Immunisation with Transgenic L. tarentolae Expressing Gamma Glutamyl Cysteine Synthetase from Pathogenic Leishmania Species Protected against L. major and L. donovani Infection in a Murine Model.

Leishmaniasis is a protozoan disease responsible for significant morbidity and mortality. There is no recommended vaccine to protect against infection. In this study, transgenic Leishmania tarentolae expressing gamma glutamyl cysteine synthetase (γGCS) from three pathogenic species were produced and their ability to protect against infection determined using models of cutaneous and visceral leishmaniasis. The ability of IL-2-producing PODS® to act as an adjuvant was also determined in L. donovani studies. Two doses of the live vaccine caused a significant reduction in L. major (p < 0.001) and L. donovani (p < 0.05) parasite burdens compared to their respective controls. In contrast, immunisation with wild type L. tarentolae, using the same immunisation protocol, had no effect on parasite burdens compared to infection controls. Joint treatment with IL-2-producing PODS® enhanced the protective effect of the live vaccine in L. donovani studies. Protection was associated with a Th1 response in L. major and a mixed Th1/Th2 response in L. donovani, based on specific IgG1 and IgG2a antibody and cytokine production from in vitro proliferation assays using antigen-stimulated splenocytes. The results of this study provide further proof that γGCS should be considered a candidate vaccine for leishmaniasis.

Absence of DEATH kinesin is fatal for Leishmania mexicana amastigotes.

Kinesins are motor proteins present in organisms from protists to mammals playing important roles in cell division, intracellular organisation and flagellum formation and maintenance. Leishmania mexicana is a protozoan parasite of the order Kinetoplastida causing human cutaneous leishmaniasis. Kinetoplastida genome sequence analyses revealed a large number of kinesins showing sequence and structure homology to eukaryotic kinesins. Here, we investigate the L. mexicana kinesin LmxKIN29 (LmxM.29.0350), also called DEATH kinesin. The activated MAP kinase LmxMPK3, a kinase affecting flagellum length in Leishmania, is able to phosphorylate recombinant full length LmxKIN29 at serine 554. Insect promastigote LmxKIN29 Leishmania null mutants showed no obvious phenotype. However, in mouse infection experiments, the null mutants were unable to cause the disease, whereas LmxKIN29 add-backs and single allele knockouts caused footpad lesions. Localisation using promastigotes expressing GFP-tagged LmxKIN29 revealed that the kinesin is predominantly found in between the nucleus and the flagellar pocket, while in dividing cells the GFP-fusion protein was found at the anterior and posterior ends of the cells indicating a role in cytokinesis. The inability to cause lesions in infected animals and the amino acid sequence divergence from mammalian kinesins suggests that LmxKIN29 is a potential drug target against leishmaniasis.

Mapping Cell Viability Quantitatively and Independently From Cell Density in 3D Gels Noninvasively.

OBJECTIVE: In biomanufacturing there is a need for quantitative methods to map cell viability and density inside 3D bioreactors to assess health and proliferation over time. Recently, noninvasive MRI readouts of cell density have been achieved. However, the ratio of live to dead cells was not varied. Herein we present an approach for measuring the viability of cells embedded in a hydrogel independently from cell density to map cell number and health. METHODS: Independent quantification of cell viability and density was achieved by calibrating the 1H magnetization transfer- (MT) and diffusion-weighted NMR signals to samples of known cell density and viability using a multivariate approach. Maps of cell viability and density were generated by weighting NMR images by these parameters post-calibration. RESULTS: Using this method, the limits of detection (LODs) of total cell density and viable cell density were found to be 3.88 ×108 cells · mL -1· Hz -1/2 and 2.36 ×109 viable cells · mL -1· Hz -1/2 respectively. CONCLUSION: This mapping technique provides a noninvasive means of visualizing cell viability and number density within optically opaque bioreactors. SIGNIFICANCE: We anticipate that such nondestructive readouts will provide valuable feedback for monitoring and controlling cell populations in bioreactors.

Spermatic cord liposarcomas incidentally found during hernia surgery: is histology of any lipoma mandatory? A review of the literature.

Purpose: Liposarcomas found incidentally during open or laparoscopic inguinal hernia surgery are extremely rare. It is unclear, whether any adipose tissue being removed during inguinal hernia surgery must be sent for histology due to the potential risk of liposarcoma of the spermatic cord. This study aims to evaluate the frequency of liposarcomas incidentally found in the inguinal canal during hernia surgery and tries to derive evidence-based recommendations regarding the optimal management of any fatty tissue found in the inguinal canal.Methods: A literature review of the PubMed/Medline electronic databases between January 1980 and January 2019 was performed using the search terms 'inguinal hernia' and 'liposarcoma'. There was only one study available on this topic. Therefore, an additional literature review was performed analyzing all reports on patients with incidentally detected liposarcomas of the spermatic cord in the inguinal canal during hernia surgery.Results: There was only one retrospective study evaluating the frequency of inguinal liposarcoma found at hernia operations with a frequency of less than 0.1%. There were 18 cases of spermatic cord liposarcomas that were truly found incidentally during operation for an unsuspected symptomatic or incarcerated inguinal hernia. These included 16 case reports with a total of 18 patients and 19 liposarcomas. All patients were male with a median age of 62.5 years (range: 24-86 years) years. Median size of liposarcoma was 10.5 cm (range: 3-30 cm). In seven patients, the inguinal liposarcoma was an extension of a retroperitoneal sarcoma. Treatment consisted of radical orchidectomy during the primary operation in 12 patients. Three out of the seven patients with retroperitoneal extension of the tumor underwent a secondary operation with complete resection of the tumor.Conclusions: Currently, there is no evidence-based recommendation available regarding the management of lipomas detected during open or laparoscopic inguinal hernia surgery. Due to the extremely low risk of the presence of a liposarcoma, routine histologic examination cannot be recommended unless the diameter exceeds 10 cm.

Leishmania donovani 90 kD Heat Shock Protein - Impact of Phosphosites on Parasite Fitness, Infectivity and Casein Kinase Affinity.

Leishmania parasites are thought to control protein activity at the post-translational level, e.g. by protein phosphorylation. In the pathogenic amastigote, the mammalian stage of Leishmania parasites, heat shock proteins show increased phosphorylation, indicating a role in stage-specific signal transduction. Here we investigate the impact of phosphosites in the L. donovani heat shock protein 90. Using a chemical knock-down/genetic complementation approach, we mutated 11 confirmed or presumed phosphorylation sites and assessed the impact on overall fitness, morphology and in vitro infectivity. Most phosphosite mutations affected the growth and morphology of promastigotes in vitro, but with one exception, none of the phosphorylation site mutants had a selective impact on the in vitro infection of macrophages. Surprisingly, aspartate replacements mimicking the negative charge of phosphorylated serines or threonines had mostly negative impacts on viability and infectivity. HSP90 is a substrate for casein kinase 1.2-catalysed phosphorylation in vitro. While several putative phosphosite mutations abrogated casein kinase 1.2 activity on HSP90, only Ser289 could be identified as casein kinase target by mass spectrometry. In summary, our data show HSP90 as a downstream client of phosphorylation-mediated signalling in an organism that depends on post-transcriptional gene regulation.

Noninvasive Quantification of Cell Density in Three-Dimensional Gels by MRI.

OBJECTIVE: For tissue engineering, there is a need for quantitative methods to map cell density inside three-dimensional (3-D) bioreactors to assess tissue growth over time. The current cell mapping methods in 2-D cultures are based on optical microscopy. However, optical methods fail in 3-D due to increased opacity of the tissue. We present an approach for measuring the density of cells embedded in a hydrogel to generate quantitative maps of cell density in a living, 3-D tissue culture sample. METHODS: Quantification of cell density was obtained by calibrating the 1H T2, magnetization transfer (MT) and diffusion-weighted nuclear magnetic resonance (NMR) signals to samples of known cell density. Maps of cell density were generated by weighting NMR images by these parameters post-calibration. RESULTS: The highest sensitivity weighting arose from MT experiments, which yielded a limit of detection (LOD) of [Formula: see text] cells/mL/ √{Hz} in a 400 MHz (9.4 T) magnet. CONCLUSION: This mapping technique provides a noninvasive means of visualizing cell growth within optically opaque bioreactors. SIGNIFICANCE: We anticipate that such readouts of tissue culture growth will provide valuable feedback for controlled cell growth in bioreactors.

Continuous hyperpolarization with parahydrogen in a membrane reactor.

Hyperpolarization methods entail a high potential to boost the sensitivity of NMR. Even though the "Signal Amplification by Reversible Exchange" (SABRE) approach uses para-enriched hydrogen, p-H2, to repeatedly achieve high polarization levels on target molecules without altering their chemical structure, such studies are often limited to batch experiments in NMR tubes. Alternatively, this work introduces a continuous flow setup including a membrane reactor for the p-H2, supply and consecutive detection in a 1 T NMR spectrometer. Two SABRE substrates pyridine and nicotinamide were hyperpolarized, and more than 1000-fold signal enhancement was found. Our strategy combines low-field NMR spectrometry and a membrane flow reactor. This enables precise control of the experimental conditions such as liquid and gas pressures, and volume flow for ensuring repeatable maximum polarization.

Efficient Concentration of an Amino Acid Using Reactive Extraction Coupled with Bipolar Electrodialysis.

One intention of the PRODIAS (processing diluted aqueous systems) project is to develop and establish a toolbox of innovative and tailored separation technologies applicable to design energy-efficient water removal and product recovery techniques. Within this project, the recovery of γ-aminobutyric acid (GABA) was investigated. Using both synthetic as well as fermented solutions, reactive extraction of GABA with the solvent di-(2-ethylhexyl)phosphoric acid + isododecane was performed. For back extraction, different mineral acids were examined. Multistage countercurrent reactive extraction using pH adjustments along the stages to increase extraction efficiency as well as back extraction were then run on pilot-plant scale with fermented GABA solutions. The resulting GABA salt from back extraction was finally split by means of bipolar electrodialysis.

An Arginine Deprivation Response Pathway Is Induced in Leishmania during Macrophage Invasion.

Amino acid sensing is an intracellular function that supports nutrient homeostasis, largely through controlled release of amino acids from lysosomal pools. The intracellular pathogen Leishmania resides and proliferates within human macrophage phagolysosomes. Here we describe a new pathway in Leishmania that specifically senses the extracellular levels of arginine, an amino acid that is essential for the parasite. During infection, the macrophage arginine pool is depleted due to its use to produce metabolites (NO and polyamines) that constitute part of the host defense response and its suppression, respectively. We found that parasites respond to this shortage of arginine by up-regulating expression and activity of the Leishmania arginine transporter (LdAAP3), as well as several other transporters. Our analysis indicates the parasite monitors arginine levels in the environment rather than the intracellular pools. Phosphoproteomics and genetic analysis indicates that the arginine-deprivation response is mediated through a mitogen-activated protein kinase-2-dependent signaling cascade.

Sorption Behavior of Compressed CO2 and CH4 on Ultrathin Hybrid Poly(POSS-imide) Layers.

Sorption of compressed gases into thin polymeric films is essential for applications including gas sensors and membrane based gas separation. For glassy polymers, the sorption behavior is dependent on the nonequilibrium status of the polymer. The uptake of molecules by a polymer is generally accompanied by dilation, or swelling, of the polymer material. In turn, this dilation can result in penetrant induced plasticization and physical aging that affect the nonequilibrium status of the polymer. Here, we investigate the dilation and sorption behavior of ultrathin membrane layers of a hybrid inorganic-organic network material that consists of alternating polyhedral oligomeric silsesquioxane and imide groups, upon exposure to compressed carbon dioxide and methane. The imide precursor contains fluoroalkene groups that provide affinity toward carbon dioxide, while the octa-functionalized silsesquioxane provides a high degree of cross-linking. This combination allows for extremely high sorption capacities, while structural rearrangements of the network are hindered. We study the simultaneous uptake of gases and dilation of the thin films at high pressures using spectroscopic ellipsometry measurements. Ellipsometry provides the changes in both the refractive index and the film thickness, and allows for accurate quantification of sorption and swelling. In contrast, gravimetric and volumetric measurements only provide a single parameter; this does not allow an accurate correction for, for instance, the changes in buoyancy because of the extensive geometrical changes of highly swelling films. The sorption behavior of the ultrathin hybrid layers depends on the fluoroalkene group content. At low pressure, the apparent molar volume of the gases is low compared to the liquid molar volume of carbon dioxide and methane, respectively. At high gas concentrations in the polymer film, the apparent molar volume of carbon dioxide and methane exceeds that of the liquid molar volume, and approaches that of the gas phase. The high sorption capacity and reversible dilation characteristics of the presented materials provide new directions for applications including gas sensors and gas separation membranes.

Transgenic analysis of the Leishmania MAP kinase MPK10 reveals an auto-inhibitory mechanism crucial for stage-regulated activity and parasite viability.

Protozoan pathogens of the genus Leishmania have evolved unique signaling mechanisms that can sense changes in the host environment and trigger adaptive stage differentiation essential for host cell infection. The signaling mechanisms underlying parasite development remain largely elusive even though Leishmania mitogen-activated protein kinases (MAPKs) have been linked previously to environmentally induced differentiation and virulence. Here, we unravel highly unusual regulatory mechanisms for Leishmania MAP kinase 10 (MPK10). Using a transgenic approach, we demonstrate that MPK10 is stage-specifically regulated, as its kinase activity increases during the promastigote to amastigote conversion. However, unlike canonical MAPKs that are activated by dual phosphorylation of the regulatory TxY motif in the activation loop, MPK10 activation is independent from the phosphorylation of the tyrosine residue, which is largely constitutive. Removal of the last 46 amino acids resulted in significantly enhanced MPK10 activity both for the recombinant and transgenic protein, revealing that MPK10 is regulated by an auto-inhibitory mechanism. Over-expression of this hyperactive mutant in transgenic parasites led to a dominant negative effect causing massive cell death during amastigote differentiation, demonstrating the essential nature of MPK10 auto-inhibition for parasite viability. Moreover, phosphoproteomics analyses identified a novel regulatory phospho-serine residue in the C-terminal auto-inhibitory domain at position 395 that could be implicated in kinase regulation. Finally, we uncovered a feedback loop that limits MPK10 activity through dephosphorylation of the tyrosine residue of the TxY motif. Together our data reveal novel aspects of protein kinase regulation in Leishmania, and propose MPK10 as a potential signal sensor of the mammalian host environment, whose intrinsic pre-activated conformation is regulated by auto-inhibition.

Scenario planning for community development in Vietnam: a new tool for integrated health approaches?

BACKGROUND: Like many countries in Southeast Asia, Vietnam's rapid population and economic growth has met challenges in infrastructure development, especially sanitation in rural areas. OBJECTIVE: As an entry point, we developed scenario planning as an action-research tool in a peri-urban community to identify first steps towards improving their complex sanitation problem and to, systemically, address emerging/re-emerging infectious diseases, as these are commonly linked to unsafe water and inadequate sanitation conditions. As an integrated approach, the process of constructing scenarios allowed us to work across sectors and stakeholders to incorporate this knowledge into a common vision. DESIGN: We conducted focus group discussions to identify and rank driving forces, orally constructed scenarios for the most uncertain drivers, discussed scenario implications and options, and examined the overall process for usefulness and sustainability. During a one-month scoping phase and in between focus group meetings, we carried out household visits which helped us understand the context of data and gather feedback from participants outside of the formal data collection process. Recorded results from these activities were used to develop subsequent tools. RESULTS AND CONCLUSIONS: The research process gave us insights into how to adapt the scenario planning tool to identify alternative options. This involved choosing boundary partners, negotiating priorities, drawing out participant learning through self-assessment of our process (a prerequisite for changing mental models and thus achieving outcomes), and understanding how conveyed messages may reinforce the status quo. These insights showed the importance of examining research results beyond outputs and outcomes, namely through process.

The Leishmania donovani chaperone cyclophilin 40 is essential for intracellular infection independent of its stage-specific phosphorylation status.

During its life cycle, the protozoan pathogen Leishmania donovani is exposed to contrasting environments inside insect vector and vertebrate host, to which the parasite must adapt for extra- and intracellular survival. Combining null mutant analysis with phosphorylation site-specific mutagenesis and functional complementation we genetically tested the requirement of the L. donovani chaperone cyclophilin 40 (LdCyP40) for infection. Targeted replacement of LdCyP40 had no effect on parasite viability, axenic amastigote differentiation, and resistance to various forms of environmental stress in culture, suggesting important functional redundancy to other parasite chaperones. However, ultrastructural analyses and video microscopy of cyp40-/- promastigotes uncovered important defects in cell shape, organization of the subpellicular tubulin network and motility at stationary growth phase. More importantly, cyp40-/- parasites were unable to establish intracellular infection in murine macrophages and were eliminated during the first 24 h post infection. Surprisingly, cyp40-/- infectivity was restored in complemented parasites expressing a CyP40 mutant of the unique S274 phosphorylation site. Together our data reveal non-redundant CyP40 functions in parasite cytoskeletal remodelling relevant for the development of infectious parasites in vitro independent of its phosphorylation status, and provide a framework for the genetic analysis of Leishmania-specific phosphorylation sites and their role in regulating parasite protein function.

Acute angioedema: recognition and management in the emergency department.

Angioedema is characterized by localized swelling of subcutaneous tissues or mucosa of the upper respiratory or gastrointestinal tract. Laryngeal involvement may threaten airway patency and can be fatal if not addressed promptly. There are several distinct subtypes of angioedema, caused by different pathological processes involving a range of proinflammatory mediators. In the emergency department, it is essential not only that acute angioedema is identified as quickly as possible but also that the likely working diagnosis is established so that the most effective treatment may be administered to resolve potentially life-threatening swelling. In this paper, we present an overview of the various types of angioedema, and offer a practical diagnostic and therapeutic approach to their management.

Reducing short-stay hospital admissions by ruling out non-ST elevation myocardial infarction and estimating coronary artery disease likelihood on an emergency department observation ward.

Chest pain is an important presentation in adult patients attending emergency departments (ED). The process of ruling out an acute coronary syndrome (ACS) conventionally requires a short in-patient stay. This places a significant burden on healthcare systems. Recent developments have encouraged us to explore the role of an ED observation ward in the management of these patients. We designed and implemented two proformas ('flowformas'). The first provides integrated guidance and documentation for the management of chest pain in the ED. In patients determined to be at low risk of short-term adverse outcomes the ACS rule-out process is now completed on the ED observation ward rather than on the cardio-respiratory admission ward. The second proforma is used before discharge to determine the likelihood of underlying coronary artery disease (CAD), thereby allowing risk-based follow-up arrangements to be made. We collected data on all patients admitted to EDU on the NSTEMI rule-out pathway over a 12-month period. Between Feb 2012 and Feb 2013, 816 patients fulfilling the criteria were admitted on the pathway. 67 patients (8%) required admission due to ACS. 15 patients were admitted on two, and one on three occasions. In conclusion, it is possible to deliver ACS rule-out on an emergency observation ward. This reduces healthcare costs and shortens hospital stay.

Modulation of Leishmania major aquaglyceroporin activity by a mitogen-activated protein kinase.

Leishmania major aquaglyceroporin (LmjAQP1) adventitiously facilitates the uptake of antimonite [Sb(III)], an active form of Pentostam® or Glucantime®, which are the first line of defence against all forms of leishmaniasis. The present paper shows that LmjAQP1 activity is modulated by the mitogen-activated protein kinase, LmjMPK2. Leishmania parasites coexpressing LmjAQP1 and LmjMPK2 show increased Sb(III) uptake and increased Sb(III) sensitivity. When subjected to a hypo-osmotic stress, these cells show faster volume recovery than cells expressing LmjAQP1 alone. LmjAQP1 is phosphorylated in vivo at Thr-197 and this phosphorylation requires LmjMPK2 activity. Lys-42 of LmjMPK2 is critical for its kinase activity. Cells expressing altered T197A LmjAQP1 or K42A LmjMPK2 showed decreased Sb(III) influx and a slower volume recovery than cells expressing wild-type proteins. Phosphorylation of LmjAQP1 led to a decrease in its turnover rate affecting LmjAQP1 activity. Although LmjAQP1 is localized to the flagellum of promastigotes, upon phosphorylation, it is relocalized to the entire surface of the parasite. Leishmania mexicana promastigotes with an MPK2 deletion showed reduced Sb(III) uptake and slower volume recovery than wild-type cells. This is the first report where a parasite aquaglyceroporin activity is post-translationally modulated by a mitogen-activated protein kinase.

Deletion of UDP-glucose pyrophosphorylase reveals a UDP-glucose independent UDP-galactose salvage pathway in Leishmania major.

The nucleotide sugar UDP-galactose (UDP-Gal) is essential for the biosynthesis of several abundant glycoconjugates forming the surface glycocalyx of the protozoan parasite Leishmania major. Current data suggest that UDP-Gal could arise de novo by epimerization of UDP-glucose (UDP-Glc) or by a salvage pathway involving phosphorylation of Gal and the action of UDP-glucose:alpha-D-galactose-1-phosphate uridylyltransferase as described by Leloir. Since both pathways require UDP-Glc, inactivation of the UDP-glucose pyrophosphorylase (UGP) catalyzing activation of glucose-1 phosphate to UDP-Glc was expected to deprive parasites of UDP-Gal required for Leishmania glycocalyx formation. Targeted deletion of the gene encoding UGP, however, only partially affected the synthesis of the Gal-rich phosphoglycans. Moreover, no alteration in the abundant Gal-containing glycoinositolphospholipids was found in the deletion mutant. Consistent with these findings, the virulence of the UGP-deficient mutant was only modestly affected. These data suggest that Leishmania elaborates a UDP-Glc independent salvage pathway for UDP-Gal biosynthesis.

LmxMPK4, an essential mitogen-activated protein kinase of Leishmania mexicana is phosphorylated and activated by the STE7-like protein kinase LmxMKK5.

The essential mitogen-activated protein kinase (MAP kinase), LmxMPK4, of Leishmania mexicana is minimally active when purified following recombinant expression in Escherichia coli and was therefore unsuitable for drug screening until now. Using an E. coli protein co-expression system we identified LmxMKK5, a STE7-like protein kinase from L. mexicana, which phosphorylates and activates recombinant LmxMPK4 in vitro. LmxMKK5 is comprised of 525 amino acids and has a calculated molecular mass of 55.9kDa. The co-expressed, purified LmxMPK4 showed strong phosphotransferase activity in radiometric kinase assays and was confirmed by immunoblot and tandem mass spectrometry analyses to be phosphorylated on threonine 190 and tyrosine 192 of the typical TXY MAP kinase activation motif. The universal protein kinase inhibitor staurosporine reduced the phosphotransferase activity of co-expressed and activated LmxMPK4 in a dose-dependent manner. To our knowledge this is the first time that an in vitro activator of an essential Leishmania MAP kinase was identified and our findings form the basis for the development of drug screening assays to identify small molecule inhibitors of LmxMPK4 in the search for new therapeutic drugs against leishmaniasis.