Egress-related osmiophilic bodies

© 2014 John Wiley & Sons Ltd. Summary: Gametogenesis is the earliest event after uptake of malaria parasites by the mosquito vector, with a decisive impact on colonization of the mosquito midgut. This process is triggered by a drop in temperature and contact with mosquito molecules. In a few minutes, male and female gametocytes escape from the host erythrocyte by rupturing the parasitophorous vacuole and the erythrocyte membranes. Electron-dense, oval-shaped organelles, the osmiophilic bodies (OB), have been implicated in the egress of female gametocytes. By comparative electron microscopy and electron tomography analyses combined with immunolocalization experiments, we here define the morphological features distinctive of male secretory organelles, hereafter named MOB (male osmiophilic bodies). These organelles appear as club-shaped, electron-dense vesicles, smaller than female OB. We found that a drop in temperature triggers MOB clustering, independently of exposure to other stimuli. MDV1/PEG3, a protein associated with OB in Plasmodium berghei females, localizes to both non-clustered and clustered MOB, suggesting that clustering precedes vesicle discharge. A P.berghei mutant lacking the OB-resident female-specific protein Pbg377 displays a dramatic reduction in size of the OB, accompanied by a delay in female gamete egress efficiency, while female gamete fertility is not affected. Immunolocalization experiments indicated that MDV1/PEG3 is still recruited to OB-remnant structures

Vaccination coverage in healthcare workers: a multicenter cross-sectional study in Italy

IntroductionIn recent years, a phenomenon known as "vaccine hesitancy" has spread throughout the world, even among health workers, determining a reduction in vaccination coverage (VC). A study aimed at evaluating VC among healthcare workers (HCWs) in 10 Italian cities (L'Aquila, Genoa, Milan, Palermo, Sassari, Catanzaro, Ferrara, Catania, Naples, Messina) was performed.Materials and methodsAnnex 3 of the Presidential Decree n. 445 of 28 December 2000 was used to collect information on the vaccination status of HCWs. The mean and standard deviation (SD) were calculated with regard to the quantitative variable (age), while absolute and relative frequencies were obtained for categorical data (sex, professional profile, working sector, vaccination status). The connection between VC and the categorical variables was evaluated by chi-square method (statistical significance at p<0.05). The statistical analyses were performed by SPSS and Stata software.ResultsA total of 3,454 HCWs participated in the project: 1,236 males and 2,218 females. The sample comprised: physicians (26.9%), trainee physicians (16.1%), nurses (17.2%) and other professional categories (9.8%). Low VC was generally recorded. Higher VC was found with regard to polio, hepatitis B, tetanus and diphtheria, while coverage was very low for measles, mumps, rubella, pertussis, chickenpox and influenza (20-30%). ConclusionsThis study revealed low VC rates among HCWs for all the vaccinations. Measures to increase VC are therefore necessary in order to prevent HCWs from becoming a source of transmission of infections with high morbidity and/or mortality both within hospitals and outside

Protein trafficking and host cell remodling in malaria parasite infection

Il Plasmodio della malaria si riproduce nell’eritrocita, una cellula che ha perso capacità di sintesi poiché è altamente specializzata nel trasporto di ossigeno. La sopravvivenza del parassita è strettamente collegata alla riorganizzazione del citoplasma e della superficie del globulo rosso tramite la generazione di strutture membranose composte sia da molecole esportate dal parassita, che da altre importate dalla cellula ospite. Plasmodio garantisce così i nutrienti necessari al suo sviluppo e la possibilità di esportare fattori di virulenza alla superficie della cellula ospite. Tre membri della famiglia genica sep di P. berghei denominate SEP (Small Exported Proteins- 13-16 KDa), che presentano similarità con le proteine ETRAMP di P. falciparum, mostrano caratteristiche peculiari: condividono quasi totalmente la sequenza codificante, contenente un peptide segnale e una regione transmembrana, mentre differiscono nella porzione C-terminale. Anche la regione del promotore è pressoché identica, mentre variano gli specifici 3’UTR. Con la generazione di anticorpi specifici della regione al C-terminale, è stato possibile definire che si tratta di proteine integrali di membrana, la cui localizzazione e stadio specificità sono sorprendentemente diverse: in particolare la SEP2 e la SEP3 vengono esportate nella cellula ospite, mentre la SEP1 risiede alla membrana del vacuolo parassitoforo, struttura di interfaccia tra il parassita ed l’eritrocita. Inoltre è stato osservato che la SEP1 e 3 sono espresse in livelli confrontabili negli stadi intraeritrocitari, mentre la SEP2 è abbondantemente espressa nei gametociti, dato significativo in quanto questi sono le uniche forme ingerite dalla zanzara anofelina durante il pasto di sangue che daranno origine a parassiti in grado di svilupparsi nell’insetto vettore. Anche nell’insetto le 3 SEP hanno un profilo di espressione differente: SEP1 e 3 sono presenti negli ookineti, mentre la SEP2 è abbondantemente espressa in ookineti, oocisti e sporozoiti. L’identificazione dei motivi proteici necessari alla localizzazione delle proteine SEP2 e SEP3 nei vari distretti dell’eritrocita infetto si è basata sulla generazione di linee transgeniche di P. berghei in grado di esprimere porzioni diverse delle proteine SEP fuse alla proteina reporter GFP: la contemporanea presenza del peptide segnale della regione transmembrana è necessaria per traslocare le proteine di fusione SEP/GFP nell’eritrocita. La seconda parte del lavoro è stata svolta presso l’Università di Montpellier 2, e si propone di caratterizzare la localizzazione di due proteine del complesso giunzionale del citoscheletro dell’eritrocita umano, la dematina e l’adducina, in quanto recenti esperimenti svolti con il parassita murino hanno suggerito la possibilità di un meccanismo di internalizzazione di entrambe. Avvalendosi dell’impiego di anticorpi specifici e dell’utilizzo del microscopio confocale e apotome, è stato osservato che entrambe sono associate a comparti del parassita. Il processo di internalizzazione di queste due proteine è stato inoltre dimostrato attraverso esperimenti di frazionamento subcellulare e successiva digestione con proteinasi K: La dematina è interamente protetta e quindi completamente internalizzata dal parassita mentre l’adducina è in parte digerita dalla proteinasi K e quindi parzialmente esposta nel citoplasma dell’eritrocita.Plasmodium endurance depends on the ability of the parasite to reorganize the cytosol of the erythrocyte, a terminally differentiated cell, and remodels its skeleton membrane immediately after invasion. In this way the parasite can organize the import/export of the molecules necessary to its survival. The comprehension of cellular trafficking mechanisms which occur during Plasmodium infection is a very important step and fundamental contribute to understand the biology of the malaria parasite. We identified in the rodent malaria parasite Plasmodium berghei the gene family sep, corresponding to etramp in P. falciparum, encoding small exported proteins conserved in the genus Plasmodium. SEP proteins (13–16 kDa) contain a predicted signal peptide at the NH2-terminus, an internal hydrophobic region while they differ in their C-terminal region; the genes share the upstream regulative region while they differ in the 3’UTR. Despite this, we showed that SEPs have a different timing of expression and a different localization: in the erythrocytic cycle PbSEP1 and PbSEP3 start to be expressed at trophozoite and the same amount of protein is detected also in schizonts and gametocytes, while PbSEP2 is highly detected in mature trophozoites and even more in gametocytes. In mosquitoes stages PbSEP1 and PbSEP3 are expressed only in ookinetes, while PbSEP2 is very abundant in ookinetes, oocysts and in sporozoites of the salivary glands. SEPs also have a different localization in the iRBC: PbSEP1 is targeted to the membrane of the parasitophorous vacuole, while PbSEP2 and 3 are exported beyond the parasite membrane and translocated to the host cell compartment in association with vesicle-like structures. In this study we identified the specific signals necessary for the correct timing of expression and to direct SEP proteins to the vacuolar membrane and to the host cell compartments. The second part of the work was carried out in Montpellier II University and aims to identify the localization of two RBC membrane skeleton components, dematin and adducin, during Plasmodium falciparum infection. Our purpose is to recognize a possible mechanism of internalization of host cytoskeleton components to the parasite compartments. In fact, IFA experiments carried on iRBCs showed that dematin and adducin start to be internalized at trophozoite stage and localize at the periphery of the parasite, most probably at the parasitophoruos vacuole (PV) membrane/lumen. Dematin and adducin internalization during Plasmodium infection is also demonstrated by subcellular fractionation and proteinase K (PK) assay: while dematin is fully internalized, adducin is partially protected and suggesting a localization of the protein at the periphery of the parasite where it can be exposed to PK degradation

Habituation of Somatosensory Evoked Potentials in Patients with Alzheimer’s Disease and Those with Vascular Dementia

Background and Objectives: The most prevalent dementia are Alzheimer’s disease and vascular dementia. There is evidence that cortical synaptic function may differ in these two conditions. Habituation of cortical responses to repeated stimuli is a well-preserved phenomenon in a normal brain cortex, related to an underlying mechanism of synaptic efficacy regulation. Lack of habituation represents a marker of synaptic dysfunction. The purpose of this study was to assess the habituation of somatosensory evoked potentials (SEPs) in 29 patients affected by mild-to-moderate Alzheimer’s disease (AD-type) or vascular (VD-type) dementia. Materials and Methods: All patients underwent a clinical history interview, neuropsychological evaluation, and neuroimaging examination. SEPs were elicited by electrical stimulation of the right median nerve at the wrist. Six-hundred stimuli were delivered, and cortical responses divided in three blocks of 200. Habituation was calculated by measuring changes of N20 amplitude from block 1 to block 3. SEP variables recorded in patients were compared with those recorded in 15 age- and gender-matched healthy volunteers. Results: SEP recordings showed similar N20 amplitudes in AD-type and VD-type patients in block 1, that were higher than those recorded in controls. N20 amplitude decreased from block 1 to block 3 (habituation) in normal subjects and in VD-type patients, whereas in AD-type patients it remained unchanged (lack of habituation). Conclusions: The findings suggest that neurophysiologic mechanisms of synaptic efficacy that underneath habituation are impaired in patients with AD-type dementia but not in patients with VD-type dementia. SEPs habituation may contribute to early distinction of Alzheimer’s disease vs. vascular dementia

Mosquito stages of <i>P. berghei</i> transgenic lines harboring <i>sep2</i> or <i>sep3</i> fused to mCherry.

<p>Transgenic lines, <i>Pbsep2-cherry</i> and <i>Pbsep3-cherry</i>, harboring an integrated copy of <i>sep2</i> or <i>sep3</i> fused with the fluorescent reporter mcherry (drawn in figure), were used to infect mosquitoes. A) SEP2 is detected in all mosquito stages with a peak of expression in mature oocysts (day 12 after infection). The protein fusion (SEP2-cherry) is detected both in the sporoblast and on the sporozoites being formed. B) Higher magnification of <i>Pbsep2-cherry</i> salivary gland sporozoite reveals that SEP2-cherry localizes to the parasite periphery. IFA with α-SEP2 immune serum on non-permeabilized wild type sporozoites shows a similar fluorescence pattern. C) In young oocysts of the <i>Pbsep3-cherry</i> line, SEP3-cherry localizes only to a pigmented area. In mature oocysts, it shows a faint diffuse signal. Salivary gland sporozoites are weakly fluorescent.</p

SEP2 is expressed in liver stages and localizes to the parasite periphery.

<p><b>A)</b> The chimeric protein SEP2-cherry localizes to the periphery of <i>P. berghei</i> liver stages at 48 hpi. B) A similar fluorescence pattern is also observed in IFA using αSEP2 antibodies on wild-type sporozoites. Parasites were co-stained with antibodies raised against the parasite 75-kDa heat-shock protein (HSP70). Bars correspond to 5 µm.</p

SEP2 is secreted during gliding motility of salivary gland sporozoites.

<p>A) Wild type salivary gland sporozoites were allowed to glide on glass microscopy slides coated with αSEP2 antibodies. Subsequent IFA with a secondary fluorescent antibody revealed that SEP2 is secreted in the trails left by the parasite during its motility. SEP2 is also detected on the surface of the sporozoite in dot-like structures. B) A similar gliding assay using the αCSP and α14-3-3 antibodies, shows that CSP is uniformly localized on the surface of the parasite and in the trails, while 14-3-3 localizes to the parasite cytoplasm.</p