A divergent protein kinase A regulatory subunit essential for morphogenesis of the human pathogen Leishmania.

Parasitic protozoa of the genus Leishmania cycle between the phagolysosome of mammalian macrophages, where they reside as rounded intracellular amastigotes, and the midgut of female sand flies, which they colonize as elongated extracellular promastigotes. Previous studies indicated that protein kinase A (PKA) plays an important role in the initial steps of promastigote differentiation into amastigotes. Here, we describe a novel regulatory subunit of PKA (which we have named PKAR3) that is unique to Leishmania and most (but not all) other Kinetoplastidae. PKAR3 is localized to subpellicular microtubules (SPMT) in the cell cortex, where it recruits a specific catalytic subunit (PKAC3). Promastigotes of pkar3 or pkac3 null mutants lose their elongated shape and become rounded but remain flagellated. Truncation of an N-terminal formin homology (FH)-like domain of PKAR3 results in its detachment from the SPMT, also leading to rounded promastigotes. Thus, the tethering of PKAC3 via PKAR3 at the cell cortex is essential for maintenance of the elongated shape of promastigotes. This role of PKAR3 is reminiscent of PKARIß and PKARIIß binding to microtubules of mammalian neurons, which is essential for the elongation of dendrites and axons, respectively. Interestingly, PKAR3 binds nucleoside analogs, but not cAMP, with a high affinity similar to the PKAR1 isoform of Trypanosoma. We propose that these early-diverged protists have re-purposed PKA for a novel signaling pathway that spatiotemporally controls microtubule remodeling and cell shape.

Surgical Ablation for Atrial Fibrillation: Risk Factors for Recurrence and Long-Term Outcome.

OBJECTIVES: Atrial fibrillation (AF) is the most common cardiac arrhythmia. Due to the insufficient efficacy of antiarrhythmic drugs and their adverse side effects, there has been considerable interest in the interventional treatment of AF, including both catheter ablation and surgical ablation. Surgical ablation or the maze procedure is a treatment option for patients with AF undergoing concomitant or isolated cardiac surgery. DESIGN: We performed a retrospective study of prospectively collected data to investigate short- and long-term outcomes of patients who underwent the surgical ablation of AF. Outcome variables included freedom from recurrent atrial arrhythmias and mortality at 1-, 3-, 5-, and 7-year follow-ups. We also identified risk factors for arrhythmia recurrence and mortality. SETTING: Israel's largest university tertiary care center. PARTICIPANTS: The study population comprised 668 patients operated on between January 1, 2006, and June 30, 2022. All patient data were extracted from our departmental database. INTERVENTIONS: Concomitant or stand-alone surgical AF ablation. MEASUREMENTS AND MAIN RESULTS: The mean duration of follow-up was 106 ± 66.7 months. Freedom from AF was 97.6% (n = 615) and mortality was 3% (n = 20) at the 1-year follow-up, 95.3% (n = 574) and 6.1% (n = 45) at 3 years, 90.1% (n = 396) and 9.1% (n = 61) at 5 years, and 77.5% (n = 308) and 10.8% (n = 72) at 7 years. According to logistic regression analysis, age and female sex determined the 7-year freedom from AF, and risk factors for 7-year mortality included diabetes mellitus, age, and valve surgery. CONCLUSIONS: Surgical ablation had a high success rate, with freedom from recurrent atrial arrhythmia at 1-, 3-, 5-, and 7-year follow-ups. Age and female sex were factors determining the 5- and 7-year recurrence of AF.

Obstetric and maternal outcomes of IVF and oocyte donation pregnancies among women ages 40-45-a large cohort study.

PURPOSE: To analyze the perinatal and maternal outcomes of women ranging in age from 40 to 45 years who gave birth after in vitro fertilization or oocyte donation, compared to spontaneous conception. METHODS: This retrospective cohort study used electronic data from a national healthcare service from 2000 through 2019. Three groups were compared: spontaneous pregnancy (SC), in vitro fertilization (IVF) utilizing autologous oocytes, and pregnancies resulting from oocyte donation (OD). The primary study outcomes were preterm labor (PTL) before 37 weeks of gestation, and infants classified as small for gestational age (SGA). RESULTS: The cohort included 26,379 SC, 2237 IVF pregnancies, and 300 OD pregnancies for women ages 40-45 years at delivery. Women with OD or IVF had a higher incidence of PTL < 37 weeks compared to women with SC (19.7% vs. 18% vs. 6.9%, p = 0.001), PTL < 34 (7% vs. 4.5% vs. 1.4%, p = 0.001), PTL < 32 (3.7 vs. 2.1 vs. 0.6, p = 0.001). A multivariable logistic regression for PTL < 37 weeks demonstrated that age (OR = 1.18) and hypertensive diseases (OR = 3.4) were statistically significant factors. The OD group had a lower rate of SGA compared to SC (1% vs. 4.3%, p = 0.001), while the IVF group had a higher rate of SGA compared to SC (9.1% vs. 4.3%, p = 0.001). Hypertensive diseases in pregnancy were significantly higher among the OD group and the IVF group compared to SP pregnancies (3.3% vs. 1%, p = 0.002; 2.3% vs. 1%, p = 0.001, respectively). CONCLUSIONS: Women ages 40-45 undergoing IVF or OD have a greater risk of PTL, possibly due to higher rates of hypertensive disorders of pregnancy.

Thawing day 3 embryos and culturing to day 5 may be a better method for frozen embryo transfer.

PURPOSE: Does thawing cleavage embryos and culturing them for transfer as blastocysts improve pregnancy and perinatal outcomes compared to transferring thawed blastocysts? METHODS: Retrospective, observational cohort study performed at two assisted reproductive technology centers, 2014 to 2020. A total of 450 patients with 463 thawed embryo transfer cycles were divided into 2 groups according to the embryonic developmental stage at cryopreservation and transfer: 231 thawed blastocysts (day 5 group) and 232 thawed cleavage embryos that were cultured for 2 days and transferred as blastocysts (day 3-5 group). The two groups were compared for demographics, routine parameters of IVF treatment, pregnancy rates, and perinatal outcomes. RESULTS: Multivariable logistic regression analysis for ongoing pregnancy and delivery demonstrated that the day 3-5 group had a greater likelihood of achieving ongoing pregnancy and delivery compared to the day 5 group (OR 1.58, 95%CI 1.062-2.361, p = 0.024). Perinatal outcomes were comparable between the three groups. CONCLUSION: Our results support culturing post-thaw cleavage embryos for 2 days and transferring them as blastocysts to increase chances of ongoing pregnancy and delivery.

Repeated GnRH agonist doses for luteal support: a proof of concept.

RESEARCH QUESTION: What are the safety and feasibility of repeated subcutaneous doses of gonadotrophin-releasing hormone (GnRH) agonist for luteal support in IVF cycles triggered by a GnRH agonist? DESIGN: In this prospective trial, patients exhibiting oestradiol concentrations of over 2500 pg/ml after use of a GnRH agonist for triggering ovulation were initially randomized to GnRH agonist luteal support (0.1 mg subcutaneously every other day, starting on day 3 after embryo transfer) or to a control group supported by 80 µg of recombinant human chorionic gonadotrophin (HCG) on day 3 after embryo transfer. All patients underwent a day 5 blastocyst transfer. Randomization to the HCG luteal support was stopped owing to two cases of ovarian hyperstimulation syndrome (OHSS) and the study was continued solely with GnRH agonist luteal support. RESULTS: The study included 39 women in the repeated GnRH agonist luteal support group and seven in the HCG micro dose group. There were no cases of OHSS among patients supported by a GnRH agonist, and no other adverse events were recorded. There were no cases of bleeding before the pregnancy test, and hence no cases of an insufficient luteal phase. A clinical pregnancy rate of 43.6% was achieved with GnRH agonist luteal support. Hormone dynamics during the stimulation cycle reflected rising LH and progesterone concentrations after the introduction of GnRH agonist support. CONCLUSIONS: Repeated doses of GnRH agonist every other day as a method of luteal support provided safe and effective luteal support for women who underwent GnRH agonist triggering in a GnRH antagonist IVF cycle.

Number of Oocytes Retrieved as a Criterion for "Freeze-All" Strategy versus a Single "Rescue" Bolus of Low-Dose Human Chorionic Gonadotropin Following GnRH Agonist for Ovulation Triggering: A Pilot Study.

AIM: To evaluate the number of oocytes retrieved as a criterion - when to use a "freeze-all" or low-dose "rescue human chorionic gonadotropin (hCG)" strategy. METHODS: A retrospective study. Instead of the classic hCG trigger, an E2 level of ≥3,000 pg/mL was used to trigger ovulation with GnRH agonist. The decision whether to "freeze all" or perform fresh embryo transfer (ET) with a bolus of hCG was made based on a maximum number of 20 oocytes retrieved. Beyond this cut off, a "freeze-all" strategy was implemented. Below this cut-off value, a fresh ET using a single bolus of 62.5 µg hCG on day 3 following oocyte pick-up was performed. The main outcome measures were clinical pregnancy rates and ovarian hyperstimulation syndrome (OHSS). RESULTS: E2 and progesterone levels increased after the rescue hCG bolus administration (E2 from 643.4 ± 311.1 to 1,086.1 ± 574.7 pg/mL, p = 0.003 and progesterone from 13.1 ± 4.8 to 39.2 ± 28.7 ng/mL, p < 0.0001). The clinical pregnancy rates were 25% in the freeze-all group and 32% in the rescue hCG group (p = 0.57). OHSS was not reported in either group. CONCLUSIONS: Both strategies seem to be efficacious and safe. An upper limit of 20 retrieved oocytes appears to be safe for applying a rescue hCG strategy.

Leishmania panamensis infection and antimonial drugs modulate expression of macrophage drug transporters and metabolizing enzymes: impact on intracellular parasite survival.

OBJECTIVES: Treatment failure is multifactorial. Despite the importance of host cell drug transporters and metabolizing enzymes in the accumulation, distribution and metabolism of drugs targeting intracellular pathogens, their impact on the efficacy of antileishmanials is unknown. We examined the contribution of pharmacologically relevant determinants in human macrophages in the antimony-mediated killing of intracellular Leishmania panamensis and its relationship with the outcome of treatment with meglumine antimoniate. METHODS: Patients with cutaneous leishmaniasis who failed (n = 8) or responded (n =8) to treatment were recruited. Gene expression profiling of pharmacological determinants in primary macrophages was evaluated by quantitative RT-PCR and correlated to the drug-mediated intracellular parasite killing. Functional validation was conducted through short hairpin RNA gene knockdown. RESULTS: Survival of L. panamensis after exposure to antimonials was significantly higher in macrophages from patients who failed treatment. Sixteen macrophage drug-response genes were modulated by infection and exposure to meglumine antimoniate. Correlation analyses of gene expression and intracellular parasite survival revealed the involvement of host cell metallothionein-2A and ABCB6 in the survival of Leishmania during exposure to antimonials. ABCB6 was functionally validated as a transporter of antimonial compounds localized in both the cell and phagolysosomal membranes of macrophages, revealing a novel mechanism of host cell-mediated regulation of intracellular drug exposure and parasite survival within phagocytes. CONCLUSIONS: These results provide insight into host cell mechanisms regulating the intracellular exposure of Leishmania to antimonials and variations among individuals that impact parasite survival. Understanding of host cell determinants of intracellular pharmacokinetics/pharmacodynamics opens new avenues to improved drug efficacy for intracellular pathogens.

Anatomical and clinical risk stratification tool for mortality risk assessment following revascularization for multivessel coronary artery disease.

OBJECTIVE: This study aimed to assess the prognostic ability of SYNTAX score II in left main and/or 3-vessel disease patients undergoing revascularization either by coronary artery bypass grafting or percutaneous coronary intervention in a national registry. METHODS: This prospective registry included consecutive patients with multivessel disease enrolled between January and April 2013 from all 22 hospitals in Israel that perform coronary angiography. Of the 1112 study patients, 368 patients (33%) had a low (<25), 372 (33%) had an intermediate (25-35) and 372 patients (33%) a high (≥35) SYNTAX score II. RESULTS: Patients with a high SYNTAX score II had higher 30-day mortality compared with those with an intermediate or low SYNTAX score II (2.8% vs 0.6% vs 0% respectively, P = .001). Each 1-unit increment in SYNTAX score II increased the odds for death at 30 days by 11% (95% CI, 1.02-1.22; P = .026). Six-year mortality was higher among patients with a high compared with an intermediate or low SYNTAX score II (34.9% vs 11% vs 3.8%; log-rank P < .001). By adding a SYNTAX score II to standard prognostic factors, we showed a significant improvement of 40.1% (P < .001) for predicting 6-year mortality. The area under the curve of the SYNTAX score II (continuous) yielded 0.79 (95% CI, 0.75-0.82) in predicting 6-year mortality. CONCLUSIONS: Our findings show that the admission SYNTAX score II is a powerful marker of short- and long-term mortality, and therefore may be used as a risk stratification tool in patients with multivessel coronary artery disease who are candidates for revascularization.

Macrophage metallothioneins participate in the antileishmanial activity of antimonials.

Host cell functions that participate in the pharmacokinetics and pharmacodynamics (PK/PD) of drugs against intracellular pathogen infections are critical for drug efficacy. In this study, we investigated whether macrophage mechanisms of xenobiotic detoxification contribute to the elimination of intracellular Leishmania upon exposure to pentavalent antimonials (SbV). Primary macrophages from patients with cutaneous leishmaniasis (CL) (n=6) were exposed ex vivo to L. V. panamensis infection and SbV, and transcriptomes were generated. Seven metallothionein (MT) genes, potent scavengers of heavy metals and central elements of the mammalian cell machinery for xenobiotic detoxification, were within the top 20 up-regulated genes. To functionally validate the participation of MTs in drug-mediated killing of intracellular Leishmania, tandem knockdown (KD) of MT2-A and MT1-E, MT1-F, and MT1-X was performed using a pan-MT shRNA approach in THP-1 cells. Parasite survival was unaffected in tandem-KD cells, as a consequence of strong transcriptional upregulation of MTs by infection and SbV, overcoming the KD effect. Gene silencing of the metal transcription factor-1 (MTF-1) abrogated expression of MT1 and MT2-A genes, but not ZnT-1. Upon exposure to SbV, intracellular survival of Leishmania in MTF-1KD cells was significantly enhanced. Results from this study highlight the participation of macrophage MTs in Sb-dependent parasite killing.

GnRH agonist-triggering ovulation in women with advanced age.

This study evaluates the effect of GnRH agonist (GnRHa) trigger for ovulation induction among women with advanced maternal age (AMA). This is a retrospective study performed at a single assisted reproductive technology centre, 2012 to 2020. A total of 306 patients with 515 IVF cycles who were triggered with GnRHa for Ovum Pick Up (OPU), were divided into two groups according to maternal age: age ≥ 40 and age < 40. The groups were compared for demographics, stimulation parameters of IVF treatment and IVF treatment outcomes. The patients in the age < 40 group were approximately 10 years younger than the patients in the age ≥ 40 group (31 ± 5.4 vs. 41.5 ± 1.3 years, p < 0.001). The age ≥ 40 group had significantly higher mean E2/retrieved oocytes ratio, compared to the age < 40 group (310.3 ± 200.6 pg/ml vs. 239 ± 168.2 pg/ml, p = 0.003), and a lower mean MII/retrieved oocyte (35 ± 37.8 vs. 43.4 ± 35.9, p = 0.05, respectively). Multivariable logistic regression analysis for E2/retrieved oocytes demonstrated that age < 40 and total dose of gonadotropins were significant variables. In conclusion, GnRHa for ovulation triggering in high responder patients prior to OPU appears to be a good option for AMA. However, this population is characterized by different parameters of ovarian response that require further evaluation.

Gliotoxin, an Immunosuppressive Fungal Metabolite, Primes Plant Immunity: Evidence from Trichoderma virens-Tomato Interaction.

Beneficial interaction of members of the fungal genus Trichoderma with plant roots primes the plant immune system, promoting systemic resistance to pathogen infection. Some strains of Trichoderma virens produce gliotoxin, a fungal epidithiodioxopiperazine (ETP)-type secondary metabolite that is toxic to animal cells. It induces apoptosis, prevents NF-κB activation via the inhibition of the proteasome, and has immunosuppressive properties. Gliotoxin is known to be involved in the antagonism of rhizosphere microorganisms. To investigate whether this metabolite has a role in the interaction of Trichoderma with plant roots, we compared gliotoxin-producing and nonproducing T. virens strains. Both colonize the root surface and outer layers, but they have differential effects on root growth and architecture. The responses of tomato plants to a pathogen challenge were followed at several levels: lesion development, levels of ethylene, and reactive oxygen species. The transcriptomic signature of the shoot tissue in response to root interaction with producing and nonproducing T. virens strains was monitored. Gliotoxin producers provided stronger protection against foliar pathogens, compared to nonproducing strains. This was reflected in the transcriptomic signature, which showed the induction of defense-related genes. Two markers of plant defense response, PR1 and Pti-5, were differentially induced in response to pure gliotoxin. Gliotoxin thus acts as a microbial signal, which the plant immune system recognizes, directly or indirectly, to promote a defense response. IMPORTANCE A single fungal metabolite induces far-reaching transcriptomic reprogramming in the plant, priming immune responses and defense, in contrast to its immunosuppressive effect on animal cells. While the negative effects of gliotoxin-producing Trichoderma strains on growth may be observed only under a particular set of laboratory conditions, gliotoxin-linked molecular patterns, including the potential for limited cell death, could strongly prime plant defense, even in mature soil-grown plants in which the same Trichoderma strain promotes growth.

Developmental Roles of the Hog1 Protein Phosphatases of the Maize Pathogen Cochliobolus heterostrophus.

Protein phosphorylation cascades are universal in cell signaling. While kinome diversity allows specific phosphorylation events, relatively few phosphatases dephosphorylate key signaling proteins. Fungal mitogen activated protein kinases (MAPK), in contrast to their mammalian counterparts, often show detectable basal phosphorylation levels. Dephosphorylation, therefore, could act as a signal. In Cochliobolus heterostrophus, the Dothideomycete causing Southern corn leaf blight, ferulic acid (FA)-an abundant phenolic found in plant host cell walls-acts as a signal to rapidly dephosphorylate the stress-activated MAP kinase Hog1 (High Osmolarity Glycerol 1). In order to identify the protein phosphatases responsible, we constructed mutants in Hog1 phosphatases predicted from the genome by homology to yeast and other species. We found that Cochliobolus heterostrophus mutants lacking PtcB, a member of the PP2C family, exhibited altered growth, sporulation, and attenuated dephosphorylation in response to FA. The loss of the dual-specificity phosphatase CDC14 led to slow growth, decreased virulence, and attenuated dephosphorylation. Mutants in two predicted tyrosine phosphatase genes PTP1 and PTP2 showed normal development and virulence. Our results suggest that a network of phosphatases modulate Hog1's dual phosphorylation levels. The mutants we constructed in this work provide a starting point to further unravel the signaling hierarchy by which exposure to FA leads to stress responses in the pathogen.

Sensing Host Arginine Is Essential for Leishmania Parasites' Intracellular Development.

Arginine homeostasis in lysosomes is critical for the growth and metabolism of mammalian cells. Phagolysosomes of macrophages are the niche where the parasitic protozoan Leishmania resides and causes human leishmaniasis. During infection, parasites encounter arginine deprivation, which is monitored by a sensor on the parasite cell surface. The sensor promptly activates a mitogen-activated protein kinase 2 (MAPK2)-mediated arginine deprivation response (ADR) pathway, resulting in upregulating the abundance and activity of the Leishmania arginine transporter (AAP3). Significantly, the ADR is also activated during macrophage infection, implying that arginine levels within the host phagolysosome are limiting for growth. We hypothesize that ADR-mediated upregulation of AAP3 activity is necessary to withstand arginine starvation, suggesting that the ADR is essential for parasite intracellular development. CRISPR/Cas9-mediated disruption of the AAP3 locus yielded mutants that retain a basal level of arginine transport but lack the ability to respond to arginine starvation. While these mutants grow normally in culture, they were impaired in their ability to develop inside THP-1 macrophages and were ∼70 to 80% less infective in BALB/c mice. Hence, inside the host macrophage, Leishmania must overcome the arginine "hunger games" by upregulating the transport of arginine via the ADR. We show that the ability to monitor and respond to changes in host metabolite levels is essential for pathogenesis.IMPORTANCE In this study, we report that the ability of the human pathogen Leishmania to sense and monitor the lack of arginine in the phagolysosome of the host macrophage is essential for disease development. Phagolysosomes of macrophages are the niche where Leishmania resides and causes human leishmaniasis. During infection, the arginine concentration in the phagolysosome decreases as part of the host innate immune response. An arginine sensor on the Leishmania cell surface activates an arginine deprivation response pathway that upregulates the expression of a parasite arginine transporter (AAP3). Here, we use CRISPR/Cas9-mediated disruption of the AAP3 locus to show that this response enables Leishmania parasites to successfully compete with the host macrophage in the "hunger games" for arginine.

Host-Free Systems for Differentiation of Axenic Leishmania.

This chapter describes, in detail, the method our laboratory developed to differentiate L. donovani promastigotes into amastigotes in a host-free culture. This method is based on previous observations that Leishmania promastigotes can combine two environmental signals, typical to lysosomes, acidic pH (~5.5) and body temperature (37 °C), into a signal that induces differentiation. Based on this concept, we have modified medium 199 to make it into an amastigote-specific medium. Shifting promastigotes to this medium, followed by incubation in a CO2 incubator, induced differentiation. Axenic amastigotes reach maturation within 5 days, resembling the time it takes in vivo. This chapter provides a complete protocol that should be useful for both Old and New World species of Leishmania.

Physicians' lack of knowledge - a possible reason for red blood cell transfusion overuse?

BACKGROUND: A significant percentage of red blood cell transfusions are inappropriately overused. This study investigated physicians from the western Galilee in terms of their knowledge of transfusion medicine as a potential reason for red blood cell overuse, and assessed the influence of personal background characteristics on their knowledge. METHODS: Data were collected via anonymous questionnaires. The questionnaires included a personal background section and a professional section. Study participants were grouped according to field of specialty, seniority, and location of medical school graduation, in order to correlate participant characteristics with knowledge. RESULTS: Scores were calculated on a 0-100 scale. The overall knowledge of the study population was low (mean score 47.8 ± 18.6). Knowledge regarding basic physiology of red blood cell transfusion was also low. Internal medicine physicians and senior physicians had significantly greater overall knowledge scores and were more familiar with a restrictive blood management policy than were surgeons and residents, respectively. Comparing knowledge scores, no difference was found regarding indications for transfusion. CONCLUSION: General and fundamental knowledge in transfusion medicine is lacking among physicians in the non-operating room setting, which may play a role in red blood cell transfusion overuse. Field of specialty and professional status influenced knowledge of transfusion medicine. Educational programs and increased physicians' awareness might help decrease unnecessary transfusions. TRIAL REGISTRATION: Not applicable.

Protein kinase A signaling during bidirectional axenic differentiation in Leishmania.

Parasitic protozoa of the genus Leishmania are obligatory intracellular parasites that cycle between the phagolysosome of mammalian macrophages, where they proliferate as intracellular amastigotes, and the midgut of female sand flies, where they proliferate as extracellular promastigotes. Shifting between the two environments induces signaling pathway-mediated developmental processes that enable adaptation to both host and vector. Developmentally regulated expression and phosphorylation of protein kinase A subunits in Leishmania and in Trypanosoma brucei point to an involvement of protein kinase A in parasite development. To assess this hypothesis in Leishmania donovani, we determined proteome-wide changes in phosphorylation of the conserved protein kinase A phosphorylation motifs RXXS and RXXT, using a phospho-specific antibody. Rapid dephosphorylation of these motifs was observed upon initiation of promastigote to amastigote differentiation in culture. No phosphorylated sites were detected in axenic amastigotes. To analyse the kinetics of (re)phosphorylation during axenic reverse differentiation from L. donovani amastigotes to promastigotes, we first established a map of this process with morphological and molecular markers. Upon initiation, the parasites rested for 6-12 h before proliferation of an asynchronous population resumed. After early changes in cell shape, the major changes in molecular marker expression and flagella biogenesis occurred between 24 and 33 h after initiation. RXXS/T re-phosphorylation and expression of the regulatory subunit PKAR1 correlated with promastigote maturation, indicating a promastigote-specific function of protein kinase A signaling. This is supported by the localization of PKAR1 to the flagellum, an organelle reduced to a remnant in amastigote forms. We conclude that a significant increase in protein kinase A-mediated phosphorylation is part of the ordered changes that characterise the amastigote to promastigote differentiation.

Quinone-amino acid conjugates targeting Leishmania amino acid transporters.

The aim of the present study was to investigate the feasibility of targeting Leishmania transporters via appropriately designed chemical probes. Leishmania donovani, the parasite that causes visceral leishmaniasis, is auxotrophic for arginine and lysine and has specific transporters (LdAAP3 and LdAAP7) to import these nutrients. Probes 1-15 were originated by conjugating cytotoxic quinone fragments (II and III) with amino acids (i.e. arginine and lysine) by means of an amide linkage. The toxicity of the synthesized conjugates against Leishmania extracellular (promastigotes) and intracellular (amastigotes) forms was investigated, as well their inhibition of the relevant amino acid transporters. We observed that some conjugates indeed displayed toxicity against the parasites; in particular, 7 was identified as the most potent derivative (at concentrations of 1 µg/mL and 2.5 µg/mL residual cell viability was reduced to 15% and 48% in promastigotes and amastigotes, respectively). Notably, 6, while retaining the cytotoxic activity of quinone II, displayed no toxicity against mammalian THP1 cells. Transport assays indicated that the novel conjugates inhibited transport activity of lysine, arginine and proline transporters. Furthermore, our analyses suggested that the toxic conjugates might be translocated by the transporters into the cells. The non-toxic probes that inhibited transport competed with the natural substrates for binding to the transporters without being translocated. Thus, it is likely that 6, by exploiting amino acid transporters, can selectively deliver its toxic effects to Leishmania cells. This work provides the first evidence that amino acid transporters of the human pathogen Leishmania might be modulated by small molecules, and warrants their further investigation from drug discovery and chemical biology perspectives.

YND1 interacts with CDC55 and is a novel mediator of E4orf4-induced toxicity.

Adenovirus E4orf4 (early region 4 open reading frame 4) protein induces protein phosphatase 2A-dependent non-classical apoptosis in mammalian cells and irreversible growth arrest in Saccharomyces cerevisiae. Oncogenic transformation sensitizes cells to E4orf4-induced cell death. To uncover additional components of the E4orf4 network required for induction of its unique mode of apoptosis, we used yeast genetics to select gene deletions conferring resistance to E4orf4. Deletion of YND1, encoding a yeast Golgi apyrase, conferred partial resistance to E4orf4. However, Ynd1p apyrase activity was not required for E4orf4-induced toxicity. Ynd1p and Cdc55p, the yeast protein phosphatase 2A-B subunit, contributed additively to E4orf4-induced toxicity. Furthermore, concomitant overexpression of one and deletion of the other was detrimental to yeast growth, demonstrating a functional interaction between the two proteins. YND1 and CDC55 also interacted genetically with CDC20 and CDH1/HCT1, encoding activating subunits of the anaphase-promoting complex/cyclosome. In addition to their functional interaction, Ynd1p and Cdc55p interacted physically, and this interaction was disrupted by E4orf4, which remained associated with both proteins. The results suggested that Ynd1p and Cdc55p share a common downstream target whose balanced modulation by the two E4orf4 partners is crucial to viability. Disruption of this balance by E4orf4 may lead to cell death. NTPDase-4/Lalp70/UDPase, the closest mammalian homologue of Ynd1p, associated with E4orf4 in mammalian cells, suggesting that the results in yeast are relevant to the mammalian system.

The scaffolding A/Tpd3 subunit and high phosphatase activity are dispensable for Cdc55 function in the Saccharomyces cerevisiae spindle checkpoint and in cytokinesis.

Protein serine/threonine phosphatase 2A (PP2A) is a multifunctional enzyme whose trimeric form consists of a scaffolding A subunit, a catalytic C subunit, and one of several regulatory B subunits (B, B', and B''). The adenovirus E4orf4 protein associates with PP2A by directly binding the B or B' subunits. An interaction with an active PP2A containing the B subunit, or its homologue in yeast, Cdc55, is required for E4orf4-induced apoptosis in mammalian cells and for induction of growth arrest in Saccharomyces cerevisiae. In this work, Cdc55 was randomly mutagenized by low-fidelity PCR amplification, and Cdc55 mutants that lost the ability to transduce the E4orf4 toxic signal in yeast were selected. The mutations obtained by this protocol inhibited the association of Cdc55 with E4orf4, or with the PP2A-AC subunits, or both. Functional analysis revealed that a mutant that does not bind Tpd3, the yeast A subunit, as well as wild type Cdc55 in a tpd3Delta background, can form a heterodimer with the catalytic subunit. This association requires C subunit carboxyl methylation. The residual phosphatase activity associated with Cdc55 in the absence of Tpd3 is sufficient to maintain a partially active spindle checkpoint and to prevent cytokinesis defects.