A hetero-multimeric chitinase-containing plasmodium falciparum and plasmodium gallinaceum ookinete-secreted protein complex involved in mosquito midgut invasion

Malaria parasites are transmitted by Anopheles mosquitoes. During its life cycle in the mosquito vector the Plasmodium ookinete escapes the proteolytic milieu of the post-blood meal midgut by traversing the midgut wall. This process requires penetration of the chitin-containing peritrophic matrix lining the midgut epithelium, which depends in part on ookinete-secreted chitinases. Plasmodium falciparum ookinetes have one chitinase (PfCHT1), whereas ookinetes of the avian-infecting parasite, P. gallinaceum, have two, a long and a short form, PgCHT1 and PgCHT2, respectively. Published data indicates that PgCHT2 forms a high molecular weight (HMW) reduction-sensitive complex; and one binding partner is the ookinete-produced von Willebrand A-domain-containing protein, WARP. Size exclusion chromatography data reported here show that P. gallinaceum PgCHT2 and its ortholog, P. falciparum PfCHT1 are covalently-linked components of a HMW chitinase-containing complex (> 1,300 kDa). Mass spectrometry of ookinete-secreted proteins isolated using a new chitin bead pull-down method identified chitinase-associated proteins in P. falciparum and P. gallinaceum ookinete-conditioned culture media. Mass spectrometry of this complex showed the presence of several micronemal proteins including von Willebrand factor A domain-related protein (WARP), ookinete surface enolase, and secreted ookinete adhesive protein (SOAP). To test the hypothesis that ookinete-produced PfCHT1 can form a high molecular homo-multimer or, alternatively, interacts with P. berghei ookinete-produced proteins to produce an HMW hetero-multimer, we created chimeric P. berghei parasites expressing PfCHT1 to replace PbCHT1, enabling the production of large numbers of PfCHT1-expressing ookinetes. We show that chimeric P. berghei ookinetes express monomeric PfCHT1, but a HMW complex containing PfCHT1 is not present. A better understanding of the chitinase-containing HMW complex may enhance development of next-generation vaccines or drugs that target malaria transmission stages.Host-parasite interactio

Screening of viral-vectored P. falciparum pre-erythrocytic candidate vaccine antigens using chimeric rodent parasites

To screen for additional vaccine candidate antigens of Plasmodium pre-erythrocytic stages, fourteen P. falciparum proteins were selected based on expression in sporozoites or their role in establishment of hepatocyte infection. For preclinical evaluation of immunogenicity of these proteins in mice, chimeric P. berghei sporozoites were created that express the P. falciparum proteins in sporozoites as an additional copy gene under control of the uis4 gene promoter. All fourteen chimeric parasites produced sporozoites but sporozoites of eight lines failed to establish a liver infection, indicating a negative impact of these P. falciparum proteins on sporozoite infectivity. Immunogenicity of the other six proteins (SPELD, ETRAMP10.3, SIAP2, SPATR, HT, RPL3) was analyzed by immunization of inbred BALB/c and outbred CD-1 mice with viral-vectored (ChAd63 or ChAdOx1, MVA) vaccines, followed by challenge with chimeric sporozoites. Protective immunogenicity was determined by analyzing parasite liver load and prepatent period of blood stage infection after challenge. Of the six proteins only SPELD immunized mice showed partial protection. We discuss both the low protective immunogenicity of these proteins in the chimeric rodent malaria challenge model and the negative effect on P. berghei sporozoite infectivity of several P. falciparum proteins expressed in the chimeric sporozoites.Host-parasite interactio

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Continuous cooling sensitization and its evaluation in austenitic stainless steel EN 1.4310

Abstract Sensitization in stainless steels is caused by chromium delpleted zones near grain boundaries which can lead to intergranular corrosion and intergranular stress corrosion cracking in service. A lot of work has been done in the past to understand sensitization behaviour under isothermal conditions. However, this study aims at studying the sensitization behaviour in continuous cooling, which is of more practical importance in steel production and in heat treatments such as welding. Conditions for sensitization were determined using experiments (Double-loop Electrochemical Potentiokinetic Reactivation tests (DL-EPR) and compared with DICTRA simulations. Using these conditions a continuous cooling sensitization (CCS) diagram is determined for EN 1.4310 (301) austentic stainless steel. DICTRA is a CALPHAD based simulation software used for diffusion related studies in multicomponent alloys. Solvus temperature for M₂₃C₆ is determined 959°C

Pd/C-mediated arylation followed by I2-catalyzed hydration strategy: Preparation of functionalized novel indanone derivatives

A simple and inexpensive synthesis of novel 2-(3-oxo-3-arylpropyl)-2,3- dihydro-1H-inden-1-one derivatives has been achieved via Pd/C-mediated arylation followed by I-mediated regioselective hydration of 2-(prop-2-ynyl)-2,3-dihydro-1H-inden-1-ones. A wide variety of 3-aryl substituted 2-propynyl indanone derivatives were conveniently prepared by using 10% Pd/C-PPh-CuI as a catalyst system, some of which were used to prepare the corresponding ketones via alkyne hydration in the presence of catalytic I. In an in vitro study a representative compound showed inhibition of PDE4B (phosphodiesterase type 4B) and binding with this protein in silic

Generation of NovelPlasmodium falciparumNF135 and NF54 Lines Expressing Fluorescent Reporter Proteins Under the Control of Strong and Constitutive Promoters

Transgenic reporter lines of malaria parasites that express fluorescent or luminescent proteins are valuable tools for drug and vaccine screening assays as well as to interrogate parasite gene function. DifferentPlasmodium falciparum(Pf) reporter lines exist, however nearly all have been created in the African NF54/3D7 laboratory strain. Here we describe the generation of novel reporter lines, using CRISPR/Cas9 gene modification, both in the standardPfNF54 background and in a recently described CambodianP. falciparumNF135.C10 line. Sporozoites of this line show more effective hepatocyte invasion and enhanced liver merozoite development compared toPfNF54. We first generatedPfNF54 reporter parasites to analyze two novel promoters for constitutive and high expression of mCherry-luciferase and GFP in blood and mosquito stages. The promoter sequences were selected based on available transcriptome data and are derived from two housekeeping genes, i.e., translation initiation factor SUI1, putative (sui1, PF3D7_1243600) and 40S ribosomal protein S30 (40s, PF3D7_0219200). We then generated and characterized reporter lines in thePfNF135.C10 line which express GFP driven by thesui1and40spromoters as well as by the previously usedef1 alpha promoter (GFP@ef1 alpha,GFP@sui1, GFP@40s). TheGFP@40sreporter line showed strongest GFP expression in liver stages as compared to the other two lines. The strength of reporter expression by the40spromoter throughout the complete life cycle, including liver stages, makes transgenic lines expressing reporters by the40spromoter valuable novel tools for analyses ofP. falciparum.Host-parasite interactio

Malaria parasite evades mosquito immunity by glutaminyl cyclase-mediated posttranslational protein modification

Glutaminyl cyclase (QC) modifies N-terminal glutamine or glutamic acid residues of target proteins into cyclic pyroglutamic acid (pGlu). Here, we report the biochemical and functional analysis of Plasmodium QC. We show that sporozoites of QC-null mutants of rodent and human malaria parasites are recognized by the mosquito immune system and melanized when they reach the hemocoel. Detailed analyses of rodent malaria QC-null mutants showed that sporozoite numbers in salivary glands are reduced in mosquitoes infected with QC-null or QC catalytically dead mutants. This phenotype can be rescued by genetic complementation or by disrupting mosquito melanization or phagocytosis by hemocytes. Mutation of a single QC-target glutamine of the major sporozoite surface protein (circumsporozoite protein; CSP) of the rodent parasite Plasmodium berghei also results in melanization of sporozoites. These findings indicate that QC-mediated posttranslational modification of surface proteins underlies evasion of killing of sporozoites by the mosquito immune system.Dermatology-oncolog