovered and characterized the mutations within the LdNT2 open reading frame that are the basis for the drug-resistance and transport-incompetent phenotype of the FBD5 line. The FBD5 cells were shown to be compound heterozygotes in which both mutant ldnt2 alleles harbor discrete point mutations, each of which impaired transport function and conferred resistance to formycin B, the drug to which the clonal FBD5 line was selected.

Identification and characterization of purine nucleoside transporters from Crithidia fasciculata.

To initiate a molecular dissection into the mechanism by which purine transport is up-regulated in Crithidia, genes encoding nucleoside transporters from Crithidia fasciculata were cloned and functionally characterized. Sequence analysis revealed CfNT1 and CfNT2 to be members of the equilibrative nucleoside transporter family, and the genes isolated encompassed polypeptides of 497 and 502 amino acids, respectively, each with 11 predicted membrane-spanning domains. Heterologous expression of CfNT1 cRNA in Xenopus laevis oocytes or CfNT2 in nucleoside transport-deficient Leishmania donovani demonstrated that CfNT1 is a novel high affinity adenosine transporter that also recognizes inosine, hypoxanthine, and pyrimidine nucleosides, while CfNT2 is a high affinity permease specific for inosine and guanosine.

Second-site suppression of a nonfunctional mutation within the Leishmania donovani inosine-guanosine transporter.

LdNT2 is a member of the equilibrative nucleoside transporter family, which possesses several conserved residues located mainly within transmembrane domains. One of these residues, Asp(389) within LdNT2, was shown previously to be critical for transporter function without affecting ligand affinity or plasma membrane targeting. To further delineate the role of Asp(389) in LdNT2 function, second-site suppressors of the ldnt2-D389N null mutation were selected in yeast deficient in purine nucleoside transport and incapable of purine biosynthesis.

Susceptibility of retinal vascular endothelium to infection with Toxoplasma gondii tachyzoites.

Purpose: Retinochoroidal infection with the protozoan parasite Toxoplasma gondii is the most common cause of posterior uveitis worldwide. Tachyzoites spread throughout the body through the blood stream and lymphatics, but preferentially encyst in the eye and other parts of the central nervous system (CNS). It is unknown whether T.

Equilibrative nucleoside transporter family members from Leishmania donovani are electrogenic proton symporters.

Leishmania donovani express two members of the equilibrative nucleoside transporter family; LdNT1 encoded by two closely related and linked genes, LdNT1.1 and LdNT1.2, that transport adenosine and pyrimidine nucleosides and LdNT2 that transports inosine and guanosine exclusively.

Functional analysis of an inosine-guanosine transporter from Leishmania donovani. The role of conserved residues, aspartate 389 and arginine 393.

Equilibrative nucleoside transporters encompass two conserved, charged residues that occur within predicted transmembrane domain 8. To assess the role of these "signature" residues in transporter function, the Asp389 and Arg393 residues within the LdNT2 nucleoside transporter from Leishmania donovani were mutated and the resultant phenotypes evaluated after transfection into Delta ldnt2 parasites. Whereas an R393K mutant retained transporter activity similar to that of wild type LdNT2, the R393L, D389E, and D389N mutations resulted in dramatic losses of transport capability.

S-adenosylmethionine decarboxylase from Leishmania donovani. Molecular, genetic, and biochemical characterization of null mutants and overproducers.

The polyamine biosynthetic enzyme, S-adenosylmethionine decarboxylase (ADOMETDC) has been advanced as a potential target for antiparasitic chemotherapy. To investigate the importance of this protein in a model parasite, the gene encoding ADOMETDC has been cloned and sequenced from Leishmania donovani. The Delta adometdc null mutants were created in the insect vector form of the parasite by double targeted gene replacement.