An ultrasensitive NanoLuc-based luminescence system for monitoring Plasmodium berghei throughout its life cycle

BACKGROUND: Bioluminescence imaging is widely used for cell-based assays and animal imaging studies, both in biomedical research and drug development. Its main advantages include its high-throughput applicability, affordability, high sensitivity, operational simplicity, and quantitative outputs. In malaria research, bioluminescence has been used for drug discovery in vivo and in vitro, exploring host-pathogen interactions, and studying multiple aspects of Plasmodium biology. While the number of fluorescent proteins available for imaging has undergone a great expansion over the last two decades, enabling simultaneous visualization of multiple molecular and cellular events, expansion of available luciferases has lagged behind. The most widely used bioluminescent probe in malaria research is the Photinus pyralis firefly luciferase, followed by the more recently introduced Click-beetle and Renilla luciferases. Ultra-sensitive imaging of Plasmodium at low parasite densities has not been previously achieved. With the purpose of overcoming these challenges, a Plasmodium berghei line expressing the novel ultra-bright luciferase enzyme NanoLuc, called PbNLuc has been generated, and is presented in this work. RESULTS: NanoLuc shows at least 150 times brighter signal than firefly luciferase in vitro, allowing single parasite detection in mosquito, liver, and sexual and asexual blood stages. As a proof-of-concept, the PbNLuc parasites were used to image parasite development in the mosquito, liver and blood stages of infection, and to specifically explore parasite liver stage egress, and pre-patency period in vivo. CONCLUSIONS: PbNLuc is a suitable parasite line for sensitive imaging of the entire Plasmodium life cycle. Its sensitivity makes it a promising line to be used as a reference for drug candidate testing, as well as the characterization of mutant parasites to explore the function of parasite proteins, host-parasite interactions, and the better understanding of Plasmodium biology. Since the substrate requirements of NanoLuc are different from those of firefly luciferase, dual bioluminescence imaging for the simultaneous characterization of two lines, or two separate biological processes, is possible, as demonstrated in this work

The machinery underlying malaria parasite virulence is conserved between rodent and human malaria parasites

Sequestration of red blood cells infected with the human malaria parasite Plasmodium falciparum in organs such as the brain is considered important for pathogenicity. A similar phenomenon has been observed in mouse models of malaria, using the rodent parasite Plasmodium berghei, but it is unclear whether the P. falciparum proteins known to be involved in this process are conserved in the rodent parasite. Here we identify the P. berghei orthologues of two such key factors of P. falciparum, SBP1 and MAHRP1. Red blood cells infected with P. berghei parasites lacking SBP1 or MAHRP1a fail to bind the endothelial receptor CD36 and show reduced sequestration and virulence in mice. Complementation of the mutant P. berghei parasites with the respective P. falciparum SBP1 and MAHRP1 orthologues restores sequestration and virulence. These findings reveal evolutionary conservation of the machinery underlying sequestration of divergent malaria parasites and support the notion that the P. berghei rodent model is an adequate tool for research on malaria virulence

Inducing Cross-Clade Neutralizing Antibodies against HIV-1 by Immunofocusing

Background: Although vaccines are important in preventing viral infections by inducing neutralizing antibodies (nAbs), HIV-1 has proven to be a difficult target and escapes humoral immunity through various mechanisms. We sought to test whether HIV-1 Env mimics may serve as immunogens. Methodology/Principal Findings: Using random peptide phage display libraries, we identified the epitopes recognized by polyclonal antibodies of a rhesus monkey that had developed high-titer, broadly reactive nAbs after infection with a simianhuman immunodeficiency virus (SHIV) encoding env of a recently transmitted HIV-1 clade C (HIV-C). Phage peptide inserts were analyzed for conformational and linear homology using computational analysis; some peptides mimicked various domains of the original HIV-C Env, such as conformational V3 loop epitopes and the conserved linear region of the gp120 C-terminus. Next, we devised a novel prime/boost strategy to test the immunogenicity of such phage-displayed peptides and primed mice only once with HIV-C gp160 DNA followed by boosting with mixtures of recombinant phages. Conclusions/Significance: This strategy, which was designed to focus the immune system on a few Env epitopes (immunofocusing), not only induced HIV-C gp160 binding antibodies and cross-clade nAbs, but also linked a conserved HIV Env region for the first time to the induction of nAbs: the C-terminus of gp120. The identification of conserved antige

Expression of the Ottoman period (Tourkokratia) in Greece: History of the Greek nation's sample

Bu çalışma, Yunanistan tarihi alanında temel çalışmalardan birisi olarak kabul edilen ve Yunanca kaleme alınmış ansiklopediden yararlanılarak Türkiye merkezli bir bakış açısı ile meydana getirilmiştir. Kaynak olarak kullanılan ve Yunan tarih yazımında en önemli çalışmalardan biri olan İstoria Tou Ellinikou Ethnous adlı ansiklopedide anlatılan ve yaklaşık 400 yıl süren Yunanistan'daki Osmanlı hâkimiyeti (Tourkokratia, 1453-1669) dönemi bu çalışmanın konusunu oluşturmuştur. Bu tezde, İslamlaştırma, devşirme, Ortodoksluğun Müslüman bir ülkede varlığını devam ettirmesi, Osmanlı'ya karşı muhalif eylemler ile Batı'ya giden Yunanların eğitim alanlarında yaptıkları değişim ve gelişimler gibi konular adı geçen çalışma doğrultusunda incelenerek değerlendirilmiştir. Bu tez içerisinde 1453-1669 yılları arasındaki dönem için İstoria Tou Ellinikou Ethnous (Yunan Ulusu Tarihi) adlı ansiklopedide verilen bilgiler önce konulara göre tasnif edilmiş ve bu bilgiler Türkiye'deki tarih yazımı ile değerlendirilerek farklılıklar ortaya konulmaya çalışılmıştır. Dört bölümden oluşan tezde her bir bölümde öncelikle ana ve alt başlıklar altında Yunan tarih yazımında (İstoria Tou Ellinikou Ethnous) konuların işleniş şekli ve yorumlanması yer almıştır. Sonuç bölümünde ise bu konuların Türk tarih yazımında nasıl işlendiği ve anlatıldığına değinilmiştir.This study was created with a Turkey-centered point of view by making use of the encyclopedia written in Greek, which is accepted as one of the main studies in the field of Greek history. The subject of this study is the period of Ottoman domination in Greece (Tourkokratia, 1453-1669), which is used as a source and described in the encyclopedia Istoria Tou Ellinikou Ethnous which is one of the most important studies in Greek historiography. In this thesis, issues such as Islamization, devshirme, the continuation of Orthodoxy in a Muslim country, opposition actions against the Ottoman Empire, and the changes and developments in the educational fields of the Greeks who went to the West were examined and evaluated in line with the aforementioned study. In this thesis, the information given in the encyclopedia Istoria Tou Ellinikou Ethnous (History of the Greek Nation) for the period between 1453 and 1669 was first classified according to the subjects and this information was evaluated with the historiography in Turkey and studied to reveal the differences. In the thesis, which consists of four chapters, in each chapter, firstly, under the main and sub-titles, the way in which the subjects were handled and interpreted in Greek historiography (Istoria Tou Ellinikou Ethnous). In the conclusion part, it is mentioned how these subjects are handled and explained in Turkish historiography

Decoy exosomes provide protection against bacterial toxins.

The production of pore-forming toxins that disrupt the plasma membrane of host cells is a common virulence strategy for bacterial pathogens such as methicillin-resistant Staphylococcus aureus (MRSA)1-3. It is unclear, however, whether host species possess innate immune mechanisms that can neutralize pore-forming toxins during infection. We previously showed that the autophagy protein ATG16L1 is necessary for protection against MRSA strains encoding α-toxin4-a pore-forming toxin that binds the metalloprotease ADAM10 on the surface of a broad range of target cells and tissues2,5,6. Autophagy typically involves the targeting of cytosolic material to the lysosome for degradation. Here we demonstrate that ATG16L1 and other ATG proteins mediate protection against α-toxin through the release of ADAM10 on exosomes-extracellular vesicles of endosomal origin. Bacterial DNA and CpG DNA induce the secretion of ADAM10-bearing exosomes from human cells as well as in mice. Transferred exosomes protect host cells in vitro by serving as scavengers that can bind multiple toxins, and improve the survival of mice infected with MRSA in vivo. These findings indicate that ATG proteins mediate a previously unknown form of defence in response to infection, facilitating the release of exosomes that serve as decoys for bacterially produced toxins

Oligothiophene-Bridged Conjugated Covalent Organic Frameworks

Two-dimensional covalent organic frameworks (2D-COFs) are crystalline, porous materials comprising aligned columns of π-stacked building blocks. With a view toward the application of these materials in organic electronics and optoelectronics, the construction of oligothiophene-based COFs would be highly desirable. The realization of such materials, however, has remained a challenge, in particular with respect to laterally conjugated imine-linked COFs. We have developed a new building block design employing an asymmetric modification on an otherwise symmetric backbone that allows us to construct a series of highly crystalline quaterthiophene-derived COFs with tunable electronic properties. Studying the optical response of these materials, we have observed for the first time the formation of a charge transfer state between the COF subunits across the imine bond. We believe that our new building block design provides a general strategy for the construction of well-ordered COFs from various extended building blocks, thus greatly expanding the range of applicable molecules

Natalizumab restores aberrant miRNA expression profile in multiple sclerosis and reveals a critical role for miR-20b

Objective: To identify microRNAs (miRNAs) regulated by anti-alpha 4 integrin monoclonal antibody therapy (natalizumab) in the peripheral blood of patients with relapsing-remitting (RR) multiple sclerosis (MS) and to confirm their role in experimental settings in vivo. Methods: In a longitudinal study of 17 RR-MS patients, we investigated blood miRNA expression profiles at baseline and after 1 year of natalizumab therapy by microarray technique and quantitative PCR validation. We compared the baseline expression profiles of these patients to those of 18 age-and sex-matched healthy controls. We confirmed the contribution of resulting candidate miRNAs in an animal model of MS, experimental autoimmune encephalomyelitis (EAE) induced by adoptive transfer of proteolipid protein (PLP)(139-151)-activated lymphocytes in SJL/J mice or by active immunization of miR-106a similar to 363-deficient C57BL/6 mice (or wildtype litter mates) with myelin oligodendrocyte glycoprotein (MOG)(35-55). Results: Our longitudinal analysis revealed that miR-18a, miR-20b, miR-29a, and miR-103 were upregulated and predominantly expressed by CD4(+) T cells, whereas miR-326 was downregulated upon natalizumab treatment. A comparison of untreated RR-MS patients at baseline with healthy controls revealed that the four natalizumab-upregulated targets were initially downregulated in MS. All confirmed targets showed disease-dependent expression in splenocytes of mice suffering from EAE. Genetic deletion of the miRNA cluster miR-106a similar to 363 (containing natalizumab-regulated miR-20b) resulted in a more severe EAE course and an in vivo upregulation of the miR-20b target genes rorgt, stat3, and vegfa. Interpretation: Our study indicates that natalizumab restores dysregulated miRNA patterns in MS and reveals the contribution of miR-20b in autoimmune demyelination in vivo

The machinery underlying malaria parasite virulence is conserved between rodent and human malaria parasites

Altres ajuts: Swiss National Foundation (grant 10030_140691/1), Deutsche Forschungsgemeinschaft (SP 1209/1)Sequestration of red blood cells infected with the human malaria parasite Plasmodium falciparum in organs such as the brain is considered important for pathogenicity. A similar phenomenon has been observed in mouse models of malaria, using the rodent parasite Plasmodium berghei, but it is unclear whether the P. falciparum proteins known to be involved in this process are conserved in the rodent parasite. Here we identify the P. berghei orthologues of two such key factors of P. falciparum, SBP1 and MAHRP1. Red blood cells infected with P. berghei parasites lacking SBP1 or MAHRP1a fail to bind the endothelial receptor CD36 and show reduced sequestration and virulence in mice. Complementation of the mutant P. berghei parasites with the respective P. falciparum SBP1 and MAHRP1 orthologues restores sequestration and virulence. These findings reveal evolutionary conservation of the machinery underlying sequestration of divergent malaria parasites and support the notion that the P. berghei rodent model is an adequate tool for research on malaria virulence