The coupled system (2)2Σ+ and (1)2Π of 7Li88Sr

We analyse rovibrational transitions of the (2)2Σ+-X(1)2Σ+ system of LiSr and find the energy levels of the (2)2Σ+ state to be perturbed by coupling between the (2)2Σ+ and (1)2Π states. We present an analysis of the coupled system yielding molecular parameters for the lowest vibrational levels of the (2)2Σ+ state and for higher vibrational levels of the (1)2Π state together with molecular coupling constants. Improved Dunham coefficients for the rovibrational levels of the X(1)2Σ+ state are also obtained, where the correlation with the parameters of the excited states is removed completely. © 2020 The Author(s). Published by IOP Publishing Ltd

An Impossible Journey? The Development of <i>Plasmodium falciparum</i> NF54 in <i>Culex quinquefasciatus</i>

<div><p>Although <i>Anopheles</i> mosquitoes are the vectors for human <i>Plasmodium</i> spp., there are also other mosquito species–among them culicines (<i>Culex</i> spp., <i>Aedes</i> spp.)–present in malaria-endemic areas. Culicine mosquitoes transmit arboviruses and filarial worms to humans and are vectors for avian <i>Plasmodium</i> spp., but have never been observed to transmit human <i>Plasmodium</i> spp. When ingested by a culicine mosquito, parasites could either face an environment that does not allow development due to biologic incompatibility or be actively killed by the mosquito’s immune system. In the latter case, the molecular mechanism of killing must be sufficiently powerful that <i>Plasmodium</i> is not able to overcome it. To investigate how human malaria parasites develop in culicine mosquitoes, we infected <i>Culex quinquefasciatus</i> with <i>Plasmodium falciparum</i> NF54 and monitored development of parasites in the blood bolus and midgut epithelium at different time points. Our results reveal that ookinetes develop in the midgut lumen of <i>C. quinquefasciatus</i> in slightly lower numbers than in <i>Anopheles gambiae</i> G3. After 30 hours, parasites have invaded the midgut and can be observed on the basal side of the midgut epithelium by confocal and transmission electron microscopy. Very few of the parasites in <i>C. quinquefasciatus</i> are alive, most of them are lysed. Eight days after the mosquito’s blood meal, no oocysts can be found in <i>C. quinquefasciatus</i>. Our results suggest that the mosquito immune system could be involved in parasite killing early in development after ookinetes have crossed the midgut epithelium and come in contact with the mosquito hemolymph.</p></div

(A) Homology model of one plasmodial Pdx1 monomer showing the analyzed amino acid residues as indicated.

<p>(B) The interface region between two <i>Pf</i>Pdx1 proteins within the same hexameric ring illustrating the amino acid residues R85, H88 and E91, which are involved in Pdx1:Pdx1 binding. The model was generated by Swiss-Model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001815#pone.0001815-Schwede1" target="_blank">[30]</a> and visualised by PyMOL (<a href="http://www.pymol.org" target="_blank">www.pymol.org</a>).</p

Systematic Identification of Plasmodium Falciparum Sporozoite Membrane Protein Interactions Reveals an Essential Role for the p24 Complex in Host Infection

Sporozoites are a motile form of malaria-causing Plasmodium falciparum parasites that migrate from the site of transmission in the dermis through the bloodstream to invade hepatocytes. Sporozoites interact with many cells within the host, but the molecular identity of these interactions and their role in the pathology of malaria is poorly understood. Parasite proteins that are secreted and embedded within membranes are known to be important for these interactions, but our understanding of how they interact with each other to form functional complexes is largely unknown. Here, we compile a library of recombinant proteins representing the repertoire of cell surface and secreted proteins from the P. falciparum sporozoite and use an assay designed to detect extracellular interactions to systematically identify complexes. We identify three protein complexes including an interaction between two components of the p24 complex that is involved in the trafficking of glycosylphosphatidylinositol-anchored proteins through the secretory pathway. Plasmodium parasites lacking either gene are strongly inhibited in the establishment of liver-stage infections. These findings reveal an important role for the p24 complex in malaria pathogenesis and show that the library of recombinant proteins represents a valuable resource to investigate P. falciparum sporozoite biology

Comparison of blood intake of <i>Anopheles gambiae</i> and <i>Culex quinquefasciatus</i> during a blood meal.

<p>Total hemoglobin content of female mosquitoes fed on a 40% hematocrit blood solution was determined by hemoglobinometry at different time points after a blood meal. Ten mosquitoes were analyzed for each time point and the average amount of ingested blood calculated using a standard curve. Values are shown as mean ± standard deviation. The volume of blood corresponding to the determined hemoglobin amount was compared between <i>An. gambiae</i> (--•--) and <i>C. quinquefasciatus</i> ( —▪— ).</p

Transmission electron microscopy of infected midguts 24 hours post feed on <i>Plasmodium falciparum</i> NF54-infected blood.

<p>Shown are overviews including the entire midgut epithelial layer (left) and magnifications of the parasite (right). (A) Parasite in the midgut epithelium of <i>Anopheles gambiae</i> G3, which is located on the basal side of the midgut epithelium underneath the basal lamina. (B) <i>P. falciparum</i> NF54 in the midgut of <i>Culex quinquefasciatus</i>. Parasites are located on the basal side of the midgut epithelium (overview left panel) outside the midgut cells. Parasite 1 (top) is located underneath the basal lamina outside the midgut cell, as it is surrounded by two membranes, one belonging to a midgut cell (arrow, M) and one of parasite origin (arrow, P) (see insets in right panel). The organelles inside the parasite are less pronounced than in <i>An. gambiae</i> (A), indicating lysis of the parasite (right panel). Parasite 2 (bottom) is located between two adjacent midgut cells toward the basal side of the epithelium. Two membranes can be seen (right panel inset, arrows M, P), showing an extracellular location of the parasite. Note here that one midgut epithelial cell (asterisk) is not connected to the basal lamina and lacks microvilli and most organelles, indicating apoptosis. MV: microvilli; BL: Basal lamina.</p

Confocal imaging of parasites in the mosquito midgut epithelium.

<p>Midgut epithelial tissue was collected 30 hours after the mosquito blood meal. (A) <i>Plasmodium falciparum</i> NF54 parasite in <i>Anopheles gambiae</i> G3. (B–D) <i>Culex quinquefasciatus</i> midgut epithelium containing (B) a live <i>P. falciparum</i> parasite and (C, D) parasites in different stages of lysis. Parasites in (C) and (D) have lost their even rim staining, which now appears dotted. Some parasites still contain nuclei (C, yellow and white arrow), but most parasites do not contain nuclei anymore (D, white arrows). A midgut cell is “budding off” into the midgut lumen (C, orange arrow). Shown is a section of the z-stack in the location of the parasite (left) and a side view of the midgut epithelium to localize the parasites (right). (E) Epifluorescence imaging of a parasite in <i>C. quinquefasciatus</i>. Note the black pigment associated with the parasite, which is visible in all fluorescent channels (arrows). Parasites were stained with a monoclonal anti-Pfs25 antibody (red), actin was stained using Phalloidin (green), and nuclei were visualized with DAPI (blue). MF: Muscle fibers on the basal side of the midgut; MV: microvilli. The scale bar indicates 5 µm.</p

Oligonucleotides which are used for cloning or site directed mutagenesis of <i>Pf</i>Pdx1 and <i>Pf</i>Pdx2. Mutation sites are underlined and in bold.

<p>Oligonucleotides which are used for cloning or site directed mutagenesis of <i>Pf</i>Pdx1 and <i>Pf</i>Pdx2. Mutation sites are underlined and in bold.</p

Western blot analysis of the co-purified <i>Pf</i>Pdx1 and <i>Pf</i>Pdx2HIS proteins.

<p>Cell homogenates of the recombinant expression of the plasmodial Pdx1 protein and Pdx2HIS protein (6× His-tag instead of a Strep-tag) were mixed and subsequently purified via Strep-tag affinity chromatography of the <i>Pf</i>Pdx1 protein. The co-purification was visualized by Western blot analysis using a monoclonal anti-Strep antibody (IBA) and the HIS-probe-HRP (Pierce) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001815#s4" target="_blank">Material and Methods</a>. (A) Co-purification of the <i>Pf</i>Pdx1 wild-type (3) and the <i>Pf</i>Pdx1 DKK (D26A, K83A and K151A) triple mutant protein (4) with the <i>Pf</i>Pdx2HIS wild-type. As a control the <i>Pf</i>Pdx1 wild-type (WT) (1) and the <i>Pf</i>Pdx2HIS (2) single proteins were purified via Strep-tag affinity chromatography and applied in Western blot analysis. (B) Co-purification of the <i>Pf</i>Pdx1 ERR (5), <i>Pf</i>Pdx1 RHE (6) as well as the <i>Pf</i>Pdx1 H88A (7) and <i>Pf</i>Pdx1 E91A (8) mutant proteins with the plasmodial Pdx2HIS wild-type. (Note: The <i>Pf</i>Pdx1 R85A mutant was not recombinantly expressible.) (C) The gate-keeper <i>Pf</i>Pdx2HIS E53Y (9) and <i>Pf</i>Pdx2HIS R154W (10) mutants were co-purified with the <i>Pf</i>Pdx1 wild-type protein via its Strep-tag.</p