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Christine Pierrot,1 Xiguang Zhang,2 Gigliola Zhangi,2 Aline Fréville,1 Angelita Rebollo,2 Jamal Khalife1 1Center for Infection and Immunity of Lille, U1019 – UMR 8204, Institut Pasteur de Lille, Université de Lille, Lille Cedex, 2CIMI Paris, UPMC/Inserm U1135, Paris, France The biogenesis of protein phosphatase 1 (PP1) holoenzyme in eukaryotes requires diverse regulatory subunit proteins (RSPs) that bind to the highly conserved PP1 catalytic subunit (PP1c) and direct its spatiotemporal activity as well as its specificity. Several studies demonstrated that most RSPs share a canonical common binding motif, the RVXF motif, which is present in ~85% of RSPs and is considered as the main contributor for the interaction to PP1c.1 In Plasmodium falciparum (Pf), our earlier studies revealed that leucine-rich repeat 1 (LRR1), one of the major RSPs of PfPP1 and an ortholog of human and yeast Sds22, lacks the RVXF motif. The amino acids sequence of PfLRR1 exhibits nine leucine-rich repeats (LRRs) and a hydrophobic region at the C-terminal end, known as the LRR cap motif.2 In this work, we identified the PP1-binding peptides of PfLRR1 and examined their capacity to affect Pf growth.

In previous studies of the infection of rats by P. berghei Anka, we have shown that primary blood stage infection induced the expansion of CD4+ T cells and CD8+ T cells in adult resistant rats while the number of CD4+CD25+ cells was found to be higher in young susceptible rats. In this work, the respective contribution of each cell population was determined in young and adult rats treated with monoclonal antibodies. Down-regulation of surface CD25 molecules, including those expressed by CD4+ cells did not significantly enhance the capacity of young rats to control the development of erythrocytic stages or modify the course of infection in adult infected rats. However, we observed a significant loss of protection when adult rats were treated with anti-CD4 mAb (W3/25) with higher blood parasitemia levels and ~ 50% of rats succumbed to infection. More importantly and in contrast to earlier studies performed in mice, we found a significant increase in blood parasite levels and a significant delay in parasite clearance in adult rats treated with anti-CD8 mAb OX8, known to deplete CD8+ cells. These results suggest that CD8+ cells play a critical role in the development of immune responses in rats to control the replication of blood stage parasites.

The activity, immunolocalization, and mRNA expression of Na, K-ATPase were studied in gills of the hyper-/ hypoosmoregulating crab Pachygrapsus marmoratus acclimated to salinities of 10, 37, and 45 ppt. Enzyme activity was significantly higher in pooled posterior gills (7-9) than in anterior gills (1-6) and increased significantly upon acclimation to diluted medium, confirming previous observations with other crab species. Immunofluorescent analysis of gill sections indicated localization of Na, K-ATPase protein in basolateral membranes of branchial epithelium adjacent to the afferent blood vessel. Using degenerate primers based on Na, K-ATPase ALPHA subunit homologies published in GenBank, we amplified related sequences in cDNA transcribed from gill mRNA and employed the resulting PCR products for sequencing and mRNA expression analyses. Immunofluorescence and mRNA quantitation produced parallel results, showing high expression of Na, K-ATPase protein and mRNA in gills 6, 7, and 8 at low salinity. In gills 6 and 8, the abundance of Na, K-ATPase protein and mRNA increased dramatically with decreasing salinity. However, gill 7 showed substantial levels of Na, K-ATPase protein and mRNA even in 45 ppt seawater, suggesting that this gill may play an important role in hypoosmoregulation. Our results offer a molecular explanation for the previously observed changes in Na, K-ATPase activity in gills of euryhaline crabs adjusting to osmoregulatory demands in salinity extremes, namely the upregulation of Na, K-ATPase gene transcription and subsequent translation of Na, K-ATPase protein. Supported by NSF (IBN-9807539).

The grapsid crab P. marmoratus is strongly euryhaline, regulating hemolymph osmolality by absorbing NaCl across the gills from low salinity and excreting NaCl into high salinity. Gene transcript levels measured in gills by quantitative PCR show significant changes with environmental salinity. Beginning within 2 hours after transfer of crabs from 36 to 10 ppt salinity, [Na.sup.++][K.sup.+]-ATPase [alpha]-subunit mRNA increases 8-to-20-fold in all analyzed gills and remains high for at least 48 hours. However, transfer from 36 to 45 ppt induces [Na.sup.++][K.sup.+]-ATPase [alpha]-subunit mRNA expression only in gill 7, increasing 10-fold by 6 hours, suggesting that gill 7 may be specialized for NaCl excretion. A closer examination of [alpha]-subunit transcripts demonstrated the presence of two isoforms, which we have labeled C and D, identical to each other except that the D isoform contains an inframe insert of 81 nucleotides near the 5 end, resulting in the encoding of 27 additional amino acids. Transcriptional response to salinity change is exhibited primarily by the C isoform, which is present at particularly low levels in animals acclimated to 36 ppt but becomes strongly induced upon salinity change. The expression of the D isoform is also enhanced but only modestly. Analysis of genomic DNA indicates that both C and D isofotms are encoded at the genomic level and are likely not the result of alternative splicing. Supported by NSF IBN-0340622.

Marbled rock crabs (Pachygrapsus marmoratus) are subjected to physiological stress due to large and rapid fluctuations in seawater salinity; conditions that we hypothesized might elicit changes in the gene expression of heat shock proteins (hsps) in gills, the most important osmoregulatory organs. We identified and sequenced an hsp70 cDNA from Pachygrapsus and quantitated hsp70 mRNA expression in anterior and posterior gills under low (10 ppt) and high (45 ppt) salinity at different time intervals following transfer from normal seawater (32 ppt). Real-time PCR analysis showed that hsp70 mRNA is expressed equally under control conditions in anterior (G5, G6) and posterior (G7, G8, G9) gills. Following transfer to low salinity, hsp70 expression increased about 2-fold in G5, G6 and G9 at 24 and 48 h. However, in G7 and G8 it started to increase by 3- to 4-fold within the first 6 h and slightly decreased by 48 h. Following transfer to high salinity, G5, G6, and G9 did not show any significant change. However, G8 showed a 2-fold increase within 4 h and by 48 h decreased gradually to the level observed under control conditions. In G7, hsp70 expression increased about 3-fold within 4 h and about 4-fold in 6 h, then slightly decreased at 24 and 48 h to a level that was still twice the amount expressed in controls. The expression of arginine kinase (AK), a putative housekeeping gene, showed nearly equal levels at all times. These results confirm that hsp70 is highly expressed under salinity-stressed conditions and that posterior gills, gill 7 in particular, play an important role in osmoregulatory adjustments of Pachygrapsus marmoratus. Supported by NSF (IBN-0340622).

Changes in gene expression in gills of the hyper-/hypo-osmoregulating grapsid crab Pachygrapsus marmoratus following transfer from seawater (35 ppt) to low and high salinities (10 and 45 ppt) were investigated by measuring mRNA content for Na/K-ATPase alpha-subunit, Na/K/2Cl cotransporter, and V-type H-ATPase B-subunit by real-time quantitative PCR. In low salinity, Na/K-ATPase mRNA increased 6- to 20-fold in all tested gills, with significant enhancement in anterior gills 5 and 6 and posterior gill 9 24h after transfer from seawater and more rapid increases in posterior gills 7 and 8 4h after transfer. No differences in cotransporter mRNA expression occurred in anterior gills, but a slight increase was measured in posterior gills 6 to 48h after transfer. V-type H-ATPase expression increased 3-fold in posterior gill 7 within 2h and increased up to 10-fold in other gills after 48 h. Because enhanced expression of V-ATPase and Na/K-ATPase is induced by low salinity, these two transporters likely play critical roles in the active uptake of NaCI in P. marmoratus. Following transfer to high salinity, only posterior gill 7 exhibited a marked increase in Na/K-ATPase mRNA, doubling 4h after transfer and becoming 4-fold higher after 6h; slight increases in gills 8 and 9 and no changes in anterior gills were observed. Similar results were obtained for V-ATPase and cotransporter with a sharp increase (6- and 3-fold respectively) in gill 7 6h after transfer. The restricted response in ion transporter mRNA expression following transfer of P. marmoratus to high salinity suggests a high degree of gill specialization for hypo-osmoregulatory NaCl excretion. Supported by MDIBL and NSF (DBI-0100394).

The article focuses on a study investigating the role of CD4+ and CD8+ cells in the control of blood parasite replication. It mentions that primary blood stage infection of Plasmodium berghei Anka induces the expansion of CD4+ T cells and CD8+ T cells in adult resistant rats while CD4+CD25+ cells is higher in young susceptible rats. It says that down regulation of surface CD25 molecules does not enhance the capacity of young rats to control the development or change the course of infection in adult infected rats. It remarks loss of protection in adult rats treated with anti-CD4 mAb with higher blood parasitemia levels. It notes the increase in blood parasite levels and delay in parasite clearance with treatment of anti-CD8 mAb OX8.

The article discusses the highlights of the fifth general assembly "Genees & Kunst 5" of the Dutch Performing Arts Medicine Association/Nederlandse Vereniging voor Dans en Muziek-Geneeskunde (NVDMG) which was held in the south of the Netherlands. The mission statement as well as the continuing growth of NVDMG was cited in the introductory remarks by Doctor A.B.M. Rietveld. Also, the abstracts of various scientific presentations prepared for the event are mentioned including one which focused on the New Multidisciplinary Performing Arts Medicine Clinic.

The article discusses the highlights of the 4th Biannual Symposium on Medical Problems of Dancers and Musicians titled "Genees & Kunst 4" held in Arnhem, the Netherlands on March 17, 2007. Among the lecturers who presented their scientific studies were Doctors W. J. Rijnberg and J. D. Speelman, and Gaby Allard, director of the ArtEZ Dance Academy in Arnhem. The meeting concluded with a performance titled "See the Music" by Introdans. It presents the abstracts of research tackled by the speakers.

Abstract: Astacus leptodactylus is a decapod crustacean fully adapted to freshwater where it spends its entire life cycle after hatching under huge osmoconcentration differences between the hemolymph and surrounding freshwater. We investigated the expression of mRNA encoding one ion transport-related protein, Na+/K+-ATPase α-subunit, and one putative housekeeping gene, β-actin, during crayfish ontogenesis using quantitative real-time PCR. A 216-amino acid part of the open reading frame region of the cDNA coding for the Na+/K+-ATPase α-subunit was sequenced from total embryo, juvenile and adult gill tissues. The predicted amino acid sequence showed a high percentage similarity to those of other invertebrates (up to 95%) and vertebrates (up to 69%). β-actin expression exhibited modest changes through embryonic development and early post-embryonic stage. The Na+/K+-ATPase α-subunit gene was expressed in all studied stages from metanauplius to juvenile. Two peaks of expression were observed: one in young embryos at 25% of embryonic development (EI=100 μm), and one in embryos just before hatching (at EI=420 μm), continuing in the freshly hatched juveniles. The Na+/K+-ATPase expression profile during embryonic development is time-correlated with the occurrence of other features, including ontogenesis of excretory antennal glands and differentiation of gill ionocytes linked to hyperosmoregulation processes and therefore involved in freshwater adaptation. [Copyright &y& Elsevier]

Toxoplasmosis is a critical health issue for immune-deficient individuals and the offspring of newly infected mothers. It is caused by a unicellular intracellular parasite called Toxoplasma gondii that is found worldwide. Although efficient drugs are commonly used to treat toxoplasmosis, serious adverse events are common. Therefore, new compounds with potent anti- T . gondii activity are needed to provide better suited treatments. We have tested compounds designed to target specifically histone deacetylase enzymes. Among the 55 compounds tested, we identified three compounds showing a concentration of drug required for 50% inhibition (IC 50 ) in the low 100 nM range with a selectivity index of more than 100. These compounds are not only active at inhibiting the growth of the parasite in vitro but also at preventing some of the consequences of the acute disease in vivo . Two of these hydroxamate based compound also induce a hyper-acetylation of the parasite histones while the parasitic acetylated tubulin level remains unchanged. These findings suggest that the enzymes regulating histone acetylation are potent therapeutic targets for the treatment of acute toxoplasmosis., Competing Interests: The authors declare no conflict of interest.

Male and female infertility has become a worldwide public health problem. The global obesity epidemic has paralleled a decrease in semen quality. Yet, the association between body mass index (BMI) and sperm parameters remains controversial. The objective of this study is to determine the link between body mass index and seminal parameters. We made an observational study and retrospective analysis. Men who made semen analysis during the period of January 2015 to September 2021 in Reims University Hospital, were included. A total of 1 655 patients were recruited and separated into five groups according to BMI values. Second- and third-degree obesity had a significantly higher risk of having a pathological sperm count (p: 0,0038). Second- and Third-degree obesity was associated with a pathologic vitality (p: 0,012). No significant differences existed between sperm mobility and body mass index. Regarding low body mass index, a significant difference exists on the sperm morphology (p: 0,013). Sperm morphology is also impacted in overweight and obesity groups. The information of the couples on weight is necessary to improve sperm parameters, spontaneous pregnancies and efficacity of assisted reproductive technic.

Protein phosphatase 1 (PP1) is a key enzyme for Plasmodium development. However, the detailed mechanisms underlying its regulation remain to be deciphered. Here, we report the functional characterization of the Plasmodium berghei leucine-rich repeat protein 1 (PbLRR1), an orthologue of SDS22, one of the most ancient and conserved PP1 interactors. Our study shows that PbLRR1 is expressed during intra-erythrocytic development of the parasite, and up to the zygote stage in mosquitoes. PbLRR1 can be found in complex with PbPP1 in both asexual and sexual stages and inhibits its phosphatase activity. Genetic analysis demonstrates that PbLRR1 depletion adversely affects the development of oocysts. PbLRR1 interactome analysis associated with phospho-proteomics studies identifies several novel putative PbLRR1/PbPP1 partners. Some of these partners have previously been characterized as essential for the parasite sexual development. Interestingly, and for the first time, Inhibitor 3 (I3), a well-known and direct interactant of Plasmodium PP1, was found to be drastically hypophosphorylated in PbLRR1-depleted parasites. These data, along with the detection of I3 with PP1 in the LRR1 interactome, strongly suggest that the phosphorylation status of PbI3 is under the control of the PP1-LRR1 complex and could contribute (in)directly to oocyst development. This study provides new insights into previously unrecognized PbPP1 fine regulation of Plasmodium oocyst development through its interaction with PbLRR1.

Parasites belonging to the Apicomplexa phylum still represent a major public health and world-wide socioeconomic burden that is greatly amplified by the spread of resistances against known therapeutic drugs. Therefore, it is essential to provide the scientific and medical communities with innovative strategies specifically targeting these organisms. In this review, we present an overview of the diversity of the phosphatome as well as the variety of functions that phosphatases display throughout the Apicomplexan parasites' life cycles. We also discuss how this diversity could be used for the design of innovative and specific new drugs/therapeutic strategies.

Introduction: Toxoplasmosis is a major health issue worldwide, especially for immune-deficient individuals and the offspring of newly infected mothers. It is caused by a unicellular intracellular parasite called Toxoplasma gondii. Although the drugs commonly used to treat toxoplasmosis are efficient, they present serious side effects and adverse events are common. Therefore, there is a need for the discovery of new compounds with potent anti-Toxoplasma gondii activity., Methods: This study tested compounds designed to target enzymes that are involved in the epigenetic regulation of gene expression., Results: Among the most active compounds, an HDAC inhibitor showing an IC 50 of 30 nM with a selectivity index above 100 was identified. MC1742 was active at inhibiting the growth of the parasite in vitro but also at preventing the consequences of the acute disease in vivo. This compound induced hyper-acetylation of histones, while the acetylated tubulin level remained unchanged. After MC1742 treatment, the parasite expression profile was profoundly changed with the activation of genes preferentially expressed in the sexual stages that are normally repressed in the tachyzoite stage., Conclusions: These findings suggest that this compound disturbs the Toxoplasma gondii gene expression program, inducing parasite death., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Malaria parasites require multiple phosphorylation and dephosphorylation steps to drive signaling pathways for proper differentiation and transformation. Several protein phosphatases, including protein phosphatase 1 (PP1), one of the main dephosphorylation enzymes, have been shown to be indispensable for the Plasmodium life cycle. The catalytic subunit of PP1 (PP1c) participates in cellular processes via dynamic interactions with a vast number of binding partners that contribute to its diversity of action. In this study, we used Plasmodium berghei transgenic parasite strains stably expressing PP1c or its inhibitor 2 (I2) tagged with mCherry, combined with the mCherry affinity pulldown of proteins from asexual and sexual stages, followed by mass spectrometry analyses. Mapped proteins were used to identify interactomes and to cluster functionally related proteins. Our findings confirm previously known physical interactions of PP1c and reveal enrichment of common biological processes linked to cellular component assembly in both schizonts and gametocytes to biosynthetic processes/translation in schizonts and to protein transport exclusively in gametocytes. Further, our analysis of PP1c and I2 interactomes revealed that nuclear export mediator factor and peptidyl-prolyl cis-trans isomerase, suggested to be essential in P. falciparum , could be potential targets of the complex PP1c/I2 in both asexual and sexual stages. Our study emphasizes the adaptability of Plasmodium PP1 and provides a fundamental study of the protein interaction landscapes involved in a myriad of events in Plasmodium , suggesting why it is crucial to the parasite and a source for alternative therapeutic strategies.

Protein phosphatase type 1 (PP1) forms a wide range of Ser/Thr-specific phosphatase holoenzymes which contain one catalytic subunit (PP1c), present in all eukaryotic cells, associated with variable subunits known as regulatory proteins. It has recently been shown that regulators take a leading role in the organization and the control of PP1 functions. Many studies have addressed the role of these regulators in diverse organisms, including humans, and investigated their link to diseases. In this review we summarize recent advances on the role of PP1c in Plasmodium, its interactome and regulators. As a proof of concept, peptides interfering with the regulator binding capacity of PP1c were shown to inhibit the growth of P. falciparum, suggesting their potential as drug precursors., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The study of host/pathogen interactions at the cellular level during Plasmodium intra-erythrocytic cycle requires differential extraction techniques aiming to analyze the different compartments of the infected cell. Various protocols have been proposed in the literature to study specific compartments and/or membranes in the infected erythrocyte. The task remains delicate despite the use of enzymes or detergents theoretically capable of degrading specific membranes inside the infected cell. The remit of this protocol is to propose a method to isolate the erythrocyte cytosol and ghosts from the other compartments of the infected cell via a percoll gradient. Also, the lysis of the erythrocyte membrane is done using equinatoxin II, which has proven to be more effective at erythrocyte lysis regardless of the cell infection status, compared to the commonly used streptolysin. The parasitophorous vacuole (PV) content is collected after saponin lysis, before recovering membrane and parasite cytosol proteins by Triton X-100 lysis. The lysates thus obtained are analyzed by Western blot to assess the accuracy of the various extraction steps. This protocol allows the separation of the host compartment from the parasite compartments (PV and parasite), leading to potential studies of host proteins as well as parasite proteins exported to the host cell., Competing Interests: Competing interestsThe authors declare no competing interests., (Copyright © 2020 The Authors; exclusive licensee Bio-protocol LLC.)

The essential and distinct functions of Protein Phosphatase type 1 (PP1) catalytic subunit in eukaryotes are exclusively achieved through its interaction with a myriad of regulatory partners. In this work, we report the molecular and functional characterization of Gametocyte EXported Protein 15 (GEXP15), a Plasmodium specific protein, as a regulator of PP1. In vitro interaction studies demonstrated that GEXP15 physically interacts with PP1 through the RVxF binding motif in P. berghei. Functional assays showed that GEXP15 was able to increase PP1 activity and the mutation of the RVxF motif completely abolished this regulation. Immunoprecipitation assays of tagged GEXP15 or PP1 in P. berghei followed by immunoblot or mass spectrometry analyses confirmed their interaction and showed that they are present both in schizont and gametocyte stages in shared protein complexes involved in the spliceosome and proteasome pathways and known to play essential role in parasite development. Phenotypic analysis of viable GEXP15 deficient P. berghei blood parasites showed that they were unable to develop lethal infection in BALB/c mice or to establish experimental cerebral malaria in C57BL/6 mice. Further, although deficient parasites produced gametocytes they did not produce any oocysts/sporozoites indicating a high fitness cost in the mosquito. Global proteomic and phosphoproteomic analyses of GEXP15 deficient schizonts revealed a profound defect with a significant decrease in the abundance and an impact on phosphorylation status of proteins involved in regulation of gene expression or invasion. Moreover, depletion of GEXP15 seemed to impact mainly the abundance of some specific proteins of female gametocytes. Our study provides the first insight into the contribution of a PP1 regulator to Plasmodium virulence and suggests that GEXP15 affects both the asexual and sexual life cycle., Competing Interests: The authors have declared that no competing interests exist.

Antimicrobial peptides (AMPs) are important components of the vertebrate and invertebrate innate immune systems. Although AMPs are widely recognized for their broad-spectrum activity against bacteria, fungi, and viruses, their activity against protozoan parasites has not been investigated in detail. In this study, we tested 10 AMPs from three different insect species: the greater wax moth Galleria mellonella (cecropin A-D), the fruit fly Drosophila melanogaster (drosocin, Mtk-1 and Mtk-2), and the blow fly Lucilia sericata (LSerPRP-2, LSerPRP-3 and stomoxyn). We tested each AMP against the protozoan parasite Plasmodium falciparum which is responsible for the most severe form of malaria in humans. We also evaluated the impact of these insect AMPs on mouse and pig erythrocytes. Whereas all AMPs showed low hemolytic effects towards mouse and pig erythrocytes, only D. melanogaster Mtk-1 and Mtk-2 significantly inhibited the growth of P. falciparum at low concentrations. Mtk-1 and Mtk-2 could therefore be considered as leads for the development of antiparasitic drugs targeting the clinically important asexual blood stage of P. falciparum.

Pseudokinases play key roles in many biological processes but they are poorly understood compared to active kinases. Eight putative pseudokinases have been predicted in Plasmodium species. We selected the unique pseudokinase belonging to tyrosine kinase like (TKL) family for detailed structural and functional analysis in P. falciparum and P. berghei. The primary structure of PfpTKL lacks residues critical for kinase activity, supporting its annotation as a pseudokinase. The recombinant pTKL pseudokinase domain was able to bind ATP, but lacked catalytic activity as predicted. The sterile alpha motif (SAM) and RVxF motifs of PfpTKL were found to interact with the P. falciparum proteins serine repeat antigen 5 (SERA5) and protein phosphatase type 1 (PP1) respectively, suggesting that pTKL has a scaffolding role. Furthermore, we found that PP1c activity in a heterologous model was modulated in an RVxF-dependent manner. During the trophozoite stages, PbpTKL was exported to infected erythrocytes where it formed complexes with proteins involved in cytoskeletal organization or host cell maturation and homeostasis. Finally, genetic analysis demonstrated that viable strains obtained by genomic deletion or knocking down PbpTKL did not affect the course of parasite intra-erythrocytic development or gametocyte emergence, indicating functional redundancy during these parasite stages.

Despite the recent reductions in the global burden of malaria, this disease remains a devastating cause of death in tropical and subtropical regions. As there is no broadly effective vaccine for malaria, prevention and treatment still rely on chemotherapy. Unfortunately, emerging resistance to the gold standard artemisinin combination therapies means that new drugs with novel modes of action are urgently needed. In this context, Plasmodium histone modifying enzymes have emerged as potential drug targets, prompting us to develop and optimize compounds directed against such epigenetic targets. A panel of 51 compounds designed to target different epigenetic enzymes were screened for activity against Plasmodium falciparum parasites. Based on in vitro activity against drug susceptible and drug-resistant P. falciparum lines, selectivity index criterion and favorable pharmacokinetic properties, four compounds, one HDAC inhibitor (1) and three DNMT inhibitors (37, 43 and 45), were selected for preclinical studies in a mouse model of malaria. In vivo data showed that 37, 43 and 45 exhibited oral efficacy in the mouse model of Plasmodium berghei infection. These compounds represent promising starting points for the development of novel antimalarial drugs., (Crown Copyright © 2018. Published by Elsevier Masson SAS. All rights reserved.)