Unusually Divergent Ubiquitin Genes and Proteins in Plasmodium Species.

Ubiquitin is an extraordinarily highly conserved 76 amino acid protein encoded by three different types of gene, where the primary translation products are fusions either of ubiquitin with one of two ribosomal proteins (RPs) or of multiple ubiquitin monomers from head to tail. Here, we investigate the evolution of ubiquitin genes in mammalian malaria parasites (Plasmodium species). The ubiquitin encoded by the RPS27a fusion gene is highly divergent, as previously found in a variety of protists. However, we also find that two other forms of divergent ubiquitin sequence, each previously thought to be extremely rare, have arisen recently during the divergence of Plasmodium subgenera. On two occasions, in two distinct lineages, the ubiquitin encoded by the RPL40 fusion gene has rapidly diverged. In addition, in one of these lineages, the polyubiquitin genes have undergone a single codon insertion, previously considered a unique feature of Rhizaria. There has been disagreement whether the multiple ubiquitin coding repeats within a genome exhibit concerted evolution or undergo a birth-and-death process; the Plasmodium ubiquitin genes show clear signs of concerted evolution, including the spread of this codon insertion to multiple repeats within the polyubiquitin gene.

The quest for control.

In 2015, we reported the development of a rapid protein degradation tool in the malaria parasite Plasmodium berghei. This commentary discusses the questions and events that led to developing this technology, as well as future outlooks.

Beyond phosphorylation: Putative roles of post-translational modifications in Plasmodium sexual stages.

Post-translational modifications (PTMs) allow proteins to regulate their structure, localisation and function in response to cell intrinsic and environmental signals. The diversity and number of modifications on proteins increase the complexity of cellular proteomes by orders of magnitude. Several proteomic and molecular studies have revealed an abundance of PTMs in malaria parasite proteome, where mediators of PTMs play crucial roles in parasite pathogenesis and transmission. In this article, we discuss recent findings in asexual stages of ten diverse PTMs and investigate whether these proteins are expressed in sexual stages. We discovered 25-50 % of proteins exhibiting post-translational modifications in asexual stages are also expressed in sexual stage gametocytes. Moreover we analyse the function of the modified proteins shared with the gametocyte proteome and try to encourage the scientific community to investigate the roles of diverse PTMs beyond phosphorylation in sexual stages which could not only reveal unique aspects of parasite biology, but also uncover new avenues for transmission blocking.

Zygote morphogenesis but not the establishment of cell polarity in Plasmodium berghei is controlled by the small GTPase, RAB11A.

Plasmodium species are apicomplexan parasites whose zoites are polarized cells with a marked apical organisation where the organelles associated with host cell invasion and colonization reside. Plasmodium gametes mate in the mosquito midgut to form the spherical and presumed apolar zygote that morphs during the following 24 hours into a polarized, elongated and motile zoite form, the ookinete. Endocytosis-mediated protein transport is generally necessary for the establishment and maintenance of polarity in epithelial cells and neurons, and the small GTPase RAB11A is an important regulator of protein transport via recycling endosomes. PbRAB11A is essential in blood stage asexual of Plasmodium. Therefore, a promoter swap strategy was employed to down-regulate PbRAB11A expression in gametocytes and zygotes of the rodent malaria parasite, Plasmodium berghei which demonstrated the essential role of RAB11A in ookinete development. The approach revealed that lack of PbRAB11A had no effect on gamete production and fertility rates however, the zygote to ookinete transition was almost totally inhibited and transmission through the mosquito was prevented. Lack of PbRAB11A did not prevent meiosis and mitosis, nor the establishment of polarity as indicated by the correct formation and positioning of the Inner Membrane Complex (IMC) and apical complex. However, morphological maturation was prevented and parasites remained spherical and immotile and furthermore, they were impaired in the secretion and distribution of microneme cargo. The data are consistent with the previously proposed model of RAB11A endosome mediated delivery of plasma membrane in Toxoplasma gondii if not its role in IMC formation and implicate it in microneme function.

Inducible developmental reprogramming redefines commitment to sexual development in the malaria parasite Plasmodium berghei.

During malaria infection, Plasmodium spp. parasites cyclically invade red blood cells and can follow two different developmental pathways. They can either replicate asexually to sustain the infection, or differentiate into gametocytes, the sexual stage that can be taken up by mosquitoes, ultimately leading to disease transmission. Despite its importance for malaria control, the process of gametocytogenesis remains poorly understood, partially due to the difficulty of generating high numbers of sexually committed parasites in laboratory conditions1. Recently, an apicomplexa-specific transcription factor (AP2-G) was identified as necessary for gametocyte production in multiple Plasmodium species2,3, and suggested to be an epigenetically regulated master switch that initiates gametocytogenesis4,5. Here we show that in a rodent malaria parasite, Plasmodium berghei, conditional overexpression of AP2-G can be used to synchronously convert the great majority of the population into fertile gametocytes. This discovery allowed us to redefine the time frame of sexual commitment, identify a number of putative AP2-G targets and chart the sequence of transcriptional changes through gametocyte development, including the observation that gender-specific transcription occurred within 6 h of induction. These data provide entry points for further detailed characterization of the key process required for malaria transmission.

Lysophosphatidylcholine Regulates Sexual Stage Differentiation in the Human Malaria Parasite Plasmodium falciparum.

Transmission represents a population bottleneck in the Plasmodium life cycle and a key intervention target of ongoing efforts to eradicate malaria. Sexual differentiation is essential for this process, as only sexual parasites, called gametocytes, are infective to the mosquito vector. Gametocyte production rates vary depending on environmental conditions, but external stimuli remain obscure. Here, we show that the host-derived lipid lysophosphatidylcholine (LysoPC) controls P. falciparum cell fate by repressing parasite sexual differentiation. We demonstrate that exogenous LysoPC drives biosynthesis of the essential membrane component phosphatidylcholine. LysoPC restriction induces a compensatory response, linking parasite metabolism to the activation of sexual-stage-specific transcription and gametocyte formation. Our results reveal that malaria parasites can sense and process host-derived physiological signals to regulate differentiation. These data close a critical knowledge gap in parasite biology and introduce a major component of the sexual differentiation pathway in Plasmodium that may provide new approaches for blocking malaria transmission.

Feasibility of prophylactic salpingectomy during vaginal hysterectomy.

BACKGROUND: The American Congress of Obstetricians and Gynecologists recommends that "the surgeon and patient discuss the potential benefits of the removal of the fallopian tubes during a hysterectomy in women at population risk of ovarian cancer who are not having an oophorectomy," resulting in an increasing rate of salpingectomy at the time of hysterectomy. Rates of salpingectomy are highest for laparoscopic and lowest for vaginal hysterectomy. OBJECTIVE: The primary objective of this study was to determine the feasibility of bilateral salpingectomy at the time of vaginal hysterectomy. Secondary objectives included identification of factors associated with unsuccessful salpingectomy and assessment of its impact on operating time, blood loss, surgical complications, and menopausal symptoms. STUDY DESIGN: This was a multicenter, prospective study of patients undergoing planned vaginal hysterectomy with bilateral salpingectomy. Baseline medical data along with operative findings, operative time, and blood loss for salpingectomy were recorded. Uterine weight and pathology reports for all fallopian tubes were reviewed. Patients completed the Menopause Rating Scale at baseline and at postoperative follow-up. Descriptive analyses were performed to characterize the sample and compare those with successful and unsuccessful completion of planned salpingectomy using Student t test, and χ2 test when appropriate. Questionnaire scores were compared using paired t tests. RESULTS: Among 77 patients offered enrollment, 74 consented (96%), and complete data were available regarding primary outcome for 69 (93%). Mean age was 51 years. Median body mass index was 29.1 kg/m2; median vaginal parity was 2, and 41% were postmenopausal. The indications for hysterectomy included prolapse (78%), heavy menstrual bleeding (20%), and fibroids (11%). When excluding conversions to alternate routes, vaginal salpingectomy was successfully performed in 52/64 (81%) women. Mean operating time for bilateral salpingectomy was 11 (±5.6) minutes, with additional estimated blood loss of 6 (±16.3) mL. There were 8 surgical complications: 3 hemorrhages >500 mL and 5 conversions to alternate routes of surgery, but none of these were due to the salpingectomy. Mean uterine weight was 102 g and there were no malignancies on fallopian tube pathology. Among the 17 patients in whom planned bilateral salpingectomy was not completed, unilateral salpingectomy was performed in 7 patients. Reasons for noncompletion included: tubes high in the pelvis (8), conversion to alternate route for pathology (4), bowel or sidewall adhesions (3), tubes absent (1), and ovarian adhesions (1). Prior adnexal surgery (odds ratio, 2.9; 95% confidence interval, 1.5-5.5; P = .006) and uterine fibroids (odds ratio, 5.8; 95% confidence interval, 1.5-22.5; P = .02) were the only significant factors associated with unsuccessful bilateral salpingectomy. Mean menopause scores improved after successful salpingectomy (12.7 vs 8.6; P < .001). CONCLUSION: Vaginal salpingectomy is feasible in the majority of women undergoing vaginal hysterectomy and increases operating time by 11 minutes and blood loss by 6 mL. Women with prior adnexal surgery or uterine fibroids should be counseled about the possibility that removal may not be feasible.

Evaluating ureteral patency in the post-indigo carmine era: a randomized controlled trial.

BACKGROUND: Many gynecologic, urologic, and pelvic reconstructive surgeries require accurate intraoperative evaluation of ureteral patency. OBJECTIVE: We performed a randomized controlled trial to compare surgeon satisfaction with 4 methods of evaluating ureteral patency during cystoscopy at the time of benign gynecologic or pelvic reconstructive surgery: oral phenazopyridine, intravenous sodium fluorescein, mannitol bladder distention, and normal saline bladder distention. STUDY DESIGN: We conducted an unblinded randomized controlled trial of the method used to evaluate ureteral patency during cystoscopy at time of benign gynecologic or pelvic reconstructive surgery. Subjects were randomized to receive 200 mg oral phenazopyridine, 25 mg intravenous sodium fluorescein, mannitol bladder distention, or normal saline bladder distention during cystoscopy. The primary outcome was surgeon satisfaction with the method, assessed via a 100-mm visual analog scale with 0 indicating strong agreement and 100 indicating strong disagreement with the statement. Secondary outcomes included comparing visual analog scale responses about ease of each method and visualization of ureteral jets, bladder mucosa and urethra, and operative information, including time to surgeon confidence in the ureteral jets. Adverse events were evaluated for at least 6 weeks after the surgical procedure, and through the end of the study. All statistical analyses were based on the intent-to-treat principle, and comparisons were 2-tailed. RESULTS: In all, 130 subjects were randomized to phenazopyridine (n = 33), sodium fluorescein (n = 32), mannitol (n = 32), or normal saline (n = 33). At randomization, patient characteristics were similar across groups. With regard to the primary outcome, mannitol was the method that physicians found most satisfactory on a visual analog scale. The median (range) scores for physicians assessing ureteral patency were 48 (0-83), 20 (0-82), 0 (0-44), and 23 (3-96) mm for phenazopyridine, sodium fluorescein, mannitol, and normal saline, respectively (P < .001). Surgery length, cystoscopy length, and time to surgeon confidence in visualization of ureteral jets were not different across the 4 randomized groups. During the 189-day follow-up, no differences in adverse events were seen among the groups, including urinary tract infections. CONCLUSION: The use of mannitol during cystoscopy to assess ureteral patency provided surgeons with the most overall satisfaction, ease of use, and superior visualization without affecting surgery or cystoscopy times. There were no differences in adverse events, including incidence of urinary tract infections.

Stage-Specific Changes in Plasmodium Metabolism Required for Differentiation and Adaptation to Different Host and Vector Environments.

Malaria parasites (Plasmodium spp.) encounter markedly different (nutritional) environments during their complex life cycles in the mosquito and human hosts. Adaptation to these different host niches is associated with a dramatic rewiring of metabolism, from a highly glycolytic metabolism in the asexual blood stages to increased dependence on tricarboxylic acid (TCA) metabolism in mosquito stages. Here we have used stable isotope labelling, targeted metabolomics and reverse genetics to map stage-specific changes in Plasmodium berghei carbon metabolism and determine the functional significance of these changes on parasite survival in the blood and mosquito stages. We show that glutamine serves as the predominant input into TCA metabolism in both asexual and sexual blood stages and is important for complete male gametogenesis. Glutamine catabolism, as well as key reactions in intermediary metabolism and CoA synthesis are also essential for ookinete to oocyst transition in the mosquito. These data extend our knowledge of Plasmodium metabolism and point towards possible targets for transmission-blocking intervention strategies. Furthermore, they highlight significant metabolic differences between Plasmodium species which are not easily anticipated based on genomics or transcriptomics studies and underline the importance of integration of metabolomics data with other platforms in order to better inform drug discovery and design.

Conditional Degradation of Plasmodium Calcineurin Reveals Functions in Parasite Colonization of both Host and Vector.

Functional analysis of essential genes in the malarial parasite, Plasmodium, is hindered by lack of efficient strategies for conditional protein regulation. We report the development of a rapid, specific, and inducible chemical-genetic tool in the rodent malaria parasite, P. berghei, in which endogenous proteins engineered to contain the auxin-inducible degron (AID) are selectively degraded upon adding auxin. Application of AID to the calcium-regulated protein phosphatase, calcineurin, revealed functions in host and vector stages of parasite development. Whereas depletion of calcineurin in late-stage schizonts demonstrated its critical role in erythrocyte attachment and invasion in vivo, stage-specific depletion uncovered roles in gamete development, fertilization, and ookinete-to-oocyst and sporozoite-to-liver stage transitions. Furthermore, AID technology facilitated concurrent generation and phenotyping of transgenic lines, allowing multiple lines to be assessed simultaneously with significant reductions in animal use. This study highlights the broad applicability of AID for functional analysis of proteins across the Plasmodium life cycle.

Intravenous immunoglobulin for hypogammaglobulinemia after lung transplantation: a randomized crossover trial.

BACKGROUND: We aimed to determine the effects of treatment with intravenous immunoglobulin on bacterial infections in patients with hypogammaglobulinemia (HGG) after lung transplantation. METHODS: We performed a randomized, double-blind, placebo-controlled two-period crossover trial of immune globulin intravenous (IVIG), 10% Purified (Gamunex, Bayer, Elkhart, IN) monthly in eleven adults who had undergone lung transplantation more than three months previously. We randomized study participants to three doses of IVIG (or 0.1% albumin solution (placebo)) given four weeks apart followed by a twelve week washout and then three doses of placebo (or IVIG). The primary outcome was the number of bacterial infections within each treatment period. RESULTS: IVIG had no effect on the number of bacterial infections during the treatment period (3 during IVIG and 1 during placebo; odds ratio 3.5, 95% confidence interval 0.4 to 27.6, p = 0.24). There were no effects on other infections, use of antibiotics, or lung function. IVIG significantly increased trough IgG levels at all time points (least square means, 765.3 mg/dl during IVIG and 486.3 mg/dl during placebo, p<0.001). Four serious adverse events (resulting in hospitalization) occurred during the treatment periods (3 during active treatment and 1 during the placebo period, p = 0.37). Chills, flushing, and nausea occurred during one infusion of IVIG. CONCLUSIONS: Treatment with IVIG did not reduce the short-term risk of bacterial infection in patients with HGG after lung transplantation. The clinical efficacy of immunoglobulin supplementation in HGG related to lung transplantation over the long term or with recurrent infections is unknown. TRIAL REGISTRATION: Clinicaltrials.gov NCT00115778.

Transfection of rodent malaria parasites.

Gene manipulation is an invaluable tool to investigate and understand the biology of an organism. Although this technology has been applied to both the human and rodent malarial parasites (RMP), Plasmodium berghei in particular offers a more robust system due to a higher and more efficient transformation rate. Here, we describe a comprehensive transfection and selection protocol using P. berghei including a variant negative selection protocol administering 5-fluorocytosine to the animals in drinking water. Additionally, we discuss and assess the latest advances in gene manipulation technologies developed in RMP to gain a better understanding of Plasmodium biology.

A unique Kelch domain phosphatase in Plasmodium regulates ookinete morphology, motility and invasion.

Signalling through post-translational modification (PTM) of proteins is a process central to cell homeostasis, development and responses to external stimuli. The best characterised PTM is protein phosphorylation which is reversibly catalysed at specific residues through the action of protein kinases (addition) and phosphatases (removal). Here, we report characterisation of an orphan protein phosphatase that possesses a domain architecture previously only described in Plantae. Through gene disruption and the production of active site mutants, the enzymatically active Protein Phosphatase containing Kelch-Like domains (PPKL, PBANKA_132950) is shown to play an essential role in the development of an infectious ookinete. PPKL is produced in schizonts and female gametocytes, is maternally inherited where its absence leads to the development of a malformed, immotile, non-infectious ookinete with an extended apical protrusion. The distribution of PPKL includes focussed localization at the ookinete apical tip implying a link between its activity and the correct deployment of the apical complex and microtubule cytoskeleton. Unlike wild type parasites, ppkl(-) ookinetes do not have a pronounced apical distribution of their micronemes yet secretion of microneme cargo is unaffected in the mutant implying that release of microneme cargo is either highly efficient at the malformed apical prominence or secretion may also occur from other points of the parasite, possibly the pellicular pores.

Translocation of sickle cell erythrocyte microRNAs into Plasmodium falciparum inhibits parasite translation and contributes to malaria resistance.

Erythrocytes carrying a variant hemoglobin allele (HbS), which causes sickle cell disease and resists infection by the malaria parasite Plasmodium falciparum. The molecular basis of this resistance, which has long been recognized as multifactorial, remains incompletely understood. Here we show that the dysregulated microRNA (miRNA) composition, of either heterozygous HbAS or homozygous HbSS erythrocytes, contributes to resistance against P. falciparum. During the intraerythrocytic life cycle of P. falciparum, a subset of erythrocyte miRNAs translocate into the parasite. Two miRNAs, miR-451 and let-7i, were highly enriched in HbAS and HbSS erythrocytes, and these miRNAs, along with miR-223, negatively regulated parasite growth. Surprisingly, we found that miR-451 and let-7i integrated into essential parasite messenger RNAs and, via impaired ribosomal loading, resulted in translational inhibition. Hence, sickle cell erythrocytes exhibit cell-intrinsic resistance to malaria in part through an atypical miRNA activity, which may represent a unique host defense strategy against complex eukaryotic pathogens.

Improved negative selection protocol for Plasmodium berghei in the rodent malarial model.

An improved methodology is presented here for transgenic Plasmodium berghei lines that express the negative selectable marker yFCU (a bifunctional protein that combines yeast cytosine deaminase and uridyl phosphoribosyl transferase (UPRT)) and substitutes delivery of selection drug 5-fluorocytosine (5FC) by intraperitoneal injection for administration via the drinking water of the mice. The improved methodology is shown to be as effective, less labour-intensive, reduces animal handling and animal numbers required for successful selection thereby contributing to two of the "three Rs" of animal experimentation, namely refinement and reduction.

Delayed access and survival in idiopathic pulmonary fibrosis: a cohort study.

RATIONALE: Idiopathic pulmonary fibrosis is often initially misdiagnosed. Delays in accessing subspecialty care could lead to worse outcomes among those with idiopathic pulmonary fibrosis. OBJECTIVES: To examine the association between delayed access to subspecialty care and survival time in idiopathic pulmonary fibrosis. METHODS: We performed a prospective cohort study of 129 adults who met American Thoracic Society criteria for idiopathic pulmonary fibrosis evaluated at a tertiary care center. Delay was defined as the time from the onset of dyspnea to the date of initial evaluation at a tertiary care center. We used competing risk survival methods to examine survival time and time to transplantation. MEASUREMENTS AND MAIN RESULTS: The mean age was 63 years and 76% were men. The median delay was 2.2 years (interquartile range 1.0­3.8 yr), and the median follow-up time was 1.1 years. Age and lung function at the time of evaluation did not vary by delay. A longer delay was associated with an increased risk of death independent of age, sex, forced vital capacity, third-party payer, and educational attainment (adjusted hazard ratio per doubling of delay was 1.3, 95% confidence interval 1.03 to 1.6). Longer delay was not associated with a lower likelihood of undergoing lung transplantation. CONCLUSIONS: Delayed access to a tertiary care center is associated with a higher mortality rate in idiopathic pulmonary fibrosis independent of disease severity. Early referral to a specialty center should be considered for those with known or suspected interstitial lung disease.

From cradle to grave: RNA biology in malaria parasites.

Malaria is caused by the unicellular apicomplexan parasites of the genus Plasmodium, some of which, including the major human parasite Plasmodium falciparum, have extreme genome compositions (A/T content > 80%). In this overview of RNA production, roles and degradation, we show that despite their unusual genome composition these parasites generally exhibit the standard eukaryotic features of these processes. Thus genes are monocistronic and transcribed by RNA polymerases that conform to the general categories of I, II, and III. Plasmodium spp. are unusual in that they possess structurally distinct rRNA genes that are expressed at different points in the complicated life cycle of the parasite. Transcription in blood stage asexual parasites follows a cascade consistent with a dependency upon plant-like apetala 2 (AP2) DNA-binding proteins. mRNA is transported to, translated and degraded in the cytoplasm and the transcription pattern is largely inflexible and responsive to temperature and glucose but not drugs. Furthermore, although Plasmodium spp. undertake controlled repression of mRNA species at a number of points in their life cycle only one mechanism, employed by female gametocytes (gamete precursor cells), is clear; it resembles that of metazoan female gametes, consisting of a complex of repression-associated proteins in an architecture formed with the mRNA 5' cap and dependent on U-rich untranslated region (UTR) elements. Extensive antisense transcription has been documented resulting in the production of both short and long transcripts of generally unknown functional significance. This review attempts to summarize what is currently known about the biology of Plasmodium RNA.

Characterization of a UBC13 kinase in Plasmodium falciparum.

Protein kinases are generally recognized as attractive drug targets to treat a variety of human diseases. Recent analysis of the Plasmodium falciparum kinome identified several kinases that are entirely unique to Plasmodium species. The specific functions and targets of most of these enzymes remain largely unknown. Here, we have identified a P. falciparum kinase (PfPK9/PF13_0085 ORF) that does not cluster with any of the typical eukaryotic protein kinases. PfPK9 protein expression was induced approximately 18 h after red blood cell infection, and was mainly localized to the parasitophorous vacuolar membrane as well as the cytosol. Recombinant PfPK9 autophosphorylated in vitro and specifically phosphorylated the exogenous substrate histone H1, indicating that it is catalytically active. Phosphopeptide mapping studies showed that autophosphorylation occurred at three residues: T082, T265, and T269. We identified a P. falciparum homolog of the E2 ubiquitin-conjugating enzyme 13 (UBC13) as an endogenous substrate for PfPK9. PfPK9 phosphorylated UBC13 at S106, a highly conserved residue among eukaryotic E2s, and suppressed its ubiquitin-conjugating activity. Our findings not only describe a previously uncharacterized Plasmodium kinase and its likely in vivo target, but also suggest that modulation of UBC13 activity by phosphorylation may be a common regulatory mechanism in eukaryotes.

Altered blood pressure responses and normal cardiac phenotype in ACE2-null mice.

The carboxypeptidase ACE2 is a homologue of angiotensin-converting enzyme (ACE). To clarify the physiological roles of ACE2, we generated mice with targeted disruption of the Ace2 gene. ACE2-deficient mice were viable, fertile, and lacked any gross structural abnormalities. We found normal cardiac dimensions and function in ACE2-deficient animals with mixed or inbred genetic backgrounds. On the C57BL/6 background, ACE2 deficiency was associated with a modest increase in blood pressure, whereas the absence of ACE2 had no effect on baseline blood pressures in 129/SvEv mice. After acute Ang II infusion, plasma concentrations of Ang II increased almost 3-fold higher in ACE2-deficient mice than in controls. In a model of Ang II-dependent hypertension, blood pressures were substantially higher in the ACE2-deficient mice than in WT. Severe hypertension in ACE2-deficient mice was associated with exaggerated accumulation of Ang II in the kidney, as determined by MALDI-TOF mass spectrometry. Although the absence of functional ACE2 causes enhanced susceptibility to Ang II-induced hypertension, we found no evidence for a role of ACE2 in the regulation of cardiac structure or function. Our data suggest that ACE2 is a functional component of the renin-angiotensin system, metabolizing Ang II and thereby contributing to regulation of blood pressure.