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Rodent malaria models serve as important preclinical antimalarial and vaccine testing tools. Evaluating treatment outcomes in these models often requires manually counting parasite-infected red blood cells (iRBCs), a time-consuming process, which can be inconsistent between individuals and laboratories. We have developed an easy-to-use machine learning (ML)-based software, Malaria Screener R, to expedite and standardize such studies by automating the counting of Plasmodium iRBCs in rodents. This software can process Giemsa-stained blood smear images captured by any camera-equipped microscope. It features an intuitive graphical user interface that facilitates image processing and visualization of the results. The software has been developed as a desktop application that processes images on standard Windows and MacOS computers. A previous ML model created by the authors designed to count Plasmodium falciparum-infected human RBCs did not perform well counting Plasmodium-infected mouse RBCs. We leveraged that model by loading the pretrained weights and training the algorithm with newly collected data to target Plasmodium yoelii- and Plasmodium berghei-infected mouse RBCs. This new model reliably measured both P. yoelii and P. berghei parasitemia (R2 = 0.9916). Additional rounds of training data to incorporate variances due to length of Giemsa staining and type of microscopes, etc., have produced a generalizable model, meeting WHO competency level 1 for the subcategory of parasite counting using independent microscopes. Reliable, automated analyses of blood-stage parasitemia will facilitate rapid and consistent evaluation of novel vaccines and antimalarials across laboratories in an easily accessible in vivo malaria model.

Rodent malaria models serve as important preclinical antimalarial and vaccine testing tools. Evaluating treatment outcomes in these models often requires manually counting parasite-infected red blood cells (iRBCs), a time-consuming process, which can be inconsistent between individuals and labs. We have developed an easy-to-use machine learning (ML)-based software, Malaria Screener R, to expedite and standardize such studies by automating the counting of Plasmodium iRBCs in rodents. This software can process Giemsa-stained blood smear images captured by any camera-equipped microscope. It features an intuitive graphical user interface that facilitates image processing and visualization of the results. The software has been developed as a desktop application that processes images on standard Windows and Mac OS computers. A previous ML model created by the authors designed to count P. falciparum -infected human RBCs did not perform well counting Plasmodium -infected mouse RBCs. We leveraged that model by loading the pre-trained weights and training the algorithm with newly collected data to target P. yoelii and P. berghei mouse iRBCs. This new model reliably measured both P. yoelii and P. berghei parasitemia (R 2 = 0.9916). Additional rounds of training data to incorporate variances due to length of Giemsa staining, microscopes etc, have produced a generalizable model, meeting WHO Competency Level 1 for the sub-category of parasite counting using independent microscopes. Reliable, automated analyses of blood-stage parasitemia will facilitate rapid and consistent evaluation of novel vaccines and antimalarials across labs in an easily accessible in vivo malaria model.

Trichomonas vaginalis is a parasitic protist that infects the human urogenital tract. During the infection, trichomonads adhere to the host mucosa, acquire nutrients from the vaginal/prostate environment, and release small extracellular vesicles (sEVs) that contribute to the trichomonad adherence and modulate the host-parasite communication. Approximately 40-70% of T. vaginalis strains harbor a double-stranded RNA virus called Trichomonasvirus (TVV). Naked TVV particles have the potential to stimulate a proinflammatory response in human cells, however, the mode of TVV release from trichomonads to the environment is not clear. In this report, we showed for the first time that TVV particles are released from T. vaginalis cells within sEVs. The sEVs loaded with TVV stimulated a higher proinflammatory response of human HaCaT cells in comparison to sEVs from TVV negative parasites. Moreover, a comparison of T. vaginalis isogenic TVV plus and TVV minus clones revealed a significant impact of TVV infection on the sEV proteome and RNA cargo. Small EVs from TVV positive trichomonads contained 12 enriched and 8 unique proteins including membrane-associated BspA adhesine, and about a 2.5-fold increase in the content of small regulatory tsRNA. As T. vaginalis isolates are frequently infected with TVV, the release of TVV via sEVs to the environment represents an important factor with the potential to enhance inflammation-related pathogenesis during trichomoniasis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rada, Hrdý, Zdrha, Narayanasamy, Smutná, Horáčková, Harant, Beneš, Ong, Tsai, Luo, Chiu, Tang and Tachezy.)

Background: Trichomonas vaginalis is the causative agent of a sexually transmitted disease in humans. The virulence of the parasite depends on multiple factors, including the presence of endosymbiotic dsRNA viruses. The presence of Trichomonasviruses (TVV) was associated with more severe genital symptoms, increased proinflammatory host reactions, and modulated parasite sensitivity to metronidazole. However, no efficient antiviral drugs are available against TVV to derive isogenic TVV-positive and TVV-negative cell lines that are essential for investigations of the TVV impact on T. vaginalis biology., Methods: 7-Deaza-2'-C-methyladenosine (7d2CMA) and 2'-C-methylcytidine (2CMC) were used for TVV inhibitory assay. TVV replication was monitored using quantitative reverse transcription PCR (RT qPCR) and western blotting. Modeling of TVV1 RNA-dependent RNA polymerase (RdRp) was performed to visualize the inhibitor-RdRp interaction. Susceptibility to metronidazole was performed under aerobic and anaerobic conditions., Results: We demonstrated that 2CMC but not 7d2CMA is a potent inhibitor of TVV replication. Molecular modeling suggested that the RdRp active site can accommodate 2CMC in the active triphosphate nucleotide form. The effect of 2CMC was shown on strains infected with a single and multiple TVV species. The optimal 2CMC concentration (10 μM) demonstrated strong selectivity for TVVs over trichomonad growth. The presence of TVV has no effect on T. vaginalis metronidazole susceptibility in derived isogenic cell lines., Conclusions: 2CMC acts against TVVs and represents a new inhibitor against Totiviridae viruses. Our isogenic clones are now available for further studies of various aspects of T. vaginalis biology related to TVV infection., Competing Interests: Declaration of competing interest The authors declare no conflict of statement., (Copyright © 2021. Published by Elsevier B.V.)

Accumulated evidence suggests that the endosymbiotic Trichomonasvirus (TVV) may play a role in the pathogenesis and drug susceptibility of Trichomonas vaginalis . Several reports have shown that extracellular vesicles (EVs) released from TVV-positive (TVV+) trichomonads can modulate the immune response in human vaginal epithelial cells and animal models. These results prompted us to examine whether EVs released from TVV+ isolates contained TVV. We isolated small extracellular vesicles (sEVs) from six T. vaginalis isolates that were either TVV free (ATCC 50143), harbored a single (ATCC 30236, ATCC 30238, T1), two (ATCC PRA-98), or three TVV subspecies (ATCC 50148). The presence of TVV subspecies in the six isolates was observed using reverse transcription-polymerase chain reaction (RT-PCR). Transmission electron microscopy (TEM) confirmed the presence of cup-shaped sEVs with a size range from 30-150 nm. Trichomonas vaginalis tetraspanin (TvTSP1; TVAG_019180), the classical exosome marker, was identified in all the sEV preparations. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that all the sEVs isolated from TVV+ isolates contain viral capsid proteins derived from the same TVV subspecies in that isolate as demonstrated by RT-PCR. To provide more comprehensive information on the TVV subspecies population in other T. vaginalis isolates, we investigated the distribution of TVV subspecies in twenty-four isolates by mining the New-Generation Sequencing (NGS) RNAseq datasets. Our results should be beneficial for future studies investigating the role of TVV on the pathogenicity of T. vaginalis and the possible transmission of virus subspecies among different isolates via sEVs.

Trichomonads, represented by the highly prevalent sexually transmitted human parasite Trichomonas vaginalis, are anaerobic eukaryotes with hydrogenosomes in the place of the standard mitochondria. Hydrogenosomes form indispensable FeS-clusters, synthesize ATP, and release molecular hydrogen as a waste product. Hydrogen formation is catalyzed by [FeFe] hydrogenase, the hallmark enzyme of all hydrogenosomes found in various eukaryotic anaerobes. Eukaryotic hydrogenases were originally thought to be exclusively localized within organelles, but today few eukaryotic anaerobes are known that possess hydrogenase in their cytosol. We identified a thus-far unknown hydrogenase in T. vaginalis cytosol that cannot use ferredoxin as a redox partner but can use cytochrome b5 as an electron acceptor. Trichomonads overexpressing the cytosolic hydrogenase, while maintaining the carbon flux through hydrogenosomes, show decreased excretion of hydrogen and increased excretion of methylated alcohols, suggesting that the cytosolic hydrogenase uses the hydrogen gas as a source of reducing power for the reactions occurring in the cytoplasm and thus accounts for the overall redox balance. This is the first evidence of hydrogen uptake in a eukaryote, although further work is needed to confirm it. Assembly of the catalytic center of [FeFe] hydrogenases (H-cluster) requires the activity of three dedicated maturases, and these proteins in T. vaginalis are exclusively localized in hydrogenosomes, where they participate in the maturation of organellar hydrogenases. Despite the different subcellular localization of cytosolic hydrogenase and maturases, the H-cluster is present in the cytosolic enzyme, suggesting the existence of an alternative mechanism of H-cluster assembly., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Entamoeba histolytica is believed to be devoid of peroxisomes, like most anaerobic protists. In this work, we provided the first evidence that peroxisomes are present in E. histolytica, although only seven proteins responsible for peroxisome biogenesis (peroxins) were identified (Pex1, Pex6, Pex5, Pex11, Pex14, Pex16, and Pex19). Targeting matrix proteins to peroxisomes is reduced to the PTS1-dependent pathway mediated via the soluble Pex5 receptor, while the PTS2 receptor Pex7 is absent. Immunofluorescence microscopy showed that peroxisomal markers (Pex5, Pex14, Pex16, Pex19) are present in vesicles distinct from mitosomes, the endoplasmic reticulum, and the endosome/phagosome system, except Pex11, which has dual localization in peroxisomes and mitosomes. Immunoelectron microscopy revealed that Pex14 localized to vesicles of approximately 90-100 nm in diameter. Proteomic analyses of affinity-purified peroxisomes and in silico PTS1 predictions provided datasets of 655 and 56 peroxisomal candidates, respectively; however, only six proteins were shared by both datasets, including myo-inositol dehydrogenase (myo-IDH). Peroxisomal NAD-dependent myo-IDH appeared to be a dimeric enzyme with high affinity to myo-inositol (Km 0.044 mM) and can utilize also scyllo-inositol, D-glucose and D-xylose as substrates. Phylogenetic analyses revealed that orthologs of myo-IDH with PTS1 are present in E. dispar, E. nutalli and E. moshkovskii but not in E. invadens, and form a monophyletic clade of mostly peroxisomal orthologs with free-living Mastigamoeba balamuthi and Pelomyxa schiedti. The presence of peroxisomes in E. histolytica and other archamoebae breaks the paradigm of peroxisome absence in anaerobes and provides a new potential target for the development of antiparasitic drugs., Competing Interests: The authors have declared that no competing interests exist.

Background: Treatment breaks during radiation for locally advanced Head and Neck Squamous Cell Carcinoma (HNSCC) is one of the important factors affecting the loco-regional control rate. We prospectively analysed the role lower pre-treatment hemoglobin (pre-T Hb) status and its influence on treatment breaks amongst patients undergoing concurrent chemoradiation (CRT)., Methods: One hundred and twenty HNSCC (T3-T4a, N1-N2c) patients treated by CRT were prospectively analysed for influence of pre-T Hb on treatment breaks. Sub-sites included oral cavity, oropharynx, hypopharynx & larynx. All patients received radiation to a total dose of 66Gy to PTV along with weekly Inj. Cisplatin 40 mg/m 2 . All patients were evaluated weekly and at the end of 6 weeks by hemogram, physician and radiological examination., Results: Our study population had a mean age (±standard deviation) of 55 (± 10.7) years (range: 27 - 69 years), 85 men and 35 women with a performance status of the Eastern Cooperative Oncology Group (ECOG) 1-2. The mean pre-T Hb calculated (using receiver operating characteristic curve [ROC]) was 10.3 g/dL. Among 120 patients, in the pre-T Hb of ≤10.3 g/dL group, 44 (75.9%) patients had treatment breaks of ≥5 days and 11 (17.7%) patients had treatment breaks < 5 days; in the pre-T Hb of >10.3 g/dL group, 14 (24.1%) patients had treatment breaks of ≥5 days and 51 (82.3%) patients had treatment breaks < 5 days (P = 0.001)., Conclusion: Lower pre-T Hb level of ≤ 10.3 g/dL is statistically significantly associated with higher treatment breaks of ≥ 5 days., Competing Interests: None

Transcription factor IID (TFIID) is a cornerstone in the transcription initiation in eukaryotes. It is composed of TBP and approximately 14 different subunits named TBP-associated factors (TAFs). TFIID has a key role in transcription of many genes involved in cell proliferation, cell growth, cell cycle, cell cycle checkpoint, and various other processes as well. Entamoeba histolytica, the protozoan parasite responsible for human amoebiasis, represents a major global health concern. Our research group has previously reported the genes coding the TATA box-binding protein (EhTBP) and TBP-related factor 1 (EhTRF1), which displayed different mRNA levels in trophozoites under different stress conditions. In this work, we identified the TBP-associated factor 1 (Ehtaf1) gene in the E. histolytica genome, which possess a well-conserved DUF domain and a Bromo domain located in the middle and C-terminus of the protein, respectively. The EhTAF1-DUF domain tertiary structure is similar to the corresponding HsTAF1 DUF domain. RT-qPCR experiments with RNA isolated from trophozoites harvested at different time points of the growth curve and under different stress conditions revealed that the Ehtaf1 gene was found slightly upregulated in the death phase of growth curve, but under heat shock stress, it was found upregulated 10 times, suggesting that Ehtaf1 might have an important role in the heat shock stress response. We also found that EhTAF1 is expressed in the nucleus and cytoplasm at 37 °C, but under heat shock stress, it is overexpressed in both the nucleus and cytoplasm, and partially colocalized with EhHSP70 in cytoplasm.

Background: Entamoeba histolytica is the protozoan parasite responsible for human amebiasis. It causes up to 100,000 deaths worldwide each year. This parasite has two closely related basal transcription factors, the TATA-box binding protein (EhTBP) and the TBP-related factor 1 (EhTRF1). TBP binds to the canonical TATTTAAA-box, as well as to different TATA variants. TRF1 also binds to the TATTTAAA-box. However, their binding capacity to diverse core promoter elements, including the GAAC-element, and their role in gene regulation in this parasite remains unknown., Methods: EMSA experiments were performed to determine the binding capacity of recombinant TBP and TRF1 to TATA variants, GAAC and GAAC-like boxes. For the functional analysis under different stress stimuli (e.g. growth curve, serum depletion, heat-shock, and UV-irradiation) and during the interaction with mammalian cells (erythrocytes, MDCK cell monolayers, and hepatocytes of hamsters), RT-qPCR, and gene knockdown were performed., Results: Both transcription factors bound to the different TATA variants tested, as well as to the GAAC-boxes, suggesting that they are GAAC-box-binding proteins. The K D values determined for TBP and TRF1 for the different TATA variants and GAAC-box were in the range of 10 -12  M to 10 -11  M. During the death phase of growth or in serum depletion, Ehtbp mRNA levels significantly increased, whereas the mRNA level of Ehtrf1 did not change under these conditions. Ehtrf1 gene expression was negatively regulated by UV-irradiation and heat-shock stress, with no changes in Ehtbp gene expression. Moreover, Ehtrf1 gene also showed a negative regulation during erythrophagocytosis, liver abscess formation, and a transient expression level increase at the initial phase of MDCK cell destruction. Finally, the Ehtbp gene knockdown displayed a drastic decrease in the efficiency of erythrophagocytosis in G3 trophozoites., Conclusions: To our knowledge, this study reveals that these basal transcription factors are able to bind multiple core promoter elements. However, their immediate change in gene expression level in response to different stimuli, as well as during the interaction with mammalian cells, and the diminishing of erythrophagocytosis by silencing the Ehtbp gene indicate the different physiological roles of these transcription factors in E. histolytica.

Many polyhydroxyalkanoates (PHAs) system genes, such as phaC, phaA, phaB, phaR, phaP and phaZ, are often found to be organised in the form of operon-like clusters. In this study, a genome survey was performed to identify such clustered PHA systems among 256 prokaryotic organisms. These data were then used to generate a comprehensive 16S rRNA species tree depicting the phylogenetic distribution of the observed clusters with diverse gene arrangements. In addition, the gene occurrences and physical linkages between PHA system genes were quantitatively estimated. From this, we identified a centrally connected hub gene, i.e. the phaC gene of PHA. Furthermore, a comparative investigation was performed between the clusters of PHA and glycogen, which decoded the role of the hub gene in the cluster organisation of both systems. Together, these findings suggest that the highly connected hub gene might contribute substantively towards the organisation and maintenance of the gene network connectivity in the clusters, particularly in the storage reserve systems., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)