Public's perspective on COVID-19 adenovirus vector vaccines after thrombosis with thrombocytopenia syndrome (TTS) reports and associated regulatory actions - A cross-sectional study in six EU member states.

OBJECTIVE: In 2021, thrombosis with thrombocytopenia syndrome (TTS) was confirmed by the European Medicines Agency (EMA) as a rare side effect of the COVID-19 adenovirus vector vaccines Vaxzevria® and Jcovden®. This study aimed to describe the public's knowledge of TTS and how it affected the willingness to be vaccinated with COVID-19 vaccines and other vaccines in six European countries. METHODS: From June to October of 2022, a multi-country cross-sectional online survey was conducted in Denmark, Greece, Latvia, Netherlands, Portugal, and Slovenia. The minimum target of participants to be recruited was based on the size of the country's population. The results were analysed descriptively. RESULTS: In total, 3794 respondents were included in the analysis; across the six countries, 33.3 %-68.3 % reported being familiar with signs and symptoms of TTS, although 3.1-61.4 % of those were able to identify the symptoms correctly. The reported changes in willingness to be vaccinated against COVID-19 and with other vaccines varied per country. The largest reported change in the willingness to be vaccinated with Vaxzevria® and Jcovden® was observed in Denmark (61.2 %), while the willingness to be vaccinated with other COVID-19 vaccines changed most in Slovenia (30.4 %). The smallest decrease in willingness towards future vaccination against COVID-19 was reported in the Netherlands (20.9 %) contrasting with the largest decrease observed in Latvia (69.1 %). CONCLUSION: Knowledge about TTS seemed to have influenced the public's opinion in Europe resulting in less willingness to be vaccinated with Vaxzevria® and Jcovden®. Willingness for vaccination against COVID-19 with other vaccines and widespread use of vaccines to prevent other diseases also differed and seemed to be determined by the approaches taken by national health authorities when reacting to and communicating about COVID-19 vaccination risks. Further investigation of optimal risk communication strategies is warranted.

Screening of the activity of sixty essential oils against plasmodium early mosquito stages in vitro and machine learning analysis reveals new putative inhibitors of malaria parasites.

Malaria, an infectious disease with a tremendous impact on human health is caused by Plasmodium parasites, and transmitted by Anopheles mosquitoes. New approaches to control the disease involve transmission blocking strategies aiming to target the parasite in the mosquito. Here, we investigated the putative inhibitory activity of essential oils and their components on the early mosquito stages of the parasite. We employed an in vitro assay of gametocyte-to-ookinete development of the rodent model parasite Plasmodium berghei combined with high content screening. 60 essential oils with known composition were tested. The results revealed that fifteen EOs had inhibitory activity. Furthermore, a machine learning approach was used to identify the putative inhibitory components. Five of the most important chemical components indicated by the machine learning-based models were actually confirmed by the experimental approach. This combined approach was used for the first time to identify the potential transmission blocking activity of essential oils and single components at the zygote and ookinete stages.

Interruption of p53-MDM2 Interaction by Nutlin-3a in Human Lymphoma Cell Models Initiates a Cell-Dependent Global Effect on Transcriptome and Proteome Level.

In most lymphomas, p53 signaling pathway is inactivated by various mechanisms independent to p53 gene mutations or deletions. In many cases, p53 function is largely regulated by alterations in the protein abundance levels by the action of E3 ubiquitin-protein ligase MDM2, targeting p53 to proteasome-mediated degradation. In the present study, an integrating transcriptomics and proteomics analysis was employed to investigate the effect of p53 activation by a small-molecule MDM2-antagonist, nutlin-3a, on three lymphoma cell models following p53 activation. Our analysis revealed a system-wide nutlin-3a-associated effect in all examined lymphoma types, identifying in total of 4037 differentially affected proteins involved in a plethora of pathways, with significant heterogeneity among lymphomas. Our findings include known p53-targets and novel p53 activation effects, involving transcription, translation, or degradation of protein components of pathways, such as a decrease in key members of PI3K/mTOR pathway, heat-shock response, and glycolysis, and an increase in key members of oxidative phoshosphorylation, autophagy and mitochondrial translation. Combined inhibition of HSP90 or PI3K/mTOR pathway with nutlin-3a-mediated p53-activation enhanced the apoptotic effects suggesting a promising strategy against human lymphomas. Integrated omic profiling after p53 activation offered novel insights on the regulatory role specific proteins and pathways may have in lymphomagenesis.

Structure and function of Plasmodium actin II in the parasite mosquito stages.

Actins are filament-forming, highly-conserved proteins in eukaryotes. They are involved in essential processes in the cytoplasm and also have nuclear functions. Malaria parasites (Plasmodium spp.) have two actin isoforms that differ from each other and from canonical actins in structure and filament-forming properties. Actin I has an essential role in motility and is fairly well characterized. The structure and function of actin II are not as well understood, but mutational analyses have revealed two essential functions in male gametogenesis and in the oocyst. Here, we present expression analysis, high-resolution filament structures, and biochemical characterization of Plasmodium actin II. We confirm expression in male gametocytes and zygotes and show that actin II is associated with the nucleus in both stages in filament-like structures. Unlike actin I, actin II readily forms long filaments in vitro, and near-atomic structures in the presence or absence of jasplakinolide reveal very similar structures. Small but significant differences compared to other actins in the openness and twist, the active site, the D-loop, and the plug region contribute to filament stability. The function of actin II was investigated through mutational analysis, suggesting that long and stable filaments are necessary for male gametogenesis, while a second function in the oocyst stage also requires fine-tuned regulation by methylation of histidine 73. Actin II polymerizes via the classical nucleation-elongation mechanism and has a critical concentration of ~0.1 µM at the steady-state, like actin I and canonical actins. Similarly to actin I, dimers are a stable form of actin II at equilibrium.

Cell penetrating peptides are versatile tools for enhancing multimodal uptake into cells from pest insects.

Cell penetrating peptides (CPPs) are small peptides defined by their ability to deliver molecular cargo into cells. While the subject of frequent investigation for pharmaceutical drug delivery, little consideration has been given to the possibility of CPPs for use as insecticides or insecticide enhancers. Here, we characterize the entry of four fluorescently tagged CPPs into two insect cell lines and dissected midgut tissues in terms of both total quantity and mode of penetration. Fluorescent microscopy showed that substantial amounts of CPPs penetrate the plasma membrane via endosomal uptake in ovarian (Sf9) and midgut derived (AW1) lepidopteran cells and that this process was sensitive to selected endocytosis inhibitors. Differences in the quantity of uptake was observed between CPPs, and further differences were found in the ability CPP-1838 to efficiently penetrate membranes through passive diffusion. These findings were extended to primary midgut derived cells and dissected tissues suggesting that CPPs show a preference for goblet cells and that CPP-1838 shows far higher rates of penetration. CPP-1838 thus shows extraordinary abilities to penetrate cells efficiency in both a diffusional and endocytotic manner. From these results more sophisticated delivery methods based on the utilization of CPPs can be developed.

Gene editing in Plasmodium berghei made easy: Development of a CRISPR/Cas9 protocol using linear donor template and ribozymes for sgRNA generation.

Efficient reverse genetics approaches are critical for the study of many organisms. The CRISPR/Cas9 gene editing system has led to a plethora of new tools for geneticists. Here, we successfully established a simplified CRISPR/Cas9 system for the malaria model parasite Plasmodium berghei. The homologous directed repair (HDR) template is provided as a linear template with homologous arms of 600-700bp while the CRISPR elements sgRNA and Cas9 are encoded from a single plasmid utilizing the Ribozyme-Guide-Ribozyme (RGR) expression strategy. Our approach eliminates the need for negative selection markers since the plasmid cannot be incorporated into the genome. As a test case we inserted the FLAG encoding sequence into the ACT2 locus using this new approach. We showed that the genetic modification of this locus had no adverse effects on the completion of the P. berghei life cycle, including transmission through the mosquito.

Targeting Smyd3 by next-generation antisense oligonucleotides suppresses liver tumor growth.

The oncogenic function of suppressor of variegation, enhancer of zeste and MYeloid-Nervy-DEAF1-domain family methyltransferase Smyd3 has been implicated in various malignancies, including hepatocellular carcinoma (HCC). Here, we show that targeting Smyd3 by next-generation antisense oligonucleotides (Smyd3-ASO) is an efficient approach to modulate its mRNA levels in vivo and to halt the growth of already initiated liver tumors. Smyd3-ASO treatment dramatically decreased tumor burden in a mouse model of chemically induced HCC and negatively affected the growth rates, migration, oncosphere formation, and xenograft growth capacity of a panel of human hepatic cancer cell lines. Smyd3-ASOs prevented the activation of oncofetal genes and the development of cancer-specific gene expression program. The results point to a mechanism by which Smyd3-ASO treatment blocks cellular de-differentiation, a hallmark feature of HCC development, and, as a result, it inhibits the expansion of hepatic cancer stem cells, a population that has been presumed to resist chemotherapy.

Erf Affects Commitment and Differentiation of Osteoprogenitor Cells in Cranial Sutures via the Retinoic Acid Pathway.

ETS2 repressor factor (ERF) haploinsufficiency causes late-onset craniosynostosis (CRS) (OMIM entry 600775; CRS4) in humans, while in mice Erf insufficiency also leads to a similar multisuture synostosis phenotype preceded by mildly reduced calvarium ossification. However, neither the cell types affected nor the effects per se have been identified so far. Here, we establish an ex vivo system for the expansion of suture-derived mesenchymal stem and progenitor cells (sdMSCs) and analyze the role of Erf levels in their differentiation. Cellular data suggest that Erf insufficiency specifically decreases osteogenic differentiation of sdMSCs, resulting in the initially delayed mineralization of the calvarium. Transcriptome analysis indicates that Erf is required for efficient osteogenic lineage commitment of sdMSCs. Elevated retinoic acid catabolism due to increased levels of the cytochrome P450 superfamily member Cyp26b1 as a result of decreased Erf levels appears to be the underlying mechanism leading to defective differentiation. Exogenous addition of retinoic acid can rescue the osteogenic differentiation defect, suggesting that Erf affects cranial bone mineralization during skull development through retinoic acid gradient regulation.

A Comprehensive Gender-related Secretome of Plasmodium berghei Sexual Stages.

Plasmodium, the malaria parasite, undergoes a complex life cycle alternating between a vertebrate host and a mosquito vector of the genus Anopheles In red blood cells of the vertebrate host, Plasmodium multiplies asexually or differentiates into gamete precursors, the male and female gametocytes, responsible for parasite transmission. Sexual stage maturation occurs in the midgut of the mosquito vector, where male and female gametes egress from the host erythrocytes to fuse and form a zygote. Gamete egress entails the successive rupture of two membranes surrounding the parasite, the parasitophorous vacuole membrane and the erythrocyte plasma membrane. In this study, we used the rodent model parasite Plasmodium berghei to design a label-free quantitative proteomic approach aimed at identifying gender-related proteins differentially released/secreted by purified mature gametocytes when activated to form gametes. We compared the abundance of molecules secreted by wild type gametocytes of both genders with that of a transgenic line defective in male gamete maturation and egress. This enabled us to provide a comprehensive data set of egress-related molecules and their gender specificity. Using specific antibodies, we validated eleven candidate molecules, predicted as either gender-specific or common to both male and female gametocytes. All of them localize to punctuate, vesicle-like structures that relocate to cell periphery upon activation, but only three of them localize to the gametocyte-specific secretory vesicles named osmiophilic bodies. Our results confirm that the egress process involves a tightly coordinated secretory apparatus that includes different types of vesicles and may put the basis for functional studies aimed at designing novel transmission-blocking molecules.

Impact of the UK Psychoactive Substances Act on awareness, use, experiences and knowledge of potential associated health risks of novel psychoactive substances.

AIMS: The risk of potential harms prompted the UK government to introduce the Psychoactive Substances Act in 2016. The aim of the present study was to evaluate the impact and effectiveness of this new legislation on patterns of novel psychoactive substance (NPS) awareness, use, experiences and risk awareness in a self-selected sample of UK consumers to inform education and policy. METHODS: The Bristol Online Survey was advertised on the Bluelight drug forum and social media Facebook pages and University email between 7 January and 7 February 2015 (168 responses) and 9 March to 18 September 2017 (726 responses). UK country of residence responses were extracted for analysis (SPSS). RESULTS: In a predominantly university-educated, young (< 25 years) self-selecting sample, 1 year after introduction of the legislation, NPS use (in males, under 18s, those educated to school/college level, P < .001) has increased, whilst health risk awareness has not changed and remains poor. Users are switching to sourcing NPSs via street dealers (49%) and the darknet (31%) and showing an increase in preference for the herbal NPS Salvia divinorum (P < .05). The main reasons for NPS use remain the influence of friends (69%) in a social setting and to get high (76%) usually in combination with alcohol, cannabis or ecstasy. CONCLUSION: Regulation alone, so far, has not impacted on health risk awareness, NPS drug demand and culture in our UK survey sample. Alongside regulation, NPS health promotion education (particularly in schools, colleges) is needed that addresses resilience and both the risks and beneficial effects of NPS.

Correction: Essential role of Plasmodium perforin-like protein 4 in ookinete midgut passage.

[This corrects the article DOI: 10.1371/journal.pone.0201651.].

Essential role of Plasmodium perforin-like protein 4 in ookinete midgut passage.

Pore forming proteins such as those belonging to the membrane attack/perforin (MACPF) family have important functions in many organisms. Of the five MACPF proteins found in Plasmodium parasites, three have functions in cell passage and one in host cell egress. Here we report an analysis of the perforin-like protein 4, PPLP4, in the rodent parasite Plasmodium berghei. We found that the protein is expressed only in the ookinete, the invasive stage of the parasite formed in the mosquito midgut. Transcriptional analysis revealed that expression of the pplp4 gene commences during ookinete development. The protein was detected in retorts and mature ookinetes. Using two antibodies, the protein was found localized in a dotted pattern, and 3-D SIM super-resolution microcopy revealed the protein in the periphery of the cell. Analysis of a C-terminal mCherry fusion of the protein however showed mainly cytoplasmic label. A pplp4 null mutant formed motile ookinetes, but these were unable to invade and traverse the midgut epithelium resulting in severely impaired oocyst formation and no transmission to naïve mice. However, when in vitro cultured ookinetes were injected into the thorax of the mosquito, thus by-passing midgut passage, sporozoites were formed and the mutant parasites were able to infect naïve mice. Taken together, our data show that PPLP4 is required only for ookinete invasion of the mosquito midgut. Thus PPLP4 has a similar role to the previously studied PPLP3 and PPLP5, raising the question why three proteins with MACPF domains are needed for invasion by the ookinete of the mosquito midgut epithelium.

Sequential Membrane Rupture and Vesiculation during Plasmodium berghei Gametocyte Egress from the Red Blood Cell.

Malaria parasites alternate between intracellular and extracellular stages and successful egress from the host cell is crucial for continuation of the life cycle. We investigated egress of Plasmodium berghei gametocytes, an essential process taking place within a few minutes after uptake of a blood meal by the mosquito. Egress entails the rupture of two membranes surrounding the parasite: the parasitophorous vacuole membrane (PVM), and the red blood cell membrane (RBCM). High-speed video microscopy of 56 events revealed that egress in both genders comprises four well-defined phases, although each event is slightly different. The first phase is swelling of the host cell, followed by rupture and immediate vesiculation of the PVM. These vesicles are extruded through a single stabilized pore of the RBCM, and the latter is subsequently vesiculated releasing the free gametes. The time from PVM vesiculation to completion of egress varies between events. These observations were supported by immunofluorescence microscopy using antibodies against proteins of the RBCM and PVM. The combined results reveal dynamic re-organization of the membranes and the cortical cytoskeleton of the erythrocyte during egress.

Sequence and functional divergence of gametocyte-specific parasitophorous vacuole membrane proteins in Plasmodium parasites.

Plasmodium parasites develop within red blood cells in a Parasitophorous Vacuole enclosed by a Membrane, the PVM. The protein family ETRAMP (Early Transcribed Membrane Protein) comprises small proteins inserted in the PVM via a single transmembrane domain. Among those, Pfs16 is specifically found in P. falciparum gametocyte PVM. The P. berghei gene PBANKA_1003900 is syntenic with pfs16. The encoded proteins have a similar domain structure but the overall protein similarity is low. A transcript of the P. berghei gene is only found in gametocytes and ookinetes and a C-terminal mCherry fusion of the protein revealed its presence only in gametocytes. A knock-out mutant of the PBANKA_1003900 gene was not affected in sexual development and ookinete formation was similar to WT. The mutation had no adverse effect on transmission through the mosquito although there was a reduction of the number of oocysts formed by the mutant parasites.

Structured illumination microscopy reveals actin I localization in discreet foci in Plasmodium berghei gametocytes.

Actin has important roles in Plasmodium parasites but its exact function in different life stages is not yet fully elucidated. Here we report the localization of ubiquitous actin I in gametocytes of the rodent model parasite P. berghei. Using an antibody specifically recognizing F-actin and deconvolution microscopy we detected actin I in a punctate pattern in gametocytes. 3D-Structured Illumination Microscopy which allows sub-diffraction limit imaging resolved the signal into structures of less than 130 nm length. A portion of actin I was soluble, but the protein was also found complexed in a stabilized form which could only be completely solubilized by treatment with SDS. An additional population of actin was pelleted at 100 000 × g, consistent with F-actin. Our results suggest that actin in this non-motile form of the parasite is present in short filaments cross-linked to other structures in a cytoskeleton.

An international survey on the awareness, use, preference, and health perception of novel psychoactive substances (NPS).

OBJECTIVE: This survey investigated the level of public awareness, preference, and motivation of novel psychoactive substances (NPS) use as well as knowledge of potential associated health risks. METHODS: A Bristol Online Survey was advertised through social media and a drug forum "Bluelight" between January 7 and February 7, 2015. RESULTS: Responses were received from 17 countries, mainly from Europe. Most responses (83%) came from university educated students. Two-thirds (65%) of the 168 respondents were aware of NPS. Awareness was significantly increased in those with bisexual or homosexual orientation (p < .05) and those in employment (p < .05). Fourteen percent of the 168 respondents were users of NPS, and use was significantly affected by age and employment (p < .01) but unaffected by level of education (p > .05). Nearly half of the NPS users perceived NPS to carry either a low risk to health (20%) or did not know whether or not they posed a health risk (29%). CONCLUSIONS: These survey data indicate that awareness of NPS and, importantly, perception of the potential health risks associated with NPS use is lacking. NPS awareness and use is higher in those in employment but is unaffected by level of education. This highlights the need for targeted drugs education intervention by policy-makers in schools and universities.

Expression of the Plasmodium berghei actin II gene is controlled by elements in a long genomic region.

Plasmodium parasites have two actin isoforms. Actin I is ubiquitously expressed, while the second actin isoform is expressed in the sexual stages and ookinetes. Reverse genetic analysis revealed two phenotypes in parasites lacking the protein: a block in male gametogenesis (exflagellation) and a second phenotype in oocyst development, dependent upon the expression of the gene in female gametocytes. Here, we report that the genetic complementation of two independent mutants lacking actin II does not fully restore wild-type function. Constructs were integrated in the c-rrna locus, previously used for expression of transgenes, in order to determine the dependence of expression on actin II flanking genomic regions. Partial restoration of male gametogenesis was achieved when the transgene contained, in addition to the coding region, 1.2 kb upstream of the actin II open reading frame. Another transgene, which comprised 2.7 kb of actin II 5' flanking regions and the cognate 3' downstream sequence, fully restored exflagellation. However, in both complemented strains, oocyst development was severely impaired compared to the WT. These data suggest that male gametocyte expression of actin II is dependent upon extensive flanking regions, while female expression requires even longer genomic sequences for correct expression of the gene.

A draft genome sequence of an invasive mosquito: an Italian Aedes albopictus.

The draft genome sequence of Italian specimens of the Asian tiger mosquito Aedes (Stegomyia) albopictus (Diptera: Culicidae) was determined using a standard NGS (next generation sequencing) approach. The size of the assembled genome is comparable to that of Aedes aegypti; the two mosquitoes are also similar as far as the high content of repetitive DNA is concerned, most of which is made up of transposable elements. Although, based on BUSCO (Benchmarking Universal Single-Copy Orthologues) analysis, the genome assembly reported here contains more than 99% of protein-coding genes, several of those are expected to be represented in the assembly in a fragmented state. We also present here the annotation of several families of genes (tRNA genes, miRNA genes, the sialome, genes involved in chromatin condensation, sex determination genes, odorant binding proteins and odorant receptors). These analyses confirm that the assembly can be used for the study of the biology of this invasive vector of disease.

Mosquito genomics. Highly evolvable malaria vectors: the genomes of 16 Anopheles mosquitoes.

Variation in vectorial capacity for human malaria among Anopheles mosquito species is determined by many factors, including behavior, immunity, and life history. To investigate the genomic basis of vectorial capacity and explore new avenues for vector control, we sequenced the genomes of 16 anopheline mosquito species from diverse locations spanning ~100 million years of evolution. Comparative analyses show faster rates of gene gain and loss, elevated gene shuffling on the X chromosome, and more intron losses, relative to Drosophila. Some determinants of vectorial capacity, such as chemosensory genes, do not show elevated turnover but instead diversify through protein-sequence changes. This dynamism of anopheline genes and genomes may contribute to their flexible capacity to take advantage of new ecological niches, including adapting to humans as primary hosts.

Distinct properties of the egress-related osmiophilic bodies in male and female gametocytes of the rodent malaria parasite Plasmodium berghei.

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.