Plasmodium falciparum replication factor C subunit 1 is involved in genotoxic stress response.

About half the world's population is at risk of malaria, with Plasmodium falciparum malaria being responsible for the most malaria related deaths globally. Antimalarial drugs such as chloroquine and artemisinin are directed towards the proliferating intra-erythrocytic stages of the parasite, which is responsible for all the clinical symptoms of the disease. These antimalarial drugs have been reported to function via multiple pathways, one of which induces DNA damage via the generation of free radicals and reactive oxygen species. An urgent need to understand the mechanistic details of drug response and resistance is highlighted by the decreasing clinical efficacy of the front line drug, Artemisinin. The replication factor C subunit 1 is an important component of the DNA replication machinery and DNA damage response mechanism. Here we show the translocation of PfRFC1 from an intranuclear localisation to the nuclear periphery, indicating an orchestrated progression of distinct patterns of replication in the developing parasites. PfRFC1 responds to genotoxic stress via elevated protein levels in soluble and chromatin bound fractions. Reduction of PfRFC1 protein levels upon treatment with antimalarials suggests an interplay of replication, apoptosis and DNA repair pathways leading to cell death. Additionally, mislocalisation of the endogenously tagged protein confirmed its essential role in parasites' replication and DNA repair. This study provides key insights into DNA replication, DNA damage response and cell death in P. falciparum.

Comparative spatial proteomics of Plasmodium-infected erythrocytes.

Plasmodium parasites contribute to one of the highest global infectious disease burdens. To achieve this success, the parasite has evolved a range of specialized subcellular compartments to extensively remodel the host cell for its survival. The information to fully understand these compartments is likely hidden in the so far poorly characterized Plasmodium species spatial proteome. To address this question, we determined the steady-state subcellular location of more than 12,000 parasite proteins across five different species by extensive subcellular fractionation of erythrocytes infected by Plasmodium falciparum, Plasmodium knowlesi, Plasmodium yoelii, Plasmodium berghei, and Plasmodium chabaudi. This comparison of the pan-species spatial proteomes and their expression patterns indicates increasing species-specific proteins associated with the more external compartments, supporting host adaptations and post-transcriptional regulation. The spatial proteome offers comprehensive insight into the different human, simian, and rodent Plasmodium species, establishing a powerful resource for understanding species-specific host adaptation processes in the parasite.

Parasite-based screening and proteome profiling reveal orlistat, an FDA-approved drug, as a potential anti Trypanosoma brucei agent.

Trypanosoma brucei is a parasite that causes African sleeping sickness in humans and nagana in livestock and is transmitted by the tsetse fly. There is an urgent need for the development of new drugs against African trypanosomiasis due to the lack of vaccines and effective drugs. Orlistat (also called tetrahydrolipstatin or THL) is an FDA-approved antiobesity drug targeting primarily the pancreatic and gastric lipases within the gastrointestinal tract. It shows potential activities against tumors, mycobacteria, and parasites. Herein, we report the synthesis and evaluation of an expanded set of orlistat-like compounds, some of which showed highly potent trypanocidal activities in both the bloodstream form (BSF) and the procyclic form (PCF) of T. brucei. Subsequent in situ parasite-based proteome profiling was carried out to elucidate potential cellular targets of the drug in both forms. Some newly identified targets were further validated by the labeling of recombinantly expressed enzymes in Escherichia coli lysates. Bioimaging experiments with a selected compound were carried out to study the cellular uptake of the drug in T. brucei. Results indicated that orlistat is much more efficiently taken up by the BSF than the PCF of T. brucei and has clear effects on the morphology of mitochondria, glycosomes, and the endoplasmic reticulum in both BSF and PCF cells. These results support specific effects of orlistat on these organelles and correlate well with our in situ proteome profiling. Given the economic challenges of de novo drug development for neglected diseases, we hope that our findings will stimulate further research towards the conversion of orlistat-like compounds into new trypanocidal drugs.

Incidence of venous thromboembolism in coronavirus disease 2019: An experience from a single large academic center.

BACKGROUND: Infection with the novel severe acute respiratory syndrome coronavirus 2 has been associated with a hypercoagulable state. Emerging data from China and Europe have consistently shown an increased incidence of venous thromboembolism (VTE). We aimed to identify the VTE incidence and early predictors of VTE at our high-volume tertiary care center. METHODS: We performed a retrospective cohort study of 147 patients who had been admitted to Temple University Hospital with coronavirus disease 2019 (COVID-19) from April 1, 2020 to April 27, 2020. We first identified the VTE (pulmonary embolism [PE] and deep vein thrombosis [DVT]) incidence in our cohort. The VTE and no-VTE groups were compared by univariable analysis for demographics, comorbidities, laboratory data, and treatment outcomes. Subsequently, multivariable logistic regression analysis was performed to identify the early predictors of VTE. RESULTS: The 147 patients (20.9% of all admissions) admitted to a designated COVID-19 unit at Temple University Hospital with a high clinical suspicion of acute VTE had undergone testing for VTE using computed tomography pulmonary angiography and/or extremity venous duplex ultrasonography. The overall incidence of VTE was 17% (25 of 147). Of the 25 patients, 16 had had acute PE, 14 had had acute DVT, and 5 had had both PE and DVT. The need for invasive mechanical ventilation (adjusted odds ratio, 3.19; 95% confidence interval, 1.07-9.55) and the admission D-dimer level ≥1500 ng/mL (adjusted odds ratio, 3.55; 95% confidence interval, 1.29-9.78) were independent markers associated with VTE. The all-cause mortality in the VTE group was greater than that in the non-VTE group (48% vs 22%; P = .007). CONCLUSIONS: Our study represents one of the earliest reported from the United States on the incidence rate of VTE in patients with COVID-19. Patients with a high clinical suspicion and the identified risk factors (invasive mechanical ventilation, admission D-dimer level ≥1500 ng/mL) should be considered for early VTE testing. We did not screen all patients admitted for VTE; therefore, the true incidence of VTE could have been underestimated. Our findings require confirmation in future prospective studies.

Tracking the biogenesis and inheritance of subpellicular microtubule in Trypanosoma brucei with inducible YFP-α-tubulin.

The microtubule cytoskeleton forms the most prominent structural system in Trypanosoma brucei, undergoing extensive modifications during the cell cycle. Visualization of tyrosinated microtubules leads to a semiconservative mode of inheritance, whereas recent studies employing microtubule plus end tracking proteins have hinted at an asymmetric pattern of cytoskeletal inheritance. To further the knowledge of microtubule synthesis and inheritance during T. brucei cell cycle, the dynamics of the microtubule cytoskeleton was visualized by inducible YFP-α-tubulin expression. During new flagellum/flagellum attachment zone (FAZ) biogenesis and cell growth, YFP-α-tubulin was incorporated mainly between the old and new flagellum/FAZ complexes. Cytoskeletal modifications at the posterior end of the cells were observed with EB1, a microtubule plus end binding protein, particularly during mitosis. Additionally, the newly formed microtubules segregated asymmetrically, with the daughter cell inheriting the new flagellum/FAZ complex retaining most of the new microtubules. Together, our results suggest an intimate connection between new microtubule formation and new FAZ assembly, consequently leading to asymmetric microtubule inheritance and cell division.