Genes differentially expressed between pathogenic and non-pathogenic Entamoeba histolytica clones influence pathogenicity-associated phenotypes by multiple mechanisms.

Recently, two genes involved in amoebic liver abscess formation in a mouse model were identified by their differential expression of non-pathogenic (A1np) and pathogenic (B2p) clones of the Entamoeba histolytica isolate HM:1-IMSS. While overexpression of a gene encoding the metallopeptidase EhMP8-2 reduces the virulence of the pathogenic clone B2p, overexpression of the gene ehi_127670 (ehhp127), encoding a hypothetical protein, increases the virulence of the non-pathogenic clone A1np, while silencing this gene in the pathogenic B2p reduces virulence. To understand the role of both molecules in determining the pathogenicity of E. histolytica, silencing, and overexpression transfectants were characterized in detail. Silencing of ehmp8-2, of the homologous gene ehmp8-1, or both in non-pathogenic A1np trophozoites significantly altered the transcript levels of 347, 216, and 58 genes, respectively. This strong change in the expression profiles caused by the silencing of ehmp8-1 and ehmp8-2 implies that these peptidases regulate the expression of numerous genes. Consequently, numerous phenotypic characteristics, including cytopathic, hemolytic, and cysteine peptidase activity, were altered in response to their silencing. Silencing of ehhp127 in pathogenic B2p trophozoites did not affect the expression of other genes, whereas its overexpression in non-pathogenic A1np trophozoites results in an altered expression of approximately 140 genes. EhHP127 is important for trophozoite motility, as its silencing reduces, while its overexpression enhances movement activity. Interestingly, the specific silencing of ehhp127 also significantly affects cytopathic, cysteine peptidase, and hemolytic activities. All three molecules characterized in this study, namely EhMP8-1, EhMP8-2, and EhHP127, are present in amoeba vesicles. The results show that ehmp8-2 and ehhp127 are not only differentially expressed between pathogenic and non-pathogenic amoebae, but that they also significantly affect amoeba pathogenicity-associated phenotypes by completely different mechanisms. This observation suggests that the regulation of amoeba pathogenicity is achieved by a complex network of molecular mechanisms rather than by single factors.

Assessing the utility of deep neural networks in detecting superficial surgical site infections from free text electronic health record data.

Background: High-quality outcomes data is crucial for continued surgical quality improvement. Outcomes are generally captured through structured administrative data or through manual curation of unstructured electronic health record (EHR) data. The aim of this study was to apply natural language processing (NLP) to chart notes in the EHR to accurately capture postoperative superficial surgical site infections (SSSIs). Methods: Deep Learning (DL) NLP models were trained on data from 389,865 surgical cases across all 11 hospitals in the Capital Region of Denmark. Surgical cases in the training dataset were performed between January 01st, 2017, and October 30th, 2021. We trained a forward reading and a backward reading universal language model on unlabeled postoperative chart notes recorded within 30 days of a surgical procedure. The two language models were subsequently finetuned on labeled data for the classification of SSSIs. Validation and testing were performed on surgical cases performed during the month of November 2021. We propose two different use cases: a stand-alone machine learning (SAM) pipeline and a human-in-the-loop (HITL) pipeline. Performances of both pipelines were compared to administrative data and to manual curation. Results: The models were trained on 3,983,864 unlabeled chart notes and finetuned on 1,231,656 labeled notes. Models had a test area under the receiver operating characteristic curves (ROC AUC) of 0.989 on individual chart notes and 0.980 on an aggregated case level. The SAM pipeline had a sensitivity of 0.604, a specificity of 0.996, a positive predictive value (PPV) of 0.763, and a negative predictive value (NPV) of 0.991. Prior to human review, the HITL pipeline had a sensitivity of 0.854, a specificity of 0.987, a PPV of 0.603, and a NPV of 0.997. Conclusion: The performance of the SAM pipeline was superior to administrative data, and significantly outperformed previously published results. The performance of the HITL pipeline approached that of manual curation.

Using machine learning for predicting intensive care unit resource use during the COVID-19 pandemic in Denmark.

The COVID-19 pandemic has put massive strains on hospitals, and tools to guide hospital planners in resource allocation during the ebbs and flows of the pandemic are urgently needed. We investigate whether machine learning (ML) can be used for predictions of intensive care requirements a fixed number of days into the future. Retrospective design where health Records from 42,526 SARS-CoV-2 positive patients in Denmark was extracted. Random Forest (RF) models were trained to predict risk of ICU admission and use of mechanical ventilation after n days (n = 1, 2, …, 15). An extended analysis was provided for n = 5 and n = 10. Models predicted n-day risk of ICU admission with an area under the receiver operator characteristic curve (ROC-AUC) between 0.981 and 0.995, and n-day risk of use of ventilation with an ROC-AUC between 0.982 and 0.997. The corresponding n-day forecasting models predicted the needed ICU capacity with a coefficient of determination (R2) between 0.334 and 0.989 and use of ventilation with an R2 between 0.446 and 0.973. The forecasting models performed worst, when forecasting many days into the future (for large n). For n = 5, ICU capacity was predicted with ROC-AUC 0.990 and R2 0.928, and use of ventilator was predicted with ROC-AUC 0.994 and R2 0.854. Random Forest-based modelling can be used for accurate n-day forecasting predictions of ICU resource requirements, when n is not too large.

Altered Cytokine Response of Human Brain Endothelial Cells after Stimulation with Malaria Patient Plasma.

Infections with the deadliest malaria parasite, Plasmodium falciparum, are accompanied by a strong immunological response of the human host. To date, more than 30 cytokines have been detected in elevated levels in plasma of malaria patients compared to healthy controls. Endothelial cells (ECs) are a potential source of these cytokines, but so far it is not known if their cytokine secretion depends on the direct contact of the P. falciparum-infected erythrocytes (IEs) with ECs in terms of cytoadhesion. Culturing ECs with plasma from malaria patients (27 returning travellers) resulted in significantly increased secretion of IL-11, CXCL5, CXCL8, CXCL10, vascular endothelial growth factor (VEGF) and angiopoietin-like protein 4 (ANGPTL4) if compared to matching controls (22 healthy individuals). The accompanying transcriptome study of the ECs identified 43 genes that were significantly increased in expression (≥1.7 fold) after co-incubation with malaria patient plasma, including cxcl5 and angptl4. Further bioinformatic analyses revealed that biological processes such as cell migration, cell proliferation and tube development were particularly affected in these ECs. It can thus be postulated that not only the cytoadhesion of IEs, but also molecules in the plasma of malaria patients exerts an influence on ECs, and that not only the immunological response but also other processes, such as angiogenesis, are altered.

Life Cycle Stage-Specific Accessibility of Leishmania donovani Chromatin at Transcription Start Regions.

Leishmania donovani is a parasitic protist that causes the lethal Kala-azar fever in India and East Africa. Gene expression in Leishmania is regulated by gene copy number variation and inducible translation while RNA synthesis initiates at a small number of sites per chromosome and proceeds through polycistronic transcription units, precluding a gene-specific regulation (C. Clayton and M. Shapira, Mol Biochem Parasitol 156:93-101, 2007, https://doi.org/10.1016/j.molbiopara.2007.07.007). Here, we analyze the dynamics of chromatin structure in both life cycle stages of the parasite and find evidence for an additional, epigenetic gene regulation pathway in this early branching eukaryote. The assay for transposase-accessible chromatin using sequencing (ATAC-seq) analysis (J. D. Buenrostro, P. G. Giresi, L. C. Zaba, H. Y. Chang, and W. J. Greenleaf, Nat Methods 10:1213-1218, 2013, https://doi.org/10.1038/nmeth.2688) predominantly shows euchromatin at transcription start regions in fast-growing promastigotes, but mostly heterochromatin in the slowly proliferating amastigotes, the mammalian stage, reflecting a previously shown increase of histone synthesis in the latter stage. IMPORTANCE Leishmania parasites are important pathogens with a global impact and cause poverty-related illness and death. They are devoid of classic cis- and trans-acting transcription regulators but use regulated translation and gene copy number variations to adapt to hosts and environments. In this work, we show that transcription start regions present as open euchromatin in fast-growing insect stages but as less-accessible heterochromatin in the slowly proliferating amastigote stage, indicating an epigenetic control of gene accessibility in this early branching eukaryotic pathogen. This finding should stimulate renewed interest in the control of RNA synthesis in Leishmania and related parasites.

Analysis of the Interaction Between Plasmodium falciparum-Infected Erythrocytes and Human Endothelial Cells Using a Laminar Flow System, Bioinformatic Tracking and Transcriptome Analysis.

During malaria infection, the endothelial lining of the small blood vessels of the brain and other vital organs is strongly stimulated. This leads to fatal complications and poor prognosis of the infection. It is believed that two main reasons are responsible for this pathology, namely the cytoadhesion of Plasmodium falciparum-infected erythrocytes (IEs) on the one hand and the proinflammatory products released by the IEs which activate the endothelial cells (ECs) on the other hand. Until recently, most of the studies that characterized the activation of ECs were performed under static conditions, which do not reflect the real sequelae in vivo. In this chapter, we present a system, which allows authentic simulation of the IEs-ECs interactions during P. falciparum infection.The main idea of the system is to provide an adequate shear stress over the ECs during the cytoadhesion and stimulation with IEs, which provides a better basis for the investigation of the cytoadhesion pathology through analyzing the ECs' transcriptome after stimulation. On the other hand, analyzing the transcriptome of the IEs might also give deeper analysis of their response to shear stress. Deep understanding of these events might help in the development of novel treatment strategies that interfere with this cell-cell interaction.

Prognostic value of the micronucleus assay for clinical endpoints in neoadjuvant radiochemotherapy for rectal cancer.

BACKGROUND: The question whether lymphocyte radiosensitivity is representative of patients' response to radiotherapy (RT) remains unsolved. We analyzed lymphocyte cytogenetic damage in patients who were homogeneously treated with preoperative radiochemotherapy (RCT) for rectal cancer within clinical trials. We tested for interindividual variation and consistent radiosensitivity after in-vivo and in-vitro irradiation, analyzed the effect of patients' and RCT characteristics on cytogenetic damage, and tested for correlations with patients' outcome in terms of tumor response, survival and treatment-related toxicity. METHODS: The cytokinesis-block micronucleus cytome (CBMNcyt) assay was performed on the peripheral blood lymphocytes (PBLCs) of 134 patients obtained before, during, at the end of RCT, and during the 2-year follow-up. A subset of PBLCs obtained before RCT was irradiated in-vitro with 3 Gy. RCT included 50.4 Gy of pelvic RT with 5-fluorouracil (5-FU) alone (n = 78) or 5-FU plus oxaliplatin (n = 56). The analyzed variables included patients' age, gender, RT characteristics (planning target volume size [PTV size], RT technique), and chemotherapy characteristics (5-FU plasma levels, addition of oxaliplatin). Outcome was analyzed as tumor regression, patient survival, and acute and late toxicity. RESULTS: Cytogenetic damage increased significantly with the radiation dose and varied substantially between individuals. Women were more sensitive than men; no significant age-dependent differences were observed. There was a significant correlation between the cytogenetic damage after in-vitro irradiation and in-vivo RCT. We found a significant effect of the PTV size on the yields of cytogenetic damage after RCT, while the RT technique had no effect. Neither the addition of oxaliplatin nor the 5-FU levels influenced cytogenetic damage. We found no correlation between patient outcome and the cytogenetic damage. CONCLUSIONS: We found consistent cytogenetic damage in lymphocytes after in-vivo RCT and in-vitro irradiation. Gender was confirmed as a well-known, and the PTV size was identified as a less well-known influencing variable on lymphocyte cytogenetic damage after partial-body irradiation. A consistent level of cytogenetic damage after in-vivo and in-vitro irradiation may indicate the importance of genetic factors for individual radiosensitivity. However, we found no evidence that in-vivo or in-vitro irradiation-induced cytogenetic damage is an adequate biomarker for the response to RCT in rectal cancer patients.

Developing and validating COVID-19 adverse outcome risk prediction models from a bi-national European cohort of 5594 patients.

Patients with severe COVID-19 have overwhelmed healthcare systems worldwide. We hypothesized that machine learning (ML) models could be used to predict risks at different stages of management and thereby provide insights into drivers and prognostic markers of disease progression and death. From a cohort of approx. 2.6 million citizens in Denmark, SARS-CoV-2 PCR tests were performed on subjects suspected for COVID-19 disease; 3944 cases had at least one positive test and were subjected to further analysis. SARS-CoV-2 positive cases from the United Kingdom Biobank was used for external validation. The ML models predicted the risk of death (Receiver Operation Characteristics-Area Under the Curve, ROC-AUC) of 0.906 at diagnosis, 0.818, at hospital admission and 0.721 at Intensive Care Unit (ICU) admission. Similar metrics were achieved for predicted risks of hospital and ICU admission and use of mechanical ventilation. Common risk factors, included age, body mass index and hypertension, although the top risk features shifted towards markers of shock and organ dysfunction in ICU patients. The external validation indicated fair predictive performance for mortality prediction, but suboptimal performance for predicting ICU admission. ML may be used to identify drivers of progression to more severe disease and for prognostication patients in patients with COVID-19. We provide access to an online risk calculator based on these findings.

Searching Novel Clock Genes Using RNAi-Based Screening.

RNA interference (RNAi) allows for the selective downregulation of gene expression by neutralizing targeted mRNA molecules and has frequently been used in high-throughput screening endeavors. Here, we describe a protocol for the highly parallel RNAi-mediated downregulation of gene expression in order to search for components involved in circadian rhythm generation. We use lentiviral gene transfer to deliver shRNA expressing plasmids into circadian reporter cells ensuring for efficient and stable knockdown. Circadian rhythms are monitored using live-cell bioluminescence recording of synchronized reporter cells over several days. In addition, we present a new software tool (ChronoStar) for efficient, parallel time-series analysis to extract rhythm parameters such as period, phase, amplitude, and damping.

Casein kinase 1.2 over expression restores stress resistance to Leishmania donovani HSP23 null mutants.

Leishmania donovani is a trypanosomatidic parasite and causes the lethal kala-azar fever, a neglected tropical disease. The Trypanosomatida are devoid of transcriptional gene regulation and rely on gene copy number variations and translational control for their adaption to changing conditions. To survive at mammalian tissue temperatures, L. donovani relies on the small heat shock protein HSP23, the loss of which renders the parasites stress sensitive and impairs their proliferation. Here, we analysed a spontaneous escape mutant with wild type-like in vitro growth. Further selection of this escape strains resulted in a complete reversion of the phenotype. Whole genome sequencing revealed a correlation between stress tolerance and the massive amplification of a six-gene cluster on chromosome 35, with further analysis showing over expression of the casein kinase 1.2 gene as responsible. In vitro phosphorylation experiments established both HSP23 and the related P23 co-chaperone as substrates and modulators of casein kinase 1.2, providing evidence for another crucial link between chaperones and signal transduction protein kinases in this early branching eukaryote.

Stringent Selection of Knobby Plasmodium falciparum-Infected Erythrocytes during Cytoadhesion at Febrile Temperature.

Changes in the erythrocyte membrane induced by Plasmodium falciparum invasion allow cytoadhesion of infected erythrocytes (IEs) to the host endothelium, which can lead to severe complications. Binding to endothelial cell receptors (ECRs) is mainly mediated by members of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family, encoded by var genes. Malaria infection causes several common symptoms, with fever being the most apparent. In this study, the effects of febrile conditions on cytoadhesion of predominately knobless erythrocytes infected with the laboratory isolate IT4 to chondroitin-4-sulfate A (CSA), intercellular adhesion molecule 1 (ICAM-1), and CD36 were investigated. IEs enriched for binding to CSA at 40 °C exhibited significantly increased binding capacity relative to parasites enriched at 37 °C. This interaction was due to increased var2csa expression and trafficking of the corresponding PfEMP1 to the IE surface as well as to a selection of knobby IEs. Furthermore, the enrichment of IEs to ICAM-1 at 40 °C also led to selection of knobby IEs over knobless IEs, whereas enrichment on CD36 did not lead to a selection. In summary, these findings demonstrate that knobs are crucial for parasitic survival in the host, especially during fever episodes, and thus, that selection pressure on the formation of knobs could be controlled by the host.

Dissecting the Gene Expression, Localization, Membrane Topology, and Function of the Plasmodium falciparum STEVOR Protein Family.

During its intraerythrocytic development, the malaria parasite Plasmodium falciparum exposes variant surface antigens (VSAs) on infected erythrocytes to establish and maintain an infection. One family of small VSAs is the polymorphic STEVOR proteins, which are marked for export to the host cell surface through their PEXEL signal peptide. Interestingly, some STEVORs have also been reported to localize to the parasite plasma membrane and apical organelles, pointing toward a putative function in host cell egress or invasion. Using deep RNA sequencing analysis, we characterized P. falciparumstevor gene expression across the intraerythrocytic development cycle, including free merozoites, in detail and used the resulting stevor expression profiles for hierarchical clustering. We found that most stevor genes show biphasic expression oscillation, with maximum expression during trophozoite stages and a second peak in late schizonts. We selected four STEVOR variants, confirmed the expected export of these proteins to the host cell membrane, and tracked them to a secondary location, either to the parasite plasma membrane or the secretory organelles of merozoites in late schizont stages. We investigated the function of a particular STEVOR that showed rhoptry localization and demonstrated its role at the parasite-host interface during host cell invasion by specific antisera and targeted gene disruption. Experimentally determined membrane topology of this STEVOR revealed a single transmembrane domain exposing the semiconserved as well as variable protein regions to the cell surface.IMPORTANCE Malaria claims about half a million lives each year. Plasmodium falciparum, the causative agent of the most severe form of the disease, uses proteins that are translocated to the surface of infected erythrocytes for immune evasion. To circumvent the detection of these gene products by the immune system, the parasite evolved a complex strategy that includes gene duplications and elaborate sequence polymorphism. STEVORs are one family of these variant surface antigens and are encoded by about 40 genes. Using deep RNA sequencing of blood-stage parasites, including free merozoites, we first established stevor expression of the cultured isolate and compared it with published transcriptomes. We reveal a biphasic expression of most stevor genes and confirm this for individual STEVORs at the protein level. The membrane topology of a rhoptry-associated variant was experimentally elucidated and linked to host cell invasion, underlining the importance of this multifunctional protein family for parasite proliferation.

Ribosome Profiling Reveals HSP90 Inhibitor Effects on Stage-Specific Protein Synthesis in Leishmania donovani.

The 90-kDa heat shock protein (HSP90) of eukaryotes is a highly abundant and essential chaperone required for the maturation of regulatory and signal proteins. In the protozoan parasite Leishmania donovani, causative agent of the fatal visceral leishmaniasis, HSP90 activity is essential for cell proliferation and survival. Even more importantly, its inhibition causes life cycle progression from the insect stage to the pathogenic, mammalian stage. To unravel the molecular impact of HSP90 activity on the parasites' gene expression, we performed a ribosome profiling analysis of L. donovani, comparing genome-wide protein synthesis patterns in the presence and absence of the HSP90-specific inhibitor radicicol and an ectopically expressed radicicol-resistant HSP90 variant. We find that ribosome-protected RNA faithfully maps open reading frames and represents 97% of the annotated protein-coding genes of L. donovani. Protein synthesis was found to correlate poorly with RNA steady-state levels, indicating a regulated translation as primary mechanism for HSP90-dependent gene expression. The results confirm inhibitory effects of HSP90 on the synthesis of Leishmania proteins that are associated with the pathogenic, intracellular stage of the parasite. Those include heat shock proteins, redox enzymes, virulence-enhancing surface proteins, proteolytic pathways, and a complete set of histones. Conversely, HSP90 promotes fatty acid synthesis enzymes. Complementing radicicol treatment with the radicicol-resistant HSP90rr variant revealed important off-target radicicol effects that control a large number of the above-listed proteins. Leishmania lacks gene-specific transcription regulation and relies on regulated translation instead. Our ribosome footprinting analysis demonstrates a controlling function of HSP90 in stage-specific protein synthesis but also significant, HSP90-independent effects of the inhibitor radicicol. IMPORTANCE Leishmania parasites cause severe illness in humans and animals. They exist in two developmental stages, insect form and mammalian form, which differ in shape and gene expression. By mapping and quantifying RNA fragments protected by protein synthesis complexes, we determined the rates of protein synthesis for >90% of all Leishmania proteins in response to the inhibition of a key regulatory protein, the 90-kDa heat shock protein. We find that Leishmania depends on a regulation of protein synthesis for controlling its gene expression and that heat shock protein 90 inhibition can trigger the developmental program from insect form to mammalian form of the pathogen.

Effects of repeated long-term psychosocial stress and acute cannabinoid exposure on mouse corticostriatal circuitries: Implications for neuropsychiatric disorders.

INTRODUCTION: Vulnerability to psychiatric manifestations is achieved by the influence of genetic and environment including stress and cannabis consumption. Here, we used a psychosocial stress model based on resident-intruder confrontations to study the brain corticostriatal-function, since deregulation of corticostriatal circuitries has been reported in many psychiatric disorders. CB1 receptors are widely expressed in the central nervous system and particularly, in both cortex and striatum brain structures. AIMS AND METHODS: The investigation presented here is addressed to assess the impact of repeated stress following acute cannabinoid exposure on behavior and corticostriatal brain physiology by assessing mice behavior, the concentration of endocannabinoid and endocannabinoid-like molecules and changes in the transcriptome. RESULTS: Stressed animals urinated frequently; showed exacerbated scratching activity, lower striatal N-arachidonylethanolamine (AEA) levels and higher cortical expression of cholinergic receptor nicotinic alpha 6. The cannabinoid agonist WIN55212.2 diminished locomotor activity while the inverse agonist increased the distance travelled in the center of the open field. Upon CB1 activation, N-oleoylethanolamide and N-palmitoylethanolamide, two AEA congeners that do not interact directly with cannabinoid receptors, were enhanced in the striatum. The co-administration with both cannabinoids induced an up-regulation of striatal FK506 binding protein 5. The inverse agonist in controls reversed the effects of WIN55212.2 on motor activity. When Rimonabant was injected under stress, the cortical levels of 2-arachidonoylglycerol were maximum. The agonist and the antagonist influenced the cortical expression of cholinergic receptor nicotinic alpha 6 and serotonin transporter neurotransmitter type 4 in opposite directions, while their co-administration tended to produce a null effect under stress. CONCLUSIONS: The endocannabinoid system had a direct effect on serotoninergic neurotransmission and glucocorticoid signaling. Cholinergic receptor nicotinic alpha-6 was shown to be deregulated in response to stress and following synthetic cannabinoid drugs thus could confer vulnerability to cannabis addiction and psychosis. Targeting the receptors of endocannabinoids and endocannabinoid-like mediators might be a valuable option for treating stress-related neuropsychiatric symptoms.

Neuronal Vacuolization in Feline Panleukopenia Virus Infection.

Feline panleukopenia virus (FPV) infections are typically associated with anorexia, vomiting, diarrhea, neutropenia, and lymphopenia. In cases of late prenatal or early neonatal infections, cerebellar hypoplasia is reported in kittens. In addition, single cases of encephalitis are described. FPV replication was recently identified in neurons, although it is mainly found in cells with high mitotic activity. A female cat, 2 months old, was submitted to necropsy after it died with neurologic deficits. Besides typical FPV intestinal tract changes, multifocal, randomly distributed intracytoplasmic vacuoles within neurons of the thoracic spinal cord were found histologically. Next-generation sequencing identified FPV-specific sequences within the central nervous system. FPV antigen was detected within central nervous system cells, including the vacuolated neurons, via immunohistochemistry. In situ hybridization confirmed the presence of FPV DNA within the vacuolated neurons. Thus, FPV should be considered a cause for neuronal vacuolization in cats presenting with ataxia.

New Avian Hepadnavirus in Palaeognathous Bird, Germany.

In 2015, we identified an avian hepatitis B virus associated with hepatitis in a group of captive elegant-crested tinamous (Eudromia elegans) in Germany. The full-length genome of this virus shares <76% sequence identity with other avihepadnaviruses. The virus may therefore be considered a new extant avian hepadnavirus.

Characterisation of Plasmodium falciparum populations selected on the human endothelial receptors P-selectin, E-selectin, CD9 and CD151.

The ability of the parasite Plasmodium falciparum to evade the immune system and be sequestered within human small blood vessels is responsible for severe forms of malaria. The sequestration depends on the interaction between human endothelial receptors and P. falciparum erythrocyte membrane protein 1 (PfEMP1) exposed on the surface of the infected erythrocytes (IEs). In this study, the transcriptomes of parasite populations enriched for parasites that bind to human P-selectin, E-selectin, CD9 and CD151 receptors were analysed. IT4_var02 and IT4_var07 were specifically expressed in IT4 parasite populations enriched for P-selectin-binding parasites; eight var genes (IT4_var02/07/09/13/17/41/44/64) were specifically expressed in isolate populations enriched for CD9-binding parasites. Interestingly, IT4 parasite populations enriched for E-selectin- and CD151-binding parasites showed identical expression profiles to those of a parasite population exposed to wild-type CHO-745 cells. The same phenomenon was observed for the 3D7 isolate population enriched for binding to P-selectin, E-selectin, CD9 and CD151. This implies that the corresponding ligands for these receptors have either weak binding capacity or do not exist on the IE surface. Conclusively, this work expanded our understanding of P. falciparum adhesive interactions, through the identification of var transcripts that are enriched within the selected parasite populations.

RNA Interference Restricts Rift Valley Fever Virus in Multiple Insect Systems.

The emerging bunyavirus Rift Valley fever virus (RVFV) is transmitted to humans and livestock by a large number of mosquito species. RNA interference (RNAi) has been characterized as an important innate immune defense mechanism used by mosquitoes to limit replication of positive-sense RNA flaviviruses and togaviruses; however, little is known about its role against negative-strand RNA viruses such as RVFV. We show that virus-specific small RNAs are produced in infected mosquito cells, in Drosophila melanogaster cells, and, most importantly, also in RVFV vector mosquitoes. By addressing the production of small RNAs in adult Aedes sp. and Culex quinquefasciatus mosquitoes, we showed the presence of virus-derived Piwi-interacting RNAs (piRNAs) not only in Aedes sp. but also in C. quinquefasciatus mosquitoes, indicating that antiviral RNA interference in C. quinquefasciatus mosquitoes is similar to the described activities of RNAi in Aedes sp. mosquitoes. We also show that these have antiviral activity, since silencing of RNAi pathway effectors enhances viral replication. Moreover, our data suggest that RVFV does not encode a suppressor of RNAi. These findings point toward a significant role of RNAi in the control of RVFV in mosquitoes. IMPORTANCE Rift Valley fever virus (RVFV; Phlebovirus, Bunyaviridae) is an emerging zoonotic mosquito-borne pathogen of high relevance for human and animal health. Successful strategies of intervention in RVFV transmission by its mosquito vectors and the prevention of human and veterinary disease rely on a better understanding of the mechanisms that govern RVFV-vector interactions. Despite its medical importance, little is known about the factors that govern RVFV replication, dissemination, and transmission in the invertebrate host. Here we studied the role of the antiviral RNA interference immune pathways in the defense against RVFV in natural vector mosquitoes and mosquito cells and draw comparisons to the model insect Drosophila melanogaster. We found that RVFV infection induces both the exogenous small interfering RNA (siRNA) and piRNA pathways, which contribute to the control of viral replication in insects. Furthermore, we demonstrate the production of virus-derived piRNAs in Culex quinquefasciatus mosquitoes. Understanding these pathways and the targets within them offers the potential of the development of novel RVFV control measures in vector-based strategies.

Differentiation of Staphylococcus argenteus (formerly: Staphylococcus aureus clonal complex 75) by mass spectrometry from S. aureus using the first strain isolated from a wild African great ape.

The species Staphylococcus argenteus was separated recently from Staphylococcus aureus (Tong S.Y., F. Schaumburg, M.J. Ellington, J. Corander, B. Pichon, F. Leendertz, S.D. Bentley, J. Parkhill, D.C. Holt, G. Peters, and P.M. Giffard, 2015). The objective of this work was to characterise the genome of a non-human S. argenteus strain, which had been isolated from the faeces of a wild-living western lowland gorilla in Gabon, and analyse the spectrum of this species in matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The full genome sequence revealed a scarcity of virulence genes and absence of resistance genes, indicating a decreased virulence potential compared to S. aureus and the human methicillin-resistant S. argenteus isolate MSHR1132T. Spectra obtained by MALDI-TOF MS and the analysis of available sequences in the genome databases identified several MALDI-TOF MS signals that clearly differentiate S. argenteus, the closely related Staphylococcus schweitzeri and S. aureus. In conclusion, in the absence of biochemical tests that identify the three species, mass spectrometry should be employed as method of choice.

Reciprocal regulation of carbon monoxide metabolism and the circadian clock.

Circadian clocks are cell-autonomous oscillators regulating daily rhythms in a wide range of physiological, metabolic and behavioral processes. Feedback of metabolic signals, such as redox state, NAD+/NADH and AMP/ADP ratios, or heme, modulate circadian rhythms and thereby optimize energy utilization across the 24-h cycle. We show that rhythmic heme degradation, which generates the signaling molecule carbon monoxide (CO), is required for normal circadian rhythms as well as circadian metabolic outputs. CO suppresses circadian transcription by attenuating CLOCK-BMAL1 binding to target promoters. Pharmacological inhibition or genetic depletion of CO-producing heme oxygenases abrogates normal daily cycles in mammalian cells and Drosophila. In mouse hepatocytes, suppression of CO production leads to a global upregulation of CLOCK-BMAL1-dependent circadian gene expression and dysregulated glucose metabolism. Together, our findings show that CO metabolism is an important link between the basic circadian-clock machinery, metabolism and behavior.